Obestatin and Rosiglitazone Differentially Modulate Lipid Metabolism Through Peroxisome Proliferator-activated Receptor-γ (PPARγ) in Pre-adipose and Mature 3T3-L1 Cells.

Obestatin is a 23-residue peptide, obtained after posttranslational modification of preproghrelin. It has been shown, in Swiss albino mice, to upregulate glycerolipid metabolism and PPARγ signaling. It was opined that the by-products of increased glycerolipid metabolism triggered PPARγ signaling. It was hypothesized that obestatin upon co-administration with a full agonist of PPARγ should reveal the comparative significance or possible synergy in PPARγ signaling. We postulated they would act synergistically by obestatin increasing PPARγ expression and rosiglitazone enhancing PPARγ activity. We evaluated the combination in DIO-C57BL/6 mice and observed that obestatin completely reversed the increase in subcutaneous fat brought about by rosiglitazone. To understand their role at the adipocyte level, 3T3-L1 cells were treated with a combination of obestatin and rosiglitazone during (1) initiation of differentiation and (2) after 14 days from initiation of differentiation when the adipocytes were mature. Interestingly, their influence was mainly adipogenic and showed double lipid accumulation when estimated 14 days after initiation of differentiation. There was an upregulation of Pparγ by fourfold, Hsl by eightfold, Glut4 by fourfold, Leptin by 2.7-fold, Atgl by sixfold, Fasn by sixfold, and Fabp4 by sevenfold at the mRNA level, whereas in mature adipocytes there was a significant decrease in fat accumulation by 20%. There was downregulation of Pparγ, Hsl, Lpl, and Fasn by 0.5-fold at the mRNA level. These results show that the combined influence of obestatin and rosiglitazone is significant and the outcome is dependent on the metabolic stage of the adipocyte.

Capsaicin And Genistein Override The Action Of Obestatin To Decrease Lipid Accumulation In 3T3-L1 Cells.

Satiety peptides convey information about short-term energy reserves in the gut to the hypothalamus and aid in regulation of appetite and food intake. Obestatin is one such gastro peptide that has been shown to upregulate glycerolipid metabolism and PPARγ signalling. Obestatin brings about moderate reduction in circulating and stored triglyceride levels and reduction in gain in body weight in mice. We wanted to test whether obestatin could be further potentiated by co-administration with nutraceuticals genistein and capsaicin that are well known to reduce triglyceride levels. Hence, we chose to administer the compounds individually and pair-wise with obestatin in 3T3-L1 cells at concentrations of 200 nM obestatin, 10 µM capsaicin and 100 µM genistein. When treated along with induction of differentiation, both capsaicin and genistein in combination with obestatin reduced triglyceride levels in 3T3-L1 cells by 25 and 20%, respectively, when accessed on day 14 after induction. The combined administrations were dominated by the effect of the nutraceuticals and showed the same effect as of capsaicin or genistein. Upregulation of Fatty acid synthase (Fasn) and Adipose triglyceride lipase (Atgl/Pnpla2) by obestatin were reversed by both capsaicin and genistein. However, their ability to upregulate Peroxisome proliferation activating receptor gamma (Pparγ), Hormone sensitive lipase (Hsl), Lipoprotein lipase (Lpl) were retained while upregulation of Uncoupling protein 1 (Ucp1) by capsaicin was unchanged upon co-administration. Over expression of the lipases and UCP1 in case of capsaicin could be resulting in net lowering of lipid accumulation in the cells.

Studies on the influence of CCK-8 on the ability of obestatin to reduce food intake, gain in body weight and related lipid parameters.

In an effort to mimic in part the redundancy of satiety peptides involved in energy homeostasis, the combined benefits of the well-established satiety peptide CCK8 and an apparently anorectic peptide obestatin were studied in Swiss albino mice. The optimal dose of obestatin that was required to give the most pronounced effect with CCK8 was worked out by varying the concentration of obestatin while keeping CCK8 concentration constant at 200 nmol/KgBW. Mice administered 160 nmol obestatin and 200 nmol CCK8 per kilogram body weight showed the most drastic reduction in food intake. Gain in body weight was arrested after day four during the eight day experiment. These studies reemphasize the beneficial effects imparted by co-administration of obestatin and CCK8 and their potential use towards countering obesity.

Obestatin and Nt8U influence glycerolipid metabolism and PPAR gamma signaling in mice.

Obestatin, its N-terminal fragment and the N-terminal fragment analog Nt8U were previously shown to reduce food intake, gain in body weight and triglyceride levels in albino mice. To establish their mode of action, mRNA profiling of the epididymal adipose tissue of mice treated with these peptides were performed. The differential expressions were markedly indicative of their involvement in lipid metabolism. Obestatin showed a significant upregulation of the genes patatin-like phospholipase domain containing 3, diacylglycerol O-acyltransferase 2, monoglyceride lipase, aldo-keto reductase family 1, member 7 which are involved in glycerolipid metabolism. It also upregulated peroxisome proliferator-activated receptor gamma, retinoid X receptor gamma, cluster of differentiation 36, adiponectin, C1Q and collagen domain containing, angiopoietin-like 4, lipoprotein lipase, stearoyl-coenzyme A and desaturase 3 involved in the peroxisome proliferator-activated receptor signaling pathway. Nt8U upregulated genes implicated in the same two pathways but with lesser significance and also upregulated APOL2. The N-terminal fragment though differentially regulated a small subset of the genes differentially regulated by obestatin and Nt8U, no conclusive evidence was obtained as to assign a specific pathway for its mode of action. We hypothesize that reduced food intake brought about by obestatin and Nt8U triggers lipid catabolism. The free fatty acids and lysophosphatidic acid thus produced in turn activates peroxisome proliferator-activated receptor gamma and the genes involved in peroxisome proliferator-activated receptor signaling. All of them together lead to reduction in gain in bodyweight, stored fat and circulating lipids. These results also correlate well with the observed efficacy of the peptides.

Fragment analogs as better mimics of obestatin.

Obestatin is a twenty three amino acid peptide produced in the stomach by post translational modification of the preproghrelin gene. Since its discovery in 2005, many studies have shown that obestatin reduces feed intake and gain in body weight in rodents. Studies from our laboratory have shown the N-terminal thirteen residues mimic obestatin the best and residues 6-18 reduce epididymal fat significantly in adult male mice. In this study we have tried to increase the efficacy of these fragments. As an initial step, we have substituted G(8) with alpha-aminoisobutyricacid(Aib,U) and F(5) with cyclohexylalanine(Cha) in the N-terminal peptide to obtain two modified peptides and modified the middle fragment (residues 6-18) by substituting both the glycine residues at position 3 and 8 with alpha-aminoisobutyricacid(U). The rationale being, unusual amino acids could protect the peptides from immediate degradation and Aib would also induce secondary structure in these unstructured peptides. The N-terminal fragment with the G(8)U substitution fared the best. It reduced food intake, gain in body weight, levels of cholesterol and triglycerides in the blood, epididymal and perirenal fat in adult male mice similar to that of obestatin. The middle fragment with G(3,8)U double substitution was the second best.

Fragments of obestatin as modulators of feed intake, circulating lipids, and stored fat.

Obestatin, shown to reduce feed intake and gain in body weight in rodents, is a very attractive candidate to be used against obesity. In this study, we aimed to investigate the relationship between the primary structure and activity of obestatin. Also of interest to us is a peptide of minimal length that closely mimics obestatin. Towards the same, we synthesized rodent obestatin and three overlapping fragments spanning residues 1-13, 6-18, and 11-23 of obestatin. These peptides subsequent to purification and characterization were tested upon adult male mice for their ability to reduce feed intake and gain in body weight. The N-terminal peptide (residues 1-13) mimicked obestatin the closest. The middle fragment (residues 6-18) significantly reduced epididymal fat without much altering feed intake or body weight.

Peptide-small molecule hybrids via orthogonal deprotection-chemoselective conjugation to cysteine-anchored scaffolds. A model study.

The feasibility of an orthogonal deprotection-conjugation protocol, holding the promise of libraries of functionally diverse chemical probes attached to cysteine-anchored peptide scaffolds, has been explored with a model system. The necessary tools for assembly of the hybrid libraries have been prepared and the tandem procedure optimized. S-alkylation and S-sulfenylation are featured as the chemoselective ligation reactions. [reaction: see text]