Jianying Dong

Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle

Insulin resistance in skeletal muscle plays a major role in the development of type 2 diabetes and may be causally associated with increases in intramuscular fatty acid metabolites. Fatty acid transport protein 1 (FATP1) is an acyl-CoA synthetase highly expressed in skeletal muscle and modulates fatty acid uptake and metabolism by converting fatty acids into fatty acyl-CoA. To investigate the role of FATP1 in glucose homeostasis and in the pathogenesis of insulin resistance, we examined the effect of acute lipid infusion or chronic high-fat feeding on insulin action in FATP1 KO mice. Whole-body adiposity, adipose tissue expression of adiponectin, intramuscular fatty acid metabolites, and insulin sensitivity were not altered in FATP1 KO mice fed a regular chow diet. In contrast, FATP1 deletion protected the KO mice from fat-induced insulin resistance and intramuscular accumulation of fatty acyl-CoA without alteration in whole-body adiposity. These findings demonstrate an important role of intramuscular fatty acid metabolites in causing insulin resistance and suggest that FATP1 may be a novel therapeutic target for the treatment of insulin resistance and type 2 diabetes.

Mitochondrial dysfunction due to long-chain Acyl-CoA dehydrogenase deficiency causes hepatic steatosis and hepatic insulin resistance

Alterations in mitochondrial function have been implicated in the pathogenesis of insulin resistance and type 2 diabetes. However, it is unclear whether the reduced mitochondrial function is a primary or acquired defect in this process. To determine whether primary defects in mitochondrial β-oxidation can cause insulin resistance, we studied mice with a deficiency of long-chain acyl-CoA dehydrogenase (LCAD), a key enzyme in mitochondrial fatty acid oxidation. Here, we show that LCAD knockout mice develop hepatic steatosis, which is associated with hepatic insulin resistance, as reflected by reduced insulin suppression of hepatic glucose production during a hyperinsulinemic-euglycemic clamp. The defects in insulin action were associated with an ≈40% reduction in insulin-stimulated insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity and an ≈50% decrease in Akt2 activation. These changes were associated with increased PKCε activity and an aberrant 4-fold increase in diacylglycerol content after insulin stimulation. The increase in diacylglycerol concentration was found to be caused by de novo synthesis of diacylglycerol from medium-chain acyl-CoA after insulin stimulation. These data demonstrate that primary defects in mitochondrial fatty acid oxidation capacity can lead to diacylglycerol accumulation, PKCε activation, and hepatic insulin resistance.

n-3 Fatty Acids Preserve Insulin Sensitivity In Vivo in a Peroxisome Proliferator–Activated Receptor-α–Dependent Manner

Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fat diet–induced insulin resistance through peroxisome proliferator–activated receptor (PPAR)-α activation and a subsequent decrease in intracellular lipid abundance. To directly test this hypothesis, we fed PPAR-α null and wild-type mice for 2 weeks with isocaloric high-fat diets containing 27% fat from either safflower oil or safflower oil with an 8% fish oil replacement (fish oil diet). In both genotypes the safflower oil diet blunted insulin-mediated suppression of hepatic glucose production (P < 0.02 vs. genotype control) and PEPCK gene expression. Feeding wild-type mice a fish oil diet restored hepatic insulin sensitivity (hepatic glucose production [HGP], P < 0.002 vs. wild-type mice fed safflower oil), whereas in contrast, in PPAR-α null mice failed to counteract hepatic insulin resistance (HGP, P = NS vs. PPAR-α null safflower oil–fed mice). In PPAR-α null mice fed the fish oil diet, safflower oil plus fish oil, hepatic insulin resistance was dissociated from increases in hepatic triacylglycerol and acyl-CoA but accompanied by a more than threefold increase in hepatic diacylglycerol concentration (P < 0.0001 vs. genotype control). These data support the hypothesis that n-3 fatty acids protect from high-fat diet–induced hepatic insulin resistance in a PPAR-α–and diacylglycerol-dependent manner.

The Role of Peroxisome Proliferator-Activated Receptor γ Coactivator-1 β in the Pathogenesis of Fructose-Induced Insulin Resistance

Peroxisome proliferator-activated receptor gamma coactivator-1 beta (PGC-1beta) is known to be a transcriptional coactivator for SREBP-1, the master regulator of hepatic lipogenesis. Here, we evaluated the role of PGC-1beta in the pathogenesis of fructose-induced insulin resistance by using an antisense oligonucletoide (ASO) to knockdown PGC-1beta in liver and adipose tissue. PGC-1beta ASO improved the metabolic phenotype induced by fructose feeding by reducing expression of SREBP-1 and downstream lipogenic genes in liver. PGC-1beta ASO also reversed hepatic insulin resistance induced by fructose in both basal and insulin-stimulated states. Furthermore, PGC-1beta ASO increased insulin-stimulated whole-body glucose disposal due to a threefold increase in glucose uptake in white adipose tissue. These data support an important role for PGC-1beta in the pathogenesis of fructose-induced insulin resistance and suggest that PGC-1beta inhibition may be a therapeutic target for treatment of NAFLD, hypertriglyceridemia, and insulin resistance associated with increased de novo lipogenesis.

Glucose toxicity and the development of diabetes in mice with muscle-specific inactivation of GLUT4

Using cre/loxP gene targeting, transgenic mice with muscle-specific inactivation of the GLUT4 gene (muscle GLUT4 KO) were generated and shown to develop a diabetes phenotype. To determine the mechanism, we examined insulin-stimulated glucose uptake and metabolism during hyperinsulinemic-euglycemic clamp in control and muscle GLUT4 KO mice before and after development of diabetes. Insulin-stimulated whole body glucose uptake was decreased by 55% in muscle GLUT4 KO mice, an effect that could be attributed to a 92% decrease in insulin-stimulated muscle glucose uptake. Surprisingly, insulins ability to stimulate adipose tissue glucose uptake and suppress hepatic glucose production was significantly impaired in muscle GLUT4 KO mice. To address whether these latter changes were caused by glucose toxicity, we treated muscle GLUT4 KO mice with phloridzin to prevent hyperglycemia and found that insulin-stimulated whole body and skeletal muscle glucose uptake were decreased substantially, whereas insulin-stimulated glucose uptake in adipose tissue and suppression of hepatic glucose production were normal after phloridzin treatment. In conclusion, these findings demonstrate that a primary defect in muscle glucose transport can lead to secondary defects in insulin action in adipose tissue and liver due to glucose toxicity. These secondary defects contribute to insulin resistance and to the development of diabetes.

N-acylphosphatidylethanolamine, a gut- derived circulating factor induced by fat ingestion, inhibits food intake.

N-acylphosphatidylethanolamines (NAPEs) are a relatively abundant group of plasma lipids of unknown physiological significance. Here, we show that NAPEs are secreted into circulation from the small intestine in response to ingested fat and that systemic administration of the most abundant circulating NAPE, at physiologic doses, decreases food intake in rats without causing conditioned taste aversion. Furthermore, (14)C-radiolabeled NAPE enters the brain and is particularly concentrated in the hypothalamus, and intracerebroventricular infusions of nanomolar amounts of NAPE reduce food intake, collectively suggesting that its effects may be mediated through direct interactions with the central nervous system. Finally, chronic NAPE infusion results in a reduction of both food intake and body weight, suggesting that NAPE and long-acting NAPE analogs may be novel therapeutic targets for the treatment of obesity.

Resistance to High-fat Diet-induced Obesity and Insulin Resistance in Mice with Very Long-chain Acyl-CoA Dehydrogenase Deficiency

Mitochondrial fatty acid oxidation provides an important energy source for cellular metabolism, and decreased mitochondrial fatty acid oxidation has been implicated in the pathogenesis of type 2 diabetes. Paradoxically, mice with an inherited deficiency of the mitochondrial fatty acid oxidation enzyme, very long-chain acyl-CoA dehydrogenase (VLCAD), were protected from high-fat diet-induced obesity and liver and muscle insulin resistance. This was associated with reduced intracellular diacylglycerol content and decreased activity of liver protein kinase Cvarepsilon and muscle protein kinase Ctheta. The increased insulin sensitivity in the VLCAD(-/-) mice were protected from diet-induced obesity and insulin resistance due to chronic activation of AMPK and PPARalpha, resulting in increased fatty acid oxidation and decreased intramyocellular and hepatocellular diacylglycerol content.

The Role of the Carbohydrate Response Element-Binding Protein in Male Fructose-Fed Rats

By 2030, nearly half of Americans will have nonalcoholic fatty liver disease. In part, this epidemic is fueled by the increasing consumption of caloric sweeteners coupled with an innate capacity to convert sugar into fat via hepatic de novo lipogenesis. In addition to serving as substrates, monosaccharides also increase the expression of key enzymes involved in de novo lipogenesis via the carbohydrate response element-binding protein (ChREBP). To determine whether ChREBP is a potential therapeutic target, we decreased hepatic expression of ChREBP with a specific antisense oligonucleotide (ASO) in male Sprague-Dawley rats fed either a high-fructose or high-fat diet. ChREBP ASO treatment decreased plasma triglyceride concentrations compared with control ASO treatment in both diet groups. The reduction was more pronounced in the fructose-fed group and attributed to decreased hepatic expression of ACC2, FAS, SCD1, and MTTP and a decrease in the rate of hepatic triglyceride secretion. This was associated with an increase in insulin-stimulated peripheral glucose uptake, as assessed by the hyperinsulinemic-euglycemic clamp. In contrast, ChREBP ASO did not alter hepatic lipid content or hepatic insulin sensitivity. Interestingly, fructose-fed rats treated with ChREBP ASO had increased plasma uric acid, alanine transaminase, and aspartate aminotransferase concentrations. This was associated with decreased expression of fructose aldolase and fructokinase, reminiscent of inherited disorders of fructose metabolism. In summary, these studies suggest that targeting ChREBP may prevent fructose-induced hypertriglyceridemia but without the improvements in hepatic steatosis and hepatic insulin responsiveness.

A Role for Mitochondrial Phosphoenolpyruvate Carboxykinase (PEPCK-M) in the Regulation of Hepatic Gluconeogenesis

Background: PEPCK-M is generally considered irrelevant for glucose production, although gluconeogenesis has never been characterized in its absence. Results: PEPCK-M loss impaired gluconeogenesis from lactate, lowered plasma glucose, insulin, and triglycerides, reduced hepatic glycogen, and increased glycerol turnover. Conclusion: Approximately a third of gluconeogenesis comes from PEPCK-M. Significance: The nutrient-sensitive PEPCK-M has been overlooked and is potentially important for metabolic diseases such as diabetes. Synthesis of phosphoenolpyruvate (PEP) from oxaloacetate is an absolute requirement for gluconeogenesis from mitochondrial substrates. Generally, this reaction has solely been attributed to the cytosolic isoform of PEPCK (PEPCK-C), although loss of the mitochondrial isoform (PEPCK-M) has never been assessed. Despite catalyzing the same reaction, to date the only significant role reported in mammals for the mitochondrial isoform is as a glucose sensor necessary for insulin secretion. We hypothesized that this nutrient-sensing mitochondrial GTP-dependent pathway contributes importantly to gluconeogenesis. PEPCK-M was acutely silenced in gluconeogenic tissues of rats using antisense oligonucleotides both in vivo and in isolated hepatocytes. Silencing PEPCK-M lowers plasma glucose, insulin, and triglycerides, reduces white adipose, and depletes hepatic glycogen, but raises lactate. There is a switch of gluconeogenic substrate preference to glycerol that quantitatively accounts for a third of glucose production. In contrast to the severe mitochondrial deficiency characteristic of PEPCK-C knock-out livers, hepatocytes from PEPCK-M-deficient livers maintained normal oxidative function. Consistent with its predicted role, gluconeogenesis rates from hepatocytes lacking PEPCK-M are severely reduced for lactate, alanine, and glutamine, but not for pyruvate and glycerol. Thus, PEPCK-M has a direct role in fasted and fed glucose homeostasis, and this mitochondrial GTP-dependent pathway should be reconsidered for its involvement in both normal and diabetic metabolism.

Hyperglucagonemia precedes a decline in insulin secretion and causes hyperglycemia in chronically glucose-infused rats

Islet damage from glucose toxicity is implicated in the pathogenesis of type 2 diabetes, but the sequence of events leading to islet cell dysfunction and hyperglycemia remains unclear. To examine the early stages of islet pathology resulting from increased basal glucose loads, normal awake rats were infused with glucose continuously for 10 days. Plasma glucose and markers of islet and liver function were monitored throughout the infusion. After initial hyperglycemia, rats adapted to the infusion and maintained euglycemia for approximately 4 days. Continued infusion led to worsening hyperglycemia in just 5% of rats after 6 days, but 69% after 8 days and 89% after 10 days, despite unchanged basal and stimulated plasma insulin and C-peptide concentrations. In contrast, plasma glucagon concentrations increased fivefold. Endogenous glucose production (EGP) was appropriately suppressed after 4 days (2.8 ± 0.7 vs. 6.1 ± 0.4 mg·kg(-1)·min(-1) on day 0, P < 0.001) but tripled between days 4 and 8 (9.9 ± 1.7 mg·kg(-1)·min(-1), P < 0.01). Surprisingly, the increase in EGP was accompanied by increased mitochondrial phosphoenolpyruvate carboxykinase expression with appropriate suppression of the cytosolic isoform. Infusion of anti-glucagon antibodies normalized plasma glucose to levels identical to those on day 4 and ∼300 mg/dl lower than controls. This improved glycemia was associated with a 60% reduction in EGP. These data support the novel concept that glucose toxicity may first manifest as α-cell dysfunction prior to any measurable deficit in insulin secretion. Such hyperglucagonemia could lead to excessive glucose production overwhelming the capacity of the β-cell to maintain glucose homeostasis.