Impaired liver regeneration and lipid homeostasis in CCl4 treated WDR13 deficient mice.

WDR13 - a WD repeat protein, is abundant in pancreas, liver, ovary and testis. Absence of this protein in mice has been seen to be associated with pancreatic ß-cell proliferation, hyperinsulinemia and age dependent mild obesity. Previously, we have reported that the absence of WDR13 in diabetic Leprdb/db mice helps in amelioration of fatty liver phenotype along with diabetes and systemic inflammation. This intrigued us to study direct liver injury and hepatic regeneration in Wdr13-/0 mice using hepatotoxin CCl4. In the present study we report slower hepatic regeneration in Wdr13-/0 mice as compared to their wild type littermates after CCl4 administration. Interestingly, during the regeneration phase, hepatic hypertriglyceridemia was observed in Wdr13-/0 mice. Further analyses revealed an upregulation of PPAR pathway in the liver of CCl4- administered Wdr13-/0 mice, causing de novo lipogenesis. The slower hepatic regeneration observed in CCl4 administered Wdr13-/0 mice, may be linked to liver hypertriglyceridemia because of activation of PPAR pathway.

Genetic deletion of Wdr13 improves the metabolic phenotype of Lepr (db/db) mice by modulating AP1 and PPARγ target genes.

AIM/HYPOTHESIS: Type 2 diabetes is a complex disease characterised by hyperglycaemia, hyperinsulinaemia, dyslipidaemia and insulin resistance accompanied by inflammation. Previously, we showed that mice lacking the Wdr13 gene had increased islet mass due to enhanced beta cell proliferation. We hypothesised that introgression of a Wdr13-null mutation, a beta cell-proliferative phenotype, into Lepr(db/db) mice, a beta cell-destructive phenotype, might rescue the diabetic phenotype of the latter. METHODS: Wdr13-deficient mice were crossed with Lepr(db/db) mice to generate mice with the double mutation. We measured various serum metabolic variables of Wdr13(+/0)Lepr(db/db) and Wdr13(-/0) Lepr(db/db) mice. Further, we analysed the histopathology and gene expression of peroxisome proliferator-activated receptor (PPAR)γ and, activator protein (AP)1 targets in various metabolic tissues. RESULTS: Lepr(db/db) mice with the Wdr13 deletion had a massively increased islet mass, hyperinsulinaemia and adipocyte hypertrophy. The increase in beta cell mass in Wdr13(-/0)Lepr(db/db) mice was due to an increase in beta cell proliferation. Hypertrophy of adipocytes may be the result of increase in transcription of Pparg and its target genes, leading in turn to increased expression of several lipogenic genes. We also observed a significant decrease in the expression of AP1 and nuclear factor κ light chain enhancer of activated B cells (NFκB) target genes involved in inflammation. CONCLUSIONS/INTERPRETATION: This study provides evidence that loss of WD repeat domain 13 (WDR13) protein in the Lepr (db/db) mouse model of diabetes is beneficial. Based on these findings, we suggest that WDR13 may be a potential drug target for ameliorating hyperglycaemia and inflammation in diabetic conditions.

C75 is converted to C75-CoA in the hypothalamus, where it inhibits carnitine palmitoyltransferase 1 and decreases food intake and body weight.

Central nervous system administration of C75 produces hypophagia and weight loss in rodents identifying C75 as a potential drug against obesity and type 2 diabetes. However, the mechanism underlying this effect is unknown. Here we show that C75-CoA is generated chemically, in vitro and in vivo from C75 and that it is a potent inhibitor of carnitine palmitoyltranferase 1 (CPT1), the rate-limiting step of fatty-acid oxidation. Three-D docking and kinetic analysis support the inhibitory effect of C75-CoA on CPT1. Central nervous system administration of C75 in rats led to C75-CoA production, inhibition of CPT1 and lower body weight and food intake. Our results suggest that inhibition of CPT1, and thus increased availability of fatty acids in the hypothalamus, contribute to the pharmacological mechanism of C75 to decrease food intake.

Definition by functional and structural analysis of two malonyl-CoA sites in carnitine palmitoyltransferase 1A.

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of l-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH2- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH2- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH2- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine Km and malonyl-CoA IC50.