Dual Exosite-binding Inhibitors of Insulin-degrading Enzyme Challenge Its Role as the Primary Mediator of Insulin Clearance in Vivo.

Insulin-degrading enzyme (IDE, insulysin) is the best characterized catabolic enzyme implicated in proteolysis of insulin. Recently, a peptide inhibitor of IDE has been shown to affect levels of insulin, amylin, and glucagon in vivo. However, IDE(-/-) mice display variable phenotypes relating to fasting plasma insulin levels, glucose tolerance, and insulin sensitivity depending on the cohort and age of animals. Here, we interrogated the importance of IDE-mediated catabolism on insulin clearance in vivo. Using a structure-based design, we linked two newly identified ligands binding at unique IDE exosites together to construct a potent series of novel inhibitors. These compounds do not interact with the catalytic zinc of the protease. Because one of these inhibitors (NTE-1) was determined to have pharmacokinetic properties sufficient to sustain plasma levels >50 times its IDE IC50 value, studies in rodents were conducted. In oral glucose tolerance tests with diet-induced obese mice, NTE-1 treatment improved the glucose excursion. Yet in insulin tolerance tests and euglycemic clamp experiments, NTE-1 did not enhance insulin action or increase plasma insulin levels. Importantly, IDE inhibition with NTE-1 did result in elevated plasma amylin levels, suggesting the in vivo role of IDE action on amylin may be more significant than an effect on insulin. Furthermore, using the inhibitors described in this report, we demonstrate that in HEK cells IDE has little impact on insulin clearance. In total, evidence from our studies supports a minimal role for IDE in insulin metabolism in vivo and suggests IDE may be more important in helping regulate amylin clearance.

Hepatic and glucagon-like peptide-1-mediated reversal of diabetes by glucagon receptor antisense oligonucleotide inhibitors.

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 beta cell function.

Novel Small Molecule Agonist of TGR5 Possesses Anti-Diabetic Effects but Causes Gallbladder Filling in Mice.

Activation of TGR5 via bile acids or bile acid analogs leads to the release of glucagon-like peptide-1 (GLP-1) from intestine, increases energy expenditure in brown adipose tissue, and increases gallbladder filling with bile. Here, we present compound 18, a non-bile acid agonist of TGR5 that demonstrates robust GLP-1 secretion in a mouse enteroendocrine cell line yet weak GLP-1 secretion in a human enteroendocrine cell line. Acute administration of compound 18 to mice increased GLP-1 and peptide YY (PYY) secretion, leading to a lowering of the glucose excursion in an oral glucose tolerance test (OGTT), while chronic administration led to weight loss. In addition, compound 18 showed a dose-dependent increase in gallbladder filling. Lastly, compound 18 failed to show similar pharmacological effects on GLP-1, PYY, and gallbladder filling in Tgr5 knockout mice. Together, these results demonstrate that compound 18 is a mouse-selective TGR5 agonist that induces GLP-1 and PYY secretion, and lowers the glucose excursion in an OGTT, but only at doses that simultaneously induce gallbladder filling. Overall, these data highlight the benefits and potential risks of using TGR5 agonists to treat diabetes and metabolic diseases.

Hepatic effects of 2-butoxyethanol in rodents.

Chronic inhalation of 2-butoxyethanol resulted in an increase in liver hemangiosarcomas and hepatic carcinomas in male mouse liver. No increase in liver neoplasia was observed in similarly exposed male and female rats or female mice. We proposed that the production of liver neoplasia in the male mouse is the result of oxidative damage secondary to the hemolytic deposition of iron in the liver. This occurs selectively in the male mouse and leads either directly or indirectly to liver neoplasia. To address this proposal, male B6C3F1 mice and male F344 rats were treated with 2-butoxyethanol (via daily gavage; five times per week) at doses of 0, 225, 450, and 900 mg/kg/day (mice) and 0, 225, and 450 mg/kg/day (rats) respectively. Following treatment for 7, 14, 28, and 90 days, DNA synthesis, oxidative damage, hematocrit, and iron deposition were measured in the livers. An increase in hemolysis (measured by a decrease in hematocrit and increase in relative spleen weight) was observed in 2-butoxyethanol-treated rats and mice in a dose-dependent manner. An increase in the percentage of iron-stained Kupffer cells was observed following treatment with 450 and 900 mg/kg of 2-butoxyethanol in mice and 225 and 450 mg/kg of 2-butoxyethanol in rats. A biphasic increase in oxidative damage (8-hydroxydeoxyguanosine and malondialdehyde) was seen in mouse liver after 7 and 90 days of treatment with 2-butoxyethanol, whereas no increases were observed in treated rat liver. Vitamin E levels were reduced by 2-butoxyethanol treatment in both mice and rat liver; however, the basal level of vitamin E was approximately 2.5-fold higher in rat than in mouse liver. A similar biphasic induction of DNA synthesis was seen following 2-butoxyethanol treatment in the mouse. In the mouse liver, increased DNA synthesis was observed in hepatocytes at 90 days and in endothelial cells at 7 and 14 days at all doses. No change in DNA synthesis was seen in 2-butoxyethanol-treated rat liver. No apparent differences in apoptosis and mitosis in the liver were observed in mouse and rat liver between 2-butoxyethanol treatment groups and untreated controls. These results suggest that DNA synthesis, possibly from oxidative stress or Kupffer cell activation, occurs selectively in the mouse liver, primarily in endothelial cells (a target of 2-butoxyethanol neoplasia), following exposure to 2-butoxyethanol.

Acute-phase serum amyloid A as a marker of insulin resistance in mice.

Acute-phase serum amyloid A (A-SAA) was shown recently to correlate with obesity and insulin resistance in humans. However, the mechanisms linking obesity-associated inflammation and elevated plasma A-SAA to insulin resistance are poorly understood. Using high-fat diet- (HFD-) fed mice, we found that plasma A-SAA was increased early upon HFD feeding and was tightly associated with systemic insulin resistance. Plasma A-SAA elevation was due to induction of Saa1 and Saa2 expression in liver but not in adipose tissue. In adipose tissue Saa3 was the predominant isoform and the earliest inflammatory marker induced, suggesting it is important for initiation of adipose tissue inflammation. To assess the potential impact of A-SAA on adipose tissue insulin resistance, we treated 3T3-L1 adipocytes with recombinant A-SAA. Intriguingly, physiological levels of A-SAA caused alterations in gene expression closely resembling those observed in HFD-fed mice. Proinflammatory genes (Ccl2, Saa3) were induced while genes critical for insulin sensitivity (Irs1, Adipoq, Glut4) were down-regulated. Our data identify HFD-fed mice as a suitable model to study A-SAA as a biomarker and a novel possible mediator of insulin resistance.

Specific reduction of hepatic glucose 6-phosphate transporter-1 ameliorates diabetes while avoiding complications of glycogen storage disease.

D-Glucose-6-phosphatase is a key regulator of endogenous glucose production, and its inhibition may improve glucose control in type 2 diabetes. Herein, 2'-O-(2-methoxy)ethyl-modified phosphorothioate antisense oligonucleotides (ASOs) specific to the glucose 6-phosphate transporter-1 (G6PT1) enabled reduction of hepatic D-Glu-6-phosphatase activity in diabetic ob/ob mice. Treatment with G6PT1 ASOs decreased G6PT1 expression, reduced G6PT1 activity, blunted glucagon-stimulated glucose production, and lowered plasma glucose concentration in a dose-dependent manner. In contrast to G6PT1 knock-out mice and patients with glycogen storage disease, excess hepatic and renal glycogen accumulation, hyperlipidemia, neutropenia, and elevations in plasma lactate and uric acid did not occur. In addition, hypoglycemia was not observed in animals during extended periods of fasting, and the ability of G6PT1 ASO-treated mice to recover from an exogenous insulin challenge was not impaired. Together, these results demonstrate that effective glucose lowering by G6PT1 inhibitors can be achieved without adversely affecting carbohydrate and lipid metabolism.

Targeted suppression of an amyloidogenic transthyretin with antisense oligonucleotides.

Transthyretin (TTR) amyloidosis, the most common form of hereditary systemic amyloidosis, is characterized clinically by adult-onset axonal neuropathy and restrictive cardiomyopathy. More than 85 mutations in transthyretin have been found to cause this hereditary disease. Since essentially all circulating TTR is of hepatic origin, orthotopic liver transplantation has been used as the only specific form of therapy. Unfortunately, in many patients amyloid deposition continues after orthotopic liver transplantation, indicating that mutant TTR is no longer required for progression of the disease after tissue deposits have been initiated. As a first step toward medical treatment of this disease, we have employed antisense oligonucleotides (ASOs) to inhibit hepatic expression of TTR. A transgenic mouse model carrying the human TTR Ile84Ser mutation was created and shown to express high levels of human mutant transthyretin. TTR ASOs suppressed hepatic TTR mRNA levels and serum TTR levels by as much as 80%. Suppression of hepatic synthesis of transthyretin may offer a medical treatment for transthyretin systemic amyloidosis.