Mario Kahn

Inhibition of protein kinase Cε prevents hepatic insulin resistance in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance and type 2 diabetes mellitus, but the molecular signals linking hepatic fat accumulation to hepatic insulin resistance are unknown. Three days of high-fat feeding in rats results specifically in hepatic steatosis and hepatic insulin resistance. In this setting, PKCepsilon, but not other isoforms of PKC, is activated. To determine whether PKCepsilon plays a causal role in the pathogenesis of hepatic insulin resistance, we treated rats with an antisense oligonucleotide against PKCepsilon and subjected them to 3 days of high-fat feeding. Knocking down PKCepsilon expression protects rats from fat-induced hepatic insulin resistance and reverses fat-induced defects in hepatic insulin signaling. Furthermore, we show that PKCepsilon associates with the insulin receptor in vivo and impairs insulin receptor kinase activity both in vivo and in vitro. These data support the hypothesis that PKCepsilon plays a critical role in mediating fat-induced hepatic insulin resistance and represents a novel therapeutic target for type 2 diabetes.

Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease

Insulin resistance is associated with nonalcoholic fatty liver disease (NAFLD) and is a major factor in the pathogenesis of type 2 diabetes. The development of hepatic insulin resistance has been ascribed to multiple causes, including inflammation, endoplasmic reticulum (ER) stress, and accumulation of hepatocellular lipids in animal models of NAFLD. However, it is unknown whether these same cellular mechanisms link insulin resistance to hepatic steatosis in humans. To examine the cellular mechanisms that link hepatic steatosis to insulin resistance, we comprehensively assessed each of these pathways by using flash-frozen liver biopsies obtained from 37 obese, nondiabetic individuals and correlating key hepatic and plasma markers of inflammation, ER stress, and lipids with the homeostatic model assessment of insulin resistance index. We found that hepatic diacylglycerol (DAG) content in cytoplasmic lipid droplets was the best predictor of insulin resistance (R = 0.80, P < 0.001), and it was responsible for 64% of the variability in insulin sensitivity. Hepatic DAG content was also strongly correlated with activation of hepatic PKCε (R = 0.67, P < 0.001), which impairs insulin signaling. In contrast, there was no significant association between insulin resistance and other putative lipid metabolites or plasma or hepatic markers of inflammation. ER stress markers were only partly correlated with insulin resistance. In conclusion, these data show that hepatic DAG content in lipid droplets is the best predictor of insulin resistance in humans, and they support the hypothesis that NAFLD-associated hepatic insulin resistance is caused by an increase in hepatic DAG content, which results in activation of PKCε.

Suppression of Diacylglycerol Acyltransferase-2 (DGAT2), but Not DGAT1, with Antisense Oligonucleotides Reverses Diet-induced Hepatic Steatosis and Insulin Resistance

Nonalcoholic fatty liver disease (NAFLD) is a major contributing factor to hepatic insulin resistance in type 2 diabetes. Diacylglycerol acyltransferase (Dgat), of which there are two isoforms (Dgat1 and Dgat2), catalyzes the final step in triglyceride synthesis. We evaluated the metabolic impact of pharmacological reduction of DGAT1 and -2 expression in liver and fat using antisense oligonucleotides (ASOs) in rats with diet-induced NAFLD. Dgat1 and Dgat2 ASO treatment selectively reduced DGAT1 and DGAT2 mRNA levels in liver and fat, but only Dgat2 ASO treatment significantly reduced hepatic lipids (diacylglycerol and triglyceride but not long chain acyl CoAs) and improved hepatic insulin sensitivity. Because Dgat catalyzes triglyceride synthesis from diacylglycerol, and because we have hypothesized that diacylglycerol accumulation triggers fat-induced hepatic insulin resistance through protein kinase Cϵ activation, we next sought to understand the paradoxical reduction in diacylglycerol in Dgat2 ASO-treated rats. Within 3 days of starting Dgat2 ASO therapy in high fat-fed rats, plasma fatty acids increased, whereas hepatic lysophosphatidic acid and diacylglycerol levels were similar to those of control rats. These changes were associated with reduced expression of lipogenic genes (SREBP1c, ACC1, SCD1, and mtGPAT) and increased expression of oxidative/thermogenic genes (CPT1 and UCP2). Taken together, these data suggest that knocking down Dgat2 protects against fat-induced hepatic insulin resistance by paradoxically lowering hepatic diacylglycerol content and protein kinase Cϵ activation through decreased SREBP1c-mediated lipogenesis and increased hepatic fatty acid oxidation.

Overexpression of uncoupling protein 3 in skeletal muscle protects against fat-induced insulin resistance

Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly associated with obesity. Increased concentrations of intracellular fatty acid metabolites have been postulated to interfere with insulin signaling by activation of a serine kinase cascade involving PKCtheta in skeletal muscle. Uncoupling protein 3 (UCP3) has been postulated to dissipate the mitochondrial proton gradient and cause metabolic inefficiency. We therefore hypothesized that overexpression of UCP3 in skeletal muscle might protect against fat-induced insulin resistance in muscle by conversion of intramyocellular fat into thermal energy. Wild-type mice fed a high-fat diet were markedly insulin resistant, a result of defects in insulin-stimulated glucose uptake in skeletal muscle and hepatic insulin resistance. Insulin resistance in these tissues was associated with reduced insulin-stimulated insulin receptor substrate 1- (IRS-1-) and IRS-2-associated PI3K activity in muscle and liver, respectively. In contrast, UCP3-overexpressing mice were completely protected against fat-induced defects in insulin signaling and action in these tissues. Furthermore, these changes were associated with a lower membrane-to-cytosolic ratio of diacylglycerol and reduced PKCtheta activity in whole-body fat-matched UCP3 transgenic mice. These results suggest that increasing mitochondrial uncoupling in skeletal muscle may be an excellent therapeutic target for type 2 diabetes mellitus.

Dissociation of Inositol-requiring Enzyme (IRE1α)-mediated c-Jun N-terminal Kinase Activation from Hepatic Insulin Resistance in Conditional X-box-binding Protein-1 (XBP1) Knock-out Mice

Background: Endoplasmic reticulum (ER) stress has been implicated in causing hepatic insulin resistance. Results: Fructose-fed XBP1 knock-out mice were protected from hepatic insulin resistance despite increased hepatic ER stress and JNK activation. Conclusion: ER stress and hepatic JNK activation can be disassociated from hepatic insulin resistance. Significance: Hepatic ER stress is not a direct causal factor in hepatic insulin resistance. Hepatic insulin resistance has been attributed to both increased endoplasmic reticulum (ER) stress and accumulation of intracellular lipids, specifically diacylglycerol (DAG). The ER stress response protein, X-box-binding protein-1 (XBP1), was recently shown to regulate hepatic lipogenesis, suggesting that hepatic insulin resistance in models of ER stress may result from defective lipid storage, as opposed to ER-specific stress signals. Studies were designed to dissociate liver lipid accumulation and activation of ER stress signaling pathways, which would allow us to delineate the individual contributions of ER stress and hepatic lipid content to the pathogenesis of hepatic insulin resistance. Conditional XBP1 knock-out (XBP1Δ) and control mice were fed fructose chow for 1 week. Determinants of whole-body energy balance, weight, and composition were determined. Hepatic lipids including triglyceride, DAGs, and ceramide were measured, alongside markers of ER stress. Whole-body and tissue-specific insulin sensitivity were determined by hyperinsulinemic-euglycemic clamp studies. Hepatic ER stress signaling was increased in fructose chow-fed XBP1Δ mice as reflected by increased phosphorylated eIF2α, HSPA5 mRNA, and a 2-fold increase in hepatic JNK activity. Despite JNK activation, XBP1Δ displayed increased hepatic insulin sensitivity during hyperinsulinemic-euglycemic clamp studies, which was associated with increased insulin-stimulated IRS2 tyrosine phosphorylation, reduced hepatic DAG content, and reduced PKCϵ activity. These studies demonstrate that ER stress and IRE1α-mediated JNK activation can be disassociated from hepatic insulin resistance and support the hypothesis that hepatic insulin resistance in models of ER stress may be secondary to ER stress modulation of hepatic lipogenesis.

Cellular mechanisms by which FGF21 improves insulin sensitivity in male mice.

Fibroblast growth factor 21 (FGF21) is a potent regulator of glucose and lipid metabolism and is currently being pursued as a therapeutic agent for insulin resistance and type 2 diabetes. However, the cellular mechanisms by which FGF21 modifies insulin action in vivo are unclear. To address this question, we assessed insulin action in regular chow- and high-fat diet (HFD)-fed wild-type mice chronically infused with FGF21 or vehicle. Here, we show that FGF21 administration results in improvements in both hepatic and peripheral insulin sensitivity in both regular chow- and HFD-fed mice. This improvement in insulin responsiveness in FGF21-treated HFD-fed mice was associated with decreased hepatocellular and myocellular diacylglycerol content and reduced protein kinase Cε activation in liver and protein kinase Cθ in skeletal muscle. In contrast, there were no effects of FGF21 on liver or muscle ceramide content. These effects may be attributed, in part, to increased energy expenditure in the liver and white adipose tissue. Taken together, these data provide a mechanism by which FGF21 protects mice from lipid-induced liver and muscle insulin resistance and support its development as a novel therapy for the treatment of nonalcoholic fatty liver disease, insulin resistance, and type 2 diabetes.

Sensitivity of Lipid Metabolism and Insulin Signaling to Genetic Alterations in Hepatic Peroxisome Proliferator–Activated Receptor-γ Coactivator-1α Expression

OBJECTIVE The peroxisome proliferator–activated receptor-γ coactivator (PGC)-1 family of transcriptional coactivators controls hepatic function by modulating the expression of key metabolic enzymes. Hepatic gain of function and complete genetic ablation of PGC-1α show that this coactivator is important for activating the programs of gluconeogenesis, fatty acid oxidation, oxidative phosphorylation, and lipid secretion during times of nutrient deprivation. However, how moderate changes in PGC-1α activity affect metabolism and energy homeostasis has yet to be determined. RESEARCH DESIGN AND METHODS To identify key metabolic pathways that may be physiologically relevant in the context of reduced hepatic PGC-1α levels, we used the Cre/Lox system to create mice heterozygous for PGC-1α specifically within the liver (LH mice). RESULTS These mice showed fasting hepatic steatosis and diminished ketogenesis associated with decreased expression of genes involved in mitochondrial β-oxidation. LH mice also exhibited high circulating levels of triglyceride that correlated with increased expression of genes involved in triglyceride-rich lipoprotein assembly. Concomitant with defects in lipid metabolism, hepatic insulin resistance was observed both in LH mice fed a high-fat diet as well as in primary hepatocytes. CONCLUSIONS These data highlight both the dose-dependent and long-term effects of reducing hepatic PGC-1α levels, underlining the importance of tightly regulated PGC-1α expression in the maintenance of lipid homeostasis and glucose metabolism.

CGI-58 knockdown sequesters diacylglycerols in lipid droplets/ER-preventing diacylglycerol-mediated hepatic insulin resistance

Comparative gene identification 58 (CGI-58) is a lipid droplet-associated protein that promotes the hydrolysis of triglyceride by activating adipose triglyceride lipase. Loss-of-function mutations in CGI-58 in humans lead to Chanarin–Dorfman syndrome, a condition in which triglyceride accumulates in various tissues, including the skin, liver, muscle, and intestines. Therefore, without adequate CGI-58 expression, lipids are stored rather than used for fuel, signaling intermediates, and membrane biosynthesis. CGI-58 knockdown in mice using antisense oligonucleotide (ASO) treatment also leads to severe hepatic steatosis as well as increased hepatocellular diacylglycerol (DAG) content, a well-documented trigger of insulin resistance. Surprisingly, CGI-58 knockdown mice remain insulin-sensitive, seemingly dissociating DAG from the development of insulin resistance. Therefore, we sought to determine the mechanism responsible for this paradox. Hyperinsulinemic-euglycemic clamp studies reveal that the maintenance of insulin sensitivity with CGI-58 ASO treatment could entirely be attributed to protection from lipid-induced hepatic insulin resistance, despite the apparent lipotoxic conditions. Analysis of the cellular compartmentation of DAG revealed that DAG increased in the membrane fraction of high fat-fed mice, leading to PKCɛ activation and hepatic insulin resistance. However, DAG increased in lipid droplets or lipid-associated endoplasmic reticulum rather than the membrane of CGI-58 ASO-treated mice, and thus prevented PKCɛ translocation to the plasma membrane and induction of insulin resistance. Taken together, these results explain the disassociation of hepatic steatosis and DAG accumulation from hepatic insulin resistance in CGI-58 ASO-treated mice, and highlight the importance of intracellular compartmentation of DAG in causing lipotoxicity and hepatic insulin resistance.

Cellular Mechanism by Which Estradiol Protects Female Ovariectomized Mice From High-Fat Diet-Induced Hepatic and Muscle Insulin Resistance

Estrogen replacement therapy reduces the incidence of type 2 diabetes in postmenopausal women; however, the mechanism is unknown. Therefore, the aim of this study was to evaluate the metabolic effects of estrogen replacement therapy in an experimental model of menopause. At 8 weeks of age, female mice were ovariectomized (OVX) or sham (SHAM) operated, and OVX mice were treated with vehicle (OVX) or estradiol (E2) (OVX+E2). After 4 weeks of high-fat diet feeding, OVX mice had increased body weight and fat mass compared with SHAM and OVX+E2 mice. OVX mice displayed reduced whole-body energy expenditure, as well as impaired glucose tolerance and whole-body insulin resistance. Differences in whole-body insulin sensitivity in OVX compared with SHAM mice were accounted for by impaired muscle insulin sensitivity, whereas both hepatic and muscle insulin sensitivity were impaired in OVX compared with OVX+E2 mice. Muscle diacylglycerol (DAG), content in OVX mice was increased relative to SHAM and OVX+E2 mice. In contrast, E2 treatment prevented the increase in hepatic DAG content observed in both SHAM and OVX mice. Increases in tissue DAG content were associated with increased protein kinase Cε activation in liver of SHAM and OVX mice compared with OVX+E2 and protein kinase Cθ activation in skeletal muscle of OVX mice compared with SHAM and OVX+E2. Taken together, these data demonstrate that E2 plays a pivotal role in the regulation of whole-body energy homeostasis, increasing O(2) consumption and energy expenditure in OVX mice, and in turn preventing diet-induced ectopic lipid (DAG) deposition and hepatic and muscle insulin resistance.

Prevention of Hepatic Steatosis and Hepatic Insulin Resistance by Knockdown of cAMP Response Element-Binding Protein

In patients with poorly controlled type 2 diabetes mellitus (T2DM), hepatic insulin resistance and increased gluconeogenesis contribute to fasting and postprandial hyperglycemia. Since cAMP response element-binding protein (CREB) is a key regulator of gluconeogenic gene expression, we hypothesized that decreasing hepatic CREB expression would reduce fasting hyperglycemia in rodent models of T2DM. In order to test this hypothesis, we used a CREB-specific antisense oligonucleotide (ASO) to knock down CREB expression in liver. CREB ASO treatment dramatically reduced fasting plasma glucose concentrations in ZDF rats, ob/ob mice, and an STZ-treated, high-fat-fed rat model of T2DM. Surprisingly, CREB ASO treatment also decreased plasma cholesterol and triglyceride concentrations, as well as hepatic triglyceride content, due to decreases in hepatic lipogenesis. These results suggest that CREB is an attractive therapeutic target for correcting both hepatic insulin resistance and dyslipidemia associated with nonalcoholic fatty liver disease (NAFLD) and T2DM.