Current understanding of microRNA (miRNA) biology is limited, and antisense oligonucleotide (ASO) inhibition of miRNAs is a powerful technique for their functionalization. To uncover the role of the liver-specific miR-122 in the adult liver, we inhibited it in mice with a 2'-O-methoxyethyl phosphorothioate ASO. miR-122 inhibition in normal mice resulted in reduced plasma cholesterol levels, increased hepatic fatty-acid oxidation, and a decrease in hepatic fatty-acid and cholesterol synthesis rates. Activation of the central metabolic sensor AMPK was also increased. miR-122 inhibition in a diet-induced obesity mouse model resulted in decreased plasma cholesterol levels and a significant improvement in liver steatosis, accompanied by reductions in several lipogenic genes. These results implicate miR-122 as a key regulator of cholesterol and fatty-acid metabolism in the adult liver and suggest that miR-122 may be an attractive therapeutic target for metabolic disease.

Uncontrolled hepatic glucose production contributes significantly to hyperglycemia in patients with type 2 diabetes. Hyperglucagonemia is implicated in the etiology of this condition; however, effective therapies to block glucagon signaling and thereby regulate glucose metabolism do not exist. To determine the extent to which blocking glucagon action would reverse hyperglycemia, we targeted the glucagon receptor (GCGR) in rodent models of type 2 diabetes using 2′-methoxyethyl-modified phosphorothioate-antisense oligonucleotide (ASO) inhibitors. Treatment with GCGR ASOs decreased GCGR expression, normalized blood glucose, improved glucose tolerance, and preserved insulin secretion. Importantly, in addition to decreasing expression of cAMP-regulated genes in liver and preventing glucagon-mediated hepatic glucose production, GCGR inhibition increased serum concentrations of active glucagon-like peptide-1 (GLP-1) and insulin levels in pancreatic islets. Together, these studies identify a novel mechanism whereby GCGR inhibitors reverse the diabetes phenotype by the dual action of decreasing hepatic glucose production and improving pancreatic β cell function.

Uncontrolled hepatic glucose production contributes significantly to hyperglycemia in patients with type 2 diabetes. Hyperglucagonemia is implicated in the etiology of this condition; however, effective therapies to block glucagon signaling and thereby regulate glucose metabolism do not exist. To determine the extent to which blocking glucagon action would reverse hyperglycemia, we targeted the glucagon receptor (GCGR) in rodent models of type 2 diabetes using 2′-methoxyethyl-modified phosphorothioate-antisense oligonucleotide (ASO) inhibitors. Treatment with GCGR ASOs decreased GCGR expression, normalized blood glucose, improved glucose tolerance, and preserved insulin secretion. Importantly, in addition to decreasing expression of cAMP-regulated genes in liver and preventing glucagon-mediated hepatic glucose production, GCGR inhibition increased serum concentrations of active glucagon-like peptide-1 (GLP-1) and insulin levels in pancreatic islets. Together, these studies identify a novel mechanism whereby GCGR inhibitors reverse the diabetes phenotype by the dual action of decreasing hepatic glucose production and improving pancreatic β cell function.

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...