Maternal obesity is thought to increase the offspring's risk of juvenile obesity and metabolic diseases; however, the mechanism(s) whereby excess maternal nutrition affects fetal development remain poorly understood. Here, we investigated in nonhuman primates the effect of chronic high-fat diet (HFD) on the development of fetal metabolic systems. We found that fetal offspring from both lean and obese mothers chronically consuming a HFD had a 3-fold increase in liver triglycerides (TGs). In addition, fetal offspring from HFD-fed mothers (O-HFD) showed increased evidence of hepatic oxidative stress early in the third trimester, consistent with the development of nonalcoholic fatty liver disease (NAFLD). O-HFD animals also exhibited elevated hepatic expression of gluconeogenic enzymes and transcription factors. Furthermore, fetal glycerol levels were 2-fold higher in O-HFD animals than in control fetal offspring and correlated with maternal levels. The increased fetal hepatic TG levels persisted at P180, concurrent with a 2-fold increase in percent body fat. Importantly, reversing the maternal HFD to a low-fat diet during a subsequent pregnancy improved fetal hepatic TG levels and partially normalized gluconeogenic enzyme expression, without changing maternal body weight. These results suggest that a developing fetus is highly vulnerable to excess lipids, independent of maternal diabetes and/or obesity, and that exposure to this may increase the risk of pediatric NAFLD.

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Acetylation has recently emerged as an important mechanism for controlling a broad array of proteins mediating cellular adaptation to metabolic fuels. Acetylation is governed, in part, by SIRTs (sirtuins), class III NAD+-dependent deacetylases that regulate lipid and glucose metabolism in liver during fasting and aging. However, the role of acetylation or SIRTs in pathogenic hepatic fuel metabolism under nutrient excess is unknown. In the present study, we isolated acetylated proteins from total liver proteome and observed 193 preferentially acetylated proteins in mice fed on an HFD (high-fat diet) compared with controls, including 11 proteins not previously identified in acetylation studies. Exposure to the HFD led to hyperacetylation of proteins involved in gluconeogenesis, mitochondrial oxidative metabolism, methionine metabolism, liver injury and the ER (endoplasmic reticulum) stress response. Livers of mice fed on the HFD had reduced SIRT3 activity, a 3-fold decrease in hepatic NAD+ levels and increased mitochondrial protein oxidation. In contrast, neither SIRT1 nor histone acetyltransferase activities were altered, implicating SIRT3 as a dominant factor contributing to the observed phenotype. In Sirt3−/− mice, exposure to the HFD further increased the acetylation status of liver proteins and reduced the activity of respiratory complexes III and IV. This is the first study to identify acetylation patterns in liver proteins of HFD-fed mice. Our results suggest that SIRT3 is an integral regulator of mitochondrial function and its depletion results in hyperacetylation of critical mitochondrial proteins that protect against hepatic lipotoxicity under conditions of nutrient excess.

Skeletal muscle insulin resistance is a key component of the etiology of type 2 diabetes. Caloric restriction (CR) enhances the sensitivity of skeletal muscle to insulin. However, the molecular signals within skeletal muscle linking CR to improved insulin action remain largely unknown. Recently, the mammalian ortholog of Sir2, sirtuin 1 (Sirt1), has been identified as a potential transducer of perturbations in cellular energy flux into subsequent metabolic adaptations, including modulation of skeletal muscle insulin action. Here, we have demonstrated that CR increases Sirt1 deacetylase activity in skeletal muscle in mice, in parallel with enhanced insulin-stimulated phosphoinositide 3-kinase (PI3K) signaling and glucose uptake. These adaptations in skeletal muscle insulin action were completely abrogated in mice lacking Sirt1 deacetylase activity. Mechanistically, Sirt1 was found to be required for the deacetylation and inactivation of the transcription factor Stat3 during CR, which resulted in decreased gene and protein expression of the p55α/p50α subunits of PI3K, thereby promoting more efficient PI3K signaling during insulin stimulation. Thus, these data demonstrate that Sirt1 is an integral signaling node in skeletal muscle linking CR to improved insulin action, primarily via modulation of PI3K signaling.

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1␣ and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1␣ expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1␣ expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1␣ after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1␣. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1␣ or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1␣ activity after exercise.Impaired mitochondrial function has been linked with physical inactivity, insulin resistance, and the pathogenesis of type 2 diabetes (1, 2). Importantly, aerobic exercise training increases mitochondrial biogenesis and oxidative capacity in skeletal muscle (3-7). The precise mechanisms, however, linking muscle contraction to mitochondrial plasticity are incompletely defined (8). Clearly, allosteric factors (e.g. Ca 2ϩ , AMP, NAD ϩ , P i ) that are increased during contraction are important because they activate signaling pathways that ultimately converge at the transcriptional co-activator, peroxisome proliferator-activated receptor-␥ coactivator 1-␣ (PGC-1␣) 2 (9 -12). Once active, PGC-1␣ targets an array of transcription factors and nuclear receptors to coordinate gene expression of nuclear and mitochondrial-encoded genes (13, 14). However, how perturbations in cellular energy stress are sensed during exercise and subsequently how this signal is transduced to regulate mitochondrial biogenesis in a coordinated manner are still under intense investigation.In recent years, lysine acetylation...

Brief calorie restriction (CR; 20 days of 60% of ad libitum [AL] intake) improves insulin-stimulated glucose transport, concomitant with enhanced phosphorylation of Akt2. The purpose of this study was to determine whether Akt2 is essential for the calorie restrictioninduced enhancement in skeletal muscle insulin sensitivity. We measured insulin-stimulated 2-deoxyglucose (2DG) uptake in isolated extensor digitorum longus (EDL) and soleus muscles from male and female wildtype (WT) and Akt2-null (knockout [KO]) mice after ad libitum or calorie-restricted (20 days at 60% of AL) feeding. In WT mice, calorie restriction significantly enhanced insulin-stimulated 2DG uptake in both muscles regardless of sex. However, in KO mice, calorie restriction did not enhance insulin-stimulated 2DG in male or female EDL or in female soleus. Only in male KO soleus did calorie restriction significantly increase insulin-stimulated 2DG through an Akt2-independent mechanism, although 2DG uptake of the KO-CR group was reduced compared with the WT-CR soleus group. Akt2 serine phosphorylation was enhanced approximately two-to threefold in insulin-stimulated WT-CR versus WT-AL muscles. Calorie restriction induced an ϳ1.5-to 2-fold elevation in Akt1 phosphorylation of insulintreated muscles, regardless of genotype, but this increase was insufficient to replace Akt2 for insulinstimulated 2DG in Akt2-deficient muscles. These results indicate that Akt2 is essential for the full effect of brief calorie restriction on insulin-stimulated glucose uptake in skeletal muscle with physiologic insulin. Diabetes 54: 1349 -1356, 2005 M oderate calorie restriction (25-40% reduction below ad libitum intake) is an effective treatment for improving insulin sensitivity in many species (1-4). Improved insulin sensitivity occurs rapidly (manifested within 20 days of calorie restriction) (1,3,4). In skeletal muscle, brief calorie restriction (5-20 days) enhances insulin-stimulated glucose uptake (5), a rate-limiting step in glucose metabolism. This increase is ultimately due to an increase in the translocation of GLUT4 glucose transporter to the cell surface after insulin stimulation (6). However, the specific cellular mechanism(s) leading to calorie-restricted enhancement of insulin-stimulated GLUT4 translocation is uncertain.Phosphatidylinositol 3-kinase (PI3K) activation is essential for insulin-stimulated glucose transport (7,8) and is therefore a possible mediator of the effects of calorie restriction on insulin sensitivity. Although we have repeatedly seen a trend for increased insulin receptor substrate (IRS)-1-associated PI3K activity, we have not found a significant change in IRS-1-, IRS-2-, or phosphotyrosineassociated PI3K activity (6,9,10). There is no change in insulin receptor number, binding affinity, or tyrosine kinase activity with brief calorie restriction (11,12). There is also no increase in IRS-1 or IRS-2 (4,6) or PI3K regulatory p85 subunit (6) abundance. Considering the lack of a significant effect of calorie restriction on PI3K and its ups...

Phosphoinositide (PI) 3-kinase is involved in insulin-mediated effects on glucose uptake, lipid deposition, and adiponectin secretion from adipocytes. Genetic disruption of the p85␣ regulatory subunit of PI 3-kinase increases insulin sensitivity, whereas elevated p85␣ levels are associated with insulin resistance through PI 3-kinase-dependent and -independent mechanisms. Adipose tissue plays a critical role in the antagonistic effects of growth hormone (GH) on insulin actions on carbohydrate and lipid metabolism through changes in gene transcription. The objective of this study was to assess the role of the p85␣ subunit of PI 3-kinase and PI 3-kinase signaling in GH-mediated insulin resistance in adipose tissue. To do this, p85␣ mRNA and protein expression and insulin receptor substrate (IRS)-1-associated PI 3-kinase activity were measured in white adipose tissue (WAT) of mice with GH excess, deficiency, and sufficiency. Additional studies using 3T3-F442A cells were conducted to confirm direct effects of GH on free p85␣ protein abundance. We found that p85␣ expression 1) is decreased in WAT from mice with isolated GH deficiency, 2) is increased in WAT from mice with chronic GH excess, 3) is acutely upregulated in WAT from GH-deficient and -sufficient mice after GH administration, and 4) is directly upregulated by GH in 3T3-F442A adipocytes. The insulininduced increase in PI 3-kinase activity was robust in mice with GH deficiency, but not in mice with GH excess. In conclusion, GH regulates p85␣ expression and PI 3-kinase activity in WAT and provides a potential explanation for 1) the insulin hypersensitivity and associated obesity and hyperadiponectinemia of GH-deficient mice and 2) the insulin resistance and associated reduced fat mass and hypoadiponectinemia of mice with GH excess. Diabetes

OBJECTIVE-Skeletal muscle-specific LPL knockout mouse (SMLPL Ϫ/Ϫ ) were created to study the systemic impact of reduced lipoprotein lipid delivery in skeletal muscle on insulin sensitivity, body weight, and composition. RESEARCH DESIGN AND METHODS-Tissue-specific insulin sensitivity was assessed using a hyperinsulinemic-euglycemic clamp and 2-deoxyglucose uptake. Gene expression and insulinsignaling molecules were compared in skeletal muscle and liver of SMLPL Ϫ/Ϫ and control mice.RESULTS-Nine-week-old SMLPL Ϫ/Ϫ mice showed no differences in body weight, fat mass, or whole-body insulin sensitivity, but older SMLPL Ϫ/Ϫ mice had greater weight gain and whole-body insulin resistance. High-fat diet feeding accelerated the development of obesity. In young SMLPL Ϫ/Ϫ mice, insulin-stimulated glucose uptake was increased 58% in the skeletal muscle, but was reduced in white adipose tissue (WAT) and heart. Insulin action was also diminished in liver: 40% suppression of hepatic glucose production in SMLPL Ϫ/Ϫ vs. 90% in control mice. Skeletal muscle triglyceride was 38% lower, and insulin-stimulated phosphorylated Akt (Ser473) was twofold greater in SMLPL Ϫ/Ϫ mice without changes in IRS-1 tyrosine phosphorylation and phosphatidylinositol 3-kinase activity. Hepatic triglyceride and liver X receptor, carbohydrate response element-binding protein, and PEPCK mRNAs were unaffected in SMLPL Ϫ/Ϫ mice, but peroxisome proliferator-activated receptor (PPAR)-␥ coactivator-1␣ and interleukin-1␤ mRNAs were higher, and stearoyl-coenzyme A desaturase-1 and PPAR␥ mRNAs were reduced. CONCLUSIONS-LPL deletion in skeletal muscle reduces lipid storage and increases insulin signaling in skeletal muscle without changes in body composition. Moreover, lack of LPL in skeletal muscle results in insulin resistance in other key metabolic tissues and ultimately leads to obesity and systemic insulin resistance. Diabetes 58:116-124, 2009 L ipoprotein lipase (LPL) (European Commission no. 3.1.1.34) is a key enzyme in lipid metabolism and is described as a "gatekeeper" for its role in partitioning lipoprotein-derived free fatty acids (FFAs) between tissues (1). Once hydrolyzed, the lipoprotein-derived FFAs are available for uptake and use by extrahepatic tissues for either storage or oxidation. LPL is most abundant in heart, adipose tissue, and skeletal muscle (2,3). The importance of LPL in fuel partitioning and utilization is underscored by observations that tissuespecific perturbations in LPL activity result in dramatic shifts in body composition and lipid and glucose metabolism (4), particularly in heart and skeletal muscle.We and others previously showed that mice with musclespecific lipoprotein lipase overexpression are insulin resistant (5,6). Insulin resistance developed selectively in muscle, while insulin sensitivity in the liver was not affected. Overexpression of LPL in the skeletal muscle also led to excessive intramyocellular lipid deposition, suggestive of the relationship between lipid storage and insulin sensitivity.To further investigate...