runathan, et al.. C75 is converted to C75-CoA in the hypothalamus, where it inhibits carnitine palmitoyltransferase 1 and decreases food intake and body weight. Biochemical Pharmacology, Elsevier, 2009, 77 (6) 20-11-2008 Please cite this article as: Mera P, Bentebibel A, López-Viñas E, Cordente AG, Gurunathan C, Sebastián D, Vázquez I, Herrero L, Ariza X, Gómez-Puertas P, Asins G, Serra D, García J, Hegardt FG, C75 is converted to C75-CoA in the hypothalamus, where it inhibits carnitine palmitoyltransferase 1 and decreases food intake and body weight, Biochemical Pharmacology (2008Pharmacology ( ), doi:10.1016Pharmacology ( /j.bcp.2008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 ABSTRACT 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.

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 NH 2 -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 NH 2 -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 NH 2 -and COOHterminal 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 (IC 50 ) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC 50 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 K m and malonyl-CoA IC 50 . Carnitine palmitoyltransferase 1 (CPT1)4 catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine. This is the first step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix, where they undergo ␤-oxidation. CPT1 is tightly regulated by its physiological inhibitor malonyl-CoA. This regulation allows CPT1 to signal the availability of lipid and carbohydrate fuels to the cell (1). Mammalian tissues express three isoforms: CPT1A (liver), CPT1B (muscle and heart), and CPT1C (brain), which are the products of different genes (2-4). CPT1A and -B have 62% amino acid identity, but they are differentially regulated by malonyl-CoA. CPT1A is inhibited to a much lesser extent than CPT1B, which may explain why fatty acid oxidation is more finely regulated in the heart than in the liver. CPT1 is a potential target for the treatment of metabolic disorders involving diabetes and coronary heart disease (5). The interaction between malonyl-CoA and CPT1C may be involved in the "malonyl-CoA signal" in hypothalamic neurons regulating food intake and peripheral energy expenditure (6).It has been postulated that there are two malonyl-CoA binding sites in the molecule of CPT1A (7,8). Kinetic studies indicate that there is a high affinity binding site and a low affinity binding site (9 -13...

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 Wdr13Lepr db/db and Wdr13Lepr 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.