Differential effects of n-3 polyunsaturated fatty acids on metabolic control and vascular reactivity in the type 2 diabetic ob/ob mouse.

Diets rich in monounsaturated fatty acids (MUFA) are recommended for individuals with type 2 diabetes mellitus (T2DM). The American Heart Association recommends increasing intakes of n-3 polyunsaturated fatty acids (PUFA) to reduce the risk of vascular disease in high-risk individuals; however, the long-term effects of these bioactive fatty acids on glucose metabolism in insulin resistance are controversial. The present studies were conducted to evaluate the effects of diets rich in both MUFA and alpha linolenic acid (C18:3n-3, ALA), eicosapentaenoic acid (C20:5n-3, EPA), or docosahexaenoic acid (C22:6n-3, DHA), on glycemic control and other parameters related to vascular health in a mouse model of T2DM and insulin resistance. Male ob/ob mice (n = 15 per treatment) were fed 1 of 4 lipid-modified formula diets (LFDs) for 4 weeks: (1) MUFA control, (2) ALA blend, (3) EPA blend, and (4) DHA blend. A portion of a MUFA-rich lipid blend in the control LFD was replaced with 11% to 14% energy as n-3 PUFA. After 4 weeks, plasma glucose response to a standard meal (1.5 g carbohydrate/kg body weight) and insulin challenge (2 U/kg body weight, IP) was assessed, and samples were collected for analysis of glucose, insulin, and lipids. Vascular reactivity of isolated aortic rings was assessed in an identical follow-up study. The results showed that insulin-resistant mice fed an LFD with EPA and/or DHA blends had significantly (P < .05) lower triglycerides and free fatty acids, but insulin sensitivity and fasting plasma glucose were not improved. However, mice fed with the ALA blend had significantly improved insulin sensitivity when compared to those fed with other LFD (P < .05). Animals fed an LFD with n-3 PUFA from marine or plant sources showed significantly improved vascular responses as compared with the MUFA-rich LFD (E(max), P < .05) and ob/ob reference mice consuming chow (E(max) and pEC(50), P < .05). In summary, long-term consumption of LFD with n-3 PUFAs improved blood lipids and vascular function in an animal model of insulin resistance and T2DM; however, only MUFA-rich LFD with ALA also improved both insulin sensitivity and glycemic responses. Further studies of MUFA-rich LFD with ALA with individuals who have T2DM are warranted.

Profile of past and current clinical trials involving endothelin receptor antagonists: the novel "-sentan" class of drug.

Since its initial characterization in 1988, over 18,236 papers, including 2,485 reviews, have been published in the endothelin (ET) field. Over this period, several generations of selective and mixed (dual) ET receptor antagonists (ERAs), from peptidic backbones to orally active potent (subnanomolar) small molecular compounds, have been developed. These agents have been studied in many experimental animal models of various pathological conditions (cardiovascular, respiratory, and neuro-immunological). Continued basic research has led to a better understanding of the complex interactions between the ET axis and other biologic systems in human pathophysiology. The first clinical trial involved patients with idiopathic pulmonary arterial hypertension and led to approval of bosentan (Tracleer) for use in the United States and Europe in 2002. Since then, bosentan, the only currently approved dual (mixed) ERA, has been used in numerous other clinical trials. In addition, more selective ET(A) receptor antagonists (ambrisentan, atrasentan, avosentan, clazosentan, darusentan, and sitaxsentan) are undergoing clinical trials. Here we outline the ERAs undergoing development and summarize the standing of completed and ongoing trials at the time of the Ninth International Conference on Endothelin and even thereafter. This review is intended to provide a useful reference for those interested in the current state of clinical trials involving ERAs, and to identify lessons that might apply to the design of future trials.

Integrated in vivo pharmacology using an instrumented, unrestrained and conscious rat platform: application to rat models of diabetes.

INTRODUCTION: We define the technical and methodological aspects that led to a practical and reproducible biological in vivo platform allowing the measurement of more than 65 physiobiochemical parameters on a daily basis in freely moving conscious animals. Such a platform presents the ability to unleash incremental information in the hands of modern-day pharmacologists and physiologists. METHODS: To validate this platform, we fully characterized three rat models of Type 1 and Type 2 diabetes and their respective controls. Control, streptozotocin- and alloxan-diabetic Wistar rats in addition to ZDF-Lean and ZDF-Fatty rats were chronically implanted with an arterial catheter and kept in metabolic cages. The catheter was connected to a minipump infusing saline at a constant rate to maintain patency and used to collect blood and measure hemodynamic parameters on a daily basis. RESULTS: Catheter implantation was successful in over 95% of animals and catheter patency was successfully maintained for 30 days in about 75% of animals. The three diabetic rat strains showed elevations in food and water consumption, urinary output, plasma glucose, blood urea nitrogen, triglycerides and cholesterol. The two Type I models also showed a depressed body weight and hemodynamic function. The STZ model differed from the alloxan-model by elevations in liver enzyme activities (AST, ALT, and bilirubin) and a more severe dyslipidemia (triglycerides and total cholesterol). The ZDF-Fatty rats distinguished themselves by higher body weight and elevated white blood cell counts. DISCUSSION: This integrated platform represents a significant improvement in standard in vivo evaluations and could greatly improve the pace of development of potential new drugs.

Endothelin antagonism improves hepatic insulin sensitivity associated with insulin signaling in Zucker fatty rats.

In the present study, we investigated the effects of long-term treatment with the endothelin (ET) antagonist atrasentan, an ET(A)-selective antagonist, on whole body glucose metabolism and insulin signaling in a commonly used model of insulin resistance, the Zucker fatty rat. Zucker lean and fatty rats were maintained for 6 weeks on either control or atrasentan-treated water. Euglycemic-hyperinsulinemic clamps (4 mU/kg per minute) were performed at the end of the 6-week treatment on a subset of rats (n=10/treatment). In another subset (n=5/treatment), an insulin tolerance test was performed; liver and muscle tissues were harvested 10 minutes following the challenge for further analysis. Results of the clamps demonstrated that long-term atrasentan treatment significantly increased whole body glucose metabolism in fatty rats compared with vehicle control subjects. Insulin-induced insulin receptor substrate 1 tyrosine and protein kinase B serine phosphorylation were significantly reduced in the liver and muscle of fatty animals compared with their lean littermates. This reduction was overcome with atrasentan treatment in the liver but not in the muscle. There was no difference between lean and fatty animals, however, in insulin receptor substrate 1 and protein kinase B protein expression in the liver and muscle and no effect by atrasentan. In contrast, expression of the regulatory subunit of PI-3 kinase (p85alpha) was significantly increased in the liver but not in the muscle of fatty animals compared with their lean littermates and this was normalized to levels of lean animals with atrasentan treatment. These findings indicate that long-standing ET antagonism improves whole body glucose metabolism in Zucker fatty rats through improvements in insulin signaling in the liver. These results indicate that therapeutic ET antagonism may assist in correcting the insulin-resistant state.

Metabolic responses with endothelin antagonism in a model of insulin resistance.

Atrasentan, an endothelin antagonist, would have beneficial effects on metabolic responses in a model of insulin resistance. Zucker lean or fatty rats were maintained either on regular (lean and fatty control, n = 12) or atrasentan-treated water (5 mg/kg/d, fatty atrasentan, n = 13) for 6 weeks. There was no significant difference in water intake and body weight with the atrasentan-treated group compared with fatty controls. Although atrasentan had no effect on 3-hour fasting glucose levels, it reduced fasting insulin levels between weeks 2 and 4 of treatment by 53% (fatty control vs fatty atrasentan, P < .01). Atrasentan decreased the incremental area under the plasma glucose response curve ( Delta AUC) after a nutritionally complete meal tolerance test (MTT), by 28% in the atrasentan-treated group compared with fatty controls ( P < .05), and decreased the MTT-induced insulin Delta AUC by 63% in treated animals compared with the fatty control group ( P < .01). In addition, atrasentan significantly decreased the MTT-induced glucose-insulin index Delta AUC by 58% in treated rats compared with fatty controls ( P < .01). In summary, in the Zucker fatty rat, atrasentan significantly reduces (1) 3-hour fasting insulin levels at 4 weeks, (2) glucose and insulin MTT-induced Delta AUCs, and (3) the MTT-induced glucose-insulin index Delta AUC. These results demonstrate an improvement in hyperinsulinemia as well as in glucose tolerance and insulin sensitivity with chronic endothelin antagonism in a model of insulin resistance and suggest that chronic endothelin antagonism may have benefits in the treatment of insulin resistance and/or diabetes.

Metabolic responses in a model of insulin resistance: comparison between oral glucose and meal tolerance tests.

The purpose of this investigation was to compare the benefits of a meal tolerance test (MTT) against those of an oral glucose tolerance test (OGTT) in one of the most commonly used models of insulin resistance, the Zucker fatty rat. Comparison of these two oral challenges will facilitate determination of the most effective means of inducing both glucose and insulin responses in this particular model and allow for possible therapeutic benefits to be examined more effectively. Eight-week-old Zucker fatty rats (n = 7 or 8) were used to perform either an OGTT or a MTT following an overnight fast. The OGTT contained a final amount of carbohydrate (CHO) of 1.2 g/kg body weight (BW). The MTT (commercially available liquid meal), in addition to having fat and protein, included a final amount of available CHO and volume to match the OGTT. A saline-treated group served as control. A greater glucose excursion was observed following the OGTT compared to the MTT. The maximal change in glucose from baseline was 140 +/- 10 mg/dL (a 2.1-fold rise) for the OGTT compared to 86.3 +/- 6.1 mg/dL (a 1.7-fold rise) for the MTT (P <.05). The MTT induced a greater change from baseline in insulin response compared to the OGTT (7.5 +/- 1.1 v 3.9 +/- 0.5 ng/mL, MTT v OGTT, respectively; P <.05). The saline challenge induced only minimal glucose and insulin responses in comparison to the other treatments. These results suggest that, in a model of insulin resistance, the MTT is a more potent insulin stimulator than glucose alone. A mixed meal, such as a MTT, provides a complete nutrient challenge (CHO, fat, and protein) that will induce both glucose and insulin responses, enabling a better capacity to detect differences in one of the most often used models of insulin resistance, the Zucker fatty rat.

Development of insulin resistance and endothelin-1 levels in the Zucker fatty rat.

In order to determine the effects of increasing insulin resistance on endothelin-1 (ET-1) levels, Zucker lean and fatty rats were studied at basal and during a complete nutrient meal tolerance test (MTT) at 7, 12, and 15 weeks of age. The fatty rats were mildly hyperglycemic, severely hyperinsulinemic and glucose-intolerant at all ages versus lean animals and this progressed with age within groups, as previously published. Basal ET-1 levels, at 7 weeks, were significantly increased in fatty versus lean rats (3.2+/-0.5 v 2.0+/-0.3 pg/mL, respectively; P<.05); however, we did not observe any significant basal difference at 12 or 15 weeks. At 7 weeks, ET-1 levels between fatty and lean rats were not different during the MTT (15 minutes: 2.9+/-0.4 v 2.7+/-0.7; 120 minutes: 6.5+/-0.8 v 6.6+/-0.5 pg/mL, fatty v lean, respectively). At 12 weeks, though there was no difference in basal levels, fatty rats had higher ET-1 levels during the MTT compared to lean animals (15 minutes: 6.9+/-1.4 v 1.8+/-0.4; 120 minutes: 9.4+/-1.7 v 3.2+/-0.5 pg/mL, respectively; P<.01). At 15 weeks, ET-1 levels during the MTT receded to levels similar to those observed at 7 weeks, which were significantly higher in fatty versus lean rats 15 minutes following the challenge (3.4+/-0.4 v 2.4+/-0.2 pg/mL, respectively; P<.05). In conclusion, ET-1 levels in the Zucker fatty rat: (1) were increased in the early stages of the progression of insulin resistance at 7 weeks, but were unchanged under basal conditions with age thereafter, and (2) were increased under nutrient challenge conditions with advanced insulin resistance up to 12 weeks, and were still significantly but to a lesser degree increased at 15 weeks of age. The explanation for these results and their relationship to the observed insulin resistance is unclear and will require further investigation.

The kinin system mediates hyperalgesia through the inducible bradykinin B1 receptor subtype: evidence in various experimental animal models of type 1 and type 2 diabetic neuropathy.

Both insulin-dependent (type 1) and insulin-independent (type 2) diabetes are complex disorders characterized by symptomatic glucose intolerance due to either defective insulin secretion, insulin action or both. Unchecked hyperglycemia leads to a series of complications among which is painful diabetic neuropathy, for which the kinin system has been implicated. Here, we review and compare the profile of several experimental models of type 1 and 2 diabetes (chemically induced versus gene-prone) and the incidence of diabetic neuropathy upon aging. We discuss the efficacy of selective antagonists of the inducible bradykinin B1 receptor (BKB1-R) subtype against hyperalgesia assessed by various nociceptive tests. In either gene-prone models of type 1 and 2 diabetes, the incidence of hyperalgesia mostly precedes the development of hyperglycemia. The administration of insulin, achieving euglycemia, does not reverse hyperalgesia. Treatment with a selective BKB1-R antagonist does not affect basal nociception in most normal control rats, whereas it induces a significant time- and dose-dependent attenuation of hyperalgesia, or even restores nociceptive responses, in experimental diabetic neuropathy models. Diabetic hyperalgesia is absent in streptozotocin-induced type 1 diabetic BKB1-R knockout mice. Thus, selective antagonism of the inducible BKB1-R subtype may constitute a novel and potential therapeutic approach for the treatment of painful diabetic neuropathy.