A choline-deficient diet exacerbates fatty liver but attenuates insulin resistance and glucose intolerance in mice fed a high-fat diet

Liver fat accumulation is proposed to link obesity and insulin resistance. To dissect the role of liver fat in the insulin resistance of diet-induced obesity, we altered liver fat using a choline-deficient diet. C57Bl/6 mice were fed a low-fat (10% of calories) or high-fat (45% of calories) diet for 8 weeks; during the final 4 weeks, diets were either choline deficient or choline supplemented. In choline replete animals, high-fat feeding induced weight gain, elevated liver triglycerides (171%), hyperinsulinemia, and glucose intolerance. Choline deficiency did not affect body or adipose depot weights but amplified liver fat accumulation with high-fat diet (281%, P < 0.01). However, choline deficiency lowered fasting plasma insulin (from 983 +/- 175 to 433 +/- 36 pmol/l, P < 0.01) and improved glucose tolerance on a high-fat diet. In mice on 30% fat diet, choline deficiency increased liver mRNA levels of the rate-limiting enzyme in phosphatidylcholine synthesis and of enzymes involved in free fatty acid esterification, without affecting those of de novo lipogenesis or fatty acid oxidation. We conclude that liver fat accumulation per se does not cause insulin resistance during high-fat feeding and that choline deficiency may shunt potentially toxic free fatty acids toward innocuous storage triglyceride in the liver.     

Adipose tissue expression of the lipid droplet-associating proteins S3-12 and perilipin is controlled by peroxisome proliferator-activated receptor-gamma

In a systematic search for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) target genes, we identified S3-12 and perilipin as novel direct PPAR-gamma target genes. Together with adipophilin and tail-interacting protein of 47 kDa, these genes are lipid droplet-associating proteins with distinct expression pattern but overlapping expression in adipose tissue. The expression of S3-12 and perilipin is tightly correlated to the expression and activation of PPAR-gamma in adipocytes, and promoter characterization revealed that the S3-12 and the perilipin promoters contain three and one evolutionarily conserved PPAR response elements, respectively. We furthermore demonstrate that the expression of S3-12 and perilipin is reduced in obese compared with lean Zucker rats, whereas the expression of adipophilin is increased. Others have shown that perilipin is an essential factor in the hormonal regulation of lipolysis of stored triglycerides within adipose tissue. The direct regulation of perilipin and S3-12 by PPAR-gamma therefore is likely to be an important mediator of the in vivo effects of prolonged treatment with PPAR-gamma activators: insulin sensitization, fatty acid trapping in adipose tissue, reduced basal adipose lipolysis, and weight gain.     

Lipin deficiency impairs diurnal metabolic fuel switching

Fatty liver is a common feature of both obesity and lipodystrophy, reflecting compromised adipose tissue function. The lipin-deficient fatty liver dystrophy (fld) mouse is an exception, as there is lipodystrophy without a fatty liver. Using a combination of indirect calorimetry and stable-isotope flux phenotyping, we determined that fld mice exhibit abnormal fuel utilization throughout the diurnal cycle, with increased glucose oxidation near the end of the fasting period and increased fatty acid oxidation during the feeding period. The mechanisms underlying these alterations include a twofold increase compared with wild-type mice in tissue glycogen storage during the fed state, a 40% reduction in hepatic glucose production in the fasted state, and a 27-fold increase in de novo fatty acid synthesis in liver during the fed state. Thus, the inability to store energy in adipose tissue in the fld mouse leads to a compensatory increase in glycogen storage for use during the fasting period and reliance upon hepatic fatty acid synthesis to provide fuel for peripheral tissues during the fed state. The increase in hepatic fatty acid synthesis and peripheral utilization provides a potential mechanism to ameliorate fatty liver in the fld that would otherwise occur as a consequence of adipose tissue dysfunction.     

Loss of stearoyl-CoA desaturase-1 improves insulin sensitivity in lean mice but worsens diabetes in leptin-deficient obese mice

The lipogenic gene stearoyl-CoA desaturase (SCD)1 appears to be a promising new target for obesity-related diabetes, as mice deficient in this enzyme are resistant to diet- and leptin deficiency-induced obesity. The BTBR mouse strain replicates many features of insulin resistance found in humans with excess visceral adiposity. Using the hyperinsulinemic-euglycemic clamp technique, we determined that insulin sensitivity was improved in heart, soleus muscle, adipose tissue, and liver of BTBR SCD1-deficient mice. We next determined whether SCD1 deficiency could prevent diabetes in leptin-deficient BTBR mice. Loss of SCD1 in leptin(ob/ob) mice unexpectedly accelerated the progression to severe diabetes; 6-week fasting glucose increased approximately 70%. In response to a glucose challenge, Scd1(-/-) leptin(ob/ob) mice had insufficient insulin secretion, resulting in glucose intolerance. A morphologically distinct class of islets isolated from the Scd1(-/-) leptin(ob/ob) mice had reduced insulin content and increased triglycerides, free fatty acids, esterified cholesterol, and free cholesterol and also a much higher content of saturated fatty acids. We believe the accumulation of lipid is due to an upregulation of lipoprotein lipase (20-fold) and Cd36 (167-fold) and downregulation of lipid oxidation genes in this class of islets. Therefore, although loss of Scd1 has beneficial effects on adiposity, this benefit may come at the expense of beta-cells, resulting in an increased risk of diabetes.     

Defect of insulin signal in peripheral tissues: important role of ceramide

In healthy people,balance between glucose production and its utilization is precisely controlled.When circulating glucose reaches a critical threshold level,pancreaticβcells secrete insulin that has two major actions:to lower circulating glucose levels by facilitating its uptake mainly into skeletal muscle while inhibiting its production by the liver.Interestingly,dietary triglycerides are the main source of fatty acids to fulfill energy needs of oxidative tissues.Normally,the unconsumed fraction of excess of fatty acids is stored in lipid droplets that are localized in adipocytes to provide energy during fasting periods.Thus,adipose tissue acts as a trap for fatty acid excess liberated from plasma triglycerides.When the buffering action of adipose tissue to store fatty acids is impaired,fatty acids that build up in othertissues are metabolized as sphingolipid derivatives such as ceramides.Several studies suggest that ceramides are among the most active lipid second messengers to inhibit the insulin signaling pathway and this review describes the major role played by ceramide accumulation in the development of insulin resistance of peripherals tissues through the targeting of specific proteins of the insulin signaling pathway.     

Peroxisome proliferator-activated receptor-alpha agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis

Abnormalities in insulin action are the characteristics of type 2 diabetes. Dominant-negative muscle-specific IGF-I receptor (MKR) mice exhibit elevated lipid levels at an early age and eventually develop type 2 diabetes. To evaluate the role of elevated lipids in the progression of the diabetic state, MKR mice were treated with WY14,643, a peroxisome proliferator-activated receptor (PPAR)-alpha agonist. WY14,643 treatment markedly reduced serum fatty acid and triglyceride levels within a few days, as well as muscle triglyceride levels, and subsequently normalized glucose and insulin levels in MKR mice. Hyperinsulinemic-euglycemic clamp analysis showed that WY14,643 treatment enhanced muscle and adipose tissue glucose uptake by improving whole-body insulin sensitivity. Insulin suppression of endogenous glucose production by the liver of MKR mice was also improved. The expression of genes involved in fatty acid oxidation was increased in liver and skeletal muscle, whereas gene expression levels of hepatic gluconeogenic enzymes were decreased in WY14,643-treated MKR mice. WY14,643 treatment also improved the pattern of glucose-stimulated insulin secretion from the perfused pancreata of MKR mice and reduced the beta-cell mass. Taken together, these findings suggest that the reduction in circulating or intracellular lipids by activation of PPAR-alpha improved insulin sensitivity and the diabetic condition of MKR mice.     

Lipin expression is attenuated in adipose tissue of insulin-resistant human subjects and increases with peroxisome proliferator-activated receptor gamma activation

Lipin-alpha and -beta are the alternatively spliced gene products of the Lpin1 gene, whose product lipin is required for adipocyte differentiation. Lipin deficiency causes lipodystrophy, fatty liver, and insulin resistance in mice, whereas adipose tissue lipin overexpression results in increased adiposity but improved insulin sensitivity. To assess lipin expression and its relation to insulin resistance in humans, we examined lipin-alpha and -beta mRNA levels in subjects with normal or impaired glucose tolerance. We found higher expression levels of both lipin isoforms in lean, insulin-sensitive subjects. When compared with normal glucose-tolerant subjects, individuals with impaired glucose tolerance were more insulin resistant, demonstrated higher levels of intramyocellular lipids (IMCLs), and expressed approximately 50% lower levels of lipin-alpha and -beta. In addition, there was a strong inverse correlation between adipose tissue lipin expression and muscle IMCLs but no evidence for an increase in muscle lipid oxidation. After treatment of the impaired glucose-tolerant subjects with insulin sensitizers for 10 weeks, pioglitazone (but not metformin) resulted in a 60% increase in the insulin sensitivity index (Si) and a 32% decrease in IMCLs (both P < 0.01), along with an increase in lipin-beta (but not lipin-alpha) expression by 200% (P < 0.005). Lipin expression in skeletal muscle, however, was not related to obesity or insulin resistance. Hence, high adipose tissue lipin expression is found in insulin-sensitive subjects, and lipin-beta expression increases following treatment with pioglitazone. These results suggest that increased adipogenesis and/or lipogenesis in subcutaneous fat, mediated by the LPIN1 gene, may prevent lipotoxicity in muscle, leading to improved insulin sensitivity.     

Skeletal Muscle Triglycerides,Diacylglycerols, and Ceramides in Insulin Resistance: Another Paradox in Endurance-Trained Athletes?

OBJECTIVE

Chronic exercise and obesity both increase intramyocellular triglycerides (IMTGs) despite having opposing effects on insulin sensitivity. We hypothesized that chronically exercise-trained muscle would be characterized by lower skeletal muscle diacylglycerols (DAGs) and ceramides despite higher IMTGs and would account for its higher insulin sensitivity. We also hypothesized that the expression of key skeletal muscle proteins involved in lipid droplet hydrolysis, DAG formation, and fatty-acid partitioning and oxidation would be associated with the lipotoxic phenotype.

RESEARCH DESIGN AND METHODS

A total of 14 normal-weight, endurance-trained athletes (NWA group) and 7 normal-weight sedentary (NWS group) and 21 obese sedentary (OBS group) volunteers were studied. Insulin sensitivity was assessed by glucose clamps. IMTGs, DAGs, ceramides, and protein expression were measured in muscle biopsies.

RESULTS

DAG content in the NWA group was approximately twofold higher than in the OBS group and ~50% higher than in the NWS group, corresponding to higher insulin sensitivity. While certain DAG moieties clearly were associated with better insulin sensitivity, other species were not. Ceramide content was higher in insulin-resistant obese muscle. The expression of OXPAT/perilipin-5, adipose triglyceride lipase, and stearoyl-CoA desaturase protein was higher in the NWA group, corresponding to a higher mitochondrial content, proportion of type 1 myocytes, IMTGs, DAGs, and insulin sensitivity.

CONCLUSIONS

Total myocellular DAGs were markedly higher in highly trained athletes, corresponding with higher insulin sensitivity, and suggest a more complex role for DAGs in insulin action. Our data also provide additional evidence in humans linking ceramides to insulin resistance. Finally, this study provides novel evidence supporting a role for specific skeletal muscle proteins involved in intramyocellular lipids, mitochondrial oxidative capacity, and insulin resistance.Skeletal muscle insulin resistance (IR) is associated with obesity and physical inactivity and is crucial for the development of type 2 diabetes (1). Unfortunately, the causes of IR within muscle are not known. Concerted efforts have been made over the past several years to understand the potential role of intramyocellular lipid (IMCL) accumulation in the development of IR (2). Studies in both animal models (3) and humans (4) provided early evidence that IMCLs, such as triglycerides, were associated with skeletal muscle IR. However, we subsequently reported on the athletes paradox in which chronically exercised humans were markedly insulin-sensitive despite having high intramyocellular triglycerides (IMTGs) (5), a phenomenon corroborated by others (6). These observations gave pause to the widely held view that IMCLs cause IR within the muscle and lent support to the concept that other potentially damaging IMCLs may play a role in the development of IR.Diacylglycerols (DAGs) and ceramides are lipid intermediates widely believed to be the true lipotoxic culprits underlying the reported associations between muscle triglycerides and IR, thereby explaining the athletes paradox and the root cause of muscle IR. Indeed, several lines of evidence from cell systems and animal models indicate that elevated DAGs (7) or ceramides (8) are associated with impaired insulin signaling and IR. Previous studies examining skeletal muscle DAG and ceramide content related to human IR, however, are limited and inconsistent (9,10). Because it has become clear that chronic exercise training increases IMTGs (5,11), recent studies have been conducted to examine whether both DAGs and ceramides are reduced with exercise (12–14). Another prevalent notion is that higher mitochondria content and capacity for fatty acid oxidation caused by exercise training are responsible for lower DAG and ceramide content (15). We recently have shown that exercise training does indeed decrease these IMCLs in conjunction with increased oxidative capacity and improved insulin sensitivity (12). The distinct roles that chronic exercise and obesity may play in the link between these potentially harmful lipid species and skeletal muscle IR, however, remain to be elucidated. Moreover, it is not known whether specific molecular species of these complex lipids are associated with skeletal muscle IR according to their fatty acid chain length or degree of saturation. Therefore, we used mass spectrometry to quantify the content and molecular species profile of both DAGs and sphingolipids within skeletal muscle biopsies in human subjects widely disparate for physical activity and obesity to test the following primary hypothesis: endurance-trained athletes, despite having higher IMTGs, would have the lowest muscular DAG and ceramide content, the highest insulin sensitivity, and the highest mitochondrial content compared with both normal weight and obese sedentary subjects and that these differences would be more pronounced for saturated DAG and ceramide species. In addition, we investigated several proteins associated with lipid droplet esterification and lipolysis: oxidative perilipin adipophilin TIP47 (OXPAT)/perilipin-5 or lipid storage protein 5 because of its reported role in lipid droplet formation and enhanced fatty acid oxidation (16,17); the lipogenic enzymes stearoyl-CoA desaturase 1 (SCD1) and diacylglycerol acyltransferase 1 (DGAT1); and adipose triglyceride lipase (ATGL). These data provide novel insight into the role of lipid-induced IR within human skeletal muscle.     

Muraglitazar, a novel dual (alpha/gamma) peroxisome proliferator-activated receptor activator, improves diabetes and other metabolic abnormalities and preserves beta-cell function in db/db mice

Muraglitazar, a novel dual (alpha/gamma) peroxisome proliferator-activated receptor (PPAR) activator, was investigated for its antidiabetic properties and its effects on metabolic abnormalities in genetically obese diabetic db/db mice. In db/db mice and normal mice, muraglitazar treatment modulates the expression of PPAR target genes in white adipose tissue and liver. In young hyperglycemic db/db mice, muraglitazar treatment (0.03-50 mg . kg(-1) . day(-1) for 2 weeks) results in dose-dependent reductions of glucose, insulin, triglycerides, free fatty acids, and cholesterol. In older hyperglycemic db/db mice, longer-term muraglitazar treatment (30 mg . kg(-1) . day(-1) for 4 weeks) prevents time-dependent deterioration of glycemic control and development of insulin deficiency. In severely hyperglycemic db/db mice, muraglitazar treatment (10 mg . kg(-1) . day(-1) for 2 weeks) improves oral glucose tolerance and reduces plasma glucose and insulin levels. In addition, treatment increases insulin content in the pancreas. Finally, muraglitazar treatment increases abnormally low plasma adiponectin levels, increases high-molecular weight adiponectin complex levels, reduces elevated plasma corticosterone levels, and lowers elevated liver lipid content in db/db mice. The overall conclusions are that in db/db mice, the novel dual (alpha/gamma) PPAR activator muraglitazar 1) exerts potent and efficacious antidiabetic effects, 2) preserves pancreatic insulin content, and 3) improves metabolic abnormalities such as hyperlipidemia, fatty liver, low adiponectin levels, and elevated corticosterone levels.     

Consequences of lipid droplet coat protein downregulation in liver cells: abnormal lipid droplet metabolism and induction of insulin resistance

OBJECTIVE—Accumulation of intracellular lipid droplets (LDs) in non-adipose tissues is recognized as a strong prognostic factor for the development of insulin resistance in obesity. LDs are coated with perilipin, adipose differentiation–related protein, tail interacting protein of 47 kd (PAT) proteins that are thought to regulate LD turnover by modulating lipolysis. Our hypothesis is that PAT proteins modulate LD metabolism and therefore insulin resistance.RESEARCH DESIGN AND METHODS—We used a cell culture model (murine AML12 loaded with oleic acid) and small interfering RNA to directly assess the impact of PAT proteins on LD accumulation, lipid metabolism, and insulin action. PAT proteins associated with excess fat deposited in livers of diet-induced obese (DIO) mice were also measured.RESULTS—Cells lacking PAT proteins exhibited a dramatic increase in LD size and a decrease in LD number. Further, the lipolytic rate increased by ∼2- to 2.5-fold in association with increased adipose triglyceride lipase (ATGL) at the LD surface. Downregulation of PAT proteins also produced insulin resistance, as indicated by decreased insulin stimulation of Akt phosphorylation (P < 0.001). Phosphoinositide-dependent kinase-1 and phosphoinositide 3-kinase decreased, and insulin receptor substrate-1 307 phosphorylation increased. Increased lipids in DIO mice livers were accompanied by changes in PAT composition but also increased ATGL, suggesting a relative PAT deficiency.CONCLUSIONS—These data establish an important role for PAT proteins as surfactant at the LD surface, packaging lipids in smaller units and restricting access of lipases and thus preventing insulin resistance. We suggest that a deficiency of PAT proteins relative to the quantity of ectopic fat could contribute to cellular dysfunction in obesity and type 2 diabetes.The surge in obesity predicts a further increase in associated complications, insulin resistance, diabetes, and heart disease (1,2). Increased fatty acid availability in obesity is associated with accumulation of ectopic fat, mainly in the form of triacylglyerol (TAG) (3). Although ectopic fat correlates with systemic and tissue insulin resistance (4–6), a number of circumstances are known in which high tissue lipid stores are not associated with insulin resistance. Endurance-trained athletes have high intramyocellular lipids yet are highly insulin sensitive. Importantly, the size and intracellular distribution of lipid droplets (LDs) differs in muscle from insulin-sensitive athletes compared with insulin-resistant patients (7). Thus, the negative consequences of high cellular lipids may be related to the ability of the cell to regulate lipid storage and utilization.LDs are energy-storage organelles but have a surprisingly complex function in lipid homeostasis. LD biogenesis is a fundamental cellular function; when exposed to nonesterified fatty acids (NEFAs), cells store them as TAG in LDs (8). Such LD accumulation maintains low intracellular NEFAs, avoiding their toxic effects on cellular physiology while supporting cellular needs by releasing NEFAs for use in β-oxidation and membrane synthesis. LDs’ function to sequester and release NEFAs is thus critical for proper cellular function. Nonadipogenic tissues in patients with metabolic syndrome are exposed to chronically elevated serum levels of NEFAs, and these tissues respond by LD accumulation. Such ectopic fat deposition protects from NEFA-mediated lipotoxicity (9), but in patients with metabolic syndrome the LD is inadequate to prevent pathological consequences. An important question arises: what molecular mechanisms regulate lipid storage in nonadipogenic tissues?To date, we have only limited information on nonadipose LDs. Recent studies (10,11) identified a proteomic “signature,” consistently including at least one member of the PAT protein family: perilipin, adipose differentiation–related protein (ADFP), tail interacting protein of 47 kDa (Tip47), S3–12, and lipid dosage droplet protein-5 (LSDP-5). Despite tissue dependence, the ubiquitous nature of the family suggests an important role in LD machinery. ADFP, Tip47, and LSDP-5 are broadly distributed, notably in nonadipogenic liver and muscle tissues that do not express perilipin (13,24). Our hypothesis is that saturation of nonadipogenic tissue''s capacity to appropriately regulate storage and release of NEFAs via LDs results from variations in the expression and/or activity of PAT proteins. To study functional consequences of downregulating two major PAT proteins, ADFP and Tip47, on insulin resistance and lipid metabolism, we used small interfering RNA (siRNA) in a cell culture model. To assess the in vivo relevance of this finding, we measured the expression of PAT proteins associated with excess lipids accumulated in the livers of high-fat–fed obese mice.     

Genetic manipulations of fatty acid metabolism in beta-cells are associated with dysregulated insulin secretion

Triacylglyceride (TG) accumulation in pancreatic beta-cells is associated with impaired insulin secretion, which is called lipotoxicity. To gain a better understanding of the pathophysiology of lipotoxicity, we generated three models of dysregulated fatty acid metabolism in beta-cells. The overexpression of sterol regulatory element binding protein-1c induced lipogenic genes and TG accumulation. Under these conditions, we observed a decrease in glucose oxidation and upregulation of uncoupling protein-2, which might be causally related to the decreased glucose-stimulated insulin secretion. The overexpression of AMP-activated protein kinase was accompanied by decreased lipogenesis, increased fatty acid oxidation, and decreased glucose oxidation; insulin secretions to glucose and depolarization stimuli were decreased, probably because of the decrease in glucose oxidation and cellular insulin content. It was notable that the secretory response to palmitate was blunted, which would suggest a role of the fatty acid synthesis pathway, but not its oxidative pathway in palmitate-stimulated insulin secretion. Finally, we studied islets of PPAR-gamma(+/-) mice that had increased insulin sensitivity and low TG content in white adipose tissue, skeletal muscle, and liver. On a high-fat diet, glucose-stimulated insulin secretion was decreased in association with increased TG content in the islets, which might be mediated through the elevated serum free fatty acid levels and their passive transport into beta-cells. These results revealed some aspects about the mechanisms by which alterations of fatty acid metabolism affect beta-cell functions.     

Effects of Sleeve Gastrectomy on Lipid Metabolism in an Obese Diabetic Rat Model

Background

Laparoscopic sleeve gastrectomy (SG) is a popular bariatric procedure for treating morbid obesity. This study aimed to investigate the relation between SG and lipid metabolism in an obese diabetic rat model.

Methods

Forty-five male Zucker diabetic fatty (ZDF) rats were divided into three groups: sham-operated (SO) control, gastric banding (GB), and SG. Six weeks after surgery, metabolic parameters, including plasma adiponectin level, small bowel transit, mRNA expression of peroxisome proliferator-activated receptor (PPAR)-α and PPARγ in the liver, skeletal muscle and white adipose tissue, and that of adiponectin in white adipose tissue, and triglyceride (TG) contents in the liver and skeletal muscle were measured.

Results

Metabolic parameters in the GB and SG groups were significantly improved compared with those in the SO group. However, plasma total cholesterol (TC) and free fatty acid (FFA) concentrations were significantly lower while the plasma adiponectin level was significantly higher in the SG group than in the GB and SO groups. In addition, small bowel transit time was significantly shorter in the SG group than in the other two groups. Furthermore, in the SG group, mRNA expression of PPARα in the liver and skeletal muscle and that of adiponection and PPARγ in white adipose tissue were significantly higher, while TG in the liver and skeletal muscle were significantly lower, compared with those in the other two groups.

Conclusions

These results suggest that SG improves lipid metabolism compared with GB, although there were no significant differences in the effect on weight loss between the two procedures.     

Downregulation of electron transport chain genes in visceral adipose tissue in type 2 diabetes independent of obesity and possibly involving tumor necrosis factor-alpha

Impaired oxidative phosphorylation is suggested as a factor behind insulin resistance of skeletal muscle in type 2 diabetes. The role of oxidative phosphorylation in adipose tissue was elucidated from results of Affymetrix gene profiling in subcutaneous and visceral adipose tissue of eight nonobese healthy, eight obese healthy, and eight obese type 2 diabetic women. Downregulation of several genes in the electron transport chain was the most prominent finding in visceral fat of type 2 diabetic women independent of obesity, but the gene pattern was distinct from that previously reported in skeletal muscle in type 2 diabetes. A similar but much weaker effect was observed in subcutaneous fat. Tumor necrosis factor-alpha (TNF-alpha) is a major factor behind inflammation and insulin resistance in adipose tissue. TNF-alpha treatment decreased mRNA expression of electron transport chain genes and also inhibited fatty acid oxidation when differentiated human preadipocytes were treated with the cytokine for 48 h. Thus, type 2 diabetes is associated with a tissue- and region-specific downregulation of oxidative phosphorylation genes that is independent of obesity and at least in part mediated by TNF-alpha, suggesting that impaired oxidative phosphorylation of visceral adipose tissue has pathogenic importance for development of type 2 diabetes.     

Improved Insulin Sensitivity 3 Months After RYGB Surgery Is Associated With Increased Subcutaneous Adipose Tissue AMPK Activity and Decreased Oxidative Stress

Morbidly obese individuals are predisposed to a wide range of disorders, including type 2 diabetes, atherosclerotic cardiovascular disease, fatty liver disease, and certain cancers. Remarkably, all of these disorders can be improved or prevented by Roux-en-Y gastric bypass (RYGB) surgery. We have reported that decreased AMPK activity, together with increased oxidative stress and inflammation in adipose tissue, is associated with insulin resistance in morbidly obese bariatric surgery patients. In the current study, we assessed how these parameters are affected by RYGB surgery. Eleven patients (average age of 46 ± 4 years) were studied immediately prior to surgery and 3 months postoperatively. We measured subcutaneous adipose tissue AMPK phosphorylation (threonine 172, an index of its activation), malonyl-CoA content, protein carbonylation (a marker of oxidative stress), plasma adiponectin, and mRNA expression of several inflammatory cytokines. After surgery, AMPK activity increased 3.5-fold and oxidative stress decreased by 50% in subcutaneous adipose tissue. In addition, malonyl-CoA levels were reduced by 80%. Furthermore, patients had improvements in their BMI and insulin sensitivity (HOMA) and had increased circulating high–molecular weight adiponectin and decreased fasting plasma insulin levels. In contrast, the expression of inflammatory markers in subcutaneous adipose tissue was unchanged postoperatively, although plasma CRP was diminished by 50%.     

Targeted Disruption of G0/G1 Switch Gene 2 Enhances Adipose Lipolysis,Alters Hepatic Energy Balance,and Alleviates High-Fat Diet–Induced Liver Steatosis

Recent biochemical and cell-based studies identified G₀/G₁ switch gene 2 (G0S2) as an inhibitor of adipose triglyceride lipase (ATGL), a key mediator of intracellular triacylglycerol (TG) mobilization. Here, we show that upon fasting, G0S2 protein expression exhibits an increase in liver and a decrease in adipose tissue. Global knockout of G0S2 in mice enhanced adipose lipolysis and attenuated gain of body weight and adiposity. More strikingly, G0S2 knockout mice displayed a drastic decrease in hepatic TG content and were resistant to high-fat diet (HFD)-induced liver steatosis, both of which were reproduced by liver-specific G0S2 knockdown. Mice with hepatic G0S2 knockdown also showed increased ketogenesis, accelerated gluconeogenesis, and decelerated glycogenolysis. Conversely, overexpression of G0S2 inhibited fatty acid oxidation in mouse primary hepatocytes and caused sustained steatosis in liver accompanied by deficient TG clearance during the fasting-refeeding transition. In response to HFD, there was a profound increase in hepatic G0S2 expression in the fed state. Global and hepatic ablation of G0S2 both led to improved insulin sensitivity in HFD-fed mice. Our findings implicate a physiological role for G0S2 in the control of adaptive energy response to fasting and as a contributor to obesity-associated liver steatosis.     

Altered Skeletal Muscle Mitochondrial Proteome As the Basis of Disruption of Mitochondrial Function in Diabetic Mice

Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and β-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of β-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a β-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress.     

Excess lipid availability increases mitochondrial fatty acid oxidative capacity in muscle: evidence against a role for reduced fatty acid oxidation in lipid-induced insulin resistance in rodents

A reduced capacity for mitochondrial fatty acid oxidation in skeletal muscle has been proposed as a major factor leading to the accumulation of intramuscular lipids and their subsequent deleterious effects on insulin action. Here, we examine markers of mitochondrial fatty acid oxidative capacity in rodent models of insulin resistance associated with an oversupply of lipids. C57BL/6J mice were fed a high-fat diet for either 5 or 20 weeks. Several markers of muscle mitochondrial fatty acid oxidative capacity were measured, including (14)C-palmitate oxidation, palmitoyl-CoA oxidation in isolated mitochondria, oxidative enzyme activity (citrate synthase, beta-hydroxyacyl CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, and carnitine palmitoyl-transferase 1), and expression of proteins involved in mitochondrial metabolism. Enzyme activity and mitochondrial protein expression were also examined in muscle from other rodent models of insulin resistance. Compared with standard diet-fed controls, muscle from fat-fed mice displayed elevated palmitate oxidation rate (5 weeks +23%, P < 0.05, and 20 weeks +29%, P < 0.05) and increased palmitoyl-CoA oxidation in isolated mitochondria (20 weeks +49%, P < 0.01). Furthermore, oxidative enzyme activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha, uncoupling protein (UCP) 3, and mitochondrial respiratory chain subunits were significantly elevated in fat-fed animals. A similar pattern was present in muscle of fat-fed rats, obese Zucker rats, and db/db mice, with increases observed for oxidative enzyme activity and expression of PGC-1alpha, UCP3, and subunits of the mitochondrial respiratory chain. These findings suggest that high lipid availability does not lead to intramuscular lipid accumulation and insulin resistance in rodents by decreasing muscle mitochondrial fatty acid oxidative capacity.