Yueshan Hu

Document Type

Dissertation - University Access Only

Award Date

2010

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

Abstract

Obesity is now widely accepted as a multi-factorial, chronic disorder, with an alarming increase in the worldwide prevalence in both adults and children. Overweight and obese persons are at risk of a number of medical conditions which can lead to further morbidity and mortality. The regulation of adipogenesis (the differentiation of preadipocytes to mature adipocytes) is a potential strategy for the prevention/treatment of obesity. Adipogenesis is regulated by a complex transcriptional cascade including key transcription factors sterol regulatory element binding protein 1 (SREBP-1 ), peroxisome proliferator-activated receptor y (PPARy), CCAAT/enhancer binding protein a (C/EBPa), and GATA binding protein 2 and 3 (GATA-2, GATA-3). In addition, weight gain/obesity is a common and potentially serious complication associated with the treatment of second generation antipsychotics such as clozapine and risperidone. Increased adipogenesis via the SREBP-1 pathway has been reported to be one critical mechanism responsible for antipsychotic drug-induced weight gain. Berberine is a botanic alkaloid which exhibits various pharmacological properties such as anti-microbial, anti-cancer, and obesity prevention/treatment effects. Other natural compounds such as evodiamine and amentoflavone have been reported to be potential agents to prevent/treat obesity. The objective of this study is to investigate the effects of berberine and its combinations (with evodiamine or amentoflavone) on adipogenesis and their transcriptional impacts during this process in vitro (cells) and in vivo (animals) with or without induction of second generation anti-psychotics ( clozapine and risperidone ). MTT (Methylthiazolyldiphenyl- tetrazolium bromide) was used to detect the cytotoxic effects of berberine, evodiamine, amentoflavone, clozapine, risperidone, and their combinations on the cell viabilities during differentiation. Differentiation /adipogenesis was monitored by Oil-Red-0 staining. The gene and protein expression of established adipogenesis markers, SREBP-1, PPARy, C/EBPu, GATA-2, GATA-3 and related adipogenic proteins was determined by Realtime RT-PCR and Western Blot. High-fat diet-induced obesity mice were treated with berberine, food intake and weight gain were recorded; and blood glucose, triglyceride, and total cholesterol levels were determined using commercially available kits. When mouse 3T3-Ll preadipocytes were treated with berberine (1-64 μM) during differentiation, the results showed that berberine had a minor effect on cell viability during differentiation induced by differentiation medium. Quantitative Oil-Red-0 staining measurements of 3T3-Ll cells which were grown in I, 2, 4, 8 μM berberine containing medium and were 8 days post differentiation induction, demonstration a reduction in fat droplets of 59.4%, 58.4%, 71.8% and 72.6% respectively. Treatment with 8 μM berberine for 8 days decreased the expression of PPARy and C/EBPa, and increased expression of GATA-2 and GA T A-3 both at the gene and protein levels. When human white preadipocytes (HWP) were treated with berberine, evodiamine, and their combination during differentiation, the results showed that individually, berberine had little effect on HWP viability, but evodiamine greatly inhibited cell viability. However, in combination, the compounds showed only a slight inhibition of viability. Individually and in combination, treatment with 8 μM berberine or 4 μM evodiamine resulted in a major inhibition of HWP differentiation accompanied by an up-regulation of both GATA-2 and 3 mRNA and protein expression. However, the expression of both PP ARy and C/EBPa remained unchanged. Nonetheless, treatment of HWP with a combination ofberberine and evodamine did not result in any additive or synergetic inhibition of the differentiation process. When the effects of berberine on the adipogenesis of high-fat diet-induced obesity (FD) or normal diet (ND) mice and possible transcriptional impacts were investigated, the results demonstrated that in FD mice, berberine reduced mouse weight gain and food intake, and decreased the weight of epididymal fat and liver relative to body weight. Serum glucose, triglyceride, and total cholesterol levels were also reduced. These changes were accompanied with a down-regulation of PPARy mRNA and protein expression and an up-regulation of both gene and protein expression of GA T A-3. Berberine had no significant effect on weight gain, epididymal fat weight, food intake, serum glucose, triglyceride, total cholesterol levels in ND mice, and did not affect the kidney, spleen and liver weight in both ND and FD mice. When mouse 3T3-Ll preadipocytes were treated with berberine during differentiation induced by differentiation medium with addition of clozapine or risperidone, the results showed that neither clozapine nor risperidone, alone or in combination with berberine had significant effects on cell viability. Eight days treatment with 15μM clozapine increased adipogenesis by 37.4% and 50 μM risperidone increased adipogenesis by 26.5% during 3T3-Ll cell differentiation accompanied by increased SREBP-1, PPARy, C/EBPa, LDLR, and Adiponectin gene expression. More importantly, the addition of 8 μM berberine diminished the induction of adipogenesis almost completely accompanied by down-regulated mRNA and protein expression levels of SREBP-1-related proteins. When mouse 3T3-Ll preadipocytes were treated with various concentration of berberine combined with amentotlavone during differentiation induced by differentiation medium and 15 μM clozapine, results showed that those combinations have no significant effects on cell viabilities, quantification of Oil-Red-0 staining demonstrated that individually, amentoflavone did not inhibit adipogenesis significantly, however interestingly, the combination of25μM amentoflavone with 1, 2, 4, 8 μM berberine produced higher significant inhibition of adipogenesis comparing to berberine alone, 8 μM berberine combined with 25μM amentoflavone inhibited adipogenesis by 97%. Realtime RT-PCR and Western Blot showed that amentoflavone enhanced the inhibitory effects of berberine on mRNA and protein expression of SREBP-1, PPARy, and C/EBPa. In summary, this study showed that berberine inhibited adipogenesis in mouse 3T3-Ll preadipocytes, in human white adipocytes, in high-fat diet-induced obesity mouse, and in 3T3-L 1 cells during differentiation with induction of second-generation anti psychotics. The combination treatment of berberine with evodiamine did not show improved inhibition of adipogenesis during HWP differentiation, however, the combination treatment of berberine with amentoflavone enhanced the inhibitory effects on adipogenesis comparing to individual treatments. The general mechanism for the pharmacological effects of berberine and its combinations is possibly by modulating the expression of some key transcriptional regulator during adipogenesis including decreased expression of adipogenic factors SREBP-1, PPARy, C/EBPa, and increased expression of anti-adipogenic factors GATA-2 and GATA-3. These encouraging findings suggest that berberine and its combination with amentoflavone have excellent pharmacological potential to prevent/treat obesity with low toxicity. Our future clinical trials will further explore the effects/mechanisms of berberine/its combinations on human subjects.

Library of Congress Subject Headings

Berberine

Fat cells

Obesity -- Prevention

Format

application/pdf

Number of Pages

175

Publisher

South Dakota State University

Download

Share

COinS