bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Suppression of Fatty Acid Oxidation by Thioesterase Superfamily
Member 2 in Skeletal Muscle Promotes Hepatic Steatosis and Insulin
Resistance
Norihiro Imai1, Hayley T. Nicholls1, Michele Alves-Bezerra1, Yingxia Li1, Anna A.
Ivanova2, Eric A. Ortlund2, and David E. Cohen1
1Division of Gastroenterology and Hepatology, Joan & Sanford I. Weill Department of
Medicine, Weill Cornell Medical College, NY 10021 USA
2Department of Biochemistry, Emory University, Atlanta, GA 30322 USA
Current addresses:
Norihiro Imai - Department of Gastroenterology and Hepatology, Nagoya University
School of Medicine, Aichi 4668560 Japan
Michele Alves-Bezerra - Department of Molecular Physiology and Biophysics, Baylor
College of Medicine, Houston, TX 77030 USA
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure number: 8 Supplemental figure number: 10 Supplemental table number: 2 References: 48
Keywords: Hepatic steatosis, obesity, acyl-CoA thioesterase, fatty acid oxidation, insulin
resistance
Conflict of interest: The authors have declared that no conflict of interest exists.
Author contributions: N.I.: designed research studies, conducted experiments,
acquired data, analyzed data and wrote manuscript. H.T.N.: conducted experiments and
analyzed data, M.A.B.: designed research studies and conducted experiments, Y.L.:
acquired data, A.A.I.: conducted experiments and analyzed data, E.A.O.: analyzed data,
D.E.C.: designed research studies, analyzed data and wrote manuscript.
Corresponding author:
David E. Cohen, M.D., Ph.D. 413 E 69th St, New York, NY 10021 Phone: 646-962-7680 Fax: 646-962-0427 Email: [email protected]
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Financial Support:
Supported by the National Institute of Diabetes and Digestive and Kidney Diseases (R37
DK048873 and R01 DK056626 to D.E.C.); the Georgia Clinical & Translational Science
Alliance of the National Institutes of Health (UL1TR002378 to E.A.O.); American Heart
Association Postdoctoral Fellowship (19POST34380692 to N.I. and 18POST33990445
to M.A-B.); the American Liver Foundation Irwin M. Arias, MD Postdoctoral Research
Fellowship Award American (to N.I.); the Liver Foundation NASH Fatty Liver Disease
Postdoctoral Research Fellowship Award (to M.A.B.).
Acknowledgements:
The authors thank Dr. Scott Rodeo, Hospital for Special Surgery for assistance with
treadmill experiments. The Weill Cornell Medicine Metabolic Phenotyping Core and the
Emory Integrated Lipidomics Core are subsidized by Weill Cornell Medicine and the
Emory University School of Medicine respectively.
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ABSTRACT
Thioesterase superfamily member 2 (Them2) is highly expressed in oxidative tissues
where it hydrolyzes long chain fatty acyl-CoA esters to free fatty acids and CoA. Although
mice globally lacking Them2 (Them2-/-) are protected against diet-induced obesity, insulin
resistance and hepatic steatosis, liver-specific Them2-/- mice remain susceptible. To
explore the contribution of Them2 in extrahepatic tissues, we created mice with Them2
deleted in skeletal muscle (S-Them2-/-), cardiac muscle (C-Them2-/-) or adipose tissue (A-
Them2-/-). When fed a high-fat diet, S-Them2-/- but not C-Them2-/- or A-Them2-/- mice
exhibited reduced weight gain. Only S-Them2-/- mice exhibited improved glucose
homeostasis together with improved insulin sensitivity in skeletal muscle. Increased rates
of fatty acid oxidation in skeletal muscle of S-Them2-/- mice were reflected in alterations
in skeletal muscle metabolites, including short chain fatty acids, branched chain amino
acids and the pentose phosphate pathway. Protection from diet-induced hepatic steatosis
in S-Them2-/- mice was attributable to increased VLDL triglyceride secretion rates in
support of demands of increased muscle fatty acid utilization. These results reveal a key
role for skeletal muscle Them2 in the pathogenesis of diet-induced obesity, insulin
resistance and hepatic steatosis.
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INTRODUCTION
Rates of obesity have increased rapidly over the past 40 years and currently affect 600
million people worldwide1. Obesity increases the risk of developing insulin resistance and
non-alcoholic fatty liver disease (NAFLD). These common conditions contribute
significantly to rising morbidity and mortality, highlighting the unmet need for effective new
therapeutic targets in NAFLD.
Thioesterase superfamily member 2 (Them2; also known as acyl-CoA thioesterase (Acot)
13) catalyzes the hydrolysis of long chain fatty acyl-CoA esters into free fatty acids and
CoA2. Them2 is abundant in the liver and primarily localized to mitochondria, where it
regulates trafficking of fatty acids to oxidative versus lipid biosynthetic pathways,
depending upon metabolic conditions2. Them2 is also expressed at high levels in other
oxidative tissues, including skeletal and cardiac muscle, as well as in adipose tissue.
However, its function in these tissues is not understood3. Mice globally lacking Them2
(Them2-/-) are protected against diet-induced obesity and hepatic steatosis, and exhibit
markedly improved glucose homeostasis4,5. The global absence of Them2 is also
associated with reduced hepatic ER stress in the setting of overnutrition6. Conversely,
Them2 liver-specific knockout (L-Them2-/-) mice exhibit similar body weights as controls,
and are not protected against hepatic steatosis or insulin resistance in response to high-
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fat diet (HFD) feeding7. Studies in L-Them2-/- mice revealed a mechanistic role for Them2
in the hepatic trafficking of acyl-CoA molecules towards triglyceride synthesis and
incorporation into VLDL particles7.
To explore the hypothesis that Them2 activity in extrahepatic tissues may be required for
the development of insulin resistance and hepatic steatosis, we generated tissue-specific
knockout mice lacking Them2 in skeletal muscle, cardiac muscle and adipose tissue. Only
skeletal muscle-specific Them2 knockout mice recapitulated the protection from insulin
resistance and hepatic steatosis that was observed in Them2-/- mice. Our results
demonstrate that Them2 limits rates of fatty acid oxidation in skeletal muscle, and its
upregulation in the setting of overnutrition promotes skeletal muscle insulin resistance
and hepatic steatosis by complementary mechanisms. In addition to reducing the demand
for hepatic VLDL secretion, the reduction in fatty acid oxidation in skeletal muscle
decreases the production of short chain fatty acids and pentose phosphate pathway
intermediates that maintain insulin sensitivity. In addition, Them2 suppresses the
expression of myokines capable of modulating skeletal muscle physiology, as well as
crosstalk with liver and adipose tissue. When taken together, these findings suggest that
Them2 in skeletal could be targeted as therapeutic strategy in the management of NAFLD.
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RESULTS
Them2 suppresses insulin sensitivity in skeletal muscle and limits exercise
capacity in HFD-fed mice
Because skeletal muscle and heart extensively utilize fatty acids to produce ATP, we
examined the potential contribution of Them2 to the regulation of insulin action in these
tissues. We first confirmed that Them2-/- mice on a C57BL6J genetic background were
protected against HFD-induced obesity (Figure 1-A,B) and glucose intolerance (Figure 1-
C), as we have observed for a mixed genetic background4. In response to exogenous
insulin administration (Figure 1-D), HFD-fed Them2-/- mice exhibited a 62 % reduction in
blood glucose concentrations compared with a non-significant 24 % decrease in Them2+/+
mice, with an associated 2.6-fold increase in Akt phosphorylation in skeletal muscle,
which together indicated improved insulin sensitivity in the absence of Them2 expression.
There was a 1.7-fold increase in Them2 expression in skeletal muscle, but not in cardiac
muscle, in HFD-fed compared to chow-fed mice (Figure 1-E).
Notwithstanding reduced body weight of Them2-/- mice, both skeletal muscle and cardiac
tissues from Them2-/- mice were proportionate in size and histologically normal (Suppl.
Figure 1-A,B). Whereas skeletal muscle of Them2-/- mice exhibited no changes in mRNA
expression levels of myosin heavy chains, there were modest decreases in the
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proportions of type I muscle fiber type relative to type II fiber type proteins (Suppl. Figure
1-C,D). Them2-/- mice also exhibited similar expression of myosin heavy chains and
hypertrophy-related genes in cardiac muscle as control mice (Suppl. Figure 1-E,F). In
chow-fed mice, Them2 expression did not influence in exercise capacity, as assessed by
treadmill testing (Figure 1-F) and lactate production (Suppl. Figure 1-G). However, in the
setting of HFD feeding, Them2-/- mice showed improved exercise capacity compared to
wild type controls (Figure 1-F). We next investigated the impact of Them2 on cardiac
function in response to chronic ionotropic stimulation. Whereas cardiac hypertrophy is
accompanied by decreased rates of fatty acid oxidation and increased glucose utilization8,
Them2 expression did not influence development of hypertrophy (Figure 1-G).
Metabolic monitoring revealed no differences in voluntary (wheel) running (Figure 1-H),
spontaneous physical activity or food intake (data not shown). Similar to Them2-/- mice on
a mixed genetic background4, chow-fed Them2-/- C57BL6J mice exhibited increased
energy expenditure (Figure 1-I). Although no differences were found in energy
expenditure during voluntary running (Figure 1-I), respiratory exchange ratios of Them2-
/- mice were elevated during the light cycle after acclimation to running wheel (Figure 1-
J), indicative of greater glucose utilization in skeletal muscle in the absence of Them2
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expression during a period when mice were more sedentary. Taken together, these results
suggest that Them2 regulates energy substrate utilization in skeletal muscle.
Skeletal muscle expression of Them2 promotes obesity and insulin resistance in
HFD-fed mice
To systematically explore contributions of extrahepatic Them2 to obesity, hepatic
steatosis and insulin resistance, we created mice lacking Them2 in skeletal muscle,
cardiac muscle and adipose tissues, each on a C57BL/6 genetic background (Figure 2-
A). Skeletal muscle deletion of Them2 with preserved cardiac expression (S-Them2-/-
mice) was accomplished using mice expressing Cre recombinase driven by the Myf5
promoter. Because Myf5 is expressed in brown adipose tissue (BAT) in addition to skeletal
muscle, mice lacking Them2 in skeletal muscle were also deficient in BAT. The Myh6
promoter was used to generate cardiac muscle deletion of Them2 with preserved skeletal
muscle expression (C-Them2-/- mice). A-Them2-/- mice lacking Them2 in BAT and white
adipose tissue (WAT), which expresses Them2 at low levels, were generated using mice
expressing Cre recombinase driven by the adiponectin promoter. Only S-Them2-/- mice
were protected against diet-induced overweight (Figure 2-B), excess fat accumulation
(Figure 2-C), glucose intolerance (Figure 2-D), and insulin resistance (Figure 2-E), as
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observed for global Them2-/- mice (Figure 1-A-D). These differences were not associated
with substantial alterations in expression of other skeletal muscle Acot or Acsl isoforms
(Suppl. Figure 2-A,B). The possibility that this phenotype was attributable to the absence
of Them2 expression in BAT was excluded by demonstrating similar weight differences
(Suppl. Figure 3-A) and body compositions (Suppl. Figure 3-B) for mice housed at 30 °C,
a thermoneutral temperature that suppresses BAT activity. This was further supported by
the phenotypes of A-Them2-/- mice (Figure 2-B-D), which also lack Them2 expression in
BAT, but were not protected against HFD-induced obesity and insulin resistance. We used
a gain of function approach to confirm the metabolic contribution of skeletal muscle
Them2: AAV8-Mck driven rescue of Them2 expression in muscle of global Them2-/- mice
(Suppl. Figure 3-C) resulted in higher body weights than AAV8- Mck-LacZ treated Them2-
/- mice (Suppl. Figure 3-D) with increases in both lean and fat mass (Suppl. Figure 3-E).
Taken together, these results indicate that skeletal muscle Them2 plays a central role in
the development of obesity and insulin resistance in response to HFD feeding.
To investigate mechanisms of improved glucose homeostasis in HFD-fed S-Them2-/- mice,
we performed hyperinsulinemic-euglycemic clamp studies. S-Them2-/- mice exhibited
higher glucose infusion rates than controls (Figure 3-A), as well as increased rates of
glucose uptake in skeletal muscle (Figure 3-B). By contrast, basal and clamped hepatic
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glucose production rates were not influenced by skeletal muscle expression of Them2
(Figure 3-C). In keeping with these findings, there were marked increases in Akt
phosphorylation by 2.5-fold in skeletal muscle of HFD-fed S-Them2-/- mice compared to
controls in response to acute administration of insulin (Figure 3-D). These results reveal
that Them2 in skeletal muscle promotes whole body glucose intolerance by reducing
skeletal muscle insulin sensitivity.
Them2 controls rates of fatty acid oxidation in skeletal muscle
We next examined the role of Them2 in the regulation of fatty acid oxidation in skeletal
muscle. Rates of fatty acid oxidation in skeletal muscle homogenates were increased in
both Them2-/- (Figure 4-A) and S-Them2-/- mice (Figure 4-B). Muscle strips isolated from
Them2-/- mice exhibited increased oxidation rates of palmitate from endogenous (pulsed)
palmitate but not from exogenous (chase) sources (Figure 4-C).
To gain mechanistic insights, we performed immunoblot analyses of gastrocnemius
muscle samples to assess the expression of key proteins that control fatty acid oxidation.
In contrast to chow-fed control mice, HFD-fed controls exhibited 2-fold increases in
Them2 expression levels (Figure 4-D,E). Suggestive of its role in regulating Them2
expression in skeletal muscle, the Them2-interacting protein PC-TP3, was reduced in
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chow-fed S-Them2-/- mice relative to controls (Figure 4-D,E). Although phosphorylation of
adenosine monophosphate-activated protein kinase (AMPK (Thr172)), which increases
fatty acid oxidation rates in skeletal muscle, showed no genotype-dependent changes,
chow-fed S-Them2-/- mice exhibited reduced phosphorylation of acetyl-CoA carboxylase
(ACC) compared to control mice (Figure 4-D,E). Phosphorylated ACC is inactive, whereas
dephosphorylated ACC enhances conversion of acetyl-CoA to malonyl-CoA, and this in
turn reduces fatty acid oxidation owing to inhibition of carnitine palmitoyltransferase I
(CPT1)9. Although CPT1 protein expression was not altered in chow-fed mice, relative
mRNA expression of CPT1 was reduced in skeletal muscle of chow-fed S-Them2-/- mice
(Suppl. Figure 4-A). Notwithstanding the absence of changes in ACC phosphorylation in
HFD-fed mice, increased CPT1 protein expression in HFD-fed S-Them2-/- mice suggests
increased mitochondrial fatty acid utilization in skeletal muscle of S-Them2-/- mice.
Consistent with a lack of effect of Them2 expression on fatty acid oxidation rates of
exogenous fatty acids, we observed no changes in skeletal muscle expression of CD36.
To further ascertain mechanisms underlying the increased skeletal muscle fatty acid
oxidation, we surveyed the expression of genes that control lipid and glucose
homeostasis. The expression of these genes was largely unchanged either in chow
(Suppl. Figure 4-A,B) or HFD-fed mice (Suppl. Figure 4-A,B), suggesting that increased
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fatty acid oxidation in S-Them2-/- mice was not compensated by fatty acid uptake or
glucose utilization.
In keeping with increased rates of fatty acid oxidation, total fatty acyl-CoAs in skeletal
muscle of chow-fed S-Them2-/- mice tended to decrease (Figure 4-F), but without
changes in individual molecular species (Suppl. Figure 5A). The absence of a change in
fatty acyl-CoAs following HFD-feeding is likely attributable to abundant cellular nutrients
in the setting of overnutrition. Acetyl-carnitine forms when acetyl-CoA concentrations
exceed what is required by the TCA cycle, and is essential molecules for maintaining
metabolic flexibility and insulin sensitivity10. Skeletal muscle of S-Them2-/- mice exhibited
increased acetyl-carnitine concentrations for mice fed HFD (Figure 4-G), without
discernible changes in individual acyl-carnitine molecular species (Suppl. Figure 5-B).
These results are consistent with increased fatty acid oxidation rates in skeletal muscle
lacking Them2 even after HFD feeding. Free fatty acid concentrations were higher in
skeletal muscle of S-Them2-/- mice (Figure 4-H), with increases being most pronounced
in short chain molecular species fatty acids (Figure 4-I, Suppl. Figure 5-C,D), which can
influence glucose homeostasis11.
To gain further insight into Them2 metabolic regulation in skeletal muscle, we conducted
targeted metabolomics profiling for 216 predefined polar metabolites. Unsupervised
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clustering of differences identified both diets and genotypes (Figure 4-J). Metabolic
pathway analysis identified significant alterations in S-Them2-/- mice fed either chow
(Suppl. Figure 6A) or HFD (Suppl. Figure 6B). Of these, amino acid metabolism and the
pentose phosphate pathway (PPP) were affected by skeletal muscle Them2 expression
in mice fed both diets.
The absence of Them2 in skeletal muscle increased tissue concentrations of 9 amino
acids in chow-fed mice (Suppl. Figure 7-A), whereas it decreased concentrations of 4
amino acids in HFD-fed mice (Suppl. Figure 7-B). For both diets, ablation of Them2 mainly
altered branched chain amino acids. Branched-chain amino acids promote protein and
glutamine synthesis in skeletal muscle, and increased levels of branched-chain amino
acids is known to correlate with an improvement in glucose metabolism12. Therefore,
increased levels of branched-chain amino acids in muscle of chow-fed S-Them2-/- mice
may have contributed to protection against diet-induced insulin resistance.
Whereas most metabolites in the PPP pathway were unchanged in both chow diet and in
HFD (Figure 4-K), ribose 5-phosphate was significantly reduced in chow-fed mice and
both ribulose 5-phosphate and ribose 5-phosphate were increased in skeletal muscle of
HFD-fed S-Them2-/- mice, suggestive of upregulation of this pathway by overnutrition in
the absence of Them2. PPP generates reducing equivalents to prevent oxidative stress13,
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and its upregulation may have contributed to improved insulin sensitivity by clearing
excess glucose-induced reactive oxygen species in skeletal muscle. Them2 expression
in skeletal muscle did not influence activity of superoxide dismutase (Suppl. Figure 8-A)
or thiobarbituric acid reactive substances (Suppl. Figure 8-B), suggesting that the ablation
of Them2 did not promote oxidative stress in skeletal muscle. Taken together, these
studies revealed that Them2 in skeletal muscle modulates tissue concentrations of short
chain fatty acids, branched chain amino acids and PPP metabolites, each of which may
play a role in the regulation of glucose homeostasis.
Them2 limits fatty acid utilization in cultured myotubes
To assess whether Them2 alters mitochondrial fatty acid utilization in skeletal muscle, we
utilized C2C12 myotubes that were transduced to overexpress mouse Them2. AAV8-
Them2 transduction increased Them2 expression by 1.5-fold (Figure 5-A). Although
increased Them2 expression did not alter myotube diameter or fusion index14, AAV8-
Them2 treated myotubes exhibited greater myotube areas, suggesting Them2 expression
enhances myotube elongation (Figure 5-B,C). Myotubes that overexpressed Them2 also
exhibited higher rates of oxygen consumption (OCR) during both basal and maximal
respiration (Figure 5-D). Of note, when palmitate was made available, there were no
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differences in basal or maximal respiration (Figure 5-D), with differences between bovine
serum albumin (BSA) treatment alone and palmitate plus BSA exposure signifying
oxidation of extracellular palmitate. As evidenced by higher OCR values following
treatment with BSA alone, AAV8-Them2 transduced myotubes utilized less extracellular
palmitic acid (Figure 5-E).
A reduction in OCR after etomoxir treatment under substrate limited conditions provides
a measure of intracellular fatty acid oxidation (Figure 5-F), which was not influenced by
Them2 expression (Figure 5-G). Taken together, these results suggest that Them2
regulates extracellular fatty acid utilization through its regulation of the intracellular fatty
acid pool that is available for oxidation.
Skeletal muscle Them2 expression enables HFD-fed mice to exceed a body weight
threshold value for hepatic steatosis and metabolic dysfunction
In human beings, hepatic steatosis is largely a function of adipose tissue insulin
resistance and serves an important barometer of metabolic dysfunction, which becomes
fully established at a threshold level of 5.5% liver fat15. Although HFD-fed mice are widely
utilized in studies of NAFLD, a threshold value for pathogenic intrahepatic triglyceride
accumulation and its relationship to body composition has not been systematically
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explored. To enable accurate weight-based comparisons between S-Them2-/- and control
mice, 87 C57BL6J male mice were fed a HFD for 12 w. Body weights exhibited a normal
distribution ranging from 31.1 g to 52.2 g (mean ± SEM; 42.3 ± 0.5 g; median 42.5g)
(Figure 6-A). Both lean and fat mass increased linearly as functions of body weight (Figure
6-B), and fat mass was a linear function of lean mass (Figure 6-C). By contrast, liver
weight exhibited two very distinct linear relationships to body weight (Figure 6-D): Up to
a body weight threshold of 41.6 g (corresponding to fat mass of 15.6 g), there was a
relatively flat slope of liver weight versus body weight (0.031 g liver/g BW). In excess of
41.6 g, the slope was 4.3-fold steeper (0.134 g liver/g BW). Additionally, there was a
similar biphasic dependence of liver weight on fat (Figure 6-E) but not lean weight of mice
(Figure 6-F).
Hepatic steatosis exhibited a biphasic relationship to body weight, with threshold body
weight of 41.6 g corresponded to 31% hepatic steatosis (Figure 6-G). By contrast, both
liver weights (Figure 6-H) and hepatic triglyceride concentrations (Figure 6-I) were linearly
correlated with histologic steatosis. The body weight threshold of 41.6 g also
discriminated plasma concentrations of free fatty acids, glucose and cholesterol (Figure
6-J), as well as hepatic mRNA expression levels of specific genes that govern the
metabolism of fatty acids (Aox and CD36) and lipid droplets (Cidea) (Figure 6-K).
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Indicative that the 41.6 g weight threshold for hepatic steatosis reflected changes in
skeletal muscle dysfunction, expression of genes that mediate fatty acid activation (Acsls)
and deactivation (Acots) were significantly altered in skeletal muscle of mice exceeding
this body weight (Figure 6-L). Additionally, five myokine genes were downregulated above
this weight threshold, as was Pgc1a, which controls mitochondrial biogenesis in skeletal
muscle (Figure 6-M). Taken together, these findings reveal two distinct phases in the
development of hepatic steatosis and metabolic dysfunction in HFD-fed C57BL/6J mice
with a threshold of 31% at a body weight of 41.6 g. After 12 w of HFD-feeding, body
weights of most (80%) of S-Them2-/- mice fell below the 41.6 g threshold, compared with
36 % of controls (Figure 6-N).
In keeping with reduced weight and improved glucose homeostasis, HFD-fed S-Them2-/-
but not L-Them2-/-7 or C-Them2-/- mice were also protected against hepatic steatosis, as
evidence by histopathology (Figure 7-A) and hepatic triglyceride concentrations (Figure
7-B), as was previously observed in Them2-/- mice4. Pyruvate tolerance tests revealed
reduced hepatic glucose production only in S-Them2-/- mice (Figure 7-C), as was also
observed in global Them2-/- mice4. There were no differences in plasma activities of
aspartate aminotransferase, alanine aminotransferase or creatine kinase or in insulin
concentrations (Suppl. Figure 9-A,B). Of note, for mice under the 41.6 g body weight
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threshold, hepatic triglyceride concentrations were also reduced in HFD-fed S-Them2-/-
mice compared to controls (Figure 7-D). These findings suggested that metabolic
improvements in S-Them2-/- mice were in part attributable to reduced body weight gain
because most mice lacking Them2 did not achieve a body weight threshold for the
development of more severe hepatic steatosis, but importantly, were also due in part to
expression of skeletal muscle Them2 per se.
Skeletal muscle Them2 expression promotes hepatic steatosis by suppressing
VLDL secretion
We next explored the mechanisms underlying skeletal muscle Them2-mediated
development of hepatic steatosis. We first examined evidence for increased rates of
hepatic fatty acid oxidation. Fasting concentrations of ß-hydroxybutyrate were reduced in
HFD-fed control mice compared with chow-fed controls, but this was not observed for S-
Them2-/- mice, in which levels remained unchanged by HFD-feeding and were
significantly higher than in HFD-fed control mice (Suppl. Figure 9-C). This suggested that
the absence of Them2 in skeletal muscle might be associated with increase rates of fatty
acid oxidation in liver. However, direct measurement of hepatic fatty acid oxidation in
HFD-fed S-Them2-/- mice did not reveal increases (Figure 7-E), suggesting that
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differences in ß-hydroxybutyrate may instead have reflected Them2-mediated changes
in rates of ketone body utilization in skeletal muscle. Chow but not HFD-fed S-Them2-/-
mice exhibited increased fasting plasma concentrations of free fatty acids (Suppl. Figure
9-D), which argues against reduced hepatic uptake of fatty acids as a mechanism by
which S-Them2-/- mice were protected against steatosis. Plasma concentrations of
triglycerides, total and free cholesterol, as well as phospholipids were unaffected by
skeletal muscle Them2 expression in either chow or HFD-fed mice (Suppl. Figure 9-D),
and despite dramatic reductions in hepatic steatosis, RNAseq analysis of liver identified
only a limited number of genes with altered expression (Suppl. Figure 10-A). Among these
was the hepatic steatosis marker Cidea, which was markedly downregulated, suggesting
reductions in triglyceride storage. This was also implicated by pathway analysis, which
identified increased degradation of hepatic triglycerides as the most upregulated pathway
(Suppl. Figure 10-B). In keeping with this possibility, S-Them2-/- mice exhibited increased
rates of VLDL triglyceride secretion (Figure 7-F). Taken together, these findings suggest
that increased demand by skeletal muscle for fatty acids in the form of secreted hepatic
triglycerides may have explained the reduced steady state concentrations in liver.
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Muscle Them2 mediates crosstalk between skeletal muscle and the liver
Lastly, we examined evidence of regulatory cross-talk between muscle and liver that may
have also contributed to protection against hepatic steatosis and metabolic dysfunction
in S-Them2-/- mice. Conditioned medium from C2C12 myotubes transduced to
overexpress Them2, suppressed basal and maximal OCR values in Hepa1-6 cells (Figure
8-A). We next assessed for Them2-dependent expression of myokines in skeletal muscle.
Ablation of Them2 altered myokine gene expression depending on dietary conditions
(Figure 8-B). Overnight fasting induced substantial changes in myokine genes expression
in S-Them2-/- mice (Figure 8-B). Skeletal muscle of chow-fed S-Them2-/- mice induced
expression of insulin like growth factor 1 (Igf-1) and interleukin 15 (IL-15), both of which
regulate muscle mass16,17, whereas IL-15 also decreases plasma triglycerides and
adipose tissue mass18. In HFD-fed mice, S-Them2-/- mice exhibited modest increases in
chemokine ligand 1 (CXCL1), which regulates angiogenesis and fatty acid metabolism19.
Independent of diet, there was consistent down-regulation in osteoclin which is
upregulated by palmitic acid and promotes insulin resistance in muscle20–22. Because
skeletal muscle of HFD-fed S-Them2-/- mice is protected against excess body weight gain,
these alterations in myokine genes might have been secondary to changes in body weight.
However, differences in relative mRNA expression were still observed in weight matched
21 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
HFD-fed mice, as defined by body weights that fell below the threshold of 41.6 g (Figure
8-C): myostatin and osteoclin were downregulated in skeletal muscle of S-Them2-/- mice
compared to control mice. These results suggested that Them2-mediated control of
skeletal muscle myokine gene expression may contribute to crosstalk among skeletal
muscle, adipose tissue and the liver (Figure 8-D).
22 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
DISCUSSION
This study identified a key role for skeletal muscle Them2 in the regulation of skeletal
muscle metabolism, as well as in the pathogenesis of HFD-induced insulin resistance and
hepatic steatosis in mice. Them2 limits rates of fatty acid oxidation by skeletal muscle,
which in turn reduces rates of insulin-mediated glucose disposal by reducing insulin
sensitizing metabolites and myokines. This also reduced demand for exogenous fatty
acids, leading to the development of hepatic steatosis.
We previously demonstrated that Them2-/- mice exhibited reduced plasma free fatty acid
concentrations, as well as reduced hepatic steatosis and glucose production5,23. However,
these effects were not recapitulated in L-Them2-/- mice, suggesting that extrahepatic
Them2 expression is required in order to recapitulate the phenotype in the setting of
overnutrition7. In both mice and humans, skeletal and cardiac muscles substantially
contribute to basal calorie consumption. Skeletal plays critical roles in energy metabolism,
glucose uptake and locomotion24. The metabolic activity of the heart is a major
determinant of the body’s oxygen consumption, predominantly utilizing long chain fatty
acids as substrates for energy production8. Consequently, glucose and lipid metabolism
in skeletal muscle and cardiac muscle contribute substantially to whole body energy
consumption.
23 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Whereas Them2 is most highly expressed in heart, its deletion from cardiac muscle did
result in discernable differences in organ function or whole body metabolism. To preserve
function, cardiac muscle has extraordinary metabolic flexibility that enable it to adapt to
changes in substrate availability8. For this reason, we speculate that alterations in this
specific enzyme of long chain fatty acid metabolism would not cause obvious functional
or metabolic consequences in C-Them2-/- mice. This notion is supported by our
observation that Them2 expression in heart of control mice was unaffected by HFD
feeding. By contrast, HFD feeding robustly upregulated Them2 expression in skeletal
muscle, suggesting a pathogenic role in skeletal muscle mechanical dysfunction and
alteration in metabolism.
As evidenced by reduced rates of glucose uptake during clamp studies and diminished
insulin-stimulated Akt phosphorylation, Them2 contributed to the development of skeletal
muscle insulin resistance, presumably as a reflection of reduced rates of fatty acid
oxidation25. Previous studies have demonstrated that the accumulation of excess free
fatty acids and their derivatives including long chain acyl-CoAs in skeletal muscle are key
determinants of insulin resistance26. Although the absence of a fatty acyl-CoA
thioesterase might have been expected to increase skeletal muscle concentrations fatty
acyl-CoAs and decrease concentrations of free fatty acids, we observed the opposite in
24 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
S-Them2-/- mice. However, the preferred substrate of Them2 is long chain fatty acyl-CoA,
and the observed increase in free fatty acid concentrations was mainly attributable to
increases in short chain fatty acids. Short chain fatty acids are predominantly generated
by colonic bacteria and are metabolized by enterocytes and hepatocytes. Short chain
fatty acids are also formed in peroxisomes during shortening of very long chain fatty acyl-
CoAs that can be hydrolyzed by peroxisomal Acots and released into the cytosol27. We
speculate, increased rates of mitochondrial ß-oxidation in skeletal muscle of S-Them2-/-
mice were also accompanied by increases in peroxisomal oxidation, which resulted in the
production of more short chain fatty acids. The reduction in skeletal muscle fatty acyl-CoA
concentrations most likely reflects their increased rates of oxidation, as evidence by the
reciprocal rise in acetyl-carnitine concentrations.
Short chain fatty acids influence muscle fatty acid oxidation, glucose uptake and insulin
sensitivity via both direct and indirect mechanisms11,28. Of note, acetate and butyrate
improve skeletal muscle glucose metabolism and insulin sensitivity11. When taken
together with observed increases in branched chain amino acids and PPP metabolites12,13,
these data suggest that elevated concentrations of short chain fatty acids in skeletal
muscle of S-Them2-/- mice contributed to the improved insulin sensitivity observed the
setting of overnutrition.
25 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Although the reduction in lean mass of S-Them2-/- mice at the thermoneutrality was similar
as at room temperature, there was less reduction in fat mass. This may have been
because Them2 in BAT also plays a role in HFD-induced obesity via adaptive
thermogenesis5. Because fat mass is tightly correlated with lean mass in HFD-fed mice,
we further postulate that reduction in lean body mass due to loss of skeletal muscle
Them2 also exerted metabolic control through associated decreases in adipose tissue.
In human NAFLD, hepatic steatosis is strongly influenced by relatively modest changes
in total body weight: An 11% loss in body weight was associated with a 52 % reduction in
hepatic triglycerides, owing largely to the restoration of insulin sensitivity29. Interestingly,
these observations in humans are of a similar magnitude to our findings in HFD-fed mice,
whereby at 16 w of age, S-Them2-/- mice exhibited a 12 % reduction in BW (Figure 2-B),
which was associated with a 52 % reduction in hepatic triglyceride concentrations.
However, unlike observations in humans15, C57BL6J mice exhibited remarkable
accelerations in hepatic triglyceride accumulation at a discrete threshold of body weight
(41.6 g). Moreover, fat weight is tightly correlated with lean weight in HFD-fed mice. Hence,
we concluded that reduced hepatic steatosis in S-Them2-/- mice is attributable in part to
the reduced body weight gain such that mice rarely exceeded body weights of 41.6 g.
Nevertheless, hepatic triglyceride concentrations were still reduced in HFD-fed S-Them2-
26 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
/- mice compared to HFD-fed control mice weighing less than 41.6 g revealing that Them2
expression per se is sufficient to regulate hepatic lipid metabolism.
A potential mechanism whereby skeletal muscle Them2 directly controls hepatic steatosis
is organ crosstalk through the secretion of myokines, which are bioactive proteins that
regulate organ function by autocrine, paracrine and endocrine actions30. Deletion of
Them2 in skeletal muscle induced expression of several myokines. Skeletal muscle of
chow-fed S-Them2-/- mice showed significantly higher gene expressions of Igf-1 and IL-
15, which regulate muscle hypertrophy16,17. IL-15 also decreases lipid deposition in pre-
adipocytes and reduces white adipose tissue mass18. In the setting of overnutrition, there
was also a modest increase in skeletal muscle expression of CXCL1 in S-Them2-/- mice.
CXCL1 is the functional homologue to human IL-831, and it plays a role in angiogenesis
and neutrophil chemoattractant activity32. Increased expression of CXCL1 attenuates diet
induced obesity in mice through enhanced fatty acid oxidation in muscle19. Osteoclin
(synonym: musclin), which was downregulated in skeletal muscle of S-Them2-/- mice, is
a myokine that is controlled by nutritional status and promotes insulin resistance. Musclin
expression inhibits glucose uptake and glycogen synthesis20, and Musclin mRNA is
regulated by the forkhead box O1 transcription factor (FOXO1) downstream of PI3K/Akt
pathway in response to insulin action22.
27 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Our findings demonstrate a central role for skeletal muscle Them2 in the control of nutrient
metabolism and in the pathogenesis of diet-induced insulin resistance and hepatic
steatosis. The inhibition of Them2 activity in muscle could offer an attractive strategy for
the management of obesity and nonalcoholic fatty liver disease.
28 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
MATERIALS AND METHODS
Animals and diets
Mixed background global Them2 knockout mice were backcrossed 20 generations with
C56BL/6J4,5. Wild type C56BL/6J mice were obtained from the Jackson Laboratory, Bar
Harbor, ME, USA (Stock #000664). Tissue specific knockout mice were created by a
LoxP/Cre system7. C56BL6 background mice with Them2 flanked by two LoxP sites
(Them2flox/flox) were crossed with mice expressing Cre recombinase driven by the Myf5
promoter to generate skeletal muscle specific deletion of Them2-/- (S-Them2-/-), the Myh6
promoter to generate cardiac muscle specific deletion of Them2-/- (C-Them2-/-) and the
Adipoq promoter was used to generate adipose tissue specific deletion of Them2-/- (A-
Them2-/-) respectively (The Jackson Laboratory, Myf5-cre; Stock #007893, Myh6-cre;
Stock #011038, Adipoq-cre; Stock #010803)33–35. These mice were viable and displayed
no apparent developmental abnormalities. The presence of Cre allele was determined by
PCR analysis using the primers specified by the Jackson Laboratory. Mice were housed
in a barrier facility on a 12 h light/dark cycle with free access to water and diet. Male mice
were weaned at 4 w of age and fed chow (PicoLab Rodent Diet 20; LabDiet, St. Louis,
MO, USA). Alternatively, 5 w old male mice were fed a HFD (D12492: protein 20 % kcal,
fat 60 % kcal, carbohydrate 20 % kcal, energy density 5.21 kcal/g; Research Diets Inc.,
29 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
New Brunswick, NJ, USA) for 12 w. Following 6 h fasting (9:00 AM to 3:00 PM) or 18 h
fasting (6:00 PM to 12:00 PM), 17 w old mice were euthanized and plasma was collected
by cardiac puncture. Tissues were harvested for immediate use or snap frozen in liquid
nitrogen and stored at -80 °C. Mice were monitored by daily health status observation by
technicians supported by veterinary care. Housing and husbandry were conducted in
facilities with a sentinel colony health monitoring program and strict biosecurity measures
to prevent, detect, and eradicate adventitious infections. Animal use and euthanasia
protocols were approved by the Institutional Animal Care and Use Committee of Weill
Cornell Medical College.
Adeno-associated virus-mediated Them2 expression in skeletal muscle
Adeno-associated virus (AAV) vectors used in this study were generated by Vigene
Biosciences, Rockville, Maryland, USA. Vectors are based on the AAV8 genome and
transgenes are driven by the muscle creatine kinase (Mck) promoter. The mouse Them2
gene (gene ID: 55856) was packaged with Flag as AAV8- Mck-Flag-Them2 and a LacZ
gene was used to generate AAV8-Mck-LacZ as a control. Mice were anesthetized with
2% isoflurane vapors prior to injection to ensure no movement. Both hindlimbs were
shaved and sterilized with isopropanol swab sticks. AAVs (1-5x1011 particles in 30μl
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saline) were injected to the gastrocnemius muscle by intramuscular injection using 30-
gauge insulin syringe.36 Mice were then return to home cages for 12 w to allow for
recombinant protein expression.
Maximal exercise capacity
Mice were acclimated to treadmill running tracks incrementally for 5 d starting from 10
min at 0.3 km/h to 60 min at 1.2 km/h. To determine maximal exercise capacity, treadmill
speed was started at 0.3 km/h and increased 0.3 km/h every 3 min until the speed
reached 1.2 km/h. The speed was kept constant (1.2 km/h) until mice reached exhaustion.
Blood lactate levels were measured from the tail tip before and after 30 min treadmill
running at 1.2 km/h using lactate pro2 LT-1730 (ARKRAY, Kyoto, Japan).
Cardiac function
Cardiac function was evaluated by echocardiography as response to 2 w chronic
isoproterenol infusion as previously described37–39. In brief, 10 w old mice were implanted
with ALZET osmotic minipumps Model 1002 (DURECT Corporation, Cupertino, CA, USA)
and infused with isoproterenol at 30 mg/kg/day for 2 w. Echocardiography was carried out
at 10 w (pre) and 12 w (post) to monitor cardiac hemodynamics using a Vevo 3100
31 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(FUJIFILM Visual sonics, Toronto, Canada). M mode was used to measure left ventricle
ejection fraction and wall thickness. Each mouse was anesthetized with inhaled isoflurane
(2%).
Metabolic monitoring
Mice were housed in individual cages for 2 w for acclimation prior to metabolic
monitoring40. Mice were then housed in temperature-controlled cabinets with a 12 h
light/dark cycle and monitored using the Promethion Metabolic Screening System (Sable
Systems International, North Las Vegas, NV). Rates of O2 consumption (VO2) and CO2
production (VCO2) were determined at 5 min intervals. Values of respiratory exchange
ratio (RER) were calculated as VCO2/VO2. After 2 d acclimation, metabolic parameters
were recorded over 24 h with or without a running wheel inside the cage. Physical
activities and voluntary running on wheel were determined according to beam breaks
within a grid of photosensors built outside the cages. Energy expenditure was calculated
by indirect calorimetry41 and adjusted by ANCOVA using VassarStats
(www.vassarstats.net) to adjust for differences in lean body mass using lean body
composition determined by magnetic resonance imaging EchoMRI (EchoMRI, Houston,
TX).
32 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Tolerance Tests
Glucose tolerance tests, insulin tolerance tests, and pyruvate tolerance tests were
performed with minor modifications7,42. In brief, mice were fasted 6 h for glucose tolerance
tests, 4 h for insulin tolerance tests, and overnight (16 h) for pyruvate tolerance tests. A
drop of blood from the tail tip was subjected to glucose measurement at baseline and at
regular intervals using GE 100 Blood Glucose Monitor (General Electric, Ontario, CA).
Glucose solution was administered by intraperitoneal injection with 1 g/kg for global
Them2-/- mice and their controls and with 1.5 g/kg for A-Them2-/- mice and their littermate
controls, or orally using gavage needle with fixed dose at 40 mg for C-Them2-/- mice, S-
Them2-/- mice and their littermate controls. Insulin solution was administered by
intraperitoneal injection with 0.75 U/kg for A-Them2-/- mice, C-Them2-/- mice, S-Them2-/-
mice and littermate controls. Pyruvate solution was administered by orally using a gavage
needle with 2 g/kg for C-Them2-/- mice, S-Them2-/- mice and littermate controls.
Hyperinsulinemic-euglycemic clamp studies
Hyperinsulinemic-euglycemic clamp studies were performed at Animal Physiology Core
(Albert Einstein College of Medicine, New York, NY, USA) as described privously43. Briefly,
33 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
vascular catheters were inserted, 8 d prior to the clamps. Hyperglycemic-euglycemic
clamp studies were performed in unrestrained mice. Mice were fasted for 5 h prior to the
study. The clamp protocol consisted of a 90 min tracer (5-uCi bolus of [3-3H]-glucose
followed by a 0.05 uCi/min infusion) equilibration period followed by a 120 min
experimental period. A blood sample (10 ul) was taken for the assessment of basal
glucose and insulin levels and glucose turnover. The insulin clamp was begun with a
primed-continuous infusion of human insulin (300 mU/kg bolus followed by 5 mU/kg/min
1; Novolin; Novo Nordisk, Princeton, NJ). The [3-3H]-glucose infusion was increased to
0.1 uCi/min for the remainder of the experiment. Euglycemia (150 mg/dl) was maintained
during clamps by measuring blood glucose every 10 min and infusing 45% dextrose as
necessary. Blood samples (60–200 µl) were taken and processed to determine glucose
specific activity. All blood samples were resuspended in heparinized saline and infused
back to mice to maintain hematocrit. Plasma samples were collected to determine
glucose levels and specific activities of [3-3H]-glucose. Tissue samples were stored at
−80°C for glucose uptake analyses43.
Insulin signaling
Mice were fasted for 6 h during the light cycle (9:00 AM to 3:00 PM). Tissues were
34 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
collected 10 min following intraperitoneal insulin (10 U/kg) injection or saline injection as
a control. Insulin signaling was evaluated by immunoblot analysis for phosphorylation of
Akt44.
Quantitative PCR
Relative mRNA expression was determined by quantitative PCR (qPCR) using SYBR
Green Real-Time PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Total RNA
was isolated from soleus muscle (10 µg) and apex of ventricle (20 µg) using RNeasy
fibrous tissue mini kit (QIAGEN, Hilden, Germany) following manufacturer’s instructions.
cDNA was synthesized with a High-Capacity cDNA Reverse transcription Kit (Applied
Biosystems, Foster City, CA, USA). Equal amount of mRNA samples were subjected to
qPCR using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster
City, CA, USA) in a 384 plate. Genes were analyzed relative to cyclophilin A. The
nucleotide sequences of oligonucleotides used for qPCR are presented in Suppl. Table 2.
Immunoblot analysis
Immunoblot analyses were performed by standard techniques7. Briefly, tissues or cells
were homogenized in a RIPA buffer containing cOmplete Protease Inhibitor Cocktail and
35 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
PhosphoSTOP Phosphate Inhibitor Cocktail Tablets (Roche, Indianapolis, IN, USA) using
a Bead Ruptor 24 Elite bead mill homogenizer (Omni International, Kennesaw, GA, USA).
Protein concentrations were determined by using a BCA reagent (Thermo Fisher
Scientific, Springfield Township, NJ). Equal amounts of protein samples were separated
by SDS-PAGE and transferred to nitrocellulose membranes for immunoblot analysis.
Membranes were incubated with primary antibodies (Suppl. Table 1) overnight and
probed with respective secondary antibodies (Dako-Agilent, Santa Clara, CA, USA) for 1
h. Bands were then developed using enhanced chemiluminescence (SuperSignal West
DURA, Thermo Fisher Scientific, Springfield Township, NJ, USA) and imaged by
ChemiDoc XRS+ (BioRad, Hercules, CA, USA).
Electrophoresis for myosin heavy chains
Frozen soleus muscle strips were homogenized in a lysis buffer containing 0.5M NaCl,
20mM NaPPi, 50mM Tris, 1mM EDTA and 1mM DTT, adjusted pH 6.8 using a Bead
Ruptor 24 Elite bead mill homogenizer (Omni International, Kennesaw, GA, USA).
Supernatants from 10 min centrifugation at 2500 x g were diluted 1:1 with glycerol.
Samples are then suspended in 1:1 in a Laemmli sample buffer and incubate at room
temperature for 10 min and then heated (70°C) for 10 min. Myosin heavy chains were
36 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
separated by SDS-PAGE in a 6% acrylamide gel containing 30% glycerol over 24 h.
Bands were visualized by Coomassie Brilliant Blue staining45.
Histopathology
Freshly harvested tissues from 17 w-old mice were immersed in 10% neutralized
formaldehyde for 2 d. Following to a paraffin embedding, sectioned tissues were stained
with Hematoxylin and Eosin by the Laboratory of Comparative Pathology, Memorial Sloan
Kettering Cancer Center, NY, USA. Slides were visualized using an Eclipse Ti microscope
(Nikon, Tokyo, Japan).
Tissue triglyceride concentrations
Lipids were extracted from frozen specimens with a mixture of chloroform/methanol (2:1)
using Folch’s method as previously described7. Concentrations of triglycerides were
assayed enzymatically (FUJIFILM Wako Diagnostics, Mountain View, CA).
Plasma assays
Enzymatic assay kits were used to measure plasma concentrations of triglycerides, free
fatty acids, total cholesterol, free cholesterol, phospholipid (FUJIFILM Wako Diagnostics,
37 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Mountain View, CA), ß-hydroxybutyrate (Stanbio Laboratories, Boerne, TX), aspartate
aminotransferase, alanine aminotransferase (Infinity AST/ALT, Thermo Scientific,
Waltham, MA, USA) and creatine kinase (Stanbio CK Liqui-UV Test, EKF Diagnostics,
Cardiff, UK). Plasma concentrations of insulin were measured by using an ELISA kit
(Crystal Chem, Downers Grove, IL), according to the manufacturer’s protocol.
Rates of fatty acid oxidation
Rates of fatty acid oxidation in muscle tissues were measured by degradation of 14C-
palmitate (American Radiolabeled Chemicals; St. Louis, MO, USA) into 14C acid soluble
14 7,46 metabolites (ASM) and C-labeled CO2 . Briefly, gastrocnemius muscle strips were
collected from 6 h fasted mice. Tissues were kept on ice no longer than 30 min. Muscle
tissues were minced and homogenized in a Dounce homogenizer followed by
centrifugation for 10 min at 420 x g. Supernatants were transferred to microtubes
containing 0.4 μCi 14C-palmitate / 500 μM palmitate conjugated with 0.7 % fatty acid-free
14 BSA and incubated at 37 °C for 30 min. C-labeled CO2 produced by TCA cycle was
captured onto filter paper soaked with 1 M NaOH and 14C-labeled ASM were separated
with 1 M perchloric acid. 14C-labeled CO2 in the filter paper and 14C-labeled ASM in the
supernatant were dissolved in Ecoscint H (National Diagnostics; Atlanta, GA, USA) and
38 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
were counted using a liquid scintillation counter (Beckman Coulter; Danvers, MA).
Fatty acid oxidation in skeletal muscle was also evaluated by applying pulse chase
design47. Soleus muscle strips were freshly dissected and incubated in preoxygenated
HBSS with 10 mM HEPES, Ca and Mg supplemented 2 mM pyruvate, 4 % fatty acid free
BSA and 1 mM palmitic acid adjusted pH 7.4. Isolated soleus muscles were then
transferred into pulse buffer (HBSS with 10 mM HEPES, Ca and Mg supplemented 2 mM
pyruvate, 4% fatty acid free BSA plus 2 uCi of 9,10 3H-palmitate). After washing following
the pulse incubation, the muscle strips with preoxygenated with incubation buffer in the
absence of 3H-palmitate. 3H-palmitate pulsed muscles were then transferred to the tubes
containing chase buffer (buffer: HBSS with 10 mM HEPES, Ca and Mg supplemented 2
14 14 mM pyruvate, 4 % fatty acid free BSA plus 0.5 uCi/ml of C-palmitate) and a CO2 trap.
3 3 H2O produced from the endogenous oxidation of [9,10- H] palmitate was separated from
14 chase buffer with a mixture of chloroform/methanol (2:1). Gaseous CO2 produced from
the exogenous oxidation of [1-14C] palmitate was measured by transferring the chase
14 buffer to 1 M H2SO4 and a paper disc with 1 M NaOH. Liberated CO2 was trapped in the
paper disc. Samples were dissolved in Ecoscint H (National Diagnostics; Atlanta, GA,
USA) and counted using a liquid scintillation counter (Beckman Coulter; Danvers, MA).
39 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Hepatic VLDL triglyceride secretion rates
Following a 12 h fast, the lipoprotein lipase inhibitor Tyloxapol (500 mg/kg of body weight)
(Sigma-Aldrich) was administered through retro-orbital injection.7 Tail tip blood samples
(25 μL) were collected into microtubes before Tyloxapol injection and at regular intervals
for up to 2 hours. Serum triglyceride concentrations were determined using the enzymatic
assay described above. Rates of hepatic triglyceride secretion were calculated from the
time-dependent linear increases in serum triglyceride concentrations.
Lipidomics
Fatty acids, fatty acyl-CoAs and acyl-carnitines were analyzed by LC MS/MS via Multiple
Reaction Monitoring on an ExionLC AC system coupled to triple quadrupole mass
spectrometer QTRAP5500 (ABSciex, Framingham, MA, USA) at the Emory Integrated
Lipidomics Core (Emory University, Atlanta, GA, USA)7. Briefly, quadriceps muscle
samples were homogenized using Bead Ruptor Elite (Omni International, Kennesaw, GA,
USA). Extracted samples were derivatized with 3-Nitrophenylhydrazine for fatty acids and
n-butanol containing 5 % acetyl chloride for carnitines. The resuspended samples were
subjected to LC MS/MS analysis. Data processing, peak determination, and peak area
integration were performed using the MultiQuant 3.0.2 software (AB Sciex, USA).
40 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Polar metabolite profiling
Predefined 216 targeted polar metabolites were analyzed at Proteomics & Metabolomics
Core Facility (Weill Cornell Medicine, New York, NY, USA). Briefly, gastrocnemius muscle
tissues were homogenized in 80 % methanol and lyophilized. Measurements were
normalized by tissue weight as described priviously48. Metabolomics pathway analysis
and clustering analysis were performed using Metabo Analyst ver.4.0
(https://www.metaboanalyst.ca/home.xhtml).
RNA sequencing
RNA sequencing was performed at Genomics Resources Core Facility (Weill Cornell
Medicine, New York, NY, USA). Briefly, RNA samples from liver tissues were extracted by
RNeasy Fibrous Tissue Mini Kit and RNeasy Mini Kit following to the manufacturer’s
instruction (QIAGEN, Venlo, Netherlands). After quality check by electrophoresis, RNA
sequencing was performed at 50 million reads per sample using Nova Seq 6000 (illumina,
San Diego, CA, USA).
Cell culture
C2C12 myoblasts (ATCC #CRL-1772) were maintained in high glucose DMEM with 10 %
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bovine serum and 1 % penicillin-streptomycin. To induce myotubes differentiation, C2C12
myoblasts were plated at 25,000 cells/cm2. Once reaching 100 % confluency, media was
changed to DMEM based differentiation medium with 2 % HS and 1 % penicillin-
streptomycin. Differentiation medium was changed 24 h later and every 48 h thereafter.
Myotubes were transduced with AAV8-Mck-Flag-Them2 or AAV8-Mck-LacZ at 1x105
genome copies after 4 d in differentiation medium. Experiments were conducted on d 6
of differentiation.
Mitochondrial respiration
Cellular metabolic flux was determined by measuring oxygen consumption rate (OCR) in
Hepa1-6 cells (ATCC #CRL-1830) with conditioned medium obtained from C2C12
myotubes reconstituted with AAVs. OCR values were measured using a Seahorse XFe
96 extracellular flux analyzer (Seahorse Bioscience; North Billerica, MA, USA). OCR
values were measured under basal conditions and following exposure to oligomycin,
carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone and antimycin A
respectively. After retrieving the plate from Seahorse instrument, live cell nuclei were
stained with NucRed™ Live 647 ReadyProbes™ Reagent (Thermo Fisher Scientific,
Waltham, MA, USA) following to the manufacturer’s instruction. Live cell nuclei were
42 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
counted in each whole well images which obtained by Spectramax i3x (Molecular Devices,
San Jose, CA, USA). Normalized OCR values were calculated according to the numbers
of live cell nuclei in each well.
Statistical Analyses
Data are presented as mean values with error bars representing SEM. Statistical
significance was determined by using two-tailed unpaired Student’s t-tests when two
groups were compared. Correlations were evaluated by Pearson’s correlation coefficient
analysis. Threshold values were determined by segmental linear regression. Multiple
group comparisons were performed using two-way ANOVA. Differences were considered
significant for P < 0.05 (GraphPad Prism 8, GraphPad Software, La Jolla, CA, USA).
43 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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FIGURE LEGENDS
Figure 1. Them2 is upregulated in skeletal muscle by HFD feeding and its ablation
improves skeletal muscle insulin sensitivity and exercise capacity in HFD-fed mice
Mice were fed a HFD (H) from 5-16 w. (A) Body weights and (B) Lean and fat mass
(determined at 16 w of age). Tolerance tests to glucose (C) were conducted between 16
and 17 w of age. Mice were injected IP with saline or insulin (10mU/g) prior to (D)
measurements of blood glucose concentrations, as well as immunoblot analysis of Akt
phosphorylation in cardiac and gastrocnemius muscle tissues. (E) Expression of Them2
in gastrocnemius muscle (skeletal muscle, SM) and heart (cardiac muscle, CM). (F)
Maximal exercise capacity was analyzed by graded treadmill test (n = 7-15/group). (G)
Cardiac ejection fraction and left ventricle wall thickness were evaluated by
echocardiography before and after isoproterenol osmotic mini pump implantation (†P <
0.05 before vs. after treatment). Chow-fed mice were individually housed in metabolic
cages for the measurement of (H) voluntary running on an exercise wheel, (I) energy
expenditure and (J) respiratory exchange ratio (n = 14/group), *P < 0.05 Them2-/- vs
Them2+/+, †P < 0.05 with running wheel vs without running wheel. Data are mean ± SEM.
Statistical analyses were conducted using Student’s t test or two-way ANOVA with
repeated-measures where appropriate.
51 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 2. Selective deletion of Them2 in skeletal muscle reduces weight gain and
improves glucose homeostasis and insulin sensitivity in HFD-fed mice
Male C57BL/6 mice with (L to R) skeletal muscle deletion of Them2 (S-Them2-/-,
Them2Flox/Flox x Myf5-CreTg/0); cardiac muscle deletion of Them2 (C-Them2-/-,
Them2Flox/Flox x Myh6-CreTg/0); and adipose tissue deletion of Them2 (A-Them2-/-,
Them2Flox/Flox x Adipoq-CreTg/0). (A) Immunoblot analysis of Them2 with HSP90 as a
loading control. Mice were fed a HFD (H) from 5-17 w. (B) Body weights and (C) lean and
fat mass (determined at 16 w). Tolerance tests to (D) glucose and (E) insulin were
conducted between 16 and 17 w of age. (B-E) Data are mean ± SEM; n=6-18/group.
Statistical analyses were conducted using 2-way ANOVA with repeated measures (B, D,
E) or Students t-test (C); *P < 0.05 compared to control.
Figure 3. Increased glucose uptake and improved insulin sensitivity in skeletal
muscle of HFD-fed S-Them2-/- mice
Hyperinsulinemic-euglycemic clamp studies were performed in mice fed a HFD for 12 w.
(A) Time course of glucose infusion rates, with bar plot presenting glucose infusion rates
during the clamp. (B) Glucose uptake rates were measured for whole body,
52 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
gastrocnemius muscle (skeletal muscle, SM), inguinal white adipose tissue (iWAT) and
brown adipose tissue (BAT). (C) Hepatic glucose production rates measured under basal
and clamp conditions. (S-Them2-/-, n = 6; Control, n = 4), (D) Gastrocnemius muscle
samples were collected from mice fed a HFD for 5-17 w, 10 min after i.p. injection with
saline or insulin (10mU/g), and subjected to immunoblot analysis (n = 4 mice/group). **P
< 0.01, S-Them2-/- vs Control. Statistical analyses were conducted using Student’s t test.
Data are mean ± SEM.
Figure 4. Increased oxidation of endogenous fatty acids and associated metabolic
changes in skeletal muscle of S-Them2-/- mice
Fatty acid oxidation rates were assessed according to degradation rates of [14C]-palmitate
14 into acid-soluble metabolites [ C]-labeled (ASM) and CO2 in homogenized
gastrocnemius (skeletal) muscle from chow-fed (A) global Them2-/- and (B) S-Them2-/-
mice (n = 8/group). (C) Fatty acid oxidation rates were measured in isolated soleus
muscle strips from chow-fed mice using a using [3H]-palmitate in a pulse-chase design
that differentiated between endogenous and exogenous fatty acids (n = 10/group). (D-E)
Gastrocnemius samples were collected from chow-fed mice (C) or HFD (H) for 12 w. (D)
Immunoblot analyses and (E) corresponding densitometry of proteins that regulate fatty
53 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
acid metabolism. Skeletal muscle concentrations of (F) fatty acyl-CoAs, (G) acetyl-
carnitine, (H) free fatty acids and (I) short chain fatty acids (n = 6-8/group). (J)
Unsupervised clustering of data obtained by targeted profiling of 216 polar metabolites in
gastrocnemius muscle. (K) Intermediates of the pentose phosphate pathway (n =
8/group). Statistical analyses were conducted using Student’s t test; *P < 0.05, **P <
0.01, ***P < 0.001, S-Them2-/- vs Control. †P < 0.05, ††P < 0.01, †††P < 0.001, chow vs
HFD. Data are mean ± SEM.
Figure 5. Them2 regulated myocellular fatty acid utilization
Cultured C2C12 myotubes were transduced with 1 x 105 GC AAV8- Mck-LacZ or AAV8-
Mck-Them2. (A) Immunoblot analysis of Them2, with GAPDH used to control for unequal
loading. (B) Representative images of transduced C2C12 myotubes. Cells were fixed and
immunostained with anti-myosin antibody (red) to reveal sarcomeres. Cell nuclei were
stained with DAPI (blue). (C) Myotube area, diameter and fusion index were evaluated
using Image J software. Oxygen consumption rates (OCR) of AAV8 transduced C2C12
myotubes were measured by Seahorse XF96e following to the sequential injections of (i)
oligomycin, (ii) FCCP and (iii) rotenone/antimycin A. (E) OCR values were in the presence
of bovine serum albumin (BSA) treated and palmitic acid (PA) plus BSA. (F) Utilization of
54 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
exogenous fatty acids (FA) was determined by subtracting OCR values in the presence
of BSA alone from PA/BSA. (F) OCR values were measured in the presence of the CPT-
1 inhibitor etomoxir or vehicle (DMSO). (G) Utilization of endogenous FA was determined
by subtracting OCR in the presence of etomoxir from vehicle. Data represent combined
results from 3 independent experiments. Bar plots present FA utilization during the
baseline (basal) and the 3 time points following the addition of FCCP (maximal). Statistical
analyses were conducted using Student’s t test; *P < 0.05, **P < 0.01. Data are mean ±
SEM.
Figure 6. Skeletal muscle Them2 expression enables HFD-fed mice to exceed a
threshold value for accelerated hepatic steatosis and metabolic dysfunction
C57BL/6J mice were fed a HFD for 12 w. (A) Body weight distribution (n = 84) and (B) fat
and lean weights. (C) Linear relationship between fat and lean weights. (D-E) Biphasic
relationships between liver weight and (D) body weight and (E) fat weight (n=64).
Threshold value of 41.6 g body weight was determined by segmental linear regression.
(F) Linear relationship between liver weight and lean weight. (G) Biphasic relationship
between hepatic steatosis determines histologically and body weight. (H) Linear
relationship of liver weight to hepatic steatosis. (I) Linear relationship of hepatic
55 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
triglycerides concentrations to hepatic steatosis. (n=64). (J) Plasma concentrations of free
fatty acids, glucose and total cholesterol. (K) Hepatic mRNA expression levels of selected
genes that govern the metabolism of fatty acids and lipid droplets (n = 8/group). Relative
mRNA expression in skeletal muscle (L) of genes that regulate fatty acid utilization and
(M) myokines. (N) Body and liver weights of HFD-fed S-Them2-/- and control mice (n =
10-14/group) relative to the biphasic linear regression derived for HFD-fed C57BL/6J mice
(panel D). Statistical analyses were conducted using Student’s t test; *P < 0.05, **P <
0.01, ***P < 0.001, body weight < 41.6 g vs body weight > 41.6 g. Data are mean ± SEM.
Abbreviations: Pctp: phosphatidylcholine transfer protein, Aox: alternative oxidase, CPT-
1: carnitine palmitoyltransferase I, Fatp: fatty acid transport protein, Fabp: fatty acid
binding protein, Cidea: cell death activator, Acot: acyl-CoA thioesterase, Acsl: acyl-CoA
synthetase long chain family member, Adipoq: adiponectin, Angptl4: angiopoietin-like 4,
Bdnf: brain derived neurotrophic factor, CXCL1: chemokine ligand 1, Epo: erythropoietin,
Fgf21: fibroblast growth factor 21, Fstl1: follistatin-like 1, Igf1: insulin like growth factor 1,
IL-6: interleukin-6, IL-15: interleukin-15, Lif: leukemia inhibitory factor, Mstn: myostatin,
Osm: oncostatin M, Ostn: osteocrin, Pgc1a: Pparg coactivator 1 alpha, Sparc: secreted
protein acidic and rich in cysteine, Vegfa: vascular endothelial growth factor A.
56 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 7. Them2 promotes hepatic steatosis by suppressing VLDL secretion
(L to R) Mice with skeletal muscle (S-Them2-/-) and cardiac muscle (C-Them2-/-) Them2
deletion were fed a HFD for 12 w and then livers were analyzed for (A) hepatic steatosis
by H&E staining (bars represent 100µm) and (B) hepatic triglyceride concentrations. (C)
Pyruvate tolerance tests conducted in HFD-fed mice at 16 w of age. Data are mean ±
SEM (n=6-12/group). Statistical analyses were conducted by Students t-test (B) or 2-way
ANOVA with repeated measures (C); *P < 0.05 vs control. Data are presented as mean
± SEM (D) Hepatic triglyceride concentrations of HFD-fed S-Them2-/- and control mice
with body weights of less than 41.6 g (n = 3-7/group). (E) Fatty acid oxidation rates in
livers of HFD-fed S-Them2-/- mice (n = 8/group). (F) Rates of triglyceride hepatic secretion
in HFD-fed mice (n = 8-10/group). Statistical analyses were conducted using Student’s t
test or two-way ANOVA with repeated-measures where appropriate. *P < 0.05, S-Them2-
/- vs Control. Data are mean ± SEM.
Figure 8. Skeletal muscle Them2 mediates crosstalk between muscle and the liver
(A) Rates of oxygen consumption (OCR) of Hepa1-6 cells cultured with conditioned
medium. Sequential additions of oligomycin (i), FCCP (ii) and rotenone/antimycin A (iii)
57 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
are indicated. Conditioned medium was collected from C2C12 myotubes reconstituted
with AAV-LacZ or AAV-Them2. Data represent combined results from 3 independent
experiments. (B) Relative mRNA expression of myokine genes in soleus (skeletal) muscle
dissected from mice fed chow diet or a HFD for 12 w. Myokine genes were also analyzed
in chow-fed mice after 18 h fasting. Heat map represents relative values to controls for
the same diet. (C) Relative mRNA expression of myokine genes in body weight matched
(<41.6g) mice fed HFD for 12 w (n = 5-7/ group). *P < 0.05, S-Them2-/- vs body weight
matched (<41.6 g) control. (D) Schematic summary of metabolic role of Them2 in skeletal
muscle and its influence on the development of hepatic steatosis. Them2 in skeletal
muscle decreases dependence on fatty acid oxidation to produce ATP, therefore utilizing
less circulating free fatty acids. Them2 limits fatty acid oxidation in skeletal muscle
consequently altering the production of short chain fatty acids, branched chain amino
acids and pentose phosphate pathway. These alterations are most evident in the setting
of over nutrition and limit insulin sensitivity in skeletal muscle. Them2 also regulates
muscle-to-organ crosstalk by multiple myokine genes depending on dietary condition,
therefore Them2 may coordinate metabolism of fatty acids in skeletal muscle, adipose
tissue and the liver. Statistical analyses were conducted using Student’s t test (B,C) or
two-way ANOVA with repeated-measures (A). Data are mean ± SEM.
58 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Supplemental Figure 1. Ablation of Them2 does not promote muscle atrophy or
hypertrophy
(A) Weights of cardiac and gastrocnemius (skeletal) muscle from mice fed chow (C) or
HFD (H) for 12 w (n = 15-17/group, C57BL6J control mice; Them2+/+, global Them2
knockout mice; Them2-/-). (B) Histology of skeletal (gastrocnemius) muscle (SM) and
cardiac muscle (CM). (C) Relative mRNA expression of myosin heavy chain genes in
soleus muscle. (D) Relative mRNA expression of myosin heavy chain and cardiac
hypertrophy genes in ventricle (n = 8/group). (E) Myosin heavy chain isoforms in soleus
muscle determined by electrophoresis separation (n = 8/group, *P < 0.05, Them2-/- vs
Them2+/+). (F) Blood lactate concentrations were measured before (sedentary) and 30
min after treadmill running (exercised mice) (n =8/group). Statistical analyses were
conducted using Student’s t test. Data are mean ± SEM. Abbreviations: Myh, myosin
heavy chain; Anf, atrial natriuretic factor; aSka, α-skeletal actin; Bnp, brain natriuretic
peptide.
59 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Supplemental Figure 2. Effect of skeletal muscle Them2 on the gene expression
of Acot and Acsl genes
(A) Relative mRNA expression levels of Acot and Acsl genes in skeletal muscle of chow-
fed mice (n = 8/group). (B) Relative mRNA expression of Acot and Acsl genes in skeletal
muscle of HFD-fed mice (n = 8/group). Heat maps represent relative gene expression
compared to C57BL6 for Them2-/- and floxed control for S-Them2-/-.
Supplemental Figure 3. Skeletal muscle expression of Them2 mediates excess
weight gain in mice fed HFD
(A) Body weights of HFD-fed mice housed at 30 °C (n=6-7/group). *P < 0.05 vs control.
(B) Body composition of mice fed a HFD for 12 w while housed at 30 °C (n=6-7/group).
Statistical comparisons were conducted using Student’s t-test; *P < 0.05 vs control. (C)
Immunoblot analysis of Them2 in gastrocnemius muscle with GAPDH as a control for
unequal loading. (D) Body weights of HFD-fed Them2-/- mice following treatment with AAV
carrying Them2 or LacZ as a control (n=7/group). P < 0.05; AAV-LacZ vs AAV-Them2. (E)
Body composition of HFD-fed mice. Mice were maintained at 30 ℃ (n=7/group). *P <
0.05; AAV-LacZ vs AAV-Them2. Statistical analyses were conducted using Student’s t test
or two-way ANOVA with repeated-measures where appropriate. Data are mean ± SEM.
60 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Supplemental Figure 4. Effect of skeletal muscle Them2 expression on the
expression of genes that control lipid and glucose homeostasis
Mice were (A,B) fed chow or (C,D) HFD for 12 w. Relative mRNA expression of genes in
skeletal muscle that (A,C) regulate fatty acid uptake and oxidation and (B,D) lipid and
glucose metabolism (n = 8/group). Statistical analyses were conducted using Student’s t
test; *P < 0.05, **P < 0.01 vs control. Data are mean ± SEM.
Supplemental Figure 5. Influence of Them2 expression in skeletal muscle on
molecular species of acyl-CoAs, acyl-carnitines and free fatty acids.
Mice were fed chow (C) or a HFD (H) for 12 w. Molecular species of (A) fatty acyl-CoAs,
(B) fatty acyl-carnitines, and (C) saturated and unsaturated long chain and short chain
free fatty acids. (n = 8/ group). Statistical analyses were conducted using Student’s t test;
*P < 0.05, **P < 0.01, ***P < 0.001 vs control. Data are mean ± SEM.
Supplemental Figure 6. Pathway analysis of metabolomic data illustrating the
influence of Them2 expression in skeletal muscle
Mice were fed (A) chow or (B) a HFD for 12 w after which gastrocnemius muscles were
subjected to targeted profiling of 216 polar metabolites. Data were analyzed by
61 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
MetaboAnalyst. Each node contains all the matched pathways arranged by p values (from
pathway enrichment analysis) on vertical-axis, and pathway impact values (from pathway
topology analysis) on horizontal-axis.
The node color is based on its p value and the node radius is determined based on their
pathway impact values.
Supplemental Figure 7. Effect of skeletal muscle Them2 expression on amino acids
contents
(A,B) Relative concentrations of amino acids in gastrocnemius muscle. (n = 8/group)
Statistical analyses were conducted using Student’s t test; *P < 0.05, **P < 0.01, ***P <
0.001 vs Control. Data are mean ± SEM.
Supplemental Figure 8. Effect of skeletal muscle Them2 expression on measures
of oxidative stress
Mice were fed chow or a HFD for 12 w. (A) Superoxide dismutase (SOD) activities were
assessed in skeletal muscle (n = 6/group). (B) Tissue-specific lipid peroxidation level was
analyzed using a thiobarbituric acid reactive substances (TBARS) assay in skeletal
62 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
muscle (n = 7/group). Statistical analyses were conducted using Student’s t test. Data are
mean ± SEM
Supplemental Figure 9. Plasma characteristics of S-Them2-/- mice
Mice were fed chow or a HFD for 12 w and the plasma was collected for (A) Activities of
aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine kinase
(CK) (n = 9 – 14/group). (B) Fasting concentrations plasma insulin (n = 8/group). (C)
Fasting concentrations of ß-hydroxybutyrate (b-HB/group) (n = 16). (D) Plasma
concentrations of triglycerides (TG), non-esterified fatty acid (NEFA), total cholesterol (T-
Cho), free cholesterol (F-Cho) and phospholipids (PL) (n = 8 – 10/group). Statistical
analyses were conducted using Student’s t test; *P < 0.05, **P < 0.01 vs control. †P<0.05,
†† P<0.01, ††† P<0.001 chow vs HFD. Data are mean ± SEM.
Supplemental Figure 10. Influence of skeletal muscle Them2 expression on hepatic
mRNA expression
(A) Volcano plot of RNAseq data comparing liver of control and S-Them2-/- mice fed a
HFD for 12 w. Red font represents significant changes in gene expression after
adjustment for false discovery rate. The top ten genes are annotated. (B) Identification of
63 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
top 10 pathways influenced by Them2 expression as assessed by Ingenuity Pathway
Analysis (Qiagen) of RNAseq data. Bar color indicates predicted directionally. Z-scores,
which represent directionality were calculated based on the data set’s correlation with the
activated state. White indicates z-score = 0 (no activation) and gray indicates no activity
pattern.
64 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Supplemental Table 1
Protein Source Reference number ACC Cell Signaling Technology (Danvers, MA, USA) 3661 Akt Cell Signaling Technology 9272 alphaGAPDH Novus Biologicals (Centennial, CL, USA) NB100-56875 AMPK Cell Signaling Technology 2532 CD36 Santa Cruz Biotechnology (Dallas, TX, USA) 7309 CPT-1 Proteintech (Rosemont, IL, USA) 15184-1-AP HSP-90 Santa Cruz Biotechnology sc-25840 Phospho-ACC (Ser79) Cell Signaling Technology 3662 Phospho-Akt (Ser473) Cell Signaling Technology 9271 Phospho-Akt (Thr308) Cell Signaling Technology 4056 Phospho-AMPK (Thr172) Cell Signaling Technology 2535 PCTP Home Shoda et al., 2001, Hepatol 33,1194-1205 Them2 Home Kanno et al., 2007, J Biol Chem 282, 30728-30736
65 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Supplemental Table 2 Gene Forward primer (5' - 3') Rverse primer (5' - 3') Acot1 CTGGCGCATGCAGGATC GGCACTTTTCTTGGATAGCTCC Acot2 CTGAAGTCAACGACGCAAAA CGGCGGAGGTACAAACAG Acot3 GCTCAGTCACCCTCAGGTAA AAGTTTCCGCCGATGTTGGA Acot4 TTGAAGAAGCAGTGCGGTACA AAAGACCCAGAAGCCCAATGT Acot5 TCAGGATGGTGCCAACAGTA GATGCTCAGCGGTTCGTC Acot6 ACGCCATCCTCAGGTGAAAG TGTCTCCAAAGGTGCTGATCTGTGCC Acot7 TGACCAATAAAGCCACCTTGTG CCTGCTCCTGCCGTAAATACAC Acot8 GATGCTGTACGAGTGTGAGAGC ACAGCAAGGACCCCATCC Acot9/10 TGCTACATGCACAACCACAA AAGGTTGCATCCAAAACAGG Acot11 (Them1) AGATCATGGCTTGGATGGAG AAAGGCGTTATTCACGATGG Acot12 GGAGATTACCACCACCTTGG TTCAACCTTAACAGATATGGCATC Acot13 (Them2) TCGACCATGGCTCTAATGTG TCCTGTGGTCTTGTTGGTCA Acsl1 TGGGGTGGAAATCATCAGCC CACAGCATTACACACTGTACAACGG Acsl3 CAATTACAGAAGTGTGGGACT CACCTTCCTCCCAGTTCTTT Acsl4 TGCCAAAAGTGACCAGTCCTATG TGTTACCAAACCAGTCTCGGGG Acsl5 TCCCAGCCCACCTCTGATGATGTG ACAAACTGTCCCGCCGAATG Acsl6 TGAATGCACAGCTGGGTGTA ATGTGGTTGCAGGGCAGAG
Myh1 (Type IIx) TCTGCAGACGGAGTCAGGT TTGAGTGAATGCCTGTTTGC Myh2 (Type IIa) GTCAAAGGAGGAGGAACAGCAGC GTTGAGTGAATGCTTGCTTCCCC Myh4 (Type IIb) TGGCCGAGCAAGAGCTAC TTGATGAGGCTGGTGTTCTG Myh6 (MHC-α) GCCCAGTACCTCCGAAAGTC GCCTTAACATACTCCTCCTTGTC Myh7 (Type I, MHC-β) CCAAGGGCCTGAATGAGGAG GCAAAGGCTCCAGGTCTGAG Anf CCATATTGGAGCAAATCCTGTG CGGCATCTTCTCCTCCAGGT aSKa TTGACGTGTACATAGATTGACTCGTTT TGGCTGGCTTTAATGCTTCA Bnp GGGAGAACACGGCATCATTG ACAGCACCTTCAGGAGATCCA
Adipoq TGTTCCTCTTAATCCTGCCCA CCAACCTGCACAAGTTCCCTT Angptl4 CATCCTGGGACGAGATGAACT TGACAAGCGTTACCACAGGC Bdnf TCATACTTCGGTTGCATGAAGG AGACCTCTCGAACCTGCCC Cxcl1 ACGAAATGCGAAATCATGTGC CTGTGTCGTCTCCAGGACAA Epo ACTCTCCTTGCTACTGATTCCT ATCGTGACATTTTCTGCCTCC Fgf21 CTGCTGGGGGTCTACCAAG CTGCGCCTACCACTGTTCC Fstl1 CACGGCGAGGAGGAACCTA TCTTGCCATTACTGCCACACA Igf-1 GTGAGCCAAAGACACACCCA ACCTCTGATTTTCCGAGTTGC IL-6 TAGTCCTTCCTACCCCAATTTCC TTGGTCCTTAGCCACTCCTTC IL-15 ACATCCATCTCGTGCTACTTGT GCCTCTGTTTTAGGGAGACCT Irisin TTGCCATCTCTCAGCAGAAGA GGCCTGCACATGGACGATA Lif ATTGTGCCCTTACTGCTGCTG GCCAGTTGATTCTTGATCTGGT Mstn AGTGGATCTAAATGAGGGCAGT GTTTCCAGGCGCAGCTTAC Osm CCCGGCACAATATCCTCGG TCTGGTGTTGTAGTGGACCGT Ostn CGTCTTGATGATCTGGTGTCC TGGGAATACCAAACCGCTTTT Sparc GTGGAAATGGGAGAATTTGAGGA CTCACACACCTTGCCATGTTT Vegfa GCACATAGAGAGAATGAGCTTCC CTCCGCTCTGAACAAGGCT
CD36 CAACCACTGTTTCTGCACTG CAGGCTTTCCTTCTTTGCAC Fatp1 CGCTTTCTGCGTATCGTCTGCAAG AAGATGCACGGGATCGTGTCT Fabp3 CACAGAGATCAACTTTCAGCTGGG CCATGAGTGAGAGTCAGGATGAG Aox TCAACAGCCCACTGTGACTTCCATTA TCAGGTAGCATTATCCATCTCTCA Cpt1 TTTGACTTTGAGAAATACCCTGACTA TGGATGAAATTCTCTCCCACAATAA Cpt2 CAGTGCACAGAAGCCTCTCTTG CTTCCCAATGCCGTTCTCAA Acc GAGAGGGCTCAAGTCCTTCC ACATCCACTTCCACACACGA Acadl CTCCGCCCGATGTTCTCATTC CTCTACTCACTTCTCCAGCTTTC
Pctp CCAGAGTATCTCGGCACCTC ACGCTTTCACCATGTCCTTC Pgc1a CAACATGCTCAAGCCAAACCAACA CGCTCAATAGTCTTGTTCTCAAATGGG Ppara GTGGCTGCTATAATTTGCTGTG GAAGGTGTCATCTGGATGGTT Pparg CACAATGCCATCAGGTTTGG GCTGGTCGATATCACTGGAGATC Ppard TCCATCGTCAACAAAGACGGG ACTTGGGCTCAATGATGTCAC Ucp2 TGCCTTCCTTTCTCCGCTTGG GAAAGGTGCCTCCCGAGATTGGTAG Ucp3 CCGATACATGAACGCTCCCCTA ATCATCACGTTCCAAGCTCCC Pdk4 AGGGAGGTCGAGCTGTTCTC GGAGTGTTCACTAAGCGGTCA Glut4 TCGTCATTGGCATTCTGGTTG AGCTCGTTCTACTAAGAGCAC Dgat1 GGCCCAAGGTAGAAGAGGAC GATCAGCATCACCACACACC Dgat2 AGGATCTGCCCTGTCACG GCCAGCCAGGTGAAGTAGAG
CyclophilinA GGCCGATGACGAGCCC TGTCTTTGGAACTTTGTCTGCAA
66
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B C
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A
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Fig 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B
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Fig 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B C D
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Fig 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B
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Fig 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B C D E
40 25 Median 42.5g Fat < 41.6 g 10 41.6 Lean 3 > 41.6 g 3
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F . A n P S V 35 40 45
A 1 4 Body weight, g Fig 6 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A
+/+
-/-
B
g /
g 150 250
m Control Control
,
s -/- 200 -/-
e S-Them2 C-Them2
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r 100
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Fig 7 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A 450 B i ii iii
) Adipoq Adipoq Adipoq
s l
l * Angptl4 Angptl4 Angptl4
e c 400 Bdnf Bdnf Bdnf 0 1.5
0 Cxcl1 Cxcl1 Cxcl1 0
, * * 5
/ Epo Epo Epo n
i 350 Fgf21 Fgf21 Fgf21 m
/ *
l Fstl1 Fstl1 Fstl1 o
m Igf1 Igf1 Igf1 p
( * * IL-6 IL-6 IL-6 R 200 *
C IL-15 IL-15 IL-15 1.0 O Irisin Ir*isin Irisin Baseline Maximal * 100 Lif Lif Lif 0 20 40 60 80 Mstn Mstn Mstn Time (minutes) Osm Osm Osm Ostn Ostn O*stn 2%HS DMEM Sparc Sp*arc Sp*arc * LacZ conditioned Vegfa Vegfa Vegfa 0.5 Them2 conditioned Chow Fasting High-fat
C Control < 41.6 g
S-Them2-/- < 41.6 g
n
o
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s
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Fig 8 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B C D
%
,
n s -/- o
250 +/+ i 3
+/+ Them2 Them2
s m
Them2 r s
g 80 o
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,
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e T T T
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l
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R R f y d 6 7 n a p r e h h k n ta s y y A S B n i M M a e rc d e e x S E
Fig S1 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B Chow High-fat 2.0 2.0 Acot1 Acot1 Acot2 Acot2 Acot3 Acot3 Acot4 Acot4
Acot5 1.5 Acot5 1.5
t t
Acot6 o Acot6
o
c
c A
A Acot7 Acot7 Acot8 Acot8 Acot9/10 1.0 Acot9/10 1.0 Acot11 Acot11 Acot12 Acot12 Acot13 Acot13
Acsl1 0.5 Acsl1 0.5 l
l Acsl3 Acsl3
s
s c
c Acsl4 Acsl4 A A Acsl5 Acsl5 Acsl6 Acsl6 0 0 Them2-/- S-Them2-/- Them2-/- S-Them2-/-
Fig S2 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B 50 30 * Control
* 25 -/- g
40 S-Them2
,
t
g
h 20
,
g
s
i s
e 30 15
a
w
M y
d 10 o 20 Control B 5 S-Them2-/- 10 0 6 8 10 12 14 16 Lean Fat Age, w
C D E
50
* 30 * AAV-LacZ g
25 AAV-Them2 ,
t 40
h g g *
i 20
,
e
s w 30 s
15
a
y
d M
o 10
B 20 AAV-LacZ AAV-Them2 5 10 0 6 8 10 12 14 16 Lean Fat Age, w
Fig S3 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B
Fatty acid uptake and oxidation Lipid and glucose homeostasis
n
n
o
o i
i 2.5 2.5 s
s Control
s
s
e
e r
2.0 -/- r 2.0 p
S-Them2 p
x
x e
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1.5 1.5 *
A
A
N
N R
1.0 R 1.0
m
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0.5 i 0.5
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0.0 e 0.0
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C D
Fatty acid uptake and oxidation Lipid and glucose homeostasis
n
n
o
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x
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Fig S4 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B
2.0 2.0
Control
e
e
u u
-/- ,
s s 1.5 S-Them2 e
, 1.5
s
s
n
i
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A g
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n
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0.0 0.0 C H C H C H C H C H C H C H C H C H l l l l l y y y y y c c c c n r o o o i i ti i o ty n n n le le i n i a a a o m o p u x c c O n l id o B e e e i a h r d L P c P H D a a x r e A H C D Saturated Unsaturated
80 , , 150 ] ***
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s
s
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F [ ** ** [ 0 0 C H C H C H C H C H C H C H C H c c c c ic ic ic ic i ti i i le le in n r s it r o l o u ri a O n o id a y lm te i in h L a S L L c M P ra A
Fig S5 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B
Fig S6 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B
Chow High-fat
L-Histidine * Control L-Histidine -/- * L-Arginine * S-Them2 L-Arginine L-Lysine * L-Lysine
c L-Glutamic acid i
l L-Glutamic acid
c
i
i
l
i
h p
L-Aspartic acid h L-Aspartic acid
o
p
r
o d
L-Glutamine r y
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y H L-Asparagine ** H L-Asparagine L-Tyrosine L-Tyrosine L-Threonine L-Threonine L-Serine L-Serine L-Proline ** L-Proline L-Tryptophan L-Tryptophan L-Phenylalanine
** L-Phenylalanine c
i L-Methionine
b
c
n
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o L-Methionine
a
b
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h
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a
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L-Valine n B
L-Alanine a r
L-Alanine B Glycine * Glycine 0.0 0.5 1.0 1.5 2.0 Relative to Control 0.0 0.5 1.0 1.5 2.0 Relative ratio to Control
Fig S7 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B
100 4 n
Control i
e
t
o ,
80 -/- r y
p 3
n S-Them2
t
i
o
i
g
v
t
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/
b
c
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μ
n
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% S R 1
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Fig S8 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B C
150
L 1.0 L
Control /
l
/ l
o †
-/- 3 o
L
m /
† S-Them2 ††† m 0.8
m
U
m
100 †††
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,
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AST ALT CK
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Fig S9 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440732; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B
10 Canonical pathways 8 Cidea Triacylglycerol Degradation ) Hsp90aa1
e α-tocopherol Degradation
u l
a PXR/RXR Activation v
4 Aqp8 Gtpbp4-ps1 Heparan Sulfate Biosynthesis P
( B430212C06RikLipe Adh6-ps1 RP23-306P12.3 Basal Cell Carcinoma Signaling
0 Fam83f Avpr1a
1 Heparan Sulfate Biosynthesis
g Osteoblasts, Osteoclasts and Chondrocytes
o 2 l
Apelin Adipocyte Signaling Pathway - LPS/IL-1 Mediated Inhibition of RXR Function 0 Prostate Cancer Signaling
-2 -1 0 1 2 0.0 0.5 1.0 ld 1.5 2.0 2.5 0 5 o . . h 4 3 h - - s -log(p-value) re h log2 (fold change) T
Fig S10