Molecular and Cellular Endocrinology 387 (2014) 1–7

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Molecular and Cellular Endocrinology

journal homepage: www.elsevier .com/locate /mce

D-chiro-inositol glycan stimulates insulin secretion in pancreatic b cells q

http://dx.doi.org/10.1016/j.mce.2014.02.0040303-7207/� 2014 Elsevier Ireland Ltd. All rights reserved.

q This work was supported by the National Institute of Diabetes and Digestiveand Kidney Diseases Grant R01 DK-078049 (C.L.) and R01 NS33583 (D.A.B.).⇑ Corresponding author. Address: P.O. Box 800735, 1300 Jefferson Park Avenue,

Department of Pharmacology, Charlottesville, VA 22908, United States. Tel.: +1 434982 6752.

E-mail address: cl4xd@virginia.edu (C. Li).1 Present address: Department of Pharmacology, Vanderbilt University Medical

Center, Nashville, TN, United States.

Roman Lazarenko 1, Jessica Geisler, Douglas Bayliss, Joseph Larner, Chien Li ⇑Department of Pharmacology, University of Virginia Health System, Charlottesville, VA, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 September 2013Received in revised form 30 January 2014Accepted 7 February 2014Available online 14 February 2014

Keywords:INS-2Insulin secretionKATP

PP2CIsletMIN6

Insulin has been shown to act on pancreatic b cells to regulate its own secretion. Currently themechanism underlying this effect is unclear. INS-2, a novel inositol glycan pseudo-disaccharide contain-ing D-chiro-inositol and galactosamine, has been shown to function as an insulin mimetic and a putativeinsulin mediator. In the present study we found that INS-2 stimulates insulin secretion in MIN6 b cellsand potentiates glucose stimulated insulin secretion in isolated mouse islets. Importantly, INS-2 failedto potentiate insulin secretion induced by tolbutamide, which stimulates insulin release by closingATP sensitive potassium channels (KATP). Electrophysiological studies showed that INS-2 inhibitedsulfonylurea-sensitive KATP conductance. The effect of INS-2 on inhibiting KATP channel is mediated byprotein phosphatase 2C (PP2C), as knocking down PP2C expression in MIN6 cells by PP2C small hairpinRNA completely abolished the effect of INS-2 on KATP and consequently attenuated INS-2 induced insulinsecretion. In conclusion, the present study identifies a novel mechanism involving PP2C in regulating KATP

channel activity and consequently insulin secretion.� 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Insulin from pancreatic b cells plays a pivotal role in regulatingglucose homeostasis. Thus, it is not surprising that insulin secre-tion is tightly regulated by multiple factors and signals (Malaisse,1990; Taborsky, 2001). The release is predominantly controlledby glucose, as glucose alone can effectively stimulate insulinsecretion (Newgard and Matschinsky, 2001). In addition to glucose,a number of secretagogues including glucagon-like peptide 1(GLP-1) and gastric inhibitory peptide (GIP) stimulate insulinrelease in a glucose dependent manner (Holst and Gromada,2004; Baggio and Drucker, 2007).

Accumulating evidence has shown that intra-islet communica-tions via both autocrine and paracrine interactions also play acritical role in insulin secretion and ultimately glucose homeosta-sis. For example, glucagon secreted from pancreatic a cellspotentiates insulin secretion, while somatostatin from d cellsinhibits both insulin and glucagon release (Samols et al., 1986).In addition to these paracrine relating hormones, a number of

factors including amylin (Cooper, 2001) and urocortin 3 (Li,2003; Li et al., 2007; Blum et al., 2012) are expressed in pancreaticb cells and functionally serve as local autocrine factors in modulat-ing insulin secretion.

In addition to b cell released local factors, several lines ofevidence support the notion that insulin itself also modulates itsown secretion. Functional insulin receptors have been identifiedon pancreatic b cells (Leibiger et al., 1998). Pharmacological studieshave shown that the insulin receptor is involved in insulinsecretion (Leibiger et al., 1998; Aspinwall et al., 1999; Argoudet al., 1987). Further, mice with insulin receptor deleted in pancre-atic b cells exhibited defective insulin secretion (Kulkarni et al.,1999; Okada et al., 2007). Currently the underlying mechanismby which insulin regulates insulin secretion remains elusive.

Early studies indicated the existence of second messengers con-taining inositols and amino hexoses in insulin target tissues tomediate some of insulin’s actions (Larner et al., 1979, 2010).4-O-(2-amino-2-deoxy-b-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol, abbreviated as INS-2, was first isolated in insulin-stimulated liver as an inositol glycan with structure determinedand later synthesized as b-1,4-galactosamine pinitol, where pinitolis the 3-O-methyl ether of D-chiro-inositol (Larner et al., 2003).Functional studies demonstrated that INS-2 is insulin mimeticand insulin sensitizing (Larner et al., 2003). Similar to insulin,INS-2 can lower blood glucose dose dependently in type 2 diabeticrats (Larner et al., 2003) and can sensitize low dose insulin in low-ering hyperglycemia in type 1 diabetic rats (Brautigan et al., 2005).

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Further, INS-2 stimulates glycogen synthesis dose dependently inhepatoma cells (Larner et al., 2003), and testosterone productionin ovarian theca cells (Nestler et al., 1998). Most importantly, anINS-2 polyclonal antibody blocks both INS-2 action and insulinaction in stimulating testosterone synthesis in ovarian theca cells(Nestler et al., 1998). This result thus suggests that some cellulareffects of insulin are mediated by an extracellularly generatedINS-2 like messenger, transported intracellularly for its action(Larner et al., 2010). Both extracellular generation of inositolglycans (Alvarez et al., 1988) as well as the presence of an ATPdependent inositol glycan transporter have been reported (Alvarezet al., 1991).

In the present study, we show that INS-2 stimulates insulinsecretion in b cells and potentiates glucose-induced insulinsecretion. We then explored the possible mechanism underlyingthe insulin releasing effect of INS-2. We found that INS-2 regulatesinsulin secretion by closing of ATP sensitive potassium channels(KATP) and that this effect of INS-2 requires protein phosphatase2C (PP2C).

2. Research design and methods

2.1. Reagents

INS-2 and C-INS-2 compounds was synthesized as describedpreviously (Larner et al., 2003; Hans et al., 2010). Chemicals anddrugs were purchased from Sigma (St. Louis, MO). Cell culturereagents were purchased from Life Technologies (Grand Island,NY).

2.2. Cell culture

MIN6 cells, a mouse clonal b cell line, were cultured in high glu-cose Dulbecco’s Modified Eagle Medium (DMEM) supplementedwith 10% fetal bovine serum, 1 mM sodium pyruvate, 1% penicil-lin/streptomycin and 86 lM b-mercaptoethanol. MIN6 cells weretransduced with lentiviral particles and cultured for 72 h prior touse in experiments. All cell culture reagents were purchased fromInvitrogen.

2.3. Islet isolation

Islets were isolated from adult male C57BL/6J mice (agedbetween 15 and 18 weeks) purchased from Jackson Laboratory(Bar Harbor, ME) via intraductal collagenase digestion and densitycentrifugation as described previously (Carter et al., 2009). Afterisolation, islets were hand-picked, size-matched and maintainedin RPMI media supplemented with 10% (v/v) FBS and 1%penicillin/streptomycin. Animal procedures were approved by theUniversity of Virginia Animal Care and Use Committee.

2.4. Lentiviral vector production

A sequence to knockdown mouse PP2C expression wasdesigned based on earlier reports (Sathe et al., 2011). A lentiviralvector expressing small hairpin RNA (shRNA) against mousePP2C was generated as previously described (Chao et al., 2012).Briefly, a PP2C shRNA expressing cassette driven by a U6 promoterwas constructed and cloned into a lentiviral packaging vector(p156RRLsinPPtCMV-GFP-PREU3Nhe, kindly provided by Dr. InderVerma, The Salk Institute). The viral vector expresses green fluores-cent protein (GFP) under the control of a CMV promoter, allowingvisual identification of cells infected by the viral vector. The shRNAPP2C vector and lentiviral packaging plasmids were co-transfectedinto HEK239FT cells. After transfection, the supernatant containing

viral particles was harvested, clarified, and concentrated by centri-fugation. In addition to the shRNA PP2C lentiviral vectors, a controlvector expressing a non-coding scrambled sequence was also gen-erated using the same method.

2.5. Insulin secretion

Ten islets were incubated in 250 lL of Kreb-Ringer bicarbonateHEPES buffer (KRB) consisting of the following in mM: NaCl (129),KCl (4.8), CaCl2 (2.5), MgCl2 (1.2), NaH2PO4 (1.2), NaHCO3 (5),HEPES (10) and 0.1% BSA per well in a 48 well plate or 80,000MIN6 cells were seeded per well in a 48 well plate. Both the cellsand islets were equilibrated for 15 min at 37 �C in KRB buffer.The pre-incubation was followed immediately by a 30 minpretreatment with testing agents then stimulated with highglucose (16.8 mM) for 60 min. Insulin concentrations in KRB bufferwere determined with an insulin ELISA kit (Millipore). Insulinsecretion assays were performed in triplicate and insulin ELISAswere averaged in duplicate.

2.6. Electrophysiology

Whole cell voltage clamp recordings were obtained from MIN6cells on a fluorescence microscope (Zeiss Axioskop FS) usingpCLAMP software interfaced with an Axopatch 200B amplifier viaa Digidata 1322A digitizer (all from Molecular Devices, Foster City,CA). In some cases, cells were infected either with noncodingscramble or PP2C shRNA viral constructs and identified by GFPfluorescence. All recordings were performed at room temperature.Pipettes were pulled from borosilicate glass capillaries to a DCresistance ranging from 3 to 5 MX and coated with Sylgard 184(Dow Corning, Midland, MA). The pipette solution contained (inmM): 120 KCH3SO3, 4 NaCl, 1 MgCl2, 0.5 CaCl2, 10 HEPES Na, 10EGTA (pH 7.2 adjusted by KOH). The bath solution contained (inmM): 140 NaCl, 3 KCl, 10 HEPES Na, 5 glucose, 2 CaCl2, and 2 MgCl2

(pH 7.3, adjusted by NaOH). ATP was omitted from the pipettesolution and glucose was omitted from bath solution to induceactivation of KATP channels. INS-2 and/or Glibenclamide were ap-plied at 10 lM concentration where indicated. Series resistancewas compensated (60–75%) and monitored throughout the record-ings to ensure adequate compensation. Cells were held at �60 mV,and hyperpolarizing ramps (70 mV/s, from �60 to �130 mV) wereapplied at 10 s intervals. For analysis, slope conductance (G) wasevaluated by linear fits to currents over the membrane potentialrange from �70 to �100 mV.

2.7. Statistics

Results are expressed as the mean ± SEM. Statistical analyseswere performed using repeated measures, two-way ANOVA withPost Hoc tests or Student’s t-tests when appropriate using Prism5 software (GraphPad).

3. Results

3.1. INS-2 stimulates insulin secretion in b cells

To investigate the effect of INS-2 on insulin secretion, MIN6cells were treated with various concentrations of INS-2 and insulinlevels in the media were determined by ELISA. As shown in Fig. 1A,INS-2 stimulates insulin secretion in MIN6 cells in a dose depen-dent manner under low glucose conditions. Importantly, INS-2potentiates glucose-stimulated insulin secretion (GSIS; Fig. 1B).The effect of INS-2 on insulin secretion was also determined usingisolated mouse islets. Similar to MIN6 cells, INS-2 potentiated GSIS

Fig. 1. INS-2 stimulates insulin release from pancreatic b cells. (A) MIN6 cells were incubated with varying concentrations of INS-2 (0.1–100 lM) or tolbutamide (Tolb;200 lM) in 2.8 mM glucose for 90 min and insulin concentrations in media determined by ELISA. (B) Insulin secretion in MIN6 cells pre-treated with INS-2 (10 lM) for 15 minbefore stimulated with basal glucose (2.8 mM) or high glucose (16.8 mM) for 90 min. Groups with different letters are significantly different (P < 0.05). (C) Insulin secretion inislets (10) treated with INS-2 (10 lM) for 15 min before stimulation with either 2.8 or 16.8 mM glucose for 60 min. (D) Insulin secretion in MIN6 cells treated with INS-2(10 lM) alone, tolbutamide (Tolb; 200 lM) alone and a combination of the two compounds in the presence of low glucose (2.8 mM). ⁄⁄P < 0.01, ⁄P < 0.05 vs. control.

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in isolated mouse islets (Fig. 1C); however, in contrast to MIN6cells, INS-2 had a mild effect on insulin release in 2.8 mM glucoseconditions (Fig. 1C).

3.2. Effect of sulfonylurea receptor agonist on INS-2-induced insulinsecretion

To investigate the possible mechanism underlying the effect ofINS-2 stimulated insulin release, we treated MIN6 cells withtolbutamide, a sulfonylurea receptor (SUR) agonist, with or with-out INS-2. As shown in Fig. 1D, INS-2 and tolbutamide alone greatlystimulated insulin secretion. Interestingly, pre-treating cells withINS-2 failed to further potentiate tolbutamide-induced insulinsecretion. The mechanism by which tolbutamide stimulates insulinsecretion is that tolbutamide binds and closes ATP-sensitivepotassium channels (KATP) (Doyle and Egan, 2003; Gribble andReimann, 2003). The lack of additional insulin releasing effect ofINS-2 in the presence of tolbutamide suggests either that the insu-lin release mechanism was saturated or that INS-2 may regulateinsulin secretion via a mechanism involving KATP channels.

3.3. INS-2 inhibits KATP channels in pancreatic ß cells

In order to test directly if INS-2 acts via inhibition of KATP chan-nels, we recorded from MIN6 cells using whole cell voltage clamps(Fig. 2). After establishing the whole-cell configuration at �60 mVwith an ATP-free pipette solution, we always observed a gradualincrease in outward holding current; this time-dependent currentwas attributed to activation of a KATP conductance since it wasabrogated in the presence of glibenclamide (Fig. 2). The glibencla-mide-sensitive outward current typically saturated at themaximum level within 6 min. Once a stable peak current wasattained, we examined the effects of INS2 and glibenclamide ineither order of application. When applied first, both INS2

(Fig. 2A) and glibenclamide (Fig. 2C) inhibited conductance rapidly(in <30 s) and strongly (72.1 ± 3.6% and 63 ± 4.7%, respectively).After INS2-induced block, glibenclamide had only modest effectson the residual current and, correspondingly, after glibenclamide-induced block, INS-2 had little further effect. In addition, the I–Vcharacteristics of INS-2- and glibenclamide-sensitive currents wereessentially identical (Fig. 2B and D and insets). These data indicatethat INS-2 inhibits glibenclamide-sensitive KATP channels in MIN6cells.

3.4. Phosphoprotein phosphatase receptor is involved in INS-2stimulated insulin secretion

It has been determined that INS-2 interacts with at least twoprominent protein targets: protein phosphatase 2C (PP2C) andpyruvate dehydrogenase phosphatase-1 (PDHP-1) (Larner et al.,2003; Brautigan et al., 2005). C-INS-2, a carbon bridge analog ofINS-2, has been shown to retain activity on PDHP-1, but wasinactive on PP2C (Hans et al., 2010). To investigate the possibledownstream target of INS-2 in insulin secretion, we treated MIN6cells with C-INS-2. As shown in Fig. 3, in contrast to INS-2(100 lM) which again stimulated insulin secretion, C-INS-2 atthe same concentration was completely inactive. The above resultssuggest that INS-2 stimulation of insulin secretion is not due toeffects on PDHP-1, and that PP2C may be the relevant target forINS-2 action.

To establish more directly that PP2C is a target of INS-2 ininsulin secretion, a lentiviral vector expressing small hairpin RNA(shRNA) against mouse PP2C was constructed to knock downexpression of PP2C in MIN6 cells (Fig. 4). We found that INS-2failed to induce insulin secretion in MIN6 cells infected withPP2C shRNA lentiviral vector compared to cells infected withcontrol noncoding viral vector (Fig. 4). Thus, PP2C is establishedas a target of INS-2 action to stimulate insulin secretion in MIN6

Fig. 2. INS-2 inhibits outward current through ATP-sensitive potassium channels (KATP) in pancreatic b cells. (A and C) Representative time-series of conductance derivedfrom slopes of the corresponding I-V curves (in B and D, between �70 and �110 mV); glibenclamide and INS-2 were applied as indicated. (B and D) Representative I–Vrelationships of currents recorded in response to hyperpolarizing ramps before drug exposure (black), during exposure to the first drug (INS-2 in B, glibenclamide in D, darkgrey) and after exposure to both drugs (glibenclamide + INS-2, light grey). Insets show averaged I–V-relationship of INS-2-sensitive current (in B) and glibenclamide-sensitivecurrent (in D, n = 6 for each). (E and F) Averaged inhibition of conductance by initial applications of INS-2 (72.1 ± 3.6%, n = 6) and glibenclamide (Glib; 63 ± 4.7%, n = 6). Notethat INS-2 and glibenclamide had similar inhibitory effects on KATP currents.

Fig. 3. Insulin secretion in MIN6 cells treated with basal, 16.8 mM glucose, INS-2(100 lM) or C-INS-2 (100 lM) for 90 min. ⁄P < 0.05 vs. 2.8 mM G.

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cells. Notably, tolbutamide retained the ability to induce insulinsecretion in PP2C-depleted cells, suggesting that loss of PP2C didnot directly affect KATP channel function and that PP2C is an inter-mediary in the signaling pathway from INS-2 to KATP channels.

3.5. INS-2 induced block of KATP channel requires PP2C

To further test this proposed role for PP2C in INS-2-mediatedKATP channel inhibition, we examined KATP channel activity inMIN6 cells treated with PP2C shRNA viral vector. As shown inFig. 5, and consistent with our earlier results in uninfected cells,INS-2 potently blocked �80% of KATP current in cells infected withcontrol viral vector (Fig. 5A and C). However, in cells infected withPP2C shRNA virus (Fig. 5B and D), INS2 was unable to inhibit KATP

current (decreased by only �4%) which could subsequently benearly completely blocked by glibenclamide. Thus, INS-2-inducedblock of KATP channels, similar to INS-2-evoked insulin secretion,requires PP2C.

Fig. 4. Abrogating PP2C in b cells attenuated INS-2 induced insulin secretion. (A)PP2C mRNA levels in MIN6 cells infected with lentiviral vector that expresses PP2CshRNA or noncoding scramble sequence (Control). (B) Insulin secretion in MIN6cells infected with lentiviral vector expression scrambled control or PP2C shRNAbefore stimulation with testing agents. ⁄P < 0.05.

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4. Discussion

The ability of insulin to regulate its own secretion has beenproposed (Kulkarni et al., 1999; Bouche et al., 2010; Leibigeret al., 2001) but a mechanism has not been established. Thepresent study demonstrates that INS-2, an insulin mimetic andputative second messenger, stimulates insulin secretion in bothMIN6 cells and isolated mouse islets. Further, we found INS-2failed to potentiate insulin secretion induced by Tolbutamide,which stimulates insulin release from b cells by closing KATP

channels (Doyle and Egan, 2003). This result argues that INS-2and tolbutamide share a similar mechanism in regulating insulinsecretion. Consistent with this notion, our electrophysiologicaldata showed that INS-2 inhibits KATP channels. Importantly, gliben-clamide, a second generation sulfonylurea, had no effect on currentwhen cells were first treated with INS-2 and vice versa. Theseresults further support our hypothesis that INS-2 stimulates insu-lin secretion, at least in part, by regulating KATP channel activity.

A key question is then how INS-2 regulates KATP channel activ-ity. Earlier studies have identified at least two targets of INS-2:PP2C and PDHP-1 (Larner et al., 2003; Brautigan et al., 2005). Theyboth contain potential allosteric binding sites for INS-2 adjacent tothe catalytic sites using in silico studies based on X-ray crystalstructures of the enzymes (Brautigan et al., 2005; Hans et al.,2010). We have further shown that an acidic amino acid is requiredfor allosteric binding of INS-2 in each enzyme; aspartic acid atposition 243 (Brautigan et al., 2005) in PP2C and glutamic acid atposition 351in PDHP-1 (Hans et al., 2010). The current study

showed that C-INS-2, a modified carbon bridge analog of INS-2(Hans et al., 2010), had no effect in insulin secretion. As C-INS-2 re-tains activity on PDHP-1, but is inactive on PP2C (Hans et al., 2010),these results argues against a role for PDHP-1 and suggests that INS-2 likely targets PP2C in b cells in regulating insulin secretion.Moreover, Yoshizaki and co-workers have determined that PP2C isinvolved in mediating the effect of insulin in fat cells (Yoshizakiet al., 2004). They found that PP2C promotes insulin action in adipo-cytes by dephosphorylating the p85 regulatory subunit of PI3 kinase(PI3K), thus facilitating the dissociation of the regulatory subunitfrom p110 catalytic subunit (Yoshizaki et al., 2004). Taken togetherwe postulate that INS-2 regulates KATP activity through PP2C.

Consistent with our hypothesis, suppressing PP2C expression inMIN6 cells by shRNA against PP2C effectively abrogated the effectof INS-2 in insulin secretion, confirming that PP2C is involved inINS-2 stimulated insulin release. Our electrophysiological studyfurther indicates that in PP2C knockdown MIN6 cells, INS-2 failedto modify KATP channel activity while, interestingly, glibenclamideremained effective in closing KATP. This result suggests that: (1)INS-2 must interact at a site distinct from the drug binding siteon the SUR subunit of the KATP channel complex, and (2) PP2C isnot required for glibenclamide binding to SUR. Our study also indi-cates that INS-2 regulates KATP channel activity through PP2C butnot SUR, or at least not on sulfonylurea binding sites (Doyle andEgan, 2003; Gribble and Reimann, 2003). Several protein phospha-tases including PP2A and PP2B have been found in b cells (Jonesand Persaud, 1998). On the other hand, there are no reports ofdetection of PP2C in b cells or islets. The present study shows forthe first time that PP2C is expressed in MIN6 cells and is involvedin regulating insulin secretion. Thus, the present study identifies anovel mechanism involving PP2C in regulating KATP channel activ-ity and consequently insulin secretion.

Currently it remains to be determined as to how PP2C regulatesKATP channel activity. PP2C has been shown to directly bind andregulate Ca2+ channels in neurons (Li et al., 2005). Flajolet andco-workers have also shown that PP2C binds and dephosphoryl-ates metabotropic glutamate receptors (Flajolet et al., 2003). Thus,it is conceivable that PP2C interacts directly with the KATP channelto modulate the channel activity by dephosphorylation of keyresidues on the channel. Both the ATP inhibited pore-forming K+

channel as well as the SUR have been reported to have kinase phos-phorylation sites (Chang et al., 2009; Lin et al., 2000). Of interest,both serine as well as threonine sites have been identified. Threo-nine is known as the preferred substrate for PP2C (Donella Deanaet al., 1990). Further experiments are needed to identify the site(s)dephosphorylated by PP2C via activation by INS-2 and the mecha-nism of increased insulin secretion. Clearly this work defines anovel mechanism of inositol glycan stimulated insulin secretion.

In the present study, we show that INS-2 stimulates insulinsecretion in MIN6 cells under basal glucose conditions. On theother hand, in isolated mouse islets the compound potentiatesGSIS without significant effect on insulin release under basal con-ditions. The discrepancy may be due to a number of issues includ-ing differences in levels of PP2C or KATP between MIN6 cells andmouse islets and may be of interest to explore. It is also notewor-thy that INS-2 potentiates GSIS in both MIN6 cells and isolatedmouse islets, suggesting the mechanism underlying INS-2 actionis still operational under high glucose conditions. Glucose inducesinsulin secretion by elevating intracellular ATP levels, which thenin turn closes KATP channels (Cook and Hales, 1984) to open voltagedependent calcium channels (Meglasson and Matschinsky, 1986;Wollheim and Sharp, 1981) and stimulates extracellular purinergicreceptors to promote insulin secretion (Petit et al., 2009; Geisleret al., 2013). It is not immediately clear as to how INS-2 remainseffective in facilitating insulin release in the face of elevated ATPlevels. It is possible that elevated ATP in cells does not bind and

Fig. 5. INS-2 block of KATP channel requires PP2C. (A and B) Averaged I–V curves of INS-2- and glibenclamide (Glib)-sensitive currents recorded in MIN6 cells infected withcontrol viral construct (A, n = 7) or infected with a PP2C shRNA construct (B, n = 7). (C) In cells infected with control virus, INS-2 blocked nearly all of the KATP current(82.0 ± 3.2%); after INS-2, glibenclamide had little further effect (15.3 ± 3.3%, n = 7). (D) In cells infected with PP2C shRNA, INS-2 was ineffective at inhibiting KATP current(4.0 ± 4.2%) which could be subsequently blocked by glibenclamide (92.4 ± 4.8%, n = 7).

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close all the KATP channels in b cells, thus allowing INS-2 toparticipate in closing additional channels. It is also possible thatin addition to KATP, INS-2 and/or PP2C acts on as yet unidentifiedtargets to regulate insulin secretion. More studies are needed tofurther resolve this issue.

In conclusion, we have determined that INS-2, an insulinmimetic and possible second messenger, stimulates insulin sectionand potentiates glucose induced insulin secretion. The mechanismby which inositol glycans stimulates insulin secretion is due, atleast in part, to the closure of KATP channels in a PP2C dependentmanner.

Acknowledgements

We thank David Lin and Michael Digruccio for providingtechnical assistance in insulin ELISA. We also thank Drs. MichaelThorner, David Brautigan and Peilin Chen for providing commentson the manuscript.

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