Role of Tyk2 in regulating energy expenditure and preventing obesity

Obesity develops when energy intake exceeds energy expenditure. Defect in the function of brown fat and skeletal muscle, two of the major tissues that contribute towards energy expenditure, lead to the development of obesity and metabolic syndrome. Our previous findings suggest that Tyk2 deficient mice become obese and develop the metabolic syndrome. Tyk2, which is a tyrosine kinase of the JAK-STAT signaling family, is important for optimal brown development and function. Since brown fat and skeletal muscle, both are derived from the Myf5+ lineage of mesenchymal stem cells, we also characterized the role of Tyk2 in the development and function of skeletal muscle. We found that Tyk2 deficient mice do not display a structural defect in skeletal muscle development; however, the function of skeletal muscle is severely impaired in these mice. Expression of troponins, which regulate the muscle contraction and muscle creatine kinase, which regulates the levels of phosphocreatine, a major fuel for skeletal muscle, is downregulated in Tyk2 deficient mice. Skeletal muscle mitochondria also display an abnormal morphology along with decreased respiration capacity, which is a function of decreased activity of complex IV of the electron transport chain. Interestingly, Tyk2 deficient mice also exhibit an increased proportion of fast, glycolytic, Type II fibers in the skeletal muscle. Using an in-vitro system for skeletal muscle differentiation, we found that Tyk2 levels increase during differentiation, suggesting a role for Tyk2 in proper development and function of the skeletal muscle. Our previous studies suggested that a kinase-inactive (Tyk2KD) form of Tyk2 is also efficient in restoring the function of Tyk2 deficient brown fat preadipocytes. We generated transgenic mice that expressed a wild type (Tyk2WT) and kinase inactive (Tyk2 KD) form of tyk2 in brown fat and skeletal muscle under Myf5 cre and in skeletal muscle using MCK cre mice. Expression of Tyk2 using the Myf5 cre (E8.0) reverts the obese and the metabolic phenotype observed in the Tyk2 deficient mice. Interestingly, expressing Tyk2 under MCK cre (E13.0) also reverts the obese phenotype, suggesting that the temporal and spatial expression of Tyk2 is critical in regulating energy expenditure. Our studies also highlight the role of Tyk2, not as a kinase, but as a component of the transcriptional assembly regulating the expression of genes inv

The Signal Transducer and Activator of Transcription 1 (STAT1) Inhibits Mitochondrial Biogenesis in Liver and Fatty Acid Oxidation in Adipocytes

The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT). Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC) and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1-/-mice utilized more lipids we examined white adipose tissue (WAT) stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. β-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA) re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice

Stress-induced dynamic regulation of mitochondrial STAT3 and its association with cyclophilin D reduces mitochondrial ROS production

Signal Transducer and Activator of Transcription 3 (STAT3) has been tied to various physiological and pathological functions, mainly as a transcription factor that translocates to the nucleus upon tyrosine phosphorylation induced by cytokine stimulation. In addition, a small pool of STAT3 resides in the mitochondria where it serves as a sensor for various metabolic stressors including reactive oxygen species (ROS). Mitochondrially-localized STAT3 largely exerts its effects through direct or indirect regulation of the activity of the electron transport chain (ETC). It has been assumed that STAT3 amounts in the mitochondria are static. We showed that various stimuli, including oxidative stress and cytokines, triggered a signaling cascade that resulted in a rapid loss of mitochondrially-localized STAT3. Recovery of the mitochondrial pool of STAT3 over time depended upon phosphorylation of Ser727 in STAT3 and new protein synthesis. Under these conditions, mitochondrially-localized STAT3 also became competent to bind to cyclophilin D (CypD). Binding of STAT3 to CypD was mediated by the N-terminus of STAT3, which was also important for reducing mitochondrial ROS production after oxidative stress. These results outline a role for mitochondrially-localized STAT3 in sensing and responding to external stimuli

Catecholamine-induced lipolysis causes mTOR complex dissociation and inhibits glucose uptake in adipocytes.

Anabolic and catabolic signaling oppose one another in adipose tissue to maintain cellular and organismal homeostasis, but these pathways are often dysregulated in metabolic disorders. Although it has long been established that stimulation of the β-adrenergic receptor inhibits insulin-stimulated glucose uptake in adipocytes, the mechanism has remained unclear. Here we report that β-adrenergic-mediated inhibition of glucose uptake requires lipolysis. We also show that lipolysis suppresses glucose uptake by inhibiting the mammalian target of rapamycin (mTOR) complexes 1 and 2 through complex dissociation. In addition, we show that products of lipolysis inhibit mTOR through complex dissociation in vitro. These findings reveal a previously unrecognized intracellular signaling mechanism whereby lipolysis blocks the phosphoinositide 3-kinase-Akt-mTOR pathway, resulting in decreased glucose uptake. This previously unidentified mechanism of mTOR regulation likely contributes to the development of insulin resistance.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

<i>STAT1</i>^<i>-/-</i> mice display increased mitochondrial biogenesis in liver.

<p>a. Amounts of PGC1α mRNA were measured by qRT-PCR and quantitated relative to tubulin in livers of fed or fasted <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> mice. Values were normalized to <i>STAT1</i>^<i>+/+</i> fed. * Significantly different p< 0.05(<i>STAT1</i>^<i>+/+</i> fed vs. <i>STAT1</i>^<i>+/+</i> fasted and <i>STAT1</i>^<i>+/+</i> fed vs. <i>STAT1</i>^<i>-/-</i> fed) as determined by two-way ANOVA + Holm-Sidak’s post-test, n = 5, 8–10 week old male mice per group. b. Mitochondrial DNA (ND4, ND6, Cytb, Cox1 and ATPase6) in livers of <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> mice was quantitated relative to actin DNA and normalized to <i>STAT1</i>^<i>+/+</i>. * Significantly different p< 0.05, Student’s T-test, correction for multiple testing c. Amount of mitochondrial protein in <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> livers, which was normalized to the weight of the liver. d. Number of mitochondria in <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> livers. n = 3 mice. * Significantly different p < 0.05, Student’s T-test. e. Mitochondrial DNA from <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> hearts. f. Amounts of mitochondrial RNAs in hearts in <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> mice. n = 6 mice per group.</p

<i>STAT1</i>^<i>-/-</i> mice display increased energy expenditure during starvation.

<p>The rates of a. energy expenditure (EE) and respiratory quotient (RER) were measured in mice over a 24 h period. The area under the circadian curves is shown to the right of the each figure. Values are the mean ± SEM, *** P<0.05 as determined by ANOVA with Bonferoni’s correction for multiple testing. b. Body composition of 24 h fasted <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> mice. Values are the mean ± SEM, * represents < 0.05. n = 6 mice per group.</p

Fasted <i>STAT1</i>^<i>-/-</i> mice have defective lipolysis of WAT.

<p>a. <i>STAT1</i>^<i>+/+</i> (left panel) and <i>STAT1</i>^<i>-/-</i> mice (right panel) were fasted and white adipose tissue depots were photographed. The images shown are representative of n = 5, 8–10 week old mice per group. b. Weight of subcutaneous and gonadal fat from <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> 24 h fasted mice. n = 4 8–10 week old male mice per group. c. H&E stains of subcutaneous fat from <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> mice were analyzed for d. size (in microns). The values represent mean size of cells (mm³) ± SEM for n = 6 mice per group. e. Quantification of the number of fat cells per mm³ from H&E stains of subcutaneous fat from <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> mice. * represents p < 0.05, Student’s T-test f. Oil Red O staining of liver from fed or fasted <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> mice. The images shown are representative of n = 4, 8–10 week old male mice per group. g. Liver triglyceride concentrations in <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> mice. The values represent mean ± SEM for n = 9 male 8–10 week old mice per group, * Significantly different p < 0.05, Student’s T-test. h. Glycerol release was measured in mature adipocytes that were isolated from subcutaneous fat and treated with various doses of isoproterenol or forskolin. n = 4, 12 week old male mice per group. The effect of genotype is significant at p<0.001; The genotype-concentration interaction is significant at p<0.05 for isoproterenol and at p<0.01 for forskolin. (<b>I</b>) Western blot and densitometric quantification of isoproterenol and forskolin induced phosphorylation of HSL normalized to total HSL.</p

Total triglycerides are increased in <i>STAT1</i>^<i>-/-</i> WAT.

<p>a. Total and b. newly synthesized triglycerides and c. the percent of the newly synthesized palmitate bound to triglyceride in tissues isolated from <i>STAT1</i>^<i>+/+</i> and <i>STAT1</i>^<i>-/-</i> mice that were treated with ²H₂O. Values are the mean ± SEM, * presents p < 0.05 as measured by Student T-test (compared <i>STAT1</i>^<i>+/+</i> liver tissue to <i>Stat1</i>^<i>-/-</i> liver tissue, <i>STAT1</i>^<i>+/+</i> white adipose tissue to <i>STAT1</i>^<i>-/-</i> white adipose tissue and WT muscle to <i>STAT1</i>^<i>-/-</i> muscle for n = 4 mice per group).</p

<i>STAT1</i>^<i>-/-</i> mice have lower oxygen consumption.

<p>Oxygen consumption was measured from isolated liver mitochondria of male mice either fed or fasted using substrates that donate to specific sites in the ETC a. Glutamate-malate for Complex I, b. Succinate (+rotenone) for complex II, and c. TMPD-ascorbate (+rotenone) for Complex IV. Significance of p < 0.05 was determined with two-way ANOVA + Holm Sidak’s post test, n = 6, 8–10 week old male mice d. Amounts of UCP2 mRNA levels were measured in male mice either fed or fasted using qRT-PCR and quantitated relative to tubulin in livers. Values were normalized to <i>STAT1</i>^<i>+/+</i> fed. Significance of p < 0.05 were determined by two-way ANOVA + Holm Sidak’s post test, n = 4, 8–10 week old male mice.</p