Page 1 of 21 Diabetes
High fat diet causes mitochondrial dysfunction as a result of impaired ADP sensitivity
Paula M Miotto1*, Paul J LeBlanc2, and Graham P Holloway1*
1Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, N1G 2W1 Ontario, Canada
2Department of Health Sciences, Brock University, St. Catharines, Ontario, Canada
Running head: High fat diet and mitochondrial dysfunction
Abstract word count (198)
Manuscript word count (1998)
* Denotes corresponding author: Graham P Holloway, Email: [email protected] Paula M Miotto, Email: [email protected] Human Health and Nutritional Sciences, University of Guelph, 491 Gordon Street, Guelph, ON, N1G 2W1, Canada
1
Diabetes Publish Ahead of Print, published online July 6, 2018 Diabetes Page 2 of 21
Key words: Insulin resistance, skeletal muscle metabolism, high fat diet, oxidative stress, reactive oxygen species, mitochondrial dysfunction, regulated membrane transport, ADP sensitivity.
Abbreviations: High fat (HF), voltage dependent anion channel (VDAC), adenine nucleotide translocase (ANT), uncoupling protein 3 (UCP3), palmitoyl CoA (P CoA), malonyl CoA (M
CoA), mitochondrial creatine kinase (Mi CK), control (CON), permeabilized muscle fibres
(PMFs), respiratory control ratio (RCR), cytosolic creatine kinase (MM CK), superoxide dismutase 2 (SOD2), area under the curve (AUC), glucose tolerance test (GTT), insulin tolerance test (ITT).
2
Page 3 of 21 Diabetes
Abstract
While molecular approaches altering mitochondrial content have implied a direct relationship
between mitochondrial bioenergetics and insulin sensitivity, paradoxically, consumption of a
high fat (HF) diet increases mitochondrial content while inducing insulin resistance. We
hypothesized that despite the induction of mitochondrial biogenesis, consumption of a HF diet
would impair mitochondrial ADP sensitivity in skeletal muscle of mice, and therefore manifest
in mitochondrial dysfunction in the presence of ADP concentrations indicative of skeletal muscle
biology. We found that HF consumption increased mitochondrial protein expression, however
absolute mitochondrial respiration and ADP sensitivity were impaired across a range of
biologically relevant ADP concentrations. In addition, HF consumption attenuated the ability of
ADP to suppress mitochondrial H2O2 emission, further suggesting impairments in ADP
sensitivity. The abundance of ADP transport proteins were not altered, while the sensitivity to
carboxyatractyloside mediated inhibition was attenuated following HF consumption, implicating
alterations in adenine nucleotide translocase (ANT) ADP sensitivity in these observations.
Moreover, palmitoyl CoA is known to inhibit ANT, and modelling intramuscular palmitoyl CoA
concentrations that occur following HF consumption exacerbated the deficiency in ADP
sensitivity. Altogether, these data suggest that a HF diet induces mitochondrial dysfunction
secondary to an intrinsic impairment in mitochondrial ADP sensitivity that is magnified by
palmitoyl CoA.
3
Diabetes Page 4 of 21
Introduction
While the etiology of type 2 diabetes is poorly defined, chronic consumption of a high fat (HF) diet is a major contributor to whole body glucose intolerance (1) and insulin resistance (2; 3).
Although the molecular explanation for these responses are not fully understood, mitochondrial dysfunction within skeletal muscle has received attention as a potential contributor, as mitochondrial content is reduced in most reports of insulin resistant/obese human skeletal muscle
(4; 5). Moreover, in various models, the induction of mitochondrial biogenesis protects against the development of insulin resistance (2; 6), and genetic approaches that decrease mitochondrial content predispose animals to HF diet induced insulin resistance (7). While these data suggest changes in mitochondrial content are related to insulin sensitivity, reductions in mitochondrial oxidative capacity are not uniformly reported in the literature with insulin resistance (8; 9), and
HF feeding has been shown to induce glucose intolerance and insulin resistance despite increasing mitochondrial content (10 12). Together, these data suggest reductions in mitochondrial respiratory capacity are not required for the development of insulin resistance.
Although short term HF experiments challenge the notion that mitochondrial dysfunction contributes to insulin resistance development, these data are difficult to rectify with molecular approaches that alter mitochondrial content and highlight a strong association with insulin sensitivity. An important consideration in this respect is that previous examinations of mitochondrial function have been performed in the presence of saturating ADP concentrations, which may not reflect the biological environment. It has been postulated that an increase in mitochondrial content improves the sensitivity of oxidative metabolism to ADP (13; 14), and therefore ADP responsiveness may represent a key process in cellular homeostasis that is not
4
Page 5 of 21 Diabetes
recapitulated in experiments conducted in the presence or absence of saturating ADP.
Importantly, the movement of ADP into the mitochondrial matrix, and the subsequent binding of
ADP to F1F0 ATP synthase, decreases membrane potential and H2O2 production while
simultaneously increasing substrate oxidation (15). In this manner, ADP sensitivity may
represent a key biological process influenced by changing mitochondrial content, as it can
influence both redox balance and reactive lipid accumulation. Intriguingly, mitochondrial ADP
sensitivity is externally regulated and inhibited by reactive lipid accumulation (i.e. palmitoyl
CoA), however surprisingly, it is not currently known how mitochondrial ADP sensitivity is
affected by HF consumption. We hypothesized that mitochondrial ADP sensitivity would be
impaired following a HF diet, independent of reductions in mitochondrial content. If accurate,
this hypothesis would rectify a prominent discrepancy in the literature regarding the notion that
mitochondrial dysfunction occurs in parallel with the development of insulin resistance.
Research design and methods
Animals and ethics
All experimental procedures were approved by the Animal Care Committee at the University of
Guelph, and conformed to the guide for the care and use of laboratory animals as indicated by
the US National Institutes of Health. Male (10 12 weeks of age) mice on a C57Bl6J background
were fed either a control (CON; 10% lard) or high fat (HF; 60% lard) diet ad libitum for 4 weeks.
All animals were anesthetized with an intraperitoneal injection of sodium pentobarbital
(60mg/kg; MTC Pharmaceuticals, Cambridge, ON) prior to red gastrocnemius extraction for
subsequent analyses.
5
Diabetes Page 6 of 21
Verification of high fat diet induced glucose and insulin-intolerance
Intraperitoneal glucose and insulin tolerance tests were performed on separate days as previously
reported (16). On a separate day, animals were anesthetised, and the red gastrocnemius muscles
removed before or 15 minutes after an intravenous injection of 1U/kg body weight of insulin
(Novorapid). The area under the curve (AUC) calculations were adjusted to account for baseline blood glucose values.
Resting whole-body metabolic measurements
Resting oxygen consumption (VO2) and carbon dioxide production (VCO2) were monitored in
metabolic caging (Columbus Instruments, Columbus, OH) and used to calculate total
carbohydrate and fat oxidation, and energy expenditure as previously reported (17).
Mitochondrial bioenergetics
Respiration in red gastrocnemius permeabilized muscle fibres was measured using high
resolution respirometry (Oroboros Oxygraph 2 K, Innsbruck, Austria) at 37ºC as previously
reported (13). Briefly, ADP (0 12000; M) was titrated in the presence of pyruvate (10mM) and
malate (5mM), followed by glutamate (10mM) and succinate (10mM), in the presence or
absence of P CoA (20 or 60 M). In separate experiments, P CoA was titrated in the presence of
L carnitine (2mM), malate (5mM), and ADP (5mM), and in the absence or presence of malonyl
CoA (M CoA; 7 M). Carboxyatractyloside (0.2 1.6 M) was used to inhibit ADP supported
respiration to investigate changes in adenine nucleotide translocase (ANT) substrate sensitivity.
Cytochrome c (10 M) did not stimulate respiration during experiments (data not shown).
6
Page 7 of 21 Diabetes
Succinate supported (20mM) H2O2 emission was determined fluorometrically in the absence and
presence of ADP (100 M) as previously reported (13).
Citrate synthase activity
Frozen red gastrocnemius (6 10mg) was homogenized in Tris buffer (100mM, pH 8.3), freeze
thawed to lyse mitochondria, and citrate synthase activity was measured spectrophotometrically
as previously reported (18).
Western blot analysis
The red gastrocnemius was homogenized and Western blotting was performed as previously
reported (17). See supplemental Figure 1 for a complete list of antibodies.
Statistical analyses
Michaelis Menton kinetics were determined by plotting data points in GraphPad Prism 5
software to estimate the apparent Km as previously described (19). Unpaired two tailed student’s
t tests were used to analyse data between CON and HF fed mice, and one way ANOVA’s were
used for P CoA ADP sensitivity comparisons followed by Student Newman Keuls post-hoc
analyses where appropriate. Statistical significance was determined at P < 0.05. Data are
expressed as mean ± standard error of the mean (SEM).
Results
Verification of HF diet-induced glucose-intolerance and insulin-resistance
We first verified the expected HF diet responses on body mass and insulin resistance. HF
consumption resulted in greater body weight (CON: 31 ± 1.78; HF: 38.6 ± 0.95; g, P<0.05),
greater epididymal fat mass (CON: 1.2g ± 0.24; HF: 2.9 ± 0.15; g, P <0.05), greater AUC in
7
Diabetes Page 8 of 21
response to a GTT and ITT (Figure 1A), and lower skeletal muscle insulin stimulated AKT phosphorylation (Figure 1B). Moreover, although mice exhibited the expected diurnal changes in
fuel metabolism (Figures 1C F), consumption of a HF diet resulted in lower carbohydrate
oxidation (Figure 1C), higher fat oxidation (Figure 1D), lower RER (Figure 1E), and greater
energy expenditure (Figure 1F), further confirming the HF diet model.
Maximal coupled respiration rates
With regards to potential mitochondrial changes influenced by HF consumption, we first
examined markers of mitochondrial content. HF consumption increased PGC 1α and several
markers of the electron transport chain, total electron transport chain abundance, and PDHE1α,
without altering CPT I, ATP synthase, and a subunit of cytochrome c oxidase (Figure 2A).
Moreover, HF consumption increased citrate synthase activity in support of a greater oxidative
capacity (Figure 2B). Despite the apparent HF diet induced mitochondrial biogenesis, HF
consumption did not alter state 2 respiration, maximal ADP stimulated respiration, or RCR
values (Figure 2C). Similarly, HF consumption did not affect maximal CPT I dependent P CoA
supported respiration (Figure 2D) or P CoA sensitivity (Figure 2E). Moreover, while the CPT I
inhibitor M CoA attenuated P CoA sensitivity, this was not affected by HF consumption (Figure
2F). While these data strongly suggest the absence of mitochondrial respiratory dysfunction
following HF consumption, given that skeletal muscle contains submaximal ADP concentrations,
we next examined mitochondrial respiration at physiological concentrations and ADP sensitivity.
Submaximal ADP-stimulated respiration rates and ADP sensitivity
Given the absence of changes in maximal ADP stimulated respiration, we next examined
mitochondrial respiration across a range of ADP concentrations. While a HF diet did not impair
8
Page 9 of 21 Diabetes
respiration in the presence of >2000 µM ADP, strikingly, respiration was attenuated ~30% at all
biologically relevant ADP concentrations (i.e., 100 1000 µM ADP; Figure 3A). In addition,
mitochondrial ADP sensitivity was decreased ~25% following HF consumption (i.e., greater
apparent Km value) (Figure 3B). Combined, despite the induction of mitochondrial biogenesis,
these data strongly suggest the development of mitochondrial dysfunction with a HF diet
secondary to impaired ADP sensitivity.
Given that ADP binding to F1F0 ATP synthase is known to suppress H2O2 emission rates
(15), we also examined mitochondrial H2O2 emissions in the absence and presence of ADP to
further solidify this relationship. While the maximal capacity for H2O2 emission did not change
following a HF diet (Figure 3C), mice fed a HF diet had an impaired ability to suppress H2O2
emission rates via ADP transport into the mitochondria; as indicated by H2O2 production in the
presence of ADP that was approaching significance (Figure 3D) and a lower % suppression of
H2O2 emission rates by ADP (Figure 3E). Further, mice fed a HF diet had greater antioxidant
expression (catalase and SOD2) and 4HNE adducts than mice fed a CON diet (Figure 3F),
supporting an increase in oxidative stress/damage and stimulation of antioxidant adaptation
during HF consumption. The impairment in ADP sensitivity occurred independent of changes in
protein expression of ADP transporters (i.e., VDAC and ANT), Mi CK, MM CK, and UCP3
(Figure 3F). We therefore examined the ability of carboxyatractyloside to inhibit ADP
stimulated respiration to gain insight into the possibility that post translational modifications on
ANT contribute to the attenuated ADP responsiveness following a HF diet. While 1.6 M
carboxyatractyloside inhibited respiration ~90% regardless of diet (data not shown), respiration
was higher following a HF diet in the presence of 0.2 M carboxyatractyloside (Figure 3G),
suggesting a decreased ability for carboxyatractyloside to interact with the ADP binding motif on
9
Diabetes Page 10 of 21
ANT. Altogether, these data suggest a HF diet attenuates ANT sensitivity to ADP, likely contributing to the apparent induction in mitochondrial dysfunction.
P-CoA-mediated inhibition on ADP sensitivity
While these data strongly suggest impaired ADP responsiveness following a HF diet, these experiments are conducted in optimal conditions. However, long chain fatty acid CoAs (e.g. P
CoA) are known to increase following HF consumption (20), and this lipid derivative has been shown to inhibit ANT and reduce ADP sensitivity in permeabilized muscle fibres (13; 17).
Therefore, given the insensitivity to carboxyatractyloside, we next examined P CoA mediated inhibition of ADP sensitivity in CON and HF fed mice. To do this, we repeated experiments on mitochondrial ADP sensitivity in the absence and presence of P CoA concentrations that better reflect control (20µM) (21) and HF diet intramuscular situations (60µM) (20). While P CoA concentrations that reflected CON conditions did not alter respiration rates at any ADP concentration (data not shown), the presence of 60µM P CoA attenuated respiration at most
ADP concentrations (Figure 4A), and further attenuated ADP sensitivity following a HF diet
(i.e., increased the apparent ADP Km: Figure 4B). Combined, these data indicate the presence of mitochondrial respiratory dysfunction following a HF diet that is exacerbated in the presence of increased intramuscular P CoA concentrations.
Discussion
We provide compelling evidence that consumption of a HF diet induced mitochondrial dysfunction as a result of impaired ADP sensitivity. Specifically, we show that despite the induction of mitochondrial biogenesis and unaltered maximal ADP stimulated respiration, respiration at physiological ADP concentrations were impaired following a HF diet. In addition,
10
Page 11 of 21 Diabetes
the ability of ADP to stimulate respiration (apparent Km) and attenuate mitochondrial H2O2
emission were decreased following a HF diet, while modelling reactive lipid levels that occur
with HF consumption exacerbated the apparent mitochondrial dysfunction. Combined, these data
strongly indicate impaired mitochondrial bioenergetics following a HF diet due to impaired ADP
sensitivity.
While genetic models/interventions that increase mitochondrial content typically have
improved insulin sensitivity and glucose tolerance (1; 2; 6), paradoxically, consumption of a HF
diet in the current study, and others (11), results in increased mitochondrial content despite the
induction of glucose intolerance and insulin resistance. Therefore, the notion that mitochondrial
dysfunction contributes to the development of insulin resistance has waned in recent years,
however our data implicates HF consumption in impairing respiration and increasing
mitochondrial H2O2 emission as a result of impaired ADP sensitivity. These derangements
appear to be amplified in the presence of higher P CoA concentrations indicative of insulin
resistant muscle, strongly suggesting the presence of mitochondrial ADP insensitivity in skeletal
muscle following HF consumption and insulin resistance. The constant VDAC/ANT and ATP
synthase protein abundance suggest altered regulation of these proteins may lead to
mitochondrial dysfunction. While an impairment on ATP synthase may also exist following a HF
diet, the insensitivity to carboxyatractyloside suggests that ANT is less sensitive to ADP binding,
a response likely amplified by increases in intramuscular P CoA concentrations (22; 23).
Altogether, our data shows that impaired mitochondrial ADP sensitivity plays an
important role in HF diet induced mitochondrial dysfunction. Although speculative, the
induction of mitochondrial biogenesis likely represents a compensatory mechanism to improve
ADP sensitivity, and potentially mitigate HF diet induced redox stress (24), although this
11
Diabetes Page 12 of 21
appears inadequate to maintain cellular homeostasis. It is currently unknown if the reduction in
ADP sensitivity directly contributes to the metabolic inflexibility observed in HF mice.
However, it is also possible that this response, similar to the induction of mitochondrial biogenesis, represents a compensatory adaptation to preserve some carbohydrate utilization in
the presence of increased intramuscular lipid availability, as a rise in cytosolic ADP could in
theory promote carbohydrate metabolism. In addition to the potential influence on fuel selection,
since the movement of ADP into the mitochondrial matrix can bind to ATP synthase to stimulate
respiration and attenuate mitochondrial derived H2O2 emission (15), an impairment in ADP
transport may represent a nexus point influencing several models implicated in the development
of insulin resistance.
Acknowledgements
None declared.
Funding
This work was supported by the Natural Sciences and Engineering Research Council (NSERC)
of Canada (G.P.H), and infrastructure was purchased with assistance from the Canadian
Foundation for Innovation/Ontario Research Fund. P.M.M is supported by an NSERC graduate
scholarship.
Disclosure statement
None declared.
Author contribution statement
12
Page 13 of 21 Diabetes
PMM and GPH designed the study, while PMM conducted experiments, analyzed data, and
drafted the manuscript. PMM, PJL, and GPH interpreted results and edited the manuscript.
GPH is guarantor of this work and takes responsibility for the integrity and accuracy of the data.
References 1. Miotto PM, Frendo-Cumbo S, Sacco SM, Wright DC, Ward WE, Holloway GP: Combined high- fat-resveratrol diet and RIP140 knockout mice reveal a novel relationship between elevated bone mitochondrial content and compromised bone microarchitecture, bone mineral mass, and bone strength in the tibia. Molecular nutrition & food research 2016;60:1994-2007 2. Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin F, Messadeq N, Milne J, Lambert P, Elliott P, Geny B, Laakso M, Puigserver P, Auwerx J: Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC- 1alpha. Cell 2006;127:1109-1122 3. Storlien LH, Jenkins AB, Chisholm DJ, Pascoe WS, Khouri S, Kraegen EW: Influence of dietary fat composition on development of insulin resistance in rats. Relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipid. Diabetes 1991;40:280-289 4. Kim JY, Hickner RC, Cortright RL, Dohm GL, Houmard JA: Lipid oxidation is reduced in obese human skeletal muscle. Am J Physiol Endocrinol Metab 2000;279:E1039-1044 5. Ritov VB, Menshikova EV, He J, Ferrell RE, Goodpaster BH, Kelley DE: Deficiency of subsarcolemmal mitochondria in obesity and type 2 diabetes. Diabetes 2005;54:8-14 6. Christian M, White R, Parker MG: Metabolic regulation by the nuclear receptor corepressor RIP140. Trends in endocrinology and metabolism: TEM 2006;17:243-250 7. Handschin C, Choi CS, Chin S, Kim S, Kawamori D, Kurpad AJ, Neubauer N, Hu J, Mootha VK, Kim YB, Kulkarni RN, Shulman GI, Spiegelman BM: Abnormal glucose homeostasis in skeletal muscle-specific PGC-1alpha knockout mice reveals skeletal muscle-pancreatic beta cell crosstalk. J Clin Invest 2007;117:3463-3474 8. Smith BK, Perry CG, Herbst EA, Ritchie IR, Beaudoin MS, Smith JC, Neufer PD, Wright DC, Holloway GP: Submaximal ADP-stimulated respiration is impaired in ZDF rats and recovered by resveratrol. J Physiol 2013;591:6089-6101 9. Holloway GP, Thrush AB, Heigenhauser GJ, Tandon NN, Dyck DJ, Bonen A, Spriet LL: Skeletal muscle mitochondrial FAT/CD36 content and palmitate oxidation are not decreased in obese women. Am J Physiol Endocrinol Metab 2007;292:E1782-1789 10. Turner N, Bruce CR, Beale SM, Hoehn KL, So T, Rolph MS, Cooney GJ: Excess lipid availability increases mitochondrial fatty acid oxidative capacity in muscle: evidence against a role for reduced fatty acid oxidation in lipid-induced insulin resistance in rodents. Diabetes 2007;56:2085-2092 11. Hancock CR, Han DH, Chen M, Terada S, Yasuda T, Wright DC, Holloszy JO: High-fat diets cause insulin resistance despite an increase in muscle mitochondria. Proceedings of the National Academy of Sciences of the United States of America 2008;105:7815-7820
13
Diabetes Page 14 of 21
12. Holloway GP, Benton C, Mullen KL, Yoshida Y, Snook LA, Han XX, Glatz JF, Luiken JJ, Lally J, Dyck DJ, Bonen A: In obese rat muscle transport of palmitate is increased and is channeled to triacylglycerol storage despite an increase in mitochondrial palmitate oxidation. Am J Physiol Endocrinol Metab 2009;296:E738-747 13. Ludzki A, Paglialunga S, Smith BK, Herbst EA, Allison MK, Heigenhauser GJ, Neufer PD, Holloway GP: Rapid Repression of ADP Transport by Palmitoyl-CoA Is Attenuated by Exercise Training in Humans: A Potential Mechanism to Decrease Oxidative Stress and Improve Skeletal Muscle Insulin Signaling. Diabetes 2015;64:2769-2779 14. Dudley GA, Tullson PC, Terjung RL: Influence of mitochondrial content on the sensitivity of respiratory control. The Journal of biological chemistry 1987;262:9109-9114 15. Anderson EJ, Lustig ME, Boyle KE, Woodlief TL, Kane DA, Lin CT, Price JW, 3rd, Kang L, Rabinovitch PS, Szeto HH, Houmard JA, Cortright RN, Wasserman DH, Neufer PD: Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans. J Clin Invest 2009;119:573-581 16. Whitfield J, Paglialunga S, Smith BK, Miotto PM, Simnett G, Robson HL, Jain SS, Herbst EAF, Desjardins EM, Dyck DJ, Spriet LL, Steinberg GR, Holloway GP: Ablating the protein TBC1D1 impairs contraction-induced sarcolemmal glucose transporter 4 redistribution but not insulin- mediated responses in rats. The Journal of biological chemistry 2017;292:16653-16664 17. Miotto PM, Holloway GP: In the absence of phosphate shuttling, exercise reveals the in vivo importance of creatine-independent mitochondrial ADP transport. Biochem J 2016;473:2831- 2843 18. Miotto PM, Horbatuk M, Proudfoot R, Matravadia S, Bakovic M, Chabowski A, Holloway GP: alpha-Linolenic acid supplementation and exercise training reveal independent and additive responses on hepatic lipid accumulation in obese rats. American journal of physiology Endocrinology and metabolism 2017;312:E461-E470 19. Perry CG, Kane DA, Herbst EA, Mukai K, Lark DS, Wright DC, Heigenhauser GJ, Neufer PD, Spriet LL, Holloway GP: Mitochondrial creatine kinase activity and phosphate shuttling are acutely regulated by exercise in human skeletal muscle. JPhysiol 2012;590:5475-5486 20. Ellis BA, Poynten A, Lowy AJ, Furler SM, Chisholm DJ, Kraegen EW, Cooney GJ: Long-chain acyl-CoA esters as indicators of lipid metabolism and insulin sensitivity in rat and human muscle. Am J Physiol Endocrinol Metab 2000;279:E554-560 21. Watt MJ, Heigenhauser GJ, O'Neill M, Spriet LL: Hormone-sensitive lipase activity and fatty acyl-CoA content in human skeletal muscle during prolonged exercise. J Appl Physiol (1985) 2003;95:314-321 22. Ho CH, Pande SV: On the specificity of the inhibition of adenine nucleotide translocase by long chain acyl-coenzyme A esters. Biochimica et biophysica acta 1974;369:86-94 23. Morel F, Lauquin G, Lunardi J, Duszynski J, Vignais PV: An appraisal of the functional significance of the inhibitory effect of long chain acyl-CoAs on mitochondrial transports. FEBS letters 1974;39:133-138 24. Jain SS, Paglialunga S, Vigna C, Ludzki A, Herbst EA, Lally JS, Schrauwen P, Hoeks J, Tupling AR, Bonen A, Holloway GP: High-fat diet-induced mitochondrial biogenesis is regulated by mitochondrial-derived reactive oxygen species activation of CaMKII. Diabetes 2014;63:1907- 1913
14
Page 15 of 21 Diabetes
Figure legends
Figure 1: Verification of HF diet-induced glucose-intolerance, insulin-resistance, and
reliance on fat oxidation
Area under the curve (AUC) during glucose tolerance and insulin tolerance testing (GTT, ITT;
A), ratio of phosphorylated / total AKT (B), carbohydrate oxidation (C), fat oxidation (D),
respiratory exchange ratio (RER; E), and energy expenditure (F) in mice fed control (CON) or
high fat (HF) diet. * indicates a significant difference from CON (P < 0.05). Data are expressed
as mean ± SEM. n = 8 11/ group.
Figure 2: HF consumption increases markers of mitochondrial content in the absence of
altered maximal respiratory capacity or CPT-I regulation
Representative images and quantification of mitochondrial and electron transport chain proteins
(A), citrate synthase activity (B), maximal ADP stimulated respiration in the presence of
complex I and II linked substrates and respiratory control ratio’s (RCR) (C), maximal palmitoyl
CoA (P CoA) supported respiration (D), P CoA sensitivity in the absence of malonyl CoA (M
CoA) (E), and P CoA sensitivity in the presence of M CoA (F) in mice fed control (CON) or
high fat (HF) diet. * indicates a significant difference from CON (P < 0.05). Data are expressed
as mean ± SEM. n=12 14/ group for mitochondrial protein content; n = 8 11/ group for
respiration.
Figure 3: HF consumption impairs submaximal ADP-stimulated respiration, ADP
sensitivity, and ADP suppression of H2O2 emission independent of substrate transporter
protein expression
15
Diabetes Page 16 of 21
ADP stimulated respiration (A), apparent Km for ADP and Michaelis Menton kinetic curve (B),
maximal H2O2 emission (C), H2O2 emission in the presence of 100µM ADP (D), % suppression
of H2O2 emission for ADP (E), western blots for proteins implicated in ADP handling/transport and redox stress (F), and ADP stimulated respiration in the presence of carboxyatractyloside
(CTA; G) in mice fed control (CON) or high fat (HF) diet. * indicates a significant difference from CON (P < 0.05). Data are expressed as mean ± SEM. n = 12 14/group for H2O2 emission;
n=8 14/group for western blots; n=8 10/group for CTA experiments.
Figure 4: HF consumption in the presence of reflective P-CoA concentrations exacerbated
impairments on mitochondrial respiration and sensitivity for ADP
ADP stimulated respiration (A) and apparent ADP sensitivity (B) in control (CON) or high fat
(HF) fed mice in the absence or presence of 60µM palmitoyl CoA (P CoA). * indicates a
significant difference from CON, # indicates a significant difference from HF (P < 0.05). Data
are expressed as mean ± SEM. n = 8 12/group.
16
Page 17 of 21CON HF CON HF Diabetes 25 9 Figure 1
20
15 6 10 C) E) 5 Blood glucose (mM) glucose Blood A) (mM) glucose Blood 0 3 0 30 60 90 120 0 30 60 90 120 Time (min) Time (min) 1500 300 1.5 Light Dark 2.0 1.0 Light Dark 1.0 * Carbohydrate oxidation * 1.5 (KJ / hour) 1000 200 1.0 0.9 0.9 RER 1.0 AUC AUC RER
500 100 hour) / (KJ 0.5 * * 0.8 0.8 0.5 * * Carbohydrate oxidation Carbohydrate
0 0 0.0 0.0 0.7 0.7 CON HF CON HF CON HF CON HF CON HF CON HF CON HF CON HF
Pre Post Pre Post Pre Post Pre Post AKT-P Thr308 D) F) B) AKT-P Ser473 AKT-Total
150 150 2.5 Light Dark 2.5 3 Light Dark 3 Energy Expenditure 2.0 * 2.0 * Fat oxidation (KJ / hour) 100 100 * 2 * 2 (KJ / hour) 1.5 1.5
* * 1.0 1.0 (KJ / hour) / (KJ (KJ / hour) / (KJ (% of CON) of (% (% of CON) of (% 50 50 1 1 Fat oxidation Fat 0.5 0.5 Energy Expenditure Energy
0 0 0.0 0.0 0 0 AKT-Phospho Ser473 /AKT-Total Ser473 AKT-Phospho AKT-Phospho Thr308 / AKT-Total / Thr308 AKT-Phospho CON HF CON HF CON HF CON HF CON HF CON HF Diabetes FigurePage 18 of 2 21 A) CON HF CON HF CON HF B) PGC-1α CON HF CPT-I CIV 250 * 50 CIII 200 * 40 * CIV * * 150 * 30 CII * * 100 20 (% of CON) of (% CI blots Western 50 weight) wet M/min/g 10 ( Citrate synthase activity synthase Citrate PDHE1α 0 0 PGC-1 CPT-I CV CIII CIV CII CI Sum (CI-V) PDHE1 COXIV CON HF COXIV α tubulin P-CoA titration P-CoA titration (+) 7μM M-CoA C) D) P-CoA VMAX E) (+) L-carnitine F) (+) L-carnitine
) 800 8 250 50 80 -1
200 40 600 6 60 dry wt) dry M P-CoA) M M P-CoA) M -1 RCR 150 30 2 2 mg 400 4 O 40 J JO mg dry weight dry mg -1 100 20 -1 sec
sec 200 2 20 50 10 (pmol Apparent Km ( Km Apparent Apparent Km ( Km Apparent
(pmol 0 0 0 0 0 PM +D +DG +DGS RCR CON HF CON HF CON HF Page 19 ofA) 21 CON DiabetesHF B) Figure 3 800 Not impaired respiration Impaired respiration 100 CON 600 HF dry wt) dry Submaximal [ADP] P=0.1 -1 1500 2 mg O 400 * J
-1 50 M ADP) M 1000 * sec CON HF 500 200 * Apparent Km ( Km Apparent * * respiration) maximal (% * 2 0 (pmol CON HF 0 JO 0 0 100 175 250 500 1000 2000 4000 6000 8000 10000 12000 0 400 800 1200 1600 2000 ADP concentration (M) ADP concentration (M) CON HF CON HF CON HF CON HF CON HF CON HF Mi-CK MM-CK 4HNE VDAC ANT1 UCP3 α tubulin Catalase (-) ADP (+) ADP E) % Suppression F) G) 0.2µM CTA C) D) SOD2 200 200 80 200 600 * * 150 150 60 150 *
* wt) dry 400
* -1
P=0.1 2 mg 100 100 40 100 O
(%) ------J -1 emission emission emission 2 2 2 O O O (% of CON) of (% 2 2 sec 2 200 Western blots Western H H H 50 50 20 50 ADP suppression of suppression ADP (pmol (pmol/min/mg dry weight) dry (pmol/min/mg (pmol/min/mg dry weight) dry (pmol/min/mg 0 0 0 0 0 CON HF CON HF CON HF Mi-CK MM-CK VDAC ANT1 UCP3 Catalase SOD2 4HNE CON HF Diabetes FigurePage 20 of 421
A) B) CON HF HF + 60µM P-CoA
Impaired respiration P-CoA impaired respiration 800 2000 *# Submaximal [ADP]
600 1500 dry wt) dry
-1 * 2
mg * * O 400 *# 1000 J -1 M ADP)
* *# (
sec *#
** Km Apparent 200 ** ** ** 500 (pmol 0 0 0 100 175 250 500 1000 2000 4000 6000 8000 10000 12000 A A A -Co -Co -Co P P P ADP concentration (M) M M M 0 0 - 0 - 6 N O HF C HF - Page 21 of 21 Diabetes
Supplementary figure 1. Antibody list
Antibody Dilution Catalogue number Supplier α tubulin 1:5000 ab7291 Abcam Mi CK 1:5000 ab131188 Abcam MM CK 1:5000 ab193292 Abcam VDAC 1:5000 ab14734 Abcam ANT 1 1:1000 ab110322 Abcam SOD2 1:5000 ab13533 Abcam Catalase 1:1000 ab16731 Abcam OXPHOS 1:500 ab110413 Abcam 4HNE 1:1000 HNE11 s Alpha Diagnostics Total AKT 1:1000 #4691 Cell Signaling Phospho AKT Ser473 1:1000 #9271 Cell Signaling Phospho AKT Thr308 1:1000 #9275 Cell Signaling UCP3 1:1000 AB3046 Millipore PGC 1α 1:1000 #516557 Calbiochem PDHE1α 1:5000 #459400 Invitrogen COXIV 1:30000 20E8C12 Invitrogen