Glucagon responses to exercise-induced hypoglycaemia are improved by somatostatin receptor type 2 antagonism in a rat model of diabetes.

AIMS/HYPOTHESIS: Regular exercise is at the cornerstone of care in type 1 diabetes. However, relative hyperinsulinaemia and a blunted glucagon response to exercise promote hypoglycaemia. Recently, a selective antagonist of somatostatin receptor 2, PRL-2903, was shown to improve glucagon counterregulation to hypoglycaemia in resting streptozotocin-induced diabetic rats. The aim of this study was to test the efficacy of PRL-2903 in enhancing glucagon counterregulation during repeated hyperinsulinaemic exercise. METHODS: Diabetic rats performed daily exercise for 1 week and were then exposed to saline (154 mmol/l NaCl) or PRL-2903, 10 mg/kg, before hyperinsulinaemic exercise on two separate occasions spaced 1 day apart. In the following week, animals crossed over to the alternate treatment for a third hyperinsulinaemic exercise protocol. RESULTS: Liver glycogen content was lower in diabetic rats compared with control rats, despite daily insulin therapy (p < 0.05). Glucagon levels failed to increase during exercise with saline but increased three-to-six fold with PRL-2903 (all p < 0.05). Glucose concentrations tended to be higher during exercise and early recovery with PRL-2903 on both days of treatment; this difference did not achieve statistical significance (p > 0.05). CONCLUSIONS/INTERPRETATION: PRL-2903 improves glucagon counterregulation during exercise. However, liver glycogen stores or other factors limit the prevention of exercise-induced hypoglycaemia in rats with streptozotocin-induced diabetes.

Exercise in ZDF rats does not attenuate weight gain, but prevents hyperglycemia concurrent with modulation of amino acid metabolism and AKT/mTOR activation in skeletal muscle.

PURPOSE: Protein metabolism is altered in obesity, accompanied by elevated plasma amino acids (AA). Previously, we showed that exercise delayed progression to type 2 diabetes in obese ZDF rats with maintenance of ß cell function and reduction in hyperglucocorticoidemia. We hypothesized that exercise would correct the abnormalities we found in circulating AA and other indices of skeletal muscle protein metabolism. METHODS: Male obese prediabetic ZDF rats (7-10/group) were exercised (swimming) 1 h/day, 5 days/week from ages 6-19 weeks, and compared with age-matched obese sedentary and lean ZDF rats. RESULTS: Food intake and weight gain were unaffected. Protein metabolism was altered in obese rats as evidenced by increased plasma concentrations of essential AA, and increased muscle phosphorylation (ph) of Akt(ser473) (187%), mTOR(ser2448) (140%), eIF4E-binding protein 1 (4E-BP1) (111%), and decreased formation of 4E-BP1*eIF4E complex (75%, 0.01 ≤ p ≤ 0.05 for all measures) in obese relative to lean rats. Exercise attenuated the increase in plasma essential AA concentrations and muscle Akt and mTOR phosphorylation. Exercise did not modify phosphorylation of S6K1, S6, and 4E-BP1, nor the formation of 4E-BP1*eIF4E complex, mRNA levels of ubiquitin or the ubiquitin ligase MAFbx. Positive correlations were observed between ph-Akt and fed circulating branched-chain AA (r = 0.56, p = 0.008), postprandial glucose (r = 0.42, p = 0.04) and glucose AUC during an IPGTT (r = 0.44, p = 0.03). CONCLUSION: Swimming exercise-induced attenuation of hyperglycemia in ZDF rats is independent of changes in body weight and could result in part from modulation of muscle AKT activation acting via alterations of systemic AA metabolism.

Somatostatin receptor type 2 antagonism improves glucagon counterregulation in biobreeding diabetic rats.

Impaired counterregulation during hypoglycemia in type 1 diabetes (T1D) is partly attributable to inadequate glucagon secretion. Intra-islet somatostatin (SST) suppression of hypoglycemia-stimulated α-cell glucagon release plays an important role. We hypothesized that hypoglycemia can be prevented in autoimmune T1D by SST receptor type 2 (SSTR2) antagonism of α-cells, which relieve SSTR2 inhibition, thereby increasing glucagon secretion. Diabetic biobreeding diabetes-prone (BBDP) rats mimic insulin-dependent human autoimmune T1D, whereas nondiabetic BBDP rats mimic prediabetes. Diabetic and nondiabetic rats underwent a 3-h infusion of vehicle compared with SSTR2 antagonist (SSTR2a) during insulin-induced hypoglycemia clamped at 3 ± 0.5 mmol/L. Diabetic rats treated with SSTR2a needed little or no glucose infusion compared with untreated rats. We attribute this effect to SSTR2a restoration of the attenuated glucagon response. Direct effects of SSTR2a on α-cells was assessed by resecting the pancreas, which was cut into fine slices and subjected to perifusion to monitor glucagon release. SSTR2a treatment enhanced low-glucose-stimulated glucagon and corticosterone secretion to normal levels in diabetic rats. SSTR2a had similar effects in vivo in nondiabetic rats and promoted glucagon secretion from nondiabetic rat and human pancreas slices. We conclude that SST contributes to impaired glucagon responsiveness to hypoglycemia in autoimmune T1D. SSTR2a treatment can fully restore hypoglycemia-stimulated glucagon release sufficient to attain normoglycemia in both diabetic and prediabetic stages.

Amelioration of hypoglycemia via somatostatin receptor type 2 antagonism in recurrently hypoglycemic diabetic rats.

Selective antagonism of somatostatin receptor type 2 (SSTR2) normalizes glucagon and corticosterone responses to hypoglycemic clamp in diabetic rats. The purpose of this study was to determine whether SSTR2 antagonism (SSTR2a) ameliorates hypoglycemia in response to overinsulinization in diabetic rats previously exposed to recurrent hypoglycemia. Streptozotocin diabetic rats (n = 19), previously subjected to five hypoglycemia events over 3 days, received an insulin bolus (10 units/kg i.v.) plus insulin infusion (50 mU/kg/min i.v.) until hypoglycemia ensued (≤3.9 mmol/L) (experimental day 1 [Expt-D1]). The next day (Expt-D2), rats were allocated to receive either placebo treatment (n = 7) or SSTR2a infusion (3,000 nmol/kg/min i.v., n = 12) 60 min prior to the same insulin regimen. On Expt-D1, all rats developed hypoglycemia by ∼90 min, while on Expt-D2, hypoglycemia was attenuated with SSTR2a treatment (nadir = 3.7 ± 0.3 vs. 2.7 ± 0.3 mmol/L in SSTR2a and controls, P < 0.01). Glucagon response to hypoglycemia on Expt-D2 deteriorated by 20-fold in the placebo group (P < 0.001) but improved in the SSTR2a group (threefold increase in area under the curve [AUC], P < 0.001). Corticosterone response deteriorated in the placebo-treated rats on Expt-D2 but increased twofold in the SSTR2a group. Catecholamine responses were not affected by SSTR2a. Thus, SSTR2 antagonism after recurrent hypoglycemia improves the glucagon and corticosterone responses and largely ameliorates insulin-induced hypoglycemia in diabetic rats.

Glucagon secretion and signaling in the development of diabetes.

Normal release of glucagon from pancreatic islet α-cells promotes glucose mobilization, which counteracts the hypoglycemic actions of insulin, thereby ensuring glucose homeostasis. In treatment of diabetes aimed at rigorously reducing hyperglycemia to avoid chronic complications, the resulting hypoglycemia triggering glucagon release from α-cells is frequently impaired, with ensuing hypoglycemic complications. This review integrates the physiology of glucagon secretion regulating glucose homeostasis in vivo to single α-cell signaling, and how both become perturbed in diabetes. α-cells within the social milieu of the islet micro-organ are regulated not only by intrinsic signaling events but also by paracrine regulation, particularly by adjacent insulin-secreting ß-cells and somatostatin-secreting δ-cells. We discuss the intrinsic α-cell signaling events, including glucose sensing and ion channel regulation leading to glucagon secretion. We then discuss the complex crosstalk between the islet cells and the breakdown of this crosstalk in diabetes contributing to the dysregulated glucagon secretion. Whereas, there are many secretory products released by ß- and δ-cells that become deficient or excess in diabetes, we discuss the major ones, including the better known insulin and lesser known somatostatin, which act as putative paracrine on/off switches that very finely regulate α-cell secretory responses in health and diabetes. Of note in several type 1 diabetes (T1D) rodent models, blockade of excess somatostatin actions on α-cell could normalize glucagon secretion sufficient to attain normoglycemia in response to hypoglycemic assaults. There has been slow progress in fully elucidating the pathophysiology of the α-cell in diabetes because of the small number of α-cells within an islet and the islet mass becomes severely reduced and inflamed in diabetes. These limitations are just now being surmounted by new approaches.

Insulin's discovery: new insights on its ninetieth birthday.

2012 marks the 90th year since the purification of insulin and the miraculous rescue from death of youngsters with type 1 diabetes. In this review, we highlight several previously unappreciated or unknown events surrounding the discovery. (i) We remind readers of the essential contributions of each of the four discoverers--Banting, Macleod, Collip, and Best. (ii) Banting and Best (each with his own inner circle) worked not only to accrue credit for himself but also to minimize credit to the other discoverers. (iii) Banting at the time of the insulin research was very likely suffering from post-traumatic stress disorder (PTSD) that originated during his heroic service as a surgeon in World War I on the Western Front in 1918, including an infected shrapnel wound that threatened amputation of his arm. His war record along with the newly discovered evidence of a suicide threat goes along with his paranoia, combativeness, alcohol excess, and depression, symptoms we associate with PTSD. (iv) Banting's eureka idea, ligation of the pancreatic duct to preserve the islets, while it energized the early research, was unnecessary and was bypassed early. (v) Post discovery, Macleod uncovered many features of insulin action that he summarized in his 1925 Nobel Lecture. Macleod closed by raising the question--what is the mechanism of insulin action in the body?--a challenge that attracted many talented investigators but remained unanswered until the latter third of the 20th century.

Somatostatin receptor type 2 antagonism improves glucagon and corticosterone counterregulatory responses to hypoglycemia in streptozotocin-induced diabetic rats.

Diminished responsiveness to hypoglycemia contributes to defective counterregulation in diabetes. Pancreatic and/or circulating somatostatin are elevated in diabetes, which may inhibit counterregulatory hormone release during hypoglycemia. Thus, a selective somatostatin receptor type 2 antagonist (SSTR2a) should improve hormone counterregulation to hypoglycemia. Nondiabetic (N) and streptozotocin-induced diabetic (D) rats underwent 4-h infusion of saline or SSTR2a with insulin-induced hypoglycemia clamped at 2.5 ± 0.5 mmol/L. To evaluate the effect of the SSTR2a in the absence of hypoglycemia, rats underwent a 4-h infusion of saline (Ctrl:N, Ctrl:D) or SSTR2a (Ctrl:D+SSTR2a) only. The attenuated glucagon response to hypoglycemia in D (P < 0.0002) was fully restored by SSTR2a (P < 0.0001). Furthermore, the attenuated corticosterone response in D (P < 0.002) was also enhanced by SSTR2a (P < 0.05). In the absence of hypoglycemia, SSTR2a did not alter basal blood glucose levels. D exhibited 62% more pancreatic somatostatin than N after hypoglycemia. In N rats, SSTR2a did not augment the glucagon or corticosterone response to hypoglycemia. Thus, somatostatin may contribute to impaired glucagon responsiveness to hypoglycemia in diabetes. We demonstrate that SSTR2 antagonism enhances hypoglycemia-stimulated glucagon and corticosterone release in D but not in N rats. SSTR2 antagonism does not affect basal glycemia in D rats.

Odyssey between Scylla and Charybdis through storms of carbohydrate metabolism and diabetes: a career retrospective.

This research perspective allows me to summarize some of my work completed over 50 years, and it is organized in seven sections. 1) The treatment of diabetes concentrates on the liver and/or the periphery. We quantified hormonal and metabolic interactions involved in physiology and the pathogenesis of diabetes by developing tracer methods to separate the effects of diabetes on both. We collaborated in the first tracer clinical studies on insulin resistance, hypertriglyceridemia, and the Cori cycle. 2) Diabetes reflects insulin deficiency and glucagon abundance. Extrapancreatic glucagon changed the prevailing dogma and permitted precise exploration of the roles of insulin and glucagon in physiology and diabetes. 3) We established the critical role of glucagon-insulin interaction and the control of glucose metabolism during moderate exercise and of catecholamines during strenuous exercise. Deficiencies of the release and effects of these hormones were quantified in diabetes. We also revealed how acute and chronic hyperglycemia affects the expression of GLUT2 gene and protein in diabetes. 4) We outlined molecular and physiological mechanisms whereby exercise training and repetitive neurogenic stress can prevent diabetes in ZDF rats. 5) We and others established that the indirect effect of insulin plays an important role in the regulation of glucose production in dogs. We confirmed this effect in humans and demonstrated that in type 2 diabetes it is mainly the indirect effect. 6) We indicated that the muscle and the liver protected against glucose changes. 7) We described molecular mechanisms responsible for increased HPA axis in diabetes and for the diminished responses of HPA axis, catecholamines, and glucagon to hypoglycemia. We proposed a new approach to decrease the threat of hypoglycemia.

Regular exercise prevents the development of hyperglucocorticoidemia via adaptations in the brain and adrenal glands in male Zucker diabetic fatty rats.

We determined the effects of voluntary wheel running on the hypothalamic-pituitary-adrenal (HPA) axis, and the peripheral determinants of glucocorticoids action, in male Zucker diabetic fatty (ZDF) rats. Six-week-old euglycemic ZDF rats were divided into Basal, Sedentary, and Exercise groups (n = 8-9 per group). Basal animals were immediately killed, whereas Sedentary and Exercising rats were monitored for 10 wk. Basal (i.e., approximately 0900 AM in the resting state) glucocorticoid levels increased 2.3-fold by week 3 in Sedentary rats where they remained elevated for the duration of the study. After an initial elevation in basal glucocorticoid levels at week 1, Exercise rats maintained low glucocorticoid levels from week 3 through week 10. Hyperglycemia was evident in Sedentary animals by week 7, whereas Exercising animals maintained euglycemia throughout. At the time of death, the Sedentary group had approximately 40% lower glucocorticoid receptor (GR) content in the hippocampus, compared with the Basal and Exercise groups (P < 0.05), suggesting that the former group had impaired negative feedback regulation of the HPA axis. Both Sedentary and Exercise groups had elevated ACTH compared with Basal rats, indicating that central drive of the axis was similar between groups. However, Sedentary, but not Exercise, animals had elevated adrenal ACTH receptor and steroidogenic acute regulatory protein content compared with the Basal animals, suggesting that regular exercise protects against elevations in glucocorticoids by a downregulation of adrenal sensitivity to ACTH. GR and 11beta-hydroxysteroid dehydrogenase type 1 content in skeletal muscle and liver were similar between groups, however, GR content in adipose tissue was elevated in the Sedentary groups compared with the Basal and Exercise (P < 0.05) groups. Thus, the gradual elevations in glucocorticoid levels associated with the development of insulin resistance in male ZDF rats can be prevented with regular exercise, likely because of adaptations that occur primarily in the adrenal glands.

Exercise maintains euglycemia in association with decreased activation of c-Jun NH2-terminal kinase and serine phosphorylation of IRS-1 in the liver of ZDF rats.

Stress-activated systems and oxidative stress are involved in insulin resistance, which, along with beta-cell failure, contribute to the development of type 2 diabetes mellitus (T2DM). Exercise improves insulin resistance and glucose tolerance, and these adaptations may, in part, be related to reductions in inflammation and oxidative stress. We investigated circulating and tissue-specific markers of inflammation and oxidative stress and insulin-signaling pathways in a rodent model of T2DM, the Zucker diabetic fatty rat, with and without voluntary exercise. At 5 wk of age, Zucker diabetic fatty rats (n = 8-9/group) were divided into basal (B), voluntary exercise (E), and sedentary control (S) groups. B rats were euthanized at 6 wk of age, and S and E rats were euthanized 10 wk later. E rats ran approximately 5 km/day, which improved insulin sensitivity and maintained fed and fasted glucose levels and glucose tolerance. Ten weeks of exercise also decreased whole body markers of inflammation and oxidative stress in plasma and liver, including lowered circulating IL-6, haptoglobin, and malondialdehyde levels, hepatic protein oxidation, and phosphorylated JNK, the latter indicating decreased JNK activity. Hepatic phosphoenolpyruvate carboxykinase levels and Ser(307)-phosphorylated insulin receptor substrate-1 were also reduced in E compared with S rats. In summary, we show that, in a rodent model of T2DM, voluntary exercise decreases circulating markers of inflammation and oxidative stress and lowers hepatic JNK activation and Ser(307)-phosphorylated insulin receptor substrate-1. These changes in oxidative stress markers and inflammation are associated with decreased hyperglycemia and insulin resistance and reduced expression of the main gluconeogenic enzyme phosphoenolpyruvate carboxykinase.

Adaptation to intermittent stress promotes maintenance of beta-cell compensation: comparison with food restriction.

Intermittent restraint stress delays hyperglycemia in ZDF rats better than pair feeding. We hypothesized that intermittent stress would preserve beta-cell mass through distinct mechanisms from food restriction. We studied temporal effects of intermittent stress on beta-cell compensation during pre-, early, and late diabetes. Six-week-old obese male ZDF rats were restraint-stressed 1 h/day, 5 days/wk for 0, 3, 6, or 13 wk and compared with age-matched obese ZDF rats that had been food restricted for 13 wk, and 19-wk-old lean ZDF rats. Thirteen weeks of stress and food restriction lowered cumulative food intake 10-15%. Obese islets were fibrotic and disorganized and not improved by stress or food restriction. Obese pancreata had islet hyperplasia and showed evidence of neogenesis, but by 19 wk old beta-cell mass was not increased, and islets had fewer beta-cells that were hypertrophic. Both stress and food restriction partially preserved beta-cell mass at 19 wk old via islet hypertrophy, whereas stress additionally lowered alpha-cell mass. Concomitant with maintenance of insulin responses to glucose, stress delayed the sixfold decline in beta-cell proliferation and reduced beta-cell hypertrophy, translating into 30% more beta-cells per islet after 13 wk. In contrast, food restriction did not improve insulin responses or beta-cell hyperplasia, exacerbated beta-cell hypertrophy, and resulted in fewer beta-cells and greater alpha-cell mass than with stress. Thus, preservation of beta-cell mass with adaptation to intermittent stress is related to beta-cell hyperplasia, maintenance of insulin responses to glucose, and reductions in alpha-cell mass that do not occur with food restriction.

The effect of long-term insulin treatment with and without antecedent hypoglycemia on neuropeptide and corticosteroid receptor expression in the brains of diabetic rats.

We previously demonstrated that while diabetic animals receiving long-term insulin treatment exhibited some impairment in their corticosterone response to hypoglycemia, the stress response to hypoglycemia was completely absent when these animals were subjected to recurrent hypoglycemia. In the current study, we examined potential mechanisms that may contribute to defects in the adrenocortical response to hypoglycemia in long-term insulin-treated diabetic animals exposed to antecedent hypoglycemia. Whereas insulin-treated diabetic animals exhibited a significant rise in corticotrophin-releasing hormone (CRH) mRNA levels during hypoglycemia, exposure to antecedent hypoglycemia completely abolished this response. Moreover, expression of hippocampal mineralocorticoid receptors (MR) mRNA, which normally act to suppress hypothalamo-pituitary-adrenal activity, decreased in the normal control and insulin-treated diabetic groups in response to hypoglycemia, whereas MR mRNA levels remained at baseline in animals subjected to antecedent hypoglycemia. Interestingly, hippocampal glucocorticoid receptor (GR) mRNA levels decreased in all three treatment groups following the hypoglycemic clamp. While GR mRNA levels in the paraventricular nucleus were lower in normal controls following hypoglycemia, this trend just failed to reach statistical significance in the two diabetic groups. These data suggest that (1) recurrent hypoglycemia, much like uncontrolled diabetes, has a pronounced effect on hippocampal mineralocorticoid receptor mRNA expression that may prevent it, and presumably also the stress axis, from responding properly to a subsequent bout of hypoglycemia, and (2) while long-term insulin treatment was sufficient to restore some of these responses in diabetic animals, tighter glycemic control may be necessary to see full restoration of the stress response.

Adaptation to mild, intermittent stress delays development of hyperglycemia in the Zucker diabetic Fatty rat independent of food intake: role of habituation of the hypothalamic-pituitary-adrenal axis.

Hypothalamic-pituitary-adrenal (HPA) axis hyperactivity occurs in type 2 diabetes, and stress is assumed to play a causal role. However, intermittent restraint stress, a model mimicking some mild stressors, delays development of hyperglycemia in Zucker diabetic fatty (ZDF) rats. We examine whether such stress delays hyperglycemia independent of stress-induced reductions in hyperphagia and is due to adaptations in gene expression of HPA-related peptides and receptors that ameliorate corticosteronemia and thus hyperglycemia. ZDF rats were intermittently restraint stressed (1 h/d, 5 d/wk) for 13 wk and compared with obese control, pair fed, and lean ZDF rats. After 13 wk, basal hormones were repeatedly measured over 24 h, and HPA-related gene expression was assessed by in situ hybridization. Although restraint initially induced hyperglycemia, this response habituated over time, and intermittent restraint delayed hyperglycemia. This delay was partly related to 5-15% decreased hyperphagia, which was not accompanied by decreased arcuate nucleus NPY or increased POMC mRNA expression, although expression was altered by obesity. Obese rats demonstrated basal hypercorticosteronemia and greater corticosterone responses to food/water removal. Basal hypercorticosteronemia was further exacerbated after 13 wk of pair feeding during the nadir. Importantly, intermittent restraint further delayed hyperglycemia independent of food intake, because glycemia was 30-40% lower than after 13 wk of pair feeding. This may be mediated by increased hippocampal MR mRNA, reduced anterior pituitary POMC mRNA levels, and lower adrenal sensitivity to ACTH, thus preventing basal and stress-induced hypercorticosteronemia. In contrast, 24-h catecholamines were unaltered. Thus, rather than playing a causal role, intermittent stress delayed deteriorations in glycemia and ameliorated HPA hyperactivity in the ZDF rat.

Oleanolic acid enhances insulin secretion in pancreatic beta-cells.

We investigated the effect of oleanolic acid, a plant-derived triterpenoid, on insulin secretion and content in pancreatic beta-cells and rat islets. Oleanolic acid significantly enhanced insulin secretion at basal and stimulatory glucose concentrations in INS-1 832/13 cells and enhanced acute glucose-stimulated insulin secretion in isolated rat islets. In the cell line the effects of oleanolic acid on insulin secretion were comparable to that of the sulfonylurea tolbutamide at basal glucose levels and with the incretin mimetic Exendin-4 under glucose-stimulated conditions, yet neither Ca(2+) nor cAMP rose in response to oleanolic acid. Chronic treatment with oleanolic acid increased total cellular insulin protein and mRNA levels. These effects may contribute to the anti-diabetic properties of this natural product.

Swim training prevents hyperglycemia in ZDF rats: mechanisms involved in the partial maintenance of beta-cell function.

Exercise improves glucose tolerance in obese rodent models and humans; however, effects with respect to mechanisms of beta-cell compensation remain unexplained. We examined exercise's effects during the progression of hyperglycemia in male Zucker diabetic fatty (ZDF) rats until 19 wk of age. At 6 wk old, rats were assigned to 1) basal--euthanized for baseline values; 2) exercise--swam individually for 1 h/day, 5 days/wk; and 3) controls (n = 8-10/group). Exercise (13 wk) resulted in maintenance of fasted hyperinsulinemia and prevented increases in fed and fasted glucose (P < 0.05) compared with sham-exercised and sedentary controls (P < 0.05). Beta-cell function calculations indicate prolonged beta-cell adaptation in exercised animals alone. During an intraperitoneal glucose tolerance test (IPGTT), exercised rats had lower 2-h glucose (P < 0.05) vs. controls. Area-under-the-curve analyses from baseline for IPGTT glucose and insulin indicate improved glucose tolerance with exercise was associated with increased insulin production and/or secretion. Beta-cell mass increased in exercised vs. basal animals; however, mass expansion was absent at 19 wk in controls (P < 0.05). Hypertrophy and replication contributed to expansion of beta-cell mass; exercised animals had increased beta-cell size and bromodeoxyuridine incorporation rates vs. controls (P < 0.05). The relative area of GLUT2 and protein kinase B was significantly elevated in exercised vs. sedentary controls (P < 0.05). Last, we show formation of ubiquitinated protein aggregates, a response to cellular/oxidative stress, occurred in nonexercised 19 wk-old ZDF rats but not in lean, 6 wk-old basal, or exercised rats. In conclusion, improved beta-cell compensation through increased beta-cell function and mass occurs in exercised but not sedentary ZDF rats and may be in part responsible for improved glucoregulation.

Recurrent intermittent restraint delays fed and fasting hyperglycemia and improves glucose return to baseline levels during glucose tolerance tests in the Zucker diabetic fatty rat--role of food intake and corticosterone.

Short-term elevations of stress hormones cause an increase in glycemia. However, the effect of intermittent stress on development of type 2 diabetes mellitus is unclear. We hypothesized that recurrent intermittent restraint stress would deteriorate glycemia. Male, prediabetic Zucker diabetic fatty (ZDF) rats were restrained 1 hour per day, 5 days per week for 13 weeks and compared with unstressed, age-matched diabetic controls and lean nondiabetic rats. To differentiate the effects of recurrent restraint stress per se vs restraint-induced inhibition of food intake, a pair-fed group of rats was included. Surprisingly, recurrent restraint and pair feeding delayed fed and fasting hyperglycemia, such that they were lowered 50% by restraint and 30% by pair feeding after 13 weeks. Rats that were previously restrained or pair fed had lower glucose levels during a glucose tolerance test, but restraint further improved the return of glucose to baseline compared to pair feeding (P<.05). This was despite pair-fed rats having slightly lowered food intake and body weights compared with restrained rats. Restraint and pair feeding did not alter insulin responses to an intraperitoneal glucose tolerance test (IPGTT) or fasting insulin, and did not lower plasma lipids. Interestingly, restraint normalized basal corticosterone to one third that in control and pair-fed rats, prevented increases in pretreatment corticosterone seen with pair feeding, and led to habituation of restraint-induced corticosterone responses. After 13 weeks of treatment, multiple regression analysis showed that elevations in basal corticosterone could explain approximately 20% of the variance in fed glucose levels. In summary, intermittent restraint and its adaptations delayed hyperglycemia and improved glucose control in Zucker diabetic fatty rats. These benefits can be partially explained by restraint-induced lowering of food intake, but additional improvements compared to pair feeding may involve lower overall corticosterone exposure with repeated restraint. Paradoxically, these novel investigations suggest some types of occasional stress may limit development of diabetes.

Attenuation of type 2 diabetes mellitus in the male Zucker diabetic fatty rat: the effects of stress and non-volitional exercise.

To date, a limited number of studies have investigated the effects of exercise on the maintenance of endocrine pancreatic adaptations to worsening insulin resistance. In particular, the roles of stress hormones that are associated with commonly used forced-exercise paradigms are not fully explained. To examine the effects of exercise per se in ameliorating pancreatic decompensation over time, we investigated the role of forced swimming and sham exercise stress on the development of type 2 diabetes mellitus in the Zucker diabetic fatty (ZDF) rat. Thirty-two male ZDF rats were obtained at 5 weeks of age and all went through a 1-week acclimatization period. They were then divided into 4 groups: basal (euthanized at 6 weeks of age), exercise (1 h/d; 5 d/wk), sham exercise (sham), and non-treated controls (n = 8 per group). After 6 weeks of treatment, an intraperitoneal glucose tolerance test was performed and animals were euthanized for tissue analysis. By 5 weeks of treatment, controls had elevated fed and fasted glycemia (>11.1 and 7.1 mmol/L, respectively; both P < .05), whereas exercise and sham rats remained euglycemic. At euthanasia, there were elevations in fed insulin levels in exercise and sham rats compared with basal animals (both P < .05). Despite improvements in fed and fasting glucose levels in sham rats, glucose tolerance in sham-treated rats (intraperitoneal glucose tolerance test) was similar to controls, whereas glucose levels were similar in exercised trained and basal rats. After 6 weeks, gastrocnemius glycogen content was higher in exercised rats and sham rats when compared with age-matched controls, whereas muscle glucose transporter 4 levels were similar between groups. Compared with controls, the exercise group had increased beta cell proliferation, beta cell mass, and partial maintenance of normal islet morphology. Sham rats also displayed beta cell compensation, as evidenced by increased fasting insulin levels and partial preservation of normal islet morphology. Finally, at the time of euthanasia, plasma corticosterone was increased in sham and control rats but was at basal levels in the exercise group. In summary, both exercise and sham treatment delay the progression of type 2 diabetes mellitus in the male ZDF rat by distinct mechanisms related to pancreatic function and improvements in peripheral glucose disposal.

Ubiquitinated-protein aggregates form in pancreatic beta-cells during diabetes-induced oxidative stress and are regulated by autophagy.

Diabetes-induced oxidative stress can lead to protein misfolding and degradation by the ubiquitin-proteasome system. This study examined protein ubiquitination in pancreatic sections from Zucker diabetic fatty rats. We observed large aggregates of ubiquitinated proteins (Ub-proteins) in insulin-expressing beta-cells and surrounding acinar cells. The formation of these aggregates was also observed in INS1 832/13 beta-cells after exposure to high glucose (30 mmol/l) for 8-72 h, allowing us to further characterize this phenotype. Oxidative stress induced by aminotriazole (ATZ) was sufficient to stimulate Ub-protein aggregate formation. Furthermore, the addition of the antioxidants N-acetyl cysteine (NAC) and taurine resulted in a significant decrease in formation of Ub-protein aggregates in high glucose. Puromycin, which induces defective ribosomal product (DRiP) formation was sufficient to induce Ub-protein aggregates in INS1 832/13 cells. However, cycloheximide (which blocks translation) did not impair Ub-protein aggregate formation at high glucose levels, suggesting that long-lived proteins are targeted to these structures. Clearance of Ub-protein aggregates was observed during recovery in normal medium (11 mmol/l glucose). Despite the fact that 20S proteasome was localized to Ub-protein aggregates, epoxomicin treatment did not affect clearance, indicating that the proteasome does not degrade proteins localized to these structures. The autophagy inhibitor 3MA blocked aggregate clearance during recovery and was sufficient to induce their formation in normal medium. Together, these findings demonstrate that diabetes-induced oxidative stress induces ubiquitination and storage of proteins into cytoplasmic aggregates that do not colocalize with insulin. Autophagy, not the proteasome, plays a key role in regulating their formation and degradation. To our knowledge, this is the first demonstration that autophagy acts as a defense to cellular damage incurred during diabetes.

Effects of insulin treatment without and with recurrent hypoglycemia on hypoglycemic counterregulation and adrenal catecholamine-synthesizing enzymes in diabetic rats.

Untreated diabetic rats show impaired counterregulation against hypoglycemia. The blunted epinephrine responses are associated with reduced adrenomedullary tyrosine hydroxylase (TH) mRNA levels. Recurrent hypoglycemia further impairs epinephrine counterregulation and is also associated with reduced phenylethanolamine N-methyltransferase mRNA. This study investigated the adaptations underlying impaired counterregulation in insulin-treated diabetic rats, a more clinically relevant model. We studied the effects of insulin treatment on counterregulatory hormones and adrenal catecholamine-synthesizing enzymes and adaptations after recurrent hypoglycemia. Groups included: normal; diabetic, insulin-treated for 3 wk (DI); and insulin-treated diabetic exposed to seven episodes (over 4 d) of hyperinsulinemic-hypoglycemia (DI-hypo) or hyperinsulinemic-hyperglycemia (DI-hyper). DI-hyper rats differentiated the effects of hyperinsulinemia from those of hypoglycemia. On d 5, rats from all groups were assessed for adrenal catecholamine-synthesizing enzyme levels or underwent hypoglycemic clamps to examine counterregulatory responses. Despite insulin treatment, fasting corticosterone levels remained increased, and corticosterone responses to hypoglycemia were impaired in DI rats. However, glucagon, epinephrine, norepinephrine, and ACTH counterregulatory defects were prevented. Recurrent hypoglycemia in DI-hypo rats blunted corticosterone but, surprisingly, not epinephrine responses. Norepinephrine and ACTH responses also were not impaired, whereas glucagon counterregulation was reduced due to repeated hyperinsulinemia. Insulin treatment prevented decreases in basal TH protein and increased PNMT and dopamine beta-hydroxylase protein. DI-hypo rats showed increases in TH, PNMT, and dopamine beta-hydroxylase. We conclude that insulin treatment of diabetic rats protects against most counterregulatory defects but not elevated fasting corticosterone and decreased corticosterone counterregulation. Protection against epinephrine defects, both without and with antecedent hypoglycemia, is associated with enhancement of adrenal catecholamine-synthesizing enzyme levels.