Zhongxiao Wan

Evidence for the role of AMPK in regulating PGC-1 alpha expression and mitochondrial proteins in mouse epididymal adipose tissue

PGC‐1α is a transcriptional co‐activator and master regulator of mitochondrial biogenesis. While extensively studied in skeletal and cardiac muscle, recent findings suggest that white adipose tissue PGC‐1α plays an important role in regulating glucose homeostasis. The purpose of the present investigation was to evaluate the role of AMPK in regulating PGC‐1α and mitochondrial enzymes in mouse epididymal and inguinal subcutaneous adipose tissue.

IL-6 indirectly modulates the induction of glyceroneogenic enzymes in adipose tissue during exercise.

Background Glyceroneogenesis is an important step in the control of fatty acid re-esterification with PEPCK and PDK4 being identified as key enzymes in this process. We have previously shown that glyceroneogenic enzymes such as PDK4 are rapidly induced in white adipose tissue during exercise. Recent studies have suggested that IL-6 regulates adipose tissue metabolism and gene expression during exercise. Interestingly, IL-6 has been reported to directly decrease PEPCK expression. The purpose of this investigation was to determine the role of IL-6 in modulating the effects of exercise on the expression of glyceroneogenic enzymes in mouse adipose tissue. We hypothesized that the exercise-mediated induction of PDK4 and PEPCK would be greater in adipose tissue from IL-6 deficient mice compared to wild type controls. Methodology and Principle Findings Treatment of cultured epididymal adipose tissue (eWAT) with IL-6 (150 ng/ml) increased the phosphorylation of AMPK, ACC and STAT3 and induced SOCS3 mRNA levels while decreasing PEPCK and PDK4 mRNA. AICAR decreased the expression of PDK4 and PEPCK. The activation of AMPK by IL-6 was independent of increases in lipolysis. An acute bout of treadmill running (15 meters/minute, 5% incline, 90 minutes) did not induce SOCS3 or increase phosphorylation of STAT3 in eWAT, indicating that IL-6 signalling was not activated. Exercise-induced increases in PEPCK and PDK4 mRNA expression were attenuated in eWAT from IL-6−/− mice in parallel with a greater relative increase in AMPK phosphorylation compared to exercised WT mice. These changes occurred independent of alterations in beta-adrenergic signalling in adipose tissue from IL-6−/− mice. Conclusions and Significance Our findings question the role of IL-6 signalling in adipose tissue during exercise and suggest an indirect effect of this cytokine in the regulation of adipose tissue gene expression during exercise.

Reductions in RIP140 are not required for exercise- and AICAR-mediated increases in skeletal muscle mitochondrial content.

Receptor interacting protein 1 (RIP140) has recently been demonstrated to be a key player in the regulation of skeletal muscle mitochondrial content. We have shown that β-guanadinopropionic acid (β-GPA) feeding reduces RIP140 protein content and mRNA levels concomitant with increases in mitochondrial content (Williams DB, Sutherland LN, Bomhof MR, Basaraba SA, Thrush AB, Dyck DJ, Field CJ, Wright DC. Am J Physiol Endocrinol Metab 296: E1400-E1408, 2009). Since β-GPA feeding reduces high-energy phosphate levels and activates AMPK, alterations reminiscent of exercise, we hypothesized that exercise training would reduce RIP140 protein content. We further postulated that an acute bout of exercise, or interventions known to induce the expression of mitochondrial enzymes or genes involved in mitochondrial biogenesis, would result in decreases in nuclear RIP140 content. Two weeks of daily swim training increased markers of mitochondrial content in rat skeletal muscle independent of reductions in RIP140 protein. Similarly, high-intensity exercise training in humans failed to reduce RIP140 content despite increasing skeletal muscle mitochondrial enzymes. We found that 6 wk of daily 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) injections had no effect on RIP140 protein content in rat skeletal muscle while RIP140 content from LKB1 knockout mice was unaltered despite reductions in mitochondria. An acute bout of exercise, AICAR treatment, and epinephrine injections increased the mRNA levels of PGC-1α, COXIV, and lipin1 independent of decreases in nuclear RIP140 protein. Surprisingly these interventions increased RIP140 mRNA expression. In conclusion our results demonstrate that decreases in RIP140 protein content are not required for exercise and AMPK-dependent increases in skeletal muscle mitochondrial content, nor do acute perturbations alter the cellular localization of RIP140 in parallel with the induction of genes involved in mitochondrial biogenesis.

Epinephrine induces PDK4 mRNA expression in adipose tissue from obese, insulin resistant rats.

Thiazolidinediones (TZDs) are a commonly prescribed class of insulin sensitizing drugs that increase fatty acid re‐esterification, in part through the induction of pyruvate dehydrogenase kinase 4 (PDK4). Owing to the deleterious side effects of TZDs the identification of alternative approaches with which to increase PDK4 is essential. We recently demonstrated that epinephrine increases PDK4 expression through p38 and peroxisome proliferator‐activated receptor γ (PPARγ) dependent pathways in cultured adipose tissue from lean rats. The purpose of this study was to determine whether acute epinephrine treatment, in vivo, can induce PDK4 mRNA expression in adipose tissue from obese, insulin resistant rats and if the reputed signaling pathways mediating this effect are intact. To this end we fed male Wistar rats a chow or high‐fat diet (HFD, 60% kcals from fat) for 6 weeks. Rats were then injected with a weight‐adjusted bolus of epinephrine and tissue harvested. Despite a blunted activation of p38 epinephrine increased PDK4 mRNA expression to a similar extent in adipose tissue from chow and HFD rats. 5′AMP‐activated protein kinase (AMPK) signaling was not altered by the HFD. Similar to epinephrine, 2 h of swim exercise, an intervention that increases plasma catecholamines, also increased PDK4 mRNA levels to a similar extent in adipose tissue from both lean and HFD rats. Collectively these findings demonstrate, for the first time, that acute elevations in catecholamines induce PDK4 in adipose tissue from HFD rats, that this effect is likely independent of p38, a reputed mediator of PDK4 expression and that exercise, similar to TZDs can induce PDK4 in adipose tissue from obese, insulin resistant rats.

IL-6 Is Not Necessary for the Regulation of Adipose Tissue Mitochondrial Content

Background Adipose tissue mitochondria have been implicated as key mediators of systemic metabolism. We have shown that IL-6 activates AMPK, a mediator of mitochondrial biogenesis, in adipose tissue; however, IL-6−/− mice fed a high fat diet have been reported to develop insulin resistance. These findings suggest that IL-6 may control adipose tissue mitochondrial content in vivo, and that reductions in adipose tissue mitochondria may be causally linked to the development of insulin resistance in IL-6−/− mice fed a high fat diet. On the other hand, IL-6 has been implicated as a negative regulator of insulin action. Given these discrepancies the purpose of the present investigation was to further evaluate the relationship between IL-6, adipose tissue mitochondrial content and whole body insulin action. Methodology and Principal Findings In cultured epididymal mouse adipose tissue IL-6 (75 ng/ml) induced the expression of the transcriptional co-activators PGC-1α and PRC, reputed mediators of mitochondrial biogenesis. Similarly, IL-6 increased the expression of COXIV and CPT-1. These effects were absent in cultured subcutaneous adipose tissue and were associated with lower levels of GP130 and IL-6 receptor alpha protein content. Markers of mitochondrial content were intact in adipose tissue from chow fed IL-6−/− mice. When fed a high fat diet IL-6−/− mice were more glucose and insulin intolerant than controls fed the same diet; however this was not explained by decreases in adipose tissue mitochondrial content or respiration. Conclusions and Significance Our findings demonstrate depot-specific differences in the ability of IL-6 to induce PGC-1α and mitochondrial enzymes and demonstrate that IL-6 is not necessary for the maintenance of adipose tissue mitochondrial content in vivo. Moreover, reductions in adipose tissue mitochondria do not explain the greater insulin resistance in IL-6−/− mice fed a high fat diet. These results question the role of adipose tissue mitochondrial dysfunction in the etiology of insulin resistance.

Dietary supplementation with vitamin E and C attenuates dexamethasone-induced glucose intolerance in rats

Glucocorticoid excess induces marked insulin resistance and glucose intolerance. A recent study has shown that antioxidants prevent dexamethasone (DEX)-induced insulin resistance in cultured adipocytes. The purpose of this investigation was to examine the effects of dietary vitamin E and C (Vit E/C) supplementation on DEX-induced glucose intolerance in rats. We hypothesized that feeding rats a diet supplemented with Vit E/C would improve glucose tolerance and restore insulin signaling in skeletal muscle, adipose, and liver and prevent alterations in AMPK signaling in these tissues. Male Wistar rats received either a control or Vit E/C-supplemented diet (0.5 g/kg diet each of L-ascorbate and DL-all rac-alpha-tocopherol) for 9 days prior to, and during, 5 days of daily DEX treatment (subcutaneous injections 0.8 mg/g body wt). DEX treatment resulted in increases in the glucose and insulin area under the curve (AUC) during an intraperitoneal glucose tolerance test. The glucose, but not insulin, AUC was lowered with Vit E/C supplementation. Improvements in glucose tolerance occurred independent of a restoration of PKB phosphorylation in tissues of rats stimulated with an intraperitoneal injection of insulin but were associated with increases in AMPK signaling in muscle and reductions in AMPK signaling and the expression of fatty acid oxidation enzymes in liver. There were no differences in mitochondrial enzymes in triceps muscles between groups. This study is the first to report that dietary Vit E/C supplementation can partially prevent DEX-induced glucose intolerance in rats.

Epinephrine and AICAR-induced PGC-1α mRNA expression is intact in skeletal muscle from rats fed a high-fat diet

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis and is controlled, at least in part, through AMP-activated protein kinase and p38-dependent pathways. There is evidence demonstrating that activation of these kinases and induction of PGC-1α in skeletal muscle are regulated by catecholamines. The purpose of the present study was to determine if consumption of a high-fat diet (HFD) impairs epinephrine and 5-aminoimidazole-4-carboxamide-1β-d-ribofuranoside (AICAR) signaling and induction of PGC-1α in rat skeletal muscle. Male Wistar rats were fed chow or a HFD for 6 wk and then given a weight-adjusted bolus injection of epinephrine (20, 10, or 5 μg/100 g body wt sc) or saline, and triceps muscles were harvested 30 min (signaling) or 2 and 4 h (gene expression) postinjection. Despite blunted increases in p38 phosphorylation, the ability of epinephrine to induce PGC-1α was intact in skeletal muscle from HFD-fed rats and was associated with normal increases in activation of PKA and phosphorylation of cAMP response element-binding protein, reputed mediators of PGC-1α expression. The attenuated epinephrine-mediated increase in p38 phosphorylation was independent of increases in MAPK phosphatase 1. At 2 h following AICAR treatment (0.5 g/kg body wt sc), AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation were similar in skeletal muscle from chow- and HFD-fed rats. Surprisingly, AICAR-induced increases in PGC-1α mRNA levels were greater in skeletal muscle from HFD-fed rats. Our results demonstrate that the ability of epinephrine and AICAR to induce PGC-1α remains intact in skeletal muscle from HFD-fed rats. These results question the existence of reduced β-adrenergic responsiveness in diet-induced obesity and demonstrate that increases in p38 phosphorylation are not required for induction of PGC-1α in muscle from obese rats.

FAT/CD36 regulates PEPCK expression in adipose tissue

Fatty acid translocase (FAT)/CD36 has been extensively studied for its role in facilitating fatty acid uptake. Recent findings have also demonstrated that this protein regulates adipocyte lipolysis and may modulate fatty acid reesterification. As FAT/CD36 has been shown to control the expression of genes involved in fatty acid oxidation in adipocytes, we reasoned that this protein might also control the expression of enzymes involved in fatty acid reesterification. In adipose tissue from FAT/CD36 knockout (KO) mice, we found that glycerol and fatty acid release were reduced and this was associated with reductions in adipose triglyceride lipase. Decreases in lipolysis were paralleled by increases in the free fatty acid-to-glycerol ratio and reductions in primary and fractional rates of fatty acid reesterfication in cultured adipose tissue from FAT/CD36 KO mice. Reductions in reesterfication were associated with decreases in the mRNA expression and protein content of phosphoenolpyruvate carboxykinase (PEPCK). To determine if reductions in lipolysis could lead to decreases in PEPCK mRNA expression, we treated cultured mouse adipose tissue with the lipase inhibitor CAY10499 (2 μM) and found that this resulted in an ∼50% reduction in PEPCK mRNA expression. Treatment with hexarelin (10 μM, 12 h), a CD36 agonist, increased PEPCK mRNA expression independent of lipolysis. Collectively, our results provide novel evidence that FAT/CD36 regulates PEPCK in adipose tissue and that this could be secondary to reductions in lipolysis.

Novel effects of rosiglitazone on SMAD2 and SMAD3 signaling in white adipose tissue of diabetic rats

The effects of the proliferator‐activated receptor gamma (PPARγ) agonist rosiglitazone (ROSI) on the transforming growth factor (TGF)‐β/SMAD signaling pathway in white adipose tissue (WAT) of diabetic rats were assessed.