Madeline M. Butler

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 HbA1C. 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 p50α, were increased and PI3-kinase p85α 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.

Evaluation of the Renal Effects of an Antisense Phosphorothioate Oligodeoxynucleotide in Monkeys

Antisense phosphorothioate oligodeoxynucleotides are therapeutic agents that provide target specificity resulting from Watson-Crick base pairing. However, there are nonspecific effects that in some instances result in toxicity. These compounds accumulate in the kidney and induce renal proximal tubular degeneration at high doses. The relationship between accumulation of phosphorothioate oligodeoxynucleotides in the kidney, indicators of renal toxicity, and histomorphology were investigated in rhesus monkeys. Monkeys received vehicle or an escalating dose regimen of 3, 10, 40, and 80 mg/kg of ISIS 2105 and were then evaluated for changes in clinical pathology indices, urinalysis parameters, and renal histopathology. Urinalysis revealed an increase in protein levels and a slight increase in blood content following the third 40 mg/kg dose and continuing through the 80 mg/kg doses, whereas other urinary markers of renal toxicity were unchanged. Creatinine clearance was slightly decreased in monkeys during the 80 mg/kg dose cycle. Granulation in the cytoplasm of proximal tubular epithelial cells was evident by microscopic examination of kidney and was present at all doses examined and increased with dose. Immunohistochemical staining localized the oligodeoxynucleotide within these granules. Histopathologic changes consisting of minimal to moderate tubular degeneration were present only at the higher doses of 40 and 80 mg/kg and at high tissue concentrations, and these changes occurred concurrent with functional alterations, whereas lower doses (≤10 mg/kg) did not affect a pathologic or functional change.

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.

Histological Localization of Phosphorothioate Oligodeoxynucleotides in Normal Rodent Tissue

Abstract The distribution of phosphorothioate oligodeoxynucleotides (P=S ODN) in rodent tissues was studied in vivo using three histological methods: direct fluorescence microscopy; immunohistochemistry; and autoradiography. All three methods gave essentially the same pattern of oligonucleotide localization in the tissues studied, and the histological results correlate well with those from radiochemical and biochemical studies of P=S ODN distribution.

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.