Micheline Kergoat

IL-1 antagonism reduces hyperglycemia and tissue inflammation in the type 2 diabetic GK rat

Recent studies suggest an inflammatory process, characterized by local cytokine/chemokine production and immune cell infiltration, regulates islet dysfunction and insulin resistance in type 2 diabetes. However, the factor initiating this inflammatory response is not known. Here, we characterized tissue inflammation in the type 2 diabetic GK rat with a focus on the pancreatic islet and investigated a role for IL-1. GK rat islets, previously characterized by increased macrophage infiltration, displayed increased expression of several inflammatory markers including IL-1β. In the periphery, increased expression of IL-1β was observed primarily in the liver. Specific blockade of IL-1 activity by the IL-1 receptor antagonist (IL-1Ra) reduced the release of inflammatory cytokines/chemokines from GK islets in vitro and from mouse islets exposed to metabolic stress. Islets from mice deficient in IL-1β or MyD88 challenged with glucose and palmitate in vitro also produced significantly less IL-6 and chemokines. In vivo, treatment of GK rats with IL-1Ra decreased hyperglycemia, reduced the proinsulin/insulin ratio, and improved insulin sensitivity. In addition, islet-derived proinflammatory cytokines/chemokines (IL-1β, IL-6, TNFα, KC, MCP-1, and MIP-1α) and islet CD68+, MHC II+, and CD53+ immune cell infiltration were reduced by IL-1Ra treatment. Treated GK rats also exhibited fewer markers of inflammation in the liver. We conclude that elevated islet IL-1β activity in the GK rat promotes cytokine and chemokine expression, leading to the recruitment of innate immune cells. Rather than being directly cytotoxic, IL-1β may drive tissue inflammation that impacts on both β cell functional mass and insulin sensitivity in type 2 diabetes.

Glucose Insensitivity and Amino-acid Hypersensitivity of Insulin Release in Rats with Non-insulin-dependent Diabetes: A Study with the Perfused Pancreas

Non-insulin-dependent diabetes (NIDDM) was obtained in adult rats following a neonatal streptozotocin injection. Rats with NIDDM exhibited slightly lowered plasma insulin, slightly elevated basal plasma glucose values (<200 mg/dl), anancreatic insulin stores (50% of the controls). Insulin secretion was studied in this model using the isolated perfused pancreas technique. Insulin response to glucose stimulation over the range 5.5–22 mM was lacking, thus indicating complete loss of B-cell sensitivity to glucose. Even in presence of theophylline, the B-cells remained insensitive to glucose. In contrast, glyceraldehyde elicited an insulin release as important as that obtained in the control pancreata. This could possibly suggest that the Bcell dysfunction in rats with NIDDM involves a block in glucose metabolism in the early steps of glycolysis prior to the triose-phosphate. Mannose stimulated insulin secretion less in the diabetics than in the controls. The insulin secretion obtained in response to isoproterenol indicated that the ability of the adenylcyclase to generate cAMP in the B-cells of the diabetics was not decreased. The insulinotropic actions of acetylcholine and tolbutamide were normal and increased, respectively, as compared with the controls. In the absence of glucose, the B-cells of the diabetics were unexpectedly hypersensitive to arginine and leucine. The α-ketoisocaproate effect in the diabetics was not significantly different from that obtained in the controls. The possibility that enhancement of insulin response to leucine in the diabetics might be related to a more active conversion of leucine to ketoisocaproate along the first steps of intraislet leucine metabolism is proposed.

The GK Rat Beta-Cell: A Prototype for the Diseased Human Beta-Cell in Type 2 Diabetes?

Increasing evidence indicates that decreased functional beta-cell mass is the hallmark of type 2 diabetes (T2D) mellitus. Nowadays, the debate focuses on the possible mechanisms responsible for abnormal islet microenvironment, decreased beta-cell number, impaired beta-cell function, and their multifactorial aetiologies. This review is aimed to illustrate to what extend the Goto-Kakizaki rat, one of the best characterized animal models of spontaneous T2D, has proved be a valuable tool offering sufficient commonalities to study these aspects. We propose that the defective beta-cell mass and function in the GK model reflect the complex interactions of multiple pathogenic players: (i) several independent loci containing genes responsible for some diabetic traits (but not decreased beta-cell mass); (ii) gestational metabolic impairment inducing an epigenetic programming of the pancreas (decreased beta-cell neogenesis and/or proliferation) which is transmitted to the next generation; and (iii) loss of beta-cell differentiation due to chronic exposure to hyperglycemia/hyperlipidemia, inflammatory mediators, oxidative stress and to perturbed islet microarchitecture.

In Vivo Hepatic and Peripheral Insulin Sensitivity in Rats with Non-insulin-dependent Diabetes Induced by Streptozocin: Assessment with the Insulin-Glucose Clamp Technique

Non-insulin-dependent diabetes mellitus (NIDDM) was obtained in adult female rats byneonatal administration of streptozocin (STZ). At 2 mo of age, the basal plasma glucose values inthe postabsorptive state were elevated, the glucose disappearance rate measured after intravenous(i.v.) glucose load was significantly lower in the diabetic than in control rats, and in vivo glucose-induced insulin release was drastically reduced. To quantify and characterize the in vivo insulin sensitivity in rats with NIDDM, we have used the insulin-glucose clamp technique. The effects of different concentrations of insulin on glucose production, glucose utilization, and glucoseclearance (measured by using 3-3H-glucose) were studied in anesthetized diabetic or control rats while in the postabsorptive state. An inherent condition to set up a valid experimental design was to take into consideration, in the diabetics, the influence of the high blood glucose concentration on glucose uptake and glucose production, since the blood glucose concentration by itself affects these two parameters by a mass action effect independent of insulin. The issue was addressed by evaluating glucose production and utilization in three experimental groups: diabetics clamped at their basal blood glucose level (170 mg/dl), controls clamped at their basal blood glucose level (110 mg/dl), and controls clamped at high blood glucose level (170 mg/dl). In the basal state, glucose production was significantly higher in the diabetics than in controls. When plasma insulin was clamped at submaximal levels (300 μU/ml), the suppression of glucose production was significantly more important in the diabetics than in the two control groups. A similar conclusion was drawn from experiments with maximal insulin levels (4500 μU/ml). In the basal state, glucose utilization was significantly higher in the diabetics as comparedwith the controls. During the clamp experiments, glucose utilization induced by a submaximal insulin level was significantly more important in the diabetics than in controls maintained at euglycemia, but it was found similar to that obtained in controls maintained at hyperglycemia. At maximal insulin level, glucose utilization was similar in the three groups. In these experiments, as well, it was found that the use of the glucose clearance is not an accurate method for the evaluation of insulin action in vivo in the rat, as glucose clearance decreases significantly in the normal rat when blood glucose concentration was increased from 110 to 170 mg/dl at plasma insulin levels of 300 μU/ml or 4500 μU/ml. Thus in the rat, the glucose clearance is not independent of blood glucose concentration. The present results obtained in vivo show that: (1) an increased sensitivity to insulins suppressive effect on hepatic glucose production is a characteristic feature of rats with NIDDM. In connection with previous data by us, this observation is consistent with a postreceptor modification of glucose metabolismin the liver of these rats. (2) The insulin-mediated glucose uptake by peripheral tissues seems to be unaffected in rats with NIDDM.

Islet Endothelial Activation and Oxidative Stress Gene Expression Is Reduced by IL-1Ra Treatment in the Type 2 Diabetic GK Rat

Background Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra). Methodology/Principal Findings Gene expression was analyzed by quantitative RT-PCR on islets isolated from 10-week-old diabetic GK and control Wistar rats. Furthermore, GK rats were treated s.c twice daily with IL-1Ra (Kineret, Amgen, 100 mg/kg/day) or saline, from 4 weeks of age onwards (onset of diabetes). Four weeks later, islet gene analysis and pancreas immunochemistry were performed. Thirty-two genes were selected encoding molecules involved in endothelial cell activation, particularly fibrinolysis, vascular tone, OS, angiogenesis and also inflammation. All genes except those encoding angiotensinogen and epoxide hydrolase (that were decreased), and 12-lipoxygenase and vascular endothelial growth factor (that showed no change), were significantly up-regulated in GK islets. After IL-1Ra treatment of GK rats in vivo, most selected genes implied in endothelium/OS/immune cells/fibrosis were significantly down-regulated. IL-1Ra also improved islet vascularization, reduced fibrosis and ameliorated glycemia. Conclusions/Significance GK rat islets have increased mRNA expression of markers of early islet endothelial cell activation, possibly triggered by several metabolic factors, and also some defense mechanisms. The beneficial effect of IL-1Ra on most islet endothelial/OS/immune cells/fibrosis parameters analyzed highlights a major endothelial-related role for IL-1 in GK islet alterations. Thus, metabolically-altered islet endothelium might affect the β-cell microenvironment and contribute to progressive type 2 diabetic β-cell dysfunction in GK rats. Counteracting islet endothelial cell inflammation might be one way to ameliorate/prevent β-cell dysfunction in type 2 diabetes.

Insulin Treatment Improves Glucose-Induced Insulin Release in Rats With NIDDM Induced by Streptozocin

Insulin-deficient diabetes in humans, as well as in the neonatal streptozocin-induced rat model of non-insulin-dependent diabetes mellitus (NIDDM), are associated with islet β-cell insensitivity to glucose. We hypothesized that the chronic hyperglycemia- -hypoinsulinemia pattern causes this impairment of the glucose influence on insulin secretion. This study was designed to determine whether the glucose defect could be counteracted by normalizing the diabetic state in rats with NIDDM after insulin therapy. Mixte lente insulin (5 U · kg−1 · day−1) was given daily at 1700 h over 24 h or 5 consecutive days. Insulin secretion was studied the morning after the last insulin injection with the isolated perfused pancreas preparation. Fed basal plasma glucose levels decreased in diabetic rats from 183 ± 8 to 136 ± 10 mg/dl after the 1-day insulin treatment and to 135 ± 5 mg/dl after the 5-day insulin treatment (vs. 116 ± 3 mg/dl in control rats). Pancreatic insulin stores were not affected by insulin therapy. Although the 1-day insulin treatment did not modify the lack of glucose response in the diabetic rats, the 5-day insulin treatment improved their glucose-induced insulin secretion. Moreover, insulin therapy improved the priming effect of glucose on a second stimulation with glucose. The return of this glucose effect was hardly detectable after the 1-day insulin therapy but was clearly present after the 5-day treatment. The hyperresponse to arginine, characteristic of the untreated diabetic rat, returned similar to that in controls after a 1-day insulin therapy, and it was again amplified at high glucose levels, although amplification remained lower than that of control rats. This indicates that the potentiating effect of glucose on the response to arginine was regained more precociously than the acute insulin response to glucose after insulin therapy. These data agree with the hypothesis that the chronic hyperglycemia-hypoinsulinemia in the NIDDM rat causes abnormal glucose influence on glucose- and arginine-stimulated insulin release.

Dynamics of Glucose-induced Insulin Release During the Spontaneous Remission of Streptozocin Diabetes Induced in the Newborn Rat

Neonatal rats injected with streptozocin (STZ, 100 mg/kg) at birth exhibited an acute diabetes followed by a spontaneous remission. We have previously shown that this recovery from neonatal diabetes is due to B-cell regeneration and reaccumulation of pancreatic insulin stores starting from 3 to 5 days after birth. The B-cell population during this period is heterogeneous with both surviving B-cells that have escaped the toxic effect of STZ and newly formed B-cells. To evaluate to what extent this B-cell population is functionally normal, we have measured in vitro the dynamics of glucose-induced insulin secretion from pancreatic fragments of rats treated with STZ at birth. The insulin responses were tested at intervals after STZ treatment, from day 1 to day 21 using perifusion of pancreatic fragments, and on day 21 and at 5 mo using perfusion of isolated pancreas. While the glucose-induced insulin release was completely obtunded (2% of the normal response)on day 1 after STZ, it could be demonstrated after day 3. Moreover, it increased as a function of age (6% and 36% of the normal responses on day 5 and day 14, respectively). This restoration of the insulin response to glucose closely paralleled the recovery of pancreatic insulin stores (6% and 51% of normal values, respectively, on day 5 and day 14). In sharp contrast with the lack of glucose response in vitro in the adult, glucose-induced insulin release was still detected on day21 regardless of the in vitro system used (perifusion or perfusion). Furthermore, on day 21 the B-cells of the STZ rats exhibited a tendency toward enhanced insulin response to arginine, which is a prominent feature in adult rats previously treated with STZ at birth. The present observations make it unlikely that STZ treatment per se causes a chronic loss of the glucosesensitive insulin secretion mechanism in the surviving B-cells or in the newly formed B-cells. Moreover, as we have previously shown that in adult rats given a neonatal STZ treatment the B-cells exhibit a selective insensitivity to glucose in vivo, the present data support the notion that the pathogenesis of this lesion is caused by factors other than a primary toxic effect of STZ.

Lack of deterioration of insulin action with aging in the GK rat: A contrasted adaptation as compared with nondiabetic rats

One of the main characteristics of non-insulin-dependent diabetes mellitus (NIDDM) is an alteration of tissue insulin sensitivity, which is also observed during the aging process in the nondiabetic. In this study, we evaluated the influence of age on insulin resistance in a genetic lean model of NIDDM, the Goto-Kakisaki (GK) rat, using the euglycemic-hyperinsulinemic clamp technique at 2, 12, and 18 months of age. In GK rats, basal hyperglycemia (11 mmol/L) and insulinemia, glucose intolerance, and the specific failure of the insulin response to glucose apparent at 2 months of age remained stable until 18 months. Whatever the age, the insulin-suppressive effect on glucose production was significantly less in GK rats than in Wistar rats. The insulin effect on whole-body glucose utilization was decreased at 2 months (15.8 +/- 1.0 mg/min/kg v 23.5 +/- 2.0, P < .001) and was only mildly aggravated between 2 and 18 months (10.3 +/- 0.9 mg/min/kg, P < .05). By contrast, in Wistar control rats, basal insulinemia and the insulin response to glucose markedly increased between 2 and 18 months (2-month delta I v 18-month delta I, 1.4 +/- 0.1 mU/ml.min v 2.9 +/- 0.3, P < .001) and glucose tolerance remained normal. In 18-month-old Wistar rats, the insulin-stimulated glucose utilization rate (GUR) was found to be markedly decreased compared with that of 2-month-old Wistar rats (9.9 +/- 0.8 mg/min/kg v 23.5 +/- 2.0, P < .001), thus demonstrating an age-related decrease of insulin action. In conclusion, we find that there is no major alteration of insulin action due to aging in the GK rat, at variance with the pattern in nondiabetic rodents. It is speculated that such an adaptation in this lean model of NIDDM could be related to the limited capacity of these rats to expand their body weight with age, since it is recognized that body weight gain is largely responsible for the age-related impairment in peripheral insulin action in nondiabetic humans and nondiabetic animal models.

Hypercholesterolaemia, signs of islet microangiopathy and altered angiogenesis precede onset of type 2 diabetes in the Goto–Kakizaki (GK) rat

Aims/hypothesisThe adult non-obese Goto–Kakizaki (GK) rat model of type 2 diabetes, particularly females, carries in addition to hyperglycaemia a genetic predisposition towards dyslipidaemia, including hypercholesterolaemia. As cholesterol-induced atherosclerosis may be programmed in utero, we looked for signs of perinatal lipid alterations and islet microangiopathy. We hypothesise that such alterations contribute towards defective pancreas/islet vascularisation that might, in turn, lead to decreased beta cell mass. Accordingly, we also evaluated islet inflammation and endothelial activation in both prediabetic and diabetic animals.MethodsBlood, liver and pancreas were collected from embryonic day (E)21 fetuses, 7-day-old prediabetic neonates and 2.5-month-old diabetic GK rats and Wistar controls for analysis/quantification of: (1) systemic variables, particularly lipids; (2) cholesterol-linked hepatic enzyme mRNA expression and/or activity; (3) pancreas (fetuses) or collagenase-isolated islet (neonates/adults) gene expression using Oligo GEArray microarrays targeted at rat endothelium, cardiovascular disease biomarkers and angiogenesis, and/or RT-PCR; and (4) pancreas endothelial immunochemistry: nestin (fetuses) or von Willebrand factor (neonates).ResultsSystemic and hepatic cholesterol anomalies already exist in GK fetuses and neonates. Hyperglycaemic GK fetuses exhibit a similar percentage decrease in total pancreas and islet vascularisation and beta cell mass. Normoglycaemic GK neonates show systemic inflammation, signs of islet pre-microangiopathy, disturbed angiogenesis, collapsed vascularisation and altered pancreas development. Concomitantly, GK neonates exhibit elevated defence mechanisms.Conclusions/interpretationThese data suggest an autoinflammatory disease, triggered by in utero programming of cholesterol-induced islet microangiopathy interacting with chronic hyperglycaemia in GK rats. During the perinatal period, GK rats show also a marked deficient islet vascularisation in conjunction with decreased beta cell mass.