Rajesh Amin

PER2 controls lipid metabolism by direct regulation of PPARγ

Accumulating evidence highlights intriguing interplays between circadian and metabolic pathways. We show that PER2 directly and specifically represses PPARγ, a nuclear receptor critical in adipogenesis, insulin sensitivity, and inflammatory response. PER2-deficient mice display altered lipid metabolism with drastic reduction of total triacylglycerol and nonesterified fatty acids. PER2 exerts its inhibitory function by blocking PPARγ recruitment to target promoters and thereby transcriptional activation. Whole-genome microarray profiling demonstrates that PER2 dictates the specificity of PPARγ transcriptional activity. Indeed, lack of PER2 results in enhanced adipocyte differentiation of cultured fibroblasts. PER2 targets S112 in PPARγ, a residue whose mutation has been associated with altered lipid metabolism. Lipidomic profiling demonstrates that PER2 is necessary for normal lipid metabolism in white adipocyte tissue. Our findings support a scenario in which PER2 controls the proadipogenic activity of PPARγ by operating as its natural modulator, thereby revealing potential avenues of pharmacological and therapeutic intervention.

Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

Selective Activation of PPARγ in Skeletal Muscle Induces Endogenous Production of Adiponectin and Protects Mice From Diet-Induced Insulin Resistance

The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma plays a key role in regulating whole body glucose homeostasis and insulin sensitivity. Although it is expressed most highly in adipose, it is also present at lower levels in many tissues, including skeletal muscle. The role muscle PPARgamma plays in metabolic regulation and in mediating the antidiabetic effects of the thiazolidinediones is not understood. The goal of this work was to examine the molecular and physiological effects of PPARgamma activation in muscle cells. We found that pharmacological activation of PPARgamma in primary cultured myocytes, and genetic activation of muscle PPARgamma in muscle tissue of transgenic mice, induced the production of adiponectin directly from muscle cells. This muscle-produced adiponectin was functional and capable of stimulating adiponectin signaling in myocytes. In addition, elevated skeletal muscle PPARgamma activity in transgenic mice provided a significant protection from high-fat diet-induced insulin resistance and associated changes in muscle phenotype, including reduced myocyte lipid content and an increase in the proportion of oxidative muscle fiber types. Our findings demonstrate that PPARgamma activation in skeletal muscle can have a significant protective effect on whole body glucose homeostasis and insulin resistance and that myocytes can produce and secrete functional adiponectin in a PPARgamma-dependent manner. We propose that activation of PPARgamma in myocytes induces a local production of adiponectin that acts on muscle tissue to improve insulin sensitivity.

Positive modulation by Ras of interleukin-1β-mediated nitric oxide generation in insulin-secreting clonal β (HIT-T15) cells

In the present study, we have shown that exposure of insulin-secreting clonal β (HIT-T15) cells to interleukin-1β (IL-1β) results in a time- and concentration-dependent increase in nitric oxide (NO) release. These effects by IL-1β on NO release were mediated by induction of inducible nitric oxide synthase (iNOS) from the cells. Preincubation of HIT cells with Clostridium sordellii lethal toxin-82, which irreversibly glucosylates and inactivates small G-proteins, such as Ras, Rap, Ral, and Rac, but not Cdc42, completely abolished IL-1β-induced NO release. Pre-exposure of HIT cells to C. sordellii lethal toxin-9048, which monoglucosylates and inhibits Ras, Cdc42, Rac, and Rap, but not Ral, also attenuated IL-1β-mediated NO release. These data indicate that activation of Ras and/or Rac may be necessary for IL-1β-mediated NO release. Preincubation of HIT cells with C. difficile toxin-B, which monoglucosylates Rac, Cdc42, and Rho, had no demonstrable effects on IL-mediated NO release, ruling out the possibility that Rac may be involved in this signaling step. Further, two structurally dissimilar inhibitors of Ras function, namely manumycin A and damnacanthal, inhibited, in a concentration-dependent manner, the IL-1β-mediated NO release from these cells. Together, our data provide evidence, for the first time, that Ras activation is an obligatory step in IL-1β-mediated NO release and, presumably, the subsequent dysfunction of the pancreatic β cell. Our data also provide a basis for future investigations to understand the mechanism of cytokine-induced β cell death leading to the onset of insulin-dependent diabetes mellitus.

Residues within the Polycationic Region of cGMP Phosphodiesterase γ Subunit Crucial for the Interaction with Transducin α Subunit IDENTIFICATION BY ENDOGENOUS ADP-RIBOSYLATION AND SITE-DIRECTED MUTAGENESIS

Interaction between the γ subunit (Pγ) of cGMP phosphodiesterase and the α subunit (Tα) of transducin is a key step for the regulation of cGMP phosphodiesterase in retinal rod outer segments. Here we have utilized a combination of specific modification by an endogenous enzyme and site-directed mutagenesis of the Pγ polycationic region to identify residues required for the interaction with Tα. Pγ, free or complexed with the αβ subunit (Pαβ) of cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of [adenylate-32P]NAD. Identification of ADP-ribose in the radiolabeled Pγ and radiolabeling of arginine-replaced mutant forms of Pγ indicate that both arginine 33 and arginine 36 are similarly ADP-ribosylated by endogenous ADP-ribosyltransferase, but only one arginine is modified at a time. Pγ complexed with Tα (both GTP- and GDP-bound forms) was not ADP-ribosylated; however, agmatine, which cannot interact with Tα, was ADP-ribosylated in the presence of Tα, suggesting that a Pγ domain containing these arginines is masked by Tα. A Pγ mutant (R33,36K), as well as wild type Pγ, inhibited both GTP hydrolysis of Tα and GTP binding to Tα. Moreover, GTP-bound Tα activated Pαβ that had been inhibited by R33,36K. However, another Pγ mutant (R33,36L) could not inhibit these Tα functions. In addition, GTP-bound Tα could not activate Pαβ inhibited by R33,36L. These results indicate that a Pγ domain containing these arginines is required for its interaction with Tα, but not with Pαβ, and that positive charges in these arginines are crucial for the interaction.