Insulin mRNA Content Research Articles

Developmental Regulation of Amylin and Insulin-Gene Expression in Lean (Fa/Fa) and obese (fa/fa) Zucker rats

Obese individuals are hyperinsulinemic and insulin resistant. Because amylin is cosecreted with insulin and may contribute to the insulin resistance of obesity, this study tested the hypothesis that insulin and amylin genes are coordinately regulated by obesity and carbohydrate feeding. Insulin and amylin gene expression were measured during the suckling/weaning transition in lean (Fa/Fa) and obese (fa/fa) Zucker rats, a period associated with marked changes in tissue insulin sensitivity. There was a decline in insulin mRNA (-90 +/- 15%, P less than 0.01) and amylin mRNA (-72 +/- 21%, P less than 0.01) content in pancreases of lean rats maintained on a high-fat diet from days 15 to 30, probably reflecting the relative increase in exocrine/endocrine development during this neonatal period and the effects of fat feeding. Weaning on high-carbohydrate versus high-fat diets resulted in enhanced expression of both insulin (P less than 0.05) and amylin (P less than 0.05) mRNAs. In contrast to the decline in pancreatic insulin and amylin mRNA content observed in lean rats, there was an increase in insulin mRNA (421.3 +/- 57.5%, P less than 0.05) and no change in amylin mRNA in obese rats maintained on a high-fat diet from days 15 to 30. There was no enhancement of insulin or amylin gene expression in obese rats with high carbohydrate relative to high-fat feeding, perhaps reflecting maximum rates of transcription in these obese insulin-resistant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure of pancreatic islets to different alkylating agents decreases mitochondrial DNA content but only streptozotocin induces long-lasting functional impairment of B-cells

Pancreatic B-cells exposed in vivo or in vitro to streptozotocin (SZ), the N-nitrosourea derivative of glucosamide, present a long-lasting impairment in the production and release of insulin while other cell functions are better preserved. This functional impairment is associated with a defective mitochondrial function. To further study the mechanisms behind SZ actions, mouse pancreatic islets were exposed in vitro to SZ (1.5 mM) or to different concentrations of methyl methanesulfonate (MMS; 2, 4 and 6 mM). The effect of the aglucone moiety of SZ, nitroso- N-methylurea (NMU; 2, 4 and 6 mM) was also tested. Islets were either studied immediately after exposure to the drugs (day 0) or after six days in culture following toxin treatment (day 6). On day 0 the islets showed a decrease in the NAD + NADH content, decreased glucose oxidation rates and an impaired insulin release in response to glucose. Six days after exposure to SZ there was still impaired glucose oxidation and insulin release, and decreased islet insulin mRNA and insulin content, but the NAD + NADH content was again similar to the control group. On the other hand, islets which survived for 6 days in culture following exposure to either MMS or NMU were able to regain normal B-cell function. The mouse islets exposed to SZ, NMU and MMS showed on day 6 a 30–40% decrease in the content of the mitochondrial DNA encoded cytochrome b mRNA and a 60–70% decrease in total mitochondrial DNA, as evaluated by dot and Southern blot analysis. Only SZ decreased the insulin mRNA content whereas both MMS and NMU decreased the glucagon mRNA content. As a whole, the data obtained indicate that SZ, NMU and MMS induce damage to the mitochondrial genome, and this may contribute to the B-cell dysfunction observed after SZ treatment. It is conceivable that the glucose moiety of SZ may direct the methylation to other intracellular sites besides the mitochondrial DNA, thus explaining the different functional responses of islets following exposure to SZ and NMU.

Liposomal Delivery of Purified Heat Shock Protein hsp70 Into Rat Pancreatic Islets as Protection Against Interleukin 1β–Induced Impaired β-Cell Function

Recently it has been demonstrated that heat shock protein 70 (hsp70) is induced in pancreatic islet cells during prolonged exposure to interleukin 1 beta (IL-1 beta). It is unclear whether this represents a cellular defense against the noxious action of IL-1 beta or whether hsp70 is involved in the suppressive action of the cytokine. To assess the role for hsp70 in isolated islets exposed to IL-1 beta, hsp70 was purified and introduced into cells of isolated rat pancreatic islets via the liposome technique. Delivery of hsp70 was efficient according to immunoblot analysis, but delivered hsp70 disappeared within 16 h. Hsp70-containing liposomes did not affect protein synthesis, insulin secretion, or islet insulin mRNA content. However, when hsp70 liposome-incubated islets were further exposed to IL-1 beta (25 U/ml) for 16 h, these islets released more insulin in response to glucose stimulation and contained more insulin mRNA than islets incubated with control liposomes and subsequently exposed to the cytokine. No protective effect of liposomes containing bovine serum albumin or ovalbumin were observed. We conclude that hsp70 may protect against IL-1 beta-induced impairment of pancreatic beta-cell function.

Increased glucose oxidation and contents of insulin and ATP in polyamine-depleted rat insulinoma cells (RINm5F)

In order to elucidate the role of polyamines in the replication and insulin production of insulin-secreting cells, we have investigated the impact of partial polyamine depletion on the proliferation, metabolism, insulin synthesis and ultrastructure of clonal rat insulinoma cells (RINm5F). For this purpose RINm5F cells were exposed for 4 days to the specific ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO). This resulted in a profound decrease in ODC activity and cytoplasmic polyamine contents. The polyamine content of cell nuclei was, however, not altered by DFMO. Addition of small amounts of putrescine during culture elevated the intracellular content of this diamine and suppressed ODC activity. The decrease in polyamine contents was accompanied by a pronounced inhibition of the cellular proliferative activity. The rates of glucose utilization, oxygen uptake and activity of the pentose cycle were decreased in DFMO-treated cells, whereas the glucose oxidation rate, oxidation/utilization ratio, ATP content and ATP/ADP ratio were increased. Insulin mRNA content and synthesis of proinsulin, insulin and total protein were not altered by DFMO. In contrast, there was a sizeable increase in the cellular insulin content, despite a lowered total protein content. Electron-microscopic analysis revealed an accumulation of insulin-secretion granules in the DFMO-treated cells. In addition, short-term insulin release was increased after DFMO exposure, but was not rendered glucose-sensitive. It is concluded that polyamines are necessary for the maintenance of rapid insulinoma-cell replication and that DFMO-treated RINm5F cells acquire an enhanced substrate oxidation and increased content of insulin and ATP.

Long-term effects of a high glucose concentration in vitro on the oxidative metabolism and insulin production of isolated rat prancreatic islets

The present study was performed to clarify whether exposure in tissue culture of pancreatic islet B cells to high glucose concentrations will lead to glucose insensitivity and/or toxicity. For this purpose, isolated rat islets were maintained in tissue culture for up to 7 days in the presence of either 5.6, 11, or 56 mmol/L glucose and subsequently analyzed with regard to oxidative metabolism, insulin release, islet content of insulin, and insulin mRNA. Islets maintained at 56 mmol/L glucose showed a decreased insulin content, but no changes in insulin mRNA content when compared with control islets (cultured at 11 mmol/L glucose). In short-term incubations of the high-glucose cultured islets, the rate of insulin release at 1.67 mmol/L glucose was enhanced and could not be further stimulated by a 16.7-mmol/L glucose challenge. However, the insulin release at 16.7 mmol/L was decreased when compared with islets cultured at 11 mmol/L glucoss. Islets cultured at 56 mmol/L glucose showed an increased oxygen uptake when incubated at 1.67 mmol/L glucose with no further stimulation at 16.7 mmol/L glucose. These islets also showed increased rates of glucose oxidation at incubation with 1.67 mmol/L glucose, but similar rates of oxidation at 16.7 mmol/L glucose as compared with islets cultured in 11 mmol/L glucose. Islets cultured at 5.6 mmol/L glucose showed decreased insulin release when incubated at either 1.67 mmol/L or 16.7 mmol/L glucose. The rates of glucose oxidation of these islets were also decreased at 16.7 mmol/L glucose when compared with the controls, whereas the oxygen uptake was decreased only during incubation at 1.67 mmol/L glucose. There was also a decreased content of insulin mRNA in these islets. When islets were cultured at 56 mmol/L glucose for 6 days followed by 5.6 mmol/L glucose for 1 day, there was a restoration of their glucose responsiveness with respect to both glucose oxidation and insulin release, although the cellular stone of insulin remained decreased. It is concluded that rat pancreatic islets maintained in culture at a high glucose concentration show no signs of lasting functional damage. The transient glucose insensitivity shown by these islets reflects a sustained high rate of insulin release also during incubation at a low glucose concentration.

Interleukin-1β Induces an Early Decrease in Insulin Release, (Pro)Insulin Biosynthesis and Insulin Mrna in Mouse Pancreatic Islets by a Mechanism Dependent on Gene Transcription and Protein Synthesis

In an attempt to further characterize the mechanisms of action of recombinant interleukin-1 beta (rIL-1 beta) on mouse pancreatic islets, islets were exposed for different periods of time (6, 12 and 24 h) to 50 U/ml rIL-1 beta. After 6 h there was already a significant decrease in glucose (16.7 mM)-induced insulin release. This was followed at 12 h by a decrease in insulin mRNA contents and (pro)insulin biosynthesis and, after 24 h, by a decrease in islet insulin contents. There was no decrease in total protein biosynthesis or DNA contents in any of the studied time points and the glucose oxidation rates were not affected by rIL-1 beta after 12 h of exposure. A similar inhibition of insulin release, (pro)insulin biosynthesis and insulin mRNA content was observed 12 h after a short (2 h) exposure of the islets to rIL-beta, suggesting that a brief exposure of mouse islets to the cytokine can modify their function for several hours. When islets were exposed for 12 h to 50 U/ml rIL-1 beta in the presence of either an inhibitor of gene transcription (actinomycin D) or an inhibitor of mRNA translation (cycloheximide) there was a complete protection against the suppressive effects of rIL-1 beta on insulin release, (pro)insulin biosynthesis and insulin mRNA contents. However, when islets were exposed for 2 h to rIL-1 beta in the presence of actinomycin D, and studied 12 h later, actinomycin counteracted the inhibitory effects of rIL-1 beta on insulin release, but not on (pro)insulin biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperglycemia-induced B cell toxicity. The fate of pancreatic islets transplanted into diabetic mice is dependent on their genetic background.

The role of pancreatic B cell dysfunction in the phase preceding clinical onset of insulin-dependent and non-insulin-dependent diabetes mellitus has been much debated. In this investigation, the impact of a prolonged diabetic environment on pancreatic islet B cells transplanted syngeneically under the kidney capsule of C57BL/6 (B6) and C57BL/Ks (BKs) mice was studied. Alloxan-diabetic mice bearing a subcapsular islet graft insufficient to normalize the blood glucose level were rendered normoglycemic by a second intrasplenic islet graft after various period of hyperglycemia to examine the reversibility of hyperglycemia-induced B cell dysfunction. Using a perfusion technique of the graft-bearing, it was found that both strains of mice exhibited a diminished glucose-induced insulin secretion after 6 wk of hyperglycemia, when compared with normoglycemic mice carrying islet grafts. When normoglycemia was restituted by the splenic graft after 4 or 12 wk, there was a normalization of glucose-stimulated insulin secretion in the renal islet grafts in B6 mice, whereas insulin secretion from the grafted BKs islets remained impaired. Morphometric measurements of the islet grafts demonstrated a 50% reduction in the graft volume in diabetic BKs mice after 12 wk, compared with normoglycemic animals, whereas no such decrease was observed in B6 mice. Islet grafts removed from hyperglycemic mice of both strains exhibited diminished insulin mRNA contents, and in the BKs mice there was also a reduced glucose oxidation rate in the islet grafts in vitro. This metabolic dysfunction can only partly be explained by a reduced graft size. The present findings emphasize the genetic constitution as a decisive factor for the survival and function during a period of sustained stress on a limited B cell mass.

Interleukin-1β Depletes Insulin Messenger Ribonucleic Acid and Increases the Heat Shock Protein hsp70 in Mouse Pancreatic Islets Without Impairing the Glucose Metabolism*

In order to further characterize the actions of recombinant interleukin-1 beta (rIL-1 beta) on the function of insulin-producing cells, the effects of different concentrations of the cytokine were studied on islets obtained from four different mouse strains (NMRI, NOD, C57BL/6, and C57BL/Ks). For this purpose the islets were exposed to rIL-1 beta (25, 50, or 100 U/ml) for a 48-h period in medium RPMI 1640 containing 10% calf serum and 11.1 mM glucose. In all groups and at the various rIL-1 beta concentrations tested, there was a similar 30-50% inhibition in glucose-induced insulin release, a 70-80% decrease in islet insulin content, and no significant differences in islet DNA content or insulin accumulation in the culture medium. To clarify the mechanisms underlying the decreased islet insulin content, rates of (pro)insulin biosynthesis and insulin messenger RNA (mRNA) contents were determined. Exposure of NMRI and C57BL/6 islets to 50 U/ml rIL-1 beta reduced the (pro)insulin biosynthesis by 40-50% and the insulin mRNA contents by 80-90%. The cytokine also induced an increased cellular content of the heat shock protein hsp70, as measured by western blot analysis, and a decrease in DNA biosynthesis, as measured by [methyl-3H]thymidine incorporation. However, exposure to rIL-1 beta did not decrease islet total protein biosynthesis, glucose oxidation, ATP content, ATP/ADP ratio, cAMP content, or polyamine contents. In conclusion, these data suggest that exposure of mouse islets to rIL-1 beta reduces DNA synthesis, insulin mRNA levels, and the biosynthesis of (pro)insulin, without equally impairing other cellular functions. The mechanisms behind these reductions seem to be different from those observed in rat islets, where a rIL-1 beta-induced impairment of substrate metabolism at the mitochondrial level seems to be related to the decrease of several cellular functions.

Increased Preproinsulin mRNA in Pancreatic Islets Incubated With Islet Cell–Stimulating Antibodies From Serums of Type I Diabetic Patients

We recently described autoantibodies that stimulate the release of insulin from pancreatic beta-cells both in vitro and in vivo. The aim of this study was to establish whether islet cell-stimulating antibodies (ICSTAs) also increase islet cell preproinsulin mRNA content. Wistar rat islets, isolated by collagenase digestion, were exposed to 2.7 and 11.1 mM glucose. Insulin release increased 10-fold in response to the higher glucose concentration, and dot-blot analysis of islet mRNA with a rat preproinsulin cDNA probe showed a concomitant increase in mRNA levels. The globulin fractions of four test serums, three from patients with type I (insulin-dependent) diabetes and one from a patient with the insulin autoimmune syndrome, showed clear (5- to 8-fold) stimulation of insulin release. The nonglobulin fractions of these serums and both fractions of three control serums failed to stimulate secretion of insulin. The insulin mRNA content of islets incubated with the ICSTA globulin fractions was greatly increased compared with levels observed in islets treated with control serum globulin fractions. We conclude that ICSTAs not only can stimulate the release of insulin but also increase the preproinsulin mRNA content of islet cells.

Genetic analysis of obesity-induced diabetes associated with a limited capacity to synthesize insulin in C57BL/KS mice: evidence for polygenic control.

Expression of obesity-induced diabetes associated with the diabetes or db mutation in mice varies in inbred strains. This study utilized a genetic analysis to evaluate the number of genes responsible for the difference in diabetes responses between mice of the susceptible C57BL/KsJ (BL/Ks) and resistant 129/J inbred strains. BL/Ks (db/+) males and 129/J (+/+) females were bred to generate F1 hybrids, and the F1 females (db/+ and +/+, distinguished by progeny testing) were backcrossed to BL/Ks (db/+) males. A total of 252 backcrossed males were obtained, of which 31 were db/db and obese. While the plasma glucose of all the fed backcrossed mice was greater than 22 mmol/l, the expression of diabetes varied considerably, as measured by fasting plasma glucose, fed plasma insulin, and pancreatic insulin and proinsulin mRNA content. That proinsulin mRNA content was a good indicator of diabetes severity and islet dysfunction was seen in the inverse correlation between proinsulin mRNA content and fasting plasma glucose (r = 0.69, p less than 0.001), and a direct correlation between proinsulin mRNA and plasma insulin (r = 0.86, p less than 0.001), and pancreatic insulin content (r = 0.61, p less than 0.01). If a single gene were responsible for severe islet dysfunction, one-half of the backcrossed mice would develop low proinsulin mRNA levels like the BL/Ks parent, and one-half would be resistant to islet destruction. Statistical evaluation (SKUMIX) of the distribution of these parameters in backcrossed mice rejected with a high degree of probability a bimodal distribution.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat Insulin II Gene Expression by Extraplacental Membranes: A Non-Pancreatic Source for Fetal Insulin

Abstract Using cDNA cloning, ribonuclease protection, and Northern hybridization analysis, we showed that insulin gene expression occurs in yolk sac-derived fetal extraplacental membranes throughout the last half of rat fetal development. The mRNA product of the ancestral rat insulin II but not the duplicated rat insulin I gene was present in high copy number, and its abundance was regulated during development. Insulin mRNA was present in extraplacental membranes before pancreatic differentiation; membrane insulin mRNA content greatly exceeded that in pancreas until the last 2 days of gestation when content in each tissue became similar. Polyadenylation and intron splicing occurred at the same sites used in pancreas, but initiation of transcription occurred at multiple sites in membranes. Minces of membranes maintained in culture produced approximately 10 ng of radioimmunoassayable insulin/mg membrane protein/day. Over a 4-day period, approximately 50 times more insulin accumulated in medium than that present in membranes at the time of isolation. These studies indicate that yolk sac is a source for insulin during fetal development and that the mechanisms regulating insulin gene expression in this tissue differ from those in pancreatic beta cells.

Polyamines and insulin production in isolated mouse pancreatic islets.

The aim of the present study was to evaluate the role of polyamines in the metabolism and insulin production of pancreatic-islet cells. For this purpose islets were prepared from adult mice and used either immediately or after tissue culture. There was a significant decrease in the islet content of spermidine during culture, although the effect was less pronounced in a high glucose concentration. Furthermore, a stimulatory effect of a high glucose concentration, as compared with low guclose, on the content of spermine was observed. To elucidate further the role of polyamaines in beta-cell physiology, the ornithine decarboxylase inhibitors difuoromethylornithine (DFMO) and methylacetylenic putrescine (MAP) and the S-adenosylmethionine decarboxylase inhibitor ethylglyoxal bis(guanylhydrazone) (EGBG) were added to the culture media. Addition of DFMO together with MAP decreased the cellular contents of putrescine and spermidine, whereas the content of sperimine was unaffected. When EGBG was added in combination with DFMO and MAP, there was a decrease in the content of spermine also. Cell viability in the islets depleted of their polyamine contents was not impaired, as assessed by determinations of oxygen-uptake rates and ATP contents. Depletion of putescine plus spermidine by addition of DFMO+MAP was associated with decreased biosynthesis of (pro)insulin and total protein. When the content of spermine was decreased also by the further addition of EGBG, the decrease in (pro) insulin biosynthesis was more pronounced and was paralleled by a decrease in the insulin-mRNA content. Under these conditions, the glucose-stimulated insulin release, the insulin content and the rates of islet DNA synthesis were also decreased. It is concluded that putrescine and spermidine are necessary for the maintenance of normal insulin and protein biosynthesis, whereas spermine may exert a role in some other cellular processes, such as DNA replication, RNA transcription and glucose-stimulated insulin release.

Preferential Reduction of Insulin Production in Mouse Pancreatic Islets Maintained in Culture after Streptozotocin Exposure*

The ability of the pancreatic beta-cell to repair itself after a cytotoxic injury and reassume its functional activities may be a key issue in affording protection from insulin-dependent diabetes mellitus. The molecular mechanisms behind the functional responses of the beta-cell after cytotoxic damage are still largely unknown. The present study in an attempt to elucidate this issue. Mouse pancreatic islets were isolated with collagenase and, after overnight culture, exposed for 30 min at 37 C to 2.2 mM streptozotocin (SZ) or vehicle alone (controls). The islets were subsequently cultured for 6 days in medium RPMI-1640 plus 10% calf serum. After the culture they were subjected to light microscopical examinations or different functional tests during short term incubations. The SZ-treated islets showed markedly diminished insulin release after stimulation with the beta-cell nutrients glucose and leucine plus glutamine. Compounds known to increase intracellular cAMP [theophylline and (Bu)2-cAMP] were able to partially counteract the SZ-induced reduction of insulin release. Stimulation with arginine could also slightly restore the impaired insulin release. Glucose-stimulated oxygen uptake, proinsulin biosynthesis, and insulin and insulin mRNA contents were also decreased, with values at about 50% of the controls. However, the cellular contents of DNA and RNA and total protein biosynthesis rates were essentially normal. Besides mild degranulation in some islets, the morphological appearance of the SZ-treated islets did not reveal any obvious differences compared to the control islets. The present observations suggest that after a toxic injury there remains a population of partially damaged beta-cells, which are able to maintain most of their basal metabolic functions, but fail to maintain adequate insulin biosynthesis and release.

Insulin mRNA content in pancreatic beta cells of coxsackievirus B4-induced diabetic mice

Molecular hybridization was used to measure poly(A)-containing mRNA and insulin mRNA, and to evaluate viral persistence, in pancreatic beta cells of coxsackievirus B4-induced diabetic mice. Cellular RNA was hybridized with [ 3H]poly(U) to measure poly(A)-containing total mRNA, 32P-labeled preproinsulin I and II probes to measure insulin mRNA, and a 32P-labeled virus-specific probe to evaluate persistence. The infected mice (80–90%) showed subnormal blood glucose at 72 h postinfection and were hyperglycemic at 6 and 8 weeks. Poly(A)-containing total mRNA decreased by about 26% at 72 h and 6 weeks and by 49% at 8 weeks, while preproinsulin I mRNA by 30% and preproinsulin II by 46% at 8 weeks postinfection compared to control. Viral sequences were abundant at 72 h and in fair amounts later. It appears that persistent viral infection produces a pathological state, which impairs beta cell function to reduce insulin mRNA and consequently insulin synthesis apparently leading to hyperglycemia.

Control of insulin gene expression in pancreatic beta-cells and in an insulin-producing cell line, RIN-5F cells. II. Regulation of insulin mRNA stability.

The half-life of insulin mRNA at various glucose concentrations was determined by filter hybridization techniques in isolated rat islets incubated with 3H-labeled uridine followed by a chase incubation at 3.3 or 17 mM glucose. High glucose induced a greater stabilization of insulin mRNA than of other poly(A) + RNAs or total cellular RNA. In RIN-5F insulinoma cells, an insulin-producing cell line, cholera toxin, but not glucose, induced a stabilization of insulin mRNA. After 24 h of culture of islets with actinomycin D or alpha-amanitin at several glucose concentrations, insulin mRNA content was decreased in comparison to controls only at higher glucose concentrations. The biosynthesis of islet proteins other than insulin was strongly decreased by actinomycin D at all glucose concentrations. Insulin biosynthesis was inhibited proportionately to the observed decreases in insulin mRNA content. We conclude that inhibition of insulin mRNA degradation is an important component in increasing the insulin mRNA content in response to glucose, thereby augmenting the effects of glucose stimulation on insulin gene transcription (5). This stabilization may be partly mediated by cAMP as evidenced by the similar responses to cholera toxin in the RIN-5F cells. Furthermore, the results of experiments with actinomycin D suggest that the degradation of insulin mRNA may require the continuous production of a factor(s) which could be either RNA or protein in nature.

Control of insulin gene expression in pancreatic beta-cells and in an insulin-producing cell line, RIN-5F cells. I. Effects of glucose and cyclic AMP on the transcription of insulin mRNA.

To define the mechanism whereby glucose regulates islet insulin mRNA content, insulin gene transcription rates were determined in islets labeled with [3H]uridine at low (3.3) or high (17 mM) glucose. Glucose stimulated the transcription of total RNA almost 2-fold and insulin mRNA 5.6-fold. Addition of dibutyryl cAMP to islets in vitro could partially mimic the effect of glucose on insulin gene-specific transcription. In the insulin-producing RIN-5F cell line, glucose did not affect transcription, while cholera toxin acted as a secretagogue and increased total RNA and insulin gene-specific transcription as well. We conclude that glucose exerts a specific stimulatory effect on the transcription of the insulin gene(s) in normal islets and that this effect may be mediated in part by cAMP.