Dehydroepiandrosterone Stimulates Glucose Uptake in …Dehydroepiandrosterone (DHEA) has been shown...

Dehydroepiandrosterone Stimulates Glucose Uptake inHuman and Murine Adipocytes by Inducing GLUT1 andGLUT4 Translocation to the Plasma MembraneSebastio Perrini,

1Annalisa Natalicchio,

1Luigi Laviola,

1Gaetana Belsanti,

1Carmela Montrone,

1

Angelo Cignarelli,1

Vincenza Minielli,2

Maria Grano,2

Giovanni De Pergola,1

Riccardo Giorgino,1

and Francesco Giorgino1

Dehydroepiandrosterone (DHEA) has been shown tomodulate glucose utilization in humans and animals, butthe mechanisms of DHEA action have not been clarified.We show that DHEA induces a dose- and time-dependentincrease in glucose transport rates in both 3T3-L1 andhuman adipocytes with maximal effects at 2 h. Exposureof adipocytes to DHEA does not result in changes oftotal GLUT4 and GLUT1 protein levels. However, it doesresult in significant increases of these glucose trans-porters in the plasma membrane. In 3T3-L1 adipocytes,DHEA increases tyrosine phosphorylation of insulinreceptor substrate (IRS)-1 and IRS-2 and stimulatesIRS-1– and IRS-2–associated phosphatidylinositol (PI)3-kinase activity with no effects on either insulin recep-tor or Akt phosphorylation. In addition, DHEA causessignificant increases of cytosolic Ca2� concentrationsand a parallel activation of protein kinase C (PKC)-�2.The effects of DHEA are abrogated by pretreatment ofadipocytes with PI 3-kinase and phospholipase C� inhib-itors, as well as by inhibitors of Ca2�-dependent PKCisoforms, including a specific PKC-� inhibitor. Thus,DHEA increases glucose uptake in both human and3T3-L1 adipocytes by stimulating GLUT4 and GLUT1translocation to the plasma membrane. PI 3-kinase,phospholipase C�, and the conventional PKC-�2 seem tobe involved in DHEA effects. Diabetes 53:41–52, 2004

Insulin enhances the rates of glucose transport inadipocytes by stimulating the translocation of theGLUT4 and, to a lesser extent, GLUT1 glucosetransporters from specific intracellular membrane

compartments to the plasma membrane (1). The cascadeof signaling events involved in glucose transporter relocal-

ization to the cell surface in response to insulin is triggeredby an increase in insulin receptor tyrosine kinase activityfollowed by tyrosine phosphorylation of the insulin recep-tor substrate (IRS) proteins and activation of a complexnetwork of downstream molecules, including phosphati-dylinositol (PI) 3-kinase and other protein kinases such asthe serine/threonine kinase Akt/protein kinase B (PKB)and PKC-�/� (1,2).

In addition to insulin, multiple other hormones or phys-iologic conditions are capable of stimulating GLUT4 trans-location to the cell surface and glucose uptake. Forexample, exercise induces GLUT4 translocation and glu-cose transport in skeletal muscle through an insulin-independent pathway (3). Also, introduction of GTPanalogs, such as GTP�S, into 3T3-L1 adipocytes andactivation of �1-adrenergic or endothelinA receptors resultin enhanced glucose uptake rates independent of insulin(4–6). Some of the signaling mechanisms that mediatethese metabolic responses are similar to those utilized byinsulin, whereas others are clearly distinct. For instance,the stimulation of glucose uptake and glucose transportertranslocation to the cell surface that occurs in adipocytestreated with arachidonic acid, peroxisome proliferator–activated receptor � agonists, or vanadate compoundsseems to involve specific and insulin-independent signal-ing molecules (7–9).

Dehydroepiandrosterone (DHEA) and its metaboliteDHEA sulfate are the most abundant circulating adrenalsteroids in humans. Other than their role as precursors ofsex steroid hormones, their physiologic functions remainunclear. A progressive decrease in circulating levels ofDHEA with age has long been recognized, with peak levelsoccurring between the third and fourth decades of life anddecreasing progressively thereafter by �90% after the ageof 85 (10). The decline in circulating DHEA levels occur-ring with aging has been linked to the gradually increasingprevalence of atherosclerosis, obesity, and diabetes inelderly individuals. In the early 1980s, Coleman et al.(11–13) reported that dietary administration of DHEA todb/db mice induced remission of hyperglycemia andlargely corrected insulin resistance in these animals. Morerecently, DHEA was shown to protect against the devel-opment of visceral obesity and muscle insulin resistance inrats fed a high-fat diet (14). Other recent studies havedemonstrated that DHEA increases glucose uptake ratesin human fibroblasts and rat adipocytes and have sug-

From the 1Department of Emergency and Organ Transplantation, Section onInternal Medicine, Endocrinology and Metabolic Diseases, Bari, Italy; and the2Department of Human Anatomy and Histology, University of Bari, Bari, Italy.

Address correspondence and reprint requests to Francesco Giorgino, MD,PhD, Department of Emergency and Organ Transplantation, Section onInternal Medicine, Endocrinology and Metabolic Diseases, University of Bari,Piazza Giulio Cesare, 11, I-70124 Bari, Italy. E-mail: [email protected].

Received for publication 31 January 2003 and accepted in revised form 24September 2003.

DHEA, dehydroepiandrosterone; DMEM, Dulbecco’s modified Eagle’s me-dium; ERK, extracellular signal–related kinase; IRS, insulin receptor sub-strate; LDM, low-density microsome; MAP, mitogen-activated protein; MEK,MAP/ERK kinase; PI, phosphatidylinositol; PIP3, PI trisphosphate; PKB,protein kinase B; PKC, protein kinase C; PLC, phospholipase C; PM, plasmamembrane; PMSF, phenylmethylsulfonyl fluoride.

© 2004 by the American Diabetes Association.

DIABETES, VOL. 53, JANUARY 2004 41

gested that this effect may be mediated by activation ofPKC and PI 3-kinase (15–17). However, the effects ofDHEA on glucose transporters and the signaling mecha-nisms that mediate DHEA regulation of cellular glucosemetabolism have not been clarified.

In this study, we examined the effects of DHEA onglucose transport and the intracellular distribution ofglucose transporters in human and 3T3-L1 adipocytes. Weshow that DHEA exerts an insulin-like effect by inducingthe translocation of GLUT4 and GLUT1 to the plasmamembrane, resulting in increased glucose transport activ-ity. Furthermore, this metabolic effect of DHEA requiresactivation of PI 3-kinase and PKC-� but is independent ofAkt/PKB and the atypical PKC isoforms.

RESEARCH DESIGN AND METHODS

Antibodies and specialized reagents. Polyclonal insulin receptor �-sub-unit, monoclonal phosphotyrosine (PY99), monoclonal PKC-�1, and poly-clonal PKC-�2 antibodies were purchased from Santa Cruz Biotechnology(Santa Cruz, CA). Polyclonal antibodies against Akt, phospho-Akt (Thr 308),phospho-Akt (Ser-473), phospho-GSK-3�/� (Ser-9/Ser-21), mitogen-activatedprotein (MAP)/extracellular signal–related kinase (ERK) kinase (MEK)-1/2,phospho-MEK-1/2 (Ser-217/Ser-221), and phospho-p42/p44 MAP kinase (Thr-202/Tyr-204) were from Cell Signaling Technology (Beverly, MA). Polyclonalantibodies against p85, GSK-3, IRS-1, and IRS-2 were purchased from UpstateBiotechnology (Saranac Lake, NY). MEK-1/2 antibodies were from ZymedLaboratories (San Francisco, CA). A monoclonal antibody against clathrinheavy chain was from Transduction Laboratories (Lexington, KY). Rabbitpolyclonal antibodies against rat GLUT4 were provided by Dr. R.J. Smith(Brown University, Providence, RI) or purchased from Diagnostic Interna-tional (Schriesheim, Germany). Sheep polyclonal antibodies against Akt2were provided by Dr. D.R. Alessi (University of Dundee, Dundee, U.K.).GLUT1 antibody was from Charles River (Southbridge, MA). Antibodiesagainst PKC isozymes (�, �, �/�) were from Life Technologies (Gibco BRL).Wortmannin, LY294002, GF109203X, U73122, Calphostin C, Go6,976, andPD098059 were from Calbiochem (La Jolla, CA). The PKC-�/� inhibitorcell-permeable myristoylated PKC-�/� pseudosubstrate (myr-PKC-�/�) wasfrom Quality Controlled Biochemicals (Hopkington, MA). Porcine insulin andLY379196, a specific PKC-� inhibitor, were gifts from Eli Lilly (Indianapolis,IN). The PKC-� inhibitor cell-permeable myristoylated PKC-� pseudosub-strate was from Calbiochem.Cell culture. 3T3-L1 fibroblasts (American Type Culture Collection, Rock-ville, MD) were grown in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10% FCS, 2 mmol/l L-glutamine, 100 units/ml penicillin, and100 mg/ml streptomycin at 37°C in an atmosphere of 5% CO2. Differentiationinto adipocytes was induced as described previously (18).Preparation of human adipocytes. Specimens of human subcutaneousadipose tissue were obtained from the abdominal region of nondiabeticsubjects undergoing elective surgery for nonmalignant diseases. The studyhad the approval of the local ethical committee. The subcutaneous adiposetissue (�1 g) was removed and placed in Krebs-Ringer buffer (136 mmol/lNaCl, 4.7 mmol/l KCl, 1.25 mmol/l MgSO4, 5 mmol/l Na2HPO4, 1.25 mmol/lCaCl2 [pH 7.4]), containing 40 mg/ml BSA (fraction V) and 5.5 mmol/l glucose,at 37°C. Isolated adipocytes were obtained using a method modified fromRodbell (19) and characterized by evaluating the insulin receptor and GLUT4content in total cell lysates.Glucose transport studies. For measuring glucose transport rates in 3T3-L1adipocytes, cells were grown in serum-free DMEM for 16 h and then incubatedin the absence or presence of insulin or DHEA for the indicated times at 37°C.Transport was started by adding 50 �mol/l [3H]2-deoxy-D-glucose (NEN,Boston, MA) and 1 �Ci in 1 ml of Krebs-Ringer phosphate buffer (pH 7.4) for5 min at 37°C and stopped by placing the cells on ice and rapidly washing themthree times with ice-cold buffer. Cells were lysed in 1 ml of lysis buffercontaining 0.1% Triton X-100 for 45 min. Aliquots of the cell lysates were usedfor liquid scintillation counting and determination of protein content by theBradford method, respectively. Nonspecific transport was assayed in thepresence of 10 �mol/l cytochalasin B. Glucose transport measurements inhuman adipocytes were performed as described by Ciaraldi et al. (20).Preparation of total and subcellular membrane fractions. For obtainingtotal membranes from 3T3-L1 adipocytes, cells were collected into 10 ml ofice-cold HES buffer (250 mmol/l sucrose, 1 mmol/l EDTA, 1 mmol/l phenyl-methylsulfonyl fluoride [PMSF], 1 �mol/l pepstatin, 1 �mol/l aprotinin, 1�mol/l leupeptin, and 20 mmol/l HEPES, pH 7.4) and subsequently homoge-

nized with 20 strokes in a glass Dounce homogenizer (Type C; Thomas,Philadelphia, PA) at 4°C. After centrifugation at 1,000g for 3 min at 4°C toremove large cell debris and unbroken cells, the supernatant was thencentrifuged at 245,000g for 90 min at 4°C to yield a pellet of total cellularmembranes and a supernatant representing the cytosolic fraction (21). Foryielding total cellular membranes from human adipocytes, cells were homog-enized with 20 strokes in a glass Dounce homogenizer in ice-cold HES buffer.The homogenate was then centrifuged at 1,000g for 3 min at 4°C, and thepostnuclear supernatant was separated from the pellet and fat cake and thencentrifuged at 245,000g for 90 min at 4°C. The resulting pellet representing thetotal cellular membrane fraction (22) was resuspended in HES buffer beforeuse. High-density microsome, low-density microsome (LDM), and plasmamembrane (PM) subcellular fractions from 3T3-L1 adipocytes were obtainedby differential ultracentrifugation, as described previously (23). PM, LDM, andhigh-density microsome fractions from human adipocytes were preparedaccording to Garvey et al. (22).

The preparation of PM lawns was performed as described by Robinson etal. (23). Briefly, after incubating cells on coverslips with the appropriatetreatment, adipocytes were sonicated, yielding a lawn of PM fragmentsattached to the coverslip. Coverslips were then incubated with GLUT4 orGLUT1 antibodies and then with Alexa 546– or Alexa 488–conjugatedsecondary antibodies, respectively. The fluorescence intensity of individualPM fragments from five random fields for each experimental condition wasanalyzed using a Leica TCS SP2 laser confocal microscope.Immunoprecipitation, immunoblotting, and measurement of PI 3-ki-

nase activity. For preparing total cell lysates, 3T3-L1 adipocytes werewashed with Ca2�/Mg2�-free PBS and then mechanically detached in ice-coldlysis buffer containing 50 mmol/l HEPES (pH 7.5), 150 mmol/l NaCl, 1 mmol/lMgCl2, 1 mmol/l CaCl2, 10% glycerol, 10 mmol/l sodium pyrophosphate, 10mmol/l sodium fluoride, 2 mmol/l EDTA, 2 mmol/l PMSF, 5 �g/ml leupeptin, 2mmol/l sodium orthovanadate, and 1% Nonidet P-40. After incubation for 45min at 4°C, the preparation was centrifuged at 12,000g for 10 min at 4°C. Theresulting supernatant was assayed for determination of protein concentrationusing the Bradford method and subjected overnight to immunoprecipitation at4°C with antibodies against the insulin receptor, IRS-1, IRS-2, PKC-�1, orPKC-�2, as indicated. The resulting immune complexes were adsorbed toprotein A–Sepharose beads for 2 h at 4°C, and the pelleted beads were washedthree times in lysis buffer and then incubated in Laemmli buffer for 5 min at100°C. Protein samples were resolved by electrophoresis on 6%, 7%, 10%, or12% SDS–polyacrylamide gels, as appropriate, directly or after immunopre-cipitation and subjected to immunoblotting with the appropriate antibodies,as described previously (24). The proteins were quantified by densitometricanalysis using Optilab image analysis software (Graftek SA, Mirmande,France). For measurements of PI 3-kinase activity, cell lysates (�1–2 mg)were subjected overnight to immunoprecipitation at 4°C with antibodiesagainst IRS-1 or IRS-2. The activity of PI 3-kinase in the immunoprecipitateswas determined as described previously (25).Measurement of [Ca2�]i. Cytosolic-free calcium concentrations were eval-uated in single adipocytes loaded with the intracellular Ca2� indicator fura-2(26). Isolated cells seeded onto 24-mm round glass coverslips were loadedwith 10 �mol/l fura-2/AM in serum-free DMEM for 1 h at 37°C. Coverslips werewashed three times with PBS and transferred to a Sykes Bellco open chamber(Bellco Biotechnology, Vineland, NJ) containing 1 ml of Krebs-Ringer-HEPESbuffer (125 mmol/l NaCl, 5 mmol/l KCl, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4,2 mmol/l CaCl2, 265 mmol/l HEPES, and 6 mmol/l glucose). [Ca2�]i-dependentfluorescence was measured with a microfluorometer (Cleveland Bioinstru-mentation, Cleveland, OH) connected with a Zeiss IM35 inverted microscopeequipped with a Nikon GFX40 fluor objective. Recordings were performed atdual excitation wavelength (340 and 380 nm, bandwidth 0.5 nm) using an airturbine high-speed rotating wheel carrying the two excitation filters. At theend of each experiment, calibration was performed by adding 5 �mol/lionomycin followed by 7.5 mmol/l EGTA to obtain Ca2�-saturated andnominally Ca2�-free fura-2 fluorescence, respectively.Statistical analysis. All data are expressed as mean SE. Statisticalanalyses were performed by unpaired Student’s t tests.

RESULTS

Effects of DHEA on the glucose transport system in

3T3-L1 and human adipocytes. For investigating theeffects of DHEA on the glucose transport system, 3T3-L1adipocytes were incubated in the presence of variousconcentrations of DHEA for different times, and glucosetransport was measured by determining the rates of [3H]2-deoxy-D-glucose uptake. DHEA induced a time- and dose-

DHEA AND GLUCOSE TRANSPORT

42 DIABETES, VOL. 53, JANUARY 2004

dependent increase in glucose transport rates in 3T3-L1adipocytes. An initial statistically significant effect ofDHEA on transport was observed at the concentration of1 �mol/l (35% of basal; P 0.05) and the maximal effect at100 �mol/l (300% of basal; P 0.05; Fig. 1A). With 100�mol/l DHEA, glucose transport was maximally increasedafter 120 min of incubation with the steroid (Fig. 1A). Theeffects of DHEA on glucose transport were specific be-cause treatment of 3T3-L1 adipocytes with equimolarconcentrations of other steroid hormones, including 17-�-estradiol, progesterone, �-4-androstenedione, testoster-one, and dihydrotestosterone, had no effects on 2-deoxy-D-glucose uptake rates (Fig. 1A). However, DHEA was lesspotent than insulin, used at maximally effective concentra-tions, in stimulating glucose transport in 3T3-L1 adipo-cytes (P 0.05 vs. insulin; Fig. 1A). For assessing whetherthe ability of DHEA to enhance glucose transport in 3T3-L1adipocytes could be explained by DHEA-induced changesin the amounts of glucose transporter protein at the cellsurface, the protein levels of GLUT1 and GLUT4, the twopredominant glucose transporter isoforms expressed in3T3-L1 adipocytes, were measured in LDM and PM frac-

tions in the basal state or after treatment with DHEA, orinsulin for comparison. PM and LDM fractions preparedfrom basal, DHEA-treated, and insulin-treated adipocytescontained comparable amounts of clathrin (27) (Fig. 2A).DHEA induced a significant increase in the PM content ofboth GLUT1 and GLUT4 proteins (respectively, 180 and160% of basal; P 0.05; Fig. 2A and B). DHEA and insulinstimulated GLUT1 translocation to the PM to a similarextent, whereas DHEA was less effective than insulin ininducing PM translocation of GLUT4 (P 0.05 vs. insulin;Fig. 2A and B). Similar results were obtained by measuringthe amounts of GLUT4 and GLUT1 in adipocyte PMfragments from 3T3-L1 adipocytes by immunofluorescenceanalysis (Fig. 2C). In addition, a modest but significantdecrease in the amount of GLUT4 and GLUT1 in the LDMfraction was observed after insulin treatment (P 0.05 vs.basal) but not in response to DHEA (Fig. 2A and B). Theincrease in PM GLUT1 and GLUT4 induced by DHEA wasnot the consequence of higher levels of these transportersin the cell because total GLUT1 and GLUT4 protein levelswere not altered by DHEA (Fig. 2D).

For assessing whether the glucose transport enhance-

FIG. 1. Effects of DHEA on adipocyte glucose transport. A: 2-deoxy-D-glucose uptake in 3T3-L1 adipocytes. Cells grown in six-well plates wereequilibrated in glucose-free Krebs-Ringer buffer for 15 min and then incubated with the indicated concentrations of DHEA (hatched bars) for 2 h(left) or with 100 �mol/l DHEA for the indicated times (center) before [3H]2-deoxy-D-glucose uptake measurements, as described in RESEARCH

DESIGN AND METHODS. Right: [3H]2-deoxy-D-glucose uptake rates in 3T3-L1 adipocytes incubated in the absence (Bas) or presence of equimolarconcentrations (10 �mol/l) of 17�-estradiol (E2), progesterone (Pg), �4-androstenedione (�4), testosterone (T), dihydrotestosterone (DHT), orDHEA for 120 min or in the presence of 100 nmol/l insulin (Ins) for 15 min. Values are mean � SE of four independent experiments performedin triplicate. *P < 0.05 vs. basal; #P < 0.05 vs. insulin. B: 2-deoxy-D-glucose uptake in human adipocytes. Human adipocytes were isolated asdescribed in RESEARCH DESIGN AND METHODS and incubated in the absence or presence of the indicated concentrations of DHEA (hatched bars) for2 h (left) or with 100 �mol/l DHEA for the indicated times (center). Right: [3H]2-deoxy-D-glucose uptake rates in human adipocytes left untreated(Bas) or incubated with 100 nmol/l insulin (Ins) for 15 min or 100 �mol/l DHEA for 120 min. [3H]2-deoxy-D-glucose uptake was measured asdescribed in RESEARCH DESIGN AND METHODS. Values are mean � SE of three independent experiments performed in triplicate. *P < 0.05 vs. basal;#P < 0.05 vs. insulin.

S. PERRINI AND ASSOCIATES


ment by DHEA was a metabolic response restricted to the3T3-L1 adipocyte cell line or could also be observed inhuman fat cells, 2-deoxy-D-glucose uptake rates weremeasured in adipocytes isolated from abdominal subcuta-neous fat tissue of normal subjects and incubated in theabsence or presence of DHEA. As shown in Fig. 1B, DHEAtreatment of human adipocytes resulted in a dose- andtime-dependent augmentation of 2-deoxy-D-glucose uptake(P 0.05 vs. basal). The glucose transport system wasmore sensitive to DHEA in human as compared with3T3-L1 adipocytes, because a significant increase in glu-cose uptake occurred with DHEA concentrations as low as0.1 �mol/l in human adipocytes (compare A and B in Fig.1). Similar to the results in 3T3-L1 adipocytes, in humanadipocytes, DHEA showed maximal effects on glucosetransport after 120 min of treatment and was less effectivethan insulin (P 0.05 vs. insulin; Fig. 1B). The DHEA-induced increase in glucose transport was associated withtwofold higher GLUT4 levels in the PM fraction (P 0.05vs. basal; Fig. 3A and B). Insulin augmented GLUT4 levelsin the PM to a greater extent than DHEA (P 0.05) and, incontrast to DHEA, significantly decreased GLUT4 in theLDM (Fig. 3A and B). The protein content of GLUT1 in thePM was slightly higher in DHEA- and insulin-treated than

in control adipocytes, but these changes did not reachstatistical significance (Fig. 3A and B). DHEA did notmodify the total levels of GLUT4 (Fig. 3C). The total levelsof GLUT1 in human adipocytes were very low, in agree-ment with previously reported findings (22), and did notshow any significant changes with DHEA treatment (Fig.3C).Effects of DHEA on insulin signaling proteins. Insulinstimulation of glucose transport in adipocytes requiresinsulin receptor–mediated tyrosine phosphorylation ofIRS-1 and IRS-2 and subsequent activation of PI 3-kinase(1,2). Thus, whether increased glucose uptake in responseto DHEA was associated with increased insulin receptorand/or IRS tyrosine phosphorylation and PI 3-kinase activ-ity was determined next. As expected, insulin stimulationof cells resulted in augmentation of insulin receptor, IRS-1and IRS-2 tyrosine phosphorylation, association of p85with IRS-1 and IRS-2, and increased PI 3-kinase activity inIRS-1 and IRS-2 immunoprecipitates (Fig. 4A and B). Bycontrast, DHEA treatment of adipocytes for various timeshad no effects on tyrosine phosphorylation of the insulinreceptor (Fig. 4A). However, DHEA significantly enhancedtyrosine phosphorylation of IRS-1 and IRS-2 (respectively,150 5% and 180 6% of basal; P 0.05), induced the

FIG. 2. Effects of DHEA and insulin on GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes. Representative immunoblots (A) andquantification (B) of GLUT1 and GLUT4 glucose transporters in LDM and PM fractions. Adipocytes were left untreated (Bas) or incubated with100 nmol/l insulin (Ins) for 15 min or 100 �mol/l DHEA (DH) for 120 min at 37°C. LDM and PM fractions were prepared as described in RESEARCH

DESIGN AND METHODS and subjected to immunoblotting with antibodies against GLUT1, GLUT4, or clathrin heavy chain, as indicated. Clathrin wasfound largely in the membrane fractions (data not shown), and its distribution was not affected after insulin or DHEA stimulation. Values aremean � SE of three independent experiments performed in triplicate. *P < 0.05 vs. PM basal; **P < 0.05 vs. LDM basal; #P < 0.05 vs. PM insulin.C: Immunofluorescence analysis of GLUT1 and GLUT4 transporters in PM fragments. Adipocytes were left untreated (Bas) or incubated with 100nmol/l insulin (Ins) for 15 min or 100 �mol/l DHEA (DH) for 120 min at 37°C. PM sheets were prepared as described in RESEARCH DESIGN AND METHODS

and analyzed by indirect immunofluorescence with antibodies against GLUT1 or GLUT4, as indicated. A representative of three experiments isshown. D: Total cell content of GLUT1 and GLUT4 glucose transporters. Adipocytes were treated with 100 �mol/l DHEA for 0–240 min. Totalmembranes were then isolated and analyzed by immunoblotting with GLUT1 or GLUT4 antibodies, as indicated. A representative of threeexperiments is shown.



association of p85 with IRS-1 and IRS-2, and increased PI3-kinase activity in IRS-1 and IRS-2 signaling complexes(Fig. 4B). As compared with insulin, DHEA was lesseffective in stimulating IRS-1 tyrosine phosphorylation(150 5% vs. 220 7% of basal in response to DHEA andinsulin, respectively; P 0.05) and the associated PI3-kinase activity but equally effective on IRS-2 tyrosinephosphorylation (180 6% and 190 10% of basal inresponse to DHEA and insulin, respectively; NS) andIRS-2–associated PI 3-kinase activity (Fig. 4B). For inves-tigating the possibility that DHEA activates insulin-signal-ing intermediates distal to PI 3-kinase, the phosphorylationof the serine/threonine kinase Akt was evaluated next.Whereas treatment of 3T3-L1 adipocytes with insulin for15 min resulted in marked stimulation of Akt phosphory-lation on both Thr-308 and Ser-473, treatment with 100�mol/l DHEA for 5–120 min did not modify Akt phosphor-ylation (Fig. 4C). Furthermore, DHEA treatment did notactivate Akt2, assessed by evaluating Thr-308 phosphory-lation specifically in Akt2 immunoprecipitates (Fig. 4D),and did not induce phosphorylation of GSK-3�, whichreflects Akt activity in intact cells (Fig. 4E). Finally, DHEAdid not change the phosphorylation state of MEK andERK-1/2 kinases, which were markedly activated in re-sponse to insulin (Fig. 4C). The levels of Erk-1 and Erk-2phosphorylation were, respectively, 750 47% and 588 49% of basal after insulin stimulation (P 0.05), 113 23%and 113 26% of basal after DHEA stimulation for 5 min(P � 0.64), and 87 25% and 85 26% of basal afterDHEA stimulation for 15 min (P � 0.66).

Effects of PI 3-kinase inhibitors on DHEA-stimulated

glucose transport. Because DHEA was found to activatePI 3-kinase in IRS-1 and IRS-2 signaling complexes, theinvolvement of PI 3-kinase in the DHEA-induced augmen-tation of glucose transport was assessed next. For thispurpose, 2-deoxy-D-glucose uptake rates were measured in3T3-L1 adipocytes after pretreatment with PI 3-kinaseinhibitors. Both Wortmannin and LY294002 completelyabrogated DHEA-stimulated glucose uptake in 3T3-L1 adi-pocytes (P 0.05 vs. DHEA control; Fig. 5A). In addition,these compounds markedly inhibited transport stimula-tion by insulin (P 0.05 vs. insulin control; Fig. 5A), asreported previously (28). Pretreatment of adipocytes withLY294002 resulted in marked inhibition of the ability ofDHEA to increase GLUT1 and GLUT4 in PM fractions (P 0.05 vs. DHEA control; Fig. 5B and C). LY294002 alsoprevented insulin-induced translocation of GLUT1 andGLUT4 from the LDM to PM fractions (P 0.05 vs. insulincontrol; Fig. 5B and C), as shown previously (28). Glucosetransport stimulation by DHEA in 3T3-L1 adipocytes wasnot affected by preincubation of cells with PD098059, aninhibitor of MEK. Glucose transport (in pmol 2-DG � �gprotein 1 � min 1) was 34 2, 54 12, 38 3, and 56 1 under basal conditions and after incubation with 100�mol/l DHEA, 20 �mol/l PD098059, and 100 �mol/l DHEAplus 20 �mol/l PD098059, respectively. In addition, it wasnot affected by 100 nmol/l rapamycin, a p70S6 kinaseinhibitor (data not shown). Thus, PI 3-kinase activity isrequired for DHEA-induced translocation of GLUT1 and

FIG. 3. Effects of DHEA and insulin on GLUT1 and GLUT4 glucose transporters in human adipocytes. Representative immunoblots (A) andquantification (B) of GLUT1 (top) and GLUT4 (bottom) glucose transporters in LDM and PM fractions. Adipocytes were left untreated (Bas) ortreated with 100 nmol/l insulin (Ins) for 15 min or 100 �mol/l DHEA (DH) for 120 min at 37°C. Cells were homogenized, and LDM and PM fractionswere obtained as described in RESEARCH DESIGN AND METHODS and subjected to immunoblotting with antibodies against GLUT1, GLUT4, or clathrinheavy chain, as indicated. Clathrin was found largely in the membrane fractions (data not shown), and its distribution was not affected followinginsulin or DHEA stimulation. Values are mean � SE of four independent experiments performed in duplicate. *P < 0.05 vs. PM basal; **P < 0.05vs. LDM basal; #P < 0.05 vs. PM insulin. C: Total cell content of GLUT1 (top) and GLUT4 (bottom) glucose transporters. Adipocytes wereincubated with 100 �mol/l DHEA for the indicated times and processed to obtain total membranes, as described in RESEARCH DESIGN AND METHODS.Equal amounts of membrane protein (5 �g) were resolved by 10% SDS-PAGE and subjected to immunoblotting with antibodies against GLUT1or GLUT4, as indicated. A representative of three experiments is shown.



GLUT4 and subsequent enhancement of glucose uptake by3T3-L1 adipocytes.Effects of DHEA on PKC isoforms. DHEA has report-edly been shown to activate PKC in rat adipocytes (16,17).For evaluating the ability of DHEA to activate specific PKCisoforms in 3T3-L1 adipocytes, cytosolic and membranefractions from DHEA- and insulin-treated cells were ana-lyzed by immunoblotting with antibodies specific toPKC-�, PKC-�, and PKC-�/�, three PKC isoforms that areexpressed in 3T3-L1 adipocytes (29,30). DHEA inducedmembrane translocation of PKC-�, with maximal effectafter 30 min (P 0.05 vs. basal), and did not modify thedistribution of either PKC-� or PKC-�/� in cytosolic andmembrane fractions (Fig. 6A). Because tyrosine phosphor-ylation reflects the activation state of specific PKCisozymes (31,32), PKC-� tyrosine phosphorylation wasassessed in these experimental conditions by immunopre-cipitation with antibodies against PKC-�1 or PKC-�2 fol-lowed by immunoblotting with phosphotyrosine antibody.DHEA induced tyrosine phosphorylation of PKC-�2 (Fig.

6B), and this effect was abrogated by pretreatment of cellswith the PKC-� inhibitor LY379196 (33) (Fig. 6B). Con-versely, no effects of DHEA on tyrosine phosphorylation ofPKC-�1 were observed (data not shown). In contrast toDHEA, insulin promoted membrane translocation ofPKC-� and PKC-�/� and did not affect the cellular distri-bution of PKC-� (Fig. 6A) or tyrosine phosphorylation ofPKC-�2 (Fig. 6B) or PKC-�1 (data not shown) in 3T3-L1adipocytes.

Because membrane translocation of conventional PKCisoforms, such as PKC-�, follows changes in the intracel-lular concentrations and localization of Ca2� (34), cytoso-lic-free Ca2� concentrations were measured at multipletime points after treatment of 3T3-L1 adipocytes with 100�mol/l DHEA. Cytosolic Ca2� concentrations were in-creased by DHEA with an initial statistically significanteffect at 33 min (P 0.05 vs. control; Fig. 6C), which wasconcomitant with the DHEA-induced membrane translo-cation of PKC-� (Fig. 6A). For assessing whether theability of DHEA to activate PKC-� required PI 3-kinase or

FIG. 4. Effects of DHEA and insulin on insulin-signaling proteins. A: Insulin receptor tyrosine phosphorylation. 3T3-L1 adipocytes were leftuntreated (Bas) or treated with 100 nmol/l insulin (Ins) for 15 min or 100 �mol/l DHEA (DH) for the indicated times. Total cell lysates weresubjected to immunoprecipitation with insulin receptor (Ins R) antibody followed by immunoblotting with phosphotyrosine (PY) or Ins Rantibodies, as indicated. B: IRS-1 and IRS-2 tyrosine phosphorylation and associated PI 3-kinase activity. Cell were left untreated (Bas) orstimulated with 100 nmol/l insulin (Ins) for 15 min or 100 �mol/l DHEA (DH) for 120 min. Cell lysates were subjected to immunoprecipitationwith IRS-1 or IRS-2 antibodies, as indicated, followed by immunoblotting with PY (top blot) or p85 (middle blot) antibodies. PI 3-kinase activityin IRS-1 and IRS-2 immunoprecipitates was measured as described in RESEARCH DESIGN AND METHODS (bottom autoradiograph). The position of PI3-phosphate (designated PI 3-P) is indicated. C: Phosphorylation of Akt, MEK, and ERK kinases. 3T3-L1 adipocytes were incubated with 100�mol/l DHEA (DH) or 100 nmol/l insulin (Ins) for the indicated times. Total cell lysates were analyzed by immunoblotting with specific antibodies,as indicated. D: Akt2 phosphorylation. 3T3-L1 adipocytes were incubated with 100 �mol/l DHEA (DH) or 100 nmol/l insulin (Ins) for the indicatedtimes. Cell lysates were subjected to immunoprecipitation with Akt2 antibodies followed by immunoblotting with antibodies to phosphorylatedAkt (Thr-308) or total Akt, as indicated. E: GSK-3 phosphorylation. Total cell lysates from 3T3-L1 adipocytes treated with 100 �mol/l DHEA (DH)or 100 nmol/l insulin (Ins) for the indicated times were analyzed by immunoblotting with antibodies to phosphorylated or total GSK-3. Arepresentative of three immunoblots for each protein is shown.



phospholipase C-� (PLC-�) activity, membrane transloca-tion of PKC-� in response to DHEA was assessed inadipocytes that had been pretreated with the PI 3-kinaseinhibitor LY294002 or the PLC-� inhibitor U73122 (35),respectively. DHEA stimulation of PKC-� translocation,which was observed in control cells, could not be demon-strated after preincubation with LY294002 or U73122 (Fig.6D). As expected, PKC-� translocation in response toDHEA was prevented by the general PKC inhibitor bisin-dolylmaleimide GF109203X (36) and the specific PKC-�inhibitor LY379196 (Fig. 6D); however, it occurred nor-mally in adipocytes that had been pretreated with the MEKinhibitor PD098059 (Fig. 6D). These results demonstratethat membrane translocation of PKC-� in response toDHEA requires the activities of PI 3-kinase and PLC-�, aswell as the activity of PKC-� itself, independent of MEK/MAP kinase activity.Effects of PKC and PLC-� inhibitors on DHEA-stim-

ulated glucose transport. Specific PKC isoforms andPLC-� have been shown to mediate, at least in part, insulinstimulation of glucose transport in adipocytes (30,37,38).For investigating the potential involvement of PKC in theDHEA-dependent enhancement of glucose uptake, theeffects of the general PKC inhibitor GF109203X on DHEA-stimulated 2-deoxy-D-glucose uptake and transportertranslocation were examined. Pretreatment of 3T3-L1 adi-pocytes with 20 �mol/l GF109203X for 30 min abolishedDHEA stimulation of glucose transport (P 0.05 vs.

control DHEA; Fig. 7A). However, insulin-stimulated glu-cose transport was inhibited 50% by this PKC inhibitor(P 0.05 vs. control insulin; Fig. 7A). In a similar manner,GF109203X abrogated DHEA-induced translocation ofGLUT4 and GLUT1 (P 0.05 vs. DHEA control; Fig. 7B

and C) and partially inhibited insulin stimulation of theseresponses (Fig. 7B and C). Insulin-stimulated GLUT4 andGLUT1 protein levels in PM fractions from GF109203X-treated adipocytes were 70 and 50%, respectively, ofcontrol (Fig. 7C). DHEA stimulation of glucose transportwas also abrogated after treatment of 3T3-L1 adipocyteswith 30 �mol/l staurosporine, another PKC inhibitor (datanot shown). Thus, PKC activity is required for DHEA-dependent glucose transporter translocation and enhance-ment of glucose uptake. The role of PLC-� in glucosetransport stimulation by DHEA was investigated by usingthe specific PLC-� inhibitor U73122. Pretreatment of3T3-L1 adipocytes with 10 �mol/l U73122 for 30 mincompletely inhibited stimulation of 2-deoxy-D-glucose up-take by DHEA (P 0.05 vs. control DHEA) and, asreported previously (35), reduced insulin-induced 2-deoxy-D-glucose uptake 50% (P 0.05 vs. control insulin; Fig.7A).

The potential contribution of distinct PKC isoforms toDHEA-induced glucose transport was evaluated by pre-treating the adipocytes with Go6976, an inhibitor of con-ventional PKCs, Calphostin C, an inhibitor of novel PKCs,myr-PKC-�/�, an inhibitor of atypical PKCs, or the PCK-�

FIG. 5. Effects of PI 3-kinase inhibition on DHEA- and insulin-stimulated glucose transport and glucose transporter translocation to the PM.3T3-L1 adipocytes were incubated in serum-free medium for 16 h and subsequently left untreated or treated with 200 nmol/l Wortmannin or 50�mol/l LY294002, as indicated, for 30 min. Then, cells were studied in the basal state (open bars) or stimulated with 100 nmol/l insulin (Ins; filledbars) for 15 min or 100 �mol/l DHEA (DH; hatched bars) for 120 min. A: [3H]2-deoxy-D-glucose uptake rates. Mean � SE of three independentexperiments performed in triplicate. *P < 0.05 vs. control basal; #P < 0.05 vs. control insulin; §P < 0.05 vs. control DHEA. B: Immunoblottinganalysis of GLUT1 and GLUT4 glucose transporters in LDM and PM fractions. A clathrin immunoblot as control for protein loading is also shown.C: Quantification of GLUT1 (top) and GLUT4 (bottom) protein levels in PM and LDM fractions. Mean � SE of three independent experiments.*P < 0.05 vs. PM control basal; **P < 0.05 vs. LDM control basal; #P < 0.05 vs. PM control insulin; §P < 0.05 vs. PM control DHEA.



inhibitor LY379196 (33,37–39). DHEA stimulation of glu-cose transport was completely blocked by pretreatmentwith 50 nmol/l Go6976 and 30 nmol/l LY379196 (P 0.05vs. control DHEA), whereas Calphostin C and myr-PKC-�/�had no effect (Fig. 8). By contrast, insulin-stimulatedglucose transport was reduced 60% after pretreatmentwith myr-PKC-�/� (P 0.05 vs. control insulin), in agree-ment with previously reported results (38,39), and notaltered by Go6976, Calphostin C, or LY379196 (Fig. 8).Furthermore, DHEA stimulation of glucose transport wasabrogated in the presence of 100 �mol/l myristoylatedPKC-� inhibitory peptide, which had no effect on theinsulin response (data not shown). Altogether, these re-sults suggest that different PKC isoforms are involved inglucose transport stimulation by insulin and DHEA in3T3-L1 adipocytes.

DISCUSSION

We show that DHEA is capable of inducing a rapidstimulation of cellular glucose uptake in both human andmurine adipocytes by activating signaling responses thatlead to glucose transporter translocation to the cell sur-face. The effects of DHEA occur within minutes of adipo-cyte exposure to this steroid hormone and do not involvechanges in cellular content of GLUT1 or GLUT4. No

stimulation of glucose uptake is observed in response toother steroid hormones. The rapid onset and specificity ofDHEA action may be explained by DHEA binding to aspecific DHEA receptor at the cell surface. Evidence forhigh-affinity DHEA binding to isolated plasma membranesfrom endothelial cells has recently been provided, and ithas been shown that DHEA increases binding of GTP�S toG�i2,3, suggesting that the DHEA receptor may be coupledto this G protein (40). However, the molecular nature andtissue distribution of such DHEA receptors are still un-known. DHEA treatment of 3T3-L1 adipocytes resulted inIRS tyrosine phosphorylation and stimulation of PI 3-ki-nase activity in the absence of changes in tyrosine phos-phorylation of the insulin receptor that could be detectedby phosphotyrosine antibody immunoblotting. PI 3-kinaseactivation in an insulin-independent manner has beendemonstrated in adipocytes after overexpression of theconstitutively active G�i2, and it has been shown that thismay occur via inhibition of protein tyrosine phosphatase1B and subsequent enhancement of IRS tyrosine phos-phorylation (41). This suggests that DHEA activation ofthe IRS/PI 3-kinase pathway may occur by DHEA bindingto specific, although yet uncharacterized, G�i2-coupledcell-surface receptors.

The glucose transport enhancement by DHEA was as-

FIG. 6. Effects of DHEA and insulin on PKC activation. A: Translocation of PKC-�, PKC-�, and PKC-�/ from the cytosol to the membrane fraction.3T3-L1 adipocytes were serum-starved overnight and then treated with 1 �mol/l insulin (Ins) for 15 min or 100 �mol/l DHEA (DH) for theindicated times. Total membranes and the cytosolic fraction were prepared and analyzed by immunoblotting with antibodies specific to differentPKC isozymes, as indicated. A representative of three independent experiments is shown. B: Tyrosine phosphorylation of PKC-�2. 3T3-L1adipocytes were serum-starved overnight and then treated with 1 �mol/l insulin (Ins) for 15 min or 100 �mol/l DHEA (DH) for 30 min. Total cellextracts were subjected to immunoprecipitation with PKC-�2 antibodies followed by immunoblotting with phosphotyrosine (PY) antibodies.Equal loading of PKC-�2 protein was confirmed by immunoblotting with PKC-�2 antibodies. C: Intracellular Ca2�. 3T3-L1 adipocytes werestimulated with 100 �mol/l DHEA and then harvested at 0, 8, 16, 25, 33, 41, and 50 min. Intracellular concentrations of Ca2� ([Ca2�]i) weremeasured as described in RESEARCH DESIGN AND METHODS. Each point is the mean � SE of five independent experiments. *P < 0.05 vs. control. D:Effects of various signaling inhibitors on DHEA-induced PKC-� translocation. After treatment with 50 �mol/l LY294002, 10 �mol/l U73122, 20�mol/l GF109203X, 30 nmol/l LY379196, or 20 �mol/l PD098059, as indicated, for 20 min, 3T3-L1 cells were exposed to 100 �mol/l DHEA for 30min. Total membranes were analyzed by immunoblotting with PKC-� antibodies. A representative of three experiments is shown.



sociated with an approximately twofold increase inGLUT4 and GLUT1 content in adipocyte PM fractions. Inaddition, DHEA treatment resulted in increased GLUT4and GLUT1 immunostaining in PM lawns from 3T3-L1adipocytes. Evidence that DHEA enhances glucose uptakein rat adipocytes has recently been provided (16,17).However, these previous studies did not assess whetherthe effects of DHEA occur through translocation of glu-cose transporters or by an increase in transporter intrinsicactivity. Oral administration of DHEA to insulin-resistantGK and OLETF rats for 2 weeks resulted in increasedglucose uptake by adipocytes compared with untreatedanimals (17). In addition, DHEA has been shown to restoreinsulin sensitivity in obese Zucker rats (42) and to protectagainst the development of insulin resistance in rats fed ahigh-fat diet (14). Whereas the data in rodents clearlyindicate a beneficial effect of DHEA on insulin sensitivity,studies in humans have yielded less clear results (43). Forthe first time, this study shows a stimulatory effect ofDHEA on glucose uptake by human fat cells that occursthrough GLUT4 translocation to the cell surface. In humanadipocytes in vitro, DHEA was effective at the concentra-tion of 100 nmol/l, which is close to the physiologiccirculating DHEA levels in humans (0.7–15 nmol/l inchildhood, 1.5–30 nmol/l in puberty, 5–50 nmol/l in adult-hood, and 1–10 nmol/l after age 50). These findings may beimportant to foster further studies in human tissues,including the investigation of DHEA actions in adipose

tissue from different body locations and/or insulin-resis-tant subjects.

In this study, maximal glucose transport stimulation byDHEA was lower compared with insulin, because theeffects of DHEA were �30% and �60% of those of insulinin 3T3-L1 and human adipocytes, respectively. In addition,whereas DHEA and insulin were equally effective in induc-ing GLUT1 translocation to the PM, DHEA was lesseffective than insulin in promoting GLUT4 translocationand, in contrast to insulin, did not decrease GLUT4 in theLDM fraction. The lower magnitude of transport stimula-tion by DHEA compared with insulin thus may be ex-plained by the lower extent of GLUT4 translocation to thePM in response to this steroid hormone, possibly as aresult of differential regulation of specific intracellulartransporter pools. Multiple other hormones and drugs,such as endothelin-1, arachidonic acid, and troglitazone,that are capable of stimulating glucose uptake by adipo-cytes also show a somewhat limited effectiveness on thismetabolic response (6,7,9). The stimulation of glucosetransport by arachidonic acid was found to be less thantwofold over basal, this compound being less efficient thaninsulin in causing GLUT4 translocation to the PM (7).Similarly, endothelin-1 was shown to induce an approxi-mately twofold increase in 2-deoxy-D-glucose uptake in3T3-L1 adipocytes and to promote GLUT4 translocation toa lesser extent than insulin (6). These results suggest thatnot all of the intracellular GLUT4 vesicle pools are re-

FIG. 7. Effects of PKC or PLC-� inhibitors on DHEA- and insulin-stimulated glucose transport and glucose transporter translocation to the PM.3T3-L1 adipocytes were incubated in serum-free medium for 16 h and subsequently left untreated or treated with 20 �mol/l GF109203X or 10�mol/l U73122, as indicated, for 30 min. Then, cells were studied in the basal state (open bars) or stimulated with 100 nmol/l insulin (Ins; filledbars) for 15 min or 100 �mol/l DHEA (DH; hatched bars) for 120 min. A: [3H]2-deoxy-D-glucose uptake rates. Mean � SE of three independentexperiments performed in triplicate. *P < 0.05 vs. control basal; #P < 0.05 vs. control insulin; §P < 0.05 vs. control DHEA. B: Immunoblottinganalysis of GLUT1 and GLUT4 glucose transporters in LDM and PM fractions. A clathrin immunoblot as control for protein loading is also shown.C: Quantitation of GLUT1 (top) and GLUT4 (bottom) protein levels in PM and LDM fractions. Mean � SE of three independent experiments. *P <0.05 vs. PM control basal; **P < 0.05 vs. LDM control basal; #P < 0.05 vs. PM control insulin; §P < 0.05 vs. PM control DHEA.



cruited by DHEA, arachidonic acid, or endothelin-1, re-spectively, and this may explain the relatively low glucosetransport response to these molecules in adipocytes. An-other potential explanation for the lower effect of DHEAon transport compared with insulin may involve differen-tial regulation of transporter intrinsic activity. Comparedwith insulin, DHEA was less effective on transport by 70%and on GLUT4 translocation by 30%. Insulin may increaseglucose transport in 3T3-L1 adipocytes by a mechanismthat is independent of GLUT4 translocation to the cellsurface (44). The lack of complete correlation betweenGLUT4 translocation and glucose transport in response toinsulin and DHEA, respectively, may be potentially ex-plained by DHEA causing GLUT4 translocation withoutincreasing transporter intrinsic activity.

In 3T3-L1 adipocytes, DHEA was found to enhance PI3-kinase activity in IRS-1 and IRS-2 signaling complexesbut did not produce detectable activation of Akt, includingAkt2 that is the presumably more relevant isoform forstimulation of GLUT4 translocation (45,46), as assessed bymeasurements of Akt and GSK-3 phosphorylation. In ad-dition, DHEA did not activate MEK or ERK kinases. Aktactivation can be triggered in the presence of very limitedPI trisphosphate (PIP3) levels, as it occurs after activationof some G protein–coupled receptors (47), or in cellstreated with PI 3-kinase inhibitors in the presence ofangiotensin (5). The mechanism of this PI 3-kinase–inde-pendent activation of Akt remains to be elucidated. Con-versely, generation of PIP3 in IRS signaling complexes maybe not sufficient for Akt activation, as was observed inresponse to DHEA. Basic fibroblast growth factor, platelet-derived growth factor, and epidermal growth factor acti-vate PI 3-kinase in vivo to a similar extent but differ in theirability to activate PI 3-kinase–dependent signaling, includ-ing Akt phosphorylation (48). These findings may bepotentially explained by the existence of distinct subcel-lular compartments of active PI 3-kinase that are linked tospecific downstream signaling molecules. Thus, the

DHEA-induced PIP3 generation may occur within a sub-cellular compartment excluding Akt, to which other PIP3-regulatable signaling molecules, such as PLC-� (49,50), arerecruited upon stimulation.

Multiple PKC isoforms are activated by insulin in adipo-cytes. Insulin was shown to activate PKC-� and, to a lesserextent, PKC-� and PKC-�/� in 3T3-L1 adipocytes (29,30)and PKC-�/� in rat adipocytes (37). However, PKC-� andPKC-� activation by insulin has not been observed in allstudies (51,52). In this study, insulin was found to affectthe subcellular distribution of PKC-� and PKC-�/�,whereas it did not influence translocation or tyrosinephosphorylation of PKC-�. By contrast, DHEA inducedactivation of PKC-�2 but did not affect the � and �/�isoforms. The ability of DHEA to activate specific classicisoforms of PKC provides an example of selective activa-tion of PKC-� in the absence of detectable PKC-� or PKC-�stimulation, and a similar selectivity has been reported inmesangial cells exposed to glucosamine (53). DHEA-in-duced tyrosine phosphorylation and membrane transloca-tion of PKC-� were blocked by the PKC inhibitorsGF109203X and LY379196, indicating that these effectsrequire PKC-� activity. Inhibitors of PI 3-kinase and PLC-�also blocked PKC-� activation by DHEA. It has recentlybeen demonstrated that insulin-induced membrane trans-location of PKC-� in rat skeletal muscle cells requires PI3-kinase activity (32), but it is the first time that PKC-�translocation is found to be downstream of both PI3-kinase and PLC-�.

Inhibition of PI 3-kinase activity in 3T3-L1 adipocytesresulted in abrogation of glucose transport stimulation byDHEA and DHEA-induced translocation of GLUT4 andGLUT1. In addition, DHEA effects on the glucose transportsystem were prevented by a general PKC inhibitor and aPLC-� inhibitor, both of which partially inhibited theeffects of insulin by 50%. Finally, inhibitors of conventionalPKCs and the specific PKC-� inhibitor blocked DHEA-induced glucose transport but had no effect on the trans-port response to insulin. By contrast, insulin stimulation ofglucose transport was inhibited 50% by the myr-PKC-�/�compound, which blocks atypical PKCs, in agreement withprevious reports (37–39). Altogether, these results suggestthat both insulin and DHEA stimulate glucose transport byactivating PI 3-kinase and PLC-�, the latter playing apartial role in the insulin effect. Signaling by these hor-mones may then diverge at the level of atypical or classicalPKCs for insulin and DHEA, respectively. However, in onestudy in rat adipocytes (16), the Go6976 compound wasshown to have no effect on DHEA-stimulated glucosetransport, which was blocked by the myr-PKC-�/� com-pound. The opposite results found in our study could bepotentially explained by species-related differences in sig-naling reactions regulating glucose transport. Indeed, in3T3-L1 adipocytes, DHEA did not induce membrane trans-location of PKC-�/�, and its effect on transport was notprevented by the myr-PKC-�/� compound.

In conclusion, DHEA treatment of human and 3T3-L1adipocytes results in enhanced glucose transport ratesthrough GLUT4 and GLUT1 transporter translocation tothe cell surface. This effect seems to involve stimulation ofIRS tyrosine phosphorylation and the associated PI 3-ki-nase activity. Then, generation of PIP3 products in specific

FIG. 8. DHEA- and insulin-stimulated glucose transport in 3T3-L1adipocytes treated with inhibitors of conventional, novel, or atypicalPKC isozymes. Adipocytes in six-well plates were serum-starved over-night and then treated with 50 nmol/l Go6976, 500 nmol/l Calphostin C,50 �mol/l myristoylated PKC-�/ pseudosubstrate (myr-PKC-�/), or 30nmol/l LY379196, as indicated, for 30 min. [3H]2-deoxy-D-glucose up-take rates were then measured in the basal state (open bars), or afterstimulation with 100 nmol/l insulin (filled bars) for 15 min or 100�mol/l DHEA (hatched bars) for 2 h. Mean � SE of three independentexperiments performed in triplicate. *P < 0.05 vs. control basal; #P <0.05 vs. control insulin; §P < 0.05 vs. control DHEA.



cell compartments may lead to the activation of PLC-�resulting in increased calcium flux and PKC-�2 activation.These processes depict a novel insulin-independent signal-ing pathway potentially relevant for the regulation ofglucose uptake by fat cells.

ACKNOWLEDGMENTS

This work was supported by grants from the Ministerodell’Istruzione, Universita e Ricerca (Italy), the Cofinlab2000-Centro di Eccellenza “Genomica comparata: genicoinvolti in processi fisiopatologici in campo biomedico eagrario” (Italy), the Ministero della Salute (Italy), theSocieta Italiana di Diabetologia (Italy), and an educationalgrant from Pfizer Italia srl (ARADO Program) to F.Giorgino.

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Dehydroepiandrosterone Stimulates Glucose Uptake in …Dehydroepiandrosterone (DHEA) has been shown...

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