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M Welsh

Publications and source records attributed to M Welsh.

At least 37 records · Page 2Linked to original sources
PubMed1991

Interleukin-1 beta increases the biosynthesis of the heat shock protein hsp70 and selectively decreases the biosynthesis of five proteins in rat pancreatic islets.

Prolonged exposure to high concentrations of human recombinant interleukin-1 beta (rIL-1 beta) has been reported to exert both suppressive and cytotoxic effects on pancreatic beta-cells during culture in vitro. In order to investigate the molecular mechanism(s) underlying the actions of rIL-1 beta on the beta-cell, we have exposed isolated rat pancreatic islets for 3 or for 24 h to 25 U/ml of rIL-1 beta. Subsequently the biosynthesis of heat shock proteins, as assessed by western blot analysis, and total protein biosynthesis patterns were studied, using one and two-dimensional gel electrophoresis of [35S]methionine labelled islet proteins from different subcellular compartments. It was found that rIL-1 beta exerted no specific effects on protein synthesis when added during a 3 h incubation period. However, after a 24 h incubation period, the synthesis of a group of acidic proteins with the approximate molecular weight of 35 kD was specifically inhibited in the rIL-1 beta treated islets. This alteration was predominantly associated with the endoplasmic reticulum fraction. The cytokine also inhibited the synthesis of four cytosolic proteins with the molecular weights 75, 85, 95 and 120 kD. In contrast, rIL-1 beta increased the expression of the heat shock protein hsp70 both in the microsomal and cytosolic fractions, in contrast to the islet nuclei in which no increase was found. These results show that exposure of pancreatic islets to rIL-1 beta is accompanied by specific alterations in the protein synthesis of the islet cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Metabolism and beta-cell function of rat pancreatic islets exposed to human interleukin-1 beta in the presence of a high glucose concentration.

It has been postulated that one of the factors causing immune-mediated pancreatic beta-cell destruction in insulin-dependent diabetes mellitus (IDDM) is interleukin-1 (IL-1). Rat pancreatic islets exposed to human recombinant IL-1 beta (rIL-1 beta) for 48 h in vitro exhibit a markedly reduced glucose-stimulated insulin secretion. Also, a deleterious effect of glucose on beta-cell function, especially under conditions of a reduced beta-cell mass, which may exist in the early phase of IDDM has been suggested. In this study the response of rat pancreatic islets in vitro to a combination of the cytokine and high glucose concentration have therefore been assessed. Thus, islets were cultured for 48 h at either 11.1 or 56 mM glucose with or without 25 U/ml rIL-1 beta. Exposure to the cytokine reduced the islet DNA content at both glucose concentrations by 20-25%. In short-term incubations in the absence of rIL-1 beta after the preceding culture with the cytokine, the glucose-stimulated insulin release was reduced by 70% in islets cultured at 11.1 mM glucose and by only 40% after culture at 56 mM glucose, when compared to the corresponding control islets. The utilization of D-[5-3H]glucose, i.e., the catabolism of glucose in the glycolytic pathway, was the same in all groups of islets. However, the D-[6-14C]glucose oxidation rate, i.e., the metabolism of glucose in the Krebs cycle, was reduced by about 65% in rIL-1 beta exposed islets kept at 11.1 mM glucose and 46% in islets cultured at 56 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Coexpression of the platelet-derived growth factor (PDGF) B chain and the PDGF beta receptor in isolated pancreatic islet cells stimulates DNA synthesis.

Suspensions rich in pancreatic beta cells were transfected by means of electroporation or by using the liposome technique with DNA constructs coding for the B chain of platelet-derived growth factor (PDGF) and the PDGF alpha and beta receptors to induce a mitotic response in this slowly replicating cell type. Transfection with the B-chain construct induced synthesis of the PDGF B-chain homodimer (PDGF-BB) as assessed by the presence of 125I-labeled PDGF-BB competing activity in the conditioned medium of the transfected islet cells. Moreover, islet cells transfected with the PDGF beta-receptor construct exhibited increased immunofluorescence staining with a PDGF beta-receptor antibody. These cells also displayed increased 125I-labeled PDGF-BB binding compared with control transfected cells. Cotransfection with the B-chain construct or the addition of 10% fetal bovine serum or purified PDGF all induced DNA synthesis in islet cells transfected with the PDGF beta-receptor construct. Islet cells transfected with the PDGF alpha-receptor construct did not respond with stimulation of [3H]thymidine incorporation to any of the PDGF isoforms (PDGF-AA, -AB, or -BB). Cotransfection of the PDGF alpha- and beta-receptor constructs resulted in a loss of the DNA synthesis response to PDGF. The beta cells exhibited elevated levels of [3H]inositol trisphosphate after transfection with the B-chain and beta-receptor constructs, indicating activation of phospholipase C. Islet cells transfected with the different receptor constructs exhibited different patterns of tyrosine phosphorylation upon ligand activation. The results demonstrate that pancreatic islet cells can be stimulated to increase DNA synthesis by transfection with the PDGF beta-receptor gene, whereas cotransfection with the alpha-receptor gene may attenuate the growth response.

Animals↗
PubMed1990

Interleukin-6 affects insulin secretion and glucose metabolism of rat pancreatic islets in vitro.

Recently it has been postulated that interleukin-1 (IL-1) locally released by infiltrating mononuclear cells may destroy the pancreatic B cells during the development of insulin-dependent diabetes mellitus. Since IL-1 is a potent inducer of interleukin-6 (IL-6) in various cells, it is conceivable that IL-6 is a second mediator of the IL-1 action. In the present study the effects of IL-6 alone or in combination with IL-1 were studied on pancreatic islet function in vitro after tissue culture and compared with the effects observed after exposure to IL-1 only. Rat pancreatic islets were cultured in medium RPMI 1640 + 10% calf serum with or without the addition of human recombinant IL-6 (500-5000 pg/ml) for 48 h. The medium insulin accumulation was increased by 40-50% after culture with 500-2000 pg/ml IL-6, but was similar to the controls at 5000 pg/ml. When islets were cultured for 18 h only, also 5000 pg/ml IL-6 stimulated the medium insulin accumulation. IL-6 did not affect the islet insulin content and the rates of islet (pro)insulin and total protein biosynthesis. It inconsistently decreased the islet DNA content. In short-term experiments after 48-h culture with IL-6, there was a dose-dependent inhibition of the glucose-stimulated insulin release. On the other hand, islets cultured with IL-6 (5000 pg/ml) exhibited an elevated glucose oxidation and oxygen uptake, but a lower ATP content at 16.7 mM glucose and an unaffected glucose utilization and glutamine oxidation compared to the controls. This raises the possibility that IL-6 had induced a condition with an increased energy expenditure, resulting in an enhanced mitochondrial metabolism of glucose. Islets cultured with human recombinant IL-1 beta (25 units/ml) showed a strong inhibition of the insulin accumulation in the culture medium and of glucose-stimulated insulin release and a marked decrease in the islet DNA and insulin content. A combination of IL-1 (25 U/ml) + IL-6 (1000 pg/ml) did not alter the inhibitory action of IL-1 alone. The present findings thus show that IL-6 induces a dissociation between insulin secretion and glucose oxidation in islets in vitro. This has not been observed in islets exposed to IL-1, which suggests that IL-6 does not solely mediate the inhibitory effects of IL-1 on islet function.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1990

Interleukin-1 beta 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.

Animals↗
PubMed1990

Liposome mediated in vitro transfection of pancreatic islet cells.

The aim of this study was to evaluate the suitability of the liposome technique for transfection of pancreatic islet cells in vitro. For this purpose, fetal islets were isolated and cultured free floating for two days after which they were further cultured, either intact or dispersed into islet cell suspensions, with different DNA-liposome preparations. The DNA-construct used were the control plasmid (pSP65) and the viral oncogene v-src contained in the plasmid pSPRIsrc. A previous study showed that islet cells transfected by means of electroporation with pSPRIsrc displayed an increased thymidine incorporation rate, making this plasmid suitable for further transfection studies. The DNA was associated with the liposomes by means of surface adhesion. The liposomes used were either conventional phosphatidylcholine-containing liposomes, phosphatidylethanolamine/oleic acid containing liposomes (pH-sensitive liposomes) or Lipofectin. After the exposure of islet cells to the DNA-liposome preparations, the transfection efficiency was assessed by determination of the uptake of the DNA-constructs (Southern blot analysis) and expression of the gene construct into an mRNA (Northern blot analysis). In addition, the impact of the different DNA-liposome preparations on islet DNA replication (thymidine incorporation rates) was determined. It was found that two days after exposure to the DNA-liposomes, the v-src construct was located in islet cell nuclei and that v-src derived transcripts were transiently expressed in the islet cells. The Lipofectin liposomes were more efficient in transfecting islet cells than the pH-sensitive liposomes as assessed by the blotting techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Maturation of fetal rat islet cells in vitro during tissue culture is associated with increased mitochondrial function.

Tissue culture for one or seven days of pancreatic islets isolated from 21-day old fetal rats was found to be associated with a marked increase in the oxidation of L-(U-14C) glutamine by intact islets and in the activity of both alanine-glutamate and aspartate-glutamate transaminases as well as glutamate dehydrogenase in islet homogenates. This coincided with an increase in the relative amount of mitochondrial DNA. The activities of glucose-phosphorylating enzymes (hexokinase and glucokinase), glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase were less markedly increased during the culture period than those of enzymes involved in amino acid catabolism and located, in part at least, in mitochondria. The combined data suggest that the functional maturation of fetal islets during the culture period is associated with and may be attributable to a preferential maturation of their mitochondria.

Alanine Transaminase↗
PubMed1989

Effects of protein kinase C activation on the regulation of the stimulus-secretion coupling in pancreatic beta-cells.

Effects of protein kinase C (PKC) activation on the insulin-secretory process were investigated, by using beta-cell-rich suspensions obtained from pancreatic islets of obese-hyperglycaemic mice. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which is known to activate PKC directly, the muscarinic-receptor agonist carbamoylcholine and high glucose concentration enhanced the phosphorylation of a specific 80 kDa PKC substrate in the beta-cells. At a non-stimulatory glucose concentration, 10 nM-TPA increased insulin release, although there were no changes in either the cytoplasmic free Ca2+ concentration ([Ca2+]i) or membrane potential, as measured with the fluorescent indicators quin-2 and bisoxonol respectively. At a stimulatory glucose concentration TPA caused a lowering in [Ca2+]i, whereas membrane potential was unaffected. Despite the decrease in [Ca2+]i, there was a large stimulation of insulin release. Addition of TPA lowered [Ca2+]i also in beta-cells stimulated by tolbutamide or high K+, although to a lesser extent than in those stimulated by glucose. There was no effect of TPA on either Ca2+ buffering or the ability of Ins(1,4,5)P3 to release Ca2+ in permeabilized beta-cells. However, the phorbol ester inhibited the rise in [Ca2+]i in response to carbamoylcholine, which stimulates the formation of InsP3, in intact beta-cells. Down-regulation of PKC influenced neither glucose-induced insulin release nor the increase in [Ca2+]i. Hence, although PKC activation is of no major importance in glucose-stimulated insulin release, this enzyme can serve as a modulator of the glucose-induced insulin-secretory response. Such a modulation involves mechanisms promoting both amplification of the secretory response and lowering of [Ca2+]i.

Animals↗
PubMed1989

Transfection of insulin-producing cells with a transforming c-Ha-ras oncogene stimulates phospholipase C activity.

Pancreatic islet beta-cells and insulin-producing RINm5F cells were electroporated in the presence of the c-Ha-ras oncogene, to assess the possible involvement of the encoded product in coupling extracellular receptors to phospholipase C. After two days the c-Ha-ras-transfected cells increased their expression of c-Ha-ras mRNA. These cells were also found to contain more [3H]InsP3, suggesting an increased basal (non-ligand-activated) phospholipase C activity. In addition, the transfected cells were unable to respond to ligand (bombesin) activation of phospholipase C. The ras-transfected insulin-producing cells showed enhanced phosphorylation of a 200 kDa substrate crossreacting with an antibody to an 80 kDa protein kinase C substrate. The phorbol ester 12-O-tetradecanoyl 13-acetate and bombesin also induced phosphorylation of the 200 kDa substrate. All of these changes occurred without changes in the rates of [3H]thymidine incorporation. The results suggest that the mutated c-Ha-ras oncogene directly or indirectly stimulates the basal phospholipase C activity of these cells.

Animals↗
PubMed1989

Failure of glucose to elicit a normal secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATP-regulated K+ channels.

Fetal pancreatic beta cells demonstrate a deficient insulin release in response to glucose, but the underlying mechanism at the cellular level is unknown. By using beta cells from 21-day fetal rats we made an attempt to clarify the mechanism(s) behind this reduced glucose response. In addition to measuring insulin release, glucose metabolism, and cellular ATP content, ATP-regulated K+ channels (G channels) and voltage-activated Ca2+ currents were investigated with the patch-clamp technique. It was thus demonstrated that the ATP-regulated K+ channels in fetal beta cells were not sensitive to glucose but otherwise had similar characteristics as those of adult beta cells. Also, the characteristics of the voltage-activated Ca2+ currents were similar in adult and fetal beta cells. However, as judged from measurements of both glucose oxidation and glucose utilization, glucose metabolism was impaired in fetal beta cells. In addition, there was no increase in the ATP content, even when the cells were stimulated for 30 min. It is therefore concluded that the attenuated glucose-induced insulin release in fetal pancreatic beta cells is due to an immature glucose metabolism resulting in impaired regulation of the ATP-sensitive K+ channels. These findings may be relevant to the understanding of the deficient stimulus-secretion coupling associated with non-insulin-dependent diabetes.

Adenosine Triphosphate↗
PubMed1989

Content of adenine nucleotide translocator mRNA in insulin-producing cells of different functional states.

This study was undertaken to characterize the expression of the gene coding for the adenine nucleotide translocator (ANT) in the insulin-producing beta-cell and to study any possible relationship between its expression and the functional state of the beta-cell. Adult and fetal rat pancreatic islets were prepared and cultured under different conditions in vitro. The total RNA from these islets and from the insulin-producing RINm5F cells was isolated and analyzed by the Northern blot technique via a cDNA clone (pAAC-9) coding for the bovine ANT. We found that a 1600-base pair (bp) mRNA hybridizing to the pAAC-9 clone could be detected in RINm5F cells, and a 1450-bp mRNA was similarly observed in the islets. These sizes correspond well to previously reported forms of mRNA for the ANT observed in other tissues. When comparing the intensities of the pAAC-9 hybridizing bands of the different islet groups, it was observed that fetal islets contained less of this mRNA than adult islets. Furthermore, the content of the ANT mRNA in adult islets cultured at a high glucose concentration was increased compared with islets cultured at a low glucose concentration. Finally, streptozocin-treated islets, which display an impaired glucose-sensitive insulin release after 6 days in culture, also contained less of this mRNA than the control islets. We conclude that pancreatic islet cells express an mRNA that appears to be highly homologous to the bovine ANT and that the contents of this mRNA increases with the functional status of the beta-cell. It is furthermore suggested that defects in the expression of this gene may be associated with impaired glucose sensitivity.

Animals↗
PubMed1989

Maternal serum and amniotic fluid alpha-fetoprotein testing: our approach to screening, diagnosis and counseling.

Maternal serum and amniotic fluid alpha-fetoprotein testing has increased significantly in our laboratory and throughout the state since 1985. The results of this test are reported in a novel fashion as multiples of the median. The calculation and reporting of the test results are highly dependent upon gestational age, maternal weight, race and presence or absence of maternal diabetes. When properly interpreted alpha-fetoprotein determinations are a good screening test for early detection of neural tube defects and Trisomy 21 (Down Syndrome). Our approach to screening, diagnosis and counseling is discussed.

Adult↗
PubMed1988

Stimulation of pancreatic islet beta-cell replication by oncogenes.

Although the growth potential of the pancreatic islet beta cells is limited, glucose, cAMP, and certain polypeptide growth factors have been reported by other workers to exert modest stimulatory effects on beta-cell replication. To further assess means through which beta-cell growth can be stimulated, selected oncogene constructs linked to a rat insulin promoter were introduced by means of electroporation into free islet cells prepared from fetal rats and adult hyperglycemic obese (ob/ob) mice. The uptake and expression of the added oncogenes were sufficiently efficient to exert effects on beta-cell physiology in short-term experiments (less than or equal to 4 days). Stimulation of islet cell [3H]thymidine incorporation was observed after transfection with src alone or the combination of myc and ras. The effect observed in the fetal islet cells with src was more pronounced than any effect previously reported. Transfection with the src oncogene resulted in phosphorylation of lipocortin I and was paralleled by an increased immunofluorescence against src-like immunoreactivity in a majority of the electroporated cells. It is concluded that electroporation can induce sufficiently efficient expression of added oncogene constructs to study their effects on cells that are not readily transformable into continuously growing cell lines. Furthermore, the results suggest that beta-cell replication might be manipulated extrinsically by inserting appropriate growth-promoting genes into these cells.

Animals↗
PubMed1988

Heat-shock treatment of mouse pancreatic islets results in a partial loss of islet cells but no remaining functional impairment among the surviving beta cells.

To elucidate the role of thermal stress on the function of pancreatic beta cells, isolated mouse pancreatic islets were incubated for 30 min at 42 degrees C. This resulted in decreased glucose-stimulated insulin secretion, inhibited total protein and pro-insulin synthesis and the induction of heat-shock proteins with molecular weights of 64 and 88 kDa. Six days later, the islets exposed to heat shock showed a lower DNA content, indicating islet cell death. However, the insulin secretory response and rates of oxygen consumption in the presence of glucose were normal. It is suggested that the induction of heat-shock proteins does not permanently impair beta-cell function, but rather protects these cells from lasting damage.

Animals↗