Chava Harel

FOXO1 Represses Peroxisome Proliferator-activated Receptor-γ1 and -γ2 Gene Promoters in Primary Adipocytes A NOVEL PARADIGM TO INCREASE INSULIN SENSITIVITY

FOXO1 and peroxisome proliferator-activated receptor-γ (PPARγ) 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 PPARγ2 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 PPARγ gene transcription are largely unknown. We studied the effects of FOXO1 on human PPARγ gene expression in primary rat adipocytes and found that both genes are endogenously expressed. FOXO1 coexpression dose-dependently repressed transcription from either the PPARγ 1 or PPARγ2 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 PPARγ1 promoter and partially lost their effects on the PPARγ2 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 PPARγ2 (but not PPARγ1) promoter. Progressive 5′-deletion and gel retardation analyses revealed that this repression involves direct and specific binding of FOXO1 to the PPARγ2 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 PPARγ, 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 PPARγ2 promoter may serve as a therapeutic target for type 2 diabetes.

Transcriptional regulation of the GLUT4 gene: from PPAR-γ 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-γ, 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.

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

Here, we examined the chronic effects of two cannabinoid receptor-1 (CB1) inverse agonists, rimonabant and ibipinabant, in hyperinsulinemic Zucker rats to determine their chronic effects on insulinemia. Rimonabant and ibipinabant (10 mg·kg⁻¹·day⁻¹) elicited body weight-independent improvements in insulinemia and glycemia during 10 wk of chronic treatment. To elucidate the mechanism of insulin lowering, acute in vivo and in vitro studies were then performed. Surprisingly, chronic treatment was not required for insulin lowering. In acute in vivo and in vitro studies, the CB1 inverse agonists exhibited acute K channel opener (KCO; e.g., diazoxide and NN414)-like effects on glucose tolerance and glucose-stimulated insulin secretion (GSIS) with approximately fivefold better potency than diazoxide. Followup studies implied that these effects were inconsistent with a CB1-mediated mechanism. Thus effects of several CB1 agonists, inverse agonists, and distomers during GTTs or GSIS studies using perifused rat islets were unpredictable from their known CB1 activities. In vivo rimonabant and ibipinabant caused glucose intolerance in CB1 but not SUR1-KO mice. Electrophysiological studies indicated that, compared with diazoxide, 3 μM rimonabant and ibipinabant are partial agonists for K channel opening. Partial agonism was consistent with data from radioligand binding assays designed to detect SUR1 K(ATP) KCOs where rimonabant and ibipinabant allosterically regulated ³H-glibenclamide-specific binding in the presence of MgATP, as did diazoxide and NN414. Our findings indicate that some CB1 ligands may directly bind and allosterically regulate Kir6.2/SUR1 K(ATP) channels like other KCOs. This mechanism appears to be compatible with and may contribute to their acute and chronic effects on GSIS and insulinemia.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.

CYP2E1 impairs GLUT4 gene expression and function: NRF2 as a possible mediator.

Impaired GLUT4 function/expression in insulin target tissues is well-documented in diabetes and obesity. Cytochrome P450 isoform 2E1 (CYP2E1) induces oxidative stress, leading to impaired insulin action. CYP2E1 knockout mice are protected against high fat diet-induced insulin resistance and obesity; however the molecular mechanisms are still unclear. We examined whether CYP2E1 impairs GLUT4 gene expression and function in adipose and muscle cells. CYP2E1 overexpression in skeletal muscle-derived L6 cells inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake, with the latter inhibition being blocked by vitamin E. CYP2E1 overexpression in L6 and primary rat adipose (PRA) cells suppressed GLUT4 gene expression at promoter and mRNA levels, whereas CYP2E1 silencing had opposite effects. In PRA, CYP2E1-induced suppression of GLUT4 expression was blocked by chlormethiazole (CYP2E1-specific inhibitor) and the antioxidants vitamin E and N-acetyl-l-cysteine. CYP2E1 effect was mediated by the transcription factor NF-E2-related factor 2 (NRF2), as evident from its complete reversal by a coexpressed dominant-negative, but not wild-type NRF2. GLUT4 transcription was suppressed by NRF2 overexpression, and enhanced by NRF2 silencing. Promoter and ChIP analysis showed a direct and specific binding of NRF2 to a 58-326 GLUT4 promoter region that was required to maintain CYP2E1 suppression; this binding was enhanced by CYP2E1 overexpression. We suggest a mechanism for CYP2E1 action that involves: a) suppression of GLUT4 gene expression that is mediated by NRF2; b) impairment of insulin-stimulated Glut4 translocation and function. CYP2E1 and NRF2 are introduced as negative regulators of GLUT4 expression and function in insulin-sensitive cells.

Insulin resistance and acanthosis nigricans: Evidence for a postbinding defect in vivo

Acanthosis nigricans (AN) with insulin resistance has been traditionally attributed to insulin receptor abnormalities. To further clarify the postbinding defects of in vivo insulin action in this state, we applied the euglycemic insulin clamp technique, combined with the glucose trace infusion method, to 26 subjects: 12 AN patients (eight normoglycemic and four hyperglycemic), eight obese, and eight lean control subjects. The normoglycemic AN group exhibited fasting hyperinsulinemia (666% of control), 160% elevated hepatic glucose production (HGP), 425% increased posthepatic insulin delivery rate, and only slightly reduced (19%) insulin clearance rates, compared with controls. Except for the latter, all these abnormalities were statistically significant (P less than .05), and could not be accounted for by body overweight. AN patients with diabetes mellitus (AN + DM) exhibited a further decreased insulin responsiveness (30%) and clearance (38%), together with a major increase in HGP (320%). All AN patients showed a significant right-shift in the insulin dose-response curve, indicating a decrease in insulin sensitivity. In conclusion, AN is characterized by increased basal rates of HGP, and peripheral insulin resistance, which can be partially attributed to postbinding defects. In AN + DM, a worsening of these abnormalities may be responsible for unmasking the existence of diabetes.

AHNAK KO mice are protected from diet-induced obesity but are glucose intolerant.

AHNAK is a 700 KD phosphoprotein primarily involved in calcium signaling in various cell types and regulating cytoskeletal organization and cell membrane architecture. AHNAK expression has also been associated with obesity. To investigate the role of AHNAK in regulating metabolic homeostasis, we studied whole body AHNAK knockout mice (KO) on either regular chow or high-fat diet (HFD). KO mice had a leaner phenotype and were resistant to high-fat diet-induced obesity (DIO), as reflected by a reduction in adipose tissue mass in conjunction with higher lean mass compared to wild-type controls (WT). However, KO mice exhibited higher fasting glucose levels, impaired glucose tolerance, and diminished serum insulin levels on either diet. Concomitantly, KO mice on HFD displayed defects in insulin signaling, as evident from reduced Akt phosphorylation and decreased cellular glucose transporter (Glut4) levels. Glucose intolerance and insulin resistance were also associated with changes in expression of genes regulating fat, glucose, and energy metabolism in adipose tissue and liver. Taken together, these data demonstrate that (a) AHNAK is involved in glucose homeostasis and weight balance (b) under normal feeding KO mice are insulin sensitive yet insulin deficient; and (c) AHNAK deletion protects against HFD-induced obesity, but not against HFD-induced insulin resistance and glucose intolerance in vivo.

PPARγ Gene Expression is Autoregulated in Primary Adipocytes: Ligand, Sumoylation, and Isoform Specificity

Being a key-factor in glucose homeostasis, PPARγ transcriptional activity (TA) is of high importance. However, its mediation by ligands and post-translational modifications in insulin target tissues are unclear. We investigated effects of rosiglitazone (Rg) and sumoylation on PPARγ-TA by overexpressing expression vectors and promoter-reporters for PPARγ1 and PPARγ2 in primary rat adipocytes. Wild type (WT) PPARγ1 and PPARγ2 dose-dependently repressed transcription from their promoters to a maximum of 40-50%. PPARγ2 mutants defective in either MAP-kinase phosphorylation (S112A) or the ligand-binding domain (LBD; P495L, L496A/E499A) exhibited decreased repression of PPARγ2 promoter. Rg enhanced repression by S112A, but not by LBD-defective mutants. Sumoylation-defective PPARγ1 mutants K77R and K365R repressed PPARγ2 promoter activity similar to WT, while Rg enhanced repression by K77R but not by K365R. Sumoylation-defective PPARγ2 mutants K107R and K395R exhibited impaired TA and impaired responsiveness to Rg. GLUT4 promoter, previously shown by us to be repressed by WT-PPARγ1 and WT-PPARγ2, was similarly repressed by both sumoylation-defective PPARγ1 mutants, while both sumoylation-defective PPARγ2 mutants exerted reduced repression. Surprisingly, Rg alleviated only WT-PPARγ2-induced repression, while augmenting that induced by WT-PPARγ1 and all sumoylation-defective mutants. Promoter and chromatin immunoprecipitation analyses revealed that PPARγ2 autorepression involves its direct binding to its promoter. In concert with effects at promoter level, Rg decreased endogenous level of PPARγ2 mRNA, while increasing that of GLUT4. We suggest a hypothetical model for PPARγ gene regulation in primary adipocytes that is isoform-specific and Rg/sumoylation-dependent. These findings are important due to the role of PPARγ and Rg in insulin sensitivity.