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K Buschard

Publications and source records attributed to K Buschard.

At least 37 records · Page 2Linked to original sources
PubMed2000

Massive parallel gene expression profiling of RINm5F pancreatic islet beta-cells stimulated with interleukin-1beta.

Interleukin 1 (IL-1) is a pleiotropic cytokine with the potential to kill pancreatic beta-cells, and this unique property is thought to be involved in the pathogenesis of type I diabetes mellitus. We therefore determined the quantitative expression of 24,000 mRNAs of RINm5F, an insulinoma cell line derived from rat pancreatic beta-cells, before and after challenge with 30 and 1,000 pg/ml of recombinant human IL-1beta. The highest concentration resulted in decreased insulin production and cell death over a period of 4 days. Using three different time points, 2, 4 and 24 hours after challenge, we found that 146 full-length genes and a large number of expressed sequence tags were differentially regulated 3-fold or more. Most of the differentially regulated transcripts have not previously been described to be regulated by IL-1beta in beta-cells. We have analysed the expression data and sorted the genes into groups according to functional relations on the basis of knowledge of the structure or function ascribed to the individual genes. Many of the differentially regulated genes are known to play a role in immune- and stress-related pathways as well as in insulin secretion and vesicle trafficking, e.g. alpha-endosulfine and K+ channel Kir6.2 are differentially regulated. A number of transcripts in the biosynthesis pathway for cholesterol are also differentially regulated.

Animals↗
PubMed2000

Neonatal treatment with beta-cell stimulatory agents reduces the incidence of diabetes in BB rats.

The aim of the study was to investigate whether various beta-cell stimulatory drugs, given neonatally, influence the incidence of diabetes in BB rats. Newborn BB rats were treated twice daily for 6 days and diabetes development was observed during the following 200-day study period. Compared to a diabetes incidence of 63.8% in 163 control BB rats which received saline or were untreated, the percentage of experimental BB rats that developed diabetes was as follows in the different subgroups: arginine-glucose: 47% (n = 73, p < 0.02); glucagon: 37% (n = 93, p < 0.0001); tolbutamide-glucose: 36% (n = 58, p < 0.0005); and theophylline-glucose: 39% (n = 41, p < 0.005). A long-term arginine-glucose treatment was not superior to the shorter neonatal treatment. Histological examination revealed a higher degree of insulitis in diabetic than in non-diabetic animals but no difference according to the kind of treatment was observed. Finally, we found that the diabetes incidence in BB rats was higher in the first litter compared to subsequent litters (p = 0.04). Thus, neonatal treatment with various beta-cell stimulatory agents reduces diabetes incidence in BB rats. The theory behind the study, that the treatment accelerates beta-cell maturation leading to increased immunological tolerance towards beta cells, is discussed.

Animals↗
PubMed1999

Selectivity of prandial glucose regulators: nateglinide, but not repaglinide, accelerates exocytosis in rat pancreatic A-cells.

The effects of the two prandial glucose regulators, repaglinide and nateglinide, on ATP-sensitive K(+) (K(ATP)) channel activity, membrane potential and exocytosis in single rat pancreatic A-cells were investigated using the patch-clamp technique. K(ATP) channel activity was reversibly blocked by repaglinide (K(d)=22 nM) and nateglinide (K(d)=410 nM) and this was associated with membrane depolarisation and initiation of electrical activity. The effect of repaglinide and nateglinide on stimulation of glucagon secretion by direct interference with the exocytotic machinery was investigated by the use of capacitance measurements. Nateglinide, but not repaglinide, at concentrations similar to those required to block K(ATP) channels potentiated Ca(2+)-evoked exocytosis 3-fold. In alphaTC1-9 glucagonoma cells addition of nateglinide, but not repaglinide, was associated with stimulation of glucagon secretion. These results indicate that the fast-acting insulin secretagogue nateglinide is glucagonotropic primarily by stimulating Ca(2+)-dependent exocytosis.

ATP-Binding Cassette Transporters↗
PubMed1999

In vitro production of cytokines is influenced by sulfatide and its precursor galactosylceramide.

Effects of sulfatide and its precursor galactosylceramide (gal-cer) on the kinetics of production of cytokines were studied. In human mononuclear leucocytes, gal-cer but not sulfatide induced significantly increased amounts of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF) mRNA. In phytohemagglutinin-stimulated cultures, gal-cer increased the levels of IL-1beta and IL-6 mRNA and secreted IL-1beta and IL-6, while sulfatide decreased the amounts of IL-6 mRNA and secreted IL-6. Gal-cer also increased TNF secretion. In lipopolysaccharide-stimulated cells, sulfatide but not gal-cer decreased the secretion of IL-1beta and IL-10, a potent suppressor of production of many cytokines. Thus, sulfatide and gal-cer affect cytokine production differently, most likely at the level of gene expression. This may have implications in diseases where inflammatory cytokines play a pathogenic role.

Adult↗
PubMed1999

Glucose induces early growth response gene (Egr-1) expression in pancreatic beta cells.

A copy deoxyribonucleic acid (cDNA) clone of the immediate early growth response gene, egr-1 (Krox-24, Zif268, NGFI-1), was isolated through subtractive hybridization screening to identify glucose-induced genes in pancreatic beta cells. Glucose rapidly and transiently induced egr-1 mRNA in the SV40-transformed murine beta-cell line, MIN6. Glucose also increased egr-1 mRNA expression in INS-1, betaTC3 and RINm5F beta-cell lines, although with different kinetics. Expression of the 82 kDa Egr-1 protein was induced both in MIN6 cells stimulated with glucose in vitro and in primary rat islet cells stimulated in vivo or in vitro. This response is unique to beta cells since glucose did not affect egr-1 expression in NIH-3T3 fibroblasts or glucose-sensitive hepatocytes. In beta cells egr-1 induction is specifically associated with insulin secretion, as it was not observed after stimulation with serum or insulin but was elicited by insulin secretagogues, including membrane depolarizing agents and cAMP agonists. Moreover, induction of egr-1 by glucose was inhibited by EDTA, indicating dependence on influx of extracellular Ca2+. Other immediate early response genes, c-fos and junB, were also induced following glucose stimulation with kinetics similar to egr-1, whereas c-jun and junD expression were not affected. Since the zinc-finger protein encoded by egr-1 is highly homologous to transcription factors that control expression of glucose-regulated genes in yeast, Egr-1 could mediate delayed adaptive responses of beta cells to sustained glucose stimulation through transcriptional regulation.

3T3 Cells↗
PubMed1999

Inhibition of insulin-specific autoreactive T-cells by sulphatide which is variably expressed in beta cells.

AIMS/HYPOTHESIS: Sulphatide and insulin are present in the secretory granules and at the surface of beta cells in islets of Langerhans. Insulin autoantibodies and T-cell reactivity against insulin exist during the development of Type I (insulin-dependent) diabetes during which active beta cells may be more vulnerable than passive. Our aims were to examine the presence of sulphatide in active and passive beta cells and to clarify whether sulphatide influences the direct autoimmunity against insulin. METHODS: We incubated rat islets in 2.8, 11.0 or 20.0 mmol/l glucose for 24 h and did an electron microscopic evaluation after labelling with a specific anti-sulphatide monoclonal antibody. The reactivity of an insulin-specific T-cell clone isolated from a patient with Type I diabetes, was examined using insulin or insulin B-chain (B11-27) peptide incubated together with sulphatide. RESULTS: We detected lower amounts of sulphatide per insulin secretory granule in active compared with passive beta cells (p = 0.003). The presence of sulphatide in vitro at doses of 43-8.3 micromol/l resulted in greatly reduced proliferation (median 3.4 % of control value, p = 0. 0004) of the insulin-specific T-cell clone. No inhibition was found using the precursor of sulphatide, galactosylceramide, or GM1. Sulphatide did not reduce non-aspecific proliferation (induced by phorbol myristate acetate plus anti-CD3) or specific proliferation induced by insulin peptide. CONCLUSION/INTERPRETATION: These results imply that sulphatide possibly affect processing of the insulin molecule. Sulphatide which has been reported to interfere with phagosome-lysosome fusion, conceivably interacts with insulin. We hypothesize a (patho)physiological role of sulphatide, variably expressed in beta cells, by reducing the antigenicity of insulin. [Diabetologia (1999) 42: 1212-1218]

Animals↗
PubMed1999

The insulinotropic effect of endothelin-1 is mediated by glucagon release from the islet alpha cells.

AIMS/HYPOTHESIS: The circulating concentrations of endothelin-1 (ET-1), a peptide derived from endothelium, are increased in hypertension and diabetes. Endothelin-1 has recently been shown to be an insulinotropic agent. The mechanism of action of endothelin-1 on the endocrine pancreas has not yet been clarified. METHODS: We investigated the action of endothelin-1 on the insulin secretion, the binding of (125)I-ET-1 to beta cells as well as its effects on purified beta and non-beta cells from normal rats. The expression of endothelin receptors in alpha- and beta-cell lines and in normal rat islets was also studied. RESULTS: First, we studied the effects of endothelin-1 on insulin secretion from beta-cell lines (INS-1, betaTC3 and MIN6). At all endothelin-1 concentrations applied (1 pmol/l to 1 micromol/l) no change in insulin secretion was found. Ligand-binding experiments on betaTC3 cells showed no specific binding of (125)I-ET-1. A prominent expression of ET(A)-receptor mRNA in an alpha-cell line (alphaTC1.9) and in normal rat islets was found whereas no expression was found in INS-1 cells. No influence of endothelin-1(1 micromol/l) on insulin secretion stimulated by glucose was detected from purified beta cells. Endothelin-1-(100 nmol/l) increased, however, both insulin and glucagon secretion from a mixture of purified beta and non-beta cells indicating that alpha cells seem to have a key role for the action of ET-1 on insulin secretion. CONCLUSION/INTERPRETATION: The insulinotropic impact of endothelin-1 is not caused by a direct action on the beta cells but seems to be mediated by a paracrine action, probably secondary to enhanced release of glucagon from the endothelin receptor positive alpha cells. [Diabetologia (1999) 42: 1302-1307]

Animals↗
PubMed1999

Characterisation of sulphonylurea and ATP-regulated K+ channels in rat pancreatic A-cells.

We have monitored whole-cell and single channel ATP-sensitive K+ (KATP) currents in isolated rat glucagon-secreting pancreatic A-cells. Tolbutamide produced a concentration-dependent decrease in the whole-cell KATP conductance (Ki = 6 microM) and initiated action potential firing. The K+ channel opener diazoxide, but not cromakalim or pinacidil, inhibited electrical activity and increased the whole-cell K+ conductance fourfold. ATP applied to the intracellular face of the membrane inhibited KATP channel activity with a Ki of 17 microM, an effect that could be counteracted by Mg-ADP and Mg-GDP. GTP and UTP did not affect KATP channel activity. Phosphatidylinositol 4,5-bisphosphate activated KATP channels inhibited by ATP after a delay of 90 s. In situ hybridisation demonstrated the expression of the mRNA encoding KATP channel subunits Kir6.2 and SUR1 but not Kir6.1 and SUR2. We conclude that rat pancreatic A-cells express KATP channels with the nucleotide-, sulphonylurea- and K+ channel-opener sensitivities expected for a channel formed by Kir6.2 and SUR1 subunits.

ATP-Binding Cassette Transporters↗
PubMed1999

Unbiased estimation of total beta-cell number and mean beta-cell volume in rodent pancreas.

We describe a method for unbiased assumption-free estimation of the total number of beta-cells and the mean beta-cell volume in mouse and rat pancreas based on light microscopy. Such a method, which takes advantage of one of the most recent developments in stereology, the fractionator, has not previously been described. It relies on repeated fractionation of the tissue using systematic uniform random sampling combined with an unbiased counting principle. The method was applied to eight BALB/cBom male mice (56 days) and six Lewis/MOL male rats (47 days). In mice, the total number of beta-cells was 1.06 +/- 0.07 x 10(6) (mean +/- SEM) per pancreas with a mean beta-cell volume of 1280 +/- 17 microm3, while in rats the total beta-cell number was 2.76 +/- 0.42 x 10(6) per pancreas with a mean beta-cell volume of 1170 +/- 65 microm3. Furthermore, the results showed that in both species the biological variability in the total beta-cell volume is due to differences in the number of beta-cells rather than variability of the mean beta-cell volume. The method can be used to give a precise description of number and volume of beta-cells at different ages, and will make it possible to estimate the contributions of hyper/hypotrophia and hyper/hypoplasia to a given induced or spontaneous change in the total beta-cell mass.

Animals↗
PubMed1999

Cytokine production in NOD mice on prophylactic insulin therapy.

We investigated whether cytokines produced primarily by monocytes/macrophages (IL-1alpha), Th1-lymphocytes (IFNgamma), or Th2-lymphocytes (IL-4) are modulated in diabetes-prone NOD mice by insulin treatment as used in prophylaxis studies. The cytokines were measured by ELISA in plasma and in supernatants of spleen cells activated ex vivo by lipopolysaccharide plus phytohemagglutinin. Insulin, 0.25-0.50 IU/day, was given subcutaneously for 8 weeks starting in 4-week-old female mice. The insulin-treated and control NOD mice showed similar weight gains and, by the end of the study, both groups exhibited cell infiltration in about 25% of their islets. IL-1alpha, IFNgamma and IL-4 were generally below detection in plasma of prediabetic animals and controls. Diabetic NOD mice, aged 28-45 weeks, had significantly elevated plasma IL-1alpha: 154+/-39 pg/ml (mean+/-SEM, p<0.0001). While ex vivo activated NOD splenocytes released similar amounts of IL-1alpha, insulin therapy increased the levels from 99+/-17 to 155+/-19 pg/10(6) cells (p<0.05). Supernatants of activated splenocytes from prediabetic NOD mice had lower levels of IL-4 (<15 pg/10(6) cells) compared with those from BALB/c mice (88+/-22 pg/10(6) cells; p<0.01), and this deficiency was partially compensated for when the NOD mice were given insulin (27+/-8; p<0.01). The levels of IFNgamma were comparable and largely unaffected by insulin treatment. Hence, insulin therapy appears to partially normalize an otherwise deficient Th2 response in NOD mice.

Animals↗
PubMed1999

Previous maternal abortion, longer gestation, and younger maternal age decrease the risk of type 1 diabetes among male offspring.

OBJECTIVE: To identify possible influences and interactions of perinatal determinants in the subsequent development of type 1 diabetes. RESEARCH DESIGN AND METHODS: The data were obtained from children born in Denmark during the periods 1978-1982 and 1984-1986 and admitted to a Danish hospital with newly diagnosed type 1 diabetes between 1978 and 1995; 857 patients fulfilled the criteria. The study was conducted by combining and analyzing two national registries: the National Patient Registry and the Medical Birth Registry. For each diabetic child, two control children were randomly selected, matched by sex, time, and district of delivery. RESULTS: By multivariate logistic regression analysis, the following significant determinants were identified. Male offspring showed decreased risk when born of mothers who had had one or more abortions (odds ratio [OR] 0.66 [95% CI 0.48-0.92]) and with long duration of gestation (linearly with OR 0.91 per week [0.85-0.99]), while increased risk was found for high maternal age (linearly with OR 1.03 per year [1.00-1.06]). Female offspring showed no such association. No significant differences between diabetic patients and control subjects were found with respect to paternal age, maternal parity, placental weight or any of the birth size parameters, or interventions and complications during delivery. CONCLUSIONS: The findings show that perinatal determinants may influence the risk of subsequent development of type 1 diabetes in a sex-specific manner.

Abortion, Spontaneous↗
PubMed1999

Intermittent feeding and fasting reduces diabetes incidence in BB rats.

Food intake may be one of several factors which influence the risk of development of insulin dependent diabetes mellitus, but the influence of the pattern of food supply has not been studied previously. The aim of the present study was to investigate the effect of intermittent feeding and fasting upon diabetes in BB rats. This study included three groups. Group 1 served as control and included 77 animals, 79% became diabetic. In groups 2 and 3, after weaning, food but not water was withdrawn from the animals: 24 h twice a week in group 2; 24 h every second day in group 3. Group 2 included 40 BB rats, 50% (p < 0.004) became diabetic. Group 3 included 44 BB rats, 52% (p < 0.01) became diabetic. No differences were seen between sexes. Degree of insulitis was not influenced by changed food supply. Regarding blood glucose, no influence was seen among diabetic animals, among non-diabetic animals changed food supply reduced blood glucose values obtained at the end of the study. Intermittent feeding and fasting tended to reduce mean age at the time of diagnosis of diabetes, significance was reached only in female animals from group 3 compared to group 1. Body weight was obtained weekly. Intermittent feeding and fasting caused a reduced weight gain in group 2 as well as in group 3 compared to control animals; however, most pronounced in group 3 and also more pronounced among males compared to females. For pre-diabetic and non-diabetic animals comparable influence on body weight was seen. The main conclusion in the study is that intermittent feeding and fasting reduced diabetes incidence in BB rats.

Age Factors↗
PubMed1999

Thyrotropin stimulates specifically the expression of the autoantibody binding domains of the thyroperoxidase molecule.

To get some insight on the in vitro effect of TSH on the expression of two thyroid specific antigens (thyroglobulin (Tg) and thyroid peroxidase (TPO)) on the cell surface of cultured human thyroid cells an indirect immunofluorescence-activated cell sorter (FACStar IV, Becton-Dickinson) was used. Only half of the cultures responded to TSH by increased surface expression of the thyroid specific antigen Tg. In these cells, TSH stimulation of TPO expression showed a difference in epitopes recognized by murine monoclonal antibodies. Epitopes of domain D, recognized by monoclonal antibody 1, 30, 40 and 53 which are not involved in autoimmunity, were unaffected by TSH stimulation, (n = 2-10). In contrast, TSH regulated the surface expression of the TPO epitopes recognized by monoclonal antibody 15, 18, 24 and 60, which are known to be related to the serum autoantibody binding domain of TPO, (n = 6-8; p < 0.05). This indicated that increased activity of the thyroid cells selectively stimulated the expression of autoantigenic epitopes on the cell surface and supports the concept that increased cellular activity might predispose to the autoimmune processes leading to autoimmune thyroid disease.

Animals↗
PubMed1998

Mitotic and meiotic instability of the CAG trinucleotide repeat in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal, dominantly inherited neurodegenerative disease caused by an unstable CAG trinucleotide repeat expansion in the ataxin-1 gene located on chromosome 6p22-p23. The expanded CAG repeat is unstable during transmission, and a variation in the CAG repeat length has been found in different tissues, including sperm samples from affected males. In order further to examine the mitotic and meiotic instability of the (CAG)n stretch we have performed single sperm and low-copy genome analysis in SCA1 patients and asymptomatic carriers. A pronounced variation in the size of the expanded allele was found in sperm cells and peripheral blood leucocytes, with a higher degree of instability seen in the sperm cells, where an allele with 50 repeat units was contracted in 11.8%, further expanded in 63.5% and unchanged in 24.6% of the single sperm analysed. We found a low instability of the normal alleles; the normal alleles from the individuals carrying a CAG repeat expansion were significantly more unstable than the normal alleles from the control individuals (P<0.001), indicating an interallelic interaction between the expanded and the normal alleles.

Alleles↗
PubMed1998

Effect of intrathymic administration of mycobacterial heat shock protein 65 and peptide p277 on the development of diabetes in NOD mice: caution required in vaccination studies.

Heat shock protein 65 (hsp65) and a derived peptide, p277, are autoantigens reported in IDDM. I.p. injection of hsp65 reduced diabetes incidence in NOD mice and administration of p277 cured already diabetic mice. Also, intrathymic (i.t.) administration of whole islets or GAD65 prevented diabetes in NOD mice. The aim of this study was to evaluate whether i.t. injection of mycobacterial hsp65 or p277 can prevent diabetes in NOD mice. Three-week-old NOD female mice were injected intrathymically with 50 microg of hsp65 (n=30), 5 microg of p277 (n=30), and PBS (n=29). Diabetes incidence was observed for the following 300 days. Pancreas was then used for histological and immunohistological evaluation. No significant differences in diabetes incidence were observed among the three groups of mice. Interestingly, hsp65-treated mice developed diabetes slightly faster at 177+/-6 days compared to 202+/-8 days (p=0.015) for the p277-treated group and 197+/-7 days (p=0.033) for controls. The insulitis score and average islet size did not differ significantly among the three groups of diabetic mice. Scattered TCR-gamma/delta positive cells were found in the pancreas of all groups of mice. In contrast, a huge infiltrate of TCR-gamma/delta positive cells was detected in four out of eight (50%) p277-diabetic NOD mice. Thus, our data show an earlier onset of diabetes in hsp65-treated mice and no improvement in the incidence with either hsp65 or p277, suggesting that hsp65 acts in a different way from what was reported with GAD65. Caution is advised in future vaccination studies as hsp65 poses a potential danger.

Animals↗
PubMed1998

Glucose stimulation of pancreatic beta-cell lines induces expression and secretion of dynorphin.

To investigate adaptive responses of pancreatic beta-cells to hyperglycemia, genes induced by glucose stimulation were identified by subtraction cloning. Among 53 clones representing differentially expressed genes, 20 encoded the endogenous opioid precursor, prodynorphin. The amino acid sequence of murine prodynorphin is identical to the rat protein in sequences comprising the opioid peptides and 86% identical in the remainder of the molecule. Stimulation of MIN6 cells increased prodynorphin RNA levels to more than 20-fold in proportion to physiological glucose concentrations. Similar induction levels were observed in murine betaTC3 and rat Rinm5F beta-cell lines. Prodynorphin RNA expression increased within 1 h of glucose stimulation, achieved maximal levels by 4 h, and remained elevated for at least 24 h. By using RIA, MIN6 cells were shown to contain and secrete increased amounts of dynorphin-A following glucose stimulation. Treatment of MIN6 cells with KCl, forskolin, or isobutyl-methyl-xanthine strongly induced prodynorphin RNA expression, suggesting that induction may be related to secretion-coupled signaling pathways. The induction of prodynorphin in several beta-cell lines is consistent with previous demonstrations of beta-cell synthesis of other endogenous opioids, including beta-endorphin, and suggests that opioids may have a potentially significant role in regulating beta-cell secretion.

Amino Acid Sequence↗
PubMed1997

Failure of high-dose insulin treatment to increase beta-cell insulin content in diabetic non obese diabetic (NOD) mice.

High-dose insulin treatment in the first period after clinical onset of insulin-dependent diabetes mellitus (IDDM) has been found to reduce diabetic manifestations in humans. The aim of the present study was to examine whether high-dose insulin treatment of newly diagnosed diabetic non obese diabetic (NOD) mice would increase beta-cell insulin content after termination of treatment in this experimental IDDM animal model. Newly diagnosed diabetic female NOD mice were randomized into three groups composed of a low-dose insulin treated group (n = 10) injected subcutaneously with 15 IU/kg per day of NPH for 14 days followed by 5 days without insulin, a high-dose insulin treated group (n = 8) injected subcutaneously with 150 IU/kg per day of Actrapid for 14 days followed by 5 days without insulin and an untreated group sacrificed 3 days after diagnosis (n = 11). A reference group of age matched non-diabetic untreated female NOD mice (n = 11) was included in the study and sacrificed at the same time as the untreated diabetic mice. No significant difference in the amount of insulin extracted from the total pancreas was found by comparison of the three diabetic groups, consisting of the newly diagnosed untreated mice, the low-dose insulin treated mice and the high-dose insulin treated mice, respectively. The level was about 100-fold less than in the non-diabetic group. Blood glucose values in the two treated diabetic groups were at a high level (median > 18 mM) throughout the study. We conclude that no increase in beta-cell insulin content could be demonstrated in newly diagnosed diabetic NOD mice after early high-dose insulin treatment, at least not in the presence of high blood glucose values.

Animals↗