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Spring 4-2014 The Role of Sirtuin 1 and in t10c12 Conjugated Linoleic Acid Induced Delipidation in 3T3-L1 Adipocytes Wei Wang University of Nebraska-Lincoln, [email protected]

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The Role of Sirtuin 1 and Ceramide in t10c12 Conjugated Linoleic Acid Induced

Delipidation in 3T3-L1 Adipocytes

By

Wei Wang

A DISSERTATION

Presented to the Faculty of

The Graduate College at the University of Nebraska

In Partial Fulfillment of Requirements

For the Degree of Doctor of Philosophy

Major: Animal Science

Under the Supervision of Professor Merlyn Nielsen

Lincoln, Nebraska

April, 2014

The Role of Sirtuin 1 and Ceramide in t10c12 Conjugated Linoleic Acid Induced

Delipidation in 3T3-L1 Adipocytes

Wei Wang, Ph.D.

University of Nebraska, 2014

Advisers: Merlyn Nielsen and Michael Fromm

Project 1: Trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) reduces triglyceride

(TG) levels in adipocytes through multiple pathways, with AMP-activated protein

(AMPK) generally facilitating, and peroxisome proliferator-activated receptor γ (PPARγ)

generally opposing these reductions. Sirtuin 1 (SIRT1), a histone/protein deacetylase that

affects energy homeostasis, often functions coordinately with AMPK, and is capable of

binding to PPARγ, thereby inhibiting its activity. This study investigated the role of

SIRT1 in the response of 3T3-L1 adipocytes to t10c12 CLA by testing the following

hypotheses: 1) SIRT1 is functionally required for robust TG reduction; and 2) SIRT1,

AMPK, and PPARγ cross regulate each other. Inhibition of SIRT1 amounts or activity

attenuated the amount of TG loss, while SIRT1 activator SRT1720 increased the TG loss.

SRT1720 increased AMPK activity while sirtuin-specific inhibitors decreased AMPK

activity. Reciprocally, an AMPK inhibitor reduced SIRT1 activity. Treatment with

t10c12 CLA increased PPARγ phosphorylation in an AMPK-dependent manner and

increased the amount of PPARγ bound to SIRT1. Reciprocally, a PPARγ agonist

attenuated AMPK and SIRT1 activity levels. These results indicated SIRT1 increased TG

loss and that cross regulation between SIRT1, AMPK, and PPARγ occurred in 3T3-L1

adipocytes treated with t10c12 CLA.

Project 2: In the current study profiling of 261 metabolites was conducted to gain new

insights into the biological pathways responding to t10c12 CLA in 3T3-L1 adipocytes.

Sphingonine, sphingosine, and ceramide levels were observed to be highly elevated in

t10c12 CLA treated adipocytes. Exogenous chemicals that increase endogenous ceramide levels decreased levels in adipocytes, and activated AMPK as well as nuclear factor kappa-B (NF-κB), both of which are normally activated in CLA treated adipocytes.

Concurrent inhibition of de novo ceramide and entry from existing

sphingoglipid pools attenuated the lipid lowering and apoptotic effects normally

associated with responses to t10c12 CLA. Our results indicate are an important

component of the lipid lowering response in t10c12 CLA treated adipocytes.

Acknowledgments

I would like to express my great appreciation to Dr. Michael Fromm and Dr. Merlyn

Nielsen for their patient guidance, constructive suggestions and enthusiastic encouragement. Thank my Ph.D. committee members, Dr. Concetta C. DiRusso and Dr.

Thomas Burkey for their time, suggestion and support assistance. My appreciation is extended to all my lab mates and facility stuff in Beadle Center for their great help during the planning and development of this research.

I also would like to thank the University of Nebraska-Lincoln, the Graduate School, the

Department of Animal Science and the Graduate Program for their support throughout my Ph.D. training.

Finally I would like to express deep gratitude to my family and friends for their support and encouragement.

i

Table of Contents

Chapter I. Literature Review 1

Introduction 2

Biological effects of CLA 3

Potential mechanism 5

t10c12 CLA alternates energy homeostasis 5

t10c12 CLA inhibits lipogenesis 7

t10c12 CLA inhibits adipogenesis 8

t10c12 CLA promotes lipolysis 8

t10c12 CLA causes insulin resistance 10

t10c12 CLA causes inflammation 11

Conclusion and implication 14

Chapter II. cross regulation of sirtuin 1, AMPK, and PPARγ in conjugated linoleic acid treated adipocytes. 29

Introduction 30

Experimental Procedures 33

Results 38

Discussion 46

References 61

Chapter III. are required for Efficient Triglyceride Loss in Conjugated

Linoleic Acid Treated 3T3-L1 Adipocytes 67

Introduction 69

Material and methods 70 ii

Results 75

Discussion 80

References 97

Chapter IV. Conclusion 101

iii

Table of Figures

Chapter II

Figure 1. SIRT activity affects TG levels in t10c12 CLA treated 3T3-L1 adipocytes. 52

Figure 2. SIRT1 affects fatty acid metabolism and inflammatory mRNA levels. 53

Figure 3. SIRT1 affects ACC and AMPK phosphorylation levels after t10c12 CLA treatment. 54

Figure 4. PPARγ antagonists and agonists affect TG levels and modulate p-AMPK activity levels in t10c12 CLA treated 3T3-L1 adipocytes. 55

Figure 5. PPARγ agonists or antagonists affect the TG loss caused by AMPK activators without t10c12 CLA being present. 57

Figure 6. SIRT1, AMPK and PPARγ affect the t10c12 CLA-dependent decrease in the amount of acetylated NF-κB. 58

Figure 7. Treatment with t10c12 CLA increases the interaction of SIRT1 with PPARγ or

NCoR1. 59

Figure 8. Summary diagram for proposed effects of AMPK, SIRT1, PPARγ, and their activators or inhibitors in 3T3-L1 adipocytes. 60

Chapter III

Figure 1. Biosynthetic pathway of ceramide and sphingolipids. 86

Figure 2. Ceramide and TG levels in treated 3T3-L1 adipocytes. 87

Figure 3. shRNA knockdown of SPTLC and S1PP: effects on TG and ceramide levels. 89

Figure 4. Inhibition of both biosynthetic entry points into ceramides affects TG and ceramide levels. 91 iv

Figure 5. Ceramide signaling is downstream of AMPK. 92

Figure 6. Ceramides activate NF-κB independently of activated AMPK. 93

Figure 7. Ceramides cause apoptosis in 3T3-L1 adipocytes. 95

Supplemental Figure 1. Monoclonal antibody to ceramides does not detect C2 ceramide.

96

Appendix. Metabolic Profile of Mouse 3T3-L1 Adipocytes 104

1

Chapter I. Literature Review

Trans-10, Cis-12 Conjugated Linoleic Acid and Delipidation

2

Introduction

Obesity has become a global problem. In 2010, 6.7% children (43 million) were

estimated to be overweight, and this incidence keeps increasing [1]. World Health

Organization (WHO) predicted that there will be about 2.3 billion overweight adults by

2015 worldwide and over 1/3 of them will be obese [2]. In the United States, obesity

incidence increased 24% from 2000 to 2005. Keeping this trend, there will be about 50%

obese adults and 30% obese children respectively in the U.S. by 2030 [3,4]. Obesity has

been associated with numerous diseases, including type 2 diabetes mellitus, breathing

difficulties during sleep, hypertension, heart disease, stroke and even certain types of

cancer [5,6]. For women, prenatal obesity is associated with gestational hypertension,

preeclampsia, large-for-gestational-age (LGA) baby and higher incidence of congenital

defects [7]. The most popular treatments for obesity are exercise-plus-diet which needs a

long term lifestyle modification or a bariatric surgery, which has an unexpected prognosis

[8]. Convenient and effective method for obesity therapeutic treatment is urgently

demanded.

Trans10, cis12 conjugated linoleic acid (t10c12 CLA) has been established to causes a

strong delipidation in vitro in mouse and human adipocytes, and in mice [9-12].

Applications in human trials have had modest results. T10c12 CLA belongs to the C18

linoleic acid fatty acid family which contain at least 28 isoforms. T10c12 CLA is a

polyunsaturated fatty acid which has a lipid number of 18 and 2 double bonds in the 10th and 12th carbons. Conjugated double bonds are separated by a single bond to form trans-

10, cis-12 octadecadienoic acid. CLA is found primarily in the meat and dairy products of 3 ruminants. Anaerobic bacteria convert linoleic acid into different isoforms of CLA via biohydrogenation [13]. Several isoforms of CLA have been identified but the cis-9, trans-

11 isoform (rumenic acid) is the predominant form in ruminant products (about 90%).

Though t10c12 CLA isoform comprises only 10%, it is specifically responsible for the

CLA induced delipidation effect [14-17].

Biological effects of CLA

CLA has been shown to have several beneficial effects such as anti-carcinogenic [18,19], anti-atherogenic [20-22], anti-diabetogenic [23] and anti-obesity [24-26] properties. CLA was initially discovered and identified as an anticarcinogen in 1987 [27]. Park’s group firstly reported the delipidation effect of CLA in 1997 [28]. In their study, both male and female mice lost nearly 60% body fat after 5 weeks of dietary supplementation with 0.5% t9c11/t10c12 CLA mixture (1:1 wt/wt). Subsequent studies showed that supplementation with a CLA mixture (t10c12 and c9t11 isoforms) or the t10c12 isoform alone decreased body fat mass in murine [28-30], chicken [31], rabbit [32], pigs [33] and some human studies [24-26]. Later studies proven this delipidation effect is attributed to the t10c12 isoform [34,35]. Our previous data demonstrated that mice lost more than 75% of white adipose tissue mass in the retroperitoneal and epididymal fat pad after 2 weeks consumption of 0.5% t10c12 CLA [9]. Yet, CLA induced body fat losses may be reversible, as removal of CLA from diet result in recover of triglyceride accumulation

[36].

4

In vitro, t10c12 CLA induced delipidation occurs in mouse 3T3-L1 and human primary adipocytes. Concentrations of 50-200µM of t10c12 CLA caused a dose dependent

triglyceride loss in 3T3-L1 adipocytes [37]. In our previous study, 50µM and 100µM

t10c12 CLA caused the robust loss of triglyceride content by 25% and 50% respectively in 3T3-L1 adipocyte compare to control group [38]. The McIntosh group recently confirmed t10c12 CLA induced delipidation in human primary adipocyte. 30 µM t10c12

CLA decreased synthesis of triglyceride, free fatty acid, diacyglycerol, cholesterol esters,

and within 3-24 h [39].

CLA induced delipidation effect in human studies are controversial. Some studies

reported that supplementation of CLA result in increased body lean mass and decreased

body fat mass [26,40,41]. However, most human studies showed that there is no

significant delipidation effect of CLA on body composition. For example,

supplementation of t9c11/t10c12 CLA mixture (1:1 wt/wt) for 14 weeks had no effect on

body composition in healthy adults [42]. In another study, supplementation of 3 gram

CLA mixture daily could not prevent body weight regain in moderately obese subjects

who had lost 10 kilogram weight previously via limiting calorie intake [43]. Finally, the

Arnaiz group did show positive delipidation effect of CLA mixture, but only in

overweight subjects, not in obese subjects [44]. Dosage differences between animal and

human studies may contribute to these contradictory findings. A typical dosage of CLA

in human study is approximately 0.05g/kg body weight. As comparison, a typical dosage

used in mouse studies is about 1.05g/kg body weight, which is 20 times the human dose.

Additionally, human studies have gender, weight, age and healthy status differences, 5

which increase the complexity of interpreting any outcomes. Our goal is to combine

t10c12 CLA with other nutritional supplements to attain synergistic interactions to induce a respectable fat loss at a lower CLA dose. This has great therapeutic potential for treating human obesity.

Potential mechanism

T10c12 CLA alters energy homeostasis

Biological homeostasis between energy intake and energy expenditure is critical for maintaining body weight. A potential mechanism by which t10c12 CLA induces

delipidation is by decreasing energy intake and/or increasing energy expenditure.

Supplementation of CLA mixture or t10c12 CLA has been shown to modestly reduce

feed intake in mice [28,35,45]. The delay in the effect of CLA on a feed intake reduction

indicates CLA might exert its effort indirectly [46,47]. A suggested mechanism for this

reduction in feed intake is CLA may regulate hypothalamic appetite-regulating genes.

The Li group demonstrated that supplementation of CLA for 27 days decreased gene

expression ratio of proopiomelanocortin (POMC) to neuropeptide Y (NPY) in

hypothalamus [45]. In another study in rat, hypothalamic injection of a CLA mixture

reduced the expression of neuropeptide Y (NPY) and agouti-related protein (AgRP)

while elevated circulating leptin level [48]. However, most studies showed that t10c12

CLA has little effect on feed intake [49-52]. In our previous study, Mice fed 0.5% t10c12

CLA for 14 days lost 85% of their white adipose tissue and didn’t change their feed

intake [9]. Additionally, CLA-mediated reduction in feed intake tends to diminish over

time while the reduction in body fat is maintained [47,53]. These results indicate that 6

CLA-mediated feed intake reduction via regulating neuronpeptide may partially

contribute to its delipidation effects.

In the case of energy expenditure, t10c12 CLA was reported to increase energy

expenditure via increasing basal metabolic rate, thermogenesis or lipid oxidation. In a

human study, consumption of 1.8 g or 3.6 g CLA/day for 14weeks increased resting

metabolic rate in both sexes compare to a placebo group [54]. Supplementation of CLA

mixture or the t10c12 isoform has been proven to induce uncoupling proteins 2 (UCP2)

transcription in white adipose tissue [9,55]. UPC2 is the most highly expressed

uncoupling protein and can be found in a variety of tissues including white adipose tissue.

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins that divert ATP

synthesis to heat production. In Balb-C mice, supplementation of CLA for 39 days

increased the percentage of energy expenditure as heat [50]. Our previous study also

demonstrated that 0.5% t10c12CLA supplementation for 2 weeks increased UCP1 and

UCP2 transcriptional levels in mice [9]. T10c12 CLA was also reported to increase

carnitine palmitoyltransferase 1 (CPT1) levels which is the rate-limiting for fatty

acid beta-oxidation in the mitochondria. Our previous study demonstrated that 0.5%

t10c12 CLA supplementation for 2 weeks increased CPT1 mRNA level in fat tissue [9].

Consistent with this findings, t10c12 CLA was reported to increase beta-oxidation in differentiating 3T3-L1 preadipocyte [56].

Activation of UCPs increases the ratio of AMP to ATP, which activates 5’-AMP- activated protein kinase (AMPK) [57]. AMPK, activated by phosphorylation, is a central 7 regulator of cellular energy levels. Activated AMPK regulates a number of substrates’ activity by phosphorylation, including activation of hormone sensitive lipase (HSL) which leads to activation of lipolysis [58], and deactivates acetyl CoA carboxylase (ACC) which result in lipogenesis suppression [59]. Activation of AMPK was observed as early as 0.5 h in response to t10c12 CLA treatment in 3T3-L1 adipocyte [38]. AMPK inhibitor compound C attenuated the ability of t10c12 CLA to induce triglyceride loss while its agonist metformin strengthened t10c12 CLA’s ability. These data suggest that the activation of AMPK plays an important role in t10c12 CLA induced delipidation.

t10c12 CLA inhibits lipogenesis

Lipogenesis is the process by which acetyl-CoA is converted to fatty acids, which are subsequently esterified with glycerol to form triglycerides. Our previous study demonstrated that t10c12 CLA decreased Sterol regulatory element-binding proteins

(SREBP) 1c expression in 3T3-L1 adipocyte [38]. SREBPs are transcription factors that regulate sterol biosynthesis. The activation of SREBPs is controlled by sterol levels through negative feedback regulation. When cell sterol levels are low, SREBPs are activated by cleavage. Activated SREBPs translocate to the nucleus where they bind to specific sterol regulatory element DNA sequence, regulating the synthesis of involved in sterol biosynthesis. Among all isoforms, SREBP-1c is responsible for regulating the genes required for lipogenesis. By down regulating SREBP-1c, t10c12

CLA decreased numerous proteins involved in lipogenesis such as ACC, fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD), lipoprotein lipase (LPL), glycerol phosphate acyltransferase and acylglycerol phosphate acyltransferase [9,60]. Additionally, 8

t10c12 CLA may also inhibits lipogenesis via suppressing peroxisome proliferator- activated receptor γ (PPARγ) activity. PPARγ is a type II nuclear receptor that regulates transcription of various downstream lipogenic genes, including glycerol-3-phosphate dehydrogenase, LPL, lipin and perilipin [61]. Our microarray data also showed that in mice or 3T3-L1 adipocytes, t10c12 CLA decreased the mRNA levels of some other lipogenic genes, including ATP citrate lyase, ACC-α and -β, caveolin I and lipin 1[9].

T10c12 CLA inhibits adipogenesis

Adipogenesis is the process of differentiation of preadipocytes to adipocytes. This process involves the activation of key transcription factors including PPARγ, PPARγ co- activators, fatty acid binding proteins (aP2) and CAAT/enhancer binging protein (C/EBP).

CLA has been proven to suppresses preadipocyte differentiation by attenuating PPARγ,

C/EBPα, SREBP 1c, liver X receptor α (LXRα) and aP2 expression [60,62-64]. Besides down regulation of PPARγ expression, t10c12 CLA treatment also decreases PPARγ activity and its target genes’ expression [65,66]. The McIntosh group demonstrated that

CLA may inhibit PPARγ activity via mitogen-activated protein kinase (MAPK) pathway which leads to PPARγ phosphorylation [12]. Because the expression of PPARγ is directly or indirectly induced by itself, deactivated PPARγ suppresses its own expression and its target genes. In preadipocytes, our previous study showed that t10c12 CLA increased the expression of preadipocyte factor 1(Pref 1), which inhibits adipogenesis via suppressing the expression of PPARγ and C/EBPα [9].

t10c12 CLA promotes lipolysis 9

Several studies have reported that t10c12 CLA increase lipolysis in 3T3-L1 and human adipocytes [28,67]. Lipolysis is the process by which stored triglyceride is mobilized and hydrolyzed into free fatty acid (FFA) and glycerol through the action of HSL. CLA may induce lipolysis via liberating FFA for uptake in metabolically active tissues like liver and muscle. Perilipin coats lipid droplets act as a protective barrier to prevent HSL accessing to neutral lipid substrates[68]. Perilipin is activated by protein kinase A (PKA, also known as cAMP-dependent protein kinase) and then exposing the stored to

HSL. Among all isoforms, perilipin A is the most abundant protein associated with adipocyte lipids [69]. Adipocyte differentiation-related protein (ADRP) is another lipid droplet-associated protein that facilitates lipid deposition or hydrolysis. Compare to perilin, ADRP is found on the surface of small lipid droplets rather than larger lipid droplets. McIntosh’s group demonstrated that t10c12 CLA increased lipolysis in human stromal vascular cells. In this research, t10c12 CLA alteration of adipocyte morphology was explained by elevated lipolysis and replacement of perilin by ADRP [67].

CLA may also promotes lipolysis via increasing HSL expression. Our previous study showed that supplementation of t10c12 CLA for 3 days in C57BL/6J mice increased HSL mRNA level [9]. Released glycerol and FFA from lipolysis can be reused to synthesize triglyceride which is an ATP dependent process. Lipolysis followed by this futile cycle may partially explain CLA induced energy expenditure increases. Consist with this mechanism, OLETF rat supplemented with CLA for 4 weeks lost 5% body weight and had lower serum FFA levels compare to control group [70]. In vitro, a study also confirmed that CLA treatment reduced glycerol release from rat adipocytes [71]. 10

t10c12 CLA causes insulin resistance

CLA mixture or t10c12 isoform has been reported to induce insulin resistance in mice and humans [72-74]. T10c12 CLA supplementation also results in hyperinsulinia, which is associated with insulin resistance [75-77]. Insulin enhances glucose uptake in muscle and adipose tissue and inhibits release of fatty acids in adipose tissue. Insulin resistant cells fail to response to insulin, cannot take in glucose, amino acid and fatty acid, leading to hyperglycemia. Additionally, insulin activates LPL and inhibits HSL in adipocyte

[78,79], which result in decreased LPL-mediated lipogenesis and ineffective inhibition of

HSL-mediated lipolysis in adipose tissue. CLA may induce insulin resistance via suppressing translocation or expression of glucose transporters (GLUT). Glucose transporters, which facilitate translocation of glucose over a plasma membrane, play an important role in insulin action. McIntosh’s group demonstrated that t10c12 CLA inhibited glucose uptake in primary human adipocytes by decreasing glucose transporter

4 (GLUT 4) gene expression [60]. In our previous study, supplementation of t10c12 CLA decreased glucose/fructose transporter GLUT 5 mRNA level in 3T3-L1 adipocyte and mice white adipose tissue [9].

CLA may also induce insulin resistance through regulating adiponectin. Besides regulating glucose level and fatty acid oxidation, adiponectin directly sensitizes the body to insulin and plays a protective role against insulin resistance [80,81]. Disruption of adiponectin caused insulin resistance in mice [82]. Supplementation of CLA decreased adiponectin mRNA level in mice, rat, 3T3-L1 adipocyte and human adipocyte [71,83-85]. 11

Because adiponectin is regulated by PPARγ, its reduction may partly result from CLA’s

suppression of PPARγ.

t10c12 CLA causes inflammation

t10c12 CLA has been reported causes inflammation in 3T3-L1 adipocytes [9].

Proinflammatory cytokines, which can be produced by white adipose tissue, promote inflammation in adipocytes by suppressing lipid synthesis and increasing lipolysis.

Nuclear factor kappa-B (NF-κB) regulates many cytokines genes like tumor necrosis factor alpha (TNFα) and interleukin (IL). T10c12 CLA has been demonstrated to increase

the expression or secretion of TNFα, IL-1β, IL-6, IL-8, IL-10, IL-15, IL-17 [9,73,83,85],

which suppress PPARγ activity and insulin sensitivity. McIntosh’s group showed that

t10c12 may up-regulate these cytokines via activation of ERK1/2 and NF-κB. These secreted cytokines activate their cognate cell surface receptors which in turn activate NF-

κB and ERK1/2. Consequently, activated NF-κB p65 results in suppression of PPARγ,

LPL, FAS, GLUT4, aP2, sterol-CoA desaturase (SCD) and perilipin [73]. Interestingly, our microarray data suggests that t10c12 CLA induced inflammatory response may be transient. Most of these gene’s expression reduced to normal or even lower level after the first week of t10c12 CLA treatment [9].

The inflammation response in t10c12 CLA treated cell may be initiated by an integrated stress response (ISR). ISR is characterized by the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) under endoplasmic reticulum stress, amino acid starvation or oxidants. Phosphorylation of eIF2α also increases the release of 12

proinflammatory cytokines by activating NF-κB and decreasing NF-κB inhibitor IkappaB

level [86,87]. In our study, phosphorylation of eIF2α by t10c12 CLA treatment increased

occurred earlier than the increase in the mRNA levels of genes involved in the ISR,

including activating transcription factor 3 (ATF3), C/EBP homologous protein (CHOP)

and pseudokinase tribbles 3 (TRB3) which are involved in cell apoptosis [88].

Microarray data analysis indicated that IRS is the earliest transcriptional response that

occurs in t10c12 CLA treated 3T3-L1 adipocytes and mice, prior to the induction of

proinflammatory cytokines such as IL-6 and IL-8 [9]. These data suggest that ISR plays

an early important role in induction of the later inflammation and apoptosis.

t10c12 CLA causes moderate apoptosis

Apoptosis was thought to be the major mechanism of t10c12 CLA induced triglyceride

loss [46,56,89]. Apoptosis is a process of programmed cell death (PCD) in organisms.

Our recent studies in mouse model showed that supplementation of t10c12 CLA reduced

adipocyte numbers by 15% and cell volumes by 90% in fat tissues. Our microarray

analysis also demonstrated that t10c12 CLA increased numerous gene expression involved in apoptotic signaling and DNA repair, including TNF ligands, TNF responsive genes, MdM-2, APAF1, BAX, PMAIP1, MCL1, PCNA,GADD45B, CDKNA1 (pRB), caspases 1 and 4 [9].

Our recent metabolite studies indicate CLA may causes apoptosis via regulating biosynthesis. Ceramide, sphingosine and shpingosine-1-phosphytate (S1P) are interconvertible sphingolipid metabolites. Ceramide and S1P exert opposite effort in 13 cell survival [90,91]. Conversion of sphingosine to S1P enhances cell growth and survival while dephosphorylation of S1P to sphingosine, which is acylated to form various ceramides, has been shown to be associated with growth arrest and apoptosis [92].

Ceramide plays an important role in initiating apoptotic events at the plasma membrane.

Ceramide can be generated from either de novo synthesis or hydrolysis

[93-95]. In some apoptosis models, ligands (FAS, TRAIL) binding to their specific death receptor or stress stimuli activate sphingomyelinase [96-98] which translocate from cytosol to outer plasma membrane [99]. Hydrolysis of sphingomyelin by sphingomyelinase turns lipid rafts, which is enriched in sphingolipids[100], to ceramide enriched platforms [98,101,102]. These platforms are considered to be instrumental in reorganization of signaling molecules and clustering of receptor molecules at the [103,104]. In Fas induced apoptosis, clustering of receptor recruit Fas- associated death domain (FADD) and caspase-8 which forming the death-inducing signalling complex (DISC) [105].

In mitochondria, ceramides synergize with Bax to increase outer membrane permeability

[106]. On one hand, ceramides faciliate Bax and/or Bak oligomerization, which form mitochondrial apoptosis channel in the outer membrane [107,108]. On the other hand, ceramides may also induce channels in the mitochondrial membrane [109-111].

Formation of these channels lead to leakage of apoptogenic factors such as cytochrome c, apoptosis inducing factor, and DIABLO into the cytosol which facilitating the formation of apoptosome complex [112]. In lysosomes, ceramide generation activates aspartic protease Cathepsin D which then cleaves and activate pro-apoptotic protein BH3 14 interacting domain death agonist (BID) [113]. Truncated BID leads to activation of cysteine-aspartic proteases (caspase), resulting in apoptosis[114].

The mechanism of how t10c12 CLA elevates ceramide levels remains unknown. Since no specific receptor for t10c12 CLA has been reported, lipid rafts may play an intermediate role in the process. Increasing evidence indicates that long chain polyunsaturated fatty acid (PUFA), like DHA, could alter lipid rafts behavior and protein function [115].

Docosahexaenoic acid (DHA) has been proven to alter basic properties of cell membranes, including elastic compressibility, ion permeability, fusion, the size and distribution of lipid rafts [115,116]. Eicosapentaenoic acid (EPA) and DHA have been demonstrated to decrease lipid raft sphingomyelin and increase lipid raft ceramide levels in human breast cancer cells[117]. These results provide a potential role of CLA in incorporating into sphingolipid rich raft domain and modulating ceramide level.

It is also unclear in t10c12 CLA induced triglyceride loss, what level of apoptosis is caused by ceramides. Our previous study based on microarray analysis indicated that apoptosis may be not the main effect on t10c12 CLA induced delipidation [9]. In our research, only moderate levels of adipocyte underwent apoptosis. These results indicated that sphingosine-1-phosphate may also play a role in attenuating cell death.

Conclusion and implication

T10c12 CLA causes robust delipidation in cultured adipocytes, animal models and some human studies. The potential mechanism responsible for its delipidation properties is 15

associated with increased energy expenditure, lipolysis, fatty acid oxidation,

inflammation, apoptosis and decreased energy intake, adipogenesis, lipogenesis.

Sirtuin 1, a member of the sirtuin family, is a NAD-dependent protein deacetylase.

Sirtuin 1 can repress PPARγ activity by interacting with its cofactors, including nuclear

receptor co-repressor (NCoR) and silencing mediator of retinoid and thyroid hormone

receptors (SMRT) [118]. Sirtuin 1 is also reported to cooperate with AMPK to regulate

energy-metabolism gene expression in mouse skeletal muscle [119]. AMPK and SIRT1

are believed to cross regulate each other and share many common target molecules [120].

Besides, Sirtuin 1 is reported regulates the transcriptional activity of NF-κB deacetylating

subunit [121]. Given that PPARγ, NF-κB and AMPK are essential in t10c12 induced

delipidation, we highly suspect that sirtuin 1 is involved in this process.

Recently, we conducted a metabolic profiling analysis of 3T3-L1 adipocytes treated with

either 100 µM LA or t10c12 CLA for 0.5 h or 12 h for the purpose of identifying early

changes in metabolites in these cells. The most striking change was a 9.8 fold increase in

sphinganine and a 3.6 fold increase in sphingosine. This indicates the sphingolipid

pathway is highly elevated after t10c12 CLA treatment. Due to the opposite effect of

sphingolipid metabolites on cell survival and apoptosis [90,91], it is important to find out

the role of the ceramide pathway in t10c12 CLA induced delipidation.

Based on these data, my thesis attempt to answer the following questions. 16

1. Whether Sirtuin 1 is required for t10c12 CLA induced delipidation and how it exerts its effort to this process.

2. What is the role of sphingolipid pathway played in t10c12 CLA induced delipidation.

17

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29

Chapter II. Cross Regulation of Sirtuin 1, AMPK, and PPARγ in Conjugated

Linoleic Acid Treated Adipocytes.

30

Introduction

Conjugated linoleic acid (CLA) reduces adiposity in human and mouse adipocytes [1-4],

and the trans-10, cis-12 CLA (t10c12 CLA) isomer is capable of causing this response

[5]. The rates of fatty acid oxidation and lipolysis increased in t10c12 CLA-treated 3T3-

L1 adipocytes [6], while lipolysis increased and fatty acid biosynthesis decreased in t10c12 CLA-treated human adipocytes [7,8]. Molecular responses to t10c12 CLA include the activation of AMP-activated protein kinase (AMPK) [9], integrated stress response

(ISR; [10,11]) or unfolded protein response (UPR; [12]), mitogen-activated protein kinase (MAPK) cascades [7], and attenuation of peroxisome proliferator-activated receptor γ (PPARγ) protein levels [3]. Treatment with t10c12 CLA requires nuclear factor kappa-B (NF-κB) for an inflammatory response [3,4,10,13-15] that includes increased prostaglandin biosynthesis in human adipocytes [11], in mouse white adipose tissue [16], and 3T3L1 adipocytes [17]. Despite this progress in understanding of the pathways involved in the early perception of t10c12 CLA and the complex regulation of the subsequent responses, much remains unknown in this process.

AMPK is a central regulator of cellular energy levels that is activated by increases in the cellular AMP/ATP ratio, various cellular stresses [18,19], or treatment of adipocytes [9] or mice [20] with t10c12 CLA or mixed isomers of CLA. AMPK activation requires phosphorylation at Thr172 [21], and two of the target proteins inhibited by phosphorylation by AMPK are acetyl-CoA carboxylase (ACC), and fatty acid synthase, two key enzymes in fatty acid biosynthesis. Through this and other mechanisms [18,19], activated AMPK decreases lipogenesis, increases fatty acid oxidation, and increases 31 lipolysis in adipocytes in vitro and in vivo [22]. Phenformin and metformin are structurally related chemicals that can be used to activate AMPK [18,23]. Metformin increases TG loss in t10c12 CLA treated adipocytes, while compound C, a potent inhibitor of AMPK, attenuates TG loss in this system [9].

PPARγ is a ligand-activated nuclear receptor that regulates lipogenesis and is a key regulatory point for controlling inflammation in adipocytes [24]. PPARγ forms a complex with nuclear receptor corepressors 1 or 2 (NCoR1 or 2) in the absence of its bound ligand

[25]. PPARγ transactivation activity is also reduced by phosphorylation at Ser112 by extracellular signal-regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), or p38

MAPKs, and a phosphorylation-dependent sumoylation at K107 [26]. In addition, a non- genomic role for PPARγ is emerging, as a number of PPARγ-dependent processes are too rapid to involve transcriptional responses [27,28]. The critical role of PPARγ in the response to t10c12 CLA is demonstrated by the attenuated responses that occur on addition of PPARγ agonists [11,29,30].

Protein deacetylation is emerging as an important mechanism for regulating energy balance [31,32]. Of the two major classes of histone/protein deacetylases in mammals, the NAD-dependent class III sirtuins are structurally and enzymatically distinct from those of the zinc-dependent class I/II deacetylases [33]. Within the seven members of the sirtuin family, SIRT1 (sirtuin 1) is particularly involved in regulating cell energy metabolism, cell stress, and cell fate [34]. SIRT1 directly binds to NCoR1 and directly or indirectly to PPARγ to repress PPARγ transactivation activity, inhibit adipogenesis, and 32 increase fat loss in adipocytes [35]. SIRT1 deacetylates liver kinase B1 (LKB1), facilitating the ability of LKB1 to phosphorylate AMPK, defining a SIRT1/LKB1/AMPK signaling pathway that provides one of the connections between SIRT1 and AMPK for regulating energy metabolism [36,37]. SIRT1 also deacetylates NF-κB, thereby modulating NF-κB transactivation activity [38,39]. Amongst the chemicals that affect the activity of sirtuins, the activator SRT1720 preferentially activates SIRT1 [40], while sirtinol and nicotinamide are used as general sirtuin inhibitors that do not inhibit class I/II deacetylases [41]. Nicotinamide is a direct product of the sirtuin deacetylation reaction that inhibits sirtuin enzymes as a non-competitive product inhibitor [42]. Etomoxir indirectly inhibits sirtuin activity as it inhibits fatty acid transport into mitochondria which prevents an increase in NAD+, an activator of sirtuin activity [43].

In this study, we analyzed the functional role of SIRT1 in the attenuation of TG levels in t10c12 CLA-treated 3T3-L1 adipocytes. Our objectives were to test whether SIRT1 was functionally required for robust triglyceride reduction, and whether SIRT1, AMPK, and

PPARγ cross regulated each other in the response. These experiments were performed with activators, inhibitors, or siRNA that affected these pathways and analyzing their effects on TG levels, fatty acid metabolism, and post-translational modifications or activity levels of SIRT1, AMPK, and PPARγ.

33

Experimental Procedures

Reagents

Compound C was purchased from Calbiochem (San Diego, CA). Bovine serum albumin

(BSA, > 99% fat free), dexamethasone, etomoxir, GW9662, insulin, isobutyl-1- methylxanthine, metformin, nicotinamide, phenformin, and sirtinol were purchased from

Sigma (St. Louis, MO). Ciglitazone, pioglitazone, rosiglitazone, and troglitazone were from Fisher (Pittsburgh, PA). SRT1720 was from Cayman Chemical (Ann Arbor, MI).

T10c12 CLA (90%, #UC-61-A) was from Nu-Chek Prep, Inc (Elysian, MN). Antibodies to acetyl-NF-κB p65 (acetyl K310), p-PPARγ (phospho S112) and negative control siRNA were from Abcam (Austin, TX). Protein A agarose beads, antibodies to β-actin,

NCoR1, NF-κB p65, PPARγ, SIRT1, anti-goat or anti-rabbit secondary antibodies coupled to horseradish peroxidase, and Sirt1 siRNA were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA). Antibodies to p-AMPK, AMPK, p-ACC, and ACC were from (Beverly, MA). T7 RNA polymerase (P2077) and rNTPs

(E6000) were purchased from Promega. SiRNA to lamin A/C, non-target siRNA, and

DharmaFECT Duo transfection reagent (T-2010-02) were from Dharmacon (Thermo

Fisher Scientific, Boulder, CO).

3T3-L1 cell culture, differentiation, and chemicals

Low passage 3T3-L1 fibroblasts [44] were obtained (H. Green, Harvard Medical School) and cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) containing 10% bovine calf serum (Fisher, Pittsburgh, PA) and differentiated as 34 described [9]. When present, chemicals were dissolved in DMSO, with the exception that

2 mmol/L metformin and 0.1 mmol/L phenformin were dissolved in water, and were added directly to the media at ≤ 0.2% of the final volume in the media. An initial chemical concentration was determined from literature values and a range of concentrations around this value were then tested for their effects in vivo. From this data, the lowest effective concentration was chosen and used at the following concentrations: 5

µmol/L ciglitazone, 10 µmol/L compound C, 10 µmol/L etomoxir, 10 µmol/L GW9662,

10 µmol/L nicotinamide, 5 µmol/L pioglitazone, 5 µmol/L rosiglitazone, 10 µmol/L sirtinol, 8 µmol/L SRT1720, or 5 µmol/L troglitazone and were added 1 h before adding fatty acids. Fatty acids, either linoleic acid or trans-10, cis-12 CLA, were dissolved in 0.1

M KOH, diluted into fatty acid free (>99%) bovine serum albumin (BSA) in phosphate buffered saline at a 1:1 ratio (2 mmol/L BSA: 2 mmol/L fatty acid), pH adjusted to 7.4, and added to the cultures containing 4 to 6 d post-differentiated 3T3-L1 adipocytes [9].

We used 50 µM t10c12 CLA if assaying chemicals that increased TG loss, but otherwise used 100 µM t10c12 CLA.

Fatty acid biosynthesis, oxidation, and lipolysis assays

Fatty acid biosynthesis was measured in differentiated adipocytes after 24 h of treatment by removing the treatment media and incubating the adipocytes in Hanks’ Balanced Salt

Solution (HBSS; Invitrogen, Carlsbad, CA) containing 37 KBq [14C]-acetate [specific activity 2.1 GBg/mmol, (PerkinElmer Radioisotopes, Waltham, MA)] for 30 min

(incorporation was linear for 60 min). Cells were washed in PBS three times, pelleted, and then resuspended in 100 µl PBS and 0.1% SDS. Lipids were extracted in 1 ml of 2:1 35 chloroform:methanol [45] and measured by scintillation counting. Cells briefly exposed to 37 KBq [14C]-acetate, followed by immediate washing and extracted as above, were used to determine background levels, which were subtracted from sample values. Fatty acid oxidation was measured in differentiated adipocytes in 3.5 cm culture plates 12 h after starting treatments by adding 37 KBq [14C]-oleic acid [specific activity 2.2

GBg/mmol, (PerkinElmer Radioisotopes, Waltham, MA) ] to the treatment media for 2 h

14 and collecting [ C]-CO2 for 1 h in collection jars as reported [46]. For lipolysis assays, the TG pool of differentiated adipocytes was labeled by adding 37 KBq [14C]-acetate

[specific activity 2.1 GBg/mmol, (PerkinElmer Radioisotopes, Waltham, MA) ] to the media for 4 h, after which time the plates were washed four times with PBS, and specific experimental media treatments were initiated. Media (0.1 ml) was collected after 24 h, lipids extracted in 1 ml of 2:1 chloroform:methanol [45] and measured by scintillation counting. The use of labeled [14C]-acetate and the 2:1 chloroform:methanol extraction step considerably reduced non-specific background to 50 DPM, as determined by using the above protocol on cells that had been briefly exposed to 37 KBq [14C]-acetate in media.

siRNA transfections

For siRNA transfections, 3T3-L1 adipocytes, 4 to 5 d post differentiation, were transfected by siQUEST transfection reagent (Mirus, Madison, WI) or DharmaFECT Duo transfection reagent (Dharmacon, Thermo Fisher Scientific, Boulder, CO) as described

[47]. For siQUEST transfections, concentrations of 2 µl of siQUEST reagent per ml of media and 40 nmol/L of siRNA were added 24 h before adding fatty acids. For 36

DharmaFECT Duo transfections, concentrations of 1.4 µl per cm2 of DharmaFECT Duo and 80 nmol/L of siRNA were added 24 h before adding fatty acids.

T7 transcription of oligonucleotide templates

In addition to commercially available siRNAs, we utilized siRNAs derived from transcription of oligonucleotide templates with T7 RNA Polymerase as described [48].

The siRNA sequences used were: siRNA control sequence: 5’-AAC AGU CGC GUU

UGC GAC UGG UCU CUU GAA CCA GUC GCA AAC GCG ACU GCC UAU AGU

GAG UCG UAU UA-3’. LaminA/C siRNA sequence: 5’-AAG GAG GAG CUU GAC

UUC CAG UCU CUU GAA CUG GAA GUC AAG CUC CUC CCC UAU AGU GAG

UCG UAU UA-3’. Sirt1 siRNA: 5’-AAG GAG ACU GCG AUG UUA UAA UCU CUU

GAA UUA UAA CAU CGC AGU CUC CCC UAU AGU GAG UCG UAU UA-3’

Immunoblot analysis

Nuclear and cytosolic extracts were isolated using a nuclear extract kit (Active Motif,

Carlsbad, CA). Equal amounts of proteins from whole cell, nuclear, or cytosolic extracts were separated by SDS-PAGE, transferred to Immun-blot PVDF membrane (Bio-Rad

Laboratories, Hercules, CA), probed with the indicated primary antibodies, and detected with secondary antibodies. Enhanced chemiluminescence (Pierce, Rockford, IL) was used for detection. Band intensities were determined from digital images from exposures in the linear range using software (Quantity One, Biorad, Hercules, CA). All western blot analyses were repeated at least three times.

37

Immunoprecipitation

Immunoprecipitations were performed according to the procedure described [35]. In brief, the collected 3T3-L1 adipocytes were sonicated, lysates were centrifuged, and aliquots of the supernatants were immunoprecipitated overnight with specific antibody or control nonspecific IgG serum. Protein A agarose beads were used to bind the specific or non- specific antibody complexes, the protein A beads containing bound proteins were washed five times, and the bound proteins were eluted in SDS sample buffer for immunoblot analysis.

Quantification of TG content

Cell isolation and TG measurements were performed according to the manufacturer’s instructions using TG reagent (T2449; Sigma, St. Louis, MO) and free glycerol reagent

(F6428; Sigma, St. Louis, MO). TG data are expressed as nmol of TG per mg of protein.

Measurement of MCP-1 and COX2 mRNA

Total RNA was extracted by TRIzol (Invitrogen) following the manufacturer’s protocol.

Total RNA (2 μg) was used for cDNA synthesis. Real-time PCR was performed by a

Bio-Rad iCycler using iQ SYBR Green Supermix reagent (Bio-Rad, Hercules, CA) using

PCR primers for MCP-1 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [9], and COX2 [9]. MCP-1 and COX2 mRNA levels were normalized to GAPDH, which showed no significant variation in microarray analyses between linoleic acid and t10c12

CLA treatments. Experiments were repeated three times, and each sample was analyzed using one RNA control and two replicates of each cDNA pool, and the relative amounts 38

of MCP-1, COX2 and GAPDH were calculated using the comparative CT method [49],

according to the manufacturer’s software (Bio-Rad, Hercules, CA). Cycle numbers were

used to calculate gene expression levels in the log linear amplification range.

Statistical Analysis

One or two-way ANOVA was used to analyze the data. Post-hoc pairwise comparisons

were calculated using Tukey’s test and were considered significant for p≤0.05. All

analyses were performed using SAS software (SAS, Cary, NC).

Results

SIRT1 is required for reduced TG levels in t10c12 CLA-treated adipocytes

We first determined whether increased SIRT1 activity would affect TG levels in t10c12

CLA-treated differentiating adipocytes. To specifically activate SIRT1, we used

SRT1720, as this chemical is known to preferentially activate SIRT1 at 8 µM [40].

SRT1720, in combination with 50 µM t10c12 CLA, significantly lowered TG to levels below those achieved by 50 µM t10c12 CLA alone (Fig. 1A). In contrast, addition of 100

µM t10c12 CLA in combination with a sirtuin inhibitor, either sirtinol or nicotinamide, significantly increased TG levels, relative to those from adipocytes treated with 100 µM t10c12 CLA alone (Fig.1. B). Etomoxir, which inhibits sirtuin activity indirectly by reducing NAD+ levels via inhibition of mitochondrial fatty acid transport and oxidation

[43], was also tested. Treatment with etomoxir and t10c12 CLA significantly increased

TG levels relative to TG levels with t10c12 CLA alone (Fig. 1C). These data supported a

hypothesis that one or more sirtuins participate in the TG loss response caused by t10c12 39

CLA treatment, and that SIRT1 involvement was likely due to the response to SRT1720,

a specific SIRT1 activator.

SiRNA was used to reduce SIRT1 expression to confirm the functional involvement of

SIRT1 in the response to t10c12 CLA. First, to verify that SIRT1 protein levels were

likely to respond to siRNAi-mediated knockdown of its mRNA levels in t10c12 CLA- treated adipocytes, the half-life of SIRT1 in cycloheximide treated adipocytes was determined to be less than 6 h (Supplemental Figure 1) and that treatment with t10c12

CLA did not change SIRT1 protein levels (Supplemental Figure 2). We additionally verified that our siRNA transfection method could deliver siRNA efficiently enough to knockdown protein levels of Lamin A/C (Supplemental Figure 3), a target known to be susceptible to siRNA knockdown in adipocytes [47].

A siRNA targeted against SIRT1 mRNA significantly reduced SIRT1 protein levels but did not affect β-actin levels, while a control non-target siRNA did not change SIRT1 or

β-actin amounts (Fig. 1D). In the presence of t10c12 CLA, siRNA knockdown of SIRT1 in adipocytes produced higher levels of TG levels than those from adipocytes treated with a non-target siRNA or reagent only controls (Fig. 1E). In the presence of LA, the siRNA

SIRT1 treated adipocytes had TG levels that were not significantly different than those from adipocytes treated with the control non-target siRNA or reagent (Fig. 1E). These results indicate that siRNA knockdown of SIRT1 attenuated the TG loss caused by t10c12 CLA. Taken together with the above inhibitor studies, these results demonstrate that inhibition of SIRT1 activity or protein levels significantly interfere with the TG loss 40 caused by t10c12 CLA. Further, the magnitude of the change in TG levels in the inhibitor studies (Figures 1b and 1c) and when SIRT1 was knocked down by siRNA (Figure 1e) indicates SIRT1 accounts for most or all of the SIRT activity involved in lipid loss in t10c12 CLA treated adipocytes.

SIRT1 affects the rates of fatty acid metabolism and the inflammatory response

The involvement of SIRT1 in t10c12 CLA-mediated changes in the rates of fatty acid biosynthesis, oxidation, and lipolysis as well as in the induction of two key inflammatory mRNAs was then measured to gain insight into how SIRT1 affected these specific pathways. Adipocytes treated with t10c12 CLA alone had a 77% reduction in their rate of lipogenesis (Fig. 2A). The combination of t10c12 CLA and sirtinol significantly changed this to a 47% reduction in the rate of lipogenesis (Fig. 2A). This result indicated SIRT1 activity was involved in inhibiting the rate of fatty acid biosynthesis. Adipocytes treated with t10c12 CLA exported significantly more radioactively-labeled lipids than LA treated cells, but the amount of t10c12 CLA-mediated lipolysis was not significantly affected by the sirtuin inhibitor sirtinol (Fig. 2B). The rate of fatty acid oxidation was significantly increased in t10c12 CLA-treated adipocytes, and although fatty acid oxidation was less when sirtinol was added, this difference was not significant (Fig. 2C). In conclusion, it appears the major change in lipid metabolism affected by SIRT1 is the rate of fatty acid biosynthesis.

The inflammatory MCP1 and COX2 mRNAs were highly induced by t10c12 CLA treatment (Fig. 2D-E). The SIRT1 activator SRT1720, in combination with t10c12 CLA, 41

modestly attenuated the induction of MCP1 to 79% of t10c12 CLA levels, and although

COX2 mRNA levels were slightly lower, this latter difference was not significant (Fig.

2D-E). In contrast, the SIRT1 inhibitors, sirtinol or nicotinamide, when used in combination with t10c12 CLA, attenuated the induction of the mRNA of MCP1 relative to t10c12 CLA control levels (Fig. 2D). Similarly, sirtinol or nicotinamide, when used in combination with t10c12 CLA, attenuated the induction of the mRNA of COX2 relative to 10c12 CLA control levels (Fig. 2E). These results indicated SIRT1 partially increased

the induction of the inflammatory MCP1 and COX2 mRNAs, possibly through its

inhibition of PPARɣ (see below), which plays an anti-inflammatory role in adipocytes

[24].

SIRT1 increases AMPK activity

We next determined whether SIRT1 affected AMPK regulation during the response to

t10c12 CLA. AMPK activity was measured by the amount of phosphorylation at AMPK

Thr172 (p-AMPK) and by the amounts of phosphorylated ACC (p-ACC), one of

AMPK’s key substrates in vivo [50]. We previously demonstrated the t10c12 CLA- stimulated increase in ACC phosphorylation was less in compound C treated adipocytes, supporting the premise that this phosphorylation was mediated by AMPK [9]. Although p-AMPK levels were increased by 2 h of treatment with t10c12 CLA, the addition of

SIRT1 activator SRT1720 or SIRT inhibitors, had no significant effect on the amount of p-AMPK or p-ACC produced after 2 h of treatment (Fig. 3A). However, when used in combination with t10c12 CLA for 12 h, SIRT1 activator SRT1720 significantly increased p-AMPK or p-ACC levels relative to the amounts present when treated by t10c12 CLA 42

alone (Fig. 3B). Conversely, when used in combination with t10c12 CLA for 12 h, SIRT1

inhibitors sirtinol or nicotinamide significantly attenuated p-AMPK levels relative to the

amounts of these proteins in adipocytes treated with t10c12 CLA (Fig. 3B). Similarly,

when used in combination with t10c12 CLA, siRNA targeting of SIRT1 significantly

reduced p-AMPK and p-ACC levels relative to the control treatment (Fig. 3C).

Collectively, these results indicated SIRT1 increased AMPK activity levels after 12 h, but

not 2 h, of exposure to t10c12 CLA.

A PPARγ agonist or antagonist affects the TG loss response to t10c12 CLA

The involvement of PPARγ in the TG loss response to t10c12 CLA was then tested through addition of a PPARγ antagonist or agonist. The PPARγ antagonist GW9662 significantly reduced TG levels when used in combination with 50 µM t10c12 CLA relative to TG levels in the t10c12 CLA treatment (Fig. 4A). Prior to examining the effects of a PPARγ agonist on the response to t10c12 CLA, we first determined that troglitazone was the most potent PPARγ agonist amongst a set of four thiazolidinedione agonists, as measured by their ability to increase the amount of TG produced in differentiating adipocytes (Fig. 4B). Troglitazone significantly attenuated t10c12 CLA‘s ability to reduce TG levels, as the combined treatment was not significantly different from the LA control (Fig. 4C). These results indicated that, in adipocytes treated with t10c12 CLA, a PPARγ antagonist facilitated TG loss while a PPARγ agonist interfered with TG loss.

Cross regulation of PPARγ and AMPK in adipocytes treated with t10c12 CLA 43

The cross regulation between PPARγ and AMPK was next determined. When used in

combination with t10c12 CLA for 12 h, PPARγ antagonist GW9662 significantly

increased AMPK and ACC phosphorylation levels (Fig. 4D). Conversely, when used in

combination with t10c12 CLA for 12 h, troglitazone significantly reduced AMPK and

ACC phosphorylation levels (Fig. 4D). However, when used in combination with t10c12

CLA for only 2 h there was no significant effect of these chemicals on AMPK or ACC

phosphorylation levels (Fig. 4D). Therefore, although AMPK activation occurred at 2 h,

cross regulation by PPARγ was not apparent at 2 h. Phosphorylation of PPARγ increased

by 140% at 12 h, but not by 2 h, after t10c12 CLA treatment (Fig. 4E-F). When compound C was used in combination with t10c12 CLA, the amount of phosphorylated

PPARγ was reduced relative to the levels when treated by t10c12 CLA. This suggests

AMPK activity was directly or indirectly involved in the phosphorylation of PPARγ (Fig.

4F). These results indicated AMPK and PPARγ cross regulated each other in the response to t10c12 CLA.

Cross regulation of PPARγ and AMPK also occurs in the absence of t10c12 CLA

We next determined whether this cross regulation between AMPK and PPARγ was a general aspect of these proteins by testing whether this occurred in the absence of t10c12

CLA. This was done by using two other chemicals to activate AMPK, alone or in combination with a PPARγ agonist or antagonist. Phenformin, a strong AMPK activator, significantly reduced TG levels to 60% of those present in the untreated control adipocytes (Fig. 5A). When phenformin was used in combination with troglitazone, significantly more TG was present than in adipocytes treated with phenformin alone (Fig. 44

5A). Metformin, a weaker AMPK activator, only slightly reduced TG levels (Fig. 5B).

The TG level was further reduced when metformin was used in combination with the

PPARγ antagonist GW9662 (Fig. 5B). These results indicated that the antagonistic cross

regulation between AMPK and PPARγ that was observed in the response to t10c12 CLA also occurred in phenformin or metformin treated adipocytes in the absence of t10c12

CLA.

NF-κB is deacetylated by one or more sirtuins in t10c12 CLA treated adipocytes

The regulation of SIRT1 activity by AMPK and PPARγ was then assessed. A known deacetylation target of SIRT1 is Lys310 of the p65 subunit of NF-κB [38]. Therefore, we determined whether the p65 subunit of NF-κB changed its acetylation levels in response to t10c12 CLA. The acetylation level of the p65 subunit of NF-κB was not significantly changed by treatment with t10c12 CLA for 2 h (Fig. 6A). In contrast, the amount of acetylated p65 subunit of NF-κB was significantly less in adipocytes treated with t10c12

CLA for 12 h (Fig. 6B). When t10c12 CLA was used in combination with sirtinol or nicotinamide inhibitors of sirtuin activity, the amount of the acetylated p65 subunit of

NF-κB was significantly higher relative to t10c12 CLA alone (Fig. 6 B). These drug inhibition results supported the premise that one or more members of the sirtuin family of protein deacetylases were involved in the deacetylation of the p65 subunit of NF-κB. Of the seven sirtuin family members, only SIRT1 and SIRT6 have a nuclear localization [51] that overlaps with the subcellular localization of acetylated NF-κB. Additionally, of the seven sirtuins, only SIRT1 is known to deacetylate the p65 subunit of NF-κB [38].

Therefore, these results established that one or more members of the sirtuins, presumably 45

at least SIRT1, were responsible for the deacetylation of the p65 subunit of NF-κB, and

verified that sirtuin deacetylase activity increased in t10c12 CLA treated adipocytes.

AMPK and PPARγ affect SIRT1 activity

The involvement of AMPK in regulating SIRT1 activity was then measured using compound C, an inhibitor of AMPK. The amount of the acetylated p65 subunit of NF-κB significantly increased in a treatment using t10c12 CLA in combination with compound

C, relative to the acetylation level produced by t10c12 CLA (Fig. 6C). This result indicated AMPK was directly or indirectly involved in regulating the SIRT1 deacetylation response to t10c12 CLA. The PPARγ agonist troglitazone was used to

determine whether PPARγ affected the levels of acetylation of the p65 subunit of NF-κB.

When used in combination with t10c12 CLA for 12 h, troglitazone-treated adipocytes

significantly increased the amount of acetylated p65 subunit of NF-κB relative to the

t10c12 CLA treatment, suggesting activated PPARγ interferes with SIRT1 deacetylation activity (Fig. 6D). Conversely, adipocytes treated with t10c12 CLA and the PPARγ

antagonist GW9662 significantly reduced the amounts of acetylated p65 subunit of NF-

κB relative to the t10c12 CLA treatment, suggesting inhibition of PPARγ increases

SIRT1 deacetylation activity (Fig. 6D). Collectively, these results indicated that AMPK

positively regulated, and PPARγ negatively regulated, SIRT1 activity in vivo, as

measured by the deacetylation of the p65 subunit of NF-κB.

SIRT1 increases its binding to PPARγ and NCoR1 in t10c12 CLA treated adipocytes 46

A possible mechanism for cross regulation of SIRT1 and PPARγ is through participation

within a common protein complex [35]. Using co-immunoprecipitation with a SIRT1

antibody, we observed that t10c12 CLA treatment resulted in a significant increase in the

amount of a SIRT1/PPARγ protein complex relative to the amount of this complex in the

LA control, despite an overall reduction in PPARγ protein levels in t10c12 CLA treated

cells (Fig. 7). Again using the same co-immunoprecipitation method, we observed that

the amount of a protein complex containing SIRT1 and NCoR1 also significantly

increased in the presence of t10c12 CLA (Fig. 7). These results demonstrated that there was increased binding of SIRT1 to PPARγ and NCoR1 in t10c12 CLA treated adipocytes.

Discussion

Here we demonstrated that SIRT1 activity was functionally involved in the TG loss

response that occurred in t10c12 CLA-treated 3T3-L1 adipocytes. Our chemical inhibitor

and activator studies indicated SIRT1 facilitated TG loss, as did AMPK, while PPARγ

stimulated TG synthesis (summarized in Figure 8). Our assays of protein activities,

modifications, and interactions supported this functional data and provided molecular

evidence of cross regulation between SIRT1, AMPK and PPARγ in the response to

t10c12 CLA. As discussed below, each of these proteins directly or indirectly affected the

activity of the others.

In order to evaluate the effects of SIRT1 on specific pathways in lipid metabolism, we

first established the effects of t10c12 CLA on lipid metabolism in our 3T3-L1 adipocyte

system. The decrease in TG levels in t10c12 CLA-treated adipocytes was caused by a 47

combination of reduced fatty acid biosynthesis, increased lipolysis, and increased fatty

acid oxidation. Our finding that t10c12 CLA-treated adipocytes have increased p-AMPK

levels and increased lipolysis and fatty acid oxidation is consistent with a report that

activated AMPK increases lipolysis and fatty acid oxidation [22]. Our results were also

consistent with the increased lipolysis and fatty acid oxidation, but not the increased fatty

acid biosynthesis, reported in t10c12 CLA-treated 3T3-L1 adipocytes [6]. The latter difference may reflect differences in the specific cell cultures. The reduction in the rate of fatty acid synthesis was consistent with the reduced transcript levels of lipid biosynthetic enzymes such as ACC1, ACC2, and fatty acid synthase in mice and 3T3-L1 adipocytes

[10,15], possibly mediated by AMPK’s ability to phosphorylate and inhibit SREBP1, a key regulatory factor of these genes [52]. Further, the reduction in the rate of fatty acid synthesis was consistent with increased phosphorylation of ACC, which inhibits ACC

(both ACC-1 and ACC-2 isoforms) and thereby inhibits production of malonyl-CoA.

Reduced concentrations of malonyl-CoA should reduce the rate of fatty acid biosynthesis and increase the rate of fatty acid oxidation, as attenuated levels of malonyl-CoA no longer inhibit carnitine palmitoyltransferase-mediated transport of fatty acids into mitochondria for β-oxidation [18,19,50]. Fatty acid synthesis was also likely reduced by

AMPK’s ability to phosphorylate and inhibit fatty acid synthase [18,19]. Our studies indicated that SIRT1 primarily affected the rate of fatty acid biosynthesis as inhibition of

SIRT1 did not significantly affect the rate of lipolysis or fatty acid oxidation in the response to t10c12 CLA. Whether SIRT1 regulates fatty acid biosynthesis primarily via differential modulation of the activities of AMPK and PPARγ or additionally through direct deacetylation of metabolic enzymes [53] requires additional research. 48

Our results strongly support an increase in SIRT1 deacetylase activity occurs in vivo in

t10c12 CLA treated adipocytes. We used the acetylation levels of Lys310 of the p65 subunit of NF-κB as an indicator of SIRT1 deacetylase activity in vivo. NF-κB/p65 had

reduced amounts of acetylation at Lys310 in t10c12 CLA-treated adipocytes, indicating

increased deacetylase activity was occurring. However, in principle Lys310 of p65/NF-

κB can be deacetylated by both class I/II or sirtuin deacetylases [38]. Results with the

sirtuin-specific inhibitors indicated the majority of NF-κB/p65 deacetylation was

accomplished by the sirtuin class of deacetylases. Of the seven sirtuin family members,

only SIRT1 and SIRT6 have the nuclear localization [51] needed to deacetylate nuclear-

localized NF-κB. Of these two candidates, SIRT1 is likely to be involved as SIRT1

physically binds to the p65 subunit of NF-κB and deacetylates it at Lys310 in human lung

cells [38]. Further, our siRNA knockdown of SIRT1 indicates it is the major contributor

to SIRT function in the t10c12 CLA response (Figure 1E). Therefore, our results strongly

support an increase in the deacetylase activity of SIRT1 in t10c12 CLA-treated

adipocytes, without ruling out minor contributions from other sirtuins.

The functional consequences of alterations in SIRT1, AMPK, and PPARγ activities

suggested there was cross-regulation between these proteins (summarized in Figure 8).

Therefore, evidence for changes in protein modifications and/or activity was investigated.

In the case of SIRT1 affecting AMPK, an activator of SIRT1 increased AMPK activity,

while inhibitors of SIRT1 reduced AMPK activity in t10c12 CLA-treated adipocytes. A

possible pathway connecting SIRT1 to AMPK is a SIRT1/LKB1/AMPK axis by which 49

SIRT1 can affect AMPK activity via deacetylation of protein kinase LKB1, which

increases LKB1’s ability to activate AMPK by phosphorylation [36,37]. In the case of

SIRT1 affecting PPARγ, we found treatment with t10c12 CLA caused more SIRT1 to bind to PPARγ and NCoR1. This is likely to inhibit PPARγ activity as the increased binding of SIRT1 to PPARγ and NCoR1 that occurred during fasting reduced PPARγ transcriptional activity [35]. Reduced PPARγ activity is consistent with the reduced transcription of lipogenic genes observed in t10c12 CLA treated adipocytes [10,13,15].

These results support a conclusion that SIRT1 stimulated AMPK activity and attenuated

PPARγ activity in t10c12 CLA treated adipocytes.

In the case of AMPK affecting SIRT1, inhibition of AMPK with compound C reduced

SIRT1 activity as measured by the deacetylation of p65/NF-κB. The mechanism is unclear but could include AMPK-mediated changes in fatty acid oxidation which affect the NAD+/NADH ratio that affects SIRT1 activity [43]. In the case of AMPK affecting

PPARγ, AMPK was directly or indirectly responsible for the increased phosphorylated at

Ser112 of PPARγ in t10c12 CLA treated adipocytes [29], as this effect was attenuated by

the AMPK inhibitor compound C. Phosphorylation of PPARγ at Ser112 facilitates its

SUMOylation at K107, and thereby decreasing its transactivation activity [26]. These

results support a conclusion that AMPK stimulated SIRT1 activity and attenuated PPARγ

activity in t10c12 CLA treated adipocytes.

In the case of PPARγ affecting AMPK and SIRT1, troglitazone, a PPARγ agonist,

reduced the activities of AMPK and SIRT1. Conversely, GW9662, an antagonist of 50

PPARγ, increased the activities of AMPK and SIRT1. These results demonstrate that

PPARγ has a repressive effect on the activities of these proteins, which is consistent with the opposing roles of PPARγ’s in stimulating lipid biosynthesis and the catabolic energy- generating roles of AMPK and SIRT1 [30,54]. The mechanisms of how PPARγ affects

AMPK and SIRT1 are unclear, despite the physical interaction between SIRT1 and

PPARγ [35,55]. Although the mechanisms are unclear, PPARγ affected the activity levels of SIRT1 and AMPK without changing the total amounts of these proteins in the response to t10c12 CLA. This suggests that PPARγ achieved these effects via a non- transcriptional mechanism. As such, our results support an emerging role for PPARγ in regulating non-genomic processes [27,28].

We also used AMPK activators in addition to t10c12 CLA to manipulate AMPK activity and explore whether cross regulation of AMPK and PPARγ occurred in the absence of t10c12 CLA (summarized in Figure 8). Phenformin, a potent AMPK activator, caused a

TG loss similar to that caused by t10c12 CLA treatment. Troglitazone, the most potent

PPARγ agonist in our 3T3-L1 adipocyte system, attenuated the TG loss caused by phenformin. Conversely, GW9662, a PPARγ antagonist, increased the amount of TG loss when used with metformin, a moderate AMPK activator. This latter finding supports a hypothesis that both AMPK activation [9] and reduced PPARγ activity [3] are important for reducing TG levels. Taken together, these results support a hypothesis that cross regulation between AMPK and PPARγ also occurs in the absence of t10c12 CLA, and is therefore likely to generally occur between these proteins in adipocytes.

51

Both AMPK and SIRT1 play major roles in regulating cellular energy homeostasis and in

response to caloric restriction [54,56,57]. The involvement of AMPK and SIRT1 in the

response to t10c12 CLA is consistent with an overall similarity to cellular energy

restriction. This is supported by the strong similarity of the whole genome transcriptional

response of adipocytes treated with t10c12 CLA to the response caused by metformin [9],

which affects the cellular AMP/ATP ratio [58,59]. Similarly, phenformin, which also

affects the cellular AMP/ATP ratio, caused TG losses similar to those caused by t10c12

CLA and caused a whole genome transcriptional response similar to that of t10c12 CLA- treated adipocytes [17]. Our results indicated SIRT1 activation and cross regulation of

AMPK occurred at 12 h but not as early as 2 h, while AMPK was activated at 2 h or earlier [9]. This is consistent with the suggestion that AMPK activation is a critical early event [9]. The signaling pathways used by t10c12 CLA to activate AMPK remain unknown.

52

Figure Legends

Figure 1. SIRT activity affects TG levels in t10c12 CLA treated 3T3-L1 adipocytes.

a-c Triglyceride (TG) levels were measured in differentiating adipocytes after incubation

with LA (L) or t10c12 CLA (C), with or without the SIRT1 activator SRT1720 (SRT), or

the sirtuin inhibitors sirtinol (SOL), nicotinamide (NAM), or etomoxir (ETO) for 24 h. d

Immunoblot analysis of whole cell extracts for the amount of SIRT1 and β-actin proteins

after exposure to 80 nmol/L of control siRNA (siCON), or siRNA against SIRT1 (siSirt1),

or transfection reagent only. Duplicate loadings of each sample were analyzed and the

lanes marked by 1/3 have one third of the indicated samples loaded. e Triglyceride (TG) amounts after treatments as in d. Each bar in panels a-c and e represents the mean + SEM

(n=3), and is representative of three independent experiments. Means not sharing a

common letter differ, P  0.05. Panel d is a representative blot of three independent

experiments. 53

Figure 2. SIRT1 affects fatty acid metabolism and inflammatory mRNA levels. a-c The effects of 100 µmol/L of t10c12 CLA, with or without the sirtuin inhibitor sirtinol (SOL), on the rates of fatty acid metabolism were measured by radioactive tracers

(disintegrations per minute: dpm) as follows: a fatty acid biosynthesis by incorporation of

[1-14C]-acetate; b lipolysis by the release of lipids previously labeled with [1-14C]-acetate;

14 c fatty acid oxidation by the release of [ C]-CO2 from lipids previously labeled with [1-

14C]-oleate. d-e The effects of the SIRT1 activator SRT1720 (SRT), or the sirtuin

inhibitors sirtinol (SOL) or nicotinamide (NAM) on RNA levels in LA or t10c12 CLA

treated adipocytes were analyzed for MCP-1 and COX2 relative to GAPDH by reverse

transcription and quantitative PCR. The relative amounts of MCP-1 and COX2 mRNA

are shown as bar graphs. Each bar in panels a-e represents the mean + SEM (n=3 for a-b

or n=2 for c-e), and is representative of three independent experiments. Means not

sharing a common letter differ, P  0.05. 54

Figure 3. SIRT1 affects ACC and AMPK phosphorylation levels after t10c12 CLA treatment.

Differentiated 3T3-L1 adipocytes were incubated with 100 µmol/L LA (L) or t10c12

CLA (C): a with or without SIRT1 activator SRT1720 (SRT), or sirtuin inhibitors sirtinol

(SOL) or nicotinamide (NAM) for 2 or b 12 h; or c with 40 nmol/L of SIRT1 siRNA

(siSIRT1) or control siRNA (siCON). Representative western blots indicate the proteins

detected in cytosolic extracts with antibodies to p-ACC (p-Ser79), or total ACC, p-

AMPK (p-Thr172), or total AMPK. Each bar in panels a-c represents the mean + SEM

(n=3) of the ratio of the phosphorylated to total form of each protein (phospho/total) for

three independent experiments. Means for p-AMPK/AMPK (a-e) or p-ACC/ACC (u-x)

not sharing a common letter differ, P  0.05. 55

Figure 4. PPARγ antagonists and agonists affect TG levels and modulate p-AMPK activity levels in t10c12 CLA treated 3T3-L1 adipocytes. a Differentiated adipocytes were incubated with 50 µmol/L LA (L) or t10c12 CLA (C) with or without PPARγ antagonist GW9662 (9662) for 24 h and TG levels were measured. b Differentiating adipocytes were treated with either control media (0), 56

troglitazone (Tro), ciglitazone (C), rosiglitazone (R), or pioglitazone (P) to determine

which PPARγ agonist was most effective for increasing TG levels. c Differentiated 3T3-

L1 adipocytes were incubated with 100 µmol/L LA or t10c12 CLA, with or without

PPARγ agonist troglitazone (Tro), and TG levels were measured after 24 h. d Adipocytes

were treated as in a or c for 2 or 12 h using 100 µmol/L LA or t10c12 CLA, and representative western blots of cytosolic extracts indicate the proteins detected with antibodies to p-ACC (p-Ser79), total ACC, p-AMPK (p-Thr172), or total AMPK. The ratio of the phosphorylated to total form of each protein (phospho/total) is shown in the bar graphs. e-f The amount of phosphorylated or total PPARγ in nuclear extracts was measured after 2 or 12 h of treatment with 100 µmol/L LA or t10c12 CLA, and with or without compound C (Comp. C) at 12 h. The ratio of the amount of phosphorylated to total PPARγ is shown in the bar graphs. Each bar in panels a-f represents the mean +

SEM (n=3), and is representative of three independent experiments (a-c) or is the mean

of three independent experiments (d-f). Means within each data type (a-e or u-x) not sharing a common letter differ, P  0.05.

57

Figure 5. PPARγ agonists or antagonists affect the TG loss caused by AMPK activators without t10c12 CLA being present. a Differentiated 3T3-L1 adipocytes were incubated with or without 0.1 mmol/L

phenformin (Phen), with or without PPARγ agonist troglitazone (Tro), and TG levels

were measured after 24 h. b TG levels were measured in differentiated 3T3-L1

adipocytes in media lacking or containing 2 mmol/L metformin (Met), with or without

PPARγ antagonist GW9662 (9662) for 24 h. Each bar represents the mean + SEM (n=3),

and is representative of three independent experiments. Means not sharing a common

letter differ, P  0.05.

58

Figure 6. SIRT1, AMPK and PPARγ affect the t10c12 CLA-dependent decrease in the

amount of acetylated NF-κB.

The amount of acetylation on the p65 subunit of NF-κB or the total amount of p65 in

nuclear extracts was detected by immunoblot analysis with antibodies specific for

acetylated p65 or total p65. The ratio of the acetylated to total p65 is shown in the bar

graphs (Acetyl-p65/p65). a-c Differentiated 3T3-L1 adipocytes were incubated with 100

µmol/L LA (L) or t10c12 CLA (C) a for 2 h, or b for 12 h, with or without sirtuin inhibitors sirtinol (SOL) or nicotinamide (NAM), or c with or without AMPK inhibitor compound C (Comp.C). d The effects of PPARγ agonist troglitazone (Tro) or PPARγ antagonist GW9662 (9662), in combination with LA or t10c12 CLA, on the acetylation of the p65 subunit of NF-κB was measured. Each bar in panels a-d represents the mean +

SEM (n=3) of three independent experiments. Means not sharing a common letter differ,

P  0.05. 59

Figure 7. Treatment with t10c12 CLA increases the interaction of SIRT1 with PPARγ or

NCoR1.

Differentiated 3T3-L1 adipocytes were incubated with 100 µmol/L LA (L) or t10c12

CLA (C) for 12 h, and a portion of the nuclear extracts were immunoprecipitated with antibody to SIRT1 (IP: SIRT1). Representative immunoblots (IB) indicate the proteins detected from the nuclear extracts (5% input) or when the immunoprecipitated proteins were probed with antibodies to SIRT1, PPARγ, or NCoR1. Each bar represents the mean

+ SEM (n=3) of three independent experiments. Means within each data type not sharing a common letter differ, P  0.05.

60

Figure 8. Summary diagram for proposed effects of AMPK, SIRT1, PPARγ, and their activators or inhibitors in 3T3-L1 adipocytes.

AMPK and SIRT1 impair lipid synthesis and PPARγ activity (red lines with stop bars), and stimulate each other (blue arrows). PPARγ impairs AMPK and SIRT1 activity and stimulates lipid synthesis. The activating (blue arrows) or inhibiting (red line with stop bar) effects of the different chemical activators and inhibitors are also shown.

61

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67

Chapter III. Sphingolipids are Required for Efficient Triglyceride Loss in

Conjugated Linoleic Acid Treated 3T3-L1 Adipocytes

68

Abstract

Conjugated linoleic acid (CLA) reduces adiposity in human and mouse adipocytes. This outcome is achieved through a variety of biological responses including increased energy expenditure and fatty acid oxidation, increased inflammation, repression of fatty acid biosynthesis, attenuated glucose transport, and apoptosis. In the current study profiling of

261 metabolites was conducted to gain new insights into the biological pathways responding to CLA in 3T3-L1 adipocytes [Appendix]. Sphingonine, sphingosine, and ceramide levels were observed to be highly elevated in CLA treated adipocytes.

Exogenous chemicals that increase endogenous ceramide levels decreased lipid levels in adipocytes, and activated AMP-activated protein kinase as well as nuclear factor kappa-B, both of which are normally activated in CLA treated adipocytes. Concurrent inhibition of de novo ceramide biosynthesis and entry from existing sphingoglipid pools attenuated the lipid lowering and apoptotic effects normally associated with responses to CLA. Our results indicate ceramides are an important component of the lipid lowering response in

CLA treated adipocytes.

69

Introduction

Conjugated linoleic acid (CLA) reduces adiposity in human and mouse adipocytes [1-4],

and the trans-10, cis-12 isomer of CLA (t10c12 CLA) is capable of causing this response

[5]. Molecular responses to t10c12 CLA treatment include the activation of AMP- activated protein kinase (AMPK) [6], Sirtuin 1 [7], and attenuation of peroxisome proliferator-activated receptor γ (PPARγ) protein levels [3]. Treatment with t10c12 CLA

requires nuclear factor kappa-B (NF-κB) for an inflammatory response [3,4,8-11] that

includes increased prostaglandin biosynthesis in human adipocytes [12], in mouse white

adipose tissue [13], and 3T3-L1 adipocytes [14]. Despite this progress in understanding

the pathways involved in the early perception of t10c12 CLA and the complex regulation

of the subsequent responses, much remains unknown in this process.

Sphingolipids are a complex class of lipids containing the sphingoid backbones of

sphinganine (dihydrosphingosine) and sphingosine. Attaching sphinganine or sphingosine

to a fatty acid via an amide linkage forms ceramide, the simplest sphingolipid that is the

starting point for more complex shingomyelins, , and [15].

Sphingolipids have structural roles in lipid membranes and can serve as sources of

signaling molecules as well. Signaling pathways affected by sphingolipids or molecules

derived from them, include apoptosis, inflammation, mitochondrial function, metabolism,

and obesity [16,17].

The role of sphinoglipids in adipocytes and obesity is still emerging. A study of

genetically obese mice (ob/ob) and high-fat diet-induced obese mice found inhibiting 70 ceramide de novo synthesis attenuated obesity symptoms, facilitating weight reduction, better energy metabolism, and improved insulin sensitivity [18]. In 3T3-L1 adipocytes, ceramide levels decreased as pre-adipocytes differentiated into adipocytes, and increased when adipocytes were treated with epigallocatechin gallate, which reduced adipocyte fat content [19].

In this study we performed metabolic profiling of t10c12 CLA treated adipocytes, and identified sphinganine and sphingosine as being elevated. The role of the ceramide pathway was then further characterized to determine its functional contribution to lowering of fat content in t10c12 CLA treated adipocytes. Elevated ceramide levels were necessary for efficient fat loss in t10c12 CLA treated adipocytes, and chemicals that increased ceramide levels were sufficient to cause efficient fat loss when added to adipocyte cultures.

Material and methods

Reagents. Insulin, Isobutyl-methylxanthine, Dexamethasone, Bovine serum, Calf serum,

N-Acetyl-D-sphingosine (C2 ceramide), monoclonal anti-ceramide antibody, ,

Fumonisin B1 and SKI-II were from Sigma (St. Louis, MO). Ampk inhibitor Compound

C was from Calbiochem (San Diego, CA). Antibodies to β-actin, Sphingosine-1- phosphate phosphatase 1(Sppase) and palmitoyltransferase (SPTLC) were from

Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to p-AMPK and AMPK were purchased from Cell Signaling (Beverly, MA). T7 RNA polymerase, RNase-Free DNase 71

I and rNTPs were purchased from Promega (Madison, WI). DharmaFECT Duo

transfection reagent was from Dharmacon (Thermo Fisher Scientific, Boulder, CO).

3T3-L1 cell culture and differentiation. 3T3-L1 fibroblasts[20] were obtained and cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) containing 10% bovine calf serum (Fisher, Pittsburgh, PA) and differentiated as described [7]. Chemicals were dissolved in DMSO, with the exception that C2-ceramide was dissolved in ethanol. The lowest effective chemical concentrations were chosen by testing a range of initial concentration and were as follows: 20 µmol/L , 50

µmol/L Myriocin, 10 µgl/mL SKI-II, 30 µmol/L C2-ceramide, and were added 1 h before adding fatty acids. Chemicals were added directly to the media at ≤0.2% of the final volume in the media.

Fatty acid preparation. Fatty acids (>99%, Nu-check Prep, Elysian, MN), either linoleic acid (LA) or trans-10, cis-12 conjugated linoleic acid (CLA), were dissolved in 0.1 M

KOH, diluted into fatty acid free (>99%) bovine serum albumin (BSA) in phosphate buffered saline at a 1:1 ratio (2 mmol/L BSA: 2 mmol/L fatty acid), pH adjusted to 7.4, and added to the cultures containing 4 to 6 d post-differentiated 3T3-L1 adipocytes[7].

All media contained 100,000U/L penicillin and 172µmol/L streptomycin (Invitrogen,

Carlsbad, CA) . 50 µM t10c12 CLA was used if assaying chemicals that increased TG loss as this facilitated observing TG loss, but otherwise 100 µM t10c12 CLA was used.

72

Metabolic profiling. Metabolite profiling of 3T3-L1 adipocytes was performed by

Metabolon (Durham, NC). Six replicates of 3T3-L1 adipocytes, treated with 100 µM LA or t10c12 CLA for 0.5 h or 12 h, were collected, frozen at -80 ◦C for later analysis.

Samples were solvent extracted and analyzed by GC/MS and LC/MS or LC/MS/MS platforms for 261 metabolites. Compounds were identified by known standards or prior characterizations of metabolite libraries. Welch’s two–sample t-tests were used to identify biochemicals that differed significantly between conjugated linoleic acid and linoleic acid treatments.

RNA interference. 3T3-L1 adipocytes, 4 to 5 days post differentiation, were transfected by DharmaFECT Duo transfection reagent as described [21]. The final transfection reagent concentration was 1.4µl/cm2. DharmaFECT Duo and 100nmol/L shRNA were added 24h before adding fatty acids. For drug and shRNA combination assays, drugs were subsequently added to media 1h before transfection.

shRNA prepration. Initial sequence against targets were obtained from literature [22,23] or online tools (http://sirna.wi.mit.edu/, http://www.thermoscientificbio.com/design- center/?redirect=true). 5 potential sequences for each target were chosen and prepared as described [24]. In brief, shRNA sequences were synthesized using the T7 polymerase

(Promega, Madison, WI) templates and purified by 12-20% denaturing polyacrylamide gel. The shRNA sequences used were: scramble (sh-Non): 5′-AAC AGU CGC GUU

UGC GAC UGG UCU CUU GAA CCA GUC GCA AAC GCG ACU GCC UAU AGU

GAG UCG UAU UA-3′; Sphingosine-1-phosphate phosphatase 1 (sh-Sppase): 5′-AAU 73

CAU CAA GCU GGA GGU CUU CUU CUC UUG AAA GAA GAC CUC CAG CUU

GAU GAC CUA UAG UGA GUC GUA UUA-3′; or Serine palmitoyltransferase 1 (sh-

Sptlc): 5′-AAG CCA UCA UUU ACU CGU AUG UCU CUU GCA CAU ACG AGU

AAA UGA UGG CCC UAU AGU GAG UCG UAU UA-3′.

Western blot. Nuclear and cytosolic extracts were isolated using a nuclear extract kit

(Active Motif, Carlsbad, CA). Equal amounts of proteins were separated by SDS-PAGE, transferred to Immun-blot PVDF membrane (Bio-Rad Laboratories, Hercules, CA), probed with the indicated primary antibodies, and detected with secondary antibodies.

Enhanced chemiluminescence (Pierce, Rockford, IL) was used for detection. Band intensities were determined from digital images from exposures in the linear range using software (Quantity One, Biorad, Hercules, CA). All western blot analyses were repeated at least three times.

Ceramide assay. Immunodot blots for ceramide levels were performed as described with modifications [25-27]. Equal amount of cells (1.5x105) in 12-well plates from each treatment were collected by spinning down at 100xg for 5 min. Cells were resuspended in

500µl ice-cold acidic methanol (acetic acid: methanol 1:50). Lipids were extracted by addition of 500µl chloroform and 500µl H2O. The organic lower-phase was collected and evaporated with nitrogen gas. After adding 100 µl of chloroform/methanol (2:1, v/v) to each tube, 2µl of of each sample was spotted on PVDF membrane and allowed to dry for

30 min. The PVDF membrane was blocked with 5% dry milk in PBS at room temperature for 1 hour, and standard immunoblot procedures followed to detect ceramide 74

levels using a monoclonal antibody that detects ceramides (Sigma, Saint Louis, MO,

USA).

Quantification of TG content. Cell isolation and TG measurements were performed according to the manufacturer’s instructions using TG reagent (T2449; Sigma, St. Louis, MO) and free glycerol reagent (F6428; Sigma, St. Louis, MO). TG data are expressed as nmol of

TG per µg of DNA.

Quantification of DNA content. DNA content assay was performed by Hoechst (33342 sigma B2261) stain according to the manufacturer’s instructions (Thermo Scientific,

Pittsburg, PA).

Statistical Analysis. One or two-way ANOVA was used to analyze the data. Posthoc pairwise comparisons were calculated using Tukey’s test and were considered significant for p ≤ 0.05. All analyses were performed using SAS software (SAS, Cary, NC).

75

Results

Metabolite profiling of t10c12 CLA treated 3T3-L1 adipocytes

A metabolic profiling analysis of 3T3-L1 adipocytes treated with either 100 µM LA or

t10c12 CLA for 0.5 h or 12 h was conducted for the purpose of identifying early changes in metabolites in these cells. A total of 261 metabolites were surveyed in this analysis

(Supplemental Table 1). At 0.5 h, the main change was the large increase in t10c12 CLA

relative to the LA controls, indicating rapid uptake of t10c12 CLA by the adipocytes. At

12 h, there were changes in various lipid levels, consistent with the ability of t10c12 CLA to perturb lipid metabolism (Supplemental Table 1). However, the most striking change and focus of the present study was a 9.8 fold increase in sphinganine and a 3.6 fold increase in sphingosine (Table 1). This indicates the sphingolipid pathway is highly elevated 12 h after adding t10c12 CLA. Sphinganine is early in the biosynthetic pathway for ceramides, while sphingosine is derived from ceramides (Figure 1). Ceramides were not measured in this metabolite profiling analysis.

Ceramide levels increase in t10c12 CLA treated 3T3-L1 adipocytes and cause triglyceride reductions.

An immunoblot analysis of ceramide levels confirmed that they are elevated in adipocytes treated with 50 µM t10c12 CLA, and are present at higher levels if treated with 100 µM t10c12 CLA (Figure 2A). Thus, t10c12 CLA strongly increases ceramide/sphingolipid pools in adipoctyes. To test the hypothesis that ceramides and/or sphinoglipids are functionally involved in t10c12 CLA-mediated TG loss in adipocytes, we tested two chemicals that enhance this pathway. C2 ceramide is a short chain 76

ceramide that can mimic some ceramide responses [28]. SKI-II (also known as

inhibitor 2) is a chemical that inhibits sphingosine kinase, which

normally depletes the sphingosine pool by producing sphingosine-1-phosphate (S1P,

Figure 1). Addition of 30 µM C2 ceramide to adipocytes results in strong reductions in

TG levels in the presence of LA, without further reductions in TG levels in the presence

of 50 µM t10c12 CLA (Figure 2B; Note this study uses 50 µM t10c12 CLA treatments

when looking for effects that lower TG levels. This is because 100 µM t10c12 CLA efficiently depletes TG levels, making further reductions difficult to measure. Conversely, we use 100 µM t10c12 CLA when analyzing effects that increase TG levels as having lower TG levels as a reference point provides a larger range of measurable TG increases).

Addition of SKI-II, which is expected to increase ceramide and sphingosine levels by inhibiting production of S1P from sphingosine (Figure 1), gives a similar result: strong loss of TGs occurs in the presence of SKI-II in LA or t10c12 CLA treated adipocytes

(Figure 2B). Measurement of ceramide levels in these treated adipocytes indicates that addition of C2 ceramide or SKI-II raised endogenous ceramide levels in LA or t10c12

CLA treated adipocytes (Figure 2A). Note this immunological assay does not detect C2 ceramide (Supplemental Figure 1), indicating exogenous C2 ceramide increases endogenous ceramide levels. These results suggest that increased ceramides/sphingolipids can mediate TG loss in the absence of t10c12 CLA (Figure 2B).

Further, there is not an additive effect on lowering TG levels when these chemicals are combined with 50 µM t10c12 CLA. 77

A remaining question is whether ceramide/sphinolipid pathways are required for TG loss

in t10c12 CLA treated adipocytes. The diversity of sphingolipid pools and the

bioconversions between these pools makes this a difficult question to answer. For

example, adding fumonisin B1 or myriocin, both inhibitors of ceramide biosynthesis

(Figure 1), had little effect on the TG levels in the presence of 100 µM t10c12 CLA

(Figure 2C). Analysis of ceramide levels in adipocytes treated with 100 µM t10c12 CLA and these chemicals indicates that ceramide levels remained high in these cells despite the presence of these inhibitors (Figure 2D). This result suggests the ceramides are likely to be derived from de novo biosynthesis and established pools within these adipocytes.

To gain more experimental control over inhibiting ceramide production, we developed short hairpin RNA (shRNA) knockdowns for serine palmitoyltransferase 1 (SPT1) and

S1P phosphatase (S1PP). SPT1 catalyzes the initial step in ceramide biosynthesis and

S1PP produces sphingosine from existing pools of S1P (Figure 1). Efficient shRNA knockdown of SPT1 was achieved (Figure 3A), but this knockdown consistently resulted in increased TG levels in both LA and t10c12 CLA treated adipocytes (Figure 3B), making interpretation difficult. ShRNA knockdown of S1PP was efficient (Figure 3C), but did not affect TG levels in t10c12 CLA treated adipocytes (Figure 3B). When ceramide levels were measured for the SPT1 and S1PP shRNA knockdowns in t10c12

CLA treated adipocytes, they were still quite high, indicating inhibition of either one of these enzymes was not sufficient to diminish ceramide levels (Figure 3D).

78

In order to investigate the effects of inhibiting production of ceramides concurrently from

both the biosynthetic and S1P entry points into the pathway, combinations of shRNA

knockdowns of S1PP and chemical inhibitors were explored. A combination of shRNA

against S1PP and myriocin, a SPT inhibitor, is expected to inhibit both de novo

biosynthesis of ceramides and their production from S1P (Figure 1). This combination

was sufficient to strongly diminish ceramide levels in 100 µM t10c12 CLA treated

adipocytes (Figure 4A). Further, this combination had higher TG levels in the presence of

100 µM t10c12 CLA, relative to 100 µM t10c12 CLA alone, indicating it was effective in attenuating the ability of t10c12 CLA to cause TG loss (Figure 4B).

A similar analysis was performed using a combination of shRNA against S1PP and fumonisin B1. Fumonisin B1 is an inhibitor of sphinganine N-acyltransferase (Figure 1), and thereby should inhibit ceramide biosynthesis. In combination with a shRNA knockdown S1PP, this should block both biosynthesis of ceramide and its generation from S1P (Figure 1). This combination was sufficient to strongly diminish ceramide levels in 100 µM t10c12 CLA treated adipocytes (Figure 4A). However, although this treatment had a higher ratio of TGCLA/TGLA than the control, part of this effect of the

combination treatment was due to the lower TG levels in the LA treated sample (Figure

4B). As this result is difficult to interpret, our analysis relies primarily on the above

results with myriocin and S1PP shRNA to support the hypothesis that ceramides mediate

much of the TG lowering effects in t10c12 CLA treated adipocytes.

Ceramide signaling is downstream of AMPK and can increase AMPK activity 79

AMPK is a key target during t10c12 CLA treatments of adipocytes, as its inhibition by compound C abolishes the effects of t10c12 CLA [6]. As demonstrated above (Figure

2B), either C2 or SKI-II is sufficient to mediate TG loss in adipocytes in the absence of t10c12 CLA, raising the question of whether ceramide-mediated TG loss requires activated AMPK. To answer this, adipocytes were treated with C2 or SKI-II in the presence or absence of 10 µM compound C (Figure 5A). For comparison, the ability of compound C to interfere with TG loss in the presence of t10c12 CLA is also shown. A considerable reduction in TG levels occurs in the presence of C2 or SKI-II, but this response is not affected by the presence of compound C (Figure 5A). Therefore, we conclude that C2 and SKI-II act downstream or independently of AMPK to mediate TG loss in adipocytes.

We also measured the activity of AMPK in the presence of these compounds. The active form of AMPK can be detected with an antibody specific for its phosphorylated active state [29]. 50 µM t10c12 CLA activates p-AMPK, relative to p-AMPK levels in LA treated adipocytes (Figure 5B). Surprisingly, both C2 and SKI-II activated AMPK in 50

µM LA or t10c12 CLA to equivalent p-AMPK levels (Figure 5B). Thus, although AMPK is not required for TG loss in C2 or SKI-II treated adipocytes (Figure 5A), these compounds activate AMPK.

C2 and SKI-II activate NF-κB independently of AMPK

An inflammatory response is an important functional component of TG loss in t10c12

CLA treated adipocytes, and NF-κB is activated by t10c12 CLA as part of this 80 inflammatory response [3,14]. As an additional comparison between the response of adipocytes to t10c12 CLA and their response to C2 or SKI-II, we examined the amount of active NF-κB present in the nucleus. The ability of 100 µM t10c12 CLA treatment to activate NF-κB is partially dependent on AMPK as compound C attenuates this response

(Figure 6A). Treatment with either C2 or SKI-II activates NF-κB in adipocytes treated with either 50 µM LA or t10c12 CLA (Figure 6B). NF-κB activation occurs equally in

C2 or SKI-II treated adipocytes whether compound C is present or absent (Figure 6C).

This indicates that C2 or SKI-II act downstream or independently of AMPK to activate

NF-κB.

Apoptosis in t10c12 CLA treated adipocytes is a ceramide-dependent response

Measurement of the amounts of cellular DNA per well in treated adipocytes supports a role for t10c12 CLA in causing apoptosis as t10c12 CLA treated adipocytes have less total DNA (Figure 7). Addition of C2 ceramide or SKI-II caused similar levels of apoptosis as reflected in the lower amounts of total cellular DNA (Figure 7). Inhibition of the ceramide pool levels in t10c12 CLA treated adipocytes by concurrent inhibition of biosynthesis and entry from S1P by combinations of myriocin or FB1 and knockdown of

SIPP by shRNA attenuated apoptosis (Figure 7). This result suggests that t10c12 CLA- mediated apoptosis in adipocytes is mediated in large part by ceramides.

Discussion

The objective of our metabolic profiling analysis was to gain insight into signaling pathways affected by t10c12 CLA. Our analysis of adipocytes after 12 h of exposure to 81

t10c12 CLA, indicated there were significant changes in lipid pathways, as well as in sphinganine and sphingosine levels in the ceramide pathway. This study focused on the ceramide pathway due to its important roles in many aspects of cell biology and cell signaling [15-17]. Our functional assays indicate ceramide/sphingolipids are necessary for efficient t10c12 CLA-mediated TG reductions in adipocytes. Surprisingly, increased

ceramide/sphinonglipid levels are sufficient for lowering TG levels in adipocytes. It is

unclear how much of these responses are due to the physical properties of

ceramide/sphingolipids in cell membranes where they can create lipid rafts, alter

membrane fluidity, and interact with key membrane proteins, or whether ceramide-

stimulated signaling pathways are also participating [30].

An earlier report on the effects of t10c12 CLA on ceramide levels in adipocytes observed

that the rate of ceramide biosynthesis was attenuated when measured from radiolabeled

acetate or pyruvate [31]. This is consistent with the reduce rate of de novo fatty acid biosynthesis in t10c12 CLA treated adipocytes [7,31]. However, the abundant pools of pre-existing fatty acids and ceramides in adipocytes should allow for ceramide synthesis, salvage, and interconversion from existing components even when de novo fatty acid biosynthesis from two-carbon units is attenuated. Therefore, as total ceramide levels were not directly measured in this earlier report, it does not contradict our findings of increased ceramide/sphingolipid levels in t10c12 CLA treated adipocytes.

We discuss some of the known responses in adipocytes treated with t10c12 CLA [5] with regards to possible roles of ceramide/sphingolipids in these responses. T10c12 CLA 82 affects a number of biological processes including: increased energy expenditure and fatty acid oxidation [7,32]; increased NFKB, inflammation, and prostaglandin levels

[3,14]; activation of AMPK and repression of fatty acid biosynthesis [6,31]; insulin resistance via suppression of glucose transporter 4 (GLUT4) expression and function [33]; and apoptosis in adipocytes and adipose tissues [11,34].

Energy metabolism and ceramide activation of AMPK

Activated AMPK is required for TG loss in t10c12 CLA-treated adipocytes [6,14].

Activated AMPK increases fatty acid oxidation and energy production while decreasing fatty acid biosynthesis [6,31]. Increasing ceramide/sphingolipid levels by addition of C2 ceramide activated AMPK in adipocytes as described here or in an INS-1 pancreatic beta cell line [35]. Sphingosine was elevated 3.6 fold in t10c12 CLA-treated adipocytes, and sphingosine can be phosphorylated by sphingosine kinase to produce S1P, which activates AMPK in hepatocytes [36]. FTY720, an analog of S1P, can stimulate AMPK activity in adipocytes and is associated with an anti-obesity effect in obese mice [37].

Ceramides occur in mitochondria and can affect mitochondrial membrane permeability, respiratory chain function, oxidative phosphorylation, and apoptosis [17,38], and may account for some of the increased fatty acid oxidation occurring in t10c12 CLA-treated adipocytes.

Increased NF-κB and inflammation

Increased NF-κB activity is observed in t10c12 CLA treated adipocytes and this increased activity is necessary for the TG loss response in adipocytes [3]. NF-κB activity 83

is associated with an inflammatory response, including prostaglandin biosynthesis, which

is required for TG loss [12,14]. Our results demonstrate that exogenous chemicals that

increased ceramide/sphingolipid levels activated NF-κB, and although not measured here,

presumably stimulate an inflammatory response. Our results suggest that increased

ceramide levels could be fully or partially responsible for NF-κB activation in t10c12

CLA treated adipocytes.

Insulin resistance via suppression of glucose transporter 4 (GLUT4) expression and

function

Insulin stimulates the translocation of GLUT4 to the plasma membrane from internal

storage vesicles. T10c12 CLA interferes with this process as plasma membrane localized

GLUT4 is reduced through a NF-κB-dependent process [3]. Lowering glycosphingolipid levels in 3T3-L1 adipocytes results in increased amounts of GLUT4 plasma membrane levels [39]. Assuming higher levels of ceramide/sphingolipids result in higher glycosphingolipid levels, the enhanced ceramide/sphingolipids levels in t10c12 CLA

treated adipocytes could account for the reduced transport of GLUT4 to plasma

membranes [3]. In white and brown adipocytes, ceramides interfere with GLUT4 gene

expression, possibly through by down regulation of C/EBP-alpha expression, a

transcription factor essential for the expression of GLUT4. This provides another possible

ceramide-mediated mechanism in t10c12 CLA-treated adipocytes for reducing GLUT4

plasma membrane levels [40,41].

Apoptosis in adipocytes and adipose tissues 84

T10c12 CLA mediates reductions in adipocyte and white adipose tissue cell numbers in

mice in part by apoptosis [11,34]. Ceramides are known to stimulate apoptosis in adipocytes [42,43]. Our results demonstrate that concurrent inhibition of ceramide de

novo biosynthesis and entry from S1P strongly attenuate t10c12 CLA-mediated apoptosis.

This result suggests the ceramide/sphinoglipid pathway is a major apoptotic signaling

pathway in t10c12 CLA-mediated apoptosis.

In conclusion, our results strongly suggest that the increased sphingosine, sphinganine,

and ceramide levels observed in t10c12 CLA treated adipocytes have functionally

important roles in the TG loss response that occurs. The TG lowering effects of

ceramides in these in vitro cultures appear to contradict instances in animals where

inhibition of ceramide de novo biosynthesis attenuated obesity symptoms, facilitating

weight reduction, better energy metabolism, and improved insulin sensitivity [18].

Additional studies are required to elucidate the differences in these in vitro and in vivo

systems.

85

Table 1. Relative sphingolipid levels in 3T3-L1 adipocytes after t10c12 CLA treatment

Metabolitea CLA vs LA 0.5 h CLA vs LA 12 h

Sphinganine 1.46 9.81*

Sphingosine 1.38 3.65* aSix replicates of 3T3-L1 adipocytes, treated with 100 µM LA or t10c12 CLA for 0.5 or

12 h, were analyzed by metabolite profiling on GC/MS, LC/MS, and LC/MS/MS systems

(Metabolon, Durham, NC).

*p ≤ 0.05, for a t-test between the LA and CLA treatments at each time.

86

Figure Legends

Figure 1. Biosynthetic pathway of ceramide and sphingolipids. Entry into the pathway occurs by biosynthesis via serine palmitoyltransferase or

dephosphorylation of S1P by sphingosine-1-phosphate phosphatase (S1PP). The enzyme

targets of myriocin, fumonisin B1, and sphingosine kinase inhibitor SKI-II are also

shown as are the targets of shRNA used in this study. 87

Figure 2. Ceramide and TG levels in treated 3T3-L1 adipocytes. (A) Ceramide levels were measured in adipocytes 24 h after addition of LA or t10c12

CLA (CLA) with or without 30µM C2 ceramide (C2) or 10µg/ml of an inhibitor of sphingosine kinase (SKI-II) that is expected to increase ceramide levels. Immunoblot monoclonal antibody detection of ceramides is shown above the bar graph displaying the quantitation of the blot. (B) Triglyceride levels 24 h after 3T3-L1 adipocytes were treated

50 µM LA or t10c12 CLA (CLA) with or without 30 µM C2 ceramide or 10µg/ml SKI-II.

(C) Triglyceride levels 24 h after 3T3-L1 adipocytes were treated 50 µM LA or t10c12

CLA (CLA) with or without 20 µM fumonisin B1 (FB1) or 50 µM myriocin (Myri.). (D)

Ceramide levels were detected and quantitated as in A. Adipocytes were treated for 24 h with 100µM LA or t10c12 CLA, with or without 20 µM FB1 or 50 µM myriocin to 88

t10c12 CLA treated adipocytes. Each bar represents the mean ± SEM (n=3), a representative experiment of at least three independent experiments is shown. Means not sharing a common letter differ, p≤0.05.

89

Figure 3. shRNA knockdown of SPTLC and S1PP: effects on TG and ceramide levels. (A) and (C). Immunoblot analysis of whole cell extracts isolated 24 h after 3T3-L1

adipocytes were treated with 100 nM of short hairpin RNA (shRNA) against non-target

(sh-Non), or against Sphingosine-1-Phosphate Phosphatase 1 (sh-S1PP), or against Serine

Palmitoyltransferase 1 long chain subunit (sh-SPTLC), or transfection reagent only

(Control). Lanes marked by 1/3 have one third of the indicated samples loaded. (B)

100µM LA or t10c12 CLA were added 24 h after adding 100 nM of shRNA against non-

target, S1PP, or SPTLC. TG levels were measured 24 hr after fatty acid additions. (D).

Adipocytes were treated as described in B and ceramide levels were determined by

immunoblot (shown at the bottom of the panel) and their quantitation is shown by the 90 bars. A representative experiment of at least three independent experiments is shown for each panel. Each bar represents the mean ± SEM (n=3). Means not sharing a common letter differ, p≤0.05.

91

Figure 4. Inhibition of both biosynthetic entry points into ceramides affects TG and ceramide levels.

(A) Immunoblot analysis 24 h after 3T3-L1 adipocytes were treated with 100 µM LA or

t10c12 CLA in the presence of 100 nM shRNA against non-target (sh-Non) or S1PP (sh-

S1PP), and with or without 20 µM Fumonisin B1 (FB1) or 50 µM Myriocin (Myri). (B)

TG levels after 3T3-L1 adipocytes were treated as in A. A representative experiment of at least three independent experiments is shown for each panel. Each bar represents the mean ± SEM (n=3). Means not sharing a common letter differ, p≤0.05.

92

Figure 5. Ceramide signaling is downstream of AMPK. (A) TG levels in 3T3-L1 adipocytes that were untreated (Control) or treated with 30 µM

C2-ceramide (C2) or 10 µg/ml SKI-II or 100 µM t10c12 CLA, with (+) or without (-) 10

µM AMPK inhibitor Compound C (Comp. C). (B) Immunoblot analyses of AMPK and activated AMPK (p-AMPK) of cytoplasmic proteins isolated 12 h after addition of 50

µM LA or t10c12 CLA, with or without 30 µM C2-ceramide or 10 µg/ml SKI-II. A

representative experiment of at least three independent experiments is shown for each

panel. Each bar represents the mean ± SEM (n=3). Means not sharing a common letter

differ, p≤0.05.

93

Figure 6. Ceramides activate NF-κB independently of activated AMPK. (A) 3T3-L1 adipocytes were treated with 100 µM LA or t10c12 CLA for 12h, with or without 10µM Compound C. Nuclear extracts were analyzed by immunoblot for NF-κB and β-actin, and their relative levels of NF-κB and β-actin are displayed. (B) As in A except 50 µM LA or t10c12 CLA was used, with or without 30 µM C2-ceramide or 10 94

µg/ml SKI-II. 12h nuclear protein immunoblot analyses for the amount of NF-κB and β- actin with or without either 30 µM C2-ceramide or 10µg/ml SKI-II. (C) 3T3-L1 adipocytes in serum were treated with or without 30 µM C2-ceramide or 10µg/ml SKI-II in the presence (+) or absence (-) of 10 µM Compound C (Comp. C). Nuclear extracts were analyzed by immunoblot for NF-κB and β-actin, and their relative levels of NF-κB and β-actin are displayed. A representative experiment of at least three independent experiments is shown for each panel. Each bar represents the mean ± SEM (n=3). Means not sharing a common letter differ, p≤0.05.

95

Figure 7. Ceramides cause apoptosis in 3T3-L1 adipocytes. 3T3-L1 adipocytes were assayed for DNA amounts, as a percentage of control levels, as an indicator of apoptosis after various treatments. (A) DNA amounts after 50 or 100 µM

LA or t10c12 CLA treatments, with or without 30 µM C2-ceramide or 10 µg/ml SKI-II.

B) DNA amounts after 100 µM LA or t10c12 CLA treatments, with non-target shRNA

(sh-Non) or shRNA targeting S1PP (Sh-S1PP) and either myriocin (Myri.) or 20 µM

Fumonisin B1 (FB1). A representative experiment of at least three independent experiments is shown for each panel. Each bar represents the mean ± SEM (n=3). Means not sharing a common letter differ, p≤0.05. 96

Supplemental Figure 1. Monoclonal antibody to ceramides does not detect C2 ceramide. 10 and 20 nmoles of C2 ceramide were spotted onto PVDF membrane as well as lipid soluble extracts from adipocytes treated with 100 µM LA (LA) or t10c12 CLA (CLA)

and analyzed by immunoblot analysis with an antibody that detects ceramides. Complete

recovery of the 60 nmoles of C2 used per well at the standard 30 µM concentration

would result in 1.2 nmoles of C2 per spot, therefore the 10 and 20 nmole amounts are

approximately 8x and 17x more than the maximum C2 levels possible.

97

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101

Chapter IV. Conclusion

102

T10c12 CLA has been shown to reduce body fat in number of models, including mice, rat, pig, cultured mice and human adipocyte. However, its delipidation effect in human studies is still controversial. Despite numerous biological process like inflammation and apoptosis have been report associated with t10c12 CLA, its mechanisms remain unclear.

Besides, high doses of t10c12 CLA may cause side effects, including lipodystrophy, insulin resistance, hyperinsulinemia and liver steatosis in mice, which impede its application in humans. The goal of this research is to discover some of the mechanisms utilized by t10c12 CLA.

Our studies demonstrated cross regulation of sirtuin 1, AMPK and PPARγ was occurring in t10c12 CLA treated adipocyte. Inhibitors or activator of Sirtuin 1 decreased or increased AMPK activity in t10c12 CLA treated adipocytes. Sirtuin 1 may affect AMPK activity via deacetylation of protein kinase LKB1, which activate AMPK by phosphorylation. T10c12 CLA treatment also caused more Sirtuin 1 binding to PPARγ and NCoRI, which reduces PPARγ activity, leading to reduced transcription of lipogenic genes. On the other hand, inhibition of AMPK reduced Sirtuin 1 activity by affecting the

NAD+/NADH ratio generated in fatty oxidation. AMPK also decreased PPARγ activity by phosphorylation. Agonists or antagonists of PPARγ caused reductions or increases in the activity of AMPK and Sirtuin 1, respectively, demonstrating the repressive effect

PPARγ has on the activities of these proteins. Given that the effect of AMPK and PPARγ on lipolysis and lipogenesis, these data suggest an approach that nutritional supplements which inhibit PPARγ activity and/or increase AMPK/Sirtuin 1 activity will promote t10c12 CLA induced delipidation. 103

Our studies also revealed the role of ceramide/sphingolipid played in t10c12 CLA

induced delipidation. Elevated ceramide/sphingolipids pathway is functionally required

for t10c12 CLA-mediated delipidation. Increased ceramide levels strongly promoted

triglyceride loss. Inhibition of de novo ceramide biosynthesis attenuated t10c12 CLA induced delipidation and apoptosis. Increased ceramide levels by C2 ceramide treatment increased AMPK and NF-κB activity in t10c12 CLA treated adipocytes. Inhibition of

AMPK did not impair triglyceride loss or NF-κB activation when ceramide levels were elevated by C2 ceramide. These data suggest that ceramide signaling is downstream of

AMPK and can increases AMPK activity. In addition, measurement of the amounts of cellular DNA indicated that increased ceramide levels caused apoptosis, which is consist with apoptosis occurring during t10c12 CLA treatment. Inhibition of the ceramide levels attenuated triglyceride loss and apoptosis induced by t10c12 CLA. This suggests that t10c12 CLA induced apoptosis is mediated in large part by ceramide. Yet, sphingolipid metabolites can have opposite effects on cell survival and apoptosis. How these sphingolipids contribute to t10c12 CLA induced delipidation needs further study.

Metabolomic Profile of Mouse 3T3-L1 Adipocytes—Raw Data

BIOCHEMICAL LA 0.5h LA 0.5h LA 0.5h LA 0.5h LA 0.5h LA 0.5h 1-arachidonoylglycerophosphoethanolamine* 43172.84 61165.02 40411.12 69282.47 98006.21 72698.64 1-arachidonoylglycerophosphoinositol* 99947.16 114886.43 98105.29 167268.98 351995.16 243062.25 1-heptadecanoylglycerol (1-monoheptadecanoin) 109048.1 192654 206021.6 263111.3 242395 230405.5 1-heptadecanoylglycerophosphocholine 308017.86 637536.39 319789.37 317668.58 297377.66 399145.48 1-heptadecanoylglycerophosphoethanolamine* 349452.2 395411.9 468979.7 583516.5 457679.9 460678 1-linoleoylglycerophosphocholine 226132.99 121378.91 55571.89 18606 74712.64 1-linoleoylglycerophosphoethanolamine* 74456.98 87378.91 56174.95 56100.72 99621.83 66761.23 1-methylnicotinamide 2614420 3020385 1915202 2179021 2387520 2385461 1-myristoylglycerol (1-monomyristin) 364191.4 602703.5 317135.3 349618.7 576127 582828 1-myristoylglycerophosphocholine 190957.9 174587.17 85717.88 107054.54 44573.76 143463.57 1-oleoylglycerol (1-monoolein) 162814.2 204697.3 147971 208752.2 561709.5 414921.3 1-oleoylglycerophosphocholine 329150 436546.6 170454.4 384423.9 172367.8 290560.6 1-oleoylglycerophosphoethanolamine 195009.06 358815.24 122702.36 251877.72 98681.6 143552.76 1-oleoylglycerophosphoinositol* 515562.85 330601.24 441467.61 542273.63 480675.98 763826.04 1-palmitoleoylglycerophosphocholine* 2352034.6 1686764.2 670219.4 696497.3 1337632.2 1-palmitoleoylglycerophosphoethanolamine* 392439 480440.2 270347.2 383248.5 590673.7 568758.8 1-palmitoleoylglycerophosphoinositol* 229625.23 134067 128516.85 108879.41 277007.82 173421.51 1-palmitoylglycerol (1-monopalmitin) 1307000.3 1934397.6 1585823.8 2097894 2239022.5 2221571.8 1-palmitoylglycerophosphocholine 2138346 2862974.2 1621846.5 2403536.1 1002065.7 1627571 1-palmitoylglycerophosphoethanolamine 526376.7 525397.4 648223.3 636770.2 531228.4 581608.2 1-palmitoylglycerophosphoinositol* 704558.8 571021.4 822572.2 664181.9 643699.9 681556.2 1-pentadecanoylglycerol (1-monopentadecanoin) 707534.1 855548.2 845202.2 1071650.7 1662767.4 1677489.1 1-pentadecanoylglycerophosphocholine* 1112786.5 1069849.5 493519.2 575041 422592.5 649948.9 1-stearoylglycerol (1-monostearin) 174913 198101 195210 209761 313957 237000.6 1-stearoylglycerophosphocholine 903178.7 1274044.6 734274.2 1079193.3 601272.8 856714.7 1-stearoylglycerophosphoethanolamine 731758.1 1176437.2 708427.1 951261 426591 580393.2 1-stearoylglycerophosphoinositol 531071.4 422921.9 647552.9 706308.5 544517.2 646954.2 1,2-dipalmitoylglycerol NA 67338 73463 102783 104

1,3-dipalmitoylglycerol NA 27281 72795 31006.44 35251 39768 10-heptadecenoate (17:1n7) 20214762 21020128 22365108 24997774 40107302 36837476 10-nonadecenoate (19:1n9) 2117608 2050622 2174597 3456128 6559436 4397829 13S-hydroxy-9Z,11E-octadecadienoic acid 855044 1108038 801654 546005 1726393 2448722 2'-deoxycytidine 15853.24 27302.84 55357.08 43732.28 2-arachidonoylglycerophosphocholine* 168229.18 279965.32 67121.9 251421.09 42151.52 159036.04 2-arachidonoylglycerophosphoethanolamine* 2358333.6 2752866.3 1156162.1 2718277.4 843489 1737348.4 2-docosahexaenoylglycerophosphoethanolamine* 1057556.9 1118783.92 508564.31 1085778.4 240413.11 756837.29 2-docosapentaenoylglycerophosphoethanolamine* 873119.67 894424.4 379526.76 834850.27 137790.02 535589.86 2-hydroxyglutarate 113516 84282.24 36302 63187 90319 2-hydroxypalmitate 1114612.6 1480934.2 910366.3 873755.1 1633298.7 1327231.5 2-hydroxystearate 180691.9 200357.96 91715.97 117766.65 227599.38 216508.98 2-linoleoylglycerophosphocholine* 666757.6 528924.9 245980.9 156701.2 133582.4 2-linoleoylglycerophosphoethanolamine* 888449.88 857846.15 390939.51 553941.66 129216.46 321230.7 2-methylbutyroylcarnitine NA 23316.96 25616.65 55695.59 62131.83 95384.83 2-methylmalonyl carnitine 32553.62 63713.5 34364.49 138011.17 249409.53 263279.35 2-myristoylglycerol (2-monomyristin) 68470 49410 38074.25 107339.54 82774 2-myristoylglycerophosphocholine* 51812.16 58683.96 41819.66 41567.3 2-oleoylglycerol (2-monoolein) NA 74643.79 2-oleoylglycerophosphocholine* 664442.3 896469.11 373632.01 766592.36 204183.62 436456.08 2-oleoylglycerophosphoethanolamine* 803259.62 994713.78 519480.71 976173.94 180475.45 502660.26 2-palmitoleoylglycerophosphocholine* 3086179.9 2764680.5 1108315.3 2104813.7 753180.9 1181459.2 2-palmitoleoylglycerophosphoethanolamine* 2034678.1 3329337.4 1861142.9 2789790.4 783856.4 1310740.1 2-palmitoylglycerol (2-monopalmitin) 466515.2 711189.8 556035.7 748167.5 1186352.4 1054388 2-palmitoylglycerophosphocholine* 193826.34 291113.37 66511.28 146093.27 73665.57 169081.99 2-palmitoylglycerophosphoethanolamine* 91843.66 22510.95 59666.14 2-phosphoglycerate 55265.89 47634 33294 38817 96195.19 79268.16 3-(4-hydroxyphenyl)lactate 477746.9 463321.1 302292.2 339869.7 416156 455724.8 3-dehydrocarnitine* 65555 71622.22 68875.93 94077.6 182832.67 200616.94 3-phosphoglycerate 1921628 2422067 2155388 2796736 3769371 3368726 3-phosphoserine 45909.63 81236.8 103460.8 178643.59 149954.35 125654.39 105

4-hydroxybutyrate (GHB) 48568 101405.4 57485 70186.64 63206 123095 4-methyl-2-oxopentanoate 15077.76 14830.31 15892.16 12713.99 13271.94 34191.67 5-dodecenoate (12:1n7) 31533.27 22183.22 24471.47 31508.27 37665.08 61202.56 5-methyltetrahydrofolate (5MeTHF) 8285.25 10102.6 6797.72 8427.54 4229.4 11908.81 5-methylthioadenosine (MTA) 814425.8 708416.5 534414.6 630173.5 683168.4 773773.2 5-oxoproline 247021.9 319042 225753.5 294792.7 314807.8 419459 6-phosphogluconate 378661 638838.2 872143.9 472586 1196715.7 786081.4 7-alpha-hydroxycholesterol NA 22336 28572 30256 7-beta-hydroxycholesterol 49080 51625.2 32615 39883.41 37725 80813 acetylcarnitine 130427.9 208720.9 125734.5 300422.6 438431.3 862831 adenine NA 149485.53 43633.68 227839.19 232222.08 adenosine 321462.9 367345.2 390951.8 523011 384162.3 767351.7 adenosine 2'-monophosphate (2'-AMP) 387512.2 578441.8 405950.4 755713.2 1017098.5 1109611.2 adenosine 3'-monophosphate (3'-AMP) 97886.08 150855.91 114654.06 410336.13 115902.61 487925.89 adenosine 5'-diphosphate (ADP) 56028.1 63488.9 25221.26 67670.14 48992.97 72679.73 adenosine 5'-monophosphate (AMP) 1165049.4 1602569 1091916.7 2325200.4 1025133.7 1845784.6 adenosine 5'diphosphoribose 94692.06 184448.2 91417.51 96680.51 80334.04 195696.83 adrenate (22:4n6) 818513.8 628686.2 770470.7 755413.6 1191836.7 1115640.5 alanine 6580915 8946321 9019816 8492301 14370015 13318728 alanylalanine 55446.03 28521.05 30204.58 16822.86 54670.29 27715.9 alanylhistidine 41385.93 29114.56 35360.72 24585.95 58415.3 41121.49 alanylleucine 203013.98 95947.05 93651.05 47252.6 145232.84 70752.79 alanylthreonine 81019.38 59094.95 60242.81 36524.73 83971.29 58001.18 alanyltyrosine 226997.43 117029.47 125017.04 47866.7 288548.32 162322.46 alanylvaline 224471.67 149255.28 160181.01 66336.08 229171.72 135423 alpha-tocopherol NA 40846.32 26930.63 43361.06 arachidonate (20:4n6) 6307297 7517877 8239614 9030442 12993049 10747203 arginine 124324.6 235519.6 207072.1 187459.1 315739.4 241891.4 asparagine 983099.8 1333989.9 851148.5 884946 1533349.9 1491116.9 aspartate 13970492 11682058 12179211 14721932 13808632 14881978 aspartylphenylalanine 284495.1 73478.39 78325.73 27340.38 91446.77 36876.9 106

beta-alanine 154850.18 152861.73 82124.2 235512.12 274747.77 583046.11 beta-hydroxyisovalerate 91870.57 60684.57 36873.73 56154.57 56764.44 60844.56 betaine 171567.8 211362.6 234753.4 468190.5 266511 347713.8 C-glycosyltryptophan* 93146.96 155592.21 94514.39 161173.93 168823.45 276622.73 caprate (10:0) 91399.73 83229.9 79471.4 85402.66 101367.25 127247.19 caproate (6:0) 22714.26 18198.87 13752.07 9375.77 15022.59 28005.32 caprylate (8:0) 31881.74 15879.27 17088.95 15068.68 22868.33 35229.06 carnitine 731148.9 765292.9 410145.5 1422373.9 1520736.3 1875764.1 cholesterol 6573284 7306678 6792976 7438346 8324228 9863363 choline 2549495 2925796 2518028 3221771 4039715 4407590 choline phosphate 380485.4 363980.4 335216.8 450440.4 313156.9 324713.6 cis-vaccenate (18:1n7) 885827.9 1063167.8 1059276.1 802462.2 2008815.2 1437801.8 citrate 258207 499460.3 314019 427848.2 533915.8 752442.6 coenzyme a 34411.29 15631.05 18059.65 20925.53 27407.18 30436.71 conjugated linoleate (18:2n7; 9Z,11E) NA 21208 creatine 4796899 5392948 4170013 5742614 4171464 6095895 creatinine 280186.35 243109.22 166570.39 298020.28 172952.81 145306.13 cysteine 421372.18 656163.03 531432.63 629567.38 778099.24 866573.66 cysteine-glutathione disulfide 282900.9 502058.9 213110.5 229980.9 281108.3 332211.1 cytidine 1397954 1440548 1232934 1418630 1488629 1713125 cytidine 5'-monophosphate (5'-CMP) 249952.4 282922 228781.8 304369.4 230666.3 359330.2 deoxycarnitine 395607.7 415812.5 216659.3 629375.8 610324.9 600826.5 dihomo-linoleate (20:2n6) 897257.5 828773.9 676828.9 754956.6 992626.7 1090950.9 dihomo-linolenate (20:3n3 or n6) 725462.3 678227.6 990861.4 866326.9 953385.3 1014367.3 dimethylarginine (SDMA + ADMA) 731610.2 901833.5 1104445.9 813151.5 1371574.3 1186893.8 docosadienoate (22:2n6) 234534.1 313413.1 248140.4 279633.2 451587.7 386976.9 docosahexaenoate (DHA; 22:6n3) 1511465.7 1542078.6 2007739.6 2328370.8 2484971.9 2399202.5 docosapentaenoate (n3 DPA; 22:5n3) 1268528 1353581 1594354 1856656 2215570 2568407 docosatrienoate (22:3n3) 413479.4 659918.4 677926.6 673914.4 530486.3 659852.3 eicosapentaenoate (EPA; 20:5n3) 1941176 2634966 3269155 3269347 5128674 3823364 eicosenoate (20:1n9 or 11) 1998082 2292899 1811500 3221112 4195979 3051874 107

erythritol NA 53556 54753.66 69251 96747.61 erythronate* NA 31977.84 18097.04 50989 46386 ethanolamine 422907.4 738074.5 405532.5 938188.8 1100834.3 1252462.1 flavin adenine dinucleotide (FAD) 135223.97 120347.22 133976.32 156872.64 181516.88 187073 fructose 68312 202238 215895 441845.2 440027.5 695855.9 fructose-6-phosphate 360432.3 996117.1 143323 270762 593568.8 1541449.1 fumarate 409140.4 315494.7 174674.6 230632.1 353694.3 842460.8 gamma-aminobutyrate (GABA) NA 39868.17 26666.46 glucose 2013502 2896870 2742807 3969212 6953599 5511642 glucose-6-phosphate (G6P) 2046649.7 3989507.5 616846.8 1613160.5 1934227 6443648.4 glutamate 3890420 3692890 2092693 3678142 2867914 5990814 glutamine 4437269 4560033 2855214 4285155 4924544 6953395 glutathione, oxidized (GSSG) 870401.8 924374.3 544710.2 693306.9 651942.6 1180799.7 glutathione, reduced (GSH) 10780567 8790416 7680213 10147310 9188021 15049884 glycerate NA 113026 112764 99620.17 270462.65 183998.21 glycerol 10091203 16806790 14920959 15992400 26001495 23599705 glycerol 2-phosphate 49236 76914 108284 65801 84287 95313 glycerol 3-phosphate (G3P) 1376486 1781551 1618281 2044850 3610142 3423982 glycerophosphorylcholine (GPC) 6156352 2797340 8411618 6514708 3091459 glycine 5048731 5813399 5843349 5964649 10085712 11376579 glycylglycine 26231 16282.56 10252 glycylisoleucine 94154.34 85242.03 67133.65 67439.31 142302.11 114134.97 glycylleucine 796550.3 776634.7 872997.2 684832.8 1486883.6 1069539.1 glycylphenylalanine 36243.37 37333.22 35117.85 27610.23 55587.63 49886.13 glycyltyrosine 169757.41 252198.9 191793.54 150740.87 347230.9 254689.56 glycylvaline 78471.98 116972.08 132411.92 79766.17 139245.72 101597.54 guanosine 71059.67 46266.28 47100.28 40810.11 81420.09 80200.59 guanosine 5'- monophosphate (GMP) 408378.7 460170.3 337270.8 449252.2 334160.6 510373.2 gulono-1,4-lactone 86534 241024.58 139939 182037 284129 372798.93 heptanoate (7:0) 16676.11 10679.97 9038.44 11226.12 10900.11 histidine 122865.1 156329.5 164356.8 175080.1 222044.7 205294 108

hydroxyisovaleroyl carnitine 480308.1 386980.7 265334.7 755490.9 531545.8 557536.7 hypotaurine 512471.9 270942.1 99860 191693.6 74525 514667.6 hypoxanthine 488459.5 485720.8 459289.6 468087.6 561466.3 642380.4 inosine 2069570.3 1893221.8 1488696.9 1603053.1 2040462.6 2347264.9 inosine 5'-monophosphate (IMP) 19254.02 35954.35 27376.8 52874.29 1-phosphate (I1P) 760623.8 1141383.4 1468050.1 1387496.2 1803904.7 1099150 Isobar: fructose 1,6-diphosphate, glucose 1,6-diphosphate 12780.31 10475.84 9745.95 11109.38 Isobar: ribulose 5-phosphate, xylulose 5-phosphate 344904.51 308751.12 388668.17 677041.37 395179.81 591896.5 isoleucine 7925480 8600536 10293504 9479519 15831298 14426919 isoleucylleucine 199728.78 120119.13 162028.64 66855.98 208862 119685.02 isovalerylcarnitine 52550.38 34529.34 18481.45 72427.3 59287.79 85239.98 lactate 4300170 3777214 1990239 2860891 3806309 9809849 laurate (12:0) 627068.3 597249.2 575670.7 541564.8 896501.6 917037.8 leucine 26284268 29560769 31983928 28229106 49602157 40149491 leucylleucine 8601.99 10894.21 26613.61 17605.86 linoleate (18:2n6) 17369600 17936208 18026304 13039514 22502860 25537586 linolenate [alpha or gamma; (18:3n3 or 6)] 1003730 1091547 1116890.4 902784.2 1623239.6 1445522.4 lysine 1307686 1849241 1568411 1818547 2939907 2189029 malate 1524336.5 1222612.3 551592.8 822809.8 839524.7 1599957.8 maltohexaose 14770.32 23642.63 22767.47 35912.8 32742.15 maltopentaose 41552.17 41499.34 64196.45 66941.88 93167.66 88006.39 maltose 328742 535469.9 772552 874279 1493778.1 1122527.1 maltotetraose 134636.5 148355.79 260233.44 286087.38 399715 356552.4 maltotriose NA 23627.55 42535 64124.79 118578.76 100501.12 mannitol 16519.16 34420 mannose 373503 534080.8 522946.4 407443 949403.4 732556 mannose-6-phosphate NA 447510.71 80105.26 129395 249570.98 701328.03 margarate (17:0) 11721739 13320338 16511685 17662825 28383892 22386853 mead acid (20:3n9) 2030818 3030521 2641714 2428387 2302781 2909733 methionine 5358786 5860585 6177193 5926887 9945117 8425949 methylphosphate 203953 436312.5 38448 436380.1 510046.7 523997 109

myo-inositol 43301299 69451190 48442003 87703819 109688273 134674488 myristate (14:0) 8571753 10924705 10232619 9093888 13570092 12803111 myristoleate (14:1n5) 5621057 6181560 6478552 5112693 10960954 10767471 N-acetylalanine 27616.83 26189.36 29440.4 23287.01 38327.84 40129.97 N-acetylasparagine 159917.57 112343.34 39044.67 75105.89 85586.72 104916.47 N-acetylglucosamine 6-phosphate 81910.2 126919 88050.36 95653 123385 168727.97 N-acetylglutamine 71866.96 52391.92 20416.98 25194.54 20625.23 46934.5 N-acetylmethionine 518717.2 496884.9 417017.3 334134 564421.3 600814.5 N-acetylthreonine 29698.14 19735.72 11285.4 14697.88 23090.89 N-formylmethionine 18837.65 19404.45 16012.02 21671.63 20596.01 28110.21 N1-methyladenosine 378387.5 547313.6 355664 526423.2 1042410.8 849542.3 nicotinamide 1681699 3147934 2330663 2557335 2603212 4297180 nicotinamide adenine dinucleotide (NAD+) 5531126.4 2626253.4 2496179 2418452.7 2739522.1 3477173.7 nicotinamide adenine dinucleotide phosphate (NADP+) 102967.09 80403.48 34406.42 73452.61 81321.13 50973.55 nicotinamide adenine dinucleotide reduced (NADH) 20068.9 9820.18 6761.86 4883.69 11343.81 nicotinamide ribonucleotide (NMN) 1049126 1472586 1465982 2184723 2432427 2469378 nicotinamide riboside* 824256.9 881372 989417.8 875197.2 1386751.6 1276878.1 nonadecanoate (19:0) 634989.2 537580.9 788365.2 1068591.9 2001226.5 1071750.1 oleate (18:1n9) 2478856 3470450 3275510 3619855 6257250 5000087 ophthalmate 63371.29 50824.98 129906.41 221485.67 71223.58 127665.31 ornithine NA 47406.29 41146 109001 86572.03 palmitate (16:0) 50021697 49488630 49602927 51408796 78096635 79745126 palmitoleate (16:1n7) 69325592 77406276 75208738 73320322 125678595 111495655 palmitoylcarnitine 87601.91 49475.64 60614.9 94008.12 pantothenate 17816198 16427186 10652296 17203432 19704703 25382293 pelargonate (9:0) 245104.6 185059.5 190754.6 167331.5 215085.8 271192.2 pentadecanoate (15:0) 4341825 5465551 5573886 5140854 13270923 9894323 phenol red 102409.46 152531.09 126930.85 214700.74 238243.47 350937.56 phenol sulfate 12761.29 9111.39 5033.3 11284.76 14403.01 23099.78 phenylacetylglycine 43713.59 33427.46 24479.53 27773.44 34902.8 45647.24 phenylalanine 22230031 23273210 24891610 24040716 37179610 35069492 110

phenyllactate (PLA) 15994.5 20524.21 14013.62 12732.19 20597.49 14745.52 phosphate 180165653 171605722 175298435 180561040 199387351 190409735 phosphoenolpyruvate (PEP) 158866.3 246025.7 181756 282212.6 270124.2 245498 phosphoethanolamine 1127572.2 1295134.3 1085559.1 1192262.2 1407164.8 1623382.5 phosphopantetheine 2219111.2 2146372.2 1976174.9 2410121.5 2245868.2 2435475 pro-hydroxy-pro 46172.71 67604.13 63934.37 112278.71 134624.65 206589.23 proline 4101663 4039345 4060008 4088498 4888037 5336920 prolylleucine 763339.7 784278.3 856345.2 690201.3 1598071.3 1231656.1 propionylcarnitine 223366.02 172884.85 126485.54 197820.42 169228.23 433538.93 pseudouridine 21688.82 19609.64 19044.78 25772.09 27836.24 25808.17 putrescine 113324 79748 48260 55155.48 127058.56 110230.4 pyridoxal 234441.8 224630 178103.7 207983.6 226011.4 263028.8 pyridoxine () 5401.03 11588.57 9606.86 12951.44 11733.53 24852.83 pyrophosphate (PPi) 1155872.1 1364584.2 574525.5 1121896 435482.8 766531.3 pyruvate NA 37980.55 12552 14488 68180.38 118072.65 riboflavin (Vitamin B2) 74894.25 130685.96 111563.59 165771.88 167597.93 195529.04 ribose 125970.4 206464.9 344887.9 245488.6 489150.8 347588.6 S-adenosylhomocysteine (SAH) 31050.54 36267.92 34458.19 41799.64 47688.06 63989.17 scyllo-inositol 476315.4 771327.5 496677.1 1026614.3 1207978.4 1665875.4 sebacate (decanedioate) NA sedoheptulose-7-phosphate 244051.2 427072 366964.7 498122.8 647501.4 736922.3 serine 9732547 12200730 11631878 12868141 18286580 15251275 sorbitol 25995 60295.69 130814.13 157888.4 261144.75 spermidine NA 86317 23214 49980 127854.94 sphinganine 77176.57 73535.13 42762.88 94565.65 87457.22 sphingosine 415354.57 390339.41 131304.05 253426.55 149129.89 270101.09 stearate (18:0) 14856645 15038296 19761563 19826313 26843397 22719580 stearidonate (18:4n3) 86416.35 95272.02 108219.38 118068.99 291111.7 262480.79 stearoyl sphingomyelin 246430.9 320569.3 261438.5 364530.1 324005.9 410931 succinate 433524.4 212863.1 145382.2 199091.7 225391.3 401708.8 succinylcarnitine 111294.69 103081.76 25892.61 75230.95 148438.87 300622.96 111

thiamin (Vitamin B1) 245101.89 149968.41 139288.01 126111.69 161193.73 156715.07 threonine 5911477 5827229 4102874 5749348 7020044 8217351 threonylphenylalanine 197819.72 89418.65 96805.8 27745.95 214820.25 113604.89 trans-4-hydroxyproline NA 21028.38 27166 45246.02 59982.58 74630.42 trizma acetate 8597 6707 10892 tryptophan 4082718 4535572 4128828 4543148 6651698 6794150 tyrosine 8687651 10051073 10376225 11353303 15608952 16784635 UDP-N-acetylglucosamine 290296.16 56303.8 42281.84 18371.77 undecanoate (11:0) NA 28697.71 25307.37 39674.05 42388.43 uracil 54731.8 49979.91 65433.88 147961.51 123319.58 113599 urate 7555 37476 29878.69 28478 60277 67329.06 uridine 794501.8 808661.9 773815.8 657340.5 1112920.2 1101738.1 uridine 5'-monophosphate (UMP) 1026738.6 1321559 934579.8 1474107.2 683294.2 1387770.5 valine 8346765 11006083 11329067 11952849 19071121 17173310 xanthine 1662506 1559362 1350637 1510395 2512449 2467736 xanthosine 37129.49 37138.64 30533.59 25006.47 116649.76 78636.09

BIOCHEMICAL CLA 0.5h CLA 0.5h CLA 0.5h CLA 0.5h CLA 0.5h CLA 0.5h 1-arachidonoylglycerophosphoethanolamine* 46384.9 106125.66 55363.18 53788.99 126256.42 98533.96 1-arachidonoylglycerophosphoinositol* 126813.29 160854.08 170761.66 105875.23 302245.28 122124.5 1-heptadecanoylglycerol (1-monoheptadecanoin) 172985.3 200445 270334 277876 244586 238266 1-heptadecanoylglycerophosphocholine 435970.05 548690.8 142066.2 363418.11 774260.24 916031.84 1-heptadecanoylglycerophosphoethanolamine* 385237.5 1385859.2 595985.9 497761.1 1609270 335231.8 1-linoleoylglycerophosphocholine 204820.15 179805 44596.08 66945.06 178365.68 108164.81 1-linoleoylglycerophosphoethanolamine* 137872.18 252137.57 138370.19 41148.51 125489.02 65955.49 1-methylnicotinamide 2981024 2352922 2603669 1952809 2541791 2403463 1-myristoylglycerol (1-monomyristin) 281088 556547.9 459720 319412 557287.6 431046.6 1-myristoylglycerophosphocholine 176171.18 209298.77 48738.33 100875.57 144754.91 158761.83 1-oleoylglycerol (1-monoolein) 153094 167890 214993 200761 242147.1 216816.9 1-oleoylglycerophosphocholine 532827.4 392333.7 110587.7 335467.2 566345.3 821870 1-oleoylglycerophosphoethanolamine 433072.1 358615.66 68250.1 200811.47 462495.83 521116.01 112

1-oleoylglycerophosphoinositol* 784761.83 768396.32 708993.38 524964.82 457664.79 384857.47 1-palmitoleoylglycerophosphocholine* 1776578 1727417 277017.1 909182.6 1286055.5 1549416.9 1-palmitoleoylglycerophosphoethanolamine* 685437.8 848323.1 478457.3 277788.5 1044779.7 342228.1 1-palmitoleoylglycerophosphoinositol* 248243.57 252113.43 217168.47 154169.22 242738.24 181987.02 1-palmitoylglycerol (1-monopalmitin) 2269698.4 2360633 2641221.1 2165747 2593068.7 2080053.6 1-palmitoylglycerophosphocholine 3341712.5 2815305.6 849269 2011968.1 2311738.7 3628772.6 1-palmitoylglycerophosphoethanolamine 849052.8 1917786 1018346 476479.3 1520083.7 635940 1-palmitoylglycerophosphoinositol* 1244284.3 1323878.8 1341748.1 688086.9 735935.7 599521.1 1-pentadecanoylglycerol (1-monopentadecanoin) 1044287.7 961559.1 1114286.8 1069770 2021653.2 1122972.6 1-pentadecanoylglycerophosphocholine* 925671 1053203.8 222540.4 659771.4 740794.1 899280.8 1-stearoylglycerol (1-monostearin) 234793 250375 295202 327433 302248 281900 1-stearoylglycerophosphocholine 1348126.6 1574997.9 388235.5 1179612.7 998830.6 1984410.8 1-stearoylglycerophosphoethanolamine 1290993.6 1598996.1 400889 1115439 1003583.9 1361430.2 1-stearoylglycerophosphoinositol 853296.4 1159271.2 1057718.2 925790 788874.8 812765.1 1,2-dipalmitoylglycerol 65305 102303 65557 65875.38 1,3-dipalmitoylglycerol 27950 27341 32997.08 30535 8027 15590.05 10-heptadecenoate (17:1n7) 14580259 21374726 20253790 21549026 39984489 20815365 10-nonadecenoate (19:1n9) 2382500 2682884 2725812 3147302 5087517 2859043 13S-hydroxy-9Z,11E-octadecadienoic acid 2276200 2117631 1840337 1099398 1084694 2198037 2'-deoxycytidine 21578.95 23190.31 15799.66 31013.45 22161.79 2-arachidonoylglycerophosphocholine* 150672.16 182010.39 17432.86 179371.03 63271.5 293523.8 2-arachidonoylglycerophosphoethanolamine* 2050915.4 2263733.5 652612.2 2558338.7 1340122.3 3701498.5 2-docosahexaenoylglycerophosphoethanolamine* 696112.71 781185.78 153421.65 1085629.72 360601.68 1295292.7 2-docosapentaenoylglycerophosphoethanolamine* 693533.15 658506.92 114610.65 797965.74 368818 1187191.31 2-hydroxyglutarate 193522.89 92577.8 50555.91 35092 130135.82 76893 2-hydroxypalmitate 1173659 2685265.9 758451 1685985.5 915889.5 1014444.7 2-hydroxystearate 163102.19 458149.91 64522.79 166120.33 136839.88 156087.51 2-linoleoylglycerophosphocholine* 225504.3 246346.6 76273.7 133263.9 145600.1 193187 2-linoleoylglycerophosphoethanolamine* 400050.89 353280.76 154043 442561.49 136621.86 548114.62 2-methylbutyroylcarnitine 55631.47 25143.67 45885.79 68824.73 110001.19 2-methylmalonyl carnitine 47189.69 53407.43 45794.88 141140.85 311795.68 188740.28 113

2-myristoylglycerol (2-monomyristin) 71787.99 35326.81 86312.34 70263 2-myristoylglycerophosphocholine* 51239.71 62951.89 2-oleoylglycerol (2-monoolein) 28225.58 31529.77 36613.52 2-oleoylglycerophosphocholine* 770820.06 552393.49 182774.32 685682.87 316410.36 1220214.53 2-oleoylglycerophosphoethanolamine* 1041462.89 699469.84 216299.41 926477.69 361512.64 1306224.07 2-palmitoleoylglycerophosphocholine* 2414351.1 2316736.5 550575.6 2259964 1096504.9 2626630.6 2-palmitoleoylglycerophosphoethanolamine* 2538268.8 2268388.4 647027 2762316.1 1052165.8 3133804.1 2-palmitoylglycerol (2-monopalmitin) 700879 902371.2 870008.8 581858.4 959653.1 724779.6 2-palmitoylglycerophosphocholine* 235843.23 226252.91 35420.01 165446.72 88128.12 334082.49 2-palmitoylglycerophosphoethanolamine* 46351.96 105353.03 62086.66 2-phosphoglycerate 80804 37915 66992.52 38415 118323.81 94974.09 3-(4-hydroxyphenyl)lactate 673977.6 489082 441396.7 289301.8 635133.7 384800.5 3-dehydrocarnitine* 85394.05 53511.04 66358.27 106022.48 235902.29 145656.36 3-phosphoglycerate 3588696 2090709 3461311 2566017 4626752 3866502 3-phosphoserine 98074.84 111470.58 157236.01 107641 108354.31 110859.08 4-hydroxybutyrate (GHB) 103382 60482.41 91627 64183.18 51252.71 4-methyl-2-oxopentanoate 42122.84 12162.11 14699.37 9240.1 26450.36 13768.95 5-dodecenoate (12:1n7) 24237.69 28193.37 32104.86 14970.01 39502.91 18318.49 5-methyltetrahydrofolate (5MeTHF) 14391.24 6711.19 6802.12 6580.3 14580.94 16966.33 5-methylthioadenosine (MTA) 791829.9 774016 615965.2 654069.8 767483.1 701143.5 5-oxoproline 295270.9 331005.1 308551.1 254604 338716.5 372031.5 6-phosphogluconate 591914.9 553821.7 1116839.8 442125.9 897149.5 597513.1 7-alpha-hydroxycholesterol 7614 21419 13291 7-beta-hydroxycholesterol 50061.57 63508.39 47289.69 53858.14 39424 45362 acetylcarnitine 258855.5 175482.3 193999.6 255116.8 599803.7 696153.2 adenine 215832.35 141107 154576 143366.03 279015.35 201461.52 adenosine 514357.7 678153.4 415194.5 619494.8 582438.4 700804.8 adenosine 2'-monophosphate (2'-AMP) 654646.3 612175 588821.2 817158.6 1184543.3 885055.9 adenosine 3'-monophosphate (3'-AMP) 391269.55 361373.6 173277.99 362583.23 417024.69 471894.82 adenosine 5'-diphosphate (ADP) 75978.12 83906.12 65282.24 63987.18 95980.66 89686.52 adenosine 5'-monophosphate (AMP) 2490004.9 2211245.2 1472283.4 2013998.3 1807421.9 2084315.5 114

adenosine 5'diphosphoribose 64481.13 184177.56 68647.11 91745.22 199261.19 181546.18 adrenate (22:4n6) 749696.5 945519.7 718336.8 703156.8 1931564.9 1154956.4 alanine 8573928 6779074 12255402 7605920 11813328 11297950 alanylalanine 33752.09 20538.87 31521.35 13199.85 43533.89 21121.5 alanylhistidine 33953.26 25728.6 35505.97 25522.43 48589.88 25556.8 alanylleucine 83744.74 58110.32 87182.65 44583.01 88910.78 48819.25 alanylthreonine 47976.06 48280.16 56427.21 26961.62 76151.11 35585.17 alanyltyrosine 137198.42 44516.05 101022.6 81615.3 280158.58 37220.87 alanylvaline 119108.34 81238.43 143595.42 47285.76 157394.3 89858.58 alpha-tocopherol 36236.65 40482 arachidonate (20:4n6) 6845567 10196727 9958568 9122326 14714935 9450908 arginine 236880.6 184292.4 285279.9 163334.1 234811 157457.2 asparagine 1568497 995624.8 1349690.1 950552.9 1856060.9 1315532.9 aspartate 15395041 10510276 14592878 12773566 14684791 13589482 aspartylphenylalanine 45164.7 49382.33 66284.25 33973.21 34234.72 28514.49 beta-alanine 191084.19 59486.16 150477.32 97400 280379.41 463205.15 beta-hydroxyisovalerate 90154.4 62057.25 71446.19 39056.84 49492.17 52149.49 betaine 250895.4 256397 357086.1 273098.5 358536.9 C-glycosyltryptophan* 144351.12 141171.49 141043.42 171397.64 211928.74 221232.02 caprate (10:0) 96782.42 95687.5 81995.41 59073.88 102179.01 89455.17 caproate (6:0) 22165.12 23389.36 17645.99 15513.76 9276.7 13523.68 caprylate (8:0) 31423.63 28095.68 29692.99 15585.16 21199.78 20791.99 carnitine 869381.9 500395.1 685123.2 1299004.3 2128034.4 1671414.9 cholesterol 6674716 7921465 6855744 7374781 8401709 9145132 choline 3376561 2444711 3737906 2843693 3347924 3961461 choline phosphate 333117.4 235845.9 360113.6 531858.5 387578.4 451873.8 cis-vaccenate (18:1n7) 879648.5 886758.4 937668.7 933106.5 1452833.7 899639.8 citrate 855959.7 444076.5 489721.1 312565.9 987162.1 784669.3 coenzyme a 29859.3 22159.38 24650.14 20514.38 48727.43 28578.29 conjugated linoleate (18:2n7; 9Z,11E) 1196321.9 1244739.2 1279777 745975.4 558545.4 606238.8 creatine 5645938 3537992 5466791 4686269 5214880 5893137 115

creatinine 163000.75 66352.98 162184.55 175030.24 140094.28 384605.17 cysteine 555949.8 269083.47 830357.61 487904.57 985116.9 614342.79 cysteine-glutathione disulfide 351260.1 275459.8 323091 157963.6 334963.2 346199.8 cytidine 1631773 1386299 1763359 1207424 1539093 1420549 cytidine 5'-monophosphate (5'-CMP) 363144 320920.1 252178.4 309867.2 370880.4 372572.9 deoxycarnitine 493256.1 353553.8 409149.8 530988.9 807671.8 533143.8 dihomo-linoleate (20:2n6) 975820.7 1135318.9 741481.2 825970.5 901222.7 1064372.1 dihomo-linolenate (20:3n3 or n6) 555733.2 1032807.8 885608.9 921203.6 1027979.3 828620.2 dimethylarginine (SDMA + ADMA) 765801.4 890149.1 1410871.1 720834.9 757404 885520.1 docosadienoate (22:2n6) 427909.7 322224.3 339820.1 327809.3 334185 281927.1 docosahexaenoate (DHA; 22:6n3) 1599532.4 2360035.7 2147105.4 2286504 2659168.8 2046293.3 docosapentaenoate (n3 DPA; 22:5n3) 1088289 2209862 1521000 2034064 2600937 1927051 docosatrienoate (22:3n3) 679724.8 1112577.9 807574.5 667692.2 721899.6 464519.1 eicosapentaenoate (EPA; 20:5n3) 2657769 3508428 4189239 2952941 5130506 2851162 eicosenoate (20:1n9 or 11) 2597585 3637626 2462741 2811664 5674790 2650063 erythritol 38941 42405.06 55795 68490 104862.1 erythronate* 42969.66 22190 17403.05 52931.99 44985.15 ethanolamine 874270.3 480507.9 1287192.4 716094.7 899596.8 920382.4 flavin adenine dinucleotide (FAD) 134695.14 139123.96 154301.62 138366.19 204055 137210.12 fructose 117112 183582 232653 367093 403068.7 687208 fructose-6-phosphate 846381.8 646886.1 279660 380222.2 1027353.3 1664098.1 fumarate 268692.7 229726 220481.6 197747.9 504995.2 495162.2 gamma-aminobutyrate (GABA) 16533.67 27949.84 glucose 1889278 2194297 4305430 3477073 4639693 4524020 glucose-6-phosphate (G6P) 4622741 2474330.3 1307160.9 1209614.7 4979990.3 6997697.9 glutamate 4020133 2579253 3163025 3317974 2957539 6034405 glutamine 5527295 2823369 3901438 3435583 7084679 7074018 glutathione, oxidized (GSSG) 1092977.5 865181.6 742263.8 1221301 826395.4 922698.3 glutathione, reduced (GSH) 13212981 8802636 11246116 8102332 12839388 11710431 glycerate 114471.11 92612.91 183050.31 124485.1 238319.83 241671.46 glycerol 16285186 13817724 22079696 13703082 22692343 16853809 116

glycerol 2-phosphate 54990 73187.3 86903 120167 124046.5 glycerol 3-phosphate (G3P) 1324803 1567992 2118717 1716013 2906749 2919733 glycerophosphorylcholine (GPC) 1886565 6373786 6779140 6766875 3033050 2971953 glycine 6636157 4465341 7595391 5662960 9342116 9932219 glycylglycine 15335 11173.09 24119 13472 28381 23355 glycylisoleucine 71601.88 58422.13 85437.79 49516.35 114264.01 82530.84 glycylleucine 786541.6 680845.9 1015533.9 619992 1020792.4 759931.2 glycylphenylalanine 34234.05 30365.97 40375.92 27020.16 46455.24 35669.28 glycyltyrosine 196887.67 205326.03 324069.24 118989.21 314722.85 243548.89 glycylvaline 85388.09 68172.46 102980.28 56773.2 99499.92 108197.56 guanosine 87260.58 38704.36 59915.62 27919.06 94823.43 42049.16 guanosine 5'- monophosphate (GMP) 657169.3 475730.8 437047.6 474934.5 549317.7 500861.7 gulono-1,4-lactone 218196.92 141169.85 218896.89 33236 321132 305633.34 heptanoate (7:0) 17147.21 14785.65 14080.53 6829.04 7907.12 7371.97 histidine 184204.2 152748.4 226075.5 163896.1 213320.1 188041.7 hydroxyisovaleroyl carnitine 424625.1 294047.5 370485.4 766352.2 524431.6 526471.2 hypotaurine 459159.5 105826 220800 205411.6 525849.4 625049.3 hypoxanthine 601970.5 507837.9 653472.1 454112.5 585177.6 512090.6 inosine 2180327.6 1679265.2 1860518.2 1466015.1 2442350.5 1953190.3 inosine 5'-monophosphate (IMP) 37401.14 42398.46 15489.87 50888.12 111613.6 97390.66 inositol 1-phosphate (I1P) 961804.2 1482567 1706259.9 1073716.7 1268283 1186391.5 Isobar: fructose 1,6-diphosphate, glucose 1,6-diphosphate 14139.72 14191.35 9628.27 15216.27 11861.93 10417.36 Isobar: ribulose 5-phosphate, xylulose 5-phosphate 215593.44 478029.48 447162.21 650345.94 428528.24 363047.5 isoleucine 8390216 8680703 14454980 8622736 12837675 11443037 isoleucylleucine 89017.27 102518.13 150600.69 40464.13 199464.72 86809.69 isovalerylcarnitine 76957.3 43479.33 52137.85 54107.68 64837.91 110814.08 lactate 5641793 2643149 2722704 2583556 5204976 9682198 laurate (12:0) 686910.6 730935.7 804060.9 505442.2 918535.4 697387.8 leucine 27489966 25948559 42587662 24020239 36900737 30796581 leucylleucine 7933.6 13472.51 linoleate (18:2n6) 23470437 38257147 27422799 19883373 18780399 17210604 117

linolenate [alpha or gamma; (18:3n3 or 6)] 1269554.6 1581411.4 1553282 1024853.2 1287466.4 1026144.6 lysine 1732563 1300282 2597306 1569000 2063839 1944835 malate 1292965.9 942090.9 523537.4 715319.5 1222714.6 1723706.5 maltohexaose 15029.15 17710.16 29284.88 17899.62 25671.66 23904.78 maltopentaose 40494.53 50144.56 82601.66 52737.13 72215.89 59085.69 maltose 310360.1 453672 933123.2 832153.9 922098.8 923464.9 maltotetraose 133665.49 189591.27 291282.58 255506.38 258023.2 302556.62 maltotriose 81366.64 74879.75 52679.32 104471.22 mannitol 22578.5 35588 38731 mannose 416109 391564 807250.8 390916 643743.4 590398 mannose-6-phosphate 452376.21 339293.13 138626 146445.35 604437.47 664476.18 margarate (17:0) 12859794 18115911 22042473 17222012 26674773 15709376 mead acid (20:3n9) 2458833 4505721 3382482 2606338 3569899 2367790 methionine 6228546 5614339 8475868 5260685 7962005 6756623 methylphosphate 309973.3 396887.3 360547 357262.2 645964.8 609064.2 myo-inositol 56680717 46389306 68341154 86500912 135265362 119724589 myristate (14:0) 11933471 15161563 13479291 9133767 12525646 10178599 myristoleate (14:1n5) 6296191 6697444 8635737 4481147 9346205 5812807 N-acetylalanine 34368.36 25997.94 37246.42 24774.79 37944.61 32507.28 N-acetylasparagine 230581.16 107562.52 59900.99 40921.28 141450.02 101020.89 N-acetylglucosamine 6-phosphate 131793 123396.42 106789 156064 248491.78 260763.29 N-acetylglutamine 84954.74 48122.99 35509.11 26924.36 52145.9 39193.97 N-acetylmethionine 493018.9 426232.4 510941.6 390166.5 591245.9 412713.9 N-acetylthreonine 63646.53 18363.24 19675.4 24591.09 36096.7 N-formylmethionine 24462.5 18830.88 23861.3 20230.16 29751.68 23274.44 N1-methyladenosine 412857.8 529945.2 390836.7 444258.9 995985.1 656710.7 nicotinamide 2955098 3067395 3271761 2082360 3147948 3669517 nicotinamide adenine dinucleotide (NAD+) 5086091.8 2676351.9 3006473.4 2399618.2 4985740.4 1873741.3 nicotinamide adenine dinucleotide phosphate (NADP+) 81171.64 67053.31 64482.73 83659.86 163323.63 72697.77 nicotinamide adenine dinucleotide reduced (NADH) 23209.39 10904.54 10039.52 5394.55 13398.11 7717.38 nicotinamide ribonucleotide (NMN) 1409445 1768832 1721665 2158456 2219944 2097923 118

nicotinamide riboside* 740954.1 920710.3 960006.5 919142.4 1118648.4 1020436.9 nonadecanoate (19:0) 742374.9 830408.8 1218013.9 1292311.9 1293348.7 577667.1 oleate (18:1n9) 2563714 2764880 3382055 3120391 4490062 3675993 ophthalmate 67018.2 33779.21 218858.35 184877.29 94854.96 119586.53 ornithine 53019 55056.06 46040 49433 palmitate (16:0) 50291038 79927078 61986635 51794001 94353558 58477245 palmitoleate (16:1n7) 74843650 95653402 85753995 70339448 117095551 75387488 palmitoylcarnitine 81655.69 33492.86 39522.63 51617.01 95491.62 pantothenate 20012983 14706037 15930907 15563287 25528084 21447963 pelargonate (9:0) 217288.9 241511.6 249473.6 152180.4 205006.5 200429.5 pentadecanoate (15:0) 4583551 4510709 7196892 3995031 10095879 5599150 phenol red 115757.59 159352.5 155107.99 180434.27 241826.89 255220.25 phenol sulfate 8894.07 8553.55 9404.72 12739.72 11997.67 phenylacetylglycine 47901.7 35341.29 33867.54 20916.15 41012.94 32692.89 phenylalanine 23899506 23017238 35150857 21511334 31713549 28186748 phenyllactate (PLA) 26711.48 18061.5 17547.92 6805.8 27044.22 7323.76 phosphate 157448323 156896527 190090595 162297511 167024830 180541975 phosphoenolpyruvate (PEP) 346715.5 184749.6 305061.8 190575.6 319787.4 242186.4 phosphoethanolamine 1482260.4 942668 1166623.8 1219681.2 2685488.5 1399795 phosphopantetheine 1856817.3 1985640.5 2365744.3 1294111.1 2748275.9 1173687.6 pro-hydroxy-pro 66927.66 46272.4 89041.44 96663.61 105026.11 164546.87 proline 4341884 3202497 6206481 3525166 4365295 4972204 prolylleucine 778768.1 713165.3 1354453.9 581622.8 1116046.1 763728 propionylcarnitine 354353.37 156715.06 212654.03 315233.15 686707.77 424565.95 pseudouridine 24335.55 23344.09 24910.59 25735.22 30541.86 22516.76 putrescine 116961.05 57771.9 90729.96 50503 89736 96134.59 pyridoxal 230084 137134.2 182781.4 189208.1 216450.6 197213.7 pyridoxine (Vitamin B6) 7996.23 8896.12 12900.36 14675.45 9579.99 23543.85 pyrophosphate (PPi) 194042 140984.9 417204.5 190677.6 860667.3 pyruvate 13815 25767 23460 61699 82471.98 riboflavin (Vitamin B2) 104586.98 113590.88 168795.63 155747.13 130167.51 158152.59 119

ribose 183123.7 218925.1 555957.9 180945 263043.8 242509.8 S-adenosylhomocysteine (SAH) 41633.78 41877.98 43843.86 37410.66 52601.9 47076.9 scyllo-inositol 645321.6 458279.3 621495.1 922101.5 1633972.4 1424452.5 sebacate (decanedioate) 15715.5 21253.29 24605.02 13753.33 7273.51 10060 sedoheptulose-7-phosphate 542272.3 464953.7 616545.8 516594.7 576909.8 588454.3 serine 13645957 9702435 16774062 11425395 15699430 13097283 sorbitol 40504 45899 90858 154924.82 273265.86 spermidine 86942.8 72028.72 63916.79 75109 101165 sphinganine 128158 163553.99 150528.97 59811.78 92044.75 sphingosine 470485.64 600076.36 56470.1 536364.97 144071.15 555267.7 stearate (18:0) 17982626 22387862 23287093 20074807 20835714 16884059 stearidonate (18:4n3) 87523.97 122683.32 169042.83 86316.99 269180.02 128370.03 stearoyl sphingomyelin 292664.3 310024.7 200112.2 326684 402550.7 475196 succinate 405038.1 213130.3 224038.8 178657.6 259168.2 368498.9 succinylcarnitine 126425.7 62872.87 28562.41 62270.63 319532.29 178681.21 thiamin (Vitamin B1) 202988.56 105267.15 253251.43 95872.3 88941.96 143659.73 threonine 7433207 4451937 6795692 5099413 8119645 7017922 threonylphenylalanine 46261.29 68960.3 89137.5 45164.73 202541.64 78208.32 trans-4-hydroxyproline 51996.66 36331 30179.39 24193.69 95923.53 102242.74 trizma acetate 11706 17513 tryptophan 4319292 4075244 6049500 4214963 5800362 5328333 tyrosine 11356066 10787777 15007126 9685236 14146898 12876423 UDP-N-acetylglucosamine 267974.8 33095.56 52253.22 18913 undecanoate (11:0) 20616.98 23233.49 39812.93 26699.24 46771.07 35106.34 uracil 111495.73 47153.83 111075.43 102605.94 63122.06 124755.38 urate 11318.62 40118.41 36047.66 15848 65463.28 69428 uridine 782132.3 686120.8 925144.6 646767.8 930432.9 850699.5 uridine 5'-monophosphate (UMP) 1401178.4 1238793.8 1209550.3 1343570.9 1222541.6 1604032.1 valine 10903856 9060760 16792438 9018113 13591862 13720868 xanthine 1848649 1346764 2260949 1328056 2240823 1862603 xanthosine 49626.58 18403.4 72652.28 35847.73 85466.18 55320.01 120

BIOCHEMICAL LA 12h LA 12h LA 12h LA 12h LA 12h LA 12h 1-arachidonoylglycerophosphoethanolamine* 45016.53 91233.82 68595.4 54914.71 43749.3 97206.34 1-arachidonoylglycerophosphoinositol* 79920.48 147610.14 48249.36 225763.65 218758.73 102243.13 1-heptadecanoylglycerol (1-monoheptadecanoin) 177819.5 177921.6 89016 253227.2 233378.1 193837 1-heptadecanoylglycerophosphocholine 306536.96 346472.58 521208.06 142170.96 51130.25 351584.38 1-heptadecanoylglycerophosphoethanolamine* 369910.6 983153.7 192978.5 340624.3 377869.1 225865.4 1-linoleoylglycerophosphocholine 160631.13 196932.38 328079.53 103093.04 204345.35 1-linoleoylglycerophosphoethanolamine* 101515.88 244116.96 112195.46 133962.65 125935.73 128286.99 1-methylnicotinamide 1794993 2593095 2118774 2655424 2767818 2331571 1-myristoylglycerol (1-monomyristin) 372129.2 359359 245796.8 492917 492339.9 185925 1-myristoylglycerophosphocholine 113724.52 216749.51 168912.75 40504.08 126994.43 1-oleoylglycerol (1-monoolein) 171772.3 177738.9 33363 245838.5 167190 230688 1-oleoylglycerophosphocholine 308961.9 414407.5 535520.4 197533.1 101113.6 447335.2 1-oleoylglycerophosphoethanolamine 179333.15 368462.48 273911.31 93636.28 35697.2 214721.21 1-oleoylglycerophosphoinositol* 374513.7 585764.48 90084.39 480118.12 565463.57 363101.08 1-palmitoleoylglycerophosphocholine* 1017784.1 1828349.5 1322862.4 207726.6 890444.8 1-palmitoleoylglycerophosphoethanolamine* 318844.1 748018.5 207784 383004.4 394887.7 232350.6 1-palmitoleoylglycerophosphoinositol* 149380.76 267143.12 39183.92 163571.18 197235.2 63016.83 1-palmitoylglycerol (1-monopalmitin) 1670901.5 1438603.8 771336.1 2177666.2 1854574.3 2152192 1-palmitoylglycerophosphocholine 2093099.5 2821280 2985455.5 1009476.4 398051.1 2805839.6 1-palmitoylglycerophosphoethanolamine 535033 1379195.6 240163.8 475655.1 537590.4 478165.4 1-palmitoylglycerophosphoinositol* 723408.9 972524 159625.5 500643.5 590336.6 585125.2 1-pentadecanoylglycerol (1-monopentadecanoin) 803968.2 861298 437946.3 1274808.4 1397091.6 721514 1-pentadecanoylglycerophosphocholine* 634486.9 1071326 869084.9 214263.5 125678 560928.1 1-stearoylglycerol (1-monostearin) 236014.9 190505 153855.7 265920 187290 343254 1-stearoylglycerophosphocholine 996708.4 1188322.4 1637332.8 483962.1 166740.5 1359382 1-stearoylglycerophosphoethanolamine 767627.7 1062010.9 863618.3 262558.4 104901 759296.4 1-stearoylglycerophosphoinositol 685783.8 650765.8 222056 697517.8 500482.5 745058.9 1,2-dipalmitoylglycerol 33183 82958 60065 93110.07 1,3-dipalmitoylglycerol 13544 28054.6 26730 42247 121

10-heptadecenoate (17:1n7) 17157462 24372310 11463730 27132362 24132898 17347394 10-nonadecenoate (19:1n9) 2260113 2158716 1244340 4018252 2527891 2283458 13S-hydroxy-9Z,11E-octadecadienoic acid 1487501 978143 1469460 1445361 647870 4649716 2'-deoxycytidine 31700.17 18449.51 34844.87 32114.58 22535.03 2-arachidonoylglycerophosphocholine* 128114.74 122869.22 435931.63 87040.64 280543.74 2-arachidonoylglycerophosphoethanolamine* 1678653.7 2087068.1 3683307.4 1022296.9 205709.4 2178994.8 2-docosahexaenoylglycerophosphoethanolamine* 680481.98 751197.99 1170955.28 299583.44 64560.96 698569.96 2-docosapentaenoylglycerophosphoethanolamine* 638107.44 633712.01 1080666.17 169069.98 69303.98 621281.45 2-hydroxyglutarate 30303.94 198636 42164 35700 48192.38 125499.02 2-hydroxypalmitate 874891.9 2363014.8 1978084.1 966052.7 529839.2 2870332.4 2-hydroxystearate 81508.03 387544.41 263516.81 108399.62 87262.02 350413.86 2-linoleoylglycerophosphocholine* 446808.8 424081.2 1215322.7 208702.9 164989 908183.1 2-linoleoylglycerophosphoethanolamine* 970937.37 1172529.44 1578015.53 357354.97 73542.52 844900 2-methylbutyroylcarnitine 52559.61 34414.52 22674.46 55464.6 33679.8 160398.17 2-methylmalonyl carnitine 30620.61 37658.26 346023.52 204717.06 179158.71 2-myristoylglycerol (2-monomyristin) 66259.02 139307 76215.44 132525 2-myristoylglycerophosphocholine* 36041.23 41636.25 98147.53 18702.28 2-oleoylglycerol (2-monoolein) 59127.52 39862.04 45637.38 2-oleoylglycerophosphocholine* 506886.49 494498.6 859102.45 221095.36 53472.46 670033.93 2-oleoylglycerophosphoethanolamine* 742345.08 799557.61 1083223.18 212925.14 31618.43 663109.48 2-palmitoleoylglycerophosphocholine* 948646.8 2177388.5 2075347 551315.2 163713.2 1264936.1 2-palmitoleoylglycerophosphoethanolamine* 2217586.7 1975743.3 2192678.9 672836.5 119427.9 959832.3 2-palmitoylglycerol (2-monopalmitin) 678514.6 543140.3 292985.4 901014 752420.5 1131237.2 2-palmitoylglycerophosphocholine* 220901.66 220122.64 511720.59 82779.65 284241.66 2-palmitoylglycerophosphoethanolamine* 66434.03 94677.5 87098.81 36874.07 2-phosphoglycerate 48447.81 108564.35 123614.18 100098.52 144796 3-(4-hydroxyphenyl)lactate 196438.5 537194.7 282367.6 334242.5 266406.7 593561.8 3-dehydrocarnitine* 62981.7 73933.02 44517.67 198819.2 129713.8 222491.77 3-phosphoglycerate 3025542 5065899 2247283 4905688 4496574 6878829 3-phosphoserine 144859 174801.14 73319 235584.23 126931 147613.14 4-hydroxybutyrate (GHB) 40406.56 112425.15 19756 86890.88 56715 187830.43 122

4-methyl-2-oxopentanoate 12291.07 29096.12 13226.07 40005.3 21446.54 22083.95 5-dodecenoate (12:1n7) 38210.06 47232.17 7746.99 18054.61 10224.75 21570.99 5-methyltetrahydrofolate (5MeTHF) 8550.96 6480.08 13400.54 18065.75 16921.39 5-methylthioadenosine (MTA) 725021.7 688312.8 523608.8 854698 851161.7 878394.3 5-oxoproline 496705.8 298075.1 219477.1 316060.8 285336.4 476603.8 6-phosphogluconate 480087.7 715138.2 287593.4 737441.7 692868.8 578244.4 7-alpha-hydroxycholesterol 21968.54 32307.16 85278.98 7-beta-hydroxycholesterol 51818.01 84865 59678 63551.06 36120.38 165970.9 acetylcarnitine 191918.3 301451.8 141355.1 852673.7 428451.3 878957.6 adenine 168527.58 180201.5 103352 271065.55 287576.37 286100 adenosine 745182.6 460755.5 455149.6 676459.2 853659.3 751870.8 adenosine 2'-monophosphate (2'-AMP) 644858.9 685887 629004.2 1269955.6 1091193.6 1135342.3 adenosine 3'-monophosphate (3'-AMP) 1102367.92 265139.69 212895.43 393694.41 758920.55 534841.01 adenosine 5'-diphosphate (ADP) 93409.33 85518.04 59753.18 75818.58 96413.67 111057.7 adenosine 5'-monophosphate (AMP) 4499619.8 1763098.4 1538806.4 1876005.4 2179543.3 2607659.6 adenosine 5'diphosphoribose 113494.26 89170.14 120869.37 183013.49 172654.77 151520.14 adrenate (22:4n6) 588877.3 1580620.7 474785.1 1104561.6 749918.8 1114643.5 alanine 7974730 11190168 5298131 16559085 10488688 23180021 alanylalanine 10938.11 28202.25 6945.63 29258.13 33511.84 12268.4 alanylhistidine 21012.3 35648.62 34890.3 31996.72 20841.36 alanylleucine 36009.73 66418.96 17258.06 84115.62 84226.14 41905.74 alanylthreonine 29381.58 49759.13 14695.09 63615.81 64508.11 20140.22 alanyltyrosine 71620.03 134983.21 31533.76 163509.97 118562.61 96404.04 alanylvaline 67718.37 112325.89 35921.75 137819.24 178943.97 59436.53 alpha-tocopherol arachidonate (20:4n6) 6110998 8849570 5190674 8317800 7364611 5793766 arginine 192320 217737.3 166872.1 219280.1 184454 279663.4 asparagine 689755.3 1800043.8 623096.4 1714526.2 1082185.6 2281563.1 aspartate 10977931 13142530 6988831 21014791 17015097 24446536 aspartylphenylalanine 15623.04 51998.58 22960.82 55944.74 52824.44 67492.47 beta-alanine 164219.46 124176.59 92806.42 560908.95 385003.79 780320.25 123

beta-hydroxyisovalerate 211370.63 75068.32 54888.1 84604.9 74314.26 115457.62 betaine 189400.6 228843.2 369977.8 386147.1 399235.6 C-glycosyltryptophan* 136832.57 143010.12 120975.35 259473.77 179847.95 356354.99 caprate (10:0) 87144.51 76762.82 46803.34 94013.18 100428.63 94460.84 caproate (6:0) 20362.63 11453.05 17904.48 8499.98 17584.64 19756.42 caprylate (8:0) 30391.97 17595.93 16203.94 23724.33 24214.37 37620.14 carnitine 475971.3 676802.9 580128.8 2034815.8 1389773.3 1946132.1 cholesterol 7013783 7605388 5225722 11149726 5918192 10278376 choline 3630442 3076391 2856773 4355495 3768036 4994869 choline phosphate 564828.8 381662.1 618779.8 360335.6 262400 491552.8 cis-vaccenate (18:1n7) 943332.7 1014275.7 580808.4 1600497 1154877.8 1321682.6 citrate 976824.3 991198.2 351987.6 815749.7 904480.1 708334.8 coenzyme a 18426.55 22629.51 10694.8 50828.07 38675.73 38289.4 conjugated linoleate (18:2n7; 9Z,11E) 180130 30579 creatine 4175811 4456746 4683566 6568842 6148734 7332047 creatinine 117318.93 202149.11 146234.6 200259.48 177144.05 290299.65 cysteine 527598.44 449939.61 241118.98 718609.17 705693.27 1028082.84 cysteine-glutathione disulfide 136787 302341.8 203239.4 322946.8 241540.6 332780.7 cytidine 1106833 1468838 1115892 1806396 1383667 2509205 cytidine 5'-monophosphate (5'-CMP) 346463.8 303734.5 255383.3 336644.5 341530.3 427936.2 deoxycarnitine 348543 401910.4 286642.9 903316.8 523442.1 788046.5 dihomo-linoleate (20:2n6) 834573.8 1375812.1 1246706.3 976247.5 914684.8 2605197.1 dihomo-linolenate (20:3n3 or n6) 777019.6 1230472.2 713349.2 833667.5 882632.6 1111092.9 dimethylarginine (SDMA + ADMA) 658847.7 737917.3 550782.7 1225495.8 860796.2 1087834.2 docosadienoate (22:2n6) 211479.5 359120.7 224008.6 345122.8 224745.3 367170 docosahexaenoate (DHA; 22:6n3) 1223117.8 1752504.2 952310.8 2074954.7 1826583.1 2383849.4 docosapentaenoate (n3 DPA; 22:5n3) 1115544 1728206 1141537 2125684 1758397 2131204 docosatrienoate (22:3n3) 443603.4 912683.8 442025.4 686546.3 577759.3 621318.7 eicosapentaenoate (EPA; 20:5n3) 2966918 2862783 1962635 3143438 3269636 1932951 eicosenoate (20:1n9 or 11) 2438046 3183999 1702702 3547771 2179896 2777273 erythritol 48072.26 55720 45986 123815.71 82073 176664.21 124

erythronate* 17478.14 67183.71 53526.4 97594.13 ethanolamine 731971.9 675542 366343.7 1609666.3 1300670.9 1399446.7 flavin adenine dinucleotide (FAD) 158509.7 127103.46 77140.4 214217.22 216180.68 182073.13 fructose 533635.1 192248 133147 496505.1 395781.2 999066.9 fructose-6-phosphate 264411.8 1219997.5 875134.5 1653906.5 890260.8 2911932.7 fumarate 212233.7 213939.5 120376 606542.8 533501.9 1140169 gamma-aminobutyrate (GABA) 9544 22066.14 14899.96 51133.8 64121 glucose 6043702 3732263 1879876 6579341 3577822 6688778 glucose-6-phosphate (G6P) 1200879.5 6030119.3 4158068.6 5006955.7 3461188.8 11961148.4 glutamate 4293976 3841204 2378242 8177091 3767005 17846199 glutamine 2711928 5048110 2465107 6299029 3713245 9525738 glutathione, oxidized (GSSG) 1475075.6 1001877 650346.8 1124852.2 939185.6 1662210.4 glutathione, reduced (GSH) 9630873 11641724 6254804 15537181 14244344 19149657 glycerate 89444.92 244618.95 120507.23 219389.57 155634.8 333255.41 glycerol 14416381 21779195 8379355 28608984 22406871 23498367 glycerol 2-phosphate 48216 73480 47435 171060.1 112899 139814.3 glycerol 3-phosphate (G3P) 1681562 1885242 1328578 3902931 2969133 4556189 glycerophosphorylcholine (GPC) 7240729 4959428 5475201 6728211 7256145 4166682 glycine 5193195 7704011 3855710 12021657 6951262 17883930 glycylglycine 26058.96 10316 17506 14628.16 29478 glycylisoleucine 69681.36 54315.84 49320.57 108931.23 102192.63 84008.41 glycylleucine 532998.9 722614.5 344819.1 1145361.6 927045.2 823226.6 glycylphenylalanine 24977.67 31882.96 10570.24 47324.61 36110.21 43058.33 glycyltyrosine 198600.85 266396.07 117488.45 233955.29 195354.58 187143.73 glycylvaline 66641.54 108664.29 34751.16 92525.33 86580.96 93194.69 guanosine 16197.07 50057.62 21167.76 92164.71 79434.93 73924.66 guanosine 5'- monophosphate (GMP) 508559.1 478343.7 663462.7 530448.7 544073 730643.9 gulono-1,4-lactone 49503 345476.07 127315 415452.83 329543.07 455698 heptanoate (7:0) 18935.5 6991.15 8025.02 12447.47 12305.51 14174.02 histidine 157293.8 179210.6 118078.6 204853.8 170377 229887.6 hydroxyisovaleroyl carnitine 548277.7 343104.2 385640.7 1163509.1 636614.2 794646.7 125

hypotaurine 175440 375301.8 209888 559698.7 356336.6 927769.5 hypoxanthine 425531.7 556311.1 408237.2 713081.6 591704.7 773234.4 inosine 1282581.9 1892357.7 1071978.1 2240576.4 2109109 2731477.9 inosine 5'-monophosphate (IMP) 37117.05 60458.11 58825.14 137766.11 inositol 1-phosphate (I1P) 918991.4 1193395 689606.2 1099799 895332 1331062.3 Isobar: fructose 1,6-diphosphate, glucose 1,6-diphosphate 19202.52 8790.64 14156.9 20062.08 9869.23 Isobar: ribulose 5-phosphate, xylulose 5-phosphate 809489.02 185024 91243.77 756075.61 862136.96 537566.6 isoleucine 8231296 10053053 6157969 16514716 10686807 18990720 isoleucylleucine 49415.97 76401.28 123202.75 118572.16 69539.76 isovalerylcarnitine 103463.08 46029.83 50412.97 71474.18 28626.38 202218.7 lactate 3377499 8735904 3502556 9348065 5040484 26703793 laurate (12:0) 716936.3 584291.5 360601 722008.6 544653.1 555524.7 leucine 24219148 27739635 17203890 45831652 34231973 47219489 leucylleucine linoleate (18:2n6) 20550420 23377048 18155512 26234583 20054910 24687157 linolenate [alpha or gamma; (18:3n3 or 6)] 1398253.7 2203208.3 2416279.3 1934159.1 1600691.1 2218235 lysine 1490472 1914024 1054770 2835433 1865036 3073553 malate 672316.1 1491437.3 824609.9 1056303.4 1135522.1 2903773.6 maltohexaose 22699.29 17163.27 9144.94 24929.86 10873.78 19731.58 maltopentaose 70019.89 46001.86 27131.49 71427.93 57333.86 64602.34 maltose 691926.9 441665 330028.9 716730 548471 894814.5 maltotetraose 340675.34 163394.14 98200.23 235775.71 230560.66 222047.53 maltotriose 67323 34869 20243 75176.14 65379.77 69741.56 mannitol 38834 21748 56302 mannose 282560 826161.3 74620 943942.3 553827 1261875.3 mannose-6-phosphate 86552 748094.08 386684.44 580620.89 361124.24 1295653.88 margarate (17:0) 11319821 12335753 8060851 19882113 16541466 12960932 mead acid (20:3n9) 2286435 3915922 2283243 2114183 2333838 1774838 methionine 5076778 6004157 3490526 8983153 6778995 9859179 methylphosphate 163232.4 442279.8 267547.4 644894.4 665047.3 1333160.2 myo-inositol 45414703 68265745 49364340 158168827 115899099 162960591 126

myristate (14:0) 10251771 10572006 5205894 10516743 9745124 9900208 myristoleate (14:1n5) 7044475 8053617 1791272 6007347 6678757 4431912 N-acetylalanine 17553.26 28923.17 14719.82 38784.8 31392.18 42577.93 N-acetylasparagine 104179.49 174086.56 94282.69 41569.76 28933.96 148907.63 N-acetylglucosamine 6-phosphate 40769 85574 133070 278511.27 134168 54513 N-acetylglutamine 25636.21 66239.31 47481.37 17102.68 18832.51 63783.12 N-acetylmethionine 226176.9 499187 206686.8 600026.5 440819.6 629528.7 N-acetylthreonine 22553.53 19827.69 56933.27 N-formylmethionine 17790.23 18997.17 11605.72 27651.18 28664.5 31786.93 N1-methyladenosine 443165.7 354464.1 201194 949712.3 680144.2 634605.3 nicotinamide 2528720 2463357 2173346 4212926 4036840 5323470 nicotinamide adenine dinucleotide (NAD+) 3099665.9 4002715.6 1356751.6 5942009.6 6587938.8 2736409.9 nicotinamide adenine dinucleotide phosphate (NADP+) 72089.96 98986.24 44781.81 93018.39 179677.52 91506.73 nicotinamide adenine dinucleotide reduced (NADH) 13651.2 14426.14 5947.71 19273.74 16365.55 nicotinamide ribonucleotide (NMN) 2303775 1333430 1310957 1912853 1769865 2315124 nicotinamide riboside* 711958 799416.6 540181.7 1379088.8 1212418.9 1187323.8 nonadecanoate (19:0) 743776.5 556789.8 387659.9 1094663.3 746067 502218.8 oleate (18:1n9) 2449578 3207658 1528365 5175471 4044144 4912832 ophthalmate 62430.13 71768.54 110135.39 185461.51 158017.69 214925.21 ornithine 70139 54688.77 115190 62690 26349 palmitate (16:0) 43233342 69828316 32526494 64806282 53030345 49317074 palmitoleate (16:1n7) 67472673 86513940 40541380 84863891 74528443 64743070 palmitoylcarnitine 55103.2 59410.32 31577.47 127047.54 pantothenate 14375294 17313810 14935148 27030857 21820901 31830267 pelargonate (9:0) 273347.8 142095.6 149440 262367.6 277985.7 249222.8 pentadecanoate (15:0) 4365036 4725581 2069117 9390710 8158420 4345891 phenol red 319850.33 142094.99 63072.75 214517.15 222524.37 317407.15 phenol sulfate 12026.17 7643.48 6739.12 11908.48 13901.84 16909.5 phenylacetylglycine 29493.49 34193.35 24127.75 37649.66 34077.22 56447.44 phenylalanine 22703898 25021217 14917199 37325578 28126026 39795690 phenyllactate (PLA) 7527.57 20718.5 8592.83 6329.36 9430.23 9902.87 127

phosphate 204565045 182685055 131319141 237758926 214533384 233721705 phosphoenolpyruvate (PEP) 265029.4 460588.1 189941.6 447172.9 268009.8 605558.9 phosphoethanolamine 988303.7 2650302.9 832133.4 2282724.1 1647371.9 3099405.1 phosphopantetheine 2396595.1 1653086.2 629166.3 2813827.8 4116201 386157.2 pro-hydroxy-pro 85754.68 59236.93 41276.83 132329.36 104319.93 320731.36 proline 2976092 4157150 2751084 5429465 3525451 7733345 prolylleucine 536188.2 863987.7 382141.1 1451382.8 853186.9 1315141.8 propionylcarnitine 156224.46 388625.66 185685.97 839753.12 436415.4 635525.28 pseudouridine 20889.06 15341.16 8077.37 32669.02 27621.16 25401.23 putrescine 61759.97 90383 26345 213114.92 105990 117569.73 pyridoxal 185480.2 227172.8 148564.4 294301.8 214140 298691.5 pyridoxine (Vitamin B6) 28457.12 8801.15 7744.25 18503.72 14205.64 38950.56 pyrophosphate (PPi) 946787.8 283781 675679.7 792039.7 1319463.4 pyruvate 18073 29606 11383 71588.01 84397.67 241262.99 riboflavin (Vitamin B2) 133308.93 85354.7 76895.8 236574.04 158493.44 168095.71 ribose 180733.3 273093.5 93174 374231.1 295623 401925.2 S-adenosylhomocysteine (SAH) 41976.78 40159.59 22871.56 57032.02 46224.45 72227 scyllo-inositol 417675.3 669446.1 432368.5 1735492.4 1302793.6 1881086 sebacate (decanedioate) 9992.06 sedoheptulose-7-phosphate 493407 624272.7 222872.1 897276.5 853850.8 723699.5 serine 10348862 14825981 8536375 20131277 11727208 22161525 sorbitol 135151.81 35261 221567.72 120353.43 305459.6 spermidine 67617 152121 75139.93 248814 132914.81 268225.47 sphinganine 152236.09 76006.46 35374.12 85059.23 sphingosine 341553.54 418122.12 361610.98 93355.88 38439.48 303163.87 stearate (18:0) 12456996 15569507 14652297 20988223 15642822 20230417 stearidonate (18:4n3) 70555.82 166267.04 70978.99 158708.7 143484.08 120980.63 stearoyl sphingomyelin 228141 275561 224644 549135.2 290571.3 740332 succinate 335893.2 300558.3 253135.8 399546.3 377417.7 595478.2 succinylcarnitine 86034.13 27095 212424.72 126748.63 252166.22 thiamin (Vitamin B1) 143593.55 167900.66 65865.56 206427.35 187497.2 234525.91 128

threonine 4750652 7465069 4141016 7808565 4964959 12098946 threonylphenylalanine 42610.74 84580.38 108263.98 152238.76 61705.9 trans-4-hydroxyproline 41620 40101.29 56799.97 159572.54 trizma acetate 22651 14366 tryptophan 4235342 4551183 2735725 7028982 5407915 8217695 tyrosine 10012112 10610110 6477413 16186292 12728090 20236573 UDP-N-acetylglucosamine 111516.82 100274.14 104635.9 15775.87 undecanoate (11:0) 37486.33 37665.31 29554.52 33278.64 32774.63 uracil 66200.53 87253.82 57343.21 156684.22 55758.49 165387.18 urate 24508 72624 22958 84855 73234 93221.64 uridine 525070 819941.6 600123.1 969900.1 750397.7 1185101.8 uridine 5'-monophosphate (UMP) 1452083.4 1012857.9 1141486.8 1230918.4 1340326.7 1758138.1 valine 9952565 11145939 7564396 18021939 13698193 20195256 xanthine 1077294 1866048 1388014 2649684 2434320 3138447 xanthosine 22246.71 60852.33 17623.85 78876.97 51544.82 104044.73

BIOCHEMICAL CLA 12h CLA 12h CLA 12h CLA 12h CLA 12h CLA 12h 1-arachidonoylglycerophosphoethanolamine* 49319.76 52164.73 49758.15 81699.53 118226.16 93500.92 1-arachidonoylglycerophosphoinositol* 102101.06 34537.58 147214.54 283333.42 90186.45 1-heptadecanoylglycerol (1-monoheptadecanoin) 335068 301827 196803.5 298253.6 395010 434310.7 1-heptadecanoylglycerophosphocholine 424642.53 791971.25 523283.82 1022029.76 2112215.68 1604410.32 1-heptadecanoylglycerophosphoethanolamine* 358801.4 287261.7 211221.8 248212.2 1704689.2 395099.4 1-linoleoylglycerophosphocholine 228505.51 254035.01 386590.01 350846.23 529506.84 631613.38 1-linoleoylglycerophosphoethanolamine* 285597.34 166607.13 106446.54 117493.62 317994.67 81321.1 1-methylnicotinamide 2186488 1323625 1650156 2403135 2212767 2133629 1-myristoylglycerol (1-monomyristin) 731138.3 459535.2 347593.6 423298.4 612944.8 575961 1-myristoylglycerophosphocholine 105300.59 371276.82 261734.87 197782.77 437007.43 537651.59 1-oleoylglycerol (1-monoolein) 217412 135142 100534.6 183324 255705 443225.6 1-oleoylglycerophosphocholine 229910.2 435208.4 487784.9 963372.3 773645.1 1717717 1-oleoylglycerophosphoethanolamine 177007.37 358330.31 300394.56 601738.9 397126.23 869256.26 1-oleoylglycerophosphoinositol* 616248.89 132887.65 173221.99 348537.85 727659.25 336028.96 129

1-palmitoleoylglycerophosphocholine* 538470.7 1566679.6 1632557.3 2297215.3 3937133.3 3054815.4 1-palmitoleoylglycerophosphoethanolamine* 426967.3 326443.6 359678.6 525394.4 990465 389586.7 1-palmitoleoylglycerophosphoinositol* 176409.2 51348.83 63249.91 122081.73 230555.88 57490.17 1-palmitoylglycerol (1-monopalmitin) 3919010.7 3590657.1 1976074.7 1852350.2 2634196 4981542.8 1-palmitoylglycerophosphocholine 1936887.5 4403489.1 4016291.2 5338412 5770170 9584036.3 1-palmitoylglycerophosphoethanolamine 877316.8 714874.8 555668.4 597945 1937112.5 1026635.8 1-palmitoylglycerophosphoinositol* 1140552.4 636367.9 464426.8 596580.9 1310258.7 860463.4 1-pentadecanoylglycerol (1-monopentadecanoin) 1214036.9 930537.4 708763.3 899273.2 1525188.7 1149981.1 1-pentadecanoylglycerophosphocholine* 406458.2 1271260.9 1125666.4 1165604.1 2589596.5 1905743.7 1-stearoylglycerol (1-monostearin) 356747 458764 304241.7 313880 381376.2 724419.5 1-stearoylglycerophosphocholine 1220121.9 3274832.1 2256626.7 3233671.5 3650079.7 6987341.2 1-stearoylglycerophosphoethanolamine 663038.5 1501439.4 1064032.9 1736322.3 2213319.1 2392645.5 1-stearoylglycerophosphoinositol 1389421.6 1215086.2 803860.9 1043359 1692081.9 1605875.1 1,2-dipalmitoylglycerol 101341 172536 48473 160719 73739 1,3-dipalmitoylglycerol 45335.79 54328.22 21287 49371 31476 10-heptadecenoate (17:1n7) 13712502 11070881 11500169 28187178 27123958 14750249 10-nonadecenoate (19:1n9) 2015447 1485442 1401746 4119525 3542823 2830764 13S-hydroxy-9Z,11E-octadecadienoic acid 2634450 1324213 859285 2044471 297905.6 2569861 2'-deoxycytidine 19598.16 27181.1 19093.3 29251.08 39198.39 50916.15 2-arachidonoylglycerophosphocholine* 66715.37 326631.53 246120.77 254253.16 160473.38 518221.28 2-arachidonoylglycerophosphoethanolamine* 1144079.8 3094546.6 2304211 2624045 3112252.9 2835321.4 2-docosahexaenoylglycerophosphoethanolamine* 309580.13 579116.56 508561.95 967176.6 1101631.82 847211.29 2-docosapentaenoylglycerophosphoethanolamine* 316073.86 856649.94 614960.76 730106.32 1010918.44 554944.28 2-hydroxyglutarate 30800.37 14464 45847 48132.19 40521.84 44303 2-hydroxypalmitate 1504250.2 2143016.7 1611470.6 2098025.2 455578.7 1485986.4 2-hydroxystearate 193948.8 340092.41 276618.1 238059.11 92891.48 216432.22 2-linoleoylglycerophosphocholine* 255916.7 277055.7 318321 325181.6 427854 496472 2-linoleoylglycerophosphoethanolamine* 385508.43 648128.22 312971.06 342511.52 417208.11 373159.67 2-methylbutyroylcarnitine 100009.44 35325.93 51800.71 28709.11 129557.13 2-methylmalonyl carnitine 58834.42 24928.95 31597.82 245852.27 135682.14 146980.08 2-myristoylglycerol (2-monomyristin) 92953.81 86127.24 67573.17 71019 105321.68 118926 130

2-myristoylglycerophosphocholine* 28897.98 107063.62 70983.96 82347.61 118611.29 2-oleoylglycerol (2-monoolein) 22374.07 48656.24 34628.35 2-oleoylglycerophosphocholine* 236928.54 729460.44 603256.16 897759.77 701597.81 1494684.84 2-oleoylglycerophosphoethanolamine* 395346.03 1144580.6 871065.37 812669.41 830934.27 650457.33 2-palmitoleoylglycerophosphocholine* 758341.4 2341734.6 1709327.2 2513819.2 2570426.9 3144029.1 2-palmitoleoylglycerophosphoethanolamine* 830859 3294603 2072871.3 2377705.8 2901645.3 1618332.5 2-palmitoylglycerol (2-monopalmitin) 1344156.7 1152759.9 674570.8 925003.2 1217602.2 2186260.2 2-palmitoylglycerophosphocholine* 138475.34 529772.27 358588.55 317757.14 395415.72 736263.3 2-palmitoylglycerophosphoethanolamine* 190660.12 133234.53 52059.1 116454.29 167599.63 2-phosphoglycerate 96460.85 20821 133365.31 77506.35 190037.86 3-(4-hydroxyphenyl)lactate 291068.9 277023.9 339543.6 415571.4 290067.2 506063.2 3-dehydrocarnitine* 61508.5 37581.27 28332.89 216335.01 106377.37 199088.73 3-phosphoglycerate 4515240 1374858 2207050 6290420 3481630 8764779 3-phosphoserine 224520.08 26861.92 44550.18 207390 120136 88644.85 4-hydroxybutyrate (GHB) 79321.93 51997.86 43160 84717 186183.17 4-methyl-2-oxopentanoate 22328.07 8396.07 12000.06 23110.6 10327.51 27489.31 5-dodecenoate (12:1n7) 24597.03 12590.63 13132.04 15929.79 38939.57 15325.56 5-methyltetrahydrofolate (5MeTHF) 19114.99 12692.38 13022.12 37348.64 5-methylthioadenosine (MTA) 905458.3 660019.4 611834.8 608261.8 696091.7 904596.4 5-oxoproline 529319.2 221764.1 197473.5 280092.1 272573.1 459829.3 6-phosphogluconate 684830.6 470243.1 262777.8 1057095.6 770402.4 753624.1 7-alpha-hydroxycholesterol 27011 22120 30594 7-beta-hydroxycholesterol 99046 84952 47458.57 47164.01 19039 29897 acetylcarnitine 201137 98353.5 216934.3 967277.3 286042 916931.8 adenine 214733.87 148297 125436.13 194189.73 189336.47 212762.56 adenosine 709544.7 263647.6 330358.5 310090.4 534440.7 515815.2 adenosine 2'-monophosphate (2'-AMP) 1084694.8 612804.5 666645.6 1199894.1 1014459.1 1433778.3 adenosine 3'-monophosphate (3'-AMP) 1081163.52 49326.88 146830.56 102194.14 215318.42 233981.37 adenosine 5'-diphosphate (ADP) 139710.24 61368.51 61279.9 72380.57 62793.24 99860.63 adenosine 5'-monophosphate (AMP) 3582078 821778.4 1185066.6 1076770.6 1603026.1 1685944.9 adenosine 5'diphosphoribose 91815.25 108108.99 89791.34 80915.14 134989.7 141480.93 131

adrenate (22:4n6) 904138.5 916692.7 660983.8 1617560.6 1938724.9 1478221.6 alanine 10256067 5473165 5074243 15601391 9212522 19689601 alanylalanine 13655.86 7384.44 5467.96 30313.93 21690.39 9313.02 alanylhistidine 26027.66 15669.42 8753.12 39825.24 28504.6 16991.04 alanylleucine 29215.65 28633.85 13408.06 61971.52 49595.21 31022.14 alanylthreonine 24230.27 25077.71 14204.67 51641.41 46136.96 21780.66 alanyltyrosine 74567.42 98799.28 75452.35 74093.92 alanylvaline 130239.16 29321.65 19386.31 113898.18 89602.01 56504.85 alpha-tocopherol 20930 34768 20122.8 arachidonate (20:4n6) 8048529 7582438 8365389 15994677 15575505 12056253 arginine 206051.6 104727.6 100480.9 283869.5 190844.6 287110.5 asparagine 1022997 656330.6 590022.2 1972088.4 976747.1 2290481.6 aspartate 14933574 5210710 6371821 16414738 13658737 19333537 aspartylphenylalanine 73469.14 23050.34 36741.4 29658.28 63061.9 beta-alanine 133574.77 26478 121050.16 177207.93 161066 496311.3 beta-hydroxyisovalerate 192718.85 33806.68 31109.54 29446.36 49851.18 101230.4 betaine 373450.1 263850.3 361808.4 C-glycosyltryptophan* 201336.91 97057.76 108735.9 233116.18 157382.33 396976.51 caprate (10:0) 84240.91 67241.1 44087.37 78661.83 81551.02 69074.83 caproate (6:0) 22376.29 15974.8 10236.17 19929.4 15653.84 15968.02 caprylate (8:0) 30383.72 20120.82 16186.69 18988.7 20730.17 25778.09 carnitine 752931.6 348738.7 398832 1700405.7 914118.3 1696694.6 cholesterol 8089663 8087829 6516300 8698916 9094226 9391763 choline 4606666 2228671 2518609 3800652 3441253 4648354 choline phosphate 640652.9 642030.3 720510.7 677957.2 563937.5 740276.6 cis-vaccenate (18:1n7) 841638.2 861839 680510 1220437.1 1299238.4 1416968.8 citrate 1293111.4 156808.5 308957.6 640116.3 631075.7 466822 coenzyme a 34860.68 9206.01 14629.75 33222.63 26948.48 32401.91 conjugated linoleate (18:2n7; 9Z,11E) 1168095.3 552518.4 538989.9 603037.3 695253.9 1108759.7 creatine 5333140 3692477 4162676 6075923 4629922 7375325 creatinine 115572.9 134945.41 233101.74 149486.57 201180.27 160273.09 132

cysteine 347105.82 77041.01 261702.04 983579.39 796429.26 714071.66 cysteine-glutathione disulfide 200376.3 123437.4 214189.8 341624.9 318995.9 197539.5 cytidine 1638712 1036120 1066153 1665128 1148525 2317906 cytidine 5'-monophosphate (5'-CMP) 393017.9 168818.9 166337.4 258267.2 226339.8 318827.2 deoxycarnitine 468194.3 191431.8 181312.8 862078.2 356416.9 655770.5 dihomo-linoleate (20:2n6) 1070511.3 402472.4 749998.8 748628 822904.7 dihomo-linolenate (20:3n3 or n6) 1219430.4 858819.9 819454 1221807.6 1364177.1 1186316.9 dimethylarginine (SDMA + ADMA) 735738.8 912828.9 522803.6 1139917.8 594046.2 1082467.4 docosadienoate (22:2n6) 562547 378716 278050.3 446814.8 355338.2 558277.8 docosahexaenoate (DHA; 22:6n3) 2065764.8 1638465.5 1618646.6 3285675.8 3170664.4 3445223.1 docosapentaenoate (n3 DPA; 22:5n3) 2072366 1941624 1695843 3317823 2850529 3216508 docosatrienoate (22:3n3) 1137673.5 1108534.3 660308.7 862916.6 1214792.7 815367.8 eicosapentaenoate (EPA; 20:5n3) 3056572 2144996 2676831 4940095 4904275 2840973 eicosenoate (20:1n9 or 11) 2780499 1874741 1864815 3923611 3867883 3229782 erythritol 69691 37955 56780 73226.96 113329.8 erythronate* 12888 25260 38187.51 ethanolamine 904667.7 362427 563188.1 1169466.9 869605.3 1496933.6 flavin adenine dinucleotide (FAD) 161906.9 78033.94 71091.12 173654.45 163643.03 175755.22 fructose 462020.6 196841.1 127467 418136.8 301440 940548 fructose-6-phosphate 855119.8 548481.9 636362.4 760066 559506.5 2556551 fumarate 432530.1 55842 109350.3 257929.7 273338.4 446248.8 gamma-aminobutyrate (GABA) 17782.44 31254.91 57850.98 64985.08 glucose 5069158 2192325 1668926 6301911 3007495 7215602 glucose-6-phosphate (G6P) 3834853.4 2473234.9 2637913.4 3024813.5 2486569 11658911.3 glutamate 4576617 2851877 2501420 4916076 2738183 15347755 glutamine 3827206 1514810 1871491 5418622 3628767 7390641 glutathione, oxidized (GSSG) 2327951.1 832840.6 523402.7 746153.2 816444.7 1935682.4 glutathione, reduced (GSH) 11884964 4348124 6586534 12332134 9587358 15462519 glycerate 150771.69 46710.44 103731.57 567672.71 192268.96 451532.43 glycerol 18637541 9407977 9013890 18963743 16859596 25967898 glycerol 2-phosphate 51239 38334 39872 26390 91421.2 129959.7 133

glycerol 3-phosphate (G3P) 2380086 1234808 1228756 2318431 2325162 5009654 glycerophosphorylcholine (GPC) 4586616 7558864 6052771 7163131 7380573 2419667 glycine 8095575 3356430 3583487 10560837 6747615 16134276 glycylglycine 18145 19089 21412 15043.68 glycylisoleucine 54464.16 67620.81 38018.99 119750.84 62293.02 79366.03 glycylleucine 600790.1 512118.4 294300.2 1191758.5 731668.6 709859.7 glycylphenylalanine 28490.1 22146.28 8587.62 53475.76 33905.68 38230.84 glycyltyrosine 177165.81 101140.64 88847.55 317273.96 233022.06 190757.59 glycylvaline 55202.28 54996.97 39288.1 131392.85 77993.91 109707.17 guanosine 26696.23 25300.89 20647.59 98183.1 71749.6 76889.78 guanosine 5'- monophosphate (GMP) 644095.4 353731.1 342642 400907.7 404208.7 662838.3 gulono-1,4-lactone 124939.19 82271.69 107392.59 371867 201999 310207 heptanoate (7:0) 9388.85 9299.54 12406.85 7493.96 13037.86 histidine 188887.8 118468.8 126234.2 254267.9 185367.8 247924.2 hydroxyisovaleroyl carnitine 599336 230494.7 225829.2 1090510.2 639633.1 829935.6 hypotaurine 452424.8 62330 240713 454706.5 232170 873877.3 hypoxanthine 647642.3 427507.7 424358.7 690333.3 582674.9 962803.8 inosine 1750618.8 1175167 998492.8 2264636 1836810.4 2758549.8 inosine 5'-monophosphate (IMP) 107072.39 56194.95 76087.38 18553.67 52264.59 168954.42 inositol 1-phosphate (I1P) 1130830.6 1286031.1 1073018.2 1488183 1233164.8 1609206 Isobar: fructose 1,6-diphosphate, glucose 1,6-diphosphate 23379.12 25402.08 28968.67 13192.61 16718.26 17850.87 Isobar: ribulose 5-phosphate, xylulose 5-phosphate 389153.98 236092.19 168895.27 122313 574514.57 497834.39 isoleucine 12143335 7519972 6169569 18051158 9367991 18747703 isoleucylleucine 27308.89 33015.71 72811.62 71078.39 47939.54 isovalerylcarnitine 141849.32 21377.81 52826.11 48509.37 36556.27 121399.86 lactate 6424075 2870009 2801579 7013662 3960630 20463776 laurate (12:0) 721441.8 396149 334583.1 534112 861757.1 510315.8 leucine 29769317 20438391 15806340 45864540 27766371 50166021 leucylleucine 4334.57 8504.93 linoleate (18:2n6) 39455131 22074757 22190578 21453204 24074551 29469841 linolenate [alpha or gamma; (18:3n3 or 6)] 4144691.2 4275974 3386013.2 2440839.9 2426123.3 3563820.5 134

lysine 1527621 1106762 1095106 2848597 1488165 3033390 malate 1329580.7 567197.8 620166.8 646342.7 935731.8 1835371.5 maltohexaose 10123.26 4290.23 9615.15 23859.47 20128.85 26118.17 maltopentaose 27504.2 16304.76 27838.18 60786.09 57252.5 60522.08 maltose 357818 336420 332639.7 874200.9 707220.2 778007.3 maltotetraose 133811.17 105965.38 98151.18 196972 225487.8 239575.97 maltotriose 42463 36494.52 27871 41014 25388 63667.23 mannitol 43327.58 13659 mannose 443775 454024.2 330150 1175152.4 567973 1565262.8 mannose-6-phosphate 408184.32 251534.33 238094.24 333362.28 263147.42 1329508.68 margarate (17:0) 14424946 13280328 12672649 25950790 28225134 18493476 mead acid (20:3n9) 4316172 4547189 3672286 3462449 4469437 2832681 methionine 6752630 4283112 3465973 10083627 6575208 10656071 methylphosphate 536696 301994.7 226477.1 535599.3 516941.8 1532418.8 myo-inositol 53298262 31227142 40853702 122681550 79636380 139126746 myristate (14:0) 12329324 8615122 7812789 11302092 13262372 9761579 myristoleate (14:1n5) 5930398 2793604 2072046 5634349 9746510 3211148 N-acetylalanine 27585.62 19058.64 13747.5 38302.9 20180.29 48723.8 N-acetylasparagine 99644.38 44886.64 24576.93 46707.37 93862.82 N-acetylglucosamine 6-phosphate 142443.4 101318.37 81859 99270 129992 215620 N-acetylglutamine 46597.21 15382.35 18268.75 20576.69 25719.78 39038.47 N-acetylmethionine 346614.3 300066.8 184839.2 548777.9 370157.5 547614.1 N-acetylthreonine 12174.93 11081.59 14596.07 56255.8 N-formylmethionine 28514.18 11600.05 12943.63 27954.93 22659.16 33500.81 N1-methyladenosine 336822.6 188322.3 206859.8 760545.4 509941.8 479793.3 nicotinamide 2630582 1957183 1831221 5010677 3404750 6095440 nicotinamide adenine dinucleotide (NAD+) 3189565.5 861429.8 1689634.5 3690575.8 3614016.6 1674995.7 nicotinamide adenine dinucleotide phosphate (NADP+) 176909.56 29916.05 32583.06 114796.4 85227.17 125480.24 nicotinamide adenine dinucleotide reduced (NADH) 14248.79 6850.61 9712.31 13345.77 nicotinamide ribonucleotide (NMN) 2821294 1448274 1208637 1944960 2003906 2393623 nicotinamide riboside* 765697.7 730236.2 599125 1250043.7 1169276.9 1331319 135

nonadecanoate (19:0) 735474.4 536562.1 576507.8 2043814.5 1483329.5 1071490.2 oleate (18:1n9) 2953218 2198837 1690129 4022979 4070274 5010811 ophthalmate 111470.2 48251.32 90591.98 147143.31 115626.37 243105.94 ornithine 20130 26272 93624.14 37835.59 98813 palmitate (16:0) 68204718 57435185 53599736 80016010 89227864 65458389 palmitoleate (16:1n7) 73403269 54081289 49155554 92400417 97475892 59552511 palmitoylcarnitine 125804.18 124747.99 81438.78 79951.61 1063359.3 pantothenate 22214700 9395728 8650780 22108646 15949994 26443868 pelargonate (9:0) 226740.3 130285.1 137725.6 208459.7 187702.2 234422.2 pentadecanoate (15:0) 3513560 1920475 2038268 5251801 6022557 3946541 phenol red 333892.54 95793.31 60649.12 138139.05 169902.86 273178.86 phenol sulfate 17870.49 4833.93 4894.03 7022.86 11292.18 8676.84 phenylacetylglycine 41375.75 14719.7 18406.77 30345.77 26378.7 39059.68 phenylalanine 30328100 18255560 13798510 39058733 24309934 43263620 phenyllactate (PLA) 7469.66 9197.38 10610.43 8845.8 phosphate 193508839 119564735 125602913 160373393 153220745 217529635 phosphoenolpyruvate (PEP) 233625.8 68021 191527 404119.8 41887 775932.2 phosphoethanolamine 1301994.9 607178.8 643306.9 2368714.2 1033752.4 2756872 phosphopantetheine 1353017.7 297137.8 250064.1 536233.5 1930256.9 154409.9 pro-hydroxy-pro 143745.48 60352.35 43179.96 146833.07 110744.02 306591.41 proline 5144805 3905105 3107801 7325768 3611314 8697377 prolylleucine 726609.8 485940.6 382780.4 1478544.3 921268.2 1359385.8 propionylcarnitine 158993.37 95803.12 113449.25 539850.57 302717.41 619900.62 pseudouridine 25420.99 16991.98 13231.73 27160.41 19392.2 26664.84 putrescine 105274.6 133290 173196.28 129784 124388.17 359255.37 pyridoxal 230718.2 133400.8 127158.3 183363.1 155085.4 267234.5 pyridoxine (Vitamin B6) 23232.8 9737.63 7730.66 13650.29 10961.17 25759 pyrophosphate (PPi) 307878.8 519449.6 431906.9 101322.3 718383.4 pyruvate 93463.98 18479 58491 66046 riboflavin (Vitamin B2) 110530.43 87933.54 77070.76 186452.94 151252.2 228028.04 ribose 193484.1 234508 112526 319675.1 146058.5 380390.7 136

S-adenosylhomocysteine (SAH) 58142.62 29779.89 26569.1 44182.76 44728.5 72298.94 scyllo-inositol 649032.4 303382 395749.9 1547403.2 855293.6 1679936.5 sebacate (decanedioate) 23383.87 19792.97 17332.33 15453.59 12285.24 19660.03 sedoheptulose-7-phosphate 557953.1 287485.3 355860 445466.7 465695.4 438865.7 serine 13307495 6212295 6768620 21724296 11815593 21730527 sorbitol 98687.08 154851.46 113160 354616.25 spermidine 100006.33 104334 225069.08 sphinganine 143574.18 1147145.49 724264.75 312245.38 374104.82 1018786.18 sphingosine 273075.17 1258515.01 709639.84 871687.32 557741.82 2042312.14 stearate (18:0) 17998289 19858274 17345177 25619929 25688304 30598779 stearidonate (18:4n3) 109498.5 47068.36 47137.81 161886.7 189294.15 104481.32 stearoyl sphingomyelin 513586.2 319368 180180 355307.3 302366 562109.4 succinate 471007.1 151579.8 187221.9 329804.9 327319.2 444617.8 succinylcarnitine 43208.59 30742.94 20868.25 138748.26 63046.74 131821.56 thiamin (Vitamin B1) 146188.41 90934.51 75564.9 142967.63 103281.27 212606.3 threonine 6523891 2643802 3197874 8134343 4771883 10822028 threonylphenylalanine 70374.45 22589.06 112265.85 98180.79 82167.25 trans-4-hydroxyproline 47990.27 109782.14 trizma acetate 325938.69 7811 17924.95 tryptophan 5660649 3054118 2760368 7997390 4623026 9029873 tyrosine 12656771 7520369 6594050 18494566 11211153 20413094 UDP-N-acetylglucosamine 94376.03 30922.3 undecanoate (11:0) 28364.01 21973.86 60808.11 18675.18 uracil 116052.08 59663.47 108351.09 184574.45 167395 210598.07 urate 34221.9 26986.5 38359.05 83859.34 117337.44 87148.19 uridine 640382.3 734604.7 633451.7 1027051.7 793552.4 1285733.9 uridine 5'-monophosphate (UMP) 1720365.5 855724.5 942671.2 765109.5 1041465.1 1520379.3 valine 12669286 7884218 6318721 18145543 11346394 22567200 xanthine 1606164 1533390 1253123 3063918 2101884 3388289 xanthosine 61509.75 30850.07 63607.52 157826.26 52946.55 198636.84

137