Samy M. Abdel-Halim

Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat

SummaryInsulin secretion and islet glucose metabolism were compared in pancreatic islets isolated from GK/Wistar (GK) rats with spontaneous Type 2 (non-insulin-dependent) diabetes mellitus and control Wistar rats. Islet insulin content was 24.5±3.1 μU/ng islet DNA in GK rats and 28.8±2.5 μU/ng islet DNA in control rats, with a mean (±SEM) islet DNA content of 17.3±1.7 and 26.5±3.4 ng (p < 0.05), respectively. Basal insulin secretion at 3.3 mmol/l glucose was 0.19±0.03 μ · ng islet DNA−1· h−1 in GK rat islets and 0.40±0.07 in control islets. Glucose (16.7 mmol/l) stimulated insulin release in GK rat islets only two-fold while in control islets five-fold. Glucose utilization at 16.7 mmol/l glucose, as measured by the formation of 3H2O from [5-3 H]glucose, was 2.4 times higher in GK rat islets (3.1±0.7 pmol · ng islet DNA−1 · h−1) than in control islets (1.3±0.1 pmol · ng islet DNA−1 · h−1; p<0.05). In contrast, glucose oxidation, estimated as the production of 14CO2 from [U-14C]glucose, was similar in both types of islets and corresponded to 15±2 and 30±3 % (p<0.001) of total glucose phosphorylated in GK and control islets, respectively. Glucose cycling, i. e. the rate of dephosphorylation of the total amount of glucose phosphorylated, (determined as production of labelled glucose from islets incubated with 3H2O) was 16.4±3.4% in GK rat and 6.4±1.0% in control islets, respectively (p<0.01). We conclude that insulin secretion stimulated by glucose is markedly impaired in GK rat islets. Glucose metabolism is also altered in GK rat islets, with diminished ratio between oxidation and utilization of glucose, and increased glucose cycling, suggesting links between impaired glucose-induced insulin release and abnormal glucose metabolism.

Impact of Diabetic Inheritance on Glucose Tolerance and Insulin Secretion in Spontaneously Diabetic GK-Wistar Rats

The impact of genetic factors and maternal diabetes on glucose tolerance and pancreatic β-cell function was studied in first generation (F1) offspring generated in crosses between the spontaneously diabetic Goto-Kakizaki (GK)-Wistar rat and normoglycemic control Wistar rats (W). The (GK × W) F1 hybrids were offspring of either male GK (mGK) and female Wistar (fW) (mGK × fW) or male Wistar (mW) and female GK (fGK) (mW × fGK) rats. Already at 8 days of age, blood glucose levels were elevated in GK (7.6 ± 0.5 vs. 4.8 ± 0.3 mM in W; P < 0.001) and in F1 rats (6.0 ± 0.3 in mGK × fW and 6.6 ± 0.4 mM in mW × fGK; both P < 0.01 vs. W). In 2-month-old male rats, glucose (2 g/kg, intraperitoneally) markedly increased blood glucose levels after 60 min in GK rats (18.1 ± 0.6 vs. 5.5 ± 0.3 mM in W; P < 0.001) and moderately increased levels in F1 rats (9.9 ± 0.9 in mGK × fW and 11.6 ± 1.0 mM in mW × fGK, both P < 0.01 vs. W). Similar patterns were obtained in female rats. Repeated backcrossing of F1 with W rats successively improved glucose tolerance. In perfused pancreases of male rats, the 20-min insulin response to 16.7 mM glucose was −7.44 ± 5.18 pmol in GK rats, 71.57 ± 12.25 pmol in W rats, 9.00 ± 0.89 pmol in mGK × fW rats, and 18.20 ± 3.97 pmol in mW × fGK rats. In female W rats, the glucose-induced insulin response was significantly lower than in males (P < 0.05). However, as in males, insulin responses to glucose were impaired in both GK and F1 female rats. Arginine-induced insulin release was similar in all groups. In mGK × fW, glucose-stimulated somatostatin release was 50% of that in W rats, whereas arginine-stimulated responses of glucagon and somatostatin were not different from W rats. Pancreatic contents of insulin and glucagon were similar in mGK × fW and W rats, whereas somatostatin content was lower in mGK × fW rats (P < 0.05). In conclusion, the diabetic state in GK and F1 rats was evident early in life. Hybrid rats were intermediate between W and GK rats with regard to glucose tolerance and glucose-stimulated insulin response in vitro, but had normal pancreatic insulin content. Results of repeated backcrossing of F1 rats with W rats showed that genes in >1 locus contribute to the diabetic state. Furthermore, the absence of significant differences between impact of maternal and paternal origin of the GK genes for glucose intolerance suggests that hyperglycemia in utero does not influence the severity of diabetes in the F1 offspring.

Glucose Metabolism in Goto-Kakizaki Rat Islets

Islets from Goto-Kakizaki (GK) rats from our colony, despite marked impairment of glucose-induced insulin release, used glucose and produced CO2 at a rate 3 times that of islets from control Wistar rats. Almost all glucose used was accounted for in CO2 and lactate production. The percentages of glucose carbon used collected in CO2 and lactate were similar for control and GK islets. GK islets also oxidized 40% more acetate and leucine to CO2 than did control islets. The fraction of carbon leaving the Krebs cycle relative to CO2 production was the same in GK and control islets. The capacities of mitochondria from GK islets to generate ATP from glutamate and malate were similar and that to generate ATP from succinate and rotenone was somewhat less from GK islets. The reason for the enhanced utilization of substrates by islets of the GK rat is not apparent. In conclusion, there is no decrease in islet glucose utilization, glucose oxidation, Krebs cycle function, or the electron transport system evident from t...

Impaired Coupling of Glucose Signal to the Exocytotic Machinery in Diabetic GK Rats

The GK rat is a spontaneous model of NIDDM. The insulin response to 16.7 mmol/l glucose was markedly impaired in both isolated perfused pancreas and isolated islets from GK rats compared with control Wistar rats. Depolarization with 30 mmol/l KC1 in the presence of 3.3 mmol/l glucose and 250 μmol/l diazoxide induced similar insulin responses in perfused pancreases of GK and control rats. In contrast, the glucose-stimulated insulin release was also severely impaired in GK pancreases in the depolarized state. Forskolin (1 μmol/l) markedly enhanced insulin release at 3.3 mmol/l glucose in GK but not control pancreases (54 ± 15 vs. 3 ± 1 pmol/l0 min, P < 0.001). Dibutyryl cAMP (1 mmol/l) exerted effects similar to forskolin on insulin release in the perfused pancreas. In depolarized pancreases of GK but not control rats, forskolin also induced a marked insulin response at 3.3 mmol/l glucose (163 ± 48 vs. 16 ± 1 pmol/20 min,< P < 0.03). Similarly, in studies on isolated islets from GK rats cultured in 5.5 or 16.7 mmol/l glucose for 48 h, forskolin (5 μmol/l) restored insulin release in response to 16.7 mmol/l glucose but had no effect on islet glucose utilization at 3.3 or 16.7 mmol/l glucose. Forskolin markedly stimulated insulin release at 3.3 mmol/l glucose in GK but not control rat islets cultured for 48 h in 5.5 mmol/l glucose, whereas 20 mmol/l arginine had an almost identical effect in both islet varieties. However, in islets cultured in 16.7 mmol/l glucose, forskolin stimulated insulin release similarly both in control and GK islets at 3.3 mmol/l glucose. In conclusion, this study suggests that the insulinotropic effects of glucose are coupled to a direct regulation of the exocytotic machinery in the pancreatic 3-cell. This pathway is markedly impaired in GK rats, contributing to defective insulin response to glucose. In this model, cAMP generation restores the insulin response to 16.7 mmol/l glucose and exerts a marked insulin release even at 3.3 mmol/l glucose.

Preserved β-Cell Density in the Endocrine Pancreas of Young, Spontaneously Diabetic Goto-Kakizaki (GK) Rats

The Goto-Kakizaki (GK) rat represents a spontaneous animal model of non-insulin-dependent diabetes mellitus (NIDDM) characterized by impaired glucose-stimulated insulin release from the pancreatic β cells. To study whether an alteration in their islet β-cell numbers occurs in parallel with the impairment of insulin secretion in this model, the relative volume density of β cells was determined by means of conventional point sampling in immunostained 4-μm-thick sections of the pancreata from 8-week-old GK rats. The pancreata of nondiabetic Wistar rats were used as control parenchyma. In the GK pancreata the majority of islets was found to have a normal structure; only a few of the islets demonstrated an irregular shape (starfish-shaped islets) with fibrosis. The relative volume of the total endocrine parenchyma was found to be 2.0 ± 0.6% (mean ± SEM) of the whole pancreatic parenchyma in GK rats. In the control rats the corresponding value was 2.3 ± 0.5%. The islet β-cell density was also similar in GK and control rat islets, amounting to 15.2 ± 8.5 and 66.9 ± 6.6%, respectively. Thus, the total relative volume of β cells was 1.5 ± 0.5% in GK rats and 1.6 ± 0.4% in controls. In conclusion, the density of β cells is preserved in the pancreata of the young, diabetic GK rats, suggesting the absence of a causal relationship between the relative pancreatic β-cell volume and the impaired glucose-induced insulin secretion in this NIDDM animal model.

Overexpression of von Hippel-Lindau protein in skeletal muscles of patients with chronic obstructive pulmonary disease

Background: A significant number of patients with chronic obstructive pulmonary disease (COPD) exhibit skeletal muscle wasting and decreased capillary area formation, which correlate with increased mortality. Aim: To determine the molecular mechanisms mediating decreased capillary formation in COPD. Methods: 24 patients with COPD and 12 matching controls were recruited. Patients with COPD were classified into mild, moderate and severe groups according to GOLD (global initiative for chronic obstructive lung disease) criteria. Biopsy specimens were obtained from the tibialis anterior muscle. Fibre typing and capillary formation, together with messenger RNA (mRNA) expression of hypoxia-inducible factors (HIF1α and HIF3α), vascular endothelial growth factors (VEGF-A, VEGF-B and VEGF-C isoforms) and von Hippel-Lindau (VHL) protein, were determined. VHL expression and localisation were further studied by immunohistochemistry. Results: Skeletal muscle capillary formation decreased significantly with increasing disease severity. Compared with controls, a tendency to mRNA overexpression of HIF1α, HIF3α and VEGF isoforms was observed in mild and moderate COPD, which decreased at the severe stage. In contrast, skeletal muscle biopsy samples from patients with COPD exhibited significant overexpression of VHL at both the mRNA and protein level by immunohistochemistry. VHL protein was further determined to be localised to satellite cells. Conclusions: Overexpression of VHL was identified in the skeletal muscle of patients with COPD. Increased VHL activity may have a negative effect on transduction of the hypoxic signal and may contribute to decreased capillarisation in skeletal muscles of patients with COPD.

Adenylyl cyclase isoform expression in non-diabetic and diabetic Goto-Kakizaki (GK) rat pancreas. Evidence for distinct overexpression of type-8 adenylyl cyclase in diabetic GK rat islets

Abstract Glucose-induced insulin release is markedly decreased in the spontaneously diabetic Goto-Kakizaki (GK) rat pancreas. This defect was recently shown to be reversed by forskolin which markedly enhances cAMP generation in GK islets. These effects of forskolin were associated with overexpression of type-3 adenylyl cyclase (AC) mRNA due to the presence of two functional point mutations in the promoter region of AC3 gene in GK rat. Nine AC isoforms have been described, but their expression pattern in relation to the main pancreatic islet cell types, as well as their involvement in the diabetic state, is still unknown. Using antibodies raised against AC1–8, we have studied by double immunofluorescence the localisation of these AC isoforms in different endocrine cell types in both normal and diabetic GK rat pancreas. Our results demonstrated a clear immunoreaction (IR) to AC1–4 and 6 in normal and GK islet β-cells, while a smaller number of ACs were expressed in α- and δ-cells. No AC-IR was observed in pancreatic polypeptide cells. Moreover, we have found an increased IR of the Ca2+-stimulated AC1, AC3 and AC8 in diabetic β- and α-cells, compared with the corresponding IR in control pancreas. Most noticeable was the eliciting of a markedly enhanced AC8-IR in GK rat β- and α-cells, in contrast to a barely discernible AC8-IR in corresponding normal cells. In conclusion, AC expression exhibits a complex pattern in the endocrine pancreas, with specific differences between the normal and diabetic state.

PACAP is expressed in secretory granules of insulin and glucagon cells in human and rodent pancreas. Evidence for generation of cAMP compartments uncoupled from hormone release in diabetic islets.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet neuropeptide with potent insulinotropic action. The current study investigates PACAP expression in normal human and rat pancreatic islets, and whether it is altered in diabetic state. To that end, PACAP immunoreactivity was studied by immunofluorescence methods enhanced by the catalyzed reporter deposition (CARD) technique. Insulin and cyclic adenosine monophosphate (cAMP) generation induced by PACAP were investigated in islets isolated from the spontaneously diabetic Goto-Kakizaki (GK) rat. PACAP immunoreactivity was observed in virtually all insulin and glucagon cells in both species, but not in somatostatin or pancreatic polypeptide (PP) cells; this co-localization pattern was unaltered in diabetic pancreata. In normal human pancreas, PACAP was further localized ultrastructurally to the secretory granules of insulin and glucagon cells. PACAP significantly potentiated glucose-stimulated insulin release in isolated islets of normal but not of GK rats. PACAP failed to enhance cAMP generation in normal islets, but induced approximately 5-folds exaggeration in the diabetic islets. In conclusion, using improved immunocytochemistry techniques and electron microscopy (EM), PACAP was shown to be expressed both in normal and diabetic islet cells and localized to secretory granules of insulin and glucagon cells. Furthermore, the insulinotropic action of PACAP was markedly impaired in diabetic islets in spite of exaggerated cAMP response.

A defective stimulus-secretion coupling rather than glucotoxicity mediates the impaired insulin secretion in the mildly diabetic F1 hybrids of GK-Wistar rats.

Adult F1 hybrids of male GK and female Wistar control rats exhibit mild, spontaneous non-insulin-dependent diabetes mellitus characterized by impaired glucose-induced insulin secretion. Using isolated pancreatic islets of hybrid rats, we first studied whether impaired glucose-induced insulin response is present not only in adult but also in neonatal rats. Furthermore, we investigated whether the impaired glucose-induced insulin response can be restored by long-term normalization of glycemia. Both 1-week- and 2- to 3-month-old hybrid rats had similar body weights but increased fed blood glucose levels (P < 0.05) compared with age-matched control rats. At 5.5 mmol/1 glucose, insulin release was two- to threefold lower in isolated islets of hybrid than in control rats of both age groups (P < 0.05). At 16.7 mmol/1 glucose, insulin secretion from hybrid islets was ∼25% of that from control islets of both 1-week- and 2- to 3-month-old rats. For the second objective, batches of 250 islets from hybrid or control rats were transplanted under the kidney capsule of athymic, normoglycemic nude mice and maintained there for 4 weeks. Perfusion of kidneys demonstrated that glucose-induced (16.7 mmol/l) insulin secretion was impaired markedly in hybrid grafts compared with that in control grafts (0.66 ± 0.23 vs. 1.8 ± 0.38 pmol/20 min; P < 0.01), whereas stimulation by 20 mmol/l arginine resulted in similar insulin responses in both groups. The volumes of the grafted islets were similar in kidneys bearing either control or hybrid islets. In conclusion, in hybrid rats the insulin response to glucose is impaired already at neonatal age. This impairment was not restored by long-term maintenance of the hybrid islets in a normoglycemic environment, whereas the arginine effect was intact. These findings suggest that the primary defect resides in the stimulus-secretion coupling for glucose in the β-cell and that hyperglycemia (glucotoxicity) does not play a major role in sustaining this selective β-cell defect.

Carbachol restores insulin release in diabetic GK rat islets by mechanisms largely involving hydrolysis of diacylglycerol and direct interaction with the exocytotic machinery.

In several models of insulin resistance, cholinergically induced insulin secretion is augmented. We studied here whether this also is present in the spontaneously diabetic GK (Goto–Kakizaki) rat pancreas. Using carbachol (50 &mgr;mol/L), enhanced insulin release was elicited in perfused pancreas under normal or depolarized conditions in GK compared with control rats at 3.3 mmol/L glucose (p < 0.03). Carbachol fully normalized insulin secretion in GK rats at 16.7 mmol/L glucose through an effect abolished by atropine. Similarly, direct stimulation of protein kinase C (PKC) with the DAG-permeable compound 1-oleoyl-2-acetyl-sn-glycerol (OAG, 300 &mgr;mol/L) induced more pronounced insulin release in GK islets than in control islets. The diacylglycerol (DAG) lipase inhibitor RHC-80267 (35 &mgr;mol/L) significantly reduced carbachol effects in control and GK islets, but had no effect on OAG-induced insulin release. The enhanced insulinotropic effects of carbachol in GK islets was not accompanied by increased cyclic adenosine monophosphate (cAMP) or arachidonic acid (AA) formation in GK when compared with control islets. In conclusion, cholinergic stimulation induced enhanced insulin release in diabetic GK islets. This is largely mediated through mechanisms involving hydrolysis of DAG to AA and interaction with exocytotic steps of insulin release.