Glucose- and arginine-induced insulin and glucagon responses from the isolated perfused pancreas of the BB-Wistar diabetic rat. Evidence for selective impairment of glucose regulation

To investigate whether preferential responsiveness of residual B-cells is a feature of a diabetic state we compared insulin-releasing effects of glucose and arginine in perfused pancreases from moderately diabetic BB-Wistar rats. BB-rats were hyperglycaemic and insulin-dependent but possessed some insulin reserves (6 per cent of pancreatic content of control Wistar rats). Glucose (27.7 mM) failed to release insulin from diabetic pancreases while, conversely, arginine (8 mM) evoked a several-fold increase in insulin secretion. Ratios between responses from diabetic and normal pancreases were 0.01 and 0.29, respectively, when glucose or arginine were used as stimuli. This difference was significant (P less than 0.05, Wilcoxon test). Glucose furthermore failed to exert a time-dependent (= priming) effect on arginine-induced insulin secretion in the diabetic animals. Also A-cell responsiveness to glucose (acute and priming effects) were lost in BB-rats. It is concluded that selective loss of glucose effects on B- and A-cell secretion are associated with the diabetic state of the BB-Wistar rats.     

Parallel changes of proinsulin and islet amyloid polypeptide in glucose intolerance

Elevated proinsulin secretion and islet amyloid deposition are both features of Type 2 diabetes but their relationship to beta-cell dysfunction is unknown. To determine if islet amyloid polypeptide (IAPP) secretion is disproportionate with other beta-cell products at any stage of glucose intolerance, 116 subjects were studied. Non-diabetic subjects with equivalent body mass index (BMI) were assigned to three groups, (i) normal fasting glucose, fpg<5.5 mmol l(-1); (ii) intermediate fasting glucose, fpg> or =5.5<6.15 mmol l(-1); (iii) impaired fasting glucose (IFG), fpg> or =6.1<7.0 mmol l(-1). Diabetic subjects were divided according to therapy (9 diet, 19 tablet, and 11 insulin). IAPP, C-peptide and proinsulin were measured fasting and at the end of a 1-h glucose infusion. Fasting C-peptide, IAPP and proinsulin were significantly elevated in the IFG group compared with the other non-diabetic groups (P<0.02); fasting IAPP/C-peptide and proinsulin/C-peptide were 1-2% in all non-diabetic groups. Fasting and 1-h proinsulin and proinsulin/C-peptide were higher in diabetic compared with non-diabetic subjects (P<0.01). IAPP and IAPP/C-peptide in diabetic groups were similar to that in non-diabetic subjects but reduced in the insulin-treated group (P<0.01). Proinsulin was disproportionately increased compared with C-peptide and IAPP in Type 2 diabetes particularly in severe beta-cell failure implying more than one concurrent beta-cell pathology.     

[Val12] HRAS downregulates GLUT2 in beta cells of transgenic mice without affecting glucose homeostasis.

Glucose-induced insulin release from pancreatic beta cells depends on the beta-cell metabolism of glucose, which generates intracellular signals for secretion. The beta-cell glucose transporter isotype GLUT2 and the glucose phosphorylating enzyme glucokinase have both been implicated in coupling insulin secretion to extracellular glucose levels. Here we present evidence that a pronounced decrease in beta-cell GLUT2 has no immediate effect on glucose homeostasis. Analysis of transgenic mice overexpressing human [Val12]HRAS oncoprotein under control of the insulin promoter reveals a great reduction in plasma-membrane GLUT2 levels. These mice are nonetheless able to maintain normal fed and fasting plasma glucose and insulin levels for a period of several months. Insulin secretion studied in isolated islets and the perfused pancreas is characterized by a normal incremental response to increasing glucose concentrations. Glucose metabolism, as measured by glucose phosphorylation and oxidation in isolated islets, shows a normal dose dependence on extracellular glucose concentrations. These findings suggest that normal GLUT2 expression in beta cells is not essential for glucose sensing. The transgenic mice provide an experimental system for studying the role of glucose phosphorylation in regulation of insulin release in the absence of GLUT2.     

Expression of yeast hexokinase in pancreatic beta cells of transgenic mice reduces blood glucose, enhances insulin secretion, and decreases diabetes.

It has been proposed that endogenous hexokinases of the pancreatic beta cell control the rate of glucose-stimulated insulin secretion and that genetic defects that reduce beta-cell hexokinase activity may lead to diabetes. To test these hypotheses, we have produced transgenic mice that have a 2-fold increase in hexokinase activity specific to the pancreatic beta cell. This increase was sufficient to significantly augment glucose-stimulated insulin secretion of isolated pancreatic islets, increase serum insulin levels in vivo, and lower the blood glucose levels of transgenic mice by 20-50% below control levels. Elevation of hexokinase activity also significantly reduced blood glucose levels of diabetic mice. These results confirm the role of beta-cell hexokinase activity in the regulation of insulin secretion and glucose homeostasis. They also provide strong support for the proposal that reductions in beta-cell hexokinase activity can produce diabetes.     

Development of the insulin secretory defect in genetically diabetic (db/db) mouse.

Genetically diabetic mice (C57BL/KsJ-db/db) were used as a model to study the development of defects of insulin secretion in relation to common metabolic indicators (body weight, serum glucose and insulin, and islet insluin contant). Consistent with the idea of a protective effect of oestrogen on the pancreatic beta-cell, the female diabetic mice survived longer than the males. In males, while serum insulin decreased in the later stages of the disease, serum glucose increased progressively with age. Perfusion of the diabetic pancreases revealed a rise and subsequent fall with age of the basal insulin released at 3 mM glucose. Despite previous reports of beta-cell hyperplasia, progressive impairment of the insulin response to 20 mM glucose, or to 20 mM glucose and 1 mM 3-isobutyl-1-methylxanthine, was seen with increasing age in experiments with perfused pancreas or microdissected islets. Islet content of insulin also decreased progressively with age in the diabetic animals.     

Metformin restores prohormone processing enzymes and normalizes aberrations in secretion of proinsulin and insulin in palmitate-exposed human islets

Aim

To elucidate how proinsulin synthesis and insulin was affected by metformin under conditions of nutrient overstimulation.

Materials and Methods

Isolated human pancreatic islets from seven donors were cultured at 5.5 mmol/L glucose and 0.5 mmol/L palmitate for 12, 24 or 72 h. Metformin (25 μmol/L) was introduced after initial 12 h with palmitate. Proinsulin and insulin were measured. Expression of prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE), was determined by western blot. Adolescents with obesity, treated with metformin and with normal glucose tolerance (n = 5), prediabetes (n = 14), or type 2 diabetes (T2DM; n = 7) were included. Fasting proinsulin, insulin, glucose, 2-h glucose and glycated haemoglobin were measured. Proinsulin/insulin ratio (PI/I) was calculated.

Results

In human islets, palmitate treatment for 12 and 24 h increased proinsulin and insulin proportionally. After 72 h, proinsulin but not insulin continued to increase which was coupled with reduced expression of PC1/3 and CPE. Metformin normalized expression of PC1/3 and CPE, and proinsulin and insulin secretion. In adolescents with obesity, before treatment, fasting proinsulin and insulin concentrations were higher in subjects with T2DM than with normal glucose tolerance. PI/I was reduced after metformin treatment in subjects with T2DM as well as in subjects with prediabetes, coupled with reduced 2-h glucose and glycated haemoglobin.

Conclusions

Metformin normalized proinsulin and insulin secretion after prolonged nutrient-overstimulation, coupled with normalization of the converting enzymes, in isolated islets. In adolescents with obesity, metformin treatment was associated with improved PI/I, which was coupled with improved glycaemic control.     

The acute effects of glucose on the insulin biosynthetic-secretory pathway in a simian virus 40-transformed hamster pancreatic islet beta-cell line

The acute regulatory effects of glucose on proinsulin synthesis at the translational level and the subsequent processing of newly made hormone through the insulin biosynthetic-secretory pathway in a clonal cell line (HIT-T15 cells) of simian virus 40-transformed hamster pancreatic islet beta-cells were examined. Proinsulin synthesis in HIT-T15 cells, assayed by immunoprecipitation, comprised 2.5-5% of the total protein synthesis during a 40-min pulse label period, though preproinsulin mRNA levels were only 0.25-0.29% of the total mRNA. There was no specific enhancement of proinsulin synthesis in response to increasing concentrations of glucose or other tested metabolites or by agents stimulating the cAMP or protein kinase-C second messenger systems during a 40-min pulse label period. Compared to that in normal beta-cells, processing of proinsulin to insulin was markedly retarded. Unlike that in normal beta-cells, newly synthesized hormone in HIT cells was secreted at a very low rate that was not enhanced by a high glucose concentration and was not preferentially secreted compared with preformed hormone. Thus, although a glucose sensor was present in HIT cells, since secretion of preformed insulin stores was stimulated 8-fold between glucose concentrations of 0 and 11 mM, regulation at several stages in the passage of newly made insulin through the biosynthetic-secretory pathway did not display the acute glucose sensitivity seen in normal nontransformed beta-cells.     

Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene.

Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The cells produce both proinsulin I and II and efficiently process each into mature insulin, in a manner comparable to normal beta cells in isolated islets. Electron microscopy reveals typical beta-cell type secretory granules, in which insulin is stored. Insulin secretion is inducible up to 30-fold by glucose, although with a lower threshold for maximal stimulation than that for normal beta cells. beta TC lines can be repeatedly derived from primary beta-cell tumors that heritably arise in the transgenic mice. Thus, targeted expression of an oncogene with a cell-specific regulatory element can be used both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype.     

Relative hyperproinsulinemia as a sign of islet dysfunction in women with impaired glucose tolerance.

Proinsulin release is increased relative to insulin secretion in subjects with type 2 diabetes, indicative of islet dysfunction. However, it has not been conclusively shown whether there is an increased relative proinsulin release in subjects with impaired glucose tolerance (IGT), i.e. whether it precedes the development of diabetes. We therefore determined the proinsulin to insulin ratios in the fasting state and after acute stimulation of insulin secretion in 23 postmenopausal women, aged 61-62 yr (mean +/- SD, 61.7 +/- 0.5 yr). Ten women had normal glucose tolerance (NGT), and 13 had IGT. The groups were matched for insulin sensitivity and did not differ in body mass index. Proinsulin and insulin secretion were measured after arginine stimulation (5 g, i.v.) at three glucose levels (fasting, 14 mmol/L, and >25 mmol/L), and the acute insulin (AIR(arg)) and proinsulin responses (APIR(arg)) were calculated as the mean 2-5 min postload increase. At fasting glucose, levels of insulin, proinsulin, or the proinsulin/insulin ratio (13.6 +/- 5.0% vs. 11.1 +/- 2.7%; P = NS) did not differ between NGT and IGT. Although the AIR(arg) values were decreased in the IGT group at all glucose levels (P < 0.05), the absolute proinsulin levels and the APIRs(arg) were similar between IGT and NGT women. Therefore, the IGT women had higher proinsulin/insulin ratios at 14 mmol/L (10.7 +/- 4.4% vs. 6.4 +/- 1.8%; P = 0.006) and more than 25 mmol/L glucose (11.4 +/- 5.2% vs. 6.7 +/- 2.1%; P = 0.007). The IGT group had increased APIR(arg)/AIR(arg) at fasting (2.2 +/- 1.4% vs. 1.3 +/- 0.6%; P = 0.047) and more than 25 mmol/L glucose (3.5 +/- 1.6% vs. 2.3 +/- 0.7%; P = 0.037). We conclude that women with IGT exhibit increased relative proinsulin secretion, suggesting a defect in the intracellular proinsulin processing before diabetes develops.     

Increasing triglyceride synthesis inhibits glucose-induced insulin secretion in isolated rat islets of langerhans: a study using adenoviral expression of diacylglycerol acyltransferase

The mechanisms by which prolonged exposure to elevated levels of fatty acids (FA) adversely affects pancreatic beta-cell function remain unclear. Studies in the Zucker diabetic fatty rat have suggested that excessive accumulation of triglycerides (TG) in islets plays a key role in the deleterious effects of FA. However, a direct relationship between TG accumulation and defective beta-cell function has not been established. The aim of the present study was therefore to determine whether increasing TG synthesis in isolated rat islets of Langerhans impairs insulin secretion. To this end, we infected isolated rat islets with an adenovirus encoding for the enzyme catalyzing the last step of triglyceride synthesis, acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT). DGAT overexpression did not modify glucose oxidation nor palmitate oxidation, but increased palmitate incorporation into triglycerides by approximately 2-fold. Islets overexpressing DGAT and cultured in elevated glucose levels for 72 h had markedly impaired insulin secretion in response to glucose, but responded normally to the nonglucose secretagogues glyburide and potassium chloride. The deleterious effects of DGAT overexpression were not additive to those of prolonged exposure to palmitate. We conclude that a selective increase in TG content impairs glucose-induced insulin secretion, a mechanism likely to mediate, at least in part, the deleterious effects of FA on pancreatic beta-cell function.     

Insulin,glucagon, and somatostatin release from the prediabetic Chinese hamster

Summary Diabetes mellitus in the adult Chinese hamster is characterized by subnormal pancreatic insulin release in vitro, decreased insulin content, and lack of obesity. The cause of the islet B-cell failure is not clear. We measured insulin, glucagon, and somatostatin release from in vitro perfused pancreases of young (mean age 10 and 20 weeks), genetically diabetic animals (subline AC, mean plasma glucose 8.0 and 16.6mmol/l, respectively). Compared to age- and sex-matched normal hamsters (subline M, mean plasma glucose 5.3 mmol/l), the younger diabetic animals had a significantly elevated mean plasma glucose level, but net in vitro pancreatic release of insulin, glucagon, and somatostatin was normal. Pancreatic content of insulin and glucagon was also not significantly different from normal. At age 20 weeks, when the plasma glucose of the diabetic animals was even more elevated, pancreatic content and release of insulin were significantly subnormal, whereas glucagon and somatostatin release were normal, and pancreatic content of glucagon was normal. In a similar group of young (mean age 10 weeks) diabetic animals, non-fasting plasma insulin levels were within the normal range, but the corresponding glucose levels were excessive in most of the animals (13 out of 19). In conclusion, 10-week-old diabetic hamsters show mild hyperglycaemia which cannot be accounted for directly by decreased pancreatic release in response to a glucose plus arginine stimulus in vitro. Decreased ability of the B cell to respond in vivo to hyperglycaemia or peripheral resistance to insulin may contribute to later B-cell failure in the older diabetic hamster.     

Streptozotocin,an O-GlcNAcase inhibitor,blunts insulin and growth hormone secretion

Type 2 diabetes mellitus results from a complex interaction between nutritional excess and multiple genes. Whereas pancreatic beta-cells normally respond to glucose challenge by rapid insulin release (first phase insulin secretion), there is a loss of this acute response in virtually all of the type 2 diabetes patients with significant fasting hyperglycemia. Our previous studies demonstrated that irreversible intracellular accumulation of a glucose metabolite, protein O-linked N-acetylglucosamine modification (O-GlcNAc), is associated with pancreatic beta-cell apoptosis. In the present study, we show that streptozotocin (STZ), a non-competitive chemical blocker of O-GlcNAcase, induces an insulin secretory defect in isolated rat islet cells. In contrast, transgenic mice with down-regulated glucose to glucosamine metabolism in beta-cells exhibited an enhanced insulin secretion capacity. Interestingly, the STZ blockade of O-GlcNAcase activity is also associated with a growth hormone secretory defect and impairment of intracellular secretory vesicle trafficking. These results provide evidence for the roles of O-GlcNAc in the insulin secretion and possible involvement of O-GlcNAc in general glucose-regulated hormone secretion pathways.     

Growth hormone and metasomatotrophic diabetes: effects on insulin and proinsulin of serum and pancreas in dogs

Summary In normal fasting dog serum, the insulin: proinsulin molar proportion was 71:29%. In response to glucose infusion, the proinsulin proportion decreased. In the pancreas, the proinsulin proportion was lower than in serum. Growth hormone treatment for one day increased serum insulin sevenfold and proinsulin 18-fold. The proinsulin proportion increased to 49%. The growth hormone injections magnified the response to glucose infusion. The rise in serum insulin was 16 times the normal, proinsulin also rose but its proportion decreased. Growth hormone treatment for 6 days decreased pancreatic insulin to 5% and proinsulin to 46% of normal. In the permanent (metasomatotrophic) diabetes produced by the prolonged administration of growth hormone, serum insulin decreased and the proinsulin proportion increased. No rises in serum insulin nor proinsulin occurred following glucose infusion. In the pancreas, insulin and proinsulin were reduced to 1.6% and 8% of normal. The reduction in the immunoreactive insulin of the pancreas was more pronounced in the tail than in the head and body regions. The results indicate that in the state of augmented insulin secretion and hyperinsulinaemia produced by growth hormone and in the reduced insulin secretion and hypoinsulinaemia of metasomatotrophic diabetes, the proportion of proinsulin in serum is increased due to beta cell secretion containing a higher proportion of proinsulin than normal.     

Glucagon and insulin from lean rats and genetically obese fatty rats: Studies by radioimmunoassay,radioreceptorassay and bioassay

Insulin, proinsulin and glucagon extracted from lean rat pancreases were studied in radioimmunoassay, radioreceptorassay and bioassay systems. Extracted insulin behaved identically to a rat insulin used as a reference standard in radioimmunoassay. On the basis of its immunoreactivity, extracted insulin was slightly less potent (about 70%) than the rat standard insulin in competing with the binding of 125I-insulin to rat liver membranes (radioreceptorassay) and in stimulating glucose oxidation by rat fat cells (bioassay). Extracted glucagon and a pork glucagon used as a reference standard were indistinguishable in two radioimmunoassay systems for glucagon, in competing with the binding of 125I-glucagon to rat liver membranes (radioreceptorassay) and in stimulating adenylate cyclase in rat liver membranes (bioassay). Genetically obese rats (Zucker, "fatty") were compared to their lean littermates with respect to insulin, proinsulin and glucagon extracted from their pancreases. Proinsulin represented the same proportion of total immunoreactive insulin in both types of rats. In the radioimmunoassays, the radioreceptorassays and the bioassays, insulin, proinsulin and glucagon from obese rats were indistinguishable from insulin, proinsulin and glucagon from lean rats. It is concluded that the pancreatic hormones of obese ("fatty") rats possess the same immunoreactivity and biological potency as those of nonobese rats. This excludes the possibility that some alteration in the biological properties of pancreas insulin and/or glucagon of fatty rats could explain the metabolic abnormalities observed in this type of obesity.     

Insulin content and insulinogenesis by the perfused rat pancreas: effects of long term glucose stimulation

The dynamic response of the perfused pancreas differed between pancreases from fed and fasted rats. Insulin secretion was significantly lower in pancreases from fasted rats during the first 40 min of perfusion at glucose levels of 200 and 300 mg/dl. Thereafter, from 40-90 min, insulin secretion was similar by pancreases from both fed and fasted rats. The typical biphasic insulin secretory profile, consisting of a transient spike of insulin secretion followed by a slowly rising secretory phase, was observed in pancreases from fasted rats. In contrast, the transition from first to second phase secretion was accelerated in pancreases from fed rats. This suggests that transport of intracellular insulin stores may be accentuated due to the fact that insulinogenic sites (e.g. Golgi) in pancreases from fed rats may be fully primed for optimal secretion. Total pancreatic insulin measurements support this concept. Total pancreatic insulin content was determined under fed and 24-h fasted conditions after various times of perfusion (0, 60, and 90 min and 6 h) and in response to various glucose levels (0, 200, and 300 mg/dl). Fasting resulted in a significant decrease in insulin content at zero time compared with pancreases from fed rats (39.2 +/- 2.4 vs. 61.6 +/- 9.8 micrograms). In the fed rat pancreases, total insulin content decreased slightly after a 60-min glucose stimulus of 300 mg/dl, but returned to the basal level after 90 min and remained at that level during a 6-h period of perfusion. In the fasted state, insulin content remained constant as a function of time until 60 min, but increased by 90 min to a level comparable to that in pancreases from fed rats. The response to lower levels of glucose stimulation (200 mg/dl) was qualitatively similar by pancreases from fed and fasted rats compared to the response to a higher glucose dose (300 mg/dl), except that secretion was less. Insulin content remained relatively constant for periods of perfusion up to 60 min. Insulinogenesis (defined as de novo synthesis and conversion of existing preproinsulin and proinsulin to insulin, less intracellular degradation of insulin) was increased as a function of glucose concentration and differed temporally as a function of the food intake of the animal. At no time of perfusion with any level of glucose stimulation did the insulin content exceed the zero time value in pancreases from fed rats. This suggests that insulin secretion is the rate-limiting step for insulinogenesis.     

Insulin and glucagon release in the diabetic Chinese hamster: Differences among inbred sublines

Summary Release of insulin and glucagon from perfused pancreases in vitro of 40 normal male and female Chinese hamsters (from one inbred subline) and 110 male and female diabetic hamsters (from three inbred sublines) was measured in response to glucose plus arginine, theophylline alone, or potassium alone, in order to determine if differences in hormone secretion exist among different diabetic sublines. Glucose plus arginine and potassium produced subnormal insulin responses in all three diabetic sublines, whereas theophylline induced normal or above normal insulin responses. Excessive glucagon release was consistently seen in only one diabetic subline. The female normal animals showed greater insulin release than the male normal hamsters in response to glucose plus arginine. This sex difference was not seen in the diabetic animals.     

Proinsulin and C-peptide: a review.

The recent work on proinsulin and C-peptide has been reviewed with major emphasis on the most significant findings since 1972. Proinsulin has now been established as the biosynthetic precursor of insulin in all species examined, including man, with a preproinsulin as a possible precursor of the prohormone. The conversion of proinsulin which appears to occur exclusively in the pancreas leads to equimolar production of insulin and C-peptide. Although proinsulin has a direct biologic effect which is one-tenth as much as that of insulin, C-peptide has no biologic activity on homologous or heterologous tissue and no ability to modify the action of insulin and/or proinsulin. Previous work on proinsulin immunoassay suggested that this prohormone, but not C-peptide, cross-reacts with insulin antiserum. On the other hand, in the C-peptide immunoassay, proinsulin but not insulin cross-reacts with the antiserum. Up to this time, therefore, it has not been possible to immunoassay human proinsulin or C-peptide specifically. The very recent work from the laboratory of Heding, however, has brought about major advances in this area in which human C-peptide and proinsulin can be separated in the plasma by the use of Sepharose particles. With this recent major advancement, it is now possible to measure human C-peptide specifically. This measurement has been shown to be a useful tool for the assessment of beta-cell function in diabetic patients treated with insulin and in insulinoma patients in whom endogenous C-peptide secretion is not suppressed with exogenous insulin-induced hypoglycemia. With the use of a specific enzyme which degrades insulin but not proinsulin, postprandial plasma proinsulin values have been measured in a large number of subjects under a variety of physiologic and pathologic conditions. These results, which are comparable to those obtained by the more laborious column chromatography, could be summarized as follows: (1) proinsulin values in lean, young normal subjects do not vary greatly in response to insulin secretagogues; (2) proinsulin secretion in response to glucose results in a greater percentage of proinsulin in the older age group than in the younger group; (3) in lean adult and juvenile diabetic patients, the percentage of proinsulin is not excessive, whereas obese diabetics and pregnant diabetics appear to secrete relatively greater proinsulin than their diabetic controls; and (4) whereas most hyperinsulinemic states (Cusing's syndrome, adult-onset diabetics, acromegaly, and glucocorticoid therapy) are not associated with an increase in percentage of proinsulin, hyperinsulinemia of insulinoma, selected cases of functional hypoglycemia, and genetic hyperproinsulinemia are associated with a greater percentage of proinsulin. Identification of a possible new proinsulin intermediate(s) in these conditions deserves further investigation...     

Role of intact proinsulin in diagnosis and treatment of type 2 diabetes mellitus

Insulin resistance in patients with type 2 diabetes is associated with an increased risk of cardiovascular events. While this can be partly explained by an impairment of direct insulin action on the endothelial cell, an independent contribution can be assigned also to the secretory dysfunction of the beta-cell. If the demand for insulin triggered by insulin resistance is arriving at a certain threshold, an insufficiency of the cleavage capacity of beta-cell carboxypeptidase H leads to an increased secretion of intact proinsulin in addition to the desired insulin molecule. Proinsulin, however, has been demonstrated to be an independent cardiovascular risk factor by stimulating plasminogen activator inhibitor-1 secretion and blocking fibrinolysis. A recently introduced intact proinsulin assay is able to distinguish between intact proinsulin and its specific and non-specific cleavage products. This assay allows for a pathophysiological staging of type 2 diabetes based on beta-cell secretion. It could be confirmed by a large epidemiological study (IRIS-2, 4,265 patients) that intact proinsulin is a highly specific marker for insulin resistance. It could also be shown in other studies that successful resistance treatment with insulin or glitazones led to a decrease in elevated proinsulin levels and, thus, to a decrease of cardiovascular risk, while the levels remained high during sulfonylurea therapy. Therefore, patients with increased fasting intact proinsulin values should be treated with a therapy focusing on insulin resistance. Assessment of beta-cell function by determination of intact proinsulin may facilitate the selection of the most promising therapy and may also serve to monitor treatment success in the further course of the disease.     

Altered glucose regulation of insulin biosynthesis in insulinoma cells: mouse beta TC3 cells secrete insulin-related peptides predominantly via a constitutive pathway.

The effects of glucose on insulin gene expression and proinsulin biosynthesis, processing, and secretion were studied in mouse beta TC3 cells, an established insulinoma cell line derived from transgenic mice carrying a hybrid insulin promotor-simian virus-40 tumor antigen. The level of insulin mRNA was maintained at high levels by culture in 11 mM glucose, but essentially disappeared after 48 h of culture without glucose. The rate of insulin biosynthesis in beta TC3 cells was also dependent on glucose concentration over periods of 24 or 48 h (but not during 3 h) of stimulation. Insulin biosynthesis decreased about 50% after 24 h and about 85% after 48 h of incubation without glucose. When beta TC3 cells were incubated without glucose for 48 h, the rate of conversion of proinsulin to insulin was decreased compared to that at 11 mM glucose. Insulin secretion was sustained by medium glucose and also exhibited a much lower threshold for maximal stimulation; 2-deoxyglucose uptake decreased about 50% after 48 h of incubation without glucose. Studies on the secretion of newly synthesized proinsulin/insulin revealed that up to 80% of the total cellular pool of labeled proinsulin was released during a 60-min chase compared to only 10% of labeled insulin. The release of immunoreactive insulin (IRI) during the chase period was stimulated by forskolin and phorbol-12-myristate-13-acetate 1.6- and 10-fold, respectively. However, the release of newly synthesized proinsulin was insensitive to these secretagogues. It is concluded that 1) as in normal islets, glucose influences the steady state levels of proinsulin mRNA in beta TC3 cells; 2) the rate of proinsulin biosynthesis reflects only the level of insulin mRNA; translational control is absent; 3) cellular conversion of proinsulin to insulin is up-regulated by glucose as in normal rat islets; 4) newly synthesized proinsulin is released predominantly via a constitutive, rather than a regulated pathway, in contrast to normal beta-cells.