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D Pipeleers

Publications and source records attributed to D Pipeleers.

At least 55 records · Page 3Linked to original sources
PubMed1996

Glucose promotes survival of rat pancreatic beta cells by activating synthesis of proteins which suppress a constitutive apoptotic program.

This study demonstrates that rat islet beta cells constitutively express an apoptotic program which is activated when mRNA or protein synthesis is blocked. Apoptotic beta cells were detectable by electron microscopy after treatment with actinomycin D or cycloheximide. With a fluorescence microscopic assay both agents were found to increase the number of apoptotic beta cells dose- and time-dependently, up to 70% after 1 wk of culture; virtually no apoptotic beta cells occurred in control preparations or in conditions leading to primary necrosis. Thus, survival of beta cells seems dependent on synthesis of proteins which suppress an endogenous suicide program. This mechanism explains earlier observed effects of glucose on survival of cultured beta cells. Glucose is known to dose-dependently increase the percentage of beta cells in active biosynthesis and the percentage that survives during culture. It is now demonstrated that the glucose-induced survival of beta cells cultured for 1 wk results from a dose-dependent reduction in the percentage of beta cells dying in apoptosis (49% at 3 mM glucose, 40% at 6 mM, 9% at 10 mM). Thus, intercellular differences in glucose sensitivity appear responsible for the heterogeneity in beta cell sensitivity to apoptotic conditions. These data indicate that glucose promotes survival of beta cells by activating synthesis of proteins which suppress apoptosis. The present model allows for further investigation of the regulation of apoptosis in beta cells and the identification of agents which induce or prevent beta cell death.

Animals↗
PubMed1996

The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells.

Expression of glucokinase in hepatocytes and pancreatic 6-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and beta-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing alpha-cells, which are negatively regulated by glucose. Purified rat alpha-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the beta-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of alpha-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic alpha-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells.

In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process.

3-O-Methylglucose↗
PubMed1996

Expression and functional activity of glucagon, glucagon-like peptide I, and glucose-dependent insulinotropic peptide receptors in rat pancreatic islet cells.

Rat pancreatic alpha- and beta-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in beta-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in beta-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide I(7-36) amide (GLP-I), while RNA from islet alpha-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in beta-cells and not in alpha-cells. Receptor activity, measured as cellular cAMP production after exposing islet beta-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/l GLP-I and 50 pmol/l GIP but required 1 nmol/l glucagon. EC50 values of GLP-I- and GIP-induced cAMP formation were comparable (0.2 nmol/l) and 45-fold lower than the EC50 of glucagon (9 nmol/l). Maximal stimulation of cAMP production was comparable for the three peptides. In purified alpha-cells, 1 nmol/l GLP-I failed to increase cAMP levels, while 10 pmol/l to 10 nmol/l GIP exerted similar stimulatory effects as in beta-cells. In conclusion, these data show that stimulation of glucagon, GLP-I, and GIP receptors in rat beta-cells causes cAMP production required for insulin release, while adenylate cyclase in alpha-cells is positively regulated by GIP.

Adenylyl Cyclases↗
PubMed1995

Differences in glucose transporter gene expression between rat pancreatic alpha- and beta-cells are correlated to differences in glucose transport but not in glucose utilization.

Glucose exerts inverse effects upon the secretory function of islet alpha- and beta-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat alpha- and beta-cells. beta-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; alpha-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from alpha- or beta-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa beta-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In beta-cells, the rates of glucose transport were 10-fold higher than in alpha-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5(3)H]glucose utilization, was comparable in alpha- and beta-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between alpha- and beta-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.

3-O-Methylglucose↗
PubMed1995

Human and rat beta cells differ in glucose transporter but not in glucokinase gene expression.

Glucose homeostasis is controlled by a glucose sensor in pancreatic beta-cells. Studies on rodent beta-cells have suggested a role for GLUT2 and glucokinase in this control function and in mechanisms leading to diabetes. Little direct evidence exists so far to implicate these two proteins in glucose recognition by human beta-cells. The present in vitro study investigates the role of glucose transport and phosphorylation in beta-cell preparations from nondiabetic human pancreata. Human beta-cells differ from rodent beta-cells in glucose transporter gene expression (predominantly GLUT1 instead of GLUT2), explaining their low Km (3 mmol/liter) and low VMAX (3 mmol/min per liter) for 3-O-methyl glucose transport. The 100-fold lower GLUT2 abundance in human versus rat beta-cells is associated with a 10-fold slower uptake of alloxan, explaining their resistance to this rodent diabetogenic agent. Human and rat beta-cells exhibit comparable glucokinase expression with similar flux-generating influence on total glucose utilization. These data underline the importance of glucokinase but not of GLUT2 in the glucose sensor of human beta-cells.

Animals↗
PubMed1995

Proliferation and hypertrophy of liver cells surrounding islet grafts in diabetic recipient rats.

The liver offers an adequate site for the metabolic function of pancreatic islet implants. Little is known about the effects of the islet grafts on the host organ. This study examines liver tissue of normal or streptozotocin (STZ)-diabetic rats at different intervals following intraportal injection of syngeneic islets. Implantation of 800-islet-grafts, containing 0.9 million beta cells, normalized overt diabetes within 14 days. This period of metabolic normalization was characterized by a specific sequence of alterations in the implant area. During the first days after transplantation, islet cells migrated into the liver lobules, whereby tight hepatocyte-islet cell contacts were established. Hepatocytes surrounding grafts showed massive lipid accumulation and hypertrophy (cellular profile area 603 +/- 72 microns 2 in diabetic islet recipients vs. 382 +/- 42 microns 2 in diabetic controls; P < .005). The implant area also contained significantly more liver cells in proliferative activity than hepatic tissue in normal controls (bromodeoxyuridine labeling index of peri-islet hepatocytes 6.2%, 4.6%, and 0.9% on posttransplantation days 2, 4, and 14, respectively, compared with 0.02% in normal controls). The cellular hypertrophy and hyperplasia explain the sudden increase in liver weight of diabetic recipients (from 8.0 +/- 1.1 g to 13.8 +/- 2.2 g on posttransplantation day 2; P < .005). Both alterations can be attributed to the massive local discharge of insulin in an insulin-deficient organ containing an excess of extra-cellular nutrients. Progressive revascularization of the implant sites and overall metabolic normalization are thought to explain the return of a normal liver histology by the third week after transplantation. In conclusion, intraportal islet grafts exert profound effects on the liver of diabetic rat recipients. The morphological features of the implant sites may serve as markers for the function of the islet grafts as well as for the adaptive capacity of the recipient liver.

Animals↗
PubMed1994

Physiologic relevance of heterogeneity in the pancreatic beta-cell population.

In vitro studies on purified rat beta cells have indicated a functional diversity among insulin-containing cells. Intercellular differences were found in the rates of glucose-induced insulin synthesis and release. They are attributed to differences in cellular thresholds for glucose utilization and oxidation, as can be caused by varying activities in rate limiting steps such as glucokinase-dependent phosphorylation. The percent of functionally active beta cells increases dose-dependently with the glucose concentration, making cellular heterogeneity and its regulation by glucose major determinants for the dose-response curves of the total beta-cell population. Beta cells which are already responsive to low glucose concentrations are characterized by a higher content in pale immature granules; their activated biosynthetic and secretory activity accounts for preferential release of newly-formed hormone by the total beta-cell population. At any glucose level, the amplitude of insulin release depends on the percent glucose-activated cells and their cyclic AMP content, an integrator of (neuro)hormonal influences. The in vitro described heterogeneity in beta-cell functions may bear physiological relevance as several of its characteristics are also detectable in intact pancreatic tissue; furthermore, in vitro signs of heterogeneity can be altered by prior in vivo treatment indicating that they express properties of the cells in their in situ configuration. Elevated basal levels of (pro)insulin may reflect the existence of an increased number of beta cells that are activated at low physiologic glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Effect of nutrients, hormones and serum on survival of rat islet beta cells in culture.

This study quantifies the survival of purified single rat beta cells under different culture conditions. Less than 10% of the cells survive 9 days of culture in Ham's F10 medium without supplements. Addition of fetal calf serum (5%) increases cell survival to 54% in the absence and to 78% in the presence of isobutylmethylxanthine (50 mumol/I). The effect of serum is explained, at least partly, by the presence of albumin and of low molecular weight constituents. In serum-free Ham's F10 with 50 mumol/l isobutylmethylxanthine, 75% of cells survive after the addition of bovine serum albumin (1%) and of ultroser (0.2%), a commercial serum substitute. Survival of at least 75% of cells is also maintained in Ham's F10 with isobutylmethylxanthine plus albumin, and supplemented by metabolizable nutrients or by the peptides glucagon (10(-8) mol/l) or growth hormone (1 micrograms/ml) plus insulin like growth factor-I (50 ng/ml). D-Glucose increases beta-cell survival in a dose-dependent manner up to 10 mmol/l; a beneficial effect is also observed with other metabolizable compounds (leucine and glutamine) but not with non-metabolizable monosaccharides. Glucose-induced survival of islet beta cells can be attributed to its dose-dependent recruitment of cells into metabolic activities; however, a 9-day exposure to excessively high nutrient concentrations (> 20 mmol/l glucose) is deleterious to the cells. These results define culture media, with or without serum, wherein at least 75% of single rat islet beta cells can survive for a minimum of 9 days. This will allow for studies on beta-cell toxic conditions and potentially protective agents.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1993

Reduced glutamate decarboxylase activity in rat islet beta cells which survived streptozotocin-induced cytotoxicity.

Rat pancreatic beta cells exhibit a 16-fold higher glutamate decarboxylase (GAD) activity than islet non-beta cells, but a similar glutamate dehydrogenase (GDH) activity. beta Cells which survive exposure to 2 mM streptozotocin only contain 10 percent of the GAD activity of control cells, but their GDH activity remains unaltered. Culture of streptozotocin-treated beta cell preparations with 2 mM nicotinamide reduces the number of dead cells and prevents in part the decline in GAD activity of surviving beta cells. These data indicate that loss in activity of the beta cell specific enzyme GAD can serve as marker for beta cells which survived a destructive process. It is furthermore demonstrated that nicotinamide increases the percent surviving cells and decreases their loss in GAD activity.

Animals↗
PubMed1993

Heterogeneity in glucose sensitivity among pancreatic beta-cells is correlated to differences in glucose phosphorylation rather than glucose transport.

Rat beta-cells differ in their individual rates of glucose-induced insulin biosynthesis and release. This functional heterogeneity has been correlated with intercellular differences in metabolic redox responsiveness to glucose. The present study compares glucose metabolism in two beta-cell subpopulations that have been separated on the basis of the presence (high responsive) or absence (low responsive) of a metabolic redox shift at 7.5 mM glucose. Mean rates of glucose utilization and glucose oxidation in high responsive beta-cells were 2- to 4-fold higher than in low responsive beta-cells, whereas their leucine and glutamine oxidation was only 10-50% higher. This heterogeneity in glucose metabolism cannot be attributed to differences in GLUT2 mRNA levels or in glucose transport. In both cell subpopulations, the rates of glucose transport (13-19 pmol/min/10(3) beta-cells) were at least 50-fold higher than corresponding rates of glucose utilization. On the other hand, rates of glucose phosphorylation (0.3-0.7 pmol/min/10(3) beta-cells) ranged within those of total glucose utilization (0.2-0.4 pmol/min/10(3) beta-cells). High responsive beta-cells exhibited a 60% higher glucokinase activity than low responsive beta-cells and their glucokinase mRNA level was 100% higher. Furthermore, glucose phosphorylation via low Km hexokinase was detected only in the high responsive beta-cell subpopulation. Heterogeneity in glucose sensitivity among pancreatic beta-cells can therefore be explained by intercellular differences in glucose phosphorylation rather than in glucose transport.

Amino Acids↗
PubMed1992

Presence of islet amyloid polypeptide in rat islet B and D cells determines parallelism and dissociation between rat pancreatic islet amyloid polypeptide and insulin content.

The islet amyloid polypeptide (IAPP) immunoreactivity of the adult rat pancreas is located in insulin-containing B cells as well as in somatostatin-containing D cells. In both cell types, the IAPP immunoreactivity is identical to rat synthetic IAPP in terms of its elution position after reversed phase HPLC and its binding to IAPP antibodies. The IAPP content per 10(6) B-cells is more than 100 fold lower than the corresponding insulin content, but comparable to the IAPP content of D cells. After induction of diabetes by streptozotocin, pancreatic IAPP seems predominantly located in somatostatin-containing cells. In normal rats, pancreatic insulin and IAPP content increase 20 fold from birth to 12 weeks of age; beyond week 12, the further rise in pancreatic insulin was not paralleled by an increase in IAPP content.

Amyloid↗
PubMed1992

Differences in glucose recognition by individual rat pancreatic B cells are associated with intercellular differences in glucose-induced biosynthetic activity.

In vitro incubated rat islet B cells differ in their individual rates of protein synthesis. The number of cells in biosynthetic activity increases with the glucose concentration. Flow cytometric monitoring of the cellular redox states indicated that islet B cells differ in their individual metabolic responsiveness to glucose. A shift from basal to increased NAD(P)H fluorescence occurred for 18% of the cells at 1 mM glucose, for 43% at 5 mM, and for 70% at 20 mM. The functional significance of this metabolic heterogeneity was assessed by comparing protein synthesis in metabolically responsive and unresponsive subpopulations, shortly after their separation by autofluorescence-activated cell sorting. The glucose-sensitive subpopulation exhibited four- to fivefold higher rates of insulin synthesis during 60-min incubations at 2.5-10 mM glucose. Its higher biosynthetic activity was mainly caused by recruitment of cells into active synthesis and, to a lesser extent, by higher biosynthetic activity per recruited cell. Cells from the glucose-sensitive subpopulation were larger, and presented a threefold higher density of a pale secretory vesicle subtype, which is thought to contain unprocessed proinsulin. It is concluded that intercellular differences in metabolic responsiveness result in functional heterogeneity of the pancreatic B cell population.

Animals↗
PubMed1992

Increased concentrations of total IgM at clinical onset of type 1 (insulin-dependent) diabetes: correlation with IgM binding to cells. The Belgian Diabetes Registry.

Eighty patients with type 1 (insulin-dependent) diabetes (ages 0-39 years) were consecutively recruited by the Belgian Diabetes Registry. Sera obtained at clinically diagnosed onset (i.e., before start of insulin therapy or within 7 days of initial treatment) were analyzed for total IgM concentrations and for IgM binding to fixed rat splenocytes (IgM-LyAb) and permeabilized rat islet cells (IgM-ICAb). Comparison of results with those in age- and sex-matched control subjects, by fluorescence-activated cell-sorter analysis, indicated greater concentrations of IgM-LyAb and IgM-ICAb in sera from the patients. IgM antibodies reacted indiscriminately with islet beta and islet endocrine non-beta cells. The prevalence of IgM-ICAb, but not of IgM-LyAB, was significantly (P less than 0.05) higher in patients than in the control subjects. Of the ICAb-positive patients, 54% were also LyAb-positive, whereas none of the control subjects were doubly positive. IgM-ICAb and IgM-LyAb binding signals were positively correlated. Serum IgM concentrations were significantly (P less than 0.001) greater in patients than in control subjects and were significantly correlated with IgM-LyAb (P less than 0.001) and IgM-ICAb (P less than 0.01). The positivity for IgM binding was not, however, merely a reflection of total IgM, because no such correlation was found in sera from seven patients with Waldenström macroglobulinemia. Clinical onset of type 1 diabetes is apparently accompanied by increased production of IgM. The correlation between IgM concentrations and IgM binding to islet cells might reflect polyclonal activation or natural autoantibodies.

Adolescent↗
PubMed1991

Transplantation of purified islet cells in diabetic BB rats.

The ability to prepare purified islet Beta-cell aggregates was used to examine the survival of this cell type after allotransplantation in diabetic BB rats. The aggregates were intraportally implanted in numbers that were previously found to correct a streptozotocin-induced diabetic state in syngeneic or allogeneic Brown Norway recipients. When the grafts were prepared from RT1u/l donors, which shared the MHC-class I antigen with the BB recipients (RT1u/u), their implant sites became diffusely infiltrated by inflammatory cells and their metabolic function was completely lost within 5 weeks. MHC-class I incompatible islet Beta-cell allografts (RT1n/n) exhibited a longer survival, in particular when combined with other islet endocrine cells and/or when covered by a 5-week cyclosporin treatment. In the latter combination, 10 of 12 BB rat recipients remained normoglycaemic over the 10-week observation period, their liver implants presenting a comparable insulin reserve and similarly discrete mononuclear cell infiltration as streptozotocin-diabetic Brown Norway rats receiving this treatment. However, administration of cyclosporin to diabetic BB rats was associated with a morbidity that was not observed in drug-treated streptozotocin-diabetic Brown Norway animals or in untreated diabetic BB rats. It is concluded that MHC-incompatible islet Beta cells can induce a long-term normalization in diabetic BB rats provided that they are implanted under conditions which allow allograft acceptance. The standardized preparation of purified islet Beta-cell grafts can help assessing the conditions for successful transplantations in diabetes with an autoimmune origin.

Animals↗