Dietary effects on (pro)insulin biosynthesis and insulin-degrading activity in islets from sand rats

Sand rats were fed either a vegetable (vegetable group) or a standard pellet diet. After 14-16 weeks, the normoglycemic subgroup selected (pellet group) from the animals that had been maintained on the standard diet showed a modest increase in body weight. Plasma immunoreactive insulin levels were not significantly increased, but glucose-stimulated insulin release was elevated from islets isolated from sand rats of the pellet group. Insulin biosynthesis was estimated in vitro by measuring [3H]leucine incorporation into (pro)insulin at 1.5 or 15 mmol/l glucose. The rate of (pro)insulin biosynthesis was elevated only at 15 mmol/l glucose in islets from those normoglycemic sand rats fed the pellet diet when compared with islets from the vegetable group. Specific insulin-degrading activity, as determined by measuring degradation of 125I-labeled insulin, was also increased for islets from the pellet group. The metabolic state of these sand rats is thus associated with normoglycemia in vivo, and increased stimulated rates of insulin biosynthesis and degradation in pancreatic islets in vitro.     

Tetracycline-regulated secretion of human (pro)insulin following plasmid-mediated transfection of human muscle

Long-term secretion of insulin by host muscle following transduction with an insulin gene construct offers the potential of gene therapy for diabetes without immunosuppression. Clinical implementation will be dependent on proof of principle in human tissue and a system for safely regulating basal insulin levels. Liposomal co-transfection with a tetracycline-responsive wild type human preproinsulin (pTRE-hppI1) or mutant construct (pTRE-hppI4), in which PC2 and PC3 cleavage sites were altered to form tetrabasic consensus sites for furin, together with pTet-off (coding for a transactivating protein) was evaluated in the C2C12 mouse myoblast cell line and human myoblasts following establishment in primary culture. In the absence of tetracycline, (pro)insulin secretion in C2C12 and human myoblasts transfected with tetracycline-responsive hppI1 and hppI4 constructs was comparable to that following transfection with equivalent constructs under the control of a constitutively active cytomegaloviral promoter. Percentage processing to mature insulin was <5% in C2C12 and human myoblasts transfected with pTet-off/pTRE-hppI1 but >90% in C2C12 cells and 45-60% in human myoblasts on transfection with pTet-off/pTRE-hppI4. Incremental dose-responsive suppression of proinsulin secretion was demonstrated in C2C12 and human myoblasts expressing pTet-off/pTRE-hppI1 following incubation with tetracycline (0-100 microg/ml) for up to 72 h. Reversibility was confirmed following tetracycline withdrawal. Dose-responsive tetracycline-inducible repression of mature insulin secretion was confirmed in C2C12 cells following transfection with pTet-off/pTRE-hppI4. Regulation of human proinsulin biosynthesis and secretion has been attained in vivo following plasmid-mediated gene transfer to rat skeletal muscle and oral tetracycline administration. In conclusion, processing to mature insulin has been confirmed following plasmid-mediated gene transfer to human muscle in addition to in vitro- and in vivo-regulated human proinsulin secretion employing the safe and well-tolerated antibiotic, tetracycline.     

Interleukin 1 dose-dependently affects the biosynthesis of (pro)insulin in isolated rat islets of Langerhans

Summary Human crude and recombinant interleukin 1 (IL-1) was found to dose- and time-dependently affect the biosynthesis of (pro)insulin in isolated rat islets of Langerhans. Incubation of rat islets with either 0.5 U/ml or 5 U/ml of crude IL-1 for 1 h had no detectable effect on (pro)insulin biosynthesis. After 24 hours of exposure 0.5 U/ml of crude or 0.6 ng/ml of recombinant IL-1 (beta) increased the (pro)insulin biosynthesis by 42% and 58%, respectively, whereas a 10-fold greater concentration of IL-1 decreased the (pro)insulin biosynthesis by 74% and 89%, respectively. The increase in (pro)insulin biosynthesis was accompanied by an increase in total protein biosynthesis indicating a nonspecific stimulatory action of low IL-1 concentrations. In contrast, high IL-1 concentrations caused a more selective decrease of the (pro)insulin biosynthesis when compared to the total protein biosynthesis. In addition, low IL-1 concentrations were found to increase and high concentrations to decrease the relative levels of pre-proinsulin mRNA suggesting that IL-1 may act both at a pre- and post-translational level of insulin biosynthesis.     

Effect of human calcitonin (hCT) on glucose- and arginine-stimulated insulin secretion

Summary Many studies have shown that in normal man salmon and porcine CT administration in bolus inhibits the release of TSH, LH, GH, and glucose- or arginine-induced insulin secretion. In the present study we investigated the effects of human synthetic calcitonin (hCT) on glucose or arginine-induced insulin secretion in man. Twenty-two subjects were submitted to i.v. administration of hCT during glucose or arginine test. In our opinion, the most interesting results are those observed with arginine plus hCT at two different dosages (25 μg and 12.5 μg infused in 30 min). In fact arginine plus hCT (25 μg in 30 min) administration induced a significant increase of glycemia at 5, 10 and 20 min (p<0.01) and at 30 min (p<0.05) and a significant decrease of IRI at 5, 10, 20 and 30 min (p<0.001) and at 45 min (p<0.005). The highest plasma CT levels were observed at 15 and 30 min (490 and 540 pg·ml⁻¹). Arginine plus hCT (12.5 μg in 30 min) infusion induced a similar significant increase in plasma glucose at 10, and 20 min (p<0.05) and at 30 min (p<0.01) and a significant decrease of plasma IRI at 10 min (p<0.05) at 20 min and 30 min (p<0.005). The highest plasma CT levels were reached at 20 min and 30 min (250 and 270 pg·ml⁻¹, respectively). Our results clearly demonstrate that physiologic doses of hCT are able to inhibit arginine induced insulin secretion in normal man. Since insulin induces hypercalcemia and food ingestion increases both insulin and CT, one could hypothesize that CT inhibits insulin secretion thus controlling post-prandial hypercalcemia by its osteotrophic effect and by its action upon calcium redistributed within the cells.     

Regulation of insulin secretion and proinsulin biosynthesis by succinate

Succinate stimulates insulin secretion and proinsulin biosynthesis. We studied the effects of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-modulating pathways on glucose- and succinate-stimulated insulin secretion and proinsulin biosynthesis in the rat and the insulin-resistant Psammomys obesus. Disruption of the anaplerotic pyruvate/malate shuttle by phenylacetic acid inhibited glucose- and succinate-stimulated insulin secretion and succinate-stimulated proinsulin biosynthesis in both species. In contrast, phenylacetic acid failed to inhibit glucose-stimulated proinsulin biosynthesis in P. obesus islets. Inhibition of the NADPH-consuming enzyme neuronal nitric oxide synthase (nNOS) with l-N(G)-nitro-l-arginine methyl ester or with N(G)-monomethyl-l-arginine(G) doubled succinate-stimulated insulin secretion in rat islets, suggesting that succinate- and nNOS-derived signals interact to regulate insulin secretion. In contrast, nNOS inhibition had no effect on succinate-stimulated proinsulin biosynthesis in both species. In P. obesus islets, insulin secretion was not stimulated by succinate in the absence of glucose, whereas proinsulin biosynthesis was increased 5-fold. Conversely, under stimulating glucose levels, succinate doubled insulin secretion, indicating glucose-dependence. Pyruvate ester and inhibition of nNOS partially mimicked the permissive effect of glucose on succinate-stimulated insulin secretion, suggesting that anaplerosis-derived signals render the beta-cells responsive to succinate. We conclude that beta-cell anaplerosis via pyruvate carboxylase is important for glucose- and succinate-stimulated insulin secretion and for succinate-stimulated proinsulin biosynthesis. In P. obesus, pyruvate/malate shuttle dependent and independent pathways that regulate proinsulin biosynthesis coexist; the latter can maintain fuel stimulated biosynthetic activity when the succinate-dependent pathway is inhibited. nNOS signaling is a negative regulator of insulin secretion, but not of proinsulin biosynthesis.     

Effect of insulin sensitivity on pulsatile insulin secretion

OBJECTIVE: The aim of the study was to determine whether derangements in insulin pulsatility are related to the presence of insulin resistance or whether these changes occur only in non-insulin-dependent diabetes mellitus (NIDDM). DESIGN AND METHODS: The study included 26 obese, 11 NIDDM and 10 control subjects. The obese group was divided into a low insulin (plasma insulin <20 mU/l, OLI, 14 subjects) and a high insulin (OHI, 12 subjects) group. For pulsatility analysis blood was sampled every 2 min for 90 min. Pulsatility analysis was carried out using the PulsDetekt program. The insulin secretion randomness was quantified using interpulse interval deviation (IpID) and approximate entropy (ApEn). ApEn and ApEn normalized by s.d. of the individual insulin time series (nApEn) were calculated. Lower values of ApEn and IpID indicate more regular secretion. Homeostasis model assessment (HOMA) was used to quantify insulin sensitivity. RESULTS: Insulin pulses were significantly less regular in the OHI and the NIDDM groups compared with the control and the OLI groups (control: ApEn 0.54+/-0.16, nApEn 0.69+/-0.19, IpID 2.53+/-0.99; OLI: ApEn 0.64+/-0.12, nApEn 0. 79+/-0.15, IpID 2.92+/-1.09; OHI: ApEn 0.88+/-0.07, nApEn 0.92+/-0. 07, IpID 3.95+/-0.84; NIDDM: ApEn 0.92+/-0.16, nApEn 0.99+/-0.09, IpID 4.41+/-0.53; means +/- s.d.). There was no difference in the pulse regularity between the OHI and the NIDDM groups. CONCLUSIONS: Decrease in insulin sensitivity was correlated with the reduction of insulin secretion regularity. Therefore irregular insulin secretion is related to a reduction in insulin sensitivity, and it is not unique to NIDDM.     

Effects of prolactin and glycosylated prolactin on (pro)insulin synthesis and insulin release from cultured rat pancreatic islets

In order to study the possible differential effects of the nonglycosylated and glycosylated forms of prolactin on insulin content and secretion in pancreatic islets, neonatal rat pancreatic islets were exposed for 6 days in vitro to 2 micrograms/ml of nonglycosylated ovine prolactin (oPRL), or to 2 micrograms/ml of glycosylated oPRL (G-oPRL). oPRL stimulated a significant increase (p less than 0.01) in the total amount of insulin released into the medium over the 6 day culture period; however, G-oPRL had no effect. Islets cultured for 6 days in the presence of oPRL showed no increase in the amount of DNA per islet. However, there was a significant (p less than 0.007) increase in the amount of total protein synthesized by the islets exposed to oPRL. These findings suggest that the effect of oPRL on neonatal rat pancreatic islet cells is a nonspecific effect. The nonglycosylated form of PRL may play a role in B-cell function by promoting protein synthesis, which results in augmented insulin synthesis.     

The effect of serotonin on in vitro insulin secretion and biosynthesis in mice

Summary The effect of serotonin on insulin secretion and biosynthesis was studied using isolated islets of mice. Serotonin produced a small stimulatory effect on insulin secretion when glucose was present in the incubation medium at a low concentration. On the other hand, an inhibition of insulin secretion was obtained with serotonin when glucose in the medium reached 3.0 mg/ml concentration. No significant effect of serotonin was obtained on insulin biosynthesis, neither in the presence of low nor with a high glucose concentration. These results suggest that the effect of this monoamine on insulin secretion is not mediated via its effect on insulin biosynthesis.Supported by Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg, SFB 87 Ulm     

Engineering of glucose-stimulated insulin secretion and biosynthesis in non-islet cells.

The high-capacity glucose transporter known as GLUT-2 and the glucose phosphorylating enzyme glucokinase are thought to be key components of the "glucose-sensing apparatus" that regulates insulin release from the beta cells of the islets of Langerhans in response to changes in external glucose concentration. AtT-20ins cells are derived from anterior pituitary cells and are like beta cells in that they express glucokinase and have been engineered to secrete correctly processed insulin in response to analogs of cAMP, but, unlike beta cells, they fail to respond to glucose and lack GLUT-2 expression. Herein we demonstrate that stable transfection of AtT-20ins cells with the GLUT-2 cDNA confers glucose-stimulated insulin secretion and glucose regulation of insulin biosynthesis and also results in glucose potentiation of the secretory response to non-glucose secretagogues. This work represents a first step toward creation of a genetically engineered "artificial beta cell."     

Fat-induced changes in mouse pancreatic islet insulin secretion,insulin biosynthesis and glucose metabolism

Insulin secretion, insulin biosynthesis and islet glucose oxidation were studied in pancreatic islets isolated from fat-fed diabetic mice of both sexes. Insulin secretion from isolated islets was studied after consecutive stimulation with -ketoisocaproic acid + glutamine, glucose, forskolin, and 12-O-tetradecanoylphorbol 13-acetate. Glucose-induced insulin secretion was impaired in islets from fat-fed mice. This was associated with a reduction of approximately 50% in islet glucose oxidation. Islet insulin secretion stimulated by the non-carbohydrate secretagogues tended to be higher in the fat-fed mice, but a statistically significant effect was not observed. Pancreatic insulin content was reduced by 50%, whereas the islet insulin and DNA content was unchanged after fat feeding. Proinsulin mRNA was reduced by 35% in islets from fat-fed mice, and was associated with a reduction of approximately 50% in glucose-stimulated (pro)insulin biosynthesis. It is concluded that the insulin secretory response of islets isolated from fat-fed mice is similar to the secretory pattern known from human type 2, non-insulin-dependent diabetics, and that a defect in islet glucose recognition, resulting in decreased glucose oxidation, may be responsible for the observed insulin secretory and biosynthetic defects seen after glucose stimulation.     

The influence off an acyl-amino-alcyl-benzoic acid (HB 699) on biosynthesis and secretion of insulin in isolated rat islets of Langerhans

HB 699 belongs to a new class of hypoglycaemic agents, the acyl-amino-alcyl-benzoic acids. Its influence on bion-synthesis and secretion of insulin was studied in collagenase-isolated rat islets. During incubations for 3 hours together with 3H-leucine at 1 and 2 mg/ml glucose, HB 699 (10 micrograms/ml) reduced biosynthesis of proinsulin and insulin (3H-leucine incorporation), whereas insulin release was stimulated. During an incubation period of 2 hours in the absence of glucose, insulin release was enhanced both by HB 699 (50 micrograms/ml) and glibenclamide (10 micrograms/ml). At 1 mg/ml glucose, no additive or potentiating effect of HB 699 to that of glibenclamide was found regarding insulin release. When calcium ions were omitted insulin output in the presence of HB 699 and glucose was reduced. In conclusion, HB 699, although not belonging to the class of sulfonylureas, behaves very similar to these drugs concerning its influence on insulin biosynthesis and secretion in vitro. It acts as an initiator of insulin release, involving probably similar mechanisms as sulfonylureas do.     

Effect of clofibrate on arginine-induced insulin and glucagon secretion

The influence of clofibrate therapy on insulin and glucagon secretion was examined in the rat. Following arginine stimulation, serum insulin and glucagon levels rose significantly, resulting an an $\text{I}\text{G}$ molar ratio of 1.0 ± 0.3. In contrast, clofibrate therapy completely suppressed the arginine-stimulated insulin secretion, but potentiated the simultaneous glucagon response. The resulting $\text{I}\text{G}$ molar ratio fell to 0.45 ± 0.06, consistent with a change in the bihormonal status in the direction of increased catabolism. These effects on hormonal balance may mediate in part the hypolipemic action of clofibrate that simultaneously occurs.