Transport of 1,5-anhydro-D-glucitol into insulinoma cells by a glucose-sensitive transport system

The uptake of 1,5-anhydro-D-glucitol (1,5-AG) occurs by passive mechanisms in cells or tissues that have passive glucose transporters. It is known that serum 1,5-AG concentrations are reduced in patients with diabetes mellitus. To elucidate the metabolism of this substance and its physiological role in pancreatic β-cells, we assayed 1,5-AG transport in the insulinoma-derived cell lines, RINr and MIN6. Both cell lines showed an insulin-insensitive, concentration-dependent uptake of 1,5-AG with a saturation time of approximately 120 min, and most of the 1,5-AG in the cytoplasm was in the free form. A biphasic saturation curve was obtained using a wide range of 1,5-AG concentrations, suggesting that accumulation was mediated by a high affinity and a low affinity transporter. The high affinity transporter had a K_m of 10.4 in RINr cells and 13.0 mM in MIN6 cells, and the low affinity transporter had a K_m of 131 in RINr cells and 211 mM in MIN6 cells. Uptake of 1,5-AG was markedly inhibited by phloretin and cytochalasin B, but was only slightly affected by phloridzin. Uptake of 1,5-AG was markedly inhibited by glucose at physiological concentrations (1.0–10 mM), as well as by galactose and mannose. The 1,5-AG concentration required to inhibit 2-deoxyglucose uptake exceeded that of glucose by >100 times, being much higher than the physiological concentrations of 1,5-AG. These results indicate that the 1,5-AG carrier system in insulinoma cells is distinct from that in either the somatic cells or renal tubular cells. These findings also suggest that a unique 1,5-AG transport system is present in pancreatic β-cells.     

Transport of 1,5-anhydro-D-glucitol into insulinoma cells by a glucose-sensitive transport system

The uptake of 1,5-anhydro-D-glucitol (1,5-AG) occurs by passive mechanisms in cells or tissues that have passive glucose transporters. It is known that serum 1,5-AG concentrations are reduced in patients with diabetes mellitus. To elucidate the metabolism of this substance and its physiological role in pancreatic beta-cells, we assayed 1,5-AG transport in the insulinoma-derived cell lines, RINr and MIN6. Both cell lines showed an insulin-insensitive, concentration-dependent uptake of 1,5-AG with a saturation time of approximately 120 min, and most of the 1,5-AG in the cytoplasm was in the free form. A biphasic saturation curve was obtained using a wide range of 1,5-AG concentrations, suggesting that accumulation was mediated by a high affinity and a low affinity transporter. The high affinity transporter had a K(m) of 10.4 in RINr cells and 13.0 mM in MIN6 cells, and the low affinity transporter had a K(m)100 times, being much higher than the physiological concentrations of 1,5-AG. These results indicate that the 1,5-AG carrier system in insulinoma cells is distinct from that in either the somatic cells or renal tubular cells. These findings also suggest that a unique 1,5-AG transport system is present in pancreatic beta-cells.     

Attenuation of expression of gamma-glutamylcysteine synthetase by ribozyme transfection enhance insulin secretion by pancreatic beta cell line, MIN6

Low levels of intracellular antioxidant enzyme activities as well as glutathione (GSH) concentrations have been described in pancreatic beta cells. We examined the effects of intracellular GSH depletion on insulin secretion and the role of intracellular GSH in signal transduction in beta cell line, MIN6 cells. Anti-gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit ribozyme was stably transfected to MIN6 cells to reduce intracellular GSH concentration. In the presence of 10 mM glucose, ribozyme-transfected cells (RTC) increased insulin secretion from 0.58 microg/10(6) cells/h in control cells (CC) to 1.48 microg/10(6) cells/h. This was associated with increased intracellular Ca(2+) concentration in RTC, detected by fluo-3 staining. Our results demonstrated that intracellular GSH concentration might influence insulin secretion by MIN6 cells, and suggest that enhanced insulin secretion by beta cells conditioned by chronic depletion of GSH is mediated by increased intracellular Ca(2+) concentration.     

Islet amyloid polypeptide inhibits glucagon release and exerts a dual action on insulin release from isolated islets

We have studied the influence of a wide concentration range of islet amyloid polypeptide (IAPP) on both glucagon and insulin release stimulated by various types of secretagogues. In an islet incubation medium devoid of glucose, the rate of glucagon release being high, we observed a marked suppressive action by low concentrations of IAPP, 10(-10) and 10(-8) M, on glucagon release. Similarly, glucagon release stimulated by L-arginine, the cholinergic agonist carbachol, or the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX), an activator of the cyclic AMP system, was inhibited by IAPP in the 10(-10) and 10(-8) M concentration range. Moreover, basal glucagon release at 7 and 10 mM glucose was suppressed by IAPP. In contrast, IAPP exerted a dual action on insulin release. Hence, low concentrations of IAPP brought about a modest increase of basal insulin secretion at 7 mM glucose and also of insulin release stimulated by carbachol. High concentrations of IAPP, however, inhibited insulin release stimulated by glucose (10 and 16.7 mM), IBMX, carbachol and L-arginine. In conclusion, our data suggest that IAPP has complex effects on islet hormone secretion serving as an inhibitor of glucagon release and having a dual action on insulin secretion exerting mainly a negative feedback on stimulated and a positive feedback on basal insulin release.     

The surface receptor is involved in annexin I-stimulated insulin secretion in MIN6N8a cells

This study investigated the effect of extracellular annexin I on regulating insulin secretion in MIN6N8a (an insulin secreting cell line) cells. The properties of annexin I receptor in MIN6N8a cells were also determined. Annexin I stimulated insulin release in MIN6N8a cells, regardless of the presence or absence of extracellular Ca(2+). Confocal microscopy revealed that annexin I bound to the surface of MIN6N8a cells. In addition, FACs analysis showed that annexin I bound to the surface of MIN6N8a cells in a dose-dependent manner. However, the annexin I-stimulated insulin secretion and the annexin I binding were abolished in MIN6N8a cells treated with proteases. Annexin I receptors were regenerated time-dependently. Furthermore, annexin I-stimulated insulin secretion was inhibited by cycloheximide but not by actinomycin D. These results showed that annexin I binds to the surface receptor in order to regulate the stimulation of insulin release in MIN6N8a cells.     

Autocrine anti-apoptotic and proliferative effects of insulin in pancreatic beta-cells

Insulin and glucose inhibited apoptosis in the MIN6 insulin-secreting cell line. The protective effect of 25 mM glucose was prevented by an anti-insulin antibody and this antibody-induced increase in apoptosis was reversed by the presence of excess insulin. Glucose stimulated MIN6 cell proliferation and this was inhibited by blockade of insulin secretion, by an anti-insulin antibody and by phosphatidylinositol-3 kinase (PI-3K) inhibition. Furthermore, MIN6 cell proliferation was stimulated by depolarising concentrations of KCl and by insulin itself. These data indicate that insulin secreted by β-cells in response to elevated glucose exerts autocrine effects to protect against apoptosis and stimulate proliferation, and suggest that the insulin signalling cascade, through the PI-3K pathway, may be an effective means of maintaining β-cell mass in diabetes.     

Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets

Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.     

Testosterone rapidly stimulates insulin release from isolated pancreatic islets through a non-genomic dependent mechanism.

The action of testosterone on the 45Ca2+ uptake and insulin secretion was studied in short-term experiments using isolated pancreatic islets of Langerhans. Testosterone (1 microM) stimulated 45Ca2+ uptake within 60 seconds of incubation on similar proportion than tolbutamide. Also, the hormone rapidly increased insulin release (34%; 180 seconds) on the presence of non-stimulatory concentrations of glucose (3 mM). Impermeant testosterone-BSA significantly stimulated the secretion of insulin to a lower percentage (10%). The action of the hormone is specific--neither 17beta-E2 nor progesterone stimulated insulin secretion in the presence of 3 mM glucose. The action of testosterone on insulin secretion was dose-dependent, and at rat plasma physiological concentrations (25 nM), stimulus was 17% (p < 0.05). In conclusion, in isolated pancreatic islets experiments, physiological concentration of testosterone rapidly stimulate insulin secretion and 45Ca2+ uptake through a membrane bound mechanism.     

High passage MIN6 cells have impaired insulin secretion with impaired glucose and lipid oxidation

Type 2 diabetes is a metabolic disorder characterized by the inability of beta-cells to secrete enough insulin to maintain glucose homeostasis. MIN6 cells secrete insulin in response to glucose and other secretagogues, but high passage (HP) MIN6 cells lose their ability to secrete insulin in response to glucose. We hypothesized that metabolism of glucose and lipids were defective in HP MIN6 cells causing impaired glucose stimulated insulin secretion (GSIS). HP MIN6 cells had no first phase and impaired second phase GSIS indicative of global functional impairment. This was coupled with a markedly reduced ATP content at basal and glucose stimulated states. Glucose uptake and oxidation were higher at basal glucose but ATP content failed to increase with glucose. HP MIN6 cells had decreased basal lipid oxidation. This was accompanied by reduced expressions of Glut1, Gck, Pfk, Srebp1c, Ucp2, Sirt3, Nampt. MIN6 cells represent an important model of beta cells which, as passage numbers increased lost first phase but retained partial second phase GSIS, similar to patients early in type 2 diabetes onset. We believe a number of gene expression changes occurred to produce this defect, with emphasis on Sirt3 and Nampt, two genes that have been implicated in maintenance of glucose homeostasis.     

Overexpression of lactate dehydrogenase A attenuates glucose-induced insulin secretion in stable MIN-6 β-cell lines

Since islet β-cells express little

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-lactate dehydrogenase (LDH) activity, we have examined the effects on these cells of LDH overexpression. In mock-transfected MIN6 β-cells, LDH activity was 38 nmol/min/mg protein, and 30 mM glucose stimulated secretion 10.4-fold. In two MIN6 cell clones stably overexpressing human LDH-A cDNA, insulin secretion was stimulated only 2.7- and 2.1-fold by high glucose. K⁺-stimulated insulin secretion was unaffected, and leucine stimulation enhanced, by LDH-A overexpression. LDH-A-overexpressing clones displayed unaltered activities of hexokinase, glucokinase, and malate dehydrogenase, slightly elevated plasma membrane lactate transport activity, and lowered insulin content. Low LDH activity would therefore appear important in β-cell glucose sensing.     

Annexin XI may be involved in Ca2+ - or GTP-gammaS-induced insulin secretion in the pancreatic beta-cell

The aim of this study was to investigate possible involvement of annexin XI in the insulin secretory machinery. In fluorescence immunocytochemistry, annexin XI was found in the cytoplasm of pancreatic endocrine cells and a pancreatic beta-cell line, MIN6, in a granular pattern. MIN6 cells also possessed weak and diffused annexin XI immunoreactivity in the cytoplasm. Immunoelectron microscopy revealed annexin XI in the insulin granules. Insulin secretion from streptolysin-O-permeabilized MIN6 cells was inhibited by anti-annexin XI antibody, when the release was stimulated by either Ca2+ or GTP-gammaS, but not by a protein kinase C-activating phorbol ester. Inhibition of insulin release by anti-annexin XI antibody was reproduced in permeabilized rat islets. These findings suggest that annexin XI may be involved in the regulation of insulin secretion from the pancreatic beta-cells.     

Characteristics of the biotin enhancement of glucose-induced insulin release in pancreatic islets of the rat

Perifused isolated rat islets were used to show that biotin plus 16.5 mM glucose evoked more insulin secretion than 16.5 mM glucose alone. Whether or not this reinforcement of glucose-induced insulin secretion by biotin is unique was studied by using perifused islets stimulated with 16.5 mM glucose plus 100 microM of one of various components of the vitamin B group. No effect of any of these vitamins was found on glucose-induced insulin secretion. These results indicate that biotin is unique among the members of the vitamin B group in enhancing glucose-induced insulin secretion. Static incubation experiments showed that biotin did not potentiate insulin release when the islets were incubated with an experimental solution containing either no or 2.8 mM glucose. The addition of biotin to 27.7 mM glucose, which is the maximal concentration for stimulating insulin release, did not significantly enhance the effect of the glucose on insulin release (although it did at 16.5 mM glucose). These findings indicate that biotin, by itself, does not stimulate insulin secretion, and does not enhance glucose-induced insulin secretion beyond the ability of glucose itself to stimulate insulin secretion.     

Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic beta-cells

Phospholipase D (PLD) has been strongly implicated in the regulation of Golgi trafficking as well as endocytosis and exocytosis. Our aim was to investigate the role of PLD in regulating the biphasic exocytosis of insulin from pancreatic beta-cells that is essential for mammalian glucose homeostasis. We observed that PLD activity in MIN6 pancreatic beta-cells is closely coupled to secretion. Cellular PLD activity was increased in response to a variety of secretagogues including the nutrient glucose and the cholinergic receptor agonist carbamoylcholine. Conversely, pharmacological or hormonal inhibition of stimulated secretion reduced PLD activity. Most importantly, blockade of PLD-catalyzed phosphatidic acid formation using butan-1-ol inhibited insulin secretion in both MIN6 cells and isolated pancreatic islets. It was further established that PLD activity was required for both the first and the second phase of glucose-stimulated insulin release, suggesting a role in the very distal steps of exocytosis, beyond granule recruitment into a readily releasable pool. Visualization of granules using green fluorescent protein-phogrin confirmed a requirement for PLD prior to granule fusion with the plasma membrane. PLD1 was shown to be the predominant isoform in MIN6 cells, and it was located at least partially on insulin granules. Overexpression of wild-type or a dominant negative catalytically inactive mutant of PLD1 augmented or inhibited secretagogue-stimulated secretion, respectively. The results suggest that phosphatidic acid formation on the granule membrane by PLD1 is essential for the regulated secretion of insulin from pancreatic beta-cells.     

Glucose regulation of arginine-induced pancreatic somatostatin release from the isolated perfused rat pancreas

The effects of glucose alone, combinations of glucose with arginine or tolbutamide and either arginine or tolbutamide alone, on somatostatin, insulin, and glucagon secretion were investigated using the isolated perfused rat pancreas. When glucose alone was raised in graded increments at 15-min intervals from an initial concentration of 0 mM to a maximum of 16.7 mM, somatostatin as well as insulin in the perfusate increased with the glucose, while glucagon decreased. The similarity of the glucose stimulated somatostatin and insulin release was especially evident when the perfusate glucose was increased from an initial dose of 4.4 mM rather than 0 mM to 8.8 mM or 16.7 mM. In addition, glucose at concentrations varying from 4.4 mM to 11 mM dose-dependently enhanced arginine-induced somatostatin and insulin release and suppressed glucagon release dose-dependently as before. Arginine in the absence of glucose was not capable of stimulating somatostatin secretion whereas tolbutamide, in contrast, was capable of stimulating somatostatin secretion even in the absence of glucose.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets.

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumulation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5--1 microgram/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 microgram/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 microgram/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 microgram/ml). Somatostatin (1 microgram/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated. The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

Acute exposure of beta-cells to troglitazone decreases insulin hypersecretion via activating AMPK

Background

It has been recognized that insulin hypersecretion can lead to the development of insulin resistance and type 2 diabetes mellitus. There is substantial evidence demonstrating that thiazolidinediones are able to delay and prevent the progression of pancreatic β-cell dysfunction. However, the mechanism underlying the protective effect of thiazolidinediones on β-cell function remains elusive.

Methods

We synchronously detected the effects of troglitazone on insulin secretion and AMP-activated protein kinase (AMPK) activity under various conditions in isolated rat islets and MIN6 cells.

Results

Long-term exposure to high glucose stimulated insulin hypersecretion and inhibited AMPK activity in rat islets. Troglitazone-suppressed insulin hypersecretion was closely related to the activation of AMPK. This action was most prominent at the moderate concentration of glucose. Glucose-stimulated insulin secretion was decreased by long-term troglitazone treatment, but significantly increased after the drug withdrawal. Compound C, an AMPK inhibitor, reversed troglitazone-suppressed insulin secretion in MIN6 cells and rat islets. Knockdown of AMPKα2 showed a similar result. In MIN6 cells, troglitazone blocked high glucose-closed ATP-sensitive K⁺ (K_ATP) channel and decreased membrane potential, along with increased voltage-dependent potassium channel currents. Troglitazone suppressed intracellular Ca^2 + response to high glucose, which was abolished by treatment with compound C.

Conclusion

Our results suggest that troglitazone provides β-cell “a rest” through activating AMPK and inhibiting insulin hypersecretion, and thus restores its response to glucose.

General significance

These data support that AMPK activation may be an important mechanism for thiazolidinediones preserving β-cell function.     

Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion

Metformin, a drug widely used in the treatment of type 2 diabetes, has recently been shown to act on skeletal muscle and liver in part through the activation of AMP-activated protein kinase (AMPK). Whether metformin or the satiety factor leptin, which also stimulates AMPK in muscle, regulates this enzyme in pancreatic islets is unknown. We have recently shown that forced increases in AMPK activity inhibit insulin secretion from MIN6 cells (da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, and Rutter GA. Biochem J 371: 761-774, 2003). Here, we explore whether 1) glucose, metformin, or leptin regulates AMPK activity in isolated islets from rodent and human and 2) whether changes in AMPK activity modulate insulin secretion from human islets. Increases in glucose concentration from 0 to 3 and from 3 to 17 mM inhibited AMPK activity in primary islets from mouse, rat, and human, confirming previous findings in insulinoma cells. Incubation with metformin (0.2-1 mM) activated AMPK in both human islets and MIN6 beta-cells in parallel with an inhibition of insulin secretion, whereas leptin (10-100 nM) was without effect in MIN6 cells. These studies demonstrate that AMPK activity is subject to regulation by both glucose and metformin in pancreatic islets and clonal beta-cells. The inhibitory effects of metformin on insulin secretion may therefore need to be considered with respect to the use of this drug for the treatment of type 2 diabetes.     

Effects of salicylate on insulin and glucagon secretion by the isolated and perfused rat pancreas

The effects of sodium salicylate, a prostaglandin synthesis inhibitor, on glucose-induced secretion of insulin and glucagon by the isolated perfused rat pancreas have been studied. Sodium salicylate inhibited both basal (2.8 mM glucose) and stimulated (16.7 mM glucose) insulin release in a dose dependent manner (1, 5 and 10 mM). This inhibition is not interpretable in terms of a simple inhibition of cyclooxygenase by sodium salicylate. Basal glucagon release was not changed by 1 mM sodium salicylate but the latter partially blocked its inhibition by 16.7 mM glucose. Higher doses of sodium salicylate (5 and 10 mM) inhibited basal glucagon secretion without affecting its response to 16.7 mM glucose. These findings suggest a predominant stimulatory action of endogenous prostaglandins on glucagon release.     

Synchronization of Ca(2+)-signals within insulin-secreting pseudoislets: effects of gap-junctional uncouplers

The secretory response of the intact islet is greater than the response of individual beta-cells in isolation, and functional coupling between cells is critical in insulin release. The changes in intracellular Ca(2+)([Ca(2+)](i)) which initiate insulin secretory responses are synchronized between groups of cells within the islet, and gap-junctions are thought to play a central role in coordinating signalling events. We have used the MIN6 insulin-secreting cell line, to examine whether uncoupling gap-junctions alters the synchronicity of nutrient- and non-nutrient-evoked Ca(2+)oscillations, or affects insulin secretion. MIN6 cells express mRNA species that can be amplified using PCR primers for connexin 36. A commonly used gap-junctional inhibitor, heptanol, inhibited glucose- and tolbutamide-induced Ca(2+)-oscillations to basal levels in MIN6 cell clusters at concentrations of 0.5 mM and greater, and it had similar effects in pseudoislets when used at 2.5 mM. Lower heptanol concentrations altered the frequency of Ca(2+)transients without affecting their synchronicity, in both monolayers and pseudoislets. Heptanol also had effects on insulin secretion from MIN6 pseudoislets such that 1 mM enhanced secretion while 2.5 mM was inhibitory. These data suggest that heptanol has multiple effects in pancreatic beta-cells, none of which appears to be related to uncoupling of synchronicity of Ca(2+)signalling between cells. A second gap-junction uncoupler, 18 alpha-glycyrrhetinic acid, also failed to uncouple synchronized Ca(2+)-oscillations, and it had no effect on insulin secretion. These data provide evidence that Ca(2+)signalling events occur simultaneously across the bulk mass of the pseudoislet, and suggest that gap-junctions are not required to coordinate the synchronicity of these events, nor is communication via gap junctions essential for integrated insulin secretory responses.