Hydrogen Sulfide and L-Cysteine IncreasePhosphatidylinositol 3,4,5-Trisphosphate (PIP3) and GlucoseUtilization by Inhibiting Phosphatase and Tensin Homolog(PTEN) Protein and Activating Phosphoinositide 3-Kinase(PI3K)/Serine/Threonine Protein Kinase (AKT)/Protein KinaseC�/� (PKC�/�) in 3T3l1 Adipocytes*

Received for publication, June 10, 2011, and in revised form, September 26, 2011 Published, JBC Papers in Press, September 27, 2011, DOI 10.1074/jbc.M111.270884

Prasenjit Manna and Sushil K. Jain1

From the Department of Pediatrics, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130

Background: H2S and L-cysteine (LC) levels are low in diabetes.Results: H2S or LC inhibits PTEN and activates IRS1, PI3K, AKT, PKC�/�, GLUT4, and PIP3 levels and glucose utilization incells exposed to high glucose.Conclusion: LC and H2S can activate insulin signaling cascades.Significance: LC has the potential to activate the insulin signaling pathway and to improve glucose metabolism.

This work examined the novel hypothesis that reduced levelsof H2S or L-cysteine (LC) play a role in the impaired glucosemetabolism seen in diabetes. 3T3L1 adipocytes were treatedwith high glucose (HG, 25mM) in the presence or absence of LCorH2S. Both LC andH2S treatments caused an increase in phos-phatidylinositol-3,4,5 trisphosphate (PIP3), AKT phosphoryla-tion, andglucoseutilization inHG-treated cells. The effect of LCon PIP3 and glucose utilization was prevented by propargylgly-cine, an inhibitor of cystathionine �-lyase that catalyzes H2Sformation from LC. This demonstrates that H2S mediates theeffect of LC on increased PIP3 and glucose utilization. H2S andLC caused phosphatidylinositol 3-kinase activation and PTENinhibition. Treatmentwith LC,H2S, or PIP3 increased the phos-phorylation of IRS1, AKT, and PKC�/� as well as GLUT4 acti-vation and glucose utilization inHG-treated cells. This providesevidence that PIP3 is involved in the increased glucose utiliza-tion observed in cells supplemented with LC or H2S. Compara-tive signal silencing studieswith siAKT2or siPKC� revealed thatPKC� phosphorylation is more effective for the GLUT4 activa-tion and glucose utilization in LC-, H2S-, or PIP3-treated cellsexposed to HG. This is the first report to demonstrate that H2Sor LC can increase cellular levels of PIP3, a positive regulator ofglucose metabolism. The PIP3 increase is mediated by PI3Kactivation and inhibition of PTEN but not of SHIP2. This studyprovides evidence for a molecular mechanism by which H2S orLC can up-regulate the insulin-signaling pathways essential formaintenance of glucose metabolism.

Hydrogen sulfide (H2S) is gaining acceptance as a signalingmolecule and has been shown to elicit a variety of biologicaleffects that may mediate the protection of cardiovascularpathophysiology (1–3). However, elucidations of the mecha-nisms of action of H2S and its molecular targets are largelylacking. H2S is produced in vivo from L-cysteine (LC)2 by theaction of two enzymes, cystathionine �-synthase and cystathi-onine �-lyase (CSE) (4, 5). Both of these enzymes are dependenton pyridoxal 5�-phosphate. CSE is expressed mainly in the tho-racic aorta, portal vein, ileum, heart, liver, kidney, and vascularsmooth muscle, whereas cystathionine �-synthase is highlyexpressed in the central and peripheral nervous systems (4–6).Recent studies have shown evidence for two otherH2S-produc-ing enzymes, 3-mercaptopyruvate sulfurtransferase and cys-teine aminotransferase, which produce H2S in the brain as wellas in vascular endothelium (7–9). H2S inhibits oxidative stress,promotes stimulation of KATP channels and relaxation andvasodilation in vascular smooth muscle cells, and relaxation ofthe human corpus cavernosum smooth muscle (4, 7–11). Invivo, H2S has been shown to inhibit leukocyte endothelial cellinteractions and ischemia-reperfusion injury in liver and heartin animal studies (5). Genetic deletion of the CSE enzyme inmice markedly reduces H2S levels in the serum, heart, aorta,and other tissues, and mice lacking CSE display pronouncedhypertension and diminished endothelium-dependent vasore-laxation (4). Furthermore, apolipoprotein E knock-out micetreated with as H2S donor showed reduced atheroscleroticplaque size compared with controls (12). These studies suggestthatH2S is a physiological neuromodulator (13), vasodilator (4),

* This work was supported, in whole or in part, by grants from NIDDK and theOffice of Dietary Supplements of the National Institutes of Health GrantR01 DK072433. This work was also supported by the Malcolm FeistEndowed Chair in Diabetes.

1 To whom correspondence should be addressed: Dept. of Pediatrics, LSUHealth Sciences Center,1501 Kings Hwy., Shreveport, LA 71130. Tel.: 1-318-675-6086; Fax: 1-318-675-6059; E-mail: sjain@lsuhsc.edu.

2 The abbreviations used are: LC, L-cysteine; AKT, serine/threonine proteinkinase; aPKC, atypical PKC; CSE, cystathionine �-lyase; GLUT4, glucosetransporter 4; HG, high glucose; IRS1, insulin receptor substrate 1; MCP-1,monocyte chemoattractant protein-1; PAG, propargylglycine; PIP3, phos-phatidylinositol 3,4,5-trisphosphate; PTEN, phosphatase and tensin hom-olog; SHIP2, SH2 domain-containing inositol 5�-phosphatase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 46, pp. 39848 –39859, November 18, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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and regulator of blood pressure (4). Human blood contains asignificant amount of H2S (10–100 �M) (4, 14).

Recent studies report lower blood levels of LC and alteredcysteine homeostasis (15, 16) as well as lower H2S levels in dia-betic patients (17, 18). Along with a host of proteins, LC is aprecursor of glutathione, which is considered essential for thereduction of cellular oxidative stress (19). Dietary supplemen-tation with N-acetylcysteine or whey protein and �-lactoalbu-min (cysteine-rich proteins) lowers the oxidative stress andinsulin resistance induced by sucrose or fructose in rats andstreptozotocin-treated diabetic mice (20–23). Oral supple-mentation with LC lowered glycemia, oxidative stress, and vas-cular inflammationmarkers in ZDF (Zucker diabetic fatty) rats,an animal model of type 2 diabetes (24). Recent studies reportthat LC supplementation lowered oxidative stress markers intype 2 diabetic patients and normal subjects (15, 25). Thesestudies led to a novel hypothesis that reduced H2S or LC levelsmay play a direct role in modulating glucose metabolism indiabetes. Using an adipocyte cell model, this study demon-strates that H2S, or its precursor LC, increases cellular phos-phatidylinositol 3,4,5-trisphosphate (PIP3), a positive regulatorof phospho-serine/threonine protein kinase (phospho-AKT)and glucose metabolism. The effect of LC supplementation onincreased levels of PIP3 and glucose utilization was attenuatedby the inhibitor of CSE that catalyzes H2S formation. In addi-tion, exogenous treatment with PIP3 similarly increased AKTphosphorylation and glucose utilization. The increase in PIP3by LC or H2S is mediated by the activation of PI3K and inhibi-tion of phosphatase and tensin homolog (PTEN) but not SH2domain containing inositol 5 phosphatase (SHIP2). This studyprovides evidence for a novel molecular mechanism by whichH2S or LC supplementation can help improve glucose metabo-lism via activation of the PI3K/PIP3/phospho-AKT insulin sig-naling pathway using an adipocyte cell model.

EXPERIMENTAL PROCEDURES

Materials—PTEN, SHIP2, and glucose transporter 4(GLUT4) antibodies were purchased from Abcam, Inc. (Cam-bridge, MA). AKT2, phospho-AKT (serine 473), phospho-NF-�B (p65) (serine 276), phospho-PKC�/� (threonine 410/403), PKC�, and PI3K (p85�) were purchased from CellSignaling Technology (Beverly,MA). Insulin receptor substrate1 (IRS1) and phospho-IRS1 (tyrosine 941) were purchased fromMillipore. NF-�B (p65) was purchased from Santa Cruz Bio-technology, Inc. (Santa Cruz, CA). All other chemicals werepurchased from Sigma unless otherwise mentioned.3T3L1 Cell Culture and Differentiation—Themurine 3T3L1

fibroblast cell line was obtained from American Type CultureCollection (ATCC,Manassas, VA). These cells were cultured inhigh glucose (HG) DMEM containing 10% (v/v) FCS, 100units/ml penicillin, and 100 �g/ml streptomycin and main-tained at 37 °C in a humidified atmosphere containing 5% (v/v)CO2. Three days after achieving confluence, to allow for differ-entiation into adipocytes, cells were incubated in HG DMEMcontaining 10% (v/v) FBS, 100 units/ml penicillin, and 100�g/ml streptomycin supplemented with 100 milliunits/mlinsulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 250 nM dex-amethasone for 2 days. The cells were then placed in the same

medium containing insulin but lacking any other supplementsfor an additional 2 days. The media were replaced every 2 daysthereafter until �85% of the cells contained lipid droplets.Seven to 10 days after the induction of differentiation, 3T3L1adipocytes were ready to be used in experiments (26). The cellswere incubatedwith serum-free low glucose DMEMduring theexperimental incubation period.Treatment with HG, Mannitol, H2S, and LC—Cells were

treated separately with normal glucose (7mM) andHG (25mM)without and with two different sources of H2S, sodium sulfide(Na2S) and LC. Mannitol was used as an osmolarity control. Inthe mannitol-treated group, cells were exposed to 18 mMman-nitol and 7mMglucose. In this study, cells were exposed to aHGconcentration of 25 mM. Many previous studies have reportedthat glucose concentrations as high as 50 mM have been foundin the blood of patients with uncontrolled diabetes (27). It istrue that blood glucose levels in patients are not likely to stay ashigh as 25 mM for 24 h. However, tissue damage in diabeticpatients occurs over many years of countless hyperglycemicepisodes. Thus, the glucose concentration of 25mMused in thiscell culture study does not seem unreasonable. Cells were pre-treated with three different concentrations (100, 500, or 1000�M) of LC and two different concentrations (10 or 100 �M) ofNa2S for 2 h, followed by HG exposure for the next 20 h.Lipopolysaccharide (LPS)was used to stimulate the secretion ofcytokines by HG in cell culture studies (28); cells were treatedwith LPS (2 ng/ml) at 37 °C either alone or along with the LC orNa2S supplementation againstHGexposure. Thus, cells treatedwith LPS alone served as a control in this study. After treat-ment, cells were lysed in radioimmunoprecipitation assaybuffer (50 mMTris, pH 8, 150mMNaCl, 1%Nonidet P-40, 0.5%deoxycholic acid, 0.1% SDS) supplemented with protease andphosphatase inhibitors (1 mM PMSF, 5 �g/ml leupeptin, 2�g/ml aprotinin, 1 mM EDTA, 10mMNaF, and 1mMNa3VO4).Lysates were cleared by centrifugation, and total protein con-centrations were determined using a BCA assay kit (Pierce/Thermo Scientific).Dose- and Time-dependent Studies with PIP3 against HG

Exposure—To investigate the role of PIP3, adipocytes weretreated with normal glucose (7 mM) and HG (25 mM) withoutand with three different concentrations (1, 5, or 10 nM) of PIP3for various time periods (2 and 4 h). After treatment, glucoseutilization, the expression of AKT phosphorylation, and theintracellular PIP3 levels were investigated in experimentalsamples.siRNA Transient Transfection Studies—siAKT2 and control

siRNA were purchased from Cell Signaling Technology (Bev-erly, MA). siPKC� was purchased from Santa Cruz Biotechnol-ogy, Inc. (Santa Cruz, CA). 3T3L1 adipocytes grown in 12-wellplates were transiently transfected with 100 nM siRNA complexaccording to the manufacturer’s instructions. Briefly, 100 nMspecified siRNA diluted in 100 �l of Opti-MEM (Invitrogen)wasmixedwith 4–6�l of Lipofectamine 2000 transfection rea-gent (Invitrogen) and incubated for 30 min at room tempera-ture to form siRNA complexes. The siRNA medium wasformed by addition of 500 �l of growth medium to the siRNAcomplexes. Cells were incubatedwith siRNAmedium for 5–6 hfollowed by incubation with fresh DMEM/10% FBS for 24 h.

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After incubation the medium was aspirated, and cells weretreated with PIP3, LC, or H2S against HG exposure as men-tioned above. After treatment, the supernatants were used forthe glucose assay, and cell lysates were used for the immuno-blotting studies with GLUT4 antibody. In parallel, lysates wereimmunoblotted with AKT2 and PKC� antibodies to evaluatethe efficiency of siRNA-mediated protein depletion.Measurement of H2S—In this study, the H2S concentrations

were measured in the cell culture medium following themethod of Zhu et al. (29), which is based upon the old methyl-ene blue methods. The limitation of this method is that thistechnique monitors not only free H2S but also other speciessuch as hydrosulfide anion (HS�) and sulfide (S2�). Briefly, 75�l of medium was mixed with 250 �l of 1% (w/v) zinc acetateand 425 �l of distilled water, followed by the addition of 133 �lN-dimethyl-p-phenylenediamine sulfate (20 mM in 7.2 M HCl)and 133�l of FeCl3 in (30mM in 1.2 MHCl) to the test tube. Themixture was incubated at room temperature for 10 min. Theprotein in the plasma was removed by adding 250 �l of 10%trichloroacetic acid to the reaction mixture which was pelletedby centrifugation. The absorbance of the resulting solution wasmeasured at 670 nm in a 96-well plate with amicroplate reader.The results are expressed as percentage change over control.Detection of PIP3 Levels—The cellular PIP3 level was meas-

ured using the PIP3 Mass ELISA kit (Echelon Biosciences, SaltLake City, UT). Appropriate controls and standards (specifiedby each manufacturer’s kit) were used each time. In addition tothe ELISAmethod, PIP3 levels were also determined using flowcytometry. After treatment cells were washed in PBS-FCSbuffer, fixed in 2% paraformaldehyde, permeabilized with 0.5%saponin in PBS, then washed and incubated overnight at 4 °Cwith anti-PIP3 antibody (Echelon) at a 1:40 dilution. The pri-mary antibody was detected with a FITC-conjugated appropri-ate secondary antibody (Abcam). In each experiment, a mini-mum of 10,000 cells was analyzed (per treatment condition) byFACSCalibur flow cytometer (BD Biosciences) equipped withmulticolor analysis capability. Gates were set to exclude nonvi-able cells, cell debris, and cells of abnormal size and shape.Glucose Utilization, Cell Viability, GSH, and Cytokine

Secretion—Glucose assays were done at 0 h and at other speci-fied times. The glucose utilization level was determined by sub-tracting glucose values at specified times (leftover glucose)from the 0 h glucose level. All assays were done in duplicate ateach time point (30). An advantage Accu-check glucometer(Roche Applied Science) was used for the glucose assay. Cellviability was determined using the Alamar Blue reduction bio-assay (Alamar Biosciences, Sacramento, CA). This method isbased upon Alamar Blue dye reduction by live cells. The intra-cellular glutathione (GSH) levels were measured using a kitfromCalbiochem.The key component of the assay is a patentedreagent, 4-chloro-1-methyl-7-trifluoromethyl-quinoliniummethylsulfate. The second step is mediated by 30% NaOH,which specifically transforms the substitution productobtained with GSH into a chromophoric thione with a maxi-mum absorbance at 400 nm. The absorbance was detected by amultidetection microplate reader (Synergy HT, BIOTEK). TheMCP-1 (monocyte chemoattractant protein-1) level in thesupernatants of treated cells was determined by the sandwich

ELISA method using a commercially available kit from R&DSystems (Minneapolis, MN). Adiponectin levels were deter-mined using a kit and reagents from ALPCO Diagnostics(Salem, NH). All appropriate controls and standards as speci-fied by eachmanufacturer’s kit were used. In the cytokine assay,control samples were analyzed each time to check the variationfrom plate to plate on different days of analyses.Immunoblotting—All samples contained approximately the

same amount of protein (�20–40 �g) and were run as 8–10%SDS-PAGE and transferred to a nitrocellulose membrane.Membraneswere blocked at room temperature for 2 h in block-ing buffer containing 1% BSA to prevent nonspecific bindingand then incubated with anti-PTEN (1:1000 dilution), anti-PI3K (p85�) (1:1000), anti-AKT (AKT2) (1:1000 dilution), anti-NF-�B (p65) (1:1000 dilution), anti-PKC� (1:1000), anti-GLUT4 (1:1000), anti-IRS1 (1:1000) and anti-phosphorylatedIRS1 (tyrosine 941) (1:500), anti-phosphorylated AKT (serine473) (1:500), anti-phosphorylated PKC�/� (1:500) (threonine410/403), and anti-phosphorylated NF-�B (p65) (serine 276)(1:500) primary antibodies at 4 °C overnight. The membraneswere washed in TBS-T (50 mmol/liter Tris-HCl, pH 7.6, 150mmol/liter NaCl, 0.1% Tween 20) for 30 min and incubatedwith the appropriate HRP-conjugated secondary antibody(1:5000 dilution) for 2 h at room temperature and developedusing the ultrasensitive ECL substrate (Millipore, MA). Theintensity of each immunoblotting bandwasmeasured using thehistogram tool of Adobe Photoshop CS5.Statistical Analysis—Data from cell culture studies were ana-

lyzed statistically using one-way analysis of variance (ANOVA)with Sigma Stat statistical software (Jandel Scientific, SanRafael, CA).When data passed a normality test, all groups werecompared using the Student-Newman-Keuls method. A differ-ence was considered significant at the p � 0.05 level.

RESULTS

Effect of LCandH2S onPTEN, PI3K, Phospho-AKT, PIP3, andGlucoseUtilization inAdipocytes—Fig. 1 illustrates the effect ofdifferent concentrations of LC and of H2S on PTEN, PI3K,PIP3, phospho-AKT and glucose utilization levels in 3T3L1adipocyte cells treated with HG. HG treatment caused a signif-icant decrease in PI3K activation (Fig. 1A) and increase inPTEN (Fig. 1B). Supplementation with LC prevented thedecrease in PI3K and increase in PTEN caused by HG. Simi-larly, treatment with H2S caused a significant increase in PI3Kand a decrease in PTEN compared with treatment with HGalone. Fig. 1E illustrates that HG treatment decreased PIP3,which was replenished in cells supplemented with LC or H2S.The increase in PIP3 mediated by LC in HG-treated cells wasgreater with increasing concentrations of LC. Notably, glucoseutilization in cells was greater in the presence of LC orH2S (Fig.1F). HG treatment caused reduction in the AKT phosphoryla-tion (Fig. 1D). Mannitol treatment did not have any significanteffect on insulin signaling cascades or glucose utilization (datanot shown). However, phosphorylation of AKT was signifi-cantly increased in the presence of different concentrations ofLC or H2S (Fig. 1D). Different treatments did not cause anychange in cell viability (data not shown).High concentrations ofsubstrate (glucose) are likely to increase glucose utilization.

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When the intracellular glucose level surpasses its thresholdvalue, cellular glucose auto-oxidation produces the reactiveintermediate, which can cause oxidative stress, impair thePI3K/PTENpathway, and suppress the cellular PIP3 level/AKTphosphorylation. These results demonstrate that supplementa-tion with H2S and LC prevents the increase in PTEN anddecrease in PI3K caused byHGand significantly increasedAKTphosphorylation. The AKT phosphorylation parallels theincrease in PIP3 levels (Fig. 1E) and glucose utilization (Fig. 1F)seen in adipocytes supplemented with LC or H2S against HGexposure.Both PIP3 and glucose utilization levels were significantly

higher in LC and H2S supplemented HG-treated adipocytes.

The PIP3 concentration is up-regulated within cells by PI3Kand is degraded by two specific phosphatases: PTEN andSHIP (31). PIP3 binds to the pleckstrin homology domain ofAKT, promoting its translocation to the membrane. At thecell membrane AKT becomes phosphorylated, resulting inits activation. Our study did not observe any effect on SHIP2levels in H2S- or LC-treated adipocytes (Fig. 1C). In additionto the ELISAmethod, the increase in PIP3 concentration wasalso confirmed using flow cytometric analysis (Fig. 1G). Ourstudies observed that LC or H2S supplementation preventedthe HG-induced loss in intracellular PIP3 and thus shiftedthe corresponding histogram more closely to the control(right side of the diagram). A shift in the curve to the right

FIGURE 1. Effect of LC and H2S supplementation on the expression of PI3K, PTEN, SHIP2, phospho-AKT (serine 473)/AKT (AKT2) and the levels of PIP3,as well as glucose utilization in adipocytes (3T3L1) exposed to HG. A, PI3K (p85�). B, PTEN levels. C, SHIP2 levels. D, phospho-AKT/AKT. E, PIP3 levels. F,glucose utilization. G, FACS analysis of PIP3 (represented in histogram format; increasing PIP3 shifts the histogram to the right side of the plot). Cells werepretreated with either LC (100, 500, or 1000 �M) or Na2S (10 or 100 �M) for 2 h followed by HG (25 mM) exposure for the next 20 h. Values are expressed asmean � S.E. (error bars; n � 3). A difference was considered significant at the p � 0.05 level.

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side corresponds to higher concentrations of cellular PIP3(Fig. 1G).

Our findings are consistent with studies in the literature,which show that inhibition of PTEN expression using PTENantisense oligonucleotides normalized blood glucose concen-trations in ob/ob (obese) mice (32) and that overexpression ofPTEN resulted in inhibition of insulin-induced PIP3 produc-tion and glucose uptake in 3T3L1 adipocytes (33).Many studiesin the literature have implicated PTEN as a negative regulatorof the insulin signaling pathway (34). Attenuation of PTENexpression by siRNA in 3T3L1 adipocytes enhanced insulin-stimulated AKT phosphorylation (35). Inhibition of PTEN hasbeen a major target in the development of new drugs for the

control of glycemia and its complications in diabetes. Theseexperiments demonstrate that LC or H2S supplementation canpotentially increase PIP3 and glucose utilization in adipocytecell model.Fig. 2 shows the effect of an inhibitor of CSE, which catalyzes

H2S formation from LC, on PI3K, AKT, PIP3, and glucose uti-lization levels in 3T3L1 adipocyte cells cultured with HGmedium. The role of H2S in PI3K activation, PIP3 generation,and glucose utilization was demonstrated when the presence ofpropargylglycine (PAG, an inhibitor of CSE) lowered both H2S(Fig. 2B) and the expression of PI3K (Fig. 2A) and PIP3 levels(Fig. 2C), AKT phosphorylation (Fig. 2D), and glucose utiliza-tion (Fig. 2E) in LC-supplemented cells. This is consistent with

FIGURE 2. Effect of H2S formation inhibitor PAG on the expression of PI3K, phospho-AKT (serine 473)/AKT (AKT2), and the levels of H2S and PIP3 aswell as glucose utilization in adipocytes (3T3L1) exposed to HG. A, PI3K (p85�). B, H2S levels. C, PIP3 levels. D, phospho-AKT/AKT. E, glucose utilization. Cellswere treated with PAG (10 mM) 5 min prior to LC (500 �M) supplementation followed by HG exposure. In PIP3 and glucose utilization assays, insulin was usedas a positive control. Insulin (100 nM) was supplemented 2 h prior to HG (25 mM) exposure. Values are expressed as mean � S.E. (error bars; n � 3). A differencewas considered significant at the p � 0.05 level.

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effects observed upon exogenous addition of standard H2S perse in adipocytes. Insulin treatment used as a positive controlincreased both PIP3 and glucose utilization levels in adipocytes(Fig. 2,C and E). According tomany reviews the concentrationsof H2S in human blood range from 10 to 100 �M (4, 12) eventhough some investigators suggest that tissue concentrations ofH2S may be lower. Human blood concentrations of LC rangefrom 200 to 300 �M (36). LC at a concentration of 500 �M and1000 �M is considered to be a pharmacological concentration.The medium used for cell culture studies does not contain anyLC according to the analyses of medium provided by Sigma.These results demonstrate that supplementation with an exog-enous source of H2S can stimulate the PI3K/AKT/PIP3 insulinsignaling pathway and glucose utilization in adipocytes.Role of PIP3 inActivation of AKT, IRS1, PKC�/�, GLUT4, and

Glucose Utilization in Adipocytes—PIP3 binds directly to thepleckstrin homology domain of AKT. This results in transloca-tion ofAKT to the plasmamembrane. Likewise, phosphoinosit-ide-dependent protein kinase 1 also contains a pleckstrinhomology domain that binds directly to PIP3, causing it totranslocate to the plasma membrane as well upon activation ofPI3K. The co-localization of activated PI3K and AKT allowsAKT to become phosphorylated on threonine 308 and serine473, leading to the activation of AKT, which is required for themaintenance of glucose homeostasis. Phosphorylation of AKTand insulin signaling are critical components in the mainte-nance of whole body glucose homeostasis. The increased AKT

activity is associated with increased PIP3 levels and diminishedPTEN (37). PIP3 induces translocation, and subsequent activa-tion of AKT is assessed by phosphorylation of its serine 473residue (38). PIP3 plays a central role in the insulin signalingpathway and the maintenance of whole body glucosehomoeostasis.Interestingly, no studies in the literature have examined

whether PIP3 increases glucose utilization at the cellular level.Fig. 3 illustrates the effect of different concentrations of exoge-nous PIP3 onAKTphosphorylation and glucose utilization lev-els in adipocytes. Treatment with HG lowered AKT phospho-rylation (Fig. 3A). However, supplementation with exogenousPIP3 increased both AKT phosphorylation (Fig. 3A) and glu-cose utilization (Fig. 3B). Fig. 3C shows that exogenous additionof PIP3 dose dependently increases the intracellular PIP3 con-centration. The higher concentration of exogenous PIP3 islower than its level in control cells. However, the decrease inintracellular PIP3 levels wasmaintained by the exogenous addi-tion of PIP3 at 5 nM and 10 nM concentrations, so the 5 nMconcentration of PIP3 was used for the subsequentexperiments.Fig. 4 illustrates the effect of PIP3, LC, or H2S supplementa-

tion on the expression of IRS1, GLUT4, and PKC�/� phospho-rylation in adipocytes. HG treatment alone significantlydecreased the phosphorylation of IRS1 (Fig. 4A), PKC�/� (Fig.4B), and GLUT4 (Fig. 4C) expression. However, supplementa-tionwith LC,H2S, or PIP3 restored the phosphorylation of both

FIGURE 3. Effect of PIP3 supplementation on the expression of phospho-AKT (serine 473)/AKT (AKT2), glucose utilization, and intracellular PIP3 levelsin adipocytes (3T3L1) exposed to HG. A, phospho-AKT/AKT. B, glucose utilization. C, PIP3 levels. Cells were pretreated with PIP3 (1, 5, or 10 nM) for 2 and 4 hfollowed by HG (25 mM) exposure for the next 20 h. Immunoblotting results and the PIP3 levels are the values after 4 h of PIP3 supplementation. Values areexpressed as mean � S.E. (error bars; n � 3). A difference was considered significant at the p � 0.05 level.

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IRS1 and PKC�/� as well as GLUT4 activation. Comparativesignal silencing studieswith either siAKT2 or siPKC� (siRNAofcorresponding proteins AKT2 and PKC�, respectively) areshown in Fig. 5. We observed that transient transfection witheither siAKT2 or siPKC� decreased glucose utilization (Fig.5A). The effect of PKC� silencing on glucose utilization wasfound to be more pronounced than the AKT2 silencing. Thesame effect was also observed in GLUT4 expression (Fig. 5B).Insulin-stimulated glucose transport is regulated by the actionsof two protein kinases, AKT/PKB (protein kinase B) (39) andatypical protein kinase C (aPKC) isoforms � and � (40). Over-expression of a dominant negative mutant of PKC� or PKC�abrogates insulin-stimulated glucose uptake andGLUT4 trans-location in adipose tissue (40, 41) and muscle cells (42, 43).Overexpression of dominant interfering PKB/AKT mutants orblocking antibodies is also reported to inhibit insulin-stimu-lated GLUT4 translocation (39, 44). It has been reported that ofthese two aPKCs serve as terminal molecular switches that par-ticipate in turning on the glucose transport response, not onlyduring insulin action but also during the actions of a number ofother important agonists, including thiazolidones (45) and car-bohydrates (46). It has been reported that binding of acidiclipids such as PIP3 with the regulatory domain of aPKC leads to

molecular unfolding and increases enzymatic activity (47).Thus, binding with PIP3 activates aPKC, which enhances morerapid GLUT4 translocalization. In our study, exogenous addi-tion of PIP3, LC, or H2S improved glucose utilization andGLUT4 activation in the adipocytes transfected with siAKT2(see Fig. 5, left); however compared with those transfected withthe siAKT2, no significant improvement was observed in theadipocytes transfected with siPKC� (Fig. 5, right).Effect of LC andH2S on NF-�B, GSH, and Secretion ofMCP-1

and Adiponectin in Adipocytes—The activation of transcrip-tion factor NF-�B has been well established in diabetic patho-physiology. Oh et al. (48) originally reported that NaHS inhib-ited the LPS-induced NF-�B activation in cultured RAW 264.7macrophages. In agreement with the earlier findings (48), wealso observed that pretreatment with either LC or H2S couldprevent transcriptional activity of NF-�B by increasing phos-phorylation of the serine 276 residue of its p65 subunit activa-tion in adipocytes treated with HG (Fig. 6A). GSH plays animportant role in scavenging intracellular reactive intermedi-ates. Recently, H2S has been found to suppress oxidative stressinmitochondria by increasing the intracellularGSHconcentra-tion (11, 49). In this study we observed the same thing andfound that either LC or H2S supplementation prevented the

FIGURE 4. Effect of PIP3, LC, and H2S on the expression of phospho-IRS1/IRS1, phospho-PKC�/�/PKC�, and GLUT4 in adipocytes (3T3L1) exposed toHG. A, phospho-IRS1 (tyrosine 941)/IRS1. B, phospho-PKC�/� (threonine 410/403)/PKC�. C, GLUT4. Cells were pretreated with PIP3 (5 nM), LC (500 mM), or H2S(100 mM) for 2 h prior to the HG (25 mM) exposure. Values are expressed as mean � S.E. (error bars; n � 3). A difference was considered significant at the p � 0.05level.

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loss in intracellular GSH pool caused by HG exposure (Fig. 6B).This observation reveals the antioxidant role of LC or H2S inoxidative pathophysiology. MCP-1 is produced constitutively,or after induction of oxidative stress or growth factors, by avariety of cell types, includingmonocytes, smoothmuscle cells,and endothelial cells (50). Increased expression of MCP-1mRNA or proteins has been observed in animals and humanswith arteriosclerosis or atherosclerosis (50). Studies using invitromonocyte-endothelial adhesion assays and in vivo studiesusing knock-outmice for the CCR2 gene (a receptor ofMCP-1)and antibodies for MCP-1 have demonstrated that MCP-1plays an important role in vascular inflammation and athero-sclerotic lesion formation (51). The data in Fig. 6C show thatHG treatment increased the MCP-1 secretion in adipocytes.However, pretreatment with either LC or H2S prevents thisphenomenon. Adiponectin, one of the adipocytokines, has sev-eral physiological actions, such as protecting against athero-sclerosis, preventing hepatic fibrosis, and improving insulin

resistance (52). It has been reported that adiponectin inhibitshepatic glucose production by down-regulating the enzymesinvolved in gluconeogenesis, such as glucose 6-phosphataseand phosphoenolpyruvate carboxykinase, thereby decreasingblood sugar levels. Adiponectin treatment up-regulated theglucose uptake and fatty acid oxidation in skeletal muscle cellsor a muscle cell line (52). Pretreatment with either LC or H2Sincreased the adiponectin secretion in a dose-dependent man-ner in adipocytes exposed to HG (Fig. 6D). These results sug-gest an antioxidant and anti-inflammatory nature for LC andH2S.In our study we used insulin as a positive control. We

observed that insulin treatment prevented the HG-inducedalterations in the expression of PI3K (Fig. 7A), PTEN (Fig. 7B),GLUT4 (Fig. 7C), and the PIP3 level (Fig. 7D) as well as glucoseutilization (Fig. 7E). Fig. 7 shows that both LC and H2S have noeffect on the cells not exposed withHG compared with control.We have also investigated the effect of PAG on the cells, eitheralone or with HG exposure (Fig. 7). It has been found that inboth cases (with or without HG exposure) PAG treatment didnot cause any alteration to the insulin signaling cascades orglucose utilization compared with the control or HG-treatedgroups, respectively.

DISCUSSION

Recent studies indicate that H2S plays a potentially signifi-cant role in biological processes and that malfunctioning H2Shomeostasis may contribute to the pathogenesis of disease(1–3). Animal studies have demonstrated that supplementa-tion with LC itself or protein rich in LC has improved glucosemetabolism (20–24) and that H2S or H2S donor supplementa-tion ameliorated atherogenic processes (23). However, elucida-tion of the mechanisms of action of H2S or LC, a precursor ofH2S, is largely lacking.This study makes the novel observation that supplementa-

tion with LC or H2S, a metabolite of LC, increases PIP3 andglucose utilization levels in an adipocyte cell model. The effectof LC or H2S on PIP3 formation was mediated through activa-tion of PI3K and inhibition of PTEN. Phosphatase PTENdephosphorylates PIP3, and the PI3K enzyme generates the sig-naling lipid PIP3. It has been established that PIP3 plays a cen-tral role in the insulin signaling pathway and the maintenanceof whole body glucose homoeostasis (31, 53). However, to ourknowledge, this study for the first time presents evidence thattreatment with standard PIP3 per se increases the utilization ofglucose at the cellular level in an adipocyte cell model. Thisstudy also demonstrated a positive effect on the activation ofIRS1 and GLUT4 translocation in adipocytes pretreated withLC or H2S. H2S is synthesized from LC in mammals by fourenzymes, cystathionine �-synthase, CSE, 3-mercaptopyruvatesulfurtransferase, and cysteine aminotransferase (8, 9, 13), ofwhich CSE is likely to be the most physiologically relevant (3).The inhibitor of CSE significantly lowered levels of H2S as wellas PIP3 and glucose utilization. Adipose tissue is crucial in reg-ulating glucose metabolism (54). Insulin increases glucoseuptake in cells by stimulating the translocation of the glucosetransporter GLUT4 from intracellular sites to the cell surface.Various studies using knock-outmice and cellmodels of PTEN,

FIGURE 5. Effect of PIP3, LC, and H2S on the glucose utilization and GLUT4expression in adipocytes (3T3L1) exposed to HG after transfection withsiAKT2 (100 nM) or siPKC� (100 nM). A, glucose utilization. B, GLUT4. Leftpanels show results with siAKT2 and the right panels with siPKC�. Values areexpressed as mean � S.E. (error bars; n � 3). A difference was consideredsignificant at the p � 0.05 level.

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GLUT4, and PI3K have documented that inhibition of PTENand activation of PI3K andAKT signalingmolecules are crucialin insulin signaling pathways and the maintenance of wholebody glucose metabolism (54). Taken together, results fromthis study demonstrate that LC or H2S supplementation canincrease cellular PIP3 levels and up-regulate insulin signalingpathways and glucose metabolism. These studies demonstratethe potential significant effect of circulating H2S and LC levelson glucose metabolism in the body.The PI3K/PTEN pathway plays a central role in mediating

insulin responses via insulin receptor tyrosine kinase, whichphosphorylates insulin receptor substrate proteins (e.g. IRS1and IRS2) that nucleate downstream signaling proteins. PI3Kconsists of a p110 catalytic subunit and a p85� regulatory sub-unit. The p85 subunit of PI3K modulates insulin sensitivity viaeffects on PTEN (55). Cells that lack PTEN show elevated PIP3levels and activated AKT-dependent signaling (37). In additionto AKT signaling, recent studies in the literature suggest thataPKC (PKC�/�) are also the downstream regulators of PI3K,and their activation is required for insulin-stimulated glucoseuptake, which involves translocation of the major insulin-re-sponsive glucose transporter GLUT4 from intracellular sites tothe cell membrane (40–42). This study also demonstrates thatLC, H2S, or PIP3 increased the phosphorylation of IRS1, AKT,and PKC�/� as well as GLUT4 activation in adipocytes. IRS1 isthe major substrate of insulin receptor kinase and has manytyrosine phosphorylation sites, which provide binding sites formany kinases including the 85-kDa subunit of PI3K. Tyrosinephosphorylation of IRS1 also binds PI3K p85, thereby activat-ing PI3K (56), leading to the activation of glucose transport into

cells through glucose transporters. GLUT4 is themajor glucosetransporter protein involved in transport of glucose across adi-pocytes, from either inside or outside depending upon the insu-lin stimulus. Comparative signal silencing studies showed thateither siAKT2 or siPKC� could reduce glucose utilization butthat transfection with siPKC� caused a pronounced decrease inglucose utilization and GLUT4 activation compared withsiAKT2 transfection. Treatment with LC, H2S, or PIP3increased the glucose utilization and GLUT4 activation againstHG exposure in the siAKT2-transfected cells, but the effect wassignificantly attenuated in the siPKC� silencing study. Thismaybe due to the high affinity of aPKC for the acidic lipid PIP3, apositive regulator of glucose metabolism, compared with theAKT/PKBbinding (47).Thus, either LCorH2S can play a role inthe metabolic actions of insulin by up-regulating the cellularPIP3, and thus any reduction in their blood levels may impairinsulin action and glucose homeostasis.Diabetes is associated with lower circulating levels of LC and

H2S (15, 17, 18). Animal studies report that supplementationwith dietary LC or compounds containing LC can improve glu-cose metabolism and lower hyperglycemia (20–24). Chemicalsthat inhibit PTEN or activate PI3K have been considered aspotential therapeutic candidates for improvement of glucosemetabolism in type 2 diabetes. Using an adipocytes cell model,this study provides evidence for a molecular mechanism bywhich impaired blood concentrations of H2S or LC can down-regulate insulin signaling pathways essential for the mainte-nance of glucose metabolism. Further studies are needed todetermine the role of PTEN/PI3K/AKT/PKC�/� and PIP3 lev-els in the liver or adipose tissue of diabetic animals after cys-

FIGURE 6. Effect of LC and H2S supplementation on the expression of phospho-NF-�B (serine 276)/NF-�B and the levels of GSH and cytokine secretionin adipocytes (3T3L1) exposed to HG. A, phospho-NF-�B/NF-�B. B, GSH. C, MCP-1. D, adiponectin. Cells were pretreated with either LC (100, 500, or 1000 �M)or Na2S (10 or 100 �M) for 2 h followed by HG (25 mM) exposure for the next 20 h. Values are expressed as mean � S.E. (error bars; n � 4). A difference wasconsidered significant at the p � 0.05 level.

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teinate supplementation. The in vivo studies are needed todemonstrate the clinical viability of our results. If this discoveryleads to tangible clinical results, it would have an impact on thedevelopment of novel molecules, containing sulfide and cys-teine moieties, for the improvement of glucose metabolism indiabetes.LC is also a precursor of GSH. This study observed increases

in cellular GSH in LC- or H2S-supplemented adipocytes, whichconfirms previous reports that both LC and H2S can decreasecellular oxidative stress. This can influence the expression ofmultiple genes in cells, including signaling molecules such asNF-�B and PIP3 (via inhibition of PTEN and PI3K activation).Some of these effects may be mediated through inhibition of

oxidative stress, and others may be independent of oxidativestress. These genes regulate the production of proinflammatorycytokines and insulin sensitivity. LC and GSH are importantcomponents of redox signaling pathways and are implicated inthe reduction of cellular oxidative stress and T cell functions(18, 57). Oxidative stress associated with diabetes activatesNF-�B, which then can activate the insulin resistance cascade(58). Overexpression ofMCP-1 causes inhibition of AKT phos-phorylation and tyrosine phosphorylation in liver and skeletalmuscle, macrophage recruitment, and insulin resistance in aP2-MCP-1 mouse model (59). MCP-1 can phosphorylate AKTindependent of activation by the PI3K pathway. Adiponectin isa positive regulator of insulin sensitivity. Thus, inhibition of

FIGURE 7. Effect of LC, H2S, PAG, and insulin on the expression of PI3K, PTEN, and GLUT4, PIP3 levels and glucose utilization in adipocytes (3T3L1).Effects of LC, H2S, and PAG were investigated without HG exposures and the effects of insulin and PAG were investigated with HG exposure. The cell treatmentprocedure was the same as described previously. A, PI3K (p85�). B, PTEN. C, GLUT4. D, PIP3. E, glucose utilization. Values are expressed as mean � S.E. (error bars;n � 4). A difference was considered significant at the p � 0.05 level.

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MCP-1 and increased adiponectin secretion can also positivelyinfluence the metabolic actions of insulin in H2S- or LC-sup-plemented adipocytes.It is not yet completely understood how H2S is metabolized

in the body (2, 3). Some reports suggest that H2S reacts readilywithmethemoglobin to form sulfhemoglobin, whichmay act asa metabolic sink for H2S. It can rapidly be oxidized to thiosul-fate (S2O3

2�) by mitochondria and is subsequently convertedto sulfite (SO3

2�) and sulfate (SO42�). H2S can also undergo

methylation by thiol-S-methyltransferase to yieldmethanethiol(CH3SH) and dimethylsulfide (CH3SCH3) and is also a sub-strate (especially in the colon) for rhodanese (thiosulfate:cya-nide sulfurtransferase), which leads to the formation of thiocy-anate (SCN�) and SO4

2�. In addition to the biochemicalmeansby which H2S is catabolyzed, H2S is a powerful reducing agentand is likely to be consumed by endogenous oxidant species inthe vasculature, such as peroxynitrite, superoxide, and hydro-gen peroxide (2, 3).It appears that H2S enters cells via active diffusion and with-

out any facilitator (60). Studies using CSE knock-out mice, orpharmacological inhibition of CSE, which catalyzes formationof H2S, demonstrate that H2S plays an important physiologicalrole in hypoxic sensing in the carotid body (61, 62). The bene-ficial role ofH2S in increasing thermotolerance and lifespan hasbeen observed in Caenorhabditis elegans (63). The studies ofElrod et al. suggest that H2S attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function(64). Recent studies demonstrate that H2S mediates the vaso-activity of garlic, which is also rich in sulfides and LC (65).Garlic supplementation has also been shown to lower hypergly-cemia in streptozotocin-treated diabetic rats (66). An increas-ing number of studies in the literature indicate a potential ben-efit of H2S with respect to some aspects of cardiovasculardisease (1–3).In conclusion, this is the first report to demonstrate that H2S

or its precursor LCcan increase cellular levels of PIP3, a positiveregulator of glucose metabolism. Fig. 8 outlines a schematic

representation of a proposed mechanism by which H2S or LCcan positively modulate glucose metabolism. Diabetes is asso-ciated with hyperglycemia and increased oxidative stress,which can up-regulate PTEN and down-regulate PI3K. Thisresults in a decrease in cellular PIP3, a positive regulator ofinsulin signaling pathways and glucose metabolism. LC or H2Scan lower oxidative stress and inhibit PTEN and activate PI3K,which leads to elevated levels of PIP3 or AKT/PKC�/� phos-phorylation, IRS1 phosphorulation, and GLUT4 activation,resulting in up-regulation of the metabolic actions of insulinand an improvement in glucose metabolism. Using an adi-pocyte cell model, this work provides evidence for a molecularmechanism by which H2S or LC may help improve glucosemetabolism. Future studies are needed to validate the proposedbiochemical mechanism by which LC or H2S may up-regulateinsulin signaling cascades and improve glucose metabolism indiabetic animals and diabetic patients.

Acknowledgment—We thank Georgia Morgan for excellent editing ofthis manuscript.

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Prasenjit Manna and Sushil K. Jain3T3l1 Adipocytes

) inλ/ζ (PKCλ/ζ(PI3K)/Serine/Threonine Protein Kinase (AKT)/Protein Kinase CTensin Homolog (PTEN) Protein and Activating Phosphoinositide 3-Kinase

3,4,5-Trisphosphate (PIP3) and Glucose Utilization by Inhibiting Phosphatase and Hydrogen Sulfide and l-Cysteine Increase Phosphatidylinositol

doi: 10.1074/jbc.M111.270884 originally published online September 27, 20112011, 286:39848-39859.J. Biol. Chem. 

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