Insulin Biosynthesis In Islets Research Articles

Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function.

Mutations in the PCSK1 gene encoding prohormone convertase 1/3 (PC1/3) are strongly associated with obesity in humans. The PC1/3(N222D) mutant mouse thus far represents the only mouse model that mimics the PC1/3 obesity phenotype in humans. The present investigation addresses the cell biology of the N222D mutation. Metabolic labeling experiments reveal a clear defect in the kinetics of insulin biosynthesis in islets from PC1/3(N222D) mutant mice, resulting in an increase in both proinsulin and its processing intermediates, predominantly lacking cleavage at the Arg-Arg site. Although the mutant PC1/3 zymogen is correctly processed to the 87-kDa form, pulse-chase immunoprecipitation experiments, labeling, and immunohistochemical experiments using uncleavable variants all demonstrate that the PC1/3-N222D protein is largely mislocalized compared with similar wild-type (WT) constructs, being predominantly retained in the endoplasmic reticulum. The PC1/3-N222D mutant also undergoes more efficient degradation via the ubiquitin-proteasome system than the WT enzyme. Lastly, the mutant PC1/3-N222D protein coimmunoprecipitates with WT PC1/3 and exerts a modest effect on intracellular retention of the WT enzyme. These profound alterations in the cell biology of PC1/3-N222D are likely to contribute to the defective insulin biosynthetic events observed in the mutant mice and may be relevant to the dramatic contributions of polymorphisms in this gene to human obesity.

Importance of oestrogen receptors to preserve functional β-cell mass in diabetes

Protecting the functional mass of insulin-producing β cells of the pancreas is a major therapeutic challenge in patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM). The gonadal hormone 17β-oestradiol (E2) is involved in reproductive, bone, cardiovascular and neuronal physiology. In rodent models of T1DM and T2DM, treatment with E2 protects pancreatic β cells against oxidative stress, amyloid polypeptide toxicity, lipotoxicity and apoptosis. Three oestrogen receptors (ERs)--ERα, ERβ and the G protein-coupled ER (GPER)--have been identified in rodent and human β cells. Whereas activation of ERα enhances glucose-stimulated insulin biosynthesis, reduces islet toxic lipid accumulation and promotes β-cell survival from proapoptotic stimuli, activation of ERβ increases glucose-stimulated insulin secretion. However, activation of GPER protects β cells from apoptosis, raises glucose-stimulated insulin secretion and lipid homeostasis without affecting insulin biosynthesis. Oestrogens are also improving islet engraftment in rodent models of pancreatic islet transplantation. This Review describes developments in the role of ERs in islet insulin biosynthesis and secretion, lipid homeostasis and survival. Moreover, we discuss why and how enhancing ER action in β cells without the undesirable effect of general oestrogen therapy is a therapeutic avenue to preserve functional β-cell mass in patients with diabetes mellitus.

Influence of vitamin D3 deficiency and 1,25 dihydroxyvitamin D3 on de novo insulin biosynthesis in the islets of the rat endocrine pancreas.

Because 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) is known to activate the biosynthesis of numerous proteins in various tissues, experiments were undertaken to compare the influence of 1,25(OH)2D3 in vitro on both the secretion and biosynthesis of insulin in islets of Langerhans from both 4-week vitamin D3-deficient rats and normal rats. Islets were either incubated or perifused after a 6-h induction period in the presence of various concentrations of 1, 25(OH)2D3 from 10(-12) M, which was inactive in controls, to 10(-6) M. Experiments were performed in the presence of a non-labelled amino acid mixture, to favour protein synthesis. Tritiated tyrosine was added as tracer during glucose stimulation. The newly synthesised proteins, labelled with [3H]tyrosine, were extracted by an acid-alcohol method and separated by gel chromatography adapted for low-molecular-weight proteins. Even in the presence of the amino acid mixture, the insulin response of the islets to 16.7 mM glucose was decreased by vitamin D3 deficiency and improved by 1,25(OH)2D3. This beneficial effect did not occur in basal conditions, but only during glucose stimulation, and was observed in both phases of insulin release. Moreover, these effects disappeared in the presence of 5x10(-4 )M cycloheximide, a protein biosynthesis inhibitor. Islets from vitamin D3-deficient rats exhibited a general decrease in the amount of de novo biosynthesised proteins and of [3H]tyrosine-labelled insulin and proinsulin fractions. A 6-h period of 1,25(OH)2D3 induction significantly improved the amount of de novo biosynthesised proteins, and particularly of newly synthesised insulin in response to a 2-h glucose stimulation. Calculation of the rate of conversion of newly synthesised proinsulin-like material to insulin as the [3H]insulin/[3H]proinsulin-like material ratio provided evidence for a dose-dependent increase, induced by 1, 25(OH)2D3, that could exceed that of normal islets. These data support the hypothesis that 1,25(OH)2D3 in vitro not only facilitated the biosynthetic capacity of the beta cell - which was highly induced during a 16.7-mM glucose stimulation, via a global activation of islets protein biosynthesis - but also produced an acceleration of the conversion of proinsulin to insulin.

Effects of porcine pancreastatin on secretion and biosynthesis of insulin and glucose oxidation of isolated rat pancreatic islets.

The effects of porcine pancreastatin were studied on insulin secretion induced by glucose and nonnutrient stimuli, insulin biosynthesis, and glucose oxidation of cultured rat islets. Pancreastatin (100 nM) significantly suppressed, by 32-52%, the insulin response to 27, 16.7, 11, and 5.5 mM but not to 50 mM glucose, whereas 10 nM pancreastatin inhibited insulin release significantly only at 11 and 5.5 mM glucose. Pancreastatin (10 and 100 nM) also suppressed release induced by 20 mM arginine (by 26 and 30%) as well as by 1 microgram/ml of glibenclamide (by 56 and 72%, respectively). Pancreastatin (10 and 100 nM) furthermore inhibited insulin release induced by 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) (by 40 and 61%, respectively) and 1.0 mM IBMX (by 44 and 76%, respectively). Neither glucose oxidation nor overall insulin biosynthesis in islets was significantly affected by pancreastatin, although a slight but significant enhancement of biosynthesis was noted at 1.7 mM glucose in the presence of 100 nM pancreastatin. In conclusion, these data demonstrate that porcine pancreastatin suppresses glucose-induced insulin response from isolated rat islets in a competitive manner. This effect seems not to be exerted through a suppression of (pro)insulin biosynthesis or glucose metabolism in the islets, and thus the effect mediated by pancreastatin must be on a step distal to the coupling between islet glucose metabolism and insulin secretion. The relatively strong inhibition by the peptide of IBMX-induced insulin release suggests that it acts on the cAMP system of islet B cells.

CRYOPRESERVATION OF MOUSE PANCREATIC ISLETS

In most previous studies of cryopreserved isolated pancreatic islets, a slow cooling rate has been employed. We recently observed that faster cooling (5 degrees C/min) resulted in better functional islet preservation than cooling at 0.5 degrees C/min. We found that a culture period after the collagenase isolation of the islets, but prior to freezing, is crucial for the preservation of the islet B cell function. In the present investigation the function of isolated mouse pancreatic islets cooled in Hanks' solution supplemented with 2 M dimethylsulphoxide was compared with that of nonfrozen, cultured islets prepared from the same donors. The islets were cultured in RPMI 1640 + 10% calf serum for 3 days before freezing, and for 3 days after rapid thawing at 37 degrees C. Islets were cooled at rates of 5, 15, or 25 degrees C/min to 70 degrees C and then plunged into liquid nitrogen. All three groups of cryopreserved islets responded with insulin secretion when challenged with high glucose concentrations in batch-type incubations. In further experiments it was found that glucose-stimulated (pro)insulin biosynthesis in islets frozen at 25 degrees C/min was the same as that in the controls. Similar observations were made with respect to glucose-stimulated insulin release in perifusion experiments. However, a 30% reduction in insulin content was observed in the rapidly frozen islets. There was no difference in the replicatory capacity of the islets cells in vitro, as determined by an autoradiographic technique, between control islets and islets cooled at 5 degrees C or 25 degrees C/min. Intrasplenic implantation of 600-800 cryopreserved syngeneic islets into alloxan-diabetic mice led to complete or partial normalization of the hyperglycemia in seven of nine mice. When splenectomy was performed in five animals the serum glucose concentrations increased promptly. We conclude that relatively rapid cooling rates may be useful for cryopreservation of isolated pancreatic islets.

Concanavalin A and alloxan interactions on glucose-induced insulin secretion and biosynthesis from islets of Langerhans.

The pretreatment of isolated islets of Langerhans with concanavalin A (Con A) completely blocks alloxan from suppressing the insulin release response to glucose. The lectin itself inhibits insulin secretion. This effect is dose dependent and reversible. Con A, however, has no protective action against the inhibition of glucose-induced insulin biosynthesis in islets exposed to alloxan. The protective action of Con A on alloxan toxicity is likely to be at the beta-cell surface at a membrane recognition site for glucose as a stimulus for secretion. The insulin biosynthetic effect of glucose appears to be mediated through a separate mechanism.

Regulation of the biosynthesis of insulin in isolated brockmann bodies of the carp ( Cyprinus carpio)

The biosynthesis of proinsulin and insulin was followed by the incorporation of radiolabeled leucine and phenylalanine into carp islet protein. Even in the absence of glucose or amino acids in the incubation medium the share of proinsulin in the total protein biosynthesis amounted to ∼ 40% of the incorporated [ 3H]leucine during 2 hr of incubation at 17°. Glucose increased only the rate of total protein synthesis by approximately 20% but did not stimulate the (pro)insulin synthesis. Furthermore, leucine did not stimulate the [ 3H]-phenylalanine incorporation into proinsulin. Cyproheptadine, a specific inhibitor of proinsulin synthesis in mammals, had no effect on carp islets. The rate of [ 3H]leucine incorporation into carp islet protein was strongly dependent on the temperature. The share of proinsulin in the total protein synthesis decreased to about 20% at 4°. The effect of temperature on the enzyme system converting proinsulin to insulin was most dramatic. Whereas the half-time of conversion was between 3 and 4 hr between 25° and 17°, it was 58 hr at 9° and undetectably slow at 4°. The results indicate that, in contrast to mammals, the regulation of insulin biosynthesis in islets of the poikilotherm carp is not determined by the supply of metabolic substrates, but by the environmental temperature.

Effects of a hypoglycemic component of ginseng radix on insulin biosynthesis in normal and diabetic animals.

DPG-3-2, a component of ginseng radix, which lowers the blood glucose level and stimulates insulin release in diabetic animals, was studied for its effects on insulin biosynthesis in different preparations of pancreas from animals with normoglycemia and with hyperglycemia (alloxan diabetic rats and genetically diabetic mice, KK-CAy). We measured incorporation of radioactive leucine into insulin and other protein fractions during a 2-h perfusion of rat pancreas, and into insulin during a 3-h incubation of mouse islets. Biosynthesis of insulin during a long-term culture of islets from KK-CAy mice was also measured. DPG-3-2 was found practically not to increase the incorporation into insulin in the pancreatic preparations from animals with normoglycemia, but in such preparations from animals with hyperglycemia DPG-3-2 (0.2-1.0 mg/ml) caused 1.5-1.8-fold incorporation into insulin. In addition, a long-term treatment of DPG-3-2 (0.5 mg/ml) was shown to stimulate insulin biosynthesis in islets from KK-CAy mice. Ginsenoside-Rb1 and -Rg1 decreased the insulin content of islet to the undetectable level. Thus, DPG-3-2 was shown to stimulate insulin biosynthesis in different preparations of pancreas from animals with hyperglycemia.

Long-term effects of glibenclamide on the insulin production, oxidative metabolism and quantitative ultrastructure of mouse pancreatic islets maintained in tissue culture at different glucose concentrations.

In order to evaluate long-term effects of sulphonylureas on pancreatic islet structure and function, isolated mouse islets were maintained in tissue culture for one week at various glucose concentrations, and in the absence or presence of glibenclamide. When the islets were cultured at 3.3 or 5.5 mmol/1, but not at 16.7 mmol/1 glucose, it was found that the drug stimulated insulin secretion into the culture medium during the initial 3 days of culture. During the remainder of the culture period no such enhancement of secretion was demonstrated. Insulin release due to glibenclamide apparently resulted in rapid depletion of intracellular insulin stores. The finding of an enlarged B-cell Golgi apparatus in the drug-treated islets was probably associated with granule discharge. The failure of glibenclamide to promote insulin secretion during the whole culture period could reflect the adverse effects of the drug on islet insulin biosynthesis as indicated by short-term experiments performed after culture. Similar experiments showed that the impaired insulin biosynthesis could not be restored by withdrawal of the drug from the culture medium for 3 days. Furthermore, the capacity for insulin release in response to an acute glucose challenge at the end of the culture period, was abolished by culture in the presence of glibenclamide. The drug effects on insulin biosynthesis and intracellular insulin stores, which were most pronounced at 5.5 mmol/1 glucose, possibly resulted from changes in B-cell metabolism as suggested by the diminished islet glucose-oxidation rate. The spatial characteristics of islet mitochondria indicated that these changes might involve an adaptation to substrates other than glucose. In conclusion, our findings suggest that sulphonylureas have an insulinotropic effect, which is however transient. Indeed, it rather seems as if long-term exposure of islet B-cells to sulphonylureas in vitro were accompanied by functional deficiency.

Insulin biosynthesis and release in isolated islets from hyperglycemic male rats.

Normal male rats were made hyperglycemic for 24 hours by the infusion of high amounts of glucose and the effect of mannose, glucose and glucose plus glucagon on insulin biosynthesis and release was studied in the isolated islets. Saline infused animals served as controls. It was found that all stimuli markedly enhanced the pro-/insulin biosynthesis in islets from hyperglycemic rats in comparison with saline infused animals. Maximal insulin release was observed in the control group with 300 mg/dl glucose plus 10 microgram/ml glucagon, while submaximal stimulation with 100 mg/dl mannose or glucose resulted in a higher insulin secretion rate in B-cells from hyperglycemic animals at 60 min, when compared with the saline infused animals. The results show that elevation of the blood glucose results in an overall stimulation of insulin biosynthesis and secretion, while lowering of the blood sugar as in starvation predominantly decreases the glucose dependent mechanisms for both insulin synthesis and release.

Effects of acetylcholine, noradrenaline, and prostaglandins on the isolated, perfused human fetal urethra.

AbstractAnderson, A., K. Asplund and R. Larkins, Insulin production by pancreuric islets of obese‐hyperglycemic mice cultured for one week in different glucose concentrations. Acta physiol. scand. 1978. 104. 377–385.Culture of pancreatic islets isolated from obese‐hyperglycemic mice (gene symbol oh) for one week in media containing widely different concentrations of glucose (3.3, 5.6 or 16.7 mM) was found to markedly influence the functional behaviour of the islet B‐cells. Thus, the insulin content of islets cultured at 3.3 or 16.7 mM glucose (subphysiological or supraphysiological glucoce concentrations respectively) was markedly reduced. Islets cultured in 5.6 or 16.7 mM glucose displayed a normal insulin secretory response when stimulated with glucose, whereas islets cultured in a subnormal glucose concentration (3.3 mM) showed a reduced insulin response to glucose stimulation in batch type incubations and also lacked a second phase of insulin secretion in islet perifusion experiments. The rate of insulin biosynthesis of non‐cultured ob/ob islets was higher than that of islets from Lheir lean siblings but cuiture for one week in 3.3 mM glucose induced a pronounced impairment of the insulin biosynthesis in islets of obese as well as lean mice. The present data indicate that the hyperfunction of the islets of the ob/ob mouse at least in part is a reversible phenomenon, suggesting that inherent properties of islet B‐cells do not act as “primary” factors in the development of the obese‐hyperglycemic syndrome.

Metabolic adaptation to pregnancy shown by increased biosynthesis of insulin in islets of Langerhans isolated from pregnant rats

MANY reports have indicated that insulin secretion is increased in pregnant women1,2 although blood sugar levels may be unchanged, indicating an increased demand for insulin in human pregnancy. Similar findings have been reported for the effects of pregnancy on insulin secretion in pregnant rats3,4 which begin to respond with a significantly increased insulin secretion at a lower glucose level than those found in the normal female5. In addition, the secretory response is considerably greater in islets of Langerhans from pregnant than in normal rats, indicating an overall increased sensitisation of the islets from pregnant rats to the effects of glucose5. Glucose has a major role in the metabolism of the mammalian β cell, in that at physiological concentrations it is capable of stimulating both insulin secretion and biosynthesis6,7. It is possible that increased insulin biosynthesis is necessary to support the increased secretory response to glucose in pregnancy, if the content of β cell insulin is to be maintained. We have therefore investigated whether the alterations in insulin secretion observed in vivo during pregnancy are paralleled by an enhanced insulin biosynthesis in isolated islets from pregnant rats, after exposure to increasing concentrations of glucose.