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C J Rhodes

Publications and source records attributed to C J Rhodes.

At least 19 recordsLinked to original sources
PubMed2006

Exendin 4 controls insulin production in rat islet beta cells predominantly by potentiation of glucose-stimulated proinsulin biosynthesis at the translational level.

AIMS/HYPOTHESIS: Ideally, a therapeutic insulin secretagogue should coordinately increase insulin production and insulin secretion to maintain islet beta cell secretory capacity. We compared the incretin mimetic exendin 4 and the sulfonylurea glibenclamide (known as glyburide in the USA and Canada) for their effects in upholding a balance between (pro)insulin biosynthesis and insulin secretion in pancreatic islets. METHODS: Isolated rat islets were incubated for 1 or 16 h over a range of glucose concentrations (2.8-16.7 mmol/l) with or without exendin 4 (10 nmol/l) or glibenclamide (1 micromol/l). Islets were then analysed for preproinsulin mRNA expression by RNase protection and quantitative real-time RT-PCR assays. Proinsulin biosynthesis was analysed by metabolic pulse-radiolabelling, immunoprecipitation and PAGE. Insulin secretion and insulin content were analysed by radioimmunoassay. RESULTS: Neither exendin 4 nor glibenclamide affected islet preproinsulin mRNA expression. However, exendin 4 significantly increased glucose-induced proinsulin biosynthesis at the translational level within 1 h, in marked contrast to glibenclamide, which inhibited proinsulin biosynthesis, especially at basal and intermediate glucose concentrations. Exendin 4 potentiated insulin secretion in a glucose-dependent manner, whereas glibenclamide stimulated insulin secretion independently of glucose. Exendin 4 better maintained rat islet insulin content compared with glibenclamide, which depleted intracellular stores of insulin in islet beta cells by 40% within 16 h. CONCLUSIONS/INTERPRETATION: Exendin 4 maintains insulin stores and beta cell secretory capacity primarily by translational control of proinsulin biosynthesis in parallel to insulin secretion. Glibenclamide does not regulate insulin production in coordination with stimulated insulin secretion, and consequently depletes islet insulin stores, compromising secretory capacity. Thus, at the level of the beta cell, incretin mimetics have an advantage over sulfonylureas for treatment of type 2 diabetes.

Animals↗
PubMed2006

Free radicals, metals and antioxidants in oxidative stress-induced cancer.

Oxygen-free radicals, more generally known as reactive oxygen species (ROS) along with reactive nitrogen species (RNS) are well recognised for playing a dual role as both deleterious and beneficial species. The "two-faced" character of ROS is substantiated by growing body of evidence that ROS within cells act as secondary messengers in intracellular signalling cascades, which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. The cumulative production of ROS/RNS through either endogenous or exogenous insults is termed oxidative stress and is common for many types of cancer cell that are linked with altered redox regulation of cellular signalling pathways. Oxidative stress induces a cellular redox imbalance which has been found to be present in various cancer cells compared with normal cells; the redox imbalance thus may be related to oncogenic stimulation. DNA mutation is a critical step in carcinogenesis and elevated levels of oxidative DNA lesions (8-OH-G) have been noted in various tumours, strongly implicating such damage in the etiology of cancer. It appears that the DNA damage is predominantly linked with the initiation process. This review examines the evidence for involvement of the oxidative stress in the carcinogenesis process. Attention is focused on structural, chemical and biochemical aspects of free radicals, the endogenous and exogenous sources of their generation, the metal (iron, copper, chromium, cobalt, vanadium, cadmium, arsenic, nickel)-mediated formation of free radicals (e.g. Fenton chemistry), the DNA damage (both mitochondrial and nuclear), the damage to lipids and proteins by free radicals, the phenomenon of oxidative stress, cancer and the redox environment of a cell, the mechanisms of carcinogenesis and the role of signalling cascades by ROS; in particular, ROS activation of AP-1 (activator protein) and NF-kappaB (nuclear factor kappa B) signal transduction pathways, which in turn lead to the transcription of genes involved in cell growth regulatory pathways. The role of enzymatic (superoxide dismutase (Cu, Zn-SOD, Mn-SOD), catalase, glutathione peroxidase) and non-enzymatic antioxidants (Vitamin C, Vitamin E, carotenoids, thiol antioxidants (glutathione, thioredoxin and lipoic acid), flavonoids, selenium and others) in the process of carcinogenesis as well as the antioxidant interactions with various regulatory factors, including Ref-1, NF-kappaB, AP-1 are also reviewed.

Animals↗
PubMed2005

Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B.

AIMS/HYPOTHESIS: The gut hormone glucagon-like peptide-1 (GLP-1) decreases beta cell apoptosis in a protein kinase B (PKB)-dependent fashion, and increases islet cell mass and function in vivo. In contrast, cytokines induce beta cell apoptosis, leading to decreased islet mass and type 1 diabetes. In the present study we used rat INS-1E beta cells and primary rat islet cells to examine the potential role of PKB as a mediator of the effect of GLP-1 on cytokine-induced apoptosis. METHODS: Cell viability was determined by MTT assay, and apoptosis and necrosis by Hoechst 33342-propidium iodide staining. Immunoblot analysis was used to detect changes in protein expression, including active (phosphorylated) and total PKB, phosphorylated and total glycogen synthase kinase-3beta, activated caspase-3 and inducible nitric oxide synthase. Reactive oxygen species were determined by 1,7-dichlorofluorescein (DCF) analysis, and mutant forms of PKB were introduced into cells using adenoviral vectors. RESULTS: Incubation of INS-1E cells with cytokines (IL-1beta, TNF-alpha and interferon-gamma; 10-50 ng/ml) for 18 h significantly decreased cell viability (by 44%, p<0.001), cell proliferation (by 80%, p<0.001), and activation of PKB (by 67%, p<0.001). Pre-treatment with exendin-4 (10(-7) mol/l), a long-acting GLP-1 receptor agonist, partially protected the cells against cytokine-induced toxicity (p<0.01) in association with a reduction in cytokine-induced inhibition of PKB phosphorylation (p<0.05). Exendin-4 pre-treatment did not change cell proliferation. Cytokine treatment increased apoptosis (by 156%, p<0.05) and necrosis (from undetectable to 2.6% of cells). These increases were both reduced by pre-treatment with exendin-4 (p<0.05-0.01). Furthermore, cytokine-induced apoptosis and necrosis were significantly increased in cells infected with kinase-dead PKB (p<0.05), and the protective effect of exendin-4 on both parameters was fully abolished in these cells. Similar changes were observed in primary islet cells. In parallel with these changes, exendin-4 decreased the cytokine-induced activation of caspase-3 (by 46%, p<0.05), and decreased levels of inducible nitric oxide synthase (by 71%, p<0.05) and reactive oxygen species (by 27%, p<0.05). CONCLUSIONS/INTERPRETATION: The results of our study indicate that GLP-1 plays a protective role against cytokine-induced apoptosis and necrosis in beta cells through a PKB-dependent signalling pathway.

Animals↗
PubMed2005

Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic beta-cells.

Several proteins play a role in the mechanism of insulin exocytosis. However, these 'exocytotic proteins' have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollin-green fluorescent protein (syncollinGFP) chimera in rat islet beta-cells as experimental tools. Syncollin is not normally expressed in beta-cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of beta-granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16.7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5 microM). Consistent with specific localization of syncollin to beta-granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in beta-cells has highlighted the importance of intralumenal beta-granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a 'fluorescent tag' to track beta-granule transport and exocytosis in real time.

Animals↗
PubMed2004

Glucagon-like peptide-1 prevents beta cell glucolipotoxicity.

AIMS/HYPOTHESIS: We have provided evidence that glucagon-like peptide-1, a potential therapeutic agent in the treatment of diabetes, activates phosphatidylinositol-3 kinase/protein kinase B signalling in the pancreatic beta cell. Since this pathway promotes cell survival in a variety of systems, we tested whether glucagon-like peptide-1 protects beta cells against cell death induced by elevated glucose and/or non-esterified fatty acids. METHODS: Human islets and INS832/13 cells were cultured at glucose concentrations of 5 or 25 mmol/l in the presence or absence of palmitate. Apoptosis was evaluated by monitoring DNA fragmentation and chromatin condensation. Wild-type and protein kinase B mutants were overexpressed in INS832/13 cells using adenoviruses. Nuclear factor-kappa B DNA binding was assayed by electrophoretic mobility shift assay. RESULTS: In human pancreatic beta cells and INS832/13 cells, glucagon-like peptide-1 prevented beta cell apoptosis induced by elevated concentrations of (i) glucose (glucotoxicity), (ii) palmitate (lipotoxicity) and (iii) both glucose and palmitate (glucolipotoxicity). Overexpression of a dominant-negative protein kinase B suppressed the anti-apoptotic action of glucagon-like peptide-1 in INS832/13 cells, whereas a constitutively active protein kinase B prevented beta cell apoptosis induced by elevated glucose and palmitate. Glucagon-like peptide-1 enhanced nuclear factor-kappa B DNA binding activity and stimulated the expression of inhibitor of apoptosis protein-2 and Bcl-2, two anti-apoptotic genes under the control of nuclear factor-kappa B. Inhibition of nuclear factor-kappa B by BAY 11-7082 abolished the prevention of glucolipotoxicity by glucagon-like peptide-1. CONCLUSIONS/INTERPRETATION: The results demonstrate a potent protective effect of glucagon-like peptide-1 on beta cell gluco-, lipo- and glucolipotoxicity. This effect is mediated via protein kinase B activation and possibly its downstream target nuclear factor-kappa B.

Apoptosis↗
PubMed2003

Fatty acid and phorbol ester-mediated interference of mitogenic signaling via novel protein kinase C isoforms in pancreatic beta-cells (INS-1).

It is possible that activation of protein kinase C (PKC) isoforms by free fatty acids (FFA) plays a role in the failure of pancreatic beta-cell mass expansion to compensate for peripheral insulin resistance in the pathogenesis of type-2 diabetes. The effect of lipid moieties on activation of conventional (PKC-alpha and -beta1), novel (PKC-delta) and atypical (PKC-zeta) PKC isoforms was evaluated in an in vitro assay, using biotinylated neurogranin as a substrate. Oleoyl-Coenzyme A (CoA) and palmitoyl-CoA, but not unesterified FFA, significantly increased the activity of all PKC isoforms (P< or =0.05), particularly that for PKC-delta. It was found that FFA (0.4 mM oleate/complexed to 0.5% bovine serum albumin) inhibited IGF-I-induced activation of protein kinase B (PKB) in the pancreatic beta-cell line (INS-1), but this was alleviated in the presence of the general PKC inhibitor (Gö6850; 1 microM). To further investigate whether conventional or novel PKC isoforms adversely affect beta-cell proliferation, the effect of phorbol ester (phorbol 12-myristate 13-acetate; PMA)-mediated activation of these PKC isoforms on glucose/IGF-I-induced INS-1 cell mitogenesis, and insulin receptor substrate (IRS)-mediated signal transduction was investigated. PMA-mediated activation of PKC (100 nM; 4 h) reduced glucose/IGF-I mediated beta-cell mitogenesis (>50%; P< or =0.05), which was reversible by the general PKC inhibitor Gö6850 (1 microM), indicating an effect of PKC and not due to a non-specific PMA toxicity. PMA inhibited IGF-I-induced activation of PKB, correlating with inhibition of IGF-I-induced association of IRS-2 with the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, in contrast, PMA activated the mitogen-activated protein kinases, Erk1/2. Titration inhibition analysis using PKC isoform inhibitors indicated that these PMA-induced effects were via novel PKC isoforms. Thus, FFA/PMA-induced activation of novel PKC isoforms can inhibit glucose/IGF-I-mediated beta-cell mitogenesis, in part by decreasing PKB activation, despite an upregulation of Erk1/2. Thus, activation of novel PKC isoforms by long-chain acyl-CoA may well contribute to decreasing beta-cell mass in the pathogenesis of type-2 diabetes, similar to their inhibition of insulin signal transduction which causes insulin resistance.

Acetophenones↗
PubMed2002

Molecular insights into insulin action and secretion.

Tightly co-ordinated control of both insulin action and secretion is required in order to maintain glucose homeostasis. Gene knockout experiments have helped to define key signalling molecules that affect insulin action, including insulin and insulin-like growth factor-1 (IGF-1) receptors, insulin receptor substrate (IRS) proteins and various downstream effector proteins. beta-cell function is also a tightly regulated process, with numerous factors (including certain signalling molecules) having an impact on insulin production, insulin secretion and beta-cell mass. While signalling molecules play important roles in insulin action and secretion under normal circumstances, abnormal insulin signalling in muscle, adipose tissue, liver and pancreas leads to insulin resistance and beta-cell dysfunction. In particular, the signalling protein IRS-2 may have a central role in linking these abnormalities, although other factors are likely to be involved.

Animals↗
PubMed2001

Cooperativity between the preproinsulin mRNA untranslated regions is necessary for glucose-stimulated translation.

Glucose regulates proinsulin biosynthesis via stimulation of the translation of the preproinsulin mRNA in pancreatic beta-cells. However, the mechanism by which this occurs has remained unclear. Using recombinant adenoviruses that express the preproinsulin mRNA with defined alterations, the untranslated regions (UTRs) of the preproinsulin mRNA were examined for elements that specifically control translation of the mRNA in rat pancreatic islets. These studies revealed that the preproinsulin 5'-UTR was necessary for glucose stimulation of preproinsulin mRNA translation, whereas the 3'-UTR appeared to suppress translation. However, together the 5'- and 3'-UTRs acted cooperatively to markedly increase glucose-induced proinsulin biosynthesis. In primary hepatocytes the presence of the preproinsulin 3'-UTR led to reduced mRNA levels compared with the presence of the SV40 3'-UTR, consistent with the presence of mRNA stability determinants in the 3'-UTR that stabilize the preproinsulin mRNA in a pancreatic beta-cell-specific manner. Translation of these mRNAs in primary hepatocytes was not stimulated by glucose, indicating that regulated translation of the preproinsulin mRNA occurs in a pancreatic beta-cell-specific manner. Thus, the untranslated regions of the preproinsulin mRNA play crucial roles in regulating insulin production and therefore glucose homeostasis by regulating the translation and the stability of the preproinsulin mRNA.

Animals↗
PubMed2001

Differential activation of protein kinase B and p70(S6)K by glucose and insulin-like growth factor 1 in pancreatic beta-cells (INS-1).

It has been shown that IGF-1-induced pancreatic beta-cell proliferation is glucose-dependent; however, the mechanisms responsible for this glucose dependence are not known. Adenoviral mediated expression of constitutively active phosphatidylinositol 3-kinase (PI3K) in the pancreatic beta-cells, INS-1, suggested that PI3K was not necessary for glucose-induced beta-cell proliferation but was required for IGF-1-induced mitogenesis. Examination of the signaling components downstream of PI3K, 3-phosphoinositide-dependent kinase 1, protein kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70(S6K)), suggested that a major part of glucose-dependent beta-cell proliferation requires activation of mammalian target of rapamycin/p70(S6K), independent of phosphoinositide-dependent kinase 1/PKB activation. Adenoviral expression of the kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced beta-cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type and constitutively active PKB-infected cells. Upon analysis of extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1 was observed in kinase-dead PKB-infected cells, but this phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of rapamycin/p70(S6K) signal transduction pathways for a full commitment to glucose-induced pancreatic beta-cell mitogenesis. However, for IGF-1-induced activation, these pathways must be carefully balanced, because chronic activation of one (PI3K/PKB) can lead to dampening of the other (ERK1/2), reducing the mitogenic response.

Adenoviridae↗
PubMed2001

Free fatty acid-induced inhibition of glucose and insulin-like growth factor I-induced deoxyribonucleic acid synthesis in the pancreatic beta-cell line INS-1.

Pancreatic beta-cell mitogenesis is increased by insulin-like growth factor I (IGF-I) in a glucose-dependent manner. In this study it was found that alternative beta-cell nutrient fuels to glucose, pyruvate, and glutamine/leucine independently induced and provided a platform for IGF-I to induce INS-1 cell DNA synthesis in the absence of serum. In contrast, long chain FFA (>/=C(12)) inhibited 15 mM glucose-induced [(3)H]thymidine incorporation (+/-10 nM IGF-I) by 95% or more within 24 h above 0.2 mM FFA complexed to 1% BSA (K(0.5) for palmitate/1% BSA = 65-85 microM for 24 h; t(0.5) for 0.2 mM palmitate/1% BSA = approximately 6 h). FFA-mediated inhibition of glucose/IGF-I-induced ss-cell DNA synthesis was reversible, and FFA oxidation did not appear to be required, nor did FFA interfere with glucose metabolism in INS-1 cells. An examination of mitogenic signal transduction pathways in INS-1 cells revealed that glucose/IGF-I induction of early signaling elements in SH2-containing protein (Shc)- and insulin receptor substrate-1/2-mediated pathways leading to downstream mitogen-activated protein kinase and phosphoinositol 3'-kinase activation, were unaffected by FFA. However, glucose-/IGF-I-induced activation of protein kinase B (PKB) was significantly inhibited, and protein kinase Czeta was chronically activated by FFA. It is possible that FFA-mediated inhibition of ss-cell mitogenesis contributes to the reduction of beta-cell mass and the subsequent failure to compensate for peripheral insulin resistance in vivo that is key to the pathogenesis of obesity-linked diabetes.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Gene and cell-replacement therapy in the treatment of type 1 diabetes: how high must the standards be set?

Recent advances in molecular and cell biology may allow for the development of novel strategies for the treatment and cure of type 1 diabetes. In particular, it is now possible to envisage restoration of insulin secretion by gene or cell-replacement therapy. The beta-cell is, however, remarkably sophisticated, and many of the features of this highly differentiated secretory cell will have to be faithfully mimicked in surrogate cells. In particular, insulin is normally secreted in a well-regulated fashion in rapid response to the metabolic needs of the individual and most specifically (but not exclusively) to changes in circulating levels of glucose. Such regulated secretion will be indispensable in order to avoid both hyper- and hypoglycemic episodes and depends on the ability of cells to store insulin in secretory granules before exocytosis in response to physiological stimuli. Furthermore, any newly created insulin-secreting cell will have to be able to adapt to alterations in insulin requirements that accompany changes with exercise, body weight, and aging. Fine tuning of insulin secretion over the longer term will also be important to avoid "clinical shifting" that could be caused by over-insulinization, including increased adiposity and cardiovascular disease. Finally, it will be necessary to ensure that newly created or implanted (surrogate) beta-cells are protected in some way from recognition by the immune system and in particular from autoimmune destruction.

Animals↗
PubMed2001

Protein kinase Czeta activation mediates glucagon-like peptide-1-induced pancreatic beta-cell proliferation.

Glucagon-like peptide-1 (GLP-1), an insulinotropic and glucoincretin hormone, is a potentially important therapeutic agent in the treatment of diabetes. We previously provided evidence that GLP-1 induces pancreatic beta-cell growth nonadditively with glucose in a phosphatidylinositol-3 kinase (PI-3K)-dependent manner. In the present study, we investigated the downstream effectors of PI-3K to determine the precise signal transduction pathways that mediate the action of GLP-1 on beta-cell proliferation. GLP-1 increased extracellular signal-related kinase 1/2, p38 mitogen-activated protein kinase (MAPK), and protein kinase B activities nonadditively with glucose in pancreatic beta(INS 832/13) cells. GLP-1 also caused nuclear translocation of the atypical protein kinase C (aPKC) zeta isoform in INS as well as in dissociated normal rat beta-cells as shown by immunolocalization and Western immunoblotting analysis. Tritiated thymidine incorporation measurements showed that the p38 MAPK inhibitor SB203580 suppressed GLP-1-induced beta-cell proliferation. Further investigation was performed using isoform-specific pseudosubstrates of classical (alpha, beta, and gamma) or zeta aPKC isoforms. The PKCzeta pseudosubstrate suppressed the proliferative action of GLP-1, whereas the inhibitor of classical PKC isoforms had no effect. Overexpression of a kinase-dead PKCzeta acting as a dominant negative protein suppressed GLP-1-induced proliferation. In addition, ectopic expression of a constitutively active PKCzeta mutant stimulated tritiated thymidine incorporation to the same extent as GLP-1, and the glucoincretin had no growth-promoting action under this condition. The data indicate that GLP-1-induced activation of PKCzeta is implicated in the beta-cell proliferative signal of the insulinotropic hormone. The results are consistent with a model in which GLP-1-induced PI-3K activation results in PKCzeta translocation to the nucleus, which may play a role in the pleiotropic effects (DNA synthesis, metabolic enzymes, and insulin gene expression) of the glucoincretin.

Animals↗
PubMed2001

Glycerol-stimulated proinsulin biosynthesis in isolated pancreatic rat islets via adenoviral-induced expression of glycerol kinase is mediated via mitochondrial metabolism.

In this study, we examined whether adenoviral-mediated glycerol kinase (AdV-CMV-GlyK) expression in isolated rat pancreatic islets could introduce glycerol-induced proinsulin biosynthesis. In AdV-CMV-GlyK-infected islets, specific glycerol-induced proinsulin biosynthesis translation and insulin secretion were observed in parallel from the same islets. The threshold concentration of glycerol required to stimulate proinsulin biosynthesis was lower (0.25-0.5 mmol/l) than that for insulin secretion (1.0-1.5 mmol/l), reminiscent of threshold differences for glucose-stimulated proinsulin biosynthesis versus insulin secretion. The dose-dependent glycerol-induced proinsulin biosynthesis correlated with the rate of glycerol oxidation in AdV-CMV-GlyK-infected islets, indicating that glycerol metabolism was required for the response. However, glycerol did not significantly increase lactate output from AdV-CMV-GlyK-infected islets, but the dihydroxyacetone phosphate (DHAP) to alpha-glycerophosphate (alpha-GP) ratio significantly increased in AdV-CMV-GlyK-infected islets incubated at 2 mmol/l glycerol compared with that at a basal level of 2.8 mmol/l glucose (P < or = 0.05). The DHAP:alpha-GP ratio was unaffected in AdV-CMV-GlyK-infected islets incubated at 2 mmol/l glycerol in the added presence of alpha-cyanohydroxycinnaminic acid (alpha-CHC), an inhibitor of the plasma membrane and mitochondrial lactate/pyruvate transporter. However, alpha-CHC inhibited glycerol-induced proinsulin biosynthesis and insulin secretion in AdV-CMV-GlyK-infected islets (>75%; P = 0.05), similarly to glucose-induced proinsulin biosynthesis and insulin secretion in AdV-CMV-GlyK-infected and control islets. These data indicated that in AdV-CMV-GlyK-infected islets, the importance of mitochondrial metabolism of glycerol was required to generate stimulus-response coupling signals to induce proinsulin biosynthesis and insulin secretion.

Adenoviridae↗
PubMed2001

A low-affinity Ca2+-dependent association of calmodulin with the Rab3A effector domain inversely correlates with insulin exocytosis.

The stimulus-response coupling pathway for glucose-regulated insulin secretion has implicated a rise in cytosolic [Ca2+]i as a key factor to induce insulin exocytosis. However, it is unclear how elevated [Ca2+]i communicates with the pancreatic beta-cell's exocytotic apparatus. As Rab3A is a model protein involved in regulated exocytosis, we have focused on its role in regulating insulin exocytosis. By using a photoactivatable cross-linking synthetic peptide that mimics the effector domain of Rab3A and microsequence analysis, we found calmodulin to be a major Rab3A target effector protein in pancreatic beta-cells. Coimmunoprecipitation analysis from pancreatic islets confirmed a Rab3A-calmodulin interaction in vivo, and that it inversely correlated with insulin exocytosis. Calmodulin affected neither GTPase nor guanine nucleotide exchange activity of Rab3A. The calmodulin-Rab3A interaction was pH- and Ca2+-dependent, and it was preferential for GTP-bound Rab3A. However, Rab3A affinity for calmodulin was relatively low (Kd = 18-22 micromol/l at 10(-5) mol/l [Ca2+]) and competed by other calmodulin-binding proteins that had higher affinity (e.g., Ca2+/calmodulin-dependent protein kinase-2 [CaMK-2] [Kd = 300-400 nmol/l at 10(-5) mol/l [Ca2+]]). Moreover, the Ca2+ dependence of the calmodulin-Rab3A interaction (K0.5 = 15-18 micromol/l [Ca2+], maximal at 100 micromol/l [Ca2+]) was significantly lower compared with that of the calmodulin-CaMK-2 association (K0.5 = 40 micromol/l [Ca2+], maximal at 1 mmol/l [Ca2+]). The data suggested that a transient Rab3A-calmodulin interaction might represent a means of directing calmodulin to the cytoplasmic face of a beta-granule, where it can be subsequently transferred for activation of other beta-granule-associated calmodulin-binding proteins as local [Ca2+]i rises to promote insulin exocytosis.

Animals↗
PubMed2000

Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI beta-cells).

Non-insulin-dependent diabetes mellitus is associated with, in addition to impaired insulin release, elevated levels of free fatty acids (FFA) in the blood. Insulin release is stimulated when beta-cells are acutely exposed to FFA, whereas chronic exposure may inhibit glucose-induced insulin secretion. In the present study we investigated the direct effects of long chain acyl-CoA (LC-CoA), the active intracellular form of FFA, on insulin exocytosis. Palmitoyl-CoA stimulated both insulin release from streptolysin-O-permeabilized HIT cells and fusion of secretory granules to the plasma membrane of mouse pancreatic beta-cells, as measured by cell capacitance. The LC-CoA effect was chain length-dependent, requiring chain lengths of at least 14 carbons. LC-CoA needed to be present to stimulate insulin release, and consequently there was no effect following its removal. The stimulatory effect was observed after inhibition of protein kinase activity and in the absence of ATP, even though both kinases and ATP, themselves, modulate exocytosis. The effect of LC-CoA was inhibited by cerulenin, which has been shown to block protein acylation. The data suggest that altered LC-CoA levels, resulting from FFA or glucose metabolism, may act directly on the exocytotic machinery to stimulate insulin release by a mechanism involving LC-CoA protein binding.

Animals↗