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H Heimberg

Publications and source records attributed to H Heimberg.

At least 19 recordsLinked to original sources
PubMed2003

AICA-riboside induces apoptosis of pancreatic beta cells through stimulation of AMP-activated protein kinase.

AIMS/HYPOTHESIS: Prolonged exposure of beta cells to low glucose concentrations triggers their apoptosis and is known to activate AMP-activated protein kinase (AMPK) in beta cell lines. We examined whether prolonged activation of AMPK can trigger apoptosis in rodent beta cells. METHODS: Primary beta cells were FACS-purified from rats, and from wild-type and AMPK(alpha2)-deficient mice. AMPK activation in beta cells was induced by the adenosine analog AICA-riboside and detected by immunoblotting using a phosphospecific antibody. Apoptosis of rodent beta cells was monitored by FACS analysis of beta cell DNA content, by direct counting of apoptotic cells using fluorescence microscopy, or by measurement of their caspase-3 activity. RESULTS: Dose-dependent and time-dependent apoptosis of the cells, concommittant with an activation of caspase-3, were suppressed by the caspase inhibitors zVAD-fmk and zDEVD-fmk. Apoptosis induction by AICA-riboside was also prevented by adding the MAPK-inhibitor SB203580 which blocked the AICA-riboside-induced phosphorylation of AMPK. Beta cells isolated from AMPK-(alpha2)-deficient mice were resistant against AICA-riboside induced apoptosis. CONCLUSION/INTERPRETATION: Sustained activation of AMPK by AICA-riboside can trigger a caspase-dependent apoptosis of pancreatic beta cells.

AMP-Activated Protein Kinases↗
PubMed2003

AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase.

We have recently shown that conditions known to activate AMP-activated protein kinase (AMPK) in primary beta-cells can trigger their apoptosis. The present study demonstrates that this is also the case in the MIN6 beta-cell line, which was used to investigate the underlying mechanism. Sustained activation of AMPK was induced by culture with the adenosine analogue AICA-riboside or at low glucose concentrations. Both conditions induced a sequential activation of AMPK, c-Jun-N-terminal kinase (JNK) and caspase-3. The effects of AMPK on JNK activation and apoptosis were demonstrated by adenoviral expression of constitutively active AMPK, a condition which reproduced the earlier-described AMPK-dependent effects on pyruvate kinase and acetyl-coA-carboxylase. The effects of JNK activation on apoptosis were demonstrated by the observations that (i). its inhibition by dicumarol prevented caspase-3 activation and apoptosis, (ii). adenoviral expression of the JNK-interacting scaffold protein JIP-1/IB-1 increased AICA-riboside-induced JNK activation and apoptosis. In primary beta-cells, AMPK activation was also found to activate JNK, involving primarily the JNK 2 (p54) isoform. It is concluded that prolonged stimulation of AMPK can induce apoptosis of insulin-producing cells through an activation pathway that involves JNK, and subsequently, caspase-3.

AMP-Activated Protein Kinases↗
PubMed2001

A comprehensive analysis of cytokine-induced and nuclear factor-kappa B-dependent genes in primary rat pancreatic beta-cells.

Type 1 diabetes mellitus results from an autoimmune destruction of pancreatic beta-cells. Cytokines, such as interleukin-1 beta and interferon-gamma, are putative mediators of immune-induced beta-cell death and, under in vitro conditions, cause beta-cell apoptosis. We have recently shown that interleukin-1 beta + interferon-gamma modifies the expression of >200 genes in beta-cells. Several of these genes are putative targets for the transcription factor nuclear factor-kappa B (NF-kappa B), and in subsequent experiments we showed that NF-kappa B activation is mostly pro-apoptotic in beta-cells. To identify cytokine-induced and NF-kappa B-regulated genes in primary rat beta-cells, we presently combined two experimental approaches: 1) blocking of NF-kappa B activation in cytokine-exposed beta-cells by a recombinant adenovirus (AdI kappa B((SA)2)) containing an inhibitor of NF-kappa B alpha (I kappa Bac) super-repressor (S32A/S36A) and 2) study of gene expression by microarray analysis. We identified 66 cytokine-modified and NF-kappa B-regulated genes in beta-cells. Cytokine-induced NF-kappa B activation decreased Pdx-1 and increased c-Myc expression. This, together with NF-kappa B-dependent inhibition of Glut-2, pro-hormone convertase-1, and Isl-1 expression, probably contributes to the loss of differentiated beta-cell functions. NF-kappa B also regulates several genes encoding for chemokines and cytokines in beta-cells. The present data suggest that NF-kappa B is a key "switch regulator" of transcription factors and gene networks controlling cytokine-induced beta-cell dysfunction and death.

Adenoviridae↗
PubMed2001

Glucose sensing in pancreatic beta-cells: a model for the study of other glucose-regulated cells in gut, pancreas, and hypothalamus.

Nutrient homeostasis is known to be regulated by pancreatic islet tissue. The function of islet beta-cells is controlled by a glucose sensor that operates at physiological glucose concentrations and acts in synergy with signals that integrate messages originating from hypothalamic neurons and endocrine cells in gut and pancreas. Evidence exists that the extrapancreatic cells producing and secreting these (neuro)endocrine signals also exhibit a glucose sensor and an ability to integrate nutrient and (neuro)hormonal messages. Similarities in these cellular and molecular pathways provide a basis for a network of coordinated functions between distant cell groups, which is necessary for an appropriate control of nutrient homeostasis. The glucose sensor seems to be a fundamental component of these control mechanisms. Its molecular characterization is most advanced in pancreatic beta-cells, with important roles for glucokinase and mitochondrial oxidative fluxes in the regulation of ATP-sensitive K+ channels. Other glucose-sensitive cells in the endocrine pancreas, hypothalamus, and gut were found to share some of these molecular characteristics. We propose that similar metabolic signaling pathways influence the function of pancreatic alpha-cells, hypothalamic neurons, and gastrointestinal endocrine and neural cells.

Animals↗
PubMed2001

Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis.

Cytokine-induced beta-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1beta (IL-1beta), and its activity promotes the expression of several beta-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1beta + gamma-interferon [IFN-gamma])-induced expression of NF-kappaB in beta-cell apoptosis, rat beta-cells were infected with the recombinant adenovirus AdIkappaB((SA)2), which contained a nondegradable mutant form of inhibitory kappaB (IkappaB((SA)2), with S32A and S36A) that locks NF-kappaB in a cytosolic protein complex, preventing its nuclear action. Expression of IkappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIkappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta-cell survival after IL-1beta + IFN-gamma treatment was significantly improved by IkappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta-cells.

Adenoviridae↗
PubMed2000

Cloning and quantitative determination of the human Ca2+/calmodulin-dependent protein kinase II (CaMK II) isoforms in human beta cells.

AIMS/HYPOTHESIS: The Ca2+/calmodulin-dependent protein kinase II (CaMK II) is highly expressed in pancreatic islets and associated with insulin secretion vesicles. The suppression of CaMK II disturbs insulin secretion and insulin gene expression. There are four isoforms of CaMK II, alpha to delta, that are expressed from different genes in mammals. Our aim was to identify the isoforms of CaMK II expressed in human beta cells by molecular cloning from a human insulinoma cDNA library and to assess its distribution in humans. METHODS: The previously unknown complete coding sequences of human CaMK IIbeta and the kinase domain of CaMK IIdelta were cloned from a human insulinoma cDNA library. Quantitative determination of CaMK II isoform mRNA was carried out in several tissues and beta cells purified by fluorescence activated cell sorting and compared to the housekeeping enzyme pyruvate dehydrogenase. RESULTS: We found CaMK IIbeta occurred in three splice variants and was highly expressed in endocrine tissues such as adrenals, pituitary and beta cells. Liver showed moderate expression but adipose tissue or lymphocytes had very low levels of CaMK IIbeta-mRNA. In human beta cells CaMK IIbeta and delta were expressed equally with pyruvate dehydrogenase whereas tenfold lower expression of CaMK IIgamma and no expression of CaMK IIalpha were found. CONCLUSION/INTERPRETATION: Although CaMK IIdelta is ubiquitously expressed, CaMK IIbeta shows preferential expression in neuroendocrine tissues. In comparison with the expression of a key regulatory enzyme in glucose oxidation, pyruvate dehydrogenase, two of the four CaM kinases investigated are expressed at equally high levels, which supports an important role in beta-cell physiology. These results provide the basis for exploring the pathophysiological relevance of CaMK IIbeta in human diabetes.

Adipose Tissue↗
PubMed2000

Modulation of rat pancreatic acinoductal transdifferentiation and expression of PDX-1 in vitro.

AIMS/HYPOTHESIS: In adult pancreatic regeneration models exocrine acini are found to transdifferentiate to duct-like complexes. This has also been associated with the formation of new endocrine islet cells. We aimed to establish an in vitro model in which this transdifferentiation process is characterised and can be modulated. METHODS: Purified rat pancreatic acini were cultured in suspension. Differentiation was analysed by immunocytochemistry, electron microscopy, western blotting and RT-PCR. RESULTS: During culture acinar cells directly transdifferentiated without dividing, the cells lost their acinar phenotype and started to express cytokeratins 20 and 7 and fetal liver kinase-1 (Flk-1) receptors for vascular endothelial growth factor. Expression of the acinar pancreatic exocrine transcription factor (PTF-1) remained and the pancreatic duodenal homeobox-containing transcription factor (PDX-1) was induced. When transdifferentiation was completed, the cells started to express protein gene product 9.5, a panneuroendocrine marker. By combining these features, the transdifferentiated cells show similar characteristics to precursor cells during active beta-cell neogenesis. We were able to modulate the differentiation state by addition of nicotinamide or sodium butyrate, agents which are known to stimulate endocrine differentiation in other models. CONCLUSION/INTERPRETATION: Here, we present an in vitro system in which the cellular differentiation of putative pancreatic endocrine precursor cells and their PDX-1 expression can be modulated, thereby providing a possible model for the study of beta-cell transdifferentiation.

Animals↗
PubMed2000

Adult human pancreatic duct and islet cells exhibit similarities in expression and differences in phosphorylation and complex formation of the homeodomain protein Ipf-1.

The homeodomain transcription factor encoded by the pancreatic and duodenal homeobox gene-1 (Ipf-1) is essential for pancreatic ontogenesis. Whether Ipf-1 is also involved in the neogenesis of beta-cells in the adult pancreas is unknown. We examined whether Ipf-1 is expressed in adult human pancreatic ducts, which are thought to generate new beta-cells. In tissue sections, virtually all duct cells were immunopositive for Ipf-1, as were the islet beta-cells but not the acinar cells. After isolation and culture, both duct and islet cell preparations contained the Ipf-1 immunoreactive proteins p42 and p45 (42 and 45 kDa, respectively) in similar proportions, but the expression levels were twofold lower in duct cells. After 4 h of labeling, the endocrine cells exhibited a sevenfold higher phosphorylation of p42 than the duct cells, whereas p45 was phosphorylated only in endocrine cells. Homeobox binding transcription factor complexes with Ipf-1 in duct cells differed from those in endocrine cells in terms of gel mobility, sequence specificity, and affinity. The observed similarities in Ipf-1 expression by adult human pancreatic duct cells and endocrine cells may reflect their common ontogenic origin, whereas the differences in Ipf-1 phosphorylation and complex formation may correlate with their divergent differentiation.

Adolescent↗
PubMed1999

Cellular origin of hexokinase in pancreatic islets.

Transgenic or tumoral pancreatic islet beta cells with enhanced expression of low K(m) hexokinases (HK) exhibit a leftward shift of the normal dose-response curve for glucose-induced insulin release. Furthermore, HK catalyzes roughly 50% of total glucose phosphorylation measured in extracts from freshly isolated rodent islets, suggesting that HK participates in the process of glucose sensing in beta cells. We previously observed that HK activity represents 20% of total glucose phosphorylation in purified rat beta cell preparations and that HK is not homogenously distributed over these cells. The present study provides several arguments for the idea that HK detected in freshly isolated rat islets or islet cell preparations originates mainly from contaminating exocrine cells. First, reverse transcriptase-polymerase chain reaction using isoform-specific primers allowed detection of hexokinase I and IV mRNA in rat beta cells, whereas the messenger levels encoding the hexokinase II and III isoforms were undetectably low. However, immunoblots indicated that hexokinase I protein was 10-fold more abundant in freshly isolated islets and flow-sorted exocrine cells than in purified rat beta cell preparations. Second, comparison of HK activity in the different pancreatic cell types resulted in 15-25-fold higher values in exocrine than in endocrine cells (acinar cells: 21 +/- 3 pmol of glucose 6-phosphate formed/h/ng of DNA; duct cells: 30 +/- 8 pmol/h/ng of DNA; islet beta cells: 1.2 +/- 0.2 pmol/h/ng DNA; alpha cells: 0.9 +/- 0.4 pmol/h/ng of DNA). Since freshly purified beta cell preparations contain 3 +/- 1% exocrine cells, at least 50% of their HK activity can be accounted for by exocrine contamination. Third, after 5 days of culture of purified islet beta cells, both HK activity and the proportion of exocrine cells decreased by more than 1 order of magnitude, while the ratio of glucokinase over hexokinase activity increased more than 10-fold. Finally, preincubating the cells with 50 mmol/liter 2-deoxyglucose did not affect glucose stimulation of insulin biosynthesis and release. In conclusion, the observation that pancreatic exocrine cells are responsible for a major part of HK activity in islet cell preparations cautions against the use of HK measurements in islet extracts in the study of these enzymes in glucose sensing by pancreatic beta cells.

Animals↗
PubMed1998

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes is caused by depletion of intracellular ATP.

Glucosamine, which enters the hexosamine pathway downstream of the rate-limiting step, has been routinely used to mimic the insulin resistance caused by high glucose and insulin. We investigated the effect of glucosamine on insulin-stimulated glucose transport in 3T3-L1 adipocytes. The Delta-insulin (insulin-stimulated minus basal) value for 2-deoxyglucose uptake was dramatically inhibited with increasing concentrations of glucosamine with an ED50 of 1.95 mM. Subcellular fractionation experiments demonstrated that reduction in insulin-stimulated 2-deoxyglucose uptake by glucosamine was due to an inhibition of translocation of both Glut 1 and Glut 4 from the low density microsomes (LDM) to the plasma membrane. Analysis of the insulin signaling cascade revealed that glucosamine impaired insulin receptor autophosphorylation, insulin receptor substrate (IRS-1) phosphorylation, IRS-1-associated PI 3-kinase activity in the LDM, and AKT-1 activation by insulin. Measurement of intracellular ATP demonstrated that the effects of glucosamine were highly correlated with its ability to reduce ATP levels. Reduction of intracellular ATP using azide inhibited Glut 1 and Glut 4 translocation from the LDM to the plasma membrane, insulin receptor autophosphorylation, and IRS-1 tyrosine phosphorylation. Additionally, both the reduction in intracellular ATP and the effects on insulin action caused by glucosamine could be prevented by the addition of inosine, which served as an alternative energy source in the medium. We conclude that direct administration of glucosamine can rapidly lower cellular ATP levels and affect insulin action in fat cells by mechanisms independent of increased intracellular UDP-N-acetylhexosamines and that increased metabolism of glucose via the hexosamine pathway may not represent the mechanism of glucose toxicity in fat cells.

3T3 Cells↗
PubMed1998

Subcellular distribution and differential expression of endogenous ADP-ribosylation factor 6 in mammalian cells.

ADP-ribosylation factor (ARF) 6 has been shown to play a role in vesicular transport; however, the expression and subcellular localization of the endogenous protein have not been clearly delineated. In this study, an ARF6-specific monoclonal antibody was raised and used to examine the subcellular distribution and expression of ARF6 in various tissues and during the differentiation of several well characterized cell types. We found that ARF6 localizes in both the cytosol and membranes of all tissues and cells tested. Moreover, ARF6 in 3T3-L1 adipocytes is principally localized on the plasma membrane, but substantial amounts are detected in the cytosolic and intracellular membrane fractions. We observed an increased expression of ARF6 during the differentiation of B lymphocytes to plasmocytes. However, the expression of ARF6 decreased during adipogenesis and monocyte differentiation. In contrast, the expression of other ARFs, detected by the monoclonal antibody 1D9, did not significantly change during differentiation of the aforementioned cell types. Taken together, our results indicate that ARF6 is a broadly expressed, differentially regulated GTPase that is present in cytoplasm and on both cell-surface and intracellular membranes and whose functions may include tissue-specific effects on vesicular trafficking during cellular differentiation.

ADP-Ribosylation Factor 6↗
PubMed1998

Differential expression of rat insulin I and II messenger ribonucleic acid after prolonged exposure of islet beta-cells to elevated glucose levels.

Prolonged exposure of rat islet beta-cells to 10 mmol/liter glucose has been previously shown to activate more cells into a glucose-responsive state (>90%) than has exposure to 6 mmol/liter glucose (50%). The present study demonstrates that this recruitment of more activated cells results in 4- to 6-fold higher levels of proinsulin I and proinsulin II messenger RNA (mRNA). However, only the rate of proinsulin I synthesis is increased. Failure to increase the rate of proinsulin II synthesis in the glucose-activated cells results in cellular depletion of the insulin II isoform, which can be responsible for degranulation of beta-cells cultured at 10 mmol/liter glucose. Higher glucose levels (20 mmol/liter) during culture did not correct this dissociation between the stimulated insulin I formation and the nonstimulated insulin II formation. On the contrary, the rise from 10 to 20 mmol/liter glucose resulted in a 2-fold reduction in the levels of proinsulin II mRNA, but not of proinsulin I mRNA; this process further increased the ratio of insulin I over insulin II to 5-fold higher values than those in freshly isolated beta-cells. The present data suggest that an elevated insulin I over insulin II ratio in pancreatic tissue is a marker for a prolonged exposure to elevated glucose levels. The increased ratio in this condition results from a transcriptional and/or a posttranscriptional failure in elevating insulin II formation while insulin I production is stimulated in the glucose-activated beta-cells.

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

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↗