The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob͞ob and db͞db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA 1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50␣, were increased and PI3-kinase p85␣ expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway is crucial for metabolic responses to insulin, and defects in PI3K signaling have been demonstrated in type 2 diabetes. PTEN (MMAC1) is a lipid/ protein phosphatase that can negatively regulate the PI3K pathway by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate, but it is unclear whether PTEN is physiologically relevant to insulin signaling in vivo. We employed an antisense oligonucleotide (ASO) strategy in an effort to specifically inhibit the expression of PTEN. Transfection of cells in culture with ASO targeting PTEN reduced PTEN mRNA and protein levels and increased insulin-stimulated Akt phosphorylation in ␣-mouse liver-12 (AML12) cells. Systemic administration of PTEN ASO once a week in mice suppressed PTEN mRNA and protein expression in liver and fat by up to 90 and 75%, respectively, and normalized blood glucose concentrations in db/db and ob/ob mice. Inhibition of PTEN expression also dramatically reduced insulin concentrations in ob/ob mice, improved the performance of db/db mice during insulin tolerance tests, and increased Akt phosphorylation in liver in response to insulin. These results suggest that PTEN plays a significant role in regulating glucose metabolism in vivo by negatively regulating insulin signaling.

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Protein tyrosine phosphatase 1B (PTP1B) has been implicated as a negative regulator of insulin action. Overexpression of PTP1B protein has been observed in insulin-resistant states associated with obesity. Mice lacking a functional PTP1B gene exhibit increased insulin sensitivity and are resistant to weight gain. To investigate the role of PTP1B in adipose tissue from obese animals, hyperglycemic obese (ob/ob) mice were treated with PTP1B antisense oligonucleotide (ISIS-113715). A significant reduction in adiposity correlated with a decrease of PTP1B protein levels in fat. Antisense treatment also influenced the triglyceride content in adipocytes, correlating with a downregulation of genes encoding proteins involved in lipogenesis, such as sterol regulatory element-binding protein 1 and their downstream targets spot14 and fatty acid synthase, as well as other adipogenic genes, lipoprotein lipase, and peroxisome proliferator-activated receptor ␥. In addition, an increase in insulin receptor substrate-2 protein and a differential regulation of the phosphatidylinositol 3-kinase regulatory subunit (p85␣) isoforms expression were found in fat from antisense-treated animals, although increased insulin sensitivity measured by protein kinase B phosphorylation was not observed. These results demonstrate that PTP1B antisense treatment can modulate fat storage and lipogenesis in adipose tissue and might implicate PTP1B in the enlargement of adipocyte energy stores and development of obesity. Diabetes 51:2405-2411, 2002 P rotein tyrosine phosphatases play an important role in the regulation of insulin signal transduction, and a number of protein tyrosine phosphatases have been reported to regulate insulin receptor (IR) signaling both under normal conditions and in the insulin-resistant state (1-3). PTP1B has been shown to serve as a negative regulator of IR and IR substrate (IRS)-1 phosphorylation (4,5). In addition, several studies have demonstrated that increased expression of PTP1B occurs in insulin-resistant states associated with obesity (6,7). Mice lacking a functional PTP1B gene exhibit increased insulin sensitivity in liver and skeletal muscle but fail to show increased insulin sensitivity in fat. They are resistant to weight gain on a high-fat diet (8) and are reported to have low adiposity due to a marked reduction in fat cell mass without a decrease in adipocyte number (9). The reason for the obesity resistance observed in PTP1BϪ/Ϫ mice is unclear. Increased leptin sensitivity in PTP1BϪ/Ϫ mice has been suggested as a mechanism for increased energy expenditure (9). Although these reports demonstrate a major role of PTP1B in the modulation of insulin sensitivity in liver and muscle, they fail to address the effect of reducing PTP1B expression in adipose tissue in obese diabetic animals.Recently, we have investigated the effects of PTP1B antisense (ISIS-113715) treatment on insulin resistance and the regulation of insulin signaling in ob/ob diabetic mice. Interestingly, hyperglycemic ob/ob mice as well as db/db mice...

Tumor necrosis factor-␣ (TNF-␣) is a key cytokine involved in the pathogenesis of inflammatory bowel disease. We have developed a second-generation antisense oligonucleotide (ISIS 25302) specific for murine TNF-␣ and have evaluated this oligonucleotide in two models of gut inflammation of distinct etiology. ISIS 25302 decreased TNF-␣ mRNA in a dose-and sequence-dependent manner in vitro in the mouse macrophage cell line P388D1. It also reduced TNF-␣ mRNA in vivo, in whole adipose tissue and in macrophages isolated from the adipose tissue of db/db mice, a strain with constitutively high expression of TNF-␣. ISIS 25302 significantly reduced disease activity index scores in mice with both an acute and a chronic form of dextran sodium sulfate (DSS)-induced colitis. It also significantly improved histopathological scores in interleukin (IL)-10-deficient mice. This was accompanied by reductions in both the basal and lipopolysaccharide-stimulated secretion of TNF-␣ and interferon-␥ in colonic organ cultures from IL-10 Ϫ/Ϫ mice. In this model, efficacy was obtained with both a prophylactic treatment regimen or a therapeutic dosing protocol begun after colitis was already present. In both the DSS and IL-10 Ϫ/Ϫ models, scrambled and mismatch control oligonucleotides were largely without effect, suggesting that ISIS 25302 was exerting its effects through a sequence-dependent antisense mechanism.