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.

Thyroid hormone receptor-ß agonists prevent hepatic steatosis in fat-fed rats but impair insulin sensitivity via discrete pathways.

Liver-specific thyroid hormone receptor-ß (TRß)-specific agonists are potent lipid-lowering drugs that also hold promise for treating nonalcoholic fatty liver disease and hepatic insulin resistance. We investigated the effect of two TRß agonists (GC-1 and KB-2115) in high-fat-fed male Sprague-Dawley rats treated for 10 days. GC-1 treatment reduced hepatic triglyceride content by 75%, but the rats developed fasting hyperglycemia and hyperinsulinemia, attributable to increased endogenous glucose production (EGP) and diminished hepatic insulin sensitivity. GC-1 also increased white adipose tissue lipolysis; the resulting increase in glycerol flux may have contributed to the increase in EGP. KB-2115, a more TRß- and liver-specific thyromimetic, also prevented hepatic steatosis but did not induce fasting hyperglycemia, increase basal EGP rate, or diminish hepatic insulin sensitivity. Surprisingly, insulin-stimulated peripheral glucose disposal was diminished because of a decrease in insulin-stimulated skeletal muscle glucose uptake. Skeletal muscle insulin signaling was unaffected. Instead, KB-2115 treatment was associated with a decrease in GLUT4 protein content. Thus, although both GC-1 and KB-2115 potently treat hepatic steatosis in fat-fed rats, they each worsen insulin action via specific and discrete mechanisms. The development of future TRß agonists must consider the potential adverse effects on insulin sensitivity.

Fasting hyperglycemia is not associated with increased expression of PEPCK or G6Pc in patients with Type 2 Diabetes.

Fasting hyperglycemia in patients with type 2 diabetes mellitus (T2DM) is attributed to increased hepatic gluconeogenesis, which has been ascribed to increased transcriptional expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase, catalytic (G6Pc). To test this hypothesis, we examined hepatic expression of these 2 key gluconeogenic enzymes in 2 rodent models of fasting hyperglycemia and in patients with T2DM. In rats, high-fat feeding (HFF) induces insulin resistance but a robust beta-cell response prevents hyperglycemia. Fasting hyperglycemia was induced in the first rat model by using nicotinamide and streptozotocin to prevent beta-cell compensation, in combination with HFF (STZ/HFF). In a second model, control and HFF rats were infused with somatostatin, followed by portal vein infusion of insulin and glucagon. Finally, the expression of these enzymes was measured in liver biopsy samples obtained from insulin sensitive, insulin resistant, and untreated T2DM patients undergoing bariatric surgery. Rats treated with STZ/HFF developed modest fasting hyperglycemia (119 +/- 4 vs. 153 +/- 6 mg/dL, P < 0.001) and increased rates of endogenous glucose production (EGP) (4.6 +/- 0.6 vs. 6.9 +/- 0.6 mg/kg/min, P = 0.02). Surprisingly, the expression of PEPCK or G6Pc was not increased. Matching plasma insulin and glucagon with portal infusions led to higher plasma glucoses in the HFF rats (147 +/- 4 vs. 161 +/- 4 mg/dL, P = 0.05) with higher rates of EGP and gluconeogenesis. However, PEPCK and G6Pc expression remained unchanged. Finally, in patients with T2DM, hepatic expression of PEPCK or G6Pc was not increased. Thus, in contrast to current dogma, these data demonstrate that increased transcriptional expression of PEPCK1 and G6Pc does not account for increased gluconeogenesis and fasting hyperglycemia in patients with T2DM.

Inhibition of protein kinase Cepsilon 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.

PEPCK1 Antisense Oligonucleotide Prevents Adiposity and Impairs Hepatic Glycogen Synthesis in High-Fat Male Fed Rats.

The increased hepatic gluconeogenesis in type 2 diabetes mellitus has often been ascribed to increased transcription of phosphoenolpyruvate carboxykinase 1, cystolic form (PEPCK1), although recent evidence has questioned this attribution. To assess the metabolic role of PEPCK1, we treated regular chow fed and high-fat fed (HFF) male Sprague-Dawley rats with a 2'-O-methoxyethyl chimeric antisense oligonucleotide (ASO) against PEPCK1 and compared them with control ASO-treated rats. PEPCK1 ASO effectively decreased PEPCK1 expression in the liver and white adipose tissue. In chow fed rats, PEPCK1 ASO did not alter adiposity, plasma glucose, or insulin. In contrast, PEPCK1 ASO decreased the white adipose tissue mass in HFF rats but without altering basal rates of lipolysis, de novo lipogenesis, or glyceroneogenesis in vivo. Despite the protection from adiposity, hepatic insulin sensitivity was impaired in HFF PEPCK1 ASO-treated rats. PEPCK1 ASO worsened hepatic steatosis, although without additional impairments in hepatic insulin signaling or activation of inflammatory signals in the liver. Instead, the development of hepatic insulin resistance and the decrease in hepatic glycogen synthesis during a hyperglycemic clamp was attributed to a decrease in hepatic glucokinase (GCK) expression and decreased synthesis of glycogen via the direct pathway. The decrease in GCK expression was associated with increased expression of activating transcription factor 3, a negative regulator of GCK transcription. These studies have demonstrated that PEPCK1 is integral to coordinating cellular metabolism in the liver and adipose tissue, although it does not directly effect hepatic glucose production or adipose glyceroneogenesis.

Hepatic glucose production pathways after three days of a high-fat diet.

OBJECTIVE: A three-day high-fat diet induces hepatic steatosis and hepatic insulin resistance in rats without altering fasting plasma glucose concentration or the rate of glucose production. However, as the nutrient profile available to the liver is substantially altered by a high-fat diet, we hypothesized that the relative fluxes supporting hepatic glucose production would be altered. MATERIALS/METHODS: To test this hypothesis, we used multiple tracers ([3,4-(13)C(2)]glucose, (2)H(2)O, and [U-(13)C(3)]propionate) followed by NMR analysis of blood glucose to quantify net glucose production and the contributions of glycogen and key gluconeogenesis precursors in 4-5-h fasted rats. RESULTS: NMR analysis demonstrated that the majority of blood glucose was derived from glycogen and the citric acid cycle, while a smaller fraction of glucose was derived from glycerol in both controls and high-fat-fed animals. High-fat feeding was associated with a two-fold increase in plasma glycerol concentration and an increase in the contribution (both fractional and absolute) of glycerol-gluconeogenesis. The increase in gluconeogenesis from glycerol tended to be balanced by a decrease in glycogenolysis. The absolute fluxes associated with the citric acid cycle including gluconeogenesis from the cycle intermediates, pyruvate cycling and the citric acid cycle flux itself, were not altered by this short high-fat diet. CONCLUSIONS: A short term high-fat diet altered the specific pathways for hepatic glucose production without influencing the overall rate of glucose production or flux in the citric acid cycle.

Targeting pyruvate carboxylase reduces gluconeogenesis and adiposity and improves insulin resistance.

We measured the mRNA and protein expression of the key gluconeogenic enzymes in human liver biopsy specimens and found that only hepatic pyruvate carboxylase protein levels related strongly with glycemia. We assessed the role of pyruvate carboxylase in regulating glucose and lipid metabolism in rats through a loss-of-function approach using a specific antisense oligonucleotide (ASO) to decrease expression predominantly in liver and adipose tissue. Pyruvate carboxylase ASO reduced plasma glucose concentrations and the rate of endogenous glucose production in vivo. Interestingly, pyruvate carboxylase ASO also reduced adiposity, plasma lipid concentrations, and hepatic steatosis in high fat-fed rats and improved hepatic insulin sensitivity. Pyruvate carboxylase ASO had similar effects in Zucker Diabetic Fatty rats. Pyruvate carboxylase ASO did not alter de novo fatty acid synthesis, lipolysis, or hepatocyte fatty acid oxidation. In contrast, the lipid phenotype was attributed to a decrease in hepatic and adipose glycerol synthesis, which is important for fatty acid esterification when dietary fat is in excess. Tissue-specific inhibition of pyruvate carboxylase is a potential therapeutic approach for nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes.

Fasting hyperglycemia in the Goto-Kakizaki rat is dependent on corticosterone: a confounding variable in rodent models of type 2 diabetes.

The Goto-Kakizaki (GK) rat is an inbred model of type 2 diabetes (T2D); GK rats are lean but have hyperglycemia and increased gluconeogenesis. However, fasting hyperglycemia in other commonly used rodent models of T2D is associated with increased corticosterone, and thus the underlying mechanism for hyperglycemia differs significantly from T2D in humans. Information regarding corticosterone in the GK rat is not readily available. We studied 14- to 16-week-old GK rats in comparison with age-matched control Wistar-Kyoto (WK) rats. GK rats had lower body weights (WK: 343±10 g vs GK: 286±9 g, P<0.01), but higher plasma glucose concentrations (WK: 132±1.5 mg/dl vs GK: 210±11.7 mg/dl, P<0.01). This was associated with an ∼twofold increase in PEPCK1 expression (P<0.05). However, these findings were also associated with elevations in plasma corticosterone and urinary corticosterone excretion. Ketoconazole (KTZ) treatment in GK rats reduced plasma corticosterone, fasting glucose (GK: 218±15 mg/dl vs GK-KTZ: 135±19 mg/dl, P<0.01) and rates of glucose production [GK: 16.5±0.6 mg/(kg-minute) vs GK-KTZ: 12.2±0.9 mg/(kg-minute), P<0.01]. This was associated with an ∼40% reduction in hepatic PEPCK1 expression as well as a 20% reduction in alanine turnover. Thus, hypercorticosteronemia might contribute to the diabetic phenotype of GK rats and should be considered as a potential confounder in rodent models of T2D.

FGF19 as a postprandial, insulin-independent activator of hepatic protein and glycogen synthesis.

Fibroblast growth factor (FGF) 19 is an enterokine synthesized and released when bile acids are taken up into the ileum. We show that FGF19 stimulates hepatic protein and glycogen synthesis but does not induce lipogenesis. The effects of FGF19 are independent of the activity of either insulin or the protein kinase Akt and, instead, are mediated through a mitogen-activated protein kinase signaling pathway that activates components of the protein translation machinery and stimulates glycogen synthase activity. Mice lacking FGF15 (the mouse FGF19 ortholog) fail to properly maintain blood concentrations of glucose and normal postprandial amounts of liver glycogen. FGF19 treatment restored the loss of glycogen in diabetic animals lacking insulin. Thus, FGF19 activates a physiologically important, insulin-independent endocrine pathway that regulates hepatic protein and glycogen metabolism.