Specific inhibition of PTEN expression reverses hyperglycemia in diabetic mice.

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 alpha-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.

Protein tyrosine phosphatase 1B reduction regulates adiposity and expression of genes involved in lipogenesis.

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 gamma. In addition, an increase in insulin receptor substrate-2 protein and a differential regulation of the phosphatidylinositol 3-kinase regulatory subunit (p85alpha) 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.

Antisense oligonucleotide blockade of alpha 4 integrin prevents and reverses clinical symptoms in murine experimental autoimmune encephalomyelitis.

We investigated the use of an antisense oligonucleotide (ASO) specific for mRNA of the alpha chain (CD49d) of mouse VLA-4 to down-regulate VLA-4 expression and alter central nervous system (CNS) inflammation. ISIS 17044 potently and specifically reduced CD49d mRNA and protein in cell lines and in ex-vivo-treated primary mouse T cells. When administered prophylactically or therapeutically, ISIS 17044 reduced the incidence and severity of paralytic symptoms in a model of experimental autoimmune encephalomyelitis (EAE). This was accompanied by a significant decrease in the number of VLA-4+ cells, CD4(+) T cells, and macrophages present in spinal cord white matter of EAE mice. ISIS 17044 was found to accumulate in lymphoid tissue of mice, and oligonucleotide was also detected in endothelial cells and macrophage-like cells in the CNS, apparently due to disruption of the blood-brain barrier during EAE. These results demonstrate the potential utility of systemically administered antisense oligonucleotides for the treatment of central nervous system inflammation.

Targeting host E-selectin expression by antisense oligodeoxynucleotides as potential antiendotoxin therapy in vivo.

This study sought to determine if antisense oligodeoxynucleotides would inhibit E-selectin expression, which mediates leukocyte adhesion on endothelial cells, otherwise induced by in vivo endotoxin challenge. Six antisense phosphorothioate oligodeoxynucleotides calculated to bind porcine E-selectin mRNA were tested in porcine aortic endothelial cells. One, ISIS9481, exerted significant inhibition of E-selectin expression induced by tumor necrosis factor-α + endotoxin [lipopolysaccharide (LPS)]. Pigs were challenged with LPS (10 µg/kg) and treated with ISIS9481 (10 mg/kg) (n = 6). Two control groups were used, an antisense inactive in porcine aortic endothelial cells (n = 6) and saline (n = 5), and were combined as control (C = 11). Control pigs challenged with LPS expressed E-selectin in heart, lung, kidneys, and liver, whereas antisense-treated pigs expressed little E-selectin in these tissues. Cardiovascular data indicated that antisense treatment attenuated pathophysiological alterations induced by LPS. Specifically, in control pigs, LPS reduced cardiac output 32% from baseline, increased pulmonary (+116%) and systemic vascular resistances (+16%), and generated neutropenia (from 51,000 at basal to 18,000 polymorphonuclear neutrophils (PMN)/µL after LPS). In antisense-treated pigs, cardiac output decreased only 18%, pulmonary vascular resistance remained unchanged, whereas systemic vascular resistance decreased compared with basal values (-37%). PMN counts remained at 45,000-54,000/µL at 3-4 hours after LPS. These data demonstrate that antisense oligodeoxynucleotides, designed and tested in vitro to interact with 1 gene product, can be developed as either therapeutics or experimental tools in vivo.

Inhibition of protein tyrosine phosphatase-1B with antisense oligonucleotides improves insulin sensitivity and increases adiponectin concentrations in monkeys.

Protein tyrosine phosphatase (PTP)-1B antagonizes insulin signaling and is a potential therapeutic target for insulin resistance associated with obesity and type 2 diabetes. To date, studies of PTP-1B have been limited by the availability of specific antagonists; however, treatment of rodents with antisense oligonucleotides (ASOs) directed against PTP-1B improves insulin sensitivity, inhibits lipogenic gene expression, and reduces triglyceride accumulation in liver and adipose tissue. Here we investigated ASO-mediated PTP-1B inhibition in primates. First, PTP-1B ASO (ISIS 113715) dose-dependently inhibited PTP-1B mRNA and protein expression in cultured monkey hepatocytes. Subcutaneous administration of ISIS 113715 reduced PTP-1B mRNA expression in liver and adipose tissue of normal-weight monkeys by 40-50% and improved insulin sensitivity during an iv glucose tolerance test (IVGTT). In obese, insulin-resistant rhesus monkeys, treatment with 20 mg/kg ISIS 113715 for 4 wk reduced fasting concentrations of insulin and glucose and reduced insulin responses during an IVGTT. In these animals, adiponectin concentrations were also increased by 70%, most of which was an increase of high-molecular-weight oligomers. These effects were not observed in monkeys on a lower, dose-escalation regimen (1-10 mg/kg over 9 wk). Overall, the increase of adiponectin concentrations during ISIS 113715 treatment was correlated with the lowering of insulin responses during IVGTT (r = -0.47, P = 0.042). These results indicate that inhibition of PTP-1B with ASOs such as ISIS 113715 may be a viable approach for the treatment and prevention of obesity-associated insulin resistance and type 2 diabetes because they potently increase adiponectin concentrations in addition to improving insulin sensitivity.

Antisense oligonucleotide blockade of tumor necrosis factor-alpha in two murine models of colitis.

Tumor necrosis factor-alpha (TNF-alpha) 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-alpha and have evaluated this oligonucleotide in two models of gut inflammation of distinct etiology. ISIS 25302 decreased TNF-alpha mRNA in a dose- and sequence-dependent manner in vitro in the mouse macrophage cell line P388D1. It also reduced TNF-alpha 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-alpha. 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-alpha and interferon-gamma in colonic organ cultures from IL-10 -/- 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 -/- models, scrambled and mismatch control oligonucleotides were largely without effect, suggesting that ISIS 25302 was exerting its effects through a sequence-dependent antisense mechanism.

Evaluation of C-5 propynyl pyrimidine-containing oligonucleotides in vitro and in vivo.

Inclusion of C-5 propynyl pyrimidines in phosphorothioate antisense oligonucleotides (ASOs) has been shown to significantly increase their potency for inhibiting gene expression in vitro. This increased potency is believed to be the result of enhanced binding affinity to target RNA. Our results show that C-5 propynyl pyrimidine-modified oligonucleotides caused an increase in the melting temperature (T(m)) of both oligodeoxynucleotides (ODNs) and 2'-O-(2-methoxy)ethyl (2'-MOE)-modified oligonucleotides. The in vitro data show a moderate increase in potency for an antisense oligodeoxynucleotide containing C-5 propynyl pyrimidines targeting the murine PTEN (MMAC1) transcript. Second-generation 2'-MOE chimeric ASOs containing C-5 propynyl pyrimidines showed no improvement in potency in PTEN target reduction in vitro or in vivo compared to their nonpropyne-modified parent. These results suggest that increasing affinity for target RNA beyond that achieved with the 2'-MOE modification does not further increase potency in cell-based assays. To evaluate whether this observation held true for in vivo applications, we evaluated both compounds in mice. We were unable to establish a dose-response relationship with C-5 propynyl pyrimidine-modified ODNs because of severe toxicity. The toxicity was characterized by mortality in animals receiving 50 mg/kg and an increase in infiltrating cells and apoptotic cells in livers of mice receiving 20 mg/kg. C-5 propynyl pyrimidine-modified chimeric oligonucleotides exhibited decreased hepatotoxicity compared with C-5 propynyl-modified ODNs but did not exhibit an increase in potency compared with unmodified chimeric oligonucleotides. The hepatotoxicity could be further limited if incorporation of propynyl pyrimidines was restricted to 2'-MOE nucleosides.

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice.

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db 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 p50alpha, were increased and PI3-kinase p85alpha 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.