Biomedical subjects

C B Chan

Publications and source records attributed to C B Chan.

At least 19 recordsLinked to original sources

PubMed2006

alpha-lipoic acid regulates AMP-activated protein kinase and inhibits insulin secretion from beta cells.

AIMS/HYPOTHESIS: The antioxidant compound alpha-lipoic acid (alpha-LA) possesses antidiabetic and anti-obesity properties. In the hypothalamus, alpha-LA suppresses appetite and prevents obesity by inhibiting AMP-activated protein kinase (AMPK). Given the therapeutic potential of alpha-LA for the treatment of type 2 diabetes and obesity, and the importance of AMPK in beta cells, we examined the effect of alpha-LA on pancreatic beta cell function. MATERIALS AND METHODS: Isolated rat islets and MIN6 beta cells were treated acutely (15-90 min) or chronically (18-24 h) with alpha-LA or the known AMPK-activating compounds 5'-amino-imidazole-4-carboxamide ribonucleoside (AICAR) and metformin. Insulin secretion, the AMPK-signalling pathway, mitochondrial function and cell growth were assessed. RESULTS: Acute or chronic treatment of islets and MIN6 cells with alpha-LA led to dose-dependent rises in phosphorylation of the AMPK alpha-subunit and acetyl CoA carboxylase. Chronic exposure to alpha-LA, AICAR or metformin caused a reduction in insulin secretion. alpha-LA inhibited the p70 s6 kinase translational control pathway, and inhibited MIN6 growth in a manner similar to rapamycin. Unlike AICAR and metformin, alpha-LA also acutely inhibited insulin secretion. Examination of the effect of alpha-LA on mitochondrial function showed that acute treatment with this compound elevated reactive oxygen species (ROS) production and enhanced mitochondrial depolarisation induced by Ca(2+). CONCLUSIONS/INTERPRETATION: This study is the first to demonstrate that alpha-LA directly affects beta cell function. The chronic effects of alpha-LA include AMPK activation and reductions in insulin secretion and content, and cell growth. Acutely, alpha-LA also inhibits insulin secretion, an effect probably involving the ROS-induced impairment of mitochondrial function.

AMP-Activated Protein Kinases↗

PubMed2006

Regulation of insulin secretion by uncoupling protein.

UCPs (uncoupling proteins) can regulate cellular ATP production by uncoupling oxidative phosphorylation. UCP2 is expressed in islet beta-cells and its induction reduces glucose-stimulated insulin secretion. Under physiological conditions, superoxide, formed as a by-product of respiration, activates UCP2. This leads to reduced ATP production, which impairs closure of the ATP-dependent K+ channels to prevent insulin secretion. It is suggested that the physiological role of UCP2 is to prevent excessive superoxide generation through a feedback loop. UCP2 induction may also alter fatty acid metabolism by altering NAD/NADH or by facilitating cycling of fatty acid anions. Recently, UCP2 has been proposed to keep insulin secretion low during starvation, a function under the control of the transcription co-repressor, surtuin-1, which has been shown to bind to the UCP2 promoter. Pathological UCP2 expression or activation may suppress glucose-stimulated insulin secretion to the extent that diabetes onset is hastened. In ob/ob mice, induction of UCP2 at age 5 weeks precedes development of insulin secretion defects and hyperglycaemia. Activating protein kinase A-dependent pathways can normalize insulin secretion in UCP2-overexpressing islets. Conversely, lowering UCP2 expression may promote increased insulin secretion. UCP2 knockout mice were protected from the diabetogenic effects of a high-fat diet and their islets exhibited increased sensitivity to glucose and elevated ATP/ADP. These results support a role for UCP2 as a gene contributing to the pathogenesis of Type 2 diabetes.

Diabetes Mellitus↗

PubMed2006

cAMP-mediated signaling normalizes glucose-stimulated insulin secretion in uncoupling protein-2 overexpressing beta-cells.

We investigated whether an increase in cAMP could normalize glucose-stimulated insulin secretion (GSIS) in uncoupling protein-2 (UCP2) overexpressing (ucp2-OE) beta-cells. Indices of beta-cell (beta-TC-6f7 cells and rodent islets) function were measured after induction of ucp2, in the presence or absence of cAMP-stimulating agents, analogs, or inhibitors. Islets of ob/ob mice had improved glucose-responsiveness in the presence of forskolin. Rat islets overexpressing ucp2 had significantly lower GSIS than controls. Acutely, the protein kinase A (PKA) and epac pathway stimulant forskolin normalized insulin secretion in ucp2-OE rat islets and beta-TC-6f7 beta-cells, an effect blocked by specific PKA inhibitors but not mimicked by epac agonists. However, there was no effect of ucp2-OE on cAMP concentrations or PKA activity. In ucp2-OE islets, forskolin inhibited ATP-dependent potassium (K(ATP)) channel currents and (86)Rb(+) efflux, indicative of K(ATP) block. Likewise, forskolin application increased intracellular Ca(2+), which could account for its stimulatory effects on insulin secretion. Chronic exposure to forskolin increased ucp2 mRNA and exaggerated basal secretion but not GSIS. In mice deficient in UCP2, there was no augmentation of either cAMP content or cAMP-dependent insulin secretion. Thus, elevating cellular cAMP can reverse the deficiency in GSIS invoked by ucp2-OE, at least partly through PKA-mediated effects on the K(ATP) channel.

Adenosine Triphosphate↗

PubMed2005

Insulin resistance causes increased beta-cell mass but defective glucose-stimulated insulin secretion in a murine model of type 2 diabetes.

AIMS/HYPOTHESIS: Although insulin resistance induces compensatory increases in beta cell mass and function to maintain normoglycaemia, it is not clear whether insulin resistance can precipitate beta cell dysfunction and hyperglycaemia without a pre-existing beta cell susceptibility. We therefore examined the beta cell phenotype in the MKR mouse, a model in which expression of a dominant-negative IGF 1 receptor (IGF1R) in skeletal muscle leads to systemic insulin resistance and diabetes. MATERIALS AND METHODS: Circulating glucose, insulin and glucagon concentrations were measured. Insulin sensitivity, glucose tolerance and insulin release in vivo were assessed by i.p. insulin and glucose tolerance tests. Beta cell function was assessed via insulin secretion from isolated islets and the glucose gradient in the perfused pancreas. Beta cell morphology was examined via immunohistochemistry. MKR mice were fed a high-fat diet containing sucrose (HFSD) to test metabolic capacity and beta cell function. RESULTS: Insulin-resistant MKR mice developed hyperglycaemia and a loss of insulin responsiveness in vivo. Basal insulin secretion from the perfused pancreas was elevated, with no response to glucose. Despite the demand on insulin secretion, MKR mice had increased pancreatic insulin content and beta cell mass mediated through hyperplasia and hypertrophy. The HFSD worsened hyperglycaemia in MKR mice but, despite increased food intake in these mice, failed to induce the obesity observed in wild-type mice. CONCLUSIONS/INTERPRETATION: Our studies demonstrate that insulin resistance of sufficient severity can impair glucose-stimulated insulin secretion, thereby undermining beta cell compensation and leading to hyperglycaemia. Moreover, because insulin stores were intact, the secretory defects reflect an early stage of beta cell dysfunction.

Animals↗

PubMed2004

Stimulation of growth hormone secretion from seabream pituitary cells in primary culture by growth hormone secretagogues is independent of growth hormone transcription.

The action of a number of growth hormone secretagogues (GHS) on growth hormone (GH) secretion and gene expression was studied in a primary culture of pituitary cells isolated from the black seabream Acanthopagrus schlegeli. The peptide GHS employed included growth hormone-releasing peptide (GHRP)-2, ipamorelin, and human ghrelin. The nonpeptide GHS employed included the benzolactam GHS L692,585 and the spiropiperidine GHS L163,540. Secreted GH was measured in the culture medium by an enzyme-linked immunosorbent assay (ELISA) method using a specific antibody against seabream GH. The GH mRNA content in the incubated cells was assessed by reverse transcription polymerase chain reaction (RT-PCR) using a pair of gene-specific primers designed from the cloned black seabream GH cDNA sequence. A dose-dependent stimulation of GH release was demonstrated by all the GHS tested, except human ghrelin, with EC(50) values in the nanomolar range. Simultaneous measurement of GH mRNA levels in the incubated seabream pituitary cells indicated that the GHS-stimulated increase in GH secretion was not paralleled by corresponding changes in GH gene expression. In contrast to the situation previously reported in the rat, no change in GH gene expression was noticed in the seabream pituitary cells even though the time of stimulation by GHS was increased up to 48 h, confirming that the GHS-stimulated GH secretion in seabream is independent of GH gene transcription.

Amino Acid Sequence↗

PubMed2003

Seabream growth hormone receptor: molecular cloning and functional studies of the full-length cDNA, and tissue expression of two alternatively spliced forms.

A full-length clone of the growth hormone receptor (GHR) was isolated from a cDNA library constructed from the liver of black seabream (Acanthopagrus schlegeli). The seabream GHR (sbGHR) cDNA sequence encodes a transmembrane protein of 640 amino acids (aa) possessing the characteristic motifs and architectural design of GHRs of other species. When compared to the other fish GHRs, it is most homologous to another marine fish species, the turbot, where the aa identity is 79.3%. But the sbGHR sequence is more remotely related to the goldfish GHR (51.6% aa identity) and the salmonid GHRs (approximately 46-48% aa identities). Phylogenetic comparison with other known GHRs indicates that the fish GHRs constitute a distinct group among the different vertebrate classes. The aa identities between sbGHR and other GHRs are low, being around 40% with mammalian GHRs, around 45% with avian and reptilian GHRs, and less than 35% with Xenopus GHR. CHO cells transfected with the sbGHR cDNA can be stimulated to proliferate by recombinant seabream growth hormone (sbGH). In addition, the transfected cells can transactivate a co-expressed mammalian serine protease inhibitor (Spi) 2.1 promoter upon stimulation by sbGH. These functional assays indicated that the fish receptor can interact with its homologous ligand to evoke the downstream post-receptor events. Reverse transcription-polymerase chain reaction (RT-PCR) and genomic PCR using a pair of gene-specific primers revealed the expression of two alternatively spliced forms of sbGHR in various tissues of the fish. A 93-bp intron, unique to the sbGHR gene and not found in any other known GHR genes, is alternatively spliced to give rise to two forms of receptor mRNA transcripts. The two forms of the receptor are differentially expressed among the different tissues of the fish.

Alternative Splicing↗

PubMed2002

Uncoupling protein-2: evidence for its function as a metabolic regulator.

Uncoupling protein-2, discovered in 1997, belongs to a family of inner mitochondrial membrane proteins that, in general, function as carriers. The function(s) of uncoupling protein-2 have not yet been definitively described. However, mounting evidence suggests that uncoupling protein-2 could act in multiple tissues as a regulator of lipid metabolism. A role as a modulator of reactive oxygen species as a defence against infection is also postulated. In this review, a brief overview of the general and specific properties of uncoupling protein-2 is given and evidence for metabolic and immune regulatory functions is summarized. Uncoupling protein-2 could have particular importance in the regulation of lipid metabolism in adipose tissue and skeletal muscle. In addition, its ability to inhibit insulin secretion could also promote fat utilization over storage. Inhibition by uncoupling protein-2 of reactive oxygen species formation in macrophages and other tissues could have implications for regulation of immune function. The possibility of functions of uncoupling protein-2 in other tissues such as the brain are beginning to emerge.

Animals↗

PubMed2002

Scattered radiation level during videofluoroscopy for swallowing study.

AIM: To evaluate the scattered radiation exposure to the surroundings during videofluoroscopy for swallowing study (VFSS). MATERIALS AND METHODS: Scattered radiation exposure was measured using an ion chamber survey meter for 17 adult patients undergoing videofluoroscopy for swallowing study. The cumulative dose area product of each case was also recorded. Data were presented as mean +/- standard deviation. RESULTS: The scattered radiation exposure at a distance of 150 cm from the patient and the dose area product recorded were 149 +/- 78 microR (range 42-308 microR) and 842 +/- 544 cGy.cm(2) (range 258-2151 cGy.cm(2)), respectively, for a single study of 18 +/- 6 minutes. A formula was then derived for estimating the scattered radiation dose to muscle tissue at an arbitrary distance based on the accumulated dose area product. With this formula, the mean scattered radiation dose to naked muscle tissue of the surrounding people at a distance of 30-100 cm from the patient were estimated to be 33.68-3.03 microSv respectively. CONCLUSION: The scattered radiation detriment associated with videofluoroscopy for swallowing study was well within acceptable levels.

Adult↗

PubMed2002

Glucose-inducible hypertrophy and suppression of anion efflux in rat beta cells.

Hypertrophy of beta cells from obese fa/fa rats is associated with increased sensitivity to basal glucose. Exposure to glucose in culture distorts insulin secretion more in beta cells from large than small islets from fa/fa rats. The aim of the present study is to investigate whether increased beta cell volume is associated with both glucose hypersensitivity and altered activity of the glucose-sensitive anion conductance. Beta cells from fa/fa rats had increased volume compared with those from lean rats after 24 h culture. Three-day exposure to 25 mM glucose in culture induced 10-15% hypertrophy in beta cells from lean rats and basal secretion from intact islets was increased tenfold. Estimates of ion channel activity were made from measurement of radiolabeled ion efflux. Taurine efflux, a marker of glucose-regulated anion channel activity, was reduced after high glucose exposure but no alterations in glucose-dependent K+ efflux were detected. The reverse hemolytic plaque assay was used to determine the contributions of the number of secreting cells (recruitment) versus secretion per cell in beta cells from enlarged (>250 microm diameter), intermediate (125-250 microm) and small (<125 microm) islets from lean and obese rats exposed to conditions mimicking hyperglycemia. After overnight culture, basal secretion was twofold greater from beta cells of large fa/fa islets compared with all other groups. Recruitment at low glucose was increased in all lean or fa/fa beta cells derived from >125 microm islets. When beta cells from small islets were exposed to supra-physiological glucose for 3 days, recruitment was increased at basal glucose and blunted at high glucose. Glucose exposure converts the recruitment profile of beta cells from small islets to resemble that of beta cells from large islets while inducing cellular hypertrophy and reduced anion conductance. However, hypertrophy alone did not predict functional characteristics of overnight-cultured beta cells from fa/fa rats.

Animals↗

PubMed2001

Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes.

beta cells sense glucose through its metabolism and the resulting increase in ATP, which subsequently stimulates insulin secretion. Uncoupling protein-2 (UCP2) mediates mitochondrial proton leak, decreasing ATP production. In the present study, we assessed UCP2's role in regulating insulin secretion. UCP2-deficient mice had higher islet ATP levels and increased glucose-stimulated insulin secretion, establishing that UCP2 negatively regulates insulin secretion. Of pathophysiologic significance, UCP2 was markedly upregulated in islets of ob/ob mice, a model of obesity-induced diabetes. Importantly, ob/ob mice lacking UCP2 had restored first-phase insulin secretion, increased serum insulin levels, and greatly decreased levels of glycemia. These results establish UCP2 as a key component of beta cell glucose sensing, and as a critical link between obesity, beta cell dysfunction, and type 2 diabetes.

Adenosine Triphosphate↗

PubMed2001

Three-dimensional MR microscopy of a transgenic mouse model of dilated cardiomyopathy.

BACKGROUND: Scientists are now able to alter the genetics of vertebrate embryos routinely to produce animal models of human developmental diseases. However, our understanding of structural changes in these animal models is limited by current methodologies. Histological techniques, although providing great anatomic detail, display only "static" data (one time point only) in two dimensions. Ultrasound may be used to generate continuous time course data, but is limited by interobserver variation, limited acoustic windows, and relatively low resolution. OBJECTIVE: To apply the high resolution, non-destructive, and three-dimensional acquisition capabilities of magnetic resonance (MR) microscopy to compare the hearts of normal mice versus an established transgenic mouse model of dilated cardiomyopathy. MATERIALS AND METHODS: Transgenic mice exhibiting dilated cardiomyopathy were developed via the introduction of a mutated, heart-specific gene (myosin light chain). Postmortem cardiac imaging was performed on the transgenic mice and normal controls. MR imaging was performed on a Bruker 3T imaging magnet using a custom radiofrequency coil following contrast perfusion of the atrial and ventricular chambers. Image resolution was 156 microm isotropic voxels. MR images were compared to gross pathologic specimens. Imaging data were post-processed using custom software to calculate the volumes of the atria and ventricles and to display the three-dimensional morphology of the chambers and myocardium. RESULTS: Of the seven mice scanned, four exhibited normal right atrial (average = 14.8 microl +/- 1.4), left atrial (average = 8.5 microl +/-0.3), right ventricular (average = 12.9 microl +/-2.7), and left ventricular (average 3.3 microl +/-0.5) volumes. Three mice exhibited dilatation of the right and left cardiac chambers (RA average = 23.9 microl +/-5.6; LA average = 15.9 microl +/-4.8; RV average = 32.5 microl +/- 6.8; LV average 24.0 microl +/-1.4). The gross morphology was verified upon autopsy of the animals and correlated with the animal's genotype. The differences in volumes between the normal and dilated cardiomyopathy mice were statistically significant (P values ranged from 0.001 to 0.024 for the different chambers). CONCLUSION: MR microscopy is a potentially useful tool for developmental biology research. The imaging of mouse hearts is feasible, and these methods provide quantitative and qualitative morphologic data of a mouse model of dilated cardiomyopathy not available using traditional methods.

Animals↗

PubMed2001

Increased uncoupling protein-2 levels in beta-cells are associated with impaired glucose-stimulated insulin secretion: mechanism of action.

In pancreatic beta-cells, glucose metabolism signals insulin secretion by altering the cellular array of messenger molecules. ATP is particularly important, given its role in regulating cation channel activity, exocytosis, and events dependent upon its hydrolysis. Uncoupling protein (UCP)-2 is proposed to catalyze a mitochondrial inner-membrane H(+) leak that bypasses ATP synthase, thereby reducing cellular ATP content. Previously, we showed that overexpression of UCP-2 suppressed glucose-stimulated insulin secretion (GSIS) in isolated islets (1). The aim of this study was to identify downstream consequences of UCP-2 overexpression and to determine whether insufficient insulin secretion in a diabetic model was correlated with increased endogenous UCP-2 expression. In isolated islets from normal rats, the degree to which GSIS was suppressed was inversely correlated with the amount of UCP-2 expression induced. Depolarizing the islets with KCl or inhibiting ATP-dependent K(+) (K(ATP)) channels with glybenclamide elicited similar insulin secretion in control and UCP-2-overexpressing islets. The glucose-stimulated mitochondrial membrane ((m)) hyperpolarization was reduced in beta-cells overexpressing UCP-2. ATP content of UCP-2-induced islets was reduced by 50%, and there was no change in the efflux of Rb(+) at high versus low glucose concentrations, suggesting that low ATP led to reduced glucose-induced depolarization, thereby causing reduced insulin secretion. Sprague-Dawley rats fed a diet with 40% fat for 3 weeks were glucose intolerant, and in vitro insulin secretion at high glucose was only increased 8.5-fold over basal, compared with 28-fold in control rats. Islet UCP-2 mRNA expression was increased twofold. These studies provide further strong evidence that UCP-2 is an important negative regulator of beta-cell insulin secretion and demonstrate that reduced (m) and increased activity of K(ATP) channels are mechanisms by which UCP-2-mediated effects are mediated. These studies also raise the possibility that a pathological upregulation of UCP-2 expression in the prediabetic state could contribute to the loss of glucose responsiveness observed in obesity-related type 2 diabetes in humans.

Adenosine Triphosphate↗

PubMed2001

Interactions between effects of adrenalectomy and diet on insulin secretion in fa/fa Zucker rats.

Our objective was to determine if a cafeteria-type diet with increased fat content would block the decrease in insulin secretion induced by adrenalectomy in obese rats. Five week old Zucker (fa/fa) rats were adrenalectomized. One week later, half of the adrenalectomized groups, and age-matched, sham-operated animals were given a diet of 16% fat and 44% carbohydrate. Control animals were maintained on standard rat chow (4.6% fat and 49% carbohydrate). After 4 weeks on the diets, in vivo measurements included caloric intake, weight gain, plasma corticosterone, triglyceride, free fatty acids, and oral glucose tolerance tests. In vitro measurements included glucose-stimulated insulin secretion, glucose phosphorylating activity, islet triglyceride content, and fatty acid oxidizing activity of cultured islets. Generally, the cafeteria diet did not block the effects of adrenalectomy on in vitro insulin secretion parameters, even though in sham-operated animals weight gain and insulin resistance was induced by the diet in vivo. Adrenalectomy and the diet exerted independent effects on glucose phosphorylation and fatty acid oxidation in islets. In conclusion, adrenalectomy decreased the elevated insulin secretion in fa/fa rats. The failure of a cafeteria diet enriched in fat to block the adrenalectomy-mediated changes in B-cell function indicates the importance of glucocorticoids and centrally-mediated effects on insulin secretion and other metabolic parameters.

Adrenalectomy↗

PubMed2000

Molecular cloning and expression studies of a prolactin receptor in goldfish (Carassius auratus).

A full-length cDNA clone, of a size of 4.6 kb, for the goldfish prolactin receptor has been isolated. This cDNA clone encodes a protein of 600 amino acids homologous to prolactin receptors of other species. A Kyte-Doolittle hydropathy analysis of the receptor indicates that the translated protein consists of a signal peptide of 22 amino acids, an extracellular domain of 228 amino acids, a single transmembrane domain of 24 amino acids, and an intracellular domain of 346 amino acids. Several characteristic landmarks of prolactin receptor could be identified in this clone. These include the four conserved cysteine residues and the WS motif within the extracellular domain, and the box 1 and box 2 regions of the intracellular domain. Among all the prolactin receptor sequences known to date, this clone bears the closest resemblance to the tilapia prolactin receptor, although homology between these two fish prolactin receptors is rather low. There are only 57.4% of nucleotide and 48.3% of amino acid sequence identities between these two fish receptors. This receptor cDNA was transfected into CHO-K1 cells for functional analysis. RT-PCR analysis with a pair of gene specific primers indicate that the receptor was transcribed in the transfected cells. Using a cell proliferation assay based on the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, the receptor transfected CHO-K1 cells can be stimulated to proliferate upon the addition of ovine prolactin in the culture medium. The tissue distribution of the prolactin receptor in goldfish was studied by RT-PCR/Southern analysis and by Northern analysis. The results indicated that the receptor is expressed mostly in the kidney, the gill and the intestine of goldfish, corroborating with the osmoregulatory role of prolactin in fish. In addition, an appreciable level of the receptor is also found in the brain and gonads of goldfish. Northern analysis showed that there are two transcript sizes, a major 4.6 kb and a minor 3.5 kb mRNAs, in the kidney, gill and intestine.

3' Untranslated Regions↗

PubMed2000

Myosin light chain replacement in the heart.

Myosin-actin cross-bridge kinetics are an important determinant for cardiac systolic and diastolic function. We compared the effects of myosin light chain substitutions on the ability of the fibers to contract in response to calcium and in their ability to produce power. Transgenesis was used to effect essentially complete replacement of the target contractile protein isoform specifically in the heart. Atrial and ventricular fibers derived from the various transgenic (TG) lines were skinned, and the force-velocity relationships, unloaded shortening velocities, and Ca(2+)-stimulated Mg(2+)-ATPase activities were determined. Replacement with an ectopic isoform resulted in significant changes in cross-bridge cycling kinetics but without any overt effects on morbidity or mortality. To confirm that this result was not light chain specific, a modified alpha-myosin heavy chain isoform that resulted in significant changes in force development was also engineered. The animals appeared healthy and have normal lifespans, and the changes in force development did not result in significant remodeling or overt hypertrophy. We conclude that myosin light chains can control aspects of cross-bridge cycling and alter force development. The myosin heavy chain data also show that changes in the kinetics of force development and power output do not necessarily lead to activation of the hypertrophic response or significant cardiac remodeling.

Aging↗

PubMed1999

Beta-cell hypertrophy in fa/fa rats is associated with basal glucose hypersensitivity and reduced SNARE protein expression.

In normal isolated beta-cells, the response to glucose is heterogeneous and characterized by an increasing number of secretory cells as glucose concentration rises (Pipeleers DG, Kiekens R, Ling Z, Wilikens A, Schuit F: Physiologic relevance of heterogeneity in the pancreatic beta-cell population. Diabetologia 37 (Suppl. 2):S57-S64, 1994). We hypothesized that fasting hyperinsulinemia in obesity might be explained by altered beta-cell heterogeneity of signal transduction mechanisms, possibly involving exocytotic proteins. Insulin secretion from individual beta-cells sorted according to the size of the islet donor (<125 microm, >250 microm, and intermediate diameter) was measured by reverse hemolytic plaque assay. Beta-cells from fa/fa rats were hypertrophied 25-40%, independent of donor islet size. This was accompanied by an increased proportion of secretory cells (recruitment) at 5.5-11.0 mmol/l glucose, increased secretion per cell at 2.8 mmol/l glucose, and decreased insulin content after acute glucose exposure without an increase in secretion per cell. Decreased expression of exocytotic (soluble N-ethylmaleimide-sensitive fusion protein receptor [SNARE]) proteins, vesicle-associated membrane protein isoform 2 (VAMP-2), synaptosomal protein of 25 kDa (SNAP-25), and syntaxin-1 and -2 in fa/fa beta-cells may contribute to the failure to sustain excessive plaque size at higher glucose concentrations. Fasting hyperinsulinemia may be maintained by increased recruitment and an exaggerated secretory response in all fa-derived islet populations. Glucose regulates beta-cell responsiveness in the short term, and these effects may involve altered expression of SNARE proteins.

Animals↗

PubMed1999

Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets.

Uncoupling protein 2 (UCP-2) mRNA expression has been shown to be altered by metabolic conditions such as obesity in humans, but its functional significance is unknown. The expression of UCP-2 mRNA and protein in normal rat islets was established by reverse transcriptase-polymerase chain reaction and immunocytochemistry in pancreatic islets and tissue, respectively. Intense immunostaining of UCP-2 correlated with insulin-positive ,-cells. Overexpression of UCP-2 in normal rat islets was accomplished by infection with an adenovirus (AdEGI-UCP-2) containing the full-length human UCP-2 coding sequence. Induction of the AdEGI-UCP-2 gene resulted in severe blunting of glucose-stimulated insulin secretion (GSIS) without affecting islet insulin content or the ability of the calcium ionophore A23187 to increase insulin secretion from AdEGI-UCP-2-expressing islets. Therefore, UCP-2 overexpression affects signal transduction proximal to Ca2+-mediated steps, including exocytosis. Insulin secretion from single beta-cells to 16.5 mmol/l glucose examined by reverse hemolytic plaque assay was nearly ablated if UCP-2 was overexpressed. Thus, a direct, causal relationship between overexpression of UCP-2 and inhibition of GSIS in normal islets has been established. These data suggest that increased expression of UCP-2 has the potential to cause the lack of a glucose effect on insulin secretion in type 2 diabetes.

Adenoviridae↗

PubMed1999

Glucose refractoriness of beta-cells from fed fa/fa rats is ameliorated by nonesterified fatty acids.

The aim of this study was to characterize the glucose responsiveness of individual beta-cells from fa/fa rats under ad libitum feeding conditions. Enlarged intact islets from fed fa/fa rats had a compressed insulin response curve to glucose compared with smaller islets. Size-sorted islets from obese rats yielded beta-cells whose glucose responsiveness was assessed by reverse hemolytic plaque assay to determine whether glucose refractoriness was caused by a decreased number of responsive cells or output per cell. In addition, the effects of palmitic acid on glucose-stimulated insulin secretion were assessed because of evidence that nonesterified fatty acids have acute beneficial effects. Two- to threefold more beta-cells from >250 microm diameter (large) islets than <125 microm diameter (small) or lean islets responded to low glucose. Increasing the glucose (8.3-16.5 mM) induced a >10-fold increase in recruitment of active cells from small islets, compared with only a 2.6-fold increase in large islets. This refractoriness was partially reversed by preincubation of the cells in low glucose for 2 h. In addition, secretion per cell of the large islet beta-cell population was significantly reduced compared with lean beta-cells, so that the overall response capacity of large but not small islet beta-cells was significantly reduced at high glucose. Therefore, continued near-normal function of the beta-cells from small islets of fa/fa rats seems crucial for glucose responsiveness. Incubation of beta-cells from large islets with palmitic acid normalized the secretory capacity to glucose mainly by increasing recruitment and secondarily by increasing secretion per cell. In conclusion, these studies demonstrate refractoriness to glucose of beta-cells from large islets of fa/fa rats under ad libitum feeding conditions. When acutely exposed to nonesterified fatty acids, islets from fa/fa rats have a potentiated insulin response despite chronic elevation of plasma lipids in vivo.

Animals↗