Lindsey Inman

Roles of insulin resistance and beta-cell dysfunction in dexamethasone-induced diabetes.

The roles of insulin resistance and beta-cell dysfunction in glucocorticoid-induced diabetes were determined in Wistar and Zucker (fa/fa) rats. All Wistar rats treated with 5 mg/kg per d of dexamethasone for 24 d exhibited increased beta-cell mass and basal and arginine-stimulated insulin secretion, indicating insulin resistance, but only 16% became diabetic. The insulin response to 20 mM glucose was normal in the perfused pancreas of all normoglycemic dexamethasone-treated rats but absent in every diabetic rat. Immunostainable high Km beta-cell transporter, GLUT-2, was present in approximately 100% of beta-cells of normoglycemic rats, but in only 25% of beta cells of diabetic rats. GLUT-2 mRNA was not reduced. All Zucker (fa/fa) rats treated with 0.2-0.4 mg/kg per d of dexamethasone for 24 d became diabetic and glucose-stimulated insulin secretion was absent in all. High Km glucose transport in islets was 50% below nondiabetic controls. Only 25% of beta cells of diabetic rats were GLUT-2-positive compared with approximately 100% in controls. Total pancreatic GLUT-2 mRNA was increased twofold suggesting a posttranscriptional abnormality. We conclude that dexamethasone induces insulin resistance, whether or not it induces hyperglycemia. Whenever hyperglycemia is present, GLUT-2-positive beta cells are reduced, high Km glucose transport into beta cells is attenuated and the insulin response to glucose is absent.

Defective Fatty Acid-mediated -Cell Compensation in Zucker Diabetic Fatty Rats PATHOGENIC IMPLICATIONS FOR OBESITY-DEPENDENT DIABETES

Although obesity is associated with insulin resistance, most obese humans and rodents remain normoglycemic because of compensatory hyperinsulinemia. This has been attributed to β-cell hyperplasia and increased low K glucose metabolism of islets. Since free fatty acids (FFA) can induce these same β-cell changes in normal islets of Wistar rats and since plasma FFA are increased in obesity, FFA could be the signal from adipocytes that elicits β-cell compensation sufficient to prevent diabetes. To determine if FFA-induced compensation is impaired in islets of rats with a diabetogenic mutation, the Zucker diabetic fatty (ZDF) rat, we cultured islets from 6-week-old obese (fa/fa) rats that had compensated for obesity and apparently normal islets from lean ZDF rats (fa/+) in 0, 1, or 2 mM FFA. Low K glucose usage rose 2.5-fold in FFA-cultured control islets from age-matched Wistar rats, but failed to rise in either the precompensated islets of ZDF rats or in islets of lean ZDF rats. Bromodeoxyuridine incorporation increased 3.2-fold in Wistar islets but not in islets from obese or lean ZDF rats. Insulin secretion doubled in normal islets cultured in 2 mM FFA (p < 0.01) but increased only slightly in islets from lean ZDF rats (not significant) and declined in islets from obese ZDF rats (p < 0.05). We conclude that, unlike the islets of age-matched Wistar rats, islets of 6-week-old heterozygous and homozygous ZDF rats lack the capacity for FFA-induced enhancement of β-cell function.

Caloric restriction in obese pre-diabetic rats prevents beta-cell depletion, loss of beta-cell GLUT 2 and glucose incompetence

SummaryPre-diabetic male Zucker diabetic fatty rats (ZDF) become diabetic between 8 and 10 weeks of age. At that time their beta cells exhibit high basal insulin secretion, absent insulin response to glucose and loss of GLUT 2 glucose transporter. Beta-cell volume, which is increased at the onset of non-insulin-dependent diabetes, declines precipitously by age 18 weeks. To determine if expression of this diabetic phenotype was dependent upon the increased food intake of these rats, they were diet-matched to lean littermates for 12 weeks beginning at 6 weeks of age. Untreated control ZDF rats received an unrestricted diet for 3 months. All of the controls became hyperglycaemic by 8 weeks of age, whereas all diet-matched rats remained euglycaemic throughout the 3 months, despite the fact that at 18 weeks of age their mean body weight equaled that of obese rats on an unrestricted diet. In the former rats glucose-stimulated insulin secretion was absent at 12 weeks of age and GLUT-2-positive beta cells had fallen below 30%. The volume fraction of their beta cells was 2.6 times normal at this age but by 18 weeks of age it had declined by 75%. Diet restriction for 3 months prevented the loss of glucose-stimulated insulin secretion and the reduction of beta-cell GLUT-2 and beta-cell volume fraction. However, neither the elevated basal insulin secretion nor the exaggerated arginine-stimulated insulin secretion of the obese rats was reversed or prevented by caloric restriction. We conclude that in diabetic ZDF rats the glucose incompetence of beta cells and the reduction of beta-cell GLUT 2, which coincide with the onset of hyperglycaemia, and the subsequent loss of beta-cell volume, occur only when the caloric intake is excessive. The increased basal insulin secretion and exaggerated insulin response to arginine appear to be relatively independent of caloric intake.

Severe Diabetes Induced in Subtotally Depancreatized Dogs by Sustained Hyperglycemia

Chronic clamping of plasma glucose levels at ≥250 mg/dl in four partially depancreatized but previously nondiabetic dogs was followed within 2 wk by persistent hyperglycemia and glycosuria of ≤500 g/day, ketonuria, and weight loss. Three of the four dogs required daily insulin injections to control these catabolic manifestations. There was no evidence of spontaneous improvement of the severe diabetic state during the 39–69 days of observation after discontinuation of intravenous glucose infusion. Impairment of intravenous glucose tolerance, loss of the insulin response to glucose and arginine, fasting hyperglucagonemia, exaggerated glucagon responsiveness to arginine, and a significant reduction in sensitivity to insulin were characteristic of all diabetic dogs. Morphometric analysis of the endocrine pancreas revealed a profound reduction in the number and size of identifiable islets of the hyperglycemic dogs compared with islets from their own pancreases resected months earlier and with those from pancreatic remnants of eight subtotally depancreatized control dogs that had not been subjected to chronic hyperglycemic clamping. The reduction in number and size of islets of the hyperglycemic dogs was largely the consequence of depletion of insulin-containing cells and was similar to that of dogs with long-standing alloxan-induced diabetes. In the eight control dogs, clinical evidence of diabetes did not develop during a follow-up period of 193–296 days. In this group, there was no evidence of diminution of intravenous glucose tolerance, of the insulin response to glucose or arginine, or of insulin sensitivity as determined by an acute hyperinsulinemic hyperglycemic clamp. The number and size of islets and number of β-cells in pancreatic remnants from these dogs did not differ morphometrically from those of the pancreatic segment that had been resected. We conclude that in subtotally depancreatized but nondiabetic dogs, maintenance of constant hyperglycemia of ≥ 250 mg/dl by means of intravenous glucose infusion causes a severe, persistent, and often insulin-requiring diabetic state that does not occur in the absence of the hyperglycemia.

GLUT2 Expression and Function in β-cells of GK rats with NIDDM: Dissociation Between Reductions in Glucose Transport and Glucose-Stimulated Insulin Secretion

GLUT2 underexpression has been reported in the +-cells of Zucker diabetic fatty rats and db/db mice, models of spontaneously occurring NIDDM with antecedent obesity. To determine whether the +-cells of a nonobese rodent model of NIDDM exhibit the same abnormalities in GLUT2, we studied Goto-Kakizaki rats. In these mildly diabetic animals glucose-stimulated insulin secretion was reduced at all ages examined from 8 to 48 wk. In normal control Wistar rats, immunostainable GLUT2 was present on all insulin-positive cells in the pancreatic islets. Only 85% of +-cells were GLUT2-positive in GK rats at 12 wk of age, and only 34% were positive at 48 wk of age. GLUT2 mRNA was 50% of normal in 12-wk-old GK rats. In the latter age-group, glucose-stimulated insulin secretion was only 28% of normal at a time when 85% of +-cells were GLUT2-positive and initial 3-O-methyl-D-glucose transport rate was 77% of the control value. We conclude that although GLUT2 is underexpressed, neither the magnitude of the underexpression of GLUT2 nor of the reduction in GLUT2 transport function in islets of GK rats is sufficient by itself to explain the profound reduction in glucose-stimulated insulin secretion.

GLUT-2 function in glucose-unresponsive beta cells of dexamethasone-induced diabetes in rats.

Spontaneous and dexamethasone-induced noninsulin-dependent diabetes mellitus (NIDDM) in rats is associated with loss of glucose-stimulated insulin secretion (GSIS) and a reduction in both GLUT-2-positive beta cells and high Km glucose transport. To determine if the chronology and correlation of these abnormalities is consistent with a causal relationship, Zucker (fa/fa) rats were studied longitudinally before and during 10 d of dexamethasone-induced (0.4 mg/kg per d i.p.) NIDDM. Within 24 h of dexamethasone treatment blood glucose rose and GSIS declined, becoming paradoxically negative (-87 +/- 12 microU/ml per min) on day 10. Blood glucose was negatively correlated with GSIS (r = -0.92; P < 0.001). 3-0-methyl-D-glucose (3MG) transport was unchanged at 12 h, 23% below normal on day 1, and declined further to a nadir 59% below normal. The GLUT-2-positive beta cell area did not decline until 48 h, reaching a nadir of 35% of normal at 10 d. The area of GLUT-2-positive beta cells was correlated with GSIS (r = 0.77; P < 0.005). We conclude that the chronology and correlation between GSIS loss and hyperglycemia is consistent with a cause-effect relationship, but that the subtotal impairment in glucose transport by itself cannot explain the total loss of GSIS if one assumes that normal beta cells are functionally homogenous.

Cocaine-induced alterations in prostaglandin production in rabbit aorta

To determine if alterations in endothelial prostaglandin production occur after long-term cocaine use, 26 New Zealand White rabbits were randomized to a low fat diet with (n = 12) or without (n = 14) daily intravenous cocaine (2 mg/kg body weight). Rabbits were killed at 6 or 12 weeks. Segments of aorta were examined in blinded manner for histologic changes. Additional slices were incubated in oxygenated Krebs buffer and release of 6-keto-prostaglandin F1 alpha, thromboxane B2 and prostaglandin E2 was assayed by radioimmunoassay. Minimal intimal histologic changes were seen in the aorta of three cocaine-treated rabbits. At 12 weeks 6-keto-prostaglandin F1 alpha was increased in the cocaine group (p = 0.063) as compared with levels in the control group. When rabbits killed at 6 and 12 weeks were considered together, increases in thromboxane B2 (p = 0.044) and a trend to increased prostaglandin E2 (p = 0.083) were seen in the cocaine group. The ratio of thromboxane B2 to 6-keto-prostaglandin F1 alpha was increased in the cocaine group compared with that in the control group (p less than 0.02). These data suggest that an increase in prostaglandin production occurs in the vascular endothelium of rabbits ingesting cocaine before gross histologic changes are evident. In addition, thromboxane B2 increases disproportionately with respect to 6-keto-prostaglandin F1 alpha, suggesting that a milieu for thrombosis may exist in users of cocaine.

Effects of hypoglycemia and prolonged fasting on insulin and glucagon gene expression. Studies with in situ hybridization

In situ hybridization of proinsulin and proglucagon mRNA was performed in rat pancreas to assess prohormone gene expression during various glucopenic conditions. During a 4-d fast mean blood glucose declined by 48 mg/dl; proinsulin mRNA signal density remained normal while proglucagon mRNA signal density more than doubled. At the end of a continuous 12-d insulin infusion blood glucose averaged 53 +/- 12 mg/dl; proinsulin mRNA signal density declined to 30% of controls while proglucagon mRNA signal density more than doubled. In insulinoma-bearing NEDH rats blood glucose averaged 34 +/- 3.5 mg/dl; the proinsulin mRNA signal was virtually undetectable and proglucagon mRNA signal density was more than twice the controls. There was no detectable change in either beta-cell area or islet number in rats subjected to fasting or insulin infusion, but in insulinoma-bearing rats beta cell area was markedly reduced. Thus compensation during 4 d of starvation involves an increase in glucagon gene expression without change in insulin gene expression or beta cell mass. In moderate insulin-induced hypoglycemia glucagon gene expression is increased and insulin gene expression decreased. In more profound insulinoma-induced hypoglycemia, in addition to the foregoing changes in hormone gene expression, there is a profound reduction in the number of insulin-expressing cells.

In vitro evidence of neutrophil-mediated lung injury after intestinal reperfusion.

ABSTRACT This study examines the hypothesis that neutrophils isolated from animals sustaining intestinal reperfusion (IIR) induce pulmonary microvascular dysfunction. Lungs were isolated from normal Sprague-Dawley rats and perfused with a physiologic buffer in vitro. Neutrophils (2 × 106) isolated from animals sustaining IIR (n = 5) or sham operation (SHAM; n = 6) were infused into the isolated lung model. A third group of lungs underwent in vitro perfusion without exposure to neutrophils (n = 5). Lung injury was assessed by measuring wet to dry weight ratios and pulmonary artery pressure (PAP). Pulmonary ultrastructure was assessed by electron microscopy. The wet:dry ratio of lungs from animals sustaining IIR was greater than that of lungs exposed to SHAM neutrophils (p = .03) or perfusate alone (p = .02). The PAP of lungs exposed to IIR neutrophils was nearly 10 times greater than that of lungs exposed to SHAM neutrophils (p = .003) or buffer alone (p = .006). Ultrastructural examination of lungs exposed to IIR neutrophils demonstrated interstitial edema with occasional focal disruptions in the alveolar capillary endothelial cell membrane whereas lungs exposed to SHAM neutrophils were normal. These experiments provide important in vitro correlation of prior in vivo studies suggesting that neutrophils are important pathogenic mediators of IIR-induced lung injury.

Effect of truncal vagotomy on parietal cell mass and antral gastrin cell mass in dogs

Vagotomy is known to reduce acid secretion and to increase serum gastrin concentrations. However, there is minimal information on the effect of vagotomy on parietal cell mass or gastrin cell mass. Basal and maximal acid secretions and fasting serum gastrin concentrations were measured in 22 gastric fistula dogs with pyloromyotomy before and up to 56 days following complete bilateral truncal vagotomy (n = 11) or sham vagotomy (n = 11). Dogs underwent total gastrectomy on postoperative days 9 (n = 5 per group) or day 56 (n = 6 per group). Parietal cells were stained with Luxol fast blue and parietal cell mass determined with computer-assisted histomorphometry. Parietal cell mass averaged 10.68 +/- 0.90 billion in control dogs and correlated significantly with maximal acid output (r = 0.76; P less than 0.01). Vagotomy reduced maximal acid output by 40%-50% (P less than 0.001) but had no significant effect on parietal cell mass (8.99 +/- 1.00 billion). Vagotomy increased serum gastrin concentrations significantly, but antral gastrin cell mass in vagotomized dogs (5.66 +/- 1.00 million) was not significantly different than that in control dogs (4.74 +/- 0.50 million). Thus, vagotomy did not lead to parietal cell hypoplasia or gastrin cell hyperplasia despite profound alterations in parietal cell and gastrin cell function.