Pancreatic Beta-cell Line MIN6 Research Articles

Transplantable cell-encapsulation device using a semipermeable ethylene-vinyl alcohol copolymer membrane in a mouse diabetic model

Cell-based therapy is an attractive approach, and encapsulation of therapeutic cells is a promising strategy because it prevents immune responses and allows transplanted cells to be retrieved in case of dysfunction. Bioartificial pancreas, in which insulin-secreting cells are encapsulated in a semipermeable membrane bag, is a new class of medical device for treating type-I diabetes. In this study, we developed a macroencapsulation device in which the pancreatic beta cell line MIN6 was encapsulated in a semipermeable bag made of an ethylene-vinyl alcohol copolymer membrane. Invitro evaluation of ATP and insulin levels revealed that MIN6 cells grown in Matrigelwithin the device secreted insulin in response to glucose levels. Transplantation of the device lowered blood glucose levels for 30 days in diabetic mice. Histological observation revealed that MIN6 cells formed spheroids in Matrigel, and no host cells were detected within the device. Blood levels of inflammatory cytokines in the transplanted mice were similar to those in non-transplanted mice, and antibody levels in the device were lower than those in the intraperitoneal fluid. These results suggest that the semipermeable ethylene-vinyl alcohol copolymer membrane developed in this study is useful for cell encapsulation in cell-based therapies, including beta-cell macroencapsulation for type-1 diabetes.

Jazf1 acts as a regulator of insulin-producing β-cell differentiation in induced pluripotent stem cells and glucose homeostasis in mice.

Genetic susceptibility of type 2 diabetes and Juxtaposed with another zinc finger protein 1 (Jazf1) has been reported; however, the precise role of Jazf1 in metabolic processes remains elusive. In this study, using Jazf1-knockout (KO)-induced pluripotent stem cells (iPSC), pancreatic beta cell line MIN6 cells, and Jazf-1 heterozygous KO (Jazf1+/- ) mice, the effect of Jazf1 on gradual differentiation was investigated. We checked the alterations of the genes related with β-cell specification, maturation, and insulin release against glucose treatment by the gain and loss of the Jazf1 gene in the MIN6 cells. Because undifferentiated Jazf1-KO iPSC were not significantly different from wild-type (WT) iPSC, the size and endoderm marker expression after embryoid body (EB) and teratoma formation were investigated. Compared to EB and teratomas formed with WT iPSC, the EB and teratomas from with Jazf1-KO iPSC were smaller, and in teratomas, the expression of proliferation markers was reduced. Moreover, the expression of the gene sets for β-cell differentiation and the levels of insulin and C-peptide secreted by insulin precursor cells were notably reduced in β-cells differentiated from Jazf1-KO iPSC compared with those differentiated from WT iPSC. A comparison of Jazf1+/- and WT mice showed that Jazf1+/- mice had lower levels of serum insulin, pancreatic insulin expression, and decreased pancreatic β-cell size, which resulted in defects in the glucose homeostasis. These findings suggest that Jazf1 plays a pivotal role in the differentiation of β-cells and glucose homeostasis.

Branched-chain amino acid supplementation restores reduced insulinotropic activity of a low-protein diet through the vagus nerve in rats

BackgroundPreviously, we reported that a low-protein diet significantly reduced insulin secretion in response to feeding within 1 h in rats, suggesting that the insulinotropic effect of dietary protein plays an important role in maintaining normal insulin release. The current study aimed to elucidate whether deficiency of certain amino acids could diminish the insulinotropic activity and to investigate whether reduced insulin secretion in response to a low-protein diet is restored by supplementation with certain amino acids.MethodsFirst, we fed male Wistar rats (5–6 rats per group) with diets deficient in every single amino acid or three branched-chain amino acids (BCAAs); within 1–2 h after the onset of feeding, we measured the plasma insulin levels by using an enzyme-linked immunosorbent assay (ELISA). As insulin secretion was reduced in BCAA-deficient groups, we fed low-protein diets supplemented with BCAAs to assess whether the reduced insulin secretion was restored. In addition, we treated the pancreatic beta cell line MIN6 with BCAAs to investigate the direct insulinotropic activity on beta cells. Lastly, we investigated the effect of the three BCAAs on sham-operated or vagotomized rats to assess involvement of the vagus nerve in restoration of the insulinotropic activity.ResultsFeeding a low-protein diet reduced essential amino acid concentrations in the plasma during an absorptive state, suggesting that reduced plasma amino acid levels can be an initial signal of protein deficiency. In normal rats, insulin secretion was reduced when leucine, valine, or three BCAAs were deficient. Insulin secretion was restored to normal levels by supplementation of the low-protein diet with three BCAAs, but not by supplementation with any single BCAA. In MIN6 cells, each BCAA alone stimulated insulin secretion but the three BCAAs did not show a synergistic stimulatory effect. The three BCAAs showed a synergistic stimulatory effect in sham-operated rats but failed to stimulate insulin secretion in vagotomized rats.ConclusionsLeucine and valine play a role in maintaining normal insulin release by directly stimulating beta cells, and supplementation with the three BCAAs is sufficient to compensate for the reduced insulinotropic activity of the low-protein diet, through the vagus nerve.

Alkaline pH induces IRR-mediated phosphorylation of IRS-1 and actin cytoskeleton remodeling in a pancreatic beta cell line

Secretion of mildly alkaline (pH 8.0–8.5) juice to intestines is one of the key functions of the pancreas. Recent reports indicate that the pancreatic duct system containing the alkaline juice may adjoin the endocrine cells of pancreatic islets. We have previously identified the insulin receptor-related receptor (IRR) that is expressed in islets as a sensor of mildly alkaline extracellular media. In this study, we show that those islet cells that are in contact with the excretory ducts are also IRR-expressing cells. We further analyzed the effects of alkaline media on pancreatic beta cell line MIN6. Activation of endogenous IRR but not of the insulin receptor was detected that could be inhibited with linsitinib. The IRR autophosphorylation correlated with pH-dependent linsitinib-sensitive activation of insulin receptor substrate 1 (IRS-1), the primary adaptor in the insulin signaling pathway. However, in contrast with insulin stimulation, no protein kinase B (Akt/PKB) phosphorylation was detected as a result of alkali treatment. We observed overexpression of several early response genes (EGR2, IER2, FOSB, EGR1 and NPAS4) upon alkali treatment of MIN6 cells but those were IRR-independent. The alkaline medium but not insulin also triggered actin cytoskeleton remodeling that was blocked by pre-incubation with linsitinib. We propose that the activation of IRR by alkali might be part of a local loop of signaling between the exocrine and endocrine parts of the pancreas where alkalinization of the juice facilitate insulin release that increases the volume of secreted juice to control its pH and bicabonate content.

Serpine1 Mediates Porphyromonas gingivalis Induced Insulin Secretion in the Pancreatic Beta Cell Line MIN6.

Periodontitis is an inflammatory disease resulting in destruction of gingiva and alveolar bone caused by an exuberant host immunological response to periodontal pathogens. Results from a number of epidemiological studies indicate a close association between diabetes and periodontitis. Results from cross-sectional studies indicate that subjects with periodontitis have a higher odds ratio of developing insulin resistance (IR). However, the mechanisms by which periodontitis influences the development of diabetes are not known. Results from our previous studies using an animal model of periodontitis suggest that periodontitis accelerates the onset of hyperinsulinemia and IR. In addition, LPS from a periodontal pathogen, Porphyromonas gingivalis (Pg), stimulates Serpine1 expression in the pancreatic beta cell line MIN6. Based on these observations, we hypothesized that a periodontal pathogen induces hyperinsulinemia and Serpine1 may be involved in this process. To test this hypothesis, we co-incubated Pg with the pancreatic beta cell line MIN6 and measured the effect on insulin secretion by MIN6 cells. We further determined the involvement of Serpine1 in insulin secretion by downregulating Serpine1 expression. Our results indicated that Pg stimulated insulin secretion by approximately 3.0 fold under normoglycemic conditions. In a hyperglycemic state, Pg increased insulin secretion by 1.5 fold. Pg significantly upregulated expression of the Serpine1 gene and this was associated with increased secretion of insulin by MIN6 cells. However, cells with downregulated Serpine1 expression were resistant to Pg stimulated insulin secretion under normoglycemic conditions. We conclude that the periodontal pathogen, Pg, induced insulin secretion by MIN6 cells and this induction was, in part, Serpine1 dependent. Thus, Serpine1 may play a pivotal role in insulin secretion during the accelerated development of hyperinsulinemia and the resulting IR in the setting of periodontitis.

Dietary gluten increases natural killer cell cytotoxicity and cytokine secretion.

Dietary gluten influences the development of type 1 diabetes in nonobese diabetic (NOD) mice and biobreeding rats, and has been shown to influence a wide range of immunological factors in the pancreas and gut. In the present study, the effects of gluten on NK cells were studied in vitro and in vivo. We demonstrated that gliadin increased direct cytotoxicity and IFN-γ secretion from murine splenocytes and NK cells toward the pancreatic beta-cell line MIN6 cells. Additionally, stimulation of MIN6 cells led to a significantly increased proportion of degranulating C57BL/6 CD107a(+) NK cells. Stimulation of C57BL/6 pancreatic islets with gliadin significantly increased secretion of IL-6 more than ninefold. In vivo, the gluten-containing diet led to a higher expression of NKG2D and CD71 on NKp46(+) cells in all lymphoid organs in BALB/c and NOD mice compared with the gluten-free diet. Collectively, our data suggest that dietary gluten increases murine NK-cell activity against pancreatic beta cells. This mechanism may contribute to development of type 1 diabetes and explain the higher disease incidence associated with gluten intake in NOD mice.

TNF-α acutely upregulates amylin expression in murine pancreatic beta cells

Amylin, a secretory protein mainly produced by pancreatic beta cells, is elevated in the circulation of patients with diseases related to acute and chronic inflammation, including acute pancreatitis, pancreas graft rejection, obesity and insulin resistance. TNF-α is involved in these disorders. We investigated the effect of TNF-α on amylin levels and the underlying mechanisms, using murine pancreatic beta cell line MIN6 and pancreatic islets. Amylin, proinsulin and prohormone convertase 1/3, 2 (Pc1/3, Pc2 [also known as Pcsk1/3 and Pcsk2, respectively]) mRNA levels, and amylin promoter and nuclear factor κB (NF-κB) activation were examined by real-time PCR and luciferase reporter assay, respectively. Amylin protein level and mitogen-activated protein kinase phosphorylation were detected by western blot. Activator protein 1 (AP1) activation was examined by electrophoretic mobility shift assay (EMSA). TNF-α acutely induced amylin expression at the transcriptional level and increased proamylin and the intermediate form of amylin in MIN6 cells and islets. However, it had no effect on proinsulin, Pc1/3 and Pc2 expression. Studies with (1) MIN6 cells treated with inhibitors of MEK1/2, c-Jun-N-terminal kinase (JNK) or protein kinase Cζ (PKC(ζ)), (2) MIN6 cells expressing a c-Jun-dominant negative construct and (3) islets from Fos knockout mice demonstrated that TNF-α induced amylin expression through the PKC(ζ)-extracellular signal-regulated kinase (ERK)/JNK pathways. EMSA showed that (PKC(ζ)), JNK and ERK1/2 were involved in TNF-α-induced AP1 activation, suggesting that TNF-α induces murine amylin expression through the (PKC(ζ)) - ERK1/2 - AP and PKC(ζ) - JNK - AP1 pathways. Further studies showed that TNF-α also induced murine amylin expression through the phosphatidylinositol 3 kinase-NF-κB signalling pathway and enhanced human amylin promoter activation through NF-κB and AP1. TNF-α acutely induces amylin gene expression in beta cells through multiple signalling pathways, possibly contributing to amylin elevation in acute inflammation-related pancreatic disorders.

Cholesterol induces mitochondrial dysfunction and apoptosis in mouse pancreatic beta-cell line MIN6 cells

Reduction of pancreatic β-cell mass is a key element leading to type 2 diabetes. Obesity and overweight with high levels of lipids including cholesterol are tightly linked to type 2 diabetes. The direct impact of cholesterol on pancreatic β-cells, however, has not been extensively studied. In this study, MIN6 mouse β-cell line was used to test the effect of cholesterol on pancreatic β-cell apoptosis over different doses and durations. It was found that cholesterol dose- and time-dependently induced cell death of MIN6 cells above 160 μM after 6 h treatment in vitro. Annexin-V staining revealed that cholesterol treatment significantly induced apoptosis in MIN6 cells. Cholesterol treatment resulted in the loss of the ability to retain Rhodamine 123, indicating mitochondrial damage in MIN6 cells. Cholesterol-induced cell apoptosis and mitochondrial damage were blocked by low-temperature condition. In addition, glutathione also protected MIN6 cells from cholesterol-induced cell death. It is concluded that high level of cholesterol induces cell apoptosis in MIN6 cells, which is in part due to mitochondrial dysfunction. We suggest that excessive uptake of cholesterol in β-cells may contribute to β-cell apoptosis and dysfunction and the deterioration of type 2 diabetes.

Multiple kinases regulate mafA expression in the pancreatic beta cell line MIN6

MafA is a basic leucine zipper transcription factor expressed within the beta cells of the pancreas and is required to maintain normal glucose homeostasis as it is involved in various aspects of beta cell biology. MafA protein levels are known to increase in response to high glucose through mechanisms that have yet to be fully characterized. We investigated whether discrete intracellular signaling events control mafA expression. We found that the general kinase inhibitor staurosporine induces mafA expression without altering the stability of the protein. Inhibition of the MAP-kinase JNK mimics the effects of staurosporine on the expression of mafA. Calmodulin kinase and calcium signaling are also important in stimulating mafA expression by high glucose. However, staurosporine, JNK, and calmodulin kinase have different effects on the induction of insulin expression. These data reveal that MafA levels are tightly controlled by the coordinated action of multiple kinase pathways.

Expression Analysis of cPLA2 Alpha Interacting TIP60 in Diabetic KKAy and Non-Diabetic C57BL Wild-Type Mice: No Impact of Transient and Stable TIP60 Overexpression on Glucose-Stimulated Insulin Secretion in Pancreatic Beta-Cells

In the present study we investigate the expression levels of cytosolic phospholipase A2 alpha (cPLA2alpha) interacting histone acetyl transferase proteins TIP60alpha and TIP60beta in non-diabetic C57BL wild-type mice and obese type 2 diabetic KKAy model mice. The aim was to test our hypothesis that TIP60 plays a regulatory role in glucose-stimulated insulin secretion from pancreatic beta-cells. Ten obese diabetic KKAy mice and ten non-diabetic C57BL mice were fed a standard chow diet. After nine weeks, islet RNA was purified and used to measure TIP60 expression. We investigated the effect of TIP60alpha and TIP60beta on glucose-stimulated insulin secretion by transient and stable overexpression in the pancreatic mouse beta-cell line MIN6 and the rat beta-cell line INS-1E. We found that non-diabetic C57BL mice and diabetic KKAy mice have the same level of both the alpha and beta splice forms of TIP60. Furthermore, we demonstrated that transient and stable expression of TIP60 in INS-1E cells affects neither glucose-stimulated insulin secretion, insulin output nor cell insulin content. Also susceptibility to developing gluco-toxicity was unaffected. TIP60 over-expression does not affect glucose stimulated insulin secretion, insulin content or abnormal beta-cell function during glucotoxicity.

Glucose-stimulated Protein Synthesis in Pancreatic β-Cells Parallels an Increase in the Availability of the Translational Ternary Complex (eIF2-GTP·Met-tRNAi) and the Dephosphorylation of eIF2α

In pancreatic beta-cells, glucose causes a rapid increase in the rate of protein synthesis. However, the mechanism by which this occurs is poorly understood. In this report, we demonstrate, in the pancreatic beta-cell line MIN6, that glucose stimulates the recruitment of ribosomes onto the mRNA, indicative of an increase in the rate of the initiation step of protein synthesis. This increase in the rate of initiation is not mediated through an increase in the availability of the initiation complex eIF4F, because glucose is unable to stimulate eIF4F assembly or, in the absence of amino acids, modulate the phosphorylation status of 4E-BP1. Moreover, in MIN6 cells and isolated islets of Langerhans, rapamycin, an inhibitor of the mammalian target of rapamycin, only partially inhibited glucose-stimulated protein synthesis. However, we show that glucose stimulates the dephosphorylation of eIF2 alpha in MIN6 cells and the assembly of the translational ternary complex, eIF2-GTP.Met-tRNAi, in both MIN6 cells and islets of Langerhans. The changes in the phosphorylation of eIF2 alpha are not mediated by the PKR-like endoplasmic reticulum eIF2 alpha kinase (PERK), because PERK is not phosphorylated at low glucose concentrations and overexpression of a dominant negative form of PERK has no significant effect on either glucose-stimulated protein synthesis or the phosphorylation of eIF2 alpha. Taken together, these results indicate that glucose-stimulated protein synthesis in pancreatic beta-cells is regulated by a mechanism largely independent of the activity of mammalian target of rapamycin, but which is likely to be dependent on the availability of the translational ternary complex, regulated by the phosphorylation status of eIF2 alpha.

GLUCOSE-STIMULATED INSULIN SECRETION FROM AN ENGINEERED HUMAN LIVER CELL LINE IS REGULATED BY ATP-SENSITIVE POTASSIUM CHANNELS (KATP) AND L-TYPE CALCIUM CHANNELS

P669 Aims: An alternative approach to the treatment of type 1 diabetes is the creation of an ‘artificial beta cell’. It has been previously shown that the stable transfection of the full length insulin cDNA into the human liver cell line HuH7 resulted in synthesis, storage and regulated release of insulin to the physiological stimulus glucose (Huh7ins cells). The aim of the present study was to determine if Huh7ins cells respond to glucose via the blockage of KATP and the activation of voltage-gated calcium channels, as is the case with pancreatic beta cells. Methods: To reveal the mechanisms underlying glucose-stimulated insulin secretion, HuH7 cells were stably transfected with insulin fused to enhanced green fluorescent protein (EGFP): HuH7-EGFPins. As a positive control the glucose responsive pancreatic beta cell line Min6 was also transfected with EGFP: Min6-EGFPins cells. The effect of 20 mM glucose and the KATP activator diazoxide (150 μM) and the blocker glibenclamide (20 μM) on acute insulin secretion from the cells lines was assessed by confocal microscopy and biochemically by static stimulation experiments. Insulin secretion was measured by radioimmunoassay. Specific primers were designed to the KATP subunits Kir 6.2 and SUR 2A together with the L-type calcium channel (D subunit) of pancreatic beta cells. RT-PCRs were perfomed on cDNA prepared from Huh7, Huh7ins, Huh7ins-EGFPins and human pancreatic beta cells (positive control). Results: Confocal microscopy revealed that Huh7ins-EGFPins and Min 6-EGFPins cells responded to a glucose stimulus via KATP. The glucose and glibenclamide responses were seen as a time-dependent loss of fluorescence as insulin granules were released by exocytosis. Cells exposed to diazoxide, accumulated brighter fluorescence. Exposure to glucose for 1 hr resulted in an increase in insulin secretion from 22.8 ± 1.5 to 52.5 ± 3.8 and glibenclamide from 19.4 ± 0.7 to 59.5 ± 3.6 μU insulin/ 106 HuH7ins cells (n=3). Diazoxide completely inhibited glucose-stimulated insulin release. Very similar results were recorded for the Huh7ins-EGFP cells. PCR analysis revealed the presence of the KATP channel subunits Kir 6.2 and SUR 2A together with the L-type calcium channel in all liver cell lines. Conclusions: These results indicate that that Huh7ins cells respond to glucose by the same mechanism seen in β cells. This provides useful information with regards to the future engineering of insulin-secreting liver cells and gives hope that the transplantation of such cells will provide a cure for Type I diabetes.

Organelle Selection Determines Agonist-specific Ca2+ Signals in Pancreatic Acinar and β Cells

How different extracellular stimuli can evoke different spatiotemporal Ca2+ signals is uncertain. We have elucidated a novel paradigm whereby different agonists use different Ca2+-storing organelles ("organelle selection") to evoke unique responses. Some agonists select the endoplasmic reticulum (ER), and others select lysosome-related (acidic) organelles, evoking spatial Ca2+ responses that mirror the organellar distribution. In pancreatic acinar cells, acetylcholine and bombesin exclusively select the ER Ca2+ store, whereas cholecystokinin additionally recruits a lysosome-related organelle. Similarly, in a pancreatic beta cell line MIN6, acetylcholine selects only the ER, whereas glucose mobilizes Ca2+ from a lysosome-related organelle. We also show that the key to organelle selection is the agonist-specific coupling messenger(s) such that the ER is selected by recruitment of inositol 1,4,5-trisphosphate (or cADP-ribose), whereas lysosome-related organelles are selected by NAADP.

Differential gene expression in well-regulated and dysregulated pancreatic beta-cell (MIN6) sublines.

To identify genes involved in regulated insulin secretion, we have established and characterized two sublines derived from the mouse pancreatic beta-cell line MIN6, designated B1 and C3. They have a similar insulin content, but differ in their secretory properties. B1 responded to glucose in a concentration- and cell confluence-dependent manner, whereas C3 did not. B1 cells were stimulated by phorbol 12-myristate 13-acetate, leucine, arginine, glibenclamide, isobutylmethylxanthine, and KCl, whereas C3 did not respond (leucine, arginine, and glibenclamide) or responded to a lesser extent (isobutylmethylxanthine, phorbol 12-myristate 13-acetate, and KCl). Although intracellular Ca(2+) rose in response to glucose in B1 but not C3 cells, KCl increased intracellular Ca(2+) in a similar manner in both sublines. GLUT-1, GLUT-2, Kir6.2, and SUR1 expression was not significantly different between B1 and C3 cells, whereas E-cadherin was more abundantly expressed in B1 cells. A more complete list of differentially expressed genes was established by suppression subtractive hybridization and high density (Affymetrix) oligonucleotide microarrays. Genes were clustered according to known or putative function. Those involved in metabolism, intracellular signaling, cytoarchitecture, and cell adhesion are of potential interest. These two sublines should be useful for identification of the genes and mechanisms involved in regulated insulin secretion of the pancreatic beta-cell.

CAMP-dependent Protein Kinase and Ca2+Influx through L-type Voltage-gated Calcium Channels Mediate Raf-independent Activation of Extracellular Regulated Kinase in Response to Glucagon-like Peptide-1 in Pancreatic β-Cells

Glucagon like peptide-1 (GLP1) is a G(s)-coupled receptor agonist that exerts multiple effects on pancreatic beta-cells, including the stimulation of insulin gene expression and secretion. In this report, we show that treatment of the mouse pancreatic beta-cell line MIN6 with GLP1 leads to the glucose-dependent activation of Erk. These effects are mimicked by forskolin, a direct activator of adenylate cyclase, and blocked by H89, an inhibitor of cAMP-dependent protein kinase. Additionally, we provide evidence that GLP1-stimulated activation of Erk requires an influx of calcium through L-type voltage-gated calcium channels and the activation of calcium/calmodulin-dependent protein kinase II. GLP1-stimulated activation of Erk is blocked by inhibitors of MEK, but GLP1 does not induce the activation of A-Raf, B-Raf, C-Raf, or Ras. Additionally, dominant negative forms of Ras(N17) and Rap1(N17) fail to block GLP1-stimulated activation of Erk. In conclusion, our results indicate that, in the presence of stimulatory concentrations of glucose, GLP1 stimulates the activation of Erk through a mechanism dependent on MEK but independent of both Raf and Ras. This requires 1) the activation of cAMP-dependent protein kinase, 2) an influx of extracellular Ca(2+) through L-type voltage-gated calcium channels, and 3) the activation of CaM kinase II.

Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.

The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic beta-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH 7.4, but not at approximately pH 5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy (5 or 10 s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50 mM KCl and 22 mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin-pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

The Rab27a/Granuphilin Complex Regulates the Exocytosis of Insulin-Containing Dense-Core Granules

Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K(+)-induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.

Insulin secretion and differential gene expression in glucose-responsive and -unresponsive MIN6 sublines.

We have established two sublines derived from the insulin-secreting mouse pancreatic beta-cell line MIN6, designated m9 and m14. m9 Cells exhibit glucose-induced insulin secretion in a concentration-dependent manner, whereas m14 cells respond poorly to glucose. In m14 cells, glucose consumption and lactate production are enhanced, and ATP production is largely through nonoxidative pathways. Moreover, lactate dehydrogenase activity is increased, and hexokinase replaces glucokinase as a glucose-phosphorylating enzyme. The ATP-sensitive K(+) channel activity and voltage-dependent calcium channel activity in m14 cells are reduced, and the resting membrane potential is significantly higher than in m9 cells. Thus, in contrast to m9, a model for beta-cells with normal insulin response, m14 is a model for beta-cells with impaired glucose-induced insulin secretion. By mRNA differential display of these sublines, we found 10 genes to be expressed at markedly different levels. These newly established MIN6 cell sublines should be useful tools in the analysis of the genetic and molecular basis of impaired glucose-induced insulin secretion.

Unresponsiveness to glibenclamide during chronic treatment induced by reduction of ATP-sensitive K+ channel activity.

The insulin response to the sulfonylurea glibenclamide was markedly impaired in pancreatic beta-cell line MIN6 cells with chronic glibenclamide treatment (MIN6-Glib). The intracellular calcium concentration increased only slightly in response to glibenclamide in MIN6-Glib. While the properties of the voltage-dependent calcium channels were not altered, the conductance of the K(ATP) channels, the primary target of glibenclamide, was significantly reduced in MIN6-Glib. The ATP-sensitive K+ (K(ATP)) channels in MIN6 cells comprise inwardly rectifying K+ channel member Kir6.2 subunits and sulfonylurea receptor (SUR) 1 subunits. MIN6 cells have both high- and low-affinity binding sites for glibenclamide. The binding affinities at these two sites were unchanged, but the maximum binding capacities at both sites were similarly increased by chronic glibenclamide treatment. Both SUR1 and Kir6.2 mRNA levels were not altered, but SUR1 protein was rather increased in MIN6-Glib. In addition, electron microscopic examination revealed a majority of the SUR1 to be present in a cluster near the plasma membrane in control MIN6, while it tends to be distributed in the cytoplasm in MIN6-Glib. These data suggest that chronic glibenclamide treatment causes the defect in acute glibenclamide-induced insulin secretion by reducing the number of functional K(ATP) channels on the plasma membrane of the beta-cells.

Long-term elevation of free fatty acids leads to delayed processing of proinsulin and prohormone convertases 2 and 3 in the pancreatic beta-cell line MIN6.

To explore the role of chronically elevated free fatty acids (FFAs) in the pathogenesis of the hyperproinsulinemia of type 2 diabetes, we have investigated the effect of FFAs on proinsulin processing and prohormone convertases PC2 and PC1/PC3 in MIN6 cells cultured in Dulbecco's modified Eagle's medium with or without 0.5 mmol/l FFA mixture (palmitic acid:oleic acid = 1:2). After 7 days of culture, the percent of proinsulin in FFA-exposed cells was increased (25.9 +/-0.3% intracellular and 75.4 +/- 1.2% in medium vs. 13.5 +/-0.2 and 56.2 +/- 4.1%, respectively, in control cells). The biosynthesis and secretion of proinsulin and insulin were analyzed by comparing the incorporation of [3H]Leu and [35S]Met. In pulse-chase studies, proinsulin-to-insulin conversion was inhibited, and proinsulin in the medium was increased by 50% after 3 h of chase, while insulin secretion was decreased by 50% after FFA exposure. Levels of cellular PC2 and PC3 analyzed by Western blotting were decreased by 23 and 15%, respectively. However, PC2, PC3, proinsulin, and 7B2 mRNA levels were not altered by FFA exposure. To test for an effect on the biosynthesis of PC2, PC3, proinsulin, and 7B2, a protein required for PC2 activation, MIN6 cells were labeled with [35S]Met for 10-15 min, followed by a prolonged chase. Most proPC2 was converted after 6 h of chase in control cells, but conversion was incomplete even after 6 h of chase in FFA-exposed MIN6 cells. Media from chase incubations showed that FFA-exposed cells secreted more proPC2 than controls. Similar inhibitory effects were noted on the processing of proPC3, proinsulin, and 7B2. In conclusion, prolonged exposure of beta-cells to FFAs may affect the biosynthesis and posttranslational processing of proinsulin, PC2, PC3, and 7B2, and thereby contribute to the hyperproinsulinemia of type 2 diabetes. The mechanism of inhibition of secretory granule processing by FFAs may be through changes in Ca2+ concentration, the pH in the secretory granules, and/or other factors that may influence the activation and function of the convertases.