The incretin hormones GIP and GLP-1 in diabetic rats: effects on insulin secretion and small bowel motility.

Incretin hormones often display inhibitory actions on gut motility. The aim of this study was to investigate if altered responsiveness to glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) as regards insulin release and small bowel motility could bring further clarity to the pathophysiology of diabetes in the Goto-Kakizaki (GK) rat. The isolated perfused pancreas was studied in male GK and Wistar rats (controls) under euglycemic and hyperglycemic conditions. Glucose-dependent insulinotropic peptide (10 nmol L(-1)) or GLP-1 (10 nmol L(-1)) were added to the medium and perfusate was collected and analysed for insulin. Moreover, GK and Wistar rats were supplied with bipolar electrodes in the small bowel and myoelectric activity was recorded during intravenous administration of GIP (1-400 pmol kg(-1) min(-1)) or GLP-1 (0.1-20 pmol kg(-1) min(-1)). Finally, tissue was collected from GK and Wistar rats for RNA extraction. Under euglycemia, GIP and GLP-1 stimulated the initial insulin response by 10-fold in GK rats (P < 0.05). At later hyperglycemia, the insulin response to GIP and GLP-1 was blunted to about one-third compared with controls (P < 0.05). In the bowel GLP-1 was about 2.6-16.7 times more potent than GIP in abolishing the migrating myoelectric complex in the GK and control rats. Polymerase chain reaction (PCR) showed GIP and GLP-1 receptor gene expression in pancreatic islets and in small bowel. The initially high, but later low insulin responsiveness to stimulation with GIP and GLP-1 along with inhibition of small bowel motility in the GK rat indicates a preserved incretin response on motility in diabetes type 2.

Physical performance and muscular characteristics in different stages of COPD.

This study has examined exercise capacity and muscle morphology in patients with different severities of chronic obstructive pulmonary disease (COPD). Twenty-three patients and 12 healthy matched controls were recruited. Based on the severity of airflow obstruction, patients were divided into two subgroups. Exercise capacity was determined using a 6-min walk test. Muscle fiber composition, fiber area and number of satellite cells/muscle fiber were determined in muscle biopsies using immunohistochemistry. A progressive decline in exercise capacity was noted with ascending disease severity. Furthermore, a correlation between reduction in exercise capacity and changes in muscle fiber composition was observed in COPD. The group with severe and very severe COPD had a lower proportion of type I and a higher proportion of type IIa fibers compared with the other groups. In severe and very severe COPD, a reduction in fiber area of type IIa fibers was also seen. The number of satellite cells/muscle fiber did not differ between the groups. In conclusion, a decline in exercise capacity occurs already in mild and moderate COPD, indicating that the 6-min walk test is a reliable indicator of disease severity. Furthermore, changes in skeletal muscle morphology are associated with disease severity while muscle regenerative capacity is not altered.

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 (HIF1alpha and HIF3alpha), 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 HIF1alpha, HIF3alpha 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.

Removal of lead ions from industrial waste water by different types of natural materials.

The adsorption capacity of some natural materials for lead such as animal bone powder, active carbon, Nile rose plant powder, commercial carbon and ceramics was studied. The V/m ratio has been chosen to be 500 ml/g. The adsorption process was affected by various parameters such as contact time, pH and concentration of lead solution. The lead uptake percent reaches equilibrium state after 15, 30, 45 and 120 min for bone powder, active carbon, plant powder and commercial carbon, respectively. The uptake percent of lead increased by increasing pH value. The sequence of lead uptake percent (% adsorption) at constant pH from certain concentration of lead nitrate solution by the different natural materials is in the order: bone powder > active carbon > plant powder > commercial carbon. The uptake percent of lead is increased by decreasing the concentration of lead at constant pH. The capacity of lead adsorbed from nitrate solution by the different natural materials increased by increasing pH value. The synthetic and industrial waste-water samples were treated by using the different natural materials (contact time 3h, pH = 4). The percent removal of lead was 100% by bone powder, 90% by active carbon, 80% by plant powder and 50% by commercial carbon. There was no removal of lead by ceramics. This may be due to the presence of high percent of lead in the constituent of ceramics (372 mg/g).

Proinsulin processing in the diabetic Goto-Kakizaki rat.

The biosynthesis and processing of proinsulin was investigated in the diabetic Goto-Kakizaki (GK) rat. Immunofluorescence microscopy comparing GK and Wistar control rat pancreata revealed marked changes in the distribution of alpha-cells and pronounced beta-cell heterogeneity in the expression patterns of insulin, prohormone convertases PC1, PC2, carboxypeptidase E (CPE) and the PC-binding proteins 7B2 and ProSAAS. Western blot analyses of isolated islets revealed little difference in PC1 and CPE expression but PC2 immunoreactivity was markedly lower in the GK islets. The processing of the PC2-dependent substrate chromogranin A was reduced as evidenced by the appearance of intermediates. No differences were seen in the biosynthesis and post-translational modification of PC1, PC2 or CPE following incubation of islets in 16.7 mM glucose, but incubation in 3.3 mM glucose resulted in decreased PC2 biosynthesis in the GK islets. The rates of biosynthesis, processing and secretion of newly synthesized (pro)insulin were comparable. Circulating insulin immunoreactivity in both Wistar and GK rats was predominantly insulin 1 and 2 in the expected ratios with no (pro)insulin evident. Thus, the marked changes in islet morphology and PC2 expression did not impact the rate or extent of proinsulin processing either in vitro or in vivo in this experimental model.

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 micromol/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 micromol/L) induced more pronounced insulin release in GK islets than in control islets. The diacylglycerol (DAG) lipase inhibitor RHC-80267 (35 micromol/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.

Differential effects of glucagon-like peptide-1 (7-36)amide versus cholecystokinin on arginine-induced islet hormone release in vivo and in vitro.

We compared the effects of two incretin hormones, glucagon-like peptide-1 (7-36)amide (GLP-1) and cholecystokinin (CCK), on islet hormone secretion. GLP-1 strongly potentiated glucose-stimulated insulin secretion in the perfused rat pancreas and in vivo in mice (p < 0.001). In contrast, GLP-1 did not enhance arginine-induced insulin release under these experimental conditions. In the perfused rat pancreas, GLP-1 also potentiated glucose-stimulated somatostatin secretion but, again, had no effect on arginine-induced somatostatin release. However, GLP-1 promptly inhibited the arginine-induced glucagon release (p < 0.02). In contrast, CCK enhanced insulin release in response to arginine both in the perfused rat pancreas and in vivo in mice (p < 0.001). In conclusion, GLP-1, in contrast to CCK, failed to enhance arginine-induced insulin release both in vitro and in vivo. This suggests that a signal generated by nutrient metabolism is required for the potentiation of insulin secretion by GLP-1. Furthermore, GLP-1 directly inhibited arginine-induced glucagon release as no concurrent increase in insulin or somatostatin release was noted.

Glucose enhances adenylyl cyclase responses in normal but not diabetic GK rat islets.

In the GK rat model of type 2 diabetes, adenylyl cyclase (AC) expression and stimulation are increased. Whether the prevalent glucose level has any effects on AC responses is, however, unclear. We have studied concurrent insulin release and cyclic adenosine monophosphate (cAMP) generation in response to 5 microM forskolin in islets cultured for 48 hours in 5.5 or 11 mM glucose. Insulin release was impaired in GK rat islets, irrespective of culture condition, in response to 3.3 and 16.7 mM glucose and was fully restored by forskolin through exaggerated insulin responses. Stimulation of normal islets with 5 microM forskolin elicited different islet cAMP responses, which were dependent on the dose of glucose in the culture medium. Thus in normal islets cultured in 11 mM glucose, forskolin increased cAMP levels fivefold to sixfold at 3.3 and 16.7 mM glucose, whereas forskolin increased cAMP levels only twofold in islets cultured at 5.5 mM glucose. In GK islets, forskolin induced a consistently exaggerated approximately eightfold increase in cAMP generation irrespective of glucose concentration in the culture medium. In conclusion, culturing normal islets at hyperglycemic glucose levels (11 mM) primed and markedly enhanced cAMP generation in response to forskolin.

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.

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 beta-cells, while a smaller number of ACs were expressed in alpha- and delta-cells. No AC-IR was observed in pancreatic polypeptide cells. Moreover, we have found an increased IR of the Ca2+-stimulated ACl, AC3 and AC8 in diabetic beta- and alpha-cells, compared with the corresponding IR in control pancreas. Most noticeable was the eliciting of a markedly enhanced AC8-IR in GK rat beta- and alpha-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.

Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat, a spontaneous model of non-insulin dependent diabetes mellitus.

Intact fibroblast function is required for normal wound healing. Although healing is generally accepted to be disturbed in non-insulin dependent diabetes mellitus, the signals modulating this disturbance are not fully understood. Therefore, we studied dermal fibroblasts from the GK rat, a non-insulin dependent diabetes mellitus model, and the Wistar rat (control) regarding growth characteristics, and L-lactate production at 5.5 mM and 25.5 mM glucose in the absence or presence of protein kinase C-inhibition, or alpha-tocopherol acetate. In addition, growth and L-lactate responses to hyaluronic acid were assessed under normal glucose conditions. At 5.5 mM glucose, the fibroblasts from the GK rat showed a lower proliferation rate during the first 24 hours, measured as DNA content, as compared to Wistar rats, i.e. at 8 hours GK was 57% of control, p < 0.01, at 24 hours GK was 60% of control, p < 0.01. The GK rat fibroblasts accumulated higher L-lactate levels in the media at 24-96 hours. Addition of glucose at a concentration of 25.5 mM decreased the total DNA content in GK rat fibroblast cultures to 74% (p < 0.05) and in control to 87% (p < 0.05), and increased L-lactate levels, measured at 48 hours. A protein kinase C-inhibitor, bisindolylmaleimide IX, increased DNA content and decreased L-lactate in both cell types during culture in high glucose, but only affected GK rat fibroblasts during normal glucose. Hyaluronic acid, increased DNA content in both types of fibroblasts, GK: 139% (p < 0.05), control: 127% (p < 0.05) and reduced L-lactate production. The above observations indicate that GK rat fibroblast proliferation is suppressed when the cells are cultured in high glucose containing media. In addition, protein kinase C and hyaluronic acid might play a role as modulators of fibroblast proliferation during the diabetic state.

Molecular cloning of a full-length cDNA for human type 3 adenylyl cyclase and its expression in human islets.

The GK (Goto-Kakizaki) rat is a lean model of type 2 diabetes in which the diabetic state was spontaneously induced. We recently demonstrated the presence in GK rats of two functional point mutations in the promoter region of the type 3 adenylyl cyclase (AC3) gene that resulted in overexpression of AC3 mRNA associated with increased cAMP generation. The AC3 gene promoter mutations are the first molecular changes to be described in any specific gene in the GK rat. Here we report cloning of a full-length cDNA encoding human AC3 from a human fetal brain cDNA library using a PCR-based screening method. This 4142-bp cDNA predicts an open reading frame encoding 1144 amino acids containing putative 12 transmembrane-spanning domains which are typically found in other mammalian AC isoforms. Comparison of the translated amino acid sequence of the AC3 gene between human and rat shows 95% homology. Using RT-PCR, clear AC3 expression was detected in isolated human islets as well as a cDNA panel containing templates from eight different tissues (brain, heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle). This wide distribution of AC3 expression may involve a number of physiological and pathophysiological metabolic processes.

Preserved initiatory and potentiatory effect of alpha-ketoisocaproate on insulin release in islets of glucose intolerant rats.

Insulin responses to glucose and non-glucose secretagogues were studied in short-term cultured pancreatic islets and perfused pancreata of the glucose intolerant F1 hybrid rats of spontaneously diabetic Goto-Kakizaki and control Wistar rats. After culture at 5.5 mmol/l glucose, hybrid islet responses to 11.1, 16.7 and 27.0 mmol/l glucose were between 60 and 40% of control islet responses. A combination of 1 mmol/l isobutylmethylxanthine and 16.7 mmol/l glucose induced a pronounced insulin release, which was of similar magnitude in hybrid and control rat islets. This response was not further augmented by addition of glibenclamide and arginine. The slope of potentiation of arginine (10 mmol/l)-stimulated insulin secretion by glucose (5.5-16.7 mmol/l) was greatly impaired in hybrid islets. In contrast to glucose, alpha-ketoisocaproate (KIC), which is metabolized in Krebs cycle, dose-dependently stimulated insulin secretion to similar levels in hybrid and control islets, cultured at 5.5 mmol/l glucose. Also in hybrid islets depolarized by potassium chloride (30 mmol/l) and with adenosine triphosphate-sensitive K+-channels kept open by diazoxide, insulin responses to glucose were greatly impaired but intact to KIC. Furthermore, KIC potentiated normally the insulin response to arginine in hybrid islets. In the isolated perfused pancreas, KIC induced similar insulin responses in hybrid rats and control rats. The potentiating effect by 5.5 mmol/l glucose on the KIC-stimulated insulin responses was, however, greatly reduced in isolated islets and absent in the perfused pancreata of hybrid rats. Taken together, these findings suggest an intact capacity for insulin release, although the initiating and potentiating effect by glucose on insulin release are defective in the Goto-Kakizaki-hybrid rats. An abnormal beta-cell glucose metabolism proximal to the Krebs cycle is likely to account for the impairment of insulin release.

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 these measurements to explain the impaired insulin release in islets from GK rats.

Mutations in the promoter of adenylyl cyclase (AC)-III gene, overexpression of AC-III mRNA, and enhanced cAMP generation in islets from the spontaneously diabetic GK rat model of type 2 diabetes.

Glucose-induced insulin release is decreased in the spontaneously diabetic GK rat, a nonobese rodent model of type 2 diabetes. Forskolin restores the impaired insulin release in both the isolated perfused pancreas and isolated islets from these rats (Abdel-Halim et al., Diabetes 45:934-940, 1996). We demonstrate here that the insulinotropic effect of forskolin in the GK rat is due to increased generation of cAMP and that it is associated with overexpression of adenylyl cyclase (AC)-III mRNA and gene mutations. The AC-III mRNA overexpression was demonstrated by in situ hybridization using oligonucleotide probes binding to different regions of the rat AC-III mRNA. It was associated with the presence of two point mutations identified at positions -28 bp (A --> G) and -358 bp (A --> C) of the promoter region of the AC-III gene and was demonstrable in both GK rat islets and peripheral blood cells. Transfection of COS cells with a luciferase reporter gene system revealed up to 25-fold increased promoter activity of GK AC-III promoter when compared with normal rat promoter (P < 0.0001). In conclusion, forskolin restores the impaired insulin release in islets of the GK rat through enhanced cAMP generation. This is linked to overexpression of AC-III mRNA in GK islets due to two functional point mutations in the promoter region of the AC-III gene.

Ultrastructural localization of insulin-like growth factor-2 (IGF-2) to the secretory granules of insulin cells: a study in normal and diabetic (GK) rats.

By using biochemical and light-microscopical techniques, insulin-like growth factor-2 (IGF-2) has recently been found in adult pancreas, co-localized immunohistochemically with insulin in the islet B-cells. The purpose of this study was to trace IGF-2 immunoreactivity (IR) at the ultrastructural level in normal and diabetic Goto-Kakizaki (GK) rats. Using a pre-embedding technique and immuno-gold-silver staining, IGF-2 antibody binding was localized exclusively to the halo of a subset of secretory beta-granules in normal rats. Insulin IR occurred more frequently in the granules. GK rats had, in addition to normal-looking islets, some islets with irregular shape and an increased amount of fibrous tissue, so-called "starfish-shaped" islets. In these, beta-granules were usually found, but most of the B-cells were also occupied by large, usually electron-translucent vesicles, some resembling crinophagic bodies, i.e., the sign of intracellular degradation of secretory granules. In starfish-shaped islets, IGF-2 IR was localized to the halo of beta-granules, as in GK islets with normal appearance. Occasionally, IGF-2 IR was also found in the cytoplasm and even in adjacent fibroblasts. Insulin IR was restricted to beta-granules. Because the lysosomes have IGF-2 receptors, the presence of IGF-2 peptide in secretory granules could explain why some granules are guided to lysosomes for degradation.