GLUT4-overexpressing engineered muscle constructs as a therapeutic platform to normalize glycemia in diabetic mice.

Skeletal muscle insulin resistance is a main defect in type 2 diabetes (T2D), which is associated with impaired function and content of glucose transporter type 4 (GLUT4). GLUT4 overexpression in skeletal muscle tissue can improve glucose homeostasis. Therefore, we created an engineered muscle construct (EMC) composed of GLUT4-overexpressing (OEG4) cells. The ability of the engineered implants to reduce fasting glucose levels was tested in diet-induced obesity mice. Decrease and stabilization of basal glucose levels were apparent up to 4 months after implantation. Analysis of the retrieved constructs showed elevated expression of myokines and proteins related to metabolic processes. In addition, we validated the efficiency of OEG4-EMCs in insulin-resistant mice. Following high glucose load administration, mice showed improved glucose tolerance. Our data indicate that OEG4-EMC implant is an efficient mode for restoring insulin sensitivity and improving glucose homeostasis in diabetic mice. Such procedure is a potential innovative modality for T2D therapy.

Cytochrome P-450 CYP2E1 knockout mice are protected against high-fat diet-induced obesity and insulin resistance.

Conventional (whole body) CYP2E1 knockout mice displayed protection against high-fat diet-induced weight gain, obesity, and hyperlipidemia with increased energy expenditure despite normal food intake and spontaneous locomotor activity. In addition, the CYP2E1 knockout mice displayed a marked improvement in glucose tolerance on both normal chow and high-fat diets. Euglycemic-hyperinsulinemic clamps demonstrated a marked protection against high-fat diet-induced insulin resistance in CYP2E1 knockout mice, with enhanced adipose tissue glucose uptake and insulin suppression of hepatic glucose output. In parallel, adipose tissue was protected against high-fat diet-induced proinflammatory cytokine production. Taken together, these data demonstrate that the CYP2E1 deletion protects mice against high-fat diet-induced insulin resistance with improved glucose homeostasis in vivo.

Transcriptional regulation of the GLUT4 gene: from PPAR-gamma and FOXO1 to FFA and inflammation.

The insulin-responsive glucose transporter 4 (GLUT4) has a major role in glucose uptake and metabolism in insulin target tissues (i.e. adipose and muscle cells). In these tissues, the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors and the winged-helix-forkhead box class O (FOXO) family of factors are two key families of transcription factors that regulate glucose homeostasis and insulin responsiveness. Type 2 diabetes mellitus and obesity are associated with impaired regulation of GLUT4 gene expression and elevated levels of free fatty acids and proinflammatory factors. Based on our studies of the interplay between PPAR-gamma, FOXO1 and free fatty acids, and inflammation in regulating GLUT4 transcription in sickness and in health, we suggest a novel paradigm to increase insulin sensitivity in bona fide insulin target cells.

FOXO1 represses peroxisome proliferator-activated receptor-gamma1 and -gamma2 gene promoters in primary adipocytes. A novel paradigm to increase insulin sensitivity.

FOXO1 and peroxisome proliferator-activated receptor-gamma (PPARgamma) are crucial transcription factors that regulate glucose metabolism and insulin responsiveness in insulin target tissues. We have shown that, in primary rat adipocytes, both factors regulate transcription of the insulin-responsive GLUT4 gene and that PPARgamma2 detachment from the GLUT4 promoter upon thiazolidinedione binding up-regulates GLUT4 gene expression, thus increasing insulin sensitivity (Armoni, M., Kritz, N., Harel, C., Bar-Yoseph, F., Chen, H., Quon, M. J., and Karnieli, E. (2003) J. Biol. Chem. 278, 30614-30623). However, the mechanisms regulating PPARgamma gene transcription are largely unknown. We studied the effects of FOXO1 on human PPARgamma gene expression in primary rat adipocytes and found that both genes are endogenously expressed. FOXO1 coexpression dose-dependently repressed transcription from either the PPARgamma 1 or PPARgamma2 promoter reporter by 65%, whereas insulin (100 nm, 20-24 h) either partially or completely reversed this effect. Phosphorylation-defective FOXO1 mutants T24A, S256A, S319A, and T24A/S256A/S319A still repressed the PPARgamma1 promoter and partially lost their effects on the PPARgamma2 promoter in either basal or insulin-stimulated cells. Use of DNA binding-defective FOXO1 (H215R) indicated that this domain is crucial for FOXO1 repression of the PPARgamma2 (but not PPARgamma1) promoter. Progressive 5'-deletion and gel retardation analyses revealed that this repression involves direct and specific binding of FOXO1 to the PPARgamma2 promoter; chromatin immunoprecipitation analysis confirmed that this binding occurs in cellulo. We suggest a novel paradigm to increase insulin sensitivity in adipocytes in which FOXO1 repression of PPARgamma, the latter being a repressor of the GLUT4 promoter, consequently leads to GLUT4 derepression/up-regulation, thus enhancing cellular insulin sensitivity. The newly identified FOXO1-binding site on the PPARgamma2 promoter may serve as a therapeutic target for type 2 diabetes.

Free fatty acids repress the GLUT4 gene expression in cardiac muscle via novel response elements.

Hyperlipidemia (HL) impairs cardiac glucose homeostasis, but the molecular mechanisms involved are yet unclear. We examined HL-regulated GLUT4 and peroxisome proliferator-activated receptor (PPAR) gamma gene expression in human cardiac muscle. Compared with control patients, GLUT4 protein levels were 30% lower in human cardiac muscle biopsies from patients with HL and/or type 2 diabetes mellitus, whereas GLUT4 mRNA levels were unchanged. PPARgamma mRNA levels were 30-50% lower in patients with HL and/or diabetes mellitus type 2 than in controls. Reporter studies in H9C2 cardiomyotubes showed that HL in vitro, induced by high levels of arachidonic (AA) stearic, linoleic, and oleic acids (24 h, 200 mum) repressed transcription from the GLUT4 promoter; AA also repressed transcription from the PPARgamma1 and PPARgamma2 promoters. Co-expression of PPARgamma2 repressed GLUT4 promoter activity, and the addition of AA further enhanced this effect. 5'-Deletion analysis revealed three GLUT4 promoter regions that accounted for AA-mediated effects: two repression-mediating sequences at -443/-423 bp and -222/-197 bp, the deletion of either or both of which led to a partial derepression of promoter activity, and a third derepression-mediating sequence at -612/-587 bp that was required for sustaining this derepression effect. Electromobility shift assay further shows that AA enhanced binding to two of the three regions of cardiac nuclear protein(s), the nature of which is still unknown. We propose that HL, exhibited as a high free fatty acid level, modulates GLUT4 gene expression in cardiac muscle via a complex mechanism that includes: (a) binding of AA mediator proteins to three newly identified response elements on the GLUT4 promoter gene and (b) repression of GLUT4 and the PPARgamma genes by AA.

Peroxisome proliferator-activated receptor-gamma represses GLUT4 promoter activity in primary adipocytes, and rosiglitazone alleviates this effect.

The synthetic thiazolidinedione ligands of peroxisome proliferator-activated receptor-gamma (PPARgamma) improve insulin sensitivity in type II diabetes and induce GLUT4 mRNA expression in fat and muscle. However, the molecular mechanisms involved are still unclear. We studied the regulatory effects of PPARgamma and its ligands on GLUT4 gene expression in primary rat adipocytes and CHO-K1 cells cotransfected with PPARgamma and the GLUT4 promoter reporter. PPARgamma1 and PPARgamma2 repressed the activity of the GLUT4 promoter in a dose-dependent manner. Whereas this repression was augmented by the natural ligand 15Delta-prostaglandin J2, it was completely alleviated by rosiglitazone (Rg). Ligand binding-defective mutants PPARgamma1-L468A/E471A and PPARgamma2-L496A/E499A retained the repression effect, which was unaffected by Rg, whereas the PPARgamma2-S112A mutant exhibited a 50% reduced capacity to repress GLUT4 promoter activity. The -66/+163 bp GLUT4 promoter region was sufficient to mediate PPARgamma inhibitory effects. The PPARgamma/retinoid X receptor-alpha heterodimer directly bound to this region, whereas binding was abolished in the presence of Rg. Thus, we show that PPARgamma represses transcriptional activity of the GLUT4 promoter via direct and specific binding of PPARgamma/retinoid X receptor-alpha to the GLUT4 promoter. This effect requires an intact Ser112 phosphorylation site on PPARgamma and is completely alleviated by Rg, acting via its ligand-binding domain. These data suggest a novel mechanism by which Rg exerts its antidiabetic effects via detaching PPARgamma from the GLUT4 gene promoter, thus leading to increased GLUT4 expression and enhanced insulin sensitivity.

PAX3/forkhead homolog in rhabdomyosarcoma oncoprotein activates glucose transporter 4 gene expression in vivo and in vitro.

Increased levels of glucose uptake and increased expression of the glucose transporter (GLUT) genes are characteristic features of tumors. In the muscle-derived tumor alveolar rhabdomyosarcoma (ARMS), a chromosomal translocation t(2:13) generates the PAX3/forkhead homolog in rhabdomyosarcoma (FKHR) oncoprotein. In muscle tissues, glucose transport is primarily mediated by GLUT4. However, the mechanisms that regulate GLUT4 gene expression in tumor tissues are largely unknown. Therefore, we evaluated the role of PAX3/FKHR in the regulation of GLUT4 gene expression in muscle tumorigenesis. GLUT4 mRNA and protein were detected in ARMS-derived human biopsies and in ARMS-derived RH30 myoblasts, which both express the PAX3/FKHR chimeric protein, but not in either C2C12 or embryonal rhabdomyosarcoma-derived myoblasts. GLUT4 was functionally active in RH30 cells, because insulin induced a 1.4-fold stimulation of basal 2-deoxyglucose uptake rates. Coexpression of PAX3/FKHR increased basal transcriptional activity from a GLUT4 promoter reporter (GLUT4-P) in C2C12, SaOS-2, and Chinese hamster ovary-K1 cells in a dose-dependent and tissue-specific manner. PAX3/FKHR mutants with deletions in either the homeodomain (DeltaHD) or the FKHR-derived activation domain (DeltaFKHR), or in which the PAX3-derived paired domain (PD) was point-mutated (PD-R56L), were unable to activate GLUT4-P. Progressive 5'-deletion analysis of GLUT4-P further identified a specific region of the promoter, -66/+163 bp, which retained about 65% of the full transactivation effect. EMSA studies established that the PAX3/FKHR protein directly and specifically binds to this region and to a shorter fragment, -4/+36 bp, that contains potential binding sites for HD and PD, but not to a -4/+36-bp fragment whose HD and PD sites have been mutated. Thus, the functional interaction of PAX3/FKHR with GLUT4-P appears to require all of the functional domains of PAX3/FKHR, as well as a -4/+36-bp region within the GLUT4 promoter. Taken together, the data suggest that the GLUT4 gene is a downstream target of PAX3/FKHR and that GLUT4 is aberrantly transactivated by this oncoprotein both in vivo and in vitro.