Estradiol stimulates adipogenesis and Slc2a4/GLUT4 expression via ESR1-mediated activation of CEBPA.

The ability of adipose tissue to expand is dependent on adipocyte differentiation and adipose tissue glucose disposal. The CCAAT/enhancer-binding protein alpha (CEBPA) enhances the expression of the Slc2a4 gene and GLUT4 protein, which are markers of adipocyte differentiation/glucose disposal. We hypothesized estradiol (E2) facilitates adipocyte differentiation/glucose disposal by an estrogen receptor 1 (ESR1)-dependent and CEBPA-mediated mechanism. Our results suggest that E2 (10 nM) has a positive effect on 3T3-L1 adipocyte differentiation (days 2-8), lipid accumulation, Slc2a4 and Cebpa mRNA expression, total GLUT4 and nuclear CEBPA contents, and CEBP/Slc2a4-binding activity. Esr1 silencing (∼50%) in mature adipocytes abrogates the 24-h E2 effects on nuclear CEBPA content, Slc2a4/GLUT4 expression and GLUT4 translocation to the cell membrane. Thus, E2 stimulates adipocyte differentiation and Slc2a4/GLUT4 expression in an ESR1/CEBPA-mediated pathway. Our data provide mechanistic insight demonstrating E2 participates in adipose-tissue differentiation and glucose transporter expression which ultimately can improve adipose tissue expandability and glycemic control.

Impact of flaxseed and soy nuts as dietary supplements on lipid profile, insulin sensitivity, and GLUT4 expression in ovariectomized rats.

We assessed the effects of a diet with flaxseed or soy nuts versus estradiol on the lipid profile, insulin sensitivity, and glucose transporter type 4 (GLUT4) expression in ovariectomized female rats. Forty-four female Wistar rats (90 days old) underwent ovariectomy and were divided into 4 groups: C (standard diet), E (standard diet + subcutaneous 17ß-estradiol pellets), L (standard diet + flaxseed + subcutaneous placebo pellets), and S (standard diet + soy nuts + subcutaneous placebo pellets). Customized diets and the insertion of pellets were started 21 days after ovariectomy and were continued for another 21 days. We measured body mass, insulin tolerance, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, and GLUT4 (in cardiac and adipose tissues). We found a lower body mass and a lower Lee index in group E and a trend toward improved insulin sensitivity in group S (p = 0.066). Groups L and S showed a better lipid profile when compared with group C. Microsomal GLUT4 increased in group L (in cardiac and adipose tissues), and plasma membrane GLUT4 increased in groups E, L, and S (in both tissues). We conclude that flaxseed and soy nuts as dietary supplements improve lipid profile and increase GLUT4 expression.

Estradiol-induced regulation of GLUT4 in 3T3-L1 cells: involvement of ESR1 and AKT activation.

Impaired insulin-stimulated glucose uptake involves reduced expression of the GLUT4 (solute carrier family 2 facilitated glucose transporter member 4, SLC2A4 gene). 17ß-estradiol (E2) modulates SLC2A4/GLUT4 expression, but the involved mechanisms are unclear. Although E2 exerts biological effects by binding to estrogen receptors 1/2 (ESR1/2), which are nuclear transcriptional factors; extranuclear effects have also been proposed. We hypothesize that E2 regulates GLUT4 through an extranuclear ESR1 mechanism. Thus, we investigated the effects of E2 upon (1) subcellular distribution of ESRs and the proto-oncogene tyrosine-protein kinases (SRC) involvement; (2) serine/threonine-protein kinase (AKT) activation; (3) Slc2a4/GLUT4 expression and (4) GLUT4 subcellular distribution and glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were cultivated or not with E2 for 24 h, and additionally treated or not with ESR1-selective agonist (PPT), ESR1-selective antagonist (MPP) or selective SRC inhibitor (PP2). Subcellular distribution of ESR1, ESR2 and GLUT4 was analyzed by immunocytochemistry; Slc2a4 mRNA and GLUT4 were quantified by qPCR and Western blotting, respectively; plasma membrane GLUT4 translocation and glucose uptake were analyzed under insulin stimulus for 20 min or not. E2 induced (1) translocation of ESR1, but not of ESR2, from nucleus to plasma membrane and AKT phosphorylation, effects mimicked by PPT and blocked by MPP and PP2; (2) increased Slc2a4/GLUT4 expression and (3) increased insulin-stimulated GLUT4 translocation and glucose uptake. In conclusion, E2 treatment promoted a SRC-mediated nucleus-plasma membrane shuttle of ESR1, and increased AKT phosphorylation, Slc2a4/GLUT4 expression and plasma membrane GLUT4 translocation; consequently, improving insulin-stimulated glucose uptake. These results unravel mechanisms through which estrogen improves insulin sensitivity.

Resveratrol Improves Glycemic Control in Type 2 Diabetic Obese Mice by Regulating Glucose Transporter Expression in Skeletal Muscle and Liver.

Insulin resistance participates in the glycaemic control disruption in type 2 diabetes mellitus (T2DM), by reducing muscle glucose influx and increasing liver glucose efflux. GLUT4 (Slc2a4 gene) and GLUT2 (Slc2a2 gene) proteins play a fundamental role in the muscle and liver glucose fluxes, respectively. Resveratrol is a polyphenol suggested to have an insulin sensitizer effect; however, this effect, and related mechanisms, have not been clearly demonstrated in T2DM. We hypothesized that resveratrol can improve glycaemic control by restoring GLUT4 and GLUT2 expression in muscle and liver. Mice were rendered obese T2DM in adult life by neonatal injection of monosodium glutamate. Then, T2DM mice were treated with resveratrol for 60 days or not. Glycaemic homeostasis, GLUT4, GLUT2, and SIRT1 (sirtuin 1) proteins (Western blotting); Slc2a4, Slc2a2, and Pck1 (key gluconeogenic enzyme codifier) mRNAs (RT-qPCR); and hepatic glucose efflux were analysed. T2DM mice revealed: high plasma concentration of glucose, fructosamine, and insulin; insulin resistance (insulin tolerance test); decreased Slc2a4/GLUT4 content in gastrocnemius and increased Slc2a2/GLUT2 content in liver; and increased Pck1 mRNA and gluconeogenic activity (pyruvate tolerance test) in liver. All alterations were restored by resveratrol treatment. Additionally, in both muscle and liver, resveratrol increased SIRT1 nuclear content, which must participate in gene expression regulations. In sum, the results indisputably reveals that resveratrol improves glycaemic control in T2DM, and that involves an increase in muscle Slc2a4/GLUT4 and a decrease in liver Slc2a2/GLUT2 expression. This study contributes to our understanding how resveratrol might be prescribed for T2DM according to the principles of evidence-based medicine.

Hormetic modulation of hepatic insulin sensitivity by advanced glycation end products.

Because of the paucity of information regarding metabolic effects of advanced glycation end products (AGEs) on liver, we evaluated effects of AGEs chronic administration in (1) insulin sensitivity; (2) hepatic expression of genes involved in AGEs, glucose and fat metabolism, oxidative stress and inflammation and; (3) hepatic morphology and glycogen content. Rats received intraperitoneally albumin modified (AlbAGE) or not by advanced glycation for 12 weeks. AlbAGE induced whole-body insulin resistance concomitantly with increased hepatic insulin sensitivity, evidenced by activation of AKT, inactivation of GSK3, increased hepatic glycogen content, and decreased expression of gluconeogenesis genes. Additionally there was reduction in hepatic fat content, in expression of lipogenic, pro-inflamatory and pro-oxidative genes and increase in reactive oxygen species and in nuclear expression of NRF2, a transcription factor essential to cytoprotective response. Although considered toxic, AGEs become protective when administered chronically, stimulating AKT signaling, which is involved in cellular defense and insulin sensitivity.

N-Acetyl Cysteine Attenuated the Deleterious Effects of Advanced Glycation End-Products on the Kidney of Non-Diabetic Rats.

AIM: To assess the renal effects of chronic exposure to advanced glycation end-products (AGEs) in the absence of diabetes and the potential impact of concomitant treatment with the antioxidant N-acetyl cysteine (NAC). METHODS: Wistar rats received intraperitoneally 20 mg/kg/day of albumin modified (AlbAGE) or not (AlbC) by advanced glycation for 12 weeks and oral NAC (600mg/L; AlbAGE+NAC and AlbC+NAC, respectively). Biochemical, urinary and renal morphological analyses; carboxymethyl-lysine (CML, an AGE), CD68 (macrophage infiltration), and 4-hydroxynonenal (4-HNE, marker of oxidative stress) immunostaining; intrarenal mRNA expression of genes belonging to pathways related to AGEs (Ager, Ddost, Nfkb1), renin-angiotensin system (Agt, Ren, Ace), fibrosis (Tgfb1, Col4a1), oxidative stress (Nox4, Txnip), and apoptosis (Bax, Bcl2); and reactive oxidative species (ROS) content were performed. RESULTS: AlbAGE significantly increased urine protein-to-creatinine ratio; glomerular area; renal CML content and macrophage infiltration; expression of Ager, Nfkb1, Agt, Ren, Tgfb1, Col4a1, Txnip, Bax/Bcl2 ratio; and 4-HNE and ROS contents. Some of these effects were attenuated by NAC concomitant treatment. CONCLUSION: Because AGEs are highly consumed in modern diets and implicated in the progression of different kidney diseases, NAC could be a therapeutic intervention to decrease renal damage, considering that long-term restriction of dietary AGEs is difficult to achieve in practice.

In type 2 diabetes mellitus glycated albumin alters macrophage gene expression impairing ABCA1-mediated cholesterol efflux.

Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE-albumin induces oxidative stress, which is linked to a reduction in ABCA-1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2-HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C- or DM2-HSA to measure the (14) C-cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real-time RT-PCR. An increased mean mass was observed in DM2-HSA compared with C-HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3 , and HDL2 was impaired in DM2-HSA-treated cells, which was related to greater intracellular lipid accumulation. DM2-HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA-1 protein content decreased. Compared with C-HAS-treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2-HSA treatment. Stearoyl-Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2-HSA. The level of glycation that occurs in vivo in DM2-HSA-treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2.

Modelo de obesidade induzida por dieta hiperlipídica e associada à resistência à ação da insulina e intolerância à glicose / Model of high-fat diet-induced obesity associated to insulin resistance and glucose intolerance

OBJETIVO: Validar um modelo de obesidade induzida por dieta hiperlipídica, de baixo custo, fácil reprodutibilidade, que mimetizasse características observadas no humano e viabilizasse posteriores proposições terapêuticas. MATERIAIS E MÉTODOS: Dezesseis camundongos Swiss receberam dieta padrão (DP) ou dieta hiperlipídica (DH), durante 10 semanas. RESULTADOS: Embora o grupo DP tenha apresentado maior consumo de água (p < 0,01) e ração (p < 0,001), o grupo DH apresentou maior ganho de peso corpóreo (p < 0,5) e aumento de coxins adiposos (p < 0,001), favorecendo maior índice de adiposidade (p < 0,001), glicemia (p < 0,01) e área sob a curva nos testes de tolerância à insulina (p < 0,001) e à glicose (p < 0,01). CONCLUSÃO: Validou-se um modelo de obesidade induzida por dieta hiperlipídica associada à resistência à ação da insulina e à intolerância à glicose, em um período de 10 semanas.

OBJECTIVE: Validate a model of high-fat diet-induced obesity, of low cost, easy reproducibility, that could express characteristics observed in human, and would enable subsequent therapy proposals. MATERIALS AND METHODS: Sixteen Swiss mice received a standard diet (DP) or high-fat diet (DH) for 10 weeks. RESULTS: Although the DP group had greater water (p < 0.01) and feed (p < 0.001) consumption, the DH group had greater body weight (p < 0.5) and adipose tissue gain (p < 0.001), favoring higher adiposity index (p < 0.001), glucose (p < 0.01), and area under the curve in the insulin (p < 0.001) and glucose (p < 0.01) tolerance tests. CONCLUSION: A high-fat diet-induced obesity model has been validated, which was also associated with insulin resistance and glucose intolerance after a period of 10 weeks.

Aerobic exercise training induces metabolic benefits in rats with metabolic syndrome independent of dietary changes

OBJECTIVES: We evaluated the effects of aerobic exercise training without dietary changes on cardiovascular and metabolic variables and on the expression of glucose transporter Type 4 in rats with metabolic syndrome. METHODS: Twenty male spontaneously hypertensive rats received monosodium glutamate during the neonatal period. The animals were allocated to the following groups: MS (sedentary metabolic syndrome), MS-T (trained on a treadmill for 1 hour/day, 5 days/week for 10 weeks), H (sedentary spontaneously hypertensive rats) and H-T (trained spontaneously hypertensive rats). The Lee index, blood pressure (tail-cuff system), insulin sensitivity (insulin tolerance test) and functional capacity were evaluated before and after 10 weeks of training. Glucose transporter Type 4 expression was analyzed using Western blotting. The data were compared using analysis of variance (ANOVA) (p<0.05). RESULTS: At baseline, the MS rats exhibited lower insulin sensitivity and increased Lee index compared with the H rats. Training decreased the body weight and Lee index of the MS rats (MS-T vs. MS), but not of the H rats (H-T vs. H). There were no differences in food intake between the groups. At the end of the experiments, the systolic blood pressure was lower in the two trained groups than in their sedentary controls. Whole-body insulin sensitivity increased in the trained groups. Glucose transporter Type 4 content increased in the heart, white adipose tissue and gastrocnemius muscle of the trained groups relative to their respective untrained groups. CONCLUSION: In conclusion, the present study shows that an isolated aerobic exercise training intervention is an efficient means of improving several components of metabolic syndrome, that is, training reduces obesity and hypertension and increases insulin sensitivity. .

Altered response of skeletal muscle to IL-6 in type 2 diabetic patients.

Interleukin-6 (IL-6) has a dual role in modulating insulin sensitivity, with evidence for this cytokine as both an enhancer and inhibitor of insulin action. We determined the effect of IL-6 exposure on glucose and lipid metabolism in cultured myotubes established from people with normal glucose tolerance or type 2 diabetes. Acute IL-6 exposure increased glycogen synthesis, glucose uptake, and signal transducer and activator of transcription 3 (STAT3) phosphorylation in cultured myotubes from normal glucose tolerant subjects. However, in type 2 diabetic patients, IL-6 was without effect on glucose metabolism and STAT3 signaling, concomitant with increased suppressor of cytokine signaling 3 (SOCS3) expression. IL-6 increased fatty acid oxidation in myotubes from type 2 diabetic and normal glucose tolerant subjects. Expression of IL-6, IL-6 receptor (IL-6R), or glycoprotein 130, as well as IL-6 secretion, was unaltered between cultured myotubes from normal glucose tolerant or type 2 diabetic subjects. Circulating serum IL-6 concentration was unaltered between normal glucose tolerant and type 2 diabetic subjects. In summary, skeletal muscle cells from type 2 diabetic patients display selective IL-6 resistance for glucose rather than lipid metabolism. In conclusion, IL-6 appears to play a differential role in regulating metabolism in type 2 diabetic patients compared with normal glucose tolerant subjects.

GLUT4 content decreases along with insulin resistance and high levels of inflammatory markers in rats with metabolic syndrome.

BACKGROUND: Metabolic syndrome is characterized by insulin resistance, which is closely related to GLUT4 content in insulin-sensitive tissues. Thus, we evaluated the GLUT4 expression, insulin resistance and inflammation, characteristics of the metabolic syndrome, in an experimental model. METHODS: Spontaneously hypertensive neonate rats (18/group) were treated with monosodium glutamate (MetS) during 9 days, and compared with Wistar-Kyoto (C) and saline-treated SHR (H). Blood pressure (BP) and lipid levels, C-reactive protein (CRP), interleukin 6 (IL-6), TNF-α and adiponectin were evaluated. GLUT4 protein was analysed in the heart, white adipose tissue and gastrocnemius. Studies were performed at 3 (3-mo), 6 (6-mo) and 9 (9-mo) months of age. RESULTS: MetS rats were more insulin resistant (p<0.001, all ages) and had higher BP (3-mo: p<0.001, 6-mo: p = 0.001, 9-mo: p = 0.015) as compared to C. At 6 months, CRP, IL-6 and TNF-α were higher (p<0.001, all comparisons) in MetS rats vs H, but adiponectin was lower in MetS at 9 months (MetS: 32 ± 2, H: 42 ± 2, C: 45 ± 2 pg/mL; p<0.001). GLUT4 protein was reduced in MetS as compared to C rats at 3, 6 and 9-mo, respectively (Heart: 54%, 50% and 57%; Gastrocnemius: 37%, 56% and 50%; Adipose tissue: 69%, 61% and 69%). CONCLUSIONS: MSG-treated SHR presented all metabolic syndrome characteristics, as well as reduced GLUT4 content, which must play a key role in the impaired glycemic homeostasis of the metabolic syndrome.

Quercetin decreases inflammatory response and increases insulin action in skeletal muscle of ob/ob mice and in L6 myotubes.

Quercetin is a potent anti-inflammatory flavonoid, but its capacity to modulate insulin sensitivity in obese insulin resistant conditions is unknown. This study investigated the effect of quercetin treatment upon insulin sensitivity of ob/ob mice and its potential molecular mechanisms. Obese ob/ob mice were treated with quercetin for 10 weeks, and L6 myotubes were treated with either palmitate or tumor necrosis factor-α (TNFα) plus quercetin. Cells and muscles were processed for analysis of glucose transporter 4 (GLUT4), TNFα and inducible nitric oxide synthase (iNOS) expression, and c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor-κB (NF-κB) kinase (IκK) phosphorylation. Myotubes were assayed for glucose uptake and NF-κB translocation. Chromatin immunoprecipitation assessed NF-κB binding to GLUT4 promoter. Quercetin treatment improved whole body insulin sensitivity by increasing GLUT4 expression and decreasing JNK phosphorylation, and TNFα and iNOS expression in skeletal muscle. Quercetin suppressed palmitate-induced upregulation of TNFα and iNOS and restored normal levels of GLUT4 in myotubes. In parallel, quercetin suppressed TNFα-induced reduction of glucose uptake in myotubes. Nuclear accumulation of NF-κB in myotubes and binding of NF-κB to GLUT4 promoter in muscles of ob/ob mice were also reduced by quercetin. We demonstrated that quercetin decreased the inflammatory status in skeletal muscle of obese mice and in L6 myotubes. This effect was followed by increased muscle GLUT4, with parallel improvement of insulin sensitivity. These results point out quercetin as a putative strategy to manage inflammatory-related insulin resistance.

SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats.

Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P < 0.001 vs. WKY), and diabetes decreased this activity (∼50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (∼30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

Anti-inflammatory effect of atorvastatin ameliorates insulin resistance in monosodium glutamate-treated obese mice.

Considering that inflammation contributes to obesity-induced insulin resistance and that statins have been reported to have other effects beyond cholesterol lowering, the present study aimed to investigate whether atorvastatin treatment has anti-inflammatory action in white adipose tissue of obese mice, consequently improving insulin sensitivity. Insulin sensitivity in vivo (by insulin tolerance test); metabolic-hormonal profile; plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and adiponectin; adipose tissue immunohistochemistry; glucose transporter (GLUT) 4; adiponectin; TNF-alpha; IL-1 beta; and IL-6 gene expression; and I kappaB kinase (IKK)-alpha/beta activity were assessed in 23-week-old monosodium glutamate-induced obese mice untreated or treated with atorvastatin for 4 weeks. Insulin-resistant obese mice had increased plasma triglyceride, insulin, TNF-alpha, and IL-6 plasma levels. Adipose tissue of obese animals showed increased macrophage infiltration, IKK-alpha (42%, P < .05) and IKK-beta (73%, P < .05) phosphorylation, and TNF-alpha and IL-6 messenger RNA (mRNA) ( approximately 15%, P < .05) levels, and decreased GLUT4 mRNA and protein (30%, P < .05) levels. Atorvastatin treatment lowered cholesterol, triglyceride, insulin, TNF-alpha, and IL-6 plasma levels, and restored whole-body insulin sensitivity. In adipose tissue, atorvastatin decreased macrophage infiltration and normalized IKK-alpha/beta phosphorylation; TNF-alpha, IL-6, and GLUT4 mRNA; and GLUT4 protein to control levels. The present findings demonstrate that atorvastatin has anti-inflammatory effects on adipose tissue of obese mice, which may be important to its local and whole-body insulin-sensitization effects.

Postpartum glycemic homeostasis in early lactating rats is accompanied by transient and specific increase of soleus insulin response through IRS2/AKT pathway.

It is known that at the moment of delivery immediate lost of conceptus (main site of glucose disposal in late pregnancy) is not able to disturb glucose homeostasis in early lactating mothers. However, the mechanism by which this adaptation takes place in early lactation is still unknown. Most studies concerning insulin sensitivity in lactating rats were carried out at 11-13 days postpartum and did not describe functional changes in insulin response in early lactation. Here we show that lactation hypersensitivity to insulin is observed as early as 3 days after delivery (L3). We show that the oxidative soleus muscle displays a transient increased maximal insulin-induced glucose uptake and CO2 production, which is temporally limited to L3. Response of soleus muscle was accompanied by an increase in glucose transporter 4 (GLUT4) content at L3. This adaptive response was not detected in the glycolytic plantaris muscle, which displayed lower content of GLUT4. We also found that soleus muscle from early lactating rats have higher insulin receptor expression and tyrosine phosphorylation. Downstream steps of insulin signaling pathway; e.g., insulin receptor substrate 2 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase were also upregulated in soleus muscle. In parallel, protein tyrosine phosphatase 1B expression, a negative regulator of insulin signal, was reduced. Importantly, all of these molecular alterations were time limited to L3 and were not observed in plantaris muscle. These results suggest that improved insulin action in oxidative, but not in glycolytic muscle might contribute to achievement of glucose homeostasis postpartum.

Muscle GLUT4 regulation by estrogen receptors ERbeta and ERalpha.

Estrogen is known to influence glucose homeostasis with dominant effects in the liver, but the role of estrogen receptors in muscle glucose metabolism is unknown. In the present study, we investigated the expression of the two estrogen receptors, ERalpha and ERbeta, and their influence on regulation of the glucose transporter, GLUT4, and its associated structural protein, caveolin-1, in mouse gastrocnemius muscle. Immunohistochemical analysis revealed that ERalpha and ERbeta are coexpressed in the nuclei of most muscle cells, and that their levels were not affected by absence of estradiol [in aromatase-knockout (ArKO) mice]. GLUT4 expression on the muscle cell membrane was not affected by loss of ERbeta but was extremely reduced in ERalpha(-/-) mice and elevated in ArKO mice. RT-PCR confirmed a parallel reduction in GLUT4 mRNA levels in ERalpha(-/-) mice. Upon treatment of ArKO mice with the ERbeta agonist 2,3-bis(4-hydroxyphenyl)propionitrile, GLUT4 expression was reduced. By immunofluorescence and Western blotting, caveolin-1 expression was higher in ArKO mice and lower in ERbeta(-/-) and ERalpha(-/-) mice than in WT littermates. GLUT4 and caveolin-1 were colocalized in WT and ArKO mice but not in ERbeta(-/-) and ERalpha(-/-) mice. These results reveal that ERalpha is a positive regulator of GLUT4 expression, whereas ERbeta has a suppressive role. Both ERbeta and ERalpha are necessary for optimal caveolin-1 expression. Taken together, these results indicate that colocalization of caveolin-1 and GLUT4 is not an absolute requirement for muscle glucose metabolism but that reduction in GLUT4 could be contributing to the insulin resistance observed in ERalpha(-/-) mice.