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 PKCe 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 PKCe 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.nonalcoholic fatty liver disease | type 2 diabetes N onalcoholic fatty liver disease (NAFLD) is now the most common chronic liver disease in the United States and is strongly associated with hepatic insulin resistance and type 2 diabetes (1, 2). Although NAFLD is characterized by excessively high triglycerides in the liver, the triglycerides do not appear to be detrimental to hepatic insulin sensitivity (3, 4). Rather, other lipid moieties, such as diacylglycerols (DAG) and ceramides, have been implicated as the molecular triggers of insulin resistance (5-7). The mechanism whereby these lipids cause insulin resistance are diverse: DAGs cause insulin resistance through activation of PKCe in liver, leading to the inhibition of insulin-receptor kinase activity (8, 9), and PKCθ in skeletal muscle, leading to insulinreceptor substrate-1 serine phosphorylation on sites that interfere with insulin action (10-12). Ceramides have been proposed to inhibit AKT2 activation by either activating PP2A, which dephosphorylates and deactivates AKT2, or activating PKCζ, which phosphorylates AKT on an inhibitory residue and prevents its translocation to the plasma ...

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.

Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. Whereas systemic GC inhibition can alleviate hyperglycemia in rodents and humans, it results in adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal axis. In the present study, we used optimized antisense oligonucleotides (ASOs) to cause selective reduction of the glucocorticoid receptor (GCCR) in liver and white adipose tissue (WAT) and evaluated the resultant changes in glucose and lipid metabolism in several rodent models of diabetes. Treatment of ob/ob mice with GCCR ASOs for 4 weeks resulted in ϳ75 and ϳ40% reduction in GCCR mRNA expression in liver and WAT, respectively. This was accompanied by ϳ65% decrease in fed and ϳ30% decrease in fasted glucose levels, a 60% decrease in plasma insulin concentration, and ϳ20 and 35% decrease in plasma resistin and tumor necrosis factor-␣ levels, respectively. Furthermore, GCCR ASO reduced hepatic glucose production and inhibited hepatic gluconeogenesis in liver slices from basal and dexamethasone-treated animals. In db/db mice, a similar reduction in GCCR expression caused ϳ40% decrease in fed and fasted glucose levels and ϳ50% reduction in plasma triglycerides. In ZDF and high-fat diet-fed streptozotocin-treated (HFD-STZ) rats, GCCR ASO treatment caused ϳ60% reduction in GCCR expression in the liver and WAT, which was accompanied by a 40 -70% decrease in fasted glucose levels and a robust reduction in plasma triglyceride, cholesterol, and free fatty acids. No change in circulating corticosterone levels was seen in any model after GCCR ASO treatment. To further demonstrate that GCCR ASO does not cause systemic GC antagonism, normal Sprague-Dawley rats were challenged with dexamethasone after treating with GCCR ASO. Dexamethasone increased the expression of GCresponsive genes such as PEPCK in the liver and decreased circulating lymphocytes. GCCR ASO treatment completely inhibited the increase in dexamethasoneinduced PEPCK expression in the liver without causing any change in the dexamethasone-induced lymphopenia. These studies demonstrate that tissue-selective GCCR antagonism with ASOs may be a viable therapeutic strategy for the treatment of the metabolic syndrome.

To investigate the role of low molecular weight proteintyrosine phosphatase (LMW-PTP) in glucose metabolism and insulin action, a specific antisense oligonucleotide (ASO) was used to reduce its expression both in vitro and in vivo. Reduction of LMW-PTP expression with the ASO in cultured mouse hepatocytes and in liver and fat tissues of diet-induced obese (DIO) mice and ob/ob mice led to increased phosphorylation and activity of key insulin signaling intermediates, including insulin receptor-␤ subunit, phosphatidylinositol 3-kinase, and Akt in response to insulin stimulation. The ASO-treated DIO and ob/ob animals showed improved insulin sensitivity, which was reflected by a lowering of both plasma insulin and glucose levels and improved glucose and insulin tolerance in DIO mice. The treatment did not decrease body weight or increase metabolic rate. These data demonstrate that LMW-PTP is a key negative regulator of insulin action and a potential novel target for the treatment of insulin resistance and type 2 diabetes.The incidence of diabetes has been steadily increasing and has become a major public health concern. Over 85% of diabetic patients have type 2 diabetes. Obesity, which can result from a sedentary life style and high calorie diet, is a major risk factor for the development of this disorder (1). A hallmark of type 2 diabetes is insulin resistance, characterized by a decreased insulin response in a variety of tissues (2), including liver, fat, and muscle. Therefore, increasing insulin sensitivity is a practical strategy for the treatment of type 2 diabetes.Insulin initiates its physiological response by binding to its membrane-bound receptor (IR, a ␣ 2 ␤ 2 -heterotetramer protein), 2 which causes autophosphorylation of the ␤-subunit and receptor activation, resulting in subsequent phosphorylation of its two major downstream substrates, IRS-1 and IRS-2 (3-5). Phosphorylated IRS-1 and -2 interact with and activate other SH2 domain-containing adapter molecules such as NCK2, Grb2, Shc, Syp (4 -8), and the regulatory subunit (p85) of phosphatidylinositol 3-kinase (PI3-K) (9, 10). Activated PI3-K stimulates Akt (or protein kinase B) that in turn phosphorylates and inactivates glycogen synthase kinase-3 (11), resulting in activation of glycogen synthase (12), thereby increasing the utilization of glucose for glycogen synthesis. In fat and muscle, activation of this pathway also causes transfer of GLUT4 from the cytoplasm to the cell membrane, resulting in increased glucose uptake (13). Therefore, IR-IRS-1/2-PI3-K-Akt signaling cascade is a key pathway in mediating the effects of insulin action on blood glucose levels. A number of studies have established a role for intracellular phosphatases in the negative regulation of insulin signaling (14 -16) such as protein-tyrosine phosphatase (PTP) 1B that negatively regulates insulin action through dephosphorylating tyrosine-phosphorylated IR (15, 16). Insulin sensitivity was enhanced in PTP1B knock-out mice, in which increased tyrosine phosphorylation of IR was found...

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.