Inhibition of calpain blocks pancreatic beta-cell spreading and insulin secretion

In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca(2+)]i. Finally, calpeptin was shown to affect the [Ca(2+)]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic beta-cells. It is therefore suggested that calpain could be a mediator of Ca(2+)-induced-insulin secretion and beta-cell spreading.     

Genetically modified rice seeds accumulating GLP-1 analogue stimulate insulin secretion from a mouse pancreatic beta-cell line

Glucagon-like peptide-1 (7-36) amide (GLP-1) is the most potent physiological insulinotropic hormone in humans. We produced large amounts of a GLP-1 analogue, [Ser8, Gln26, Asp34]-GLP-1, which is resistant to trypsin-digestion, as part of a chimeric rice seed storage protein, a 26 kDa globulin, in genetically modified rice seeds. Junction sites between GLP-1 analogue and globulin were replaced by tryptic cleavage sites. The highest level of GLP-1 analogue accumulation was approximately 20-50 microg per seed. We found that GLP-1 analogue derived from trypsin-digested genetically modified rice seeds stimulated insulin secretion from a mouse pancreatic beta-cell line, MIN6.     

Extracellular acidification parallels insulin secretion in INS-1 and HIT-T15 beta-cell lines

As an alternative to manual assays that track insulin secretion, we tested a silicon-based biosensor that allows automated monitoring of extracellular acidification. Glucose stimulation of INS-1 and HIT-T15 cells resulted in a rapid increase in extracellular acidification in a biphasic and concentration-dependent fashion much like insulin secretion (EC(50) INS-1=5 mM and HIT-T15=1 mM). This response was attenuated by verapamil (10 microM) and stimulated by administration of glybenclamide (100 nM) or KCl-induced (40 mM) depolarization. These experiments suggest that automated monitoring of extracellular pH may be a useful assay and support the relevance of linking metabolic activity to insulin secretion.     

Standardized biosynthesis of flavan-3-ols with effects on pancreatic beta-cell insulin secretion

Flavan-3-ols, such as green tea catechins represent a major group of phenolic compounds with significant medicinal properties. We describe the construction and optimization of Escherichia coli recombinant strains for the production of mono- and dihydroxylated catechins from their flavanone and phenylpropanoid acid precursors. Use of glucose minimal medium, Fe(II), and control of oxygen availability during shake-flask experiments resulted in production yield increases. Additional production improvement resulted from the use of medium rather than high-copy number plasmids and, in the case of mono-hydroxylated compounds, the addition of extracellular cofactors in the culture medium. The established metabolic engineering approach allowed the biosynthesis of natural catechins at high purity for assessing their possible insulinotropic effects in pancreatic beta-cell cultures. We demonstrated that (+)-afzelechin and (+)-catechin modulated the secretion of insulin by pancreatic beta-cells. These results indicate the potential of applying metabolic engineering approaches for the synthesis of natural and non-natural catechin analogues as drug candidates in diabetes treatments.     

Phorbol ester stimulation of pancreatic beta-cell replication, polyamine content and insulin secretion.

Long-term effects of the protein kinase C activating phorbol ester, TPA, on pancreatic beta-cell proliferation and insulin production were investigated. It was found that beta-cell replication and long-term insulin secretion were enhanced in TPA-treated islets. This was not accompanied by a corresponding increase in (pro)insulin biosynthesis, presumably contributing to the lowered islet insulin content. TPA also increased islet polyamine content but when this increase was prevented by blocking polyamine synthesis, DNA replication and insulin secretion remained elevated. These findings indicate that TPA stimulates beta-cell replication and insulin secretion and suggest a stimulatory role for protein kinase C, but not for polyamines, in these processes.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Cannabinoid receptors regulate Ca(2+) signals and insulin secretion in pancreatic beta-cell

Insulin is the main hormone involved in the regulation of glycaemia, its impaired secretion is a hallmark of type I and type II diabetic individuals. Additionally, insulin is involved in lipogenesis and weight gain, provoking an anorexigenic action. The endocannabinoid system contributes to the physiological regulation of energy balance, food intake and lipid and glucose metabolisms. Despite that, an experimental link between the endocannabinoid system and the endocrine pancreas has not yet been described. Using quantitative real-time PCR and immunocytochemistry, we have demonstrated the existence of both CB1 and CB2 receptors in the endocrine pancreas. While the CB1 receptor is mainly expressed in non-beta-cells, the CB2 type exists in beta- and non-beta-cells within the islet. The endocannabinoid 2-arachidonylglycerol (2-AG) through CB2 receptors regulates [Ca(2+)](i) signals in beta-cells and as a consequence, it decreases insulin secretion. This effect may be a new component involved in the orexigenic effect of endocannabinoids and constitutes a potential target for pharmacologic manipulation of the energy balance.     

Alpha-adrenergic inhibition of rat pancreatic beta-cell replication and insulin secretion is mediated through a pertussis toxin-sensitive G-protein regulating islet cAMP content.

The rate of DNA synthesis, insulin secretion and cAMP content in isolated pancreatic islets were markedly inhibited by long-term exposure to the alpha 1-adrenoceptor agonist phenylephrine, the alpha 2-adrenoceptor agonist clonidine and the beta-adrenoceptor antagonist propranolol. Pertussis toxin or the stimulatory cAMP analog Sp-cAMPS increased DNA synthesis and insulin secretion in the absence of the adrenergic agents. Pertussis toxin blocked the inhibitory actions of these agents on DNA synthesis, insulin secretion and cAMP content, and a similar protection was imposed by Sp-cAMPS. Thus, long-term alpha-adrenergic stimulation interferes with signaling through pertussis toxin-sensitive G-protein(s) and, by decreasing the islet cAMP content, inhibits beta-cell DNA synthesis and insulin secretion.     

Ultrastructural changes of the pancreatic beta-cell and the insulin secretion by islets from lactating and non-lactating rats

Islets isolated from lactating rats, as compared to islets from non-lactating rats, release less insulin when incubated in the absence of exogenous nutrient or presence of either D-glucose (11.1 mM) or the association of L-leucine and L-glutamine (10.0 mM each). The insulin content of the islets is not different in lactating and non-lactating rats. The volume density of the dark granules in the beta-cells is not at variance in both groups. However the volume density of the light (pale) granules is significantly lower in the lactating rats. The reduced amount of light granules is in keeping with the reduced secretory capacity of the beta-cells from lactating rats.     

Dehydroepiandrosterone increases beta-cell mass and improves the glucose-induced insulin secretion by pancreatic islets from aged rats

The effect of dehydroepiandrosterone (DHEA) on pancreatic islet function of aged rats, an animal model with impaired glucose-induced insulin secretion, was investigated. The following parameters were examined: morphological analysis of endocrine pancreata by immunohistochemistry; protein levels of insulin receptor, IRS-1, IRS-2, PI 3-kinase, Akt-1, and Akt-2; and static insulin secretion in isolated pancreatic islets. Pancreatic islets from DHEA-treated rats showed an increased beta-cell mass accompanied by increased Akt-1 protein level but reduced IR, IRS-1, and IRS-2 protein levels and enhanced glucose-stimulated insulin secretion. The present results suggest that DHEA may be a promising drug to prevent diabetes during aging.     

Regulatory roles for small G proteins in the pancreatic beta-cell: lessons from models of impaired insulin secretion

Emerging evidence suggests that GTP-binding proteins (G proteins) play important regulatory roles in physiological insulin secretion from the islet beta-cell. Such conclusions were drawn primarily from experimental data derived through the use of specific inhibitors of G protein function. Data from gene depletion experiments appear to further substantiate key roles for these signaling proteins in the islet metabolism. The first part of this review will focus on findings supporting the hypothesis that activation of specific G proteins is essential for insulin secretion, including regulation of their function by posttranslational modifications at their COOH-terminal cysteines (e.g., isoprenylation). The second part will overview novel, non-receptor-dependent mechanism(s) whereby glucose might activate specific G proteins via protein histidine phosphorylation. The third section will review findings that appear to link abnormalities in the expression and/or functional activation of these key signaling proteins to impaired insulin secretion. It is hoped that this review will establish a basis for future research in this area of islet signal transduction, which presents a significant potential, not only in identifying key signaling proteins that are involved in physiological insulin secretion, but also in examining potential abnormalities in this signaling cascade that lead to islet dysfunction and onset of diabetes.     

Impact of mitochondrial calcium on the coupling of metabolism to insulin secretion in the pancreatic beta-cell

Mitochondria play an essential role in metabolism-secretion coupling in the pancreatic beta-cell. Dysfunction of the organelle leads to impaired glucose-stimulated insulin secretion, as exemplified by the rare disease mitochondrial diabetes, which is caused by mutations in the mitochondrial DNA. In the excitable beta-cell, mitochondria generate ATP and possibly other coupling factors that promote plasma membrane depolarization and calcium influx triggering insulin exocytosis. Cytosolic calcium signals are relayed into the mitochondria, where the ion potentiates oxidative metabolism. Hormones such as glucagon-like peptide 1 (GLP-1) or neurotransmitter secretagogues stimulate the beta-cell by activating different signal transduction pathways eventually also raising mitochondrial calcium. Likewise, pharmacological inhibition of the Na(+)/Ca(2+) exchanger of the inner mitochondrial membrane augments intra-organellar calcium and insulin secretion. Islets obtained after autopsy from type 2 diabetic patients have altered mitochondrial morphology impaired glucose oxidation and reduced ATP generation, explaining defective insulin secretion. We hypothesize that the improvement of glucose-stimulated insulin secretion by sulfonylurea compounds in type 2 diabetic patients is in part due to their capacity to raise mitochondrial calcium, which is beneficial for the generation of metabolic coupling factors.     

Type 2 diabetes, insulin secretion and beta-cell mass

In nondiabetic subjects, insulin secretion is sufficiently increased as a compensatory adaptation to insulin resistance whereas in subjects with type 2 diabetes, the adaptation is insufficient. Evidences for the islet dysfunction in type 2 diabetes are a)impaired insulin response to various challenges such as glucose, arginine and isoproterenol, b)defective dynamic of insulin secretion resulting in preferential reduction on first phase insulin secretion and irregular oscillations of plasma insulin and c)defective conversion of proinsulin to insulin leading to elevated proinsulin to insulin ratio. In addition, recent studies have also presented evidence of a reduced beta cell mass in diabetes, caused predominantly by enhanced islet apoptosis, although this needs to be confirmed in more studies. These defects may be caused by primary beta cell defects, such as seen in the monogenic diabetes forms of MODY, or by secondary beta cell defects, caused by glucotoxicity, lipotoxicity or islet amyloid aggregation. The defects may also be secondary to defective beta cell stimulation by incretin hormones or the autonomic nerves. The appreciation of islet dysfunction as a key factor underlying the progression from an insulin resistant state into type 2 diabetes has therapeutic implications, since besides improvement of insulin sensitivity, treatment should also aim at improving the islet compensation. This may possibly be achieved by stimulating insulin secretion, supporting islet stimulating mechanisms, removing toxic beta-cell insults and inhibiting beta cell apoptosis.     

Calcium and pancreatic beta-cell function. The mechanism of insulin secretion studied with the aid of lanthanum.

La3+ was used to study the involvement of Ca2+ in insulin secretion in beta-cell-rich pancreatic islets micro-dissected from non-inbred ob/ob mice. Ultrastructural studies revealed that the localization of La3+ was entirely restricted to the exterior of the cells. Consistent with a membrane action, exposure to La3+ failed to affect glucose oxidation and either the sucrose space or the general ultrastructure of the islets. In contrast, La3+ had marked effects on insulin release and 45Ca fluxes. Exposure to La3+ resulted in pronounced inhibition of insulin release irrespective of the presence or absence of Ca2+, 3-isobutyl-1-methylxanthine or glucose. Perifusion experiments revealed that the inhibitory action was prompt, sustained and readily reversible. Removal of La3+ was associated with a subsequent prolonged stimulatory phase of insulin release even in medium deficient in Ca2+. This action could not be attributed to an increase in cyclic AMP, but was potentiated by 3-isobutyl-1-methylxanthine and abolished by L-adrenaline. La3+ displaced 45Ca from superficially located binding sites and inhibited the uptake and efflux of 45Ca. The stimulatory and inhibitory actions of glucose on 45Ca efflux were also abolished in the presence of 2 mM-La3+ Removal of La3+ was associated with the preferential mobilization of 45Ca incorporated in response to glucose. The results indicate that binding of La3+ to superficial sites in the plasma membrane leads to inhibition of insulin release by suppression of transmembrane Ca2+ fluxes. It is suggested that accumulation of Ca2+ in the cytoplasm accounts for the stimulation of insulin release seen after removal of La3+ from inhibitory binding sites in the beta-cell plasma membrane.     

Slow interaction of islet-activating protein with pancreatic islets during primary culture to cause reversal of alpha-adrenergic inhibition of insulin secretion

The manner in which islet-activating protein (IAP), a protein purified from the culture medium of Bordetella pertussis, interacts with the islet B-cell was studied by following the progressive development of IAP-induced reversal of alpha-adrenergic inhibition of insulin release during maintenance of islets in culture with glucose and epinephrine. This action of IAP developed in an exponential manner dependent on its concentration after a true lag period of about 1 h. The lag period was not grossly dependent on the concentration of IAP added but highly dependent on temperature of culture, and was still seen upon adding a second dose of IAP to partially stimulated cells. After 24-h culture significantly more insulin was secreted with IAP at a concentration as low as 1 pg/ml and the half-maximal effect was observed at 0.1 ng/ml. The development of IAP action occurred even in the islets that had been exposed to IAP for only 30 s, but was significantly prevented by anti-IAP serum added before the end of the lag period. IAP was effective in the presence of cycloheximide, an inhibitor of protein synthesis, or of vinblastine or cytochalasin B, microtubular-microfilamentous modifiers. It is suggested that the IAP molecule is rapidly bound to the receptor area of the islet B-cell and then is gradually inserted into the cell membrane before appearance of its action to activate native calcium ionophores. This slow interaction of IAP with the membrane may be responsible for potentiation of insulin secretory and cAMP responses of the cell to various stimuli as well as for reversal of alpha-adrenergic inhibition.     

The effect of starvation on insulin secretion and glucose metabolism in mouse pancreatic islets

1. Crude extracts of seeds of Pinus radiata catalysed acetate-, propionate-, n-butyrate- and n-valerate-dependent PP(i)-ATP exchange in the presence of MgCl(2), which was apparently due to a single enzyme. Propionate was the preferred substrate. Crude extracts did not catalyse medium-chain or long-chain fatty acid-dependent exchange. 2. Ungerminated dry seeds contained short-chain fatty acyl-CoA synthetase activity. The activity per seed was approximately constant for 11 days after imbibition and then declined. The enzyme was located only in the female gametophyte tissue. 3. The synthetase was purified 70-fold. 4. Some properties of the enzyme were studied by [(32)P]PP(i)-ATP exchange. K(m) values for acetate, propionate, n-butyrate and n-valerate were 4.7, 0.21, 0.33 and 2.1mm respectively. Competition experiments between acetate and propionate demonstrated that only one enzyme was involved and confirmed that the affinity of the enzyme for propionate was greater than that for acetate. CoA inhibited fatty acid-dependent PP(i)-ATP exchange. The enzyme catalysed fatty acid-dependent [(32)P]PP(i)-dATP exchange. 5. The enzyme also catalysed the fatty acyl-AMP-dependent synthesis of [(32)P]ATP from [(32)P]PP(i). Apparent K(m) (acetyl-AMP) and apparent K(m) (propionyl-AMP) were 57mum and 7.5mum respectively. The reaction was inhibited by AMP and CoA. 6. Purified enzyme catalysed the synthesis of acetyl-CoA and propionyl-CoA. Apparent K(m) (acetate) and apparent K(m) (propionate) were 16mm and 7.5mm respectively. The rate of formation of acetyl-CoA was enhanced by pyrophosphatase. 7. It was concluded that fatty acyl adenylates are intermediates in the formation of the corresponding fatty acyl-CoA.     

Effect of perchlorate on calcium uptake and insulin secretion in mouse pancreatic islets.

Microdissected beta-cell-rich pancreatic islets of non-inbred ob/ob mice were used in studies of how perchlorate (CIO4-) affects stimulus-secretion coupling in beta-cells. CIO4- at 16 mM potentiated D-glucose-induced insulin release, without inducing secretion at non-stimulatory glucose concentrations. The potentiation mainly applied to the first phase of stimulated insulin release. In the presence of 20 mM-glucose, the half-maximum effect of CIO4- was reached at 5.5 mM and maximum effect at 12 mM of the anion. The potentiation was reversible and inhibitable by D-mannoheptulose (20 mM) or Ca2+ deficiency. CIO4- at 1-8 mM did not affect glucose oxidation. The effects on secretion were paralleled by a potentiation of glucose-induced 45Ca2+ influx during 3 min. K+-induced insulin secretion and 45Ca2+ uptake were potentiated by 8-16 mM-CIO4-. The spontaneous inactivation of K+-induced (20.9 mM-K+) insulin release was delayed by 8 mM-CIO4-. The anion potentiated the 45Ca2+ uptake induced by glibenclamide, which is known to depolarize the beta-cell. Insulin release was not affected by 1-10 mM-trichloroacetate. It is suggested that CIO4- stimulates the beta-cell by affecting the gating of voltage-controlled Ca2+ channels.