Body weight and abdominal fat gene expression profile in response to a novel hydroxycitric acid-based dietary supplement

Abstract

Obesity is a global public health problem, with about 315 million people worldwide estimated to fall into the WHO-defined obesity categories. Traditional herbal medicines may have some potential in managing obesity. Botanical dietary supplements often contain complex mixtures of phytochemicals that have additive or synergistic interactions. The dried fruit rind of Garcinia cambogia, also known as Malabar tamarind, is a unique source of (-)-hydroxycitric acid (HCA), which exhibits a distinct sour taste and has been safely used for centuries in Southeastern Asia to make meals more filling. Recently it has been demonstrated that HCA-SX or Super Citrimax, a novel derivative of HCA, is safe when taken orally and that HCA-SX is bioavailable in the human plasma as studied by GC-MS. Although HCA-SX has been observed to be conditionally effective in weight management in experimental animals as well as in humans, its mechanism of action remains to be understood. We sought to determine the effects of low-dose oral HCA-SX on the body weight and abdominal fat gene expression profile of Sprague-Dawley rats. We observed that at doses relevant for human consumption dietary HCA-SX significantly contained body weight growth. This response was associated with lowered abdominal fat leptin expression while plasma leptin levels remained unaffected. Repeated high-density microarray analysis of 9960 genes and ESTs present in the fat tissue identified a small set (approximately 1% of all genes screened) of specific genes sensitive to dietary HCA-SX. Other genes, including vital genes transcribing for mitochondrial/nuclear proteins and which are necessary for fundamental support of the tissue, were not affected by HCA-SX. Under the current experimental conditions, HCA-SX proved to be effective in restricting body weight gain in adult rats. Functional characterization of HCA-SX-sensitive genes revealed that upregulation of genes encoding serotonin receptors represent a distinct effect of dietary HCA-SX supplementation.

Figures

Figure 1

Body weights and plasma leptin levels following dietary supplementation of HCA-SX. (A) Body weight (kg) of male Sprague-Dawley rats supplemented intragastric with HCA-SX (10 mg/kg body weight) once every day, 5 times a week for 8 weeks. The rats had free access to regular diet and water ad libitum. Control group was gavaged with matching volumes of water and were given regular diet and water ad libitum. Body weights of each rat were recorded every week. Data shown are mean ± SD (n = 7 in each group). *p < 0.05 compared with the control group. #p = 0.06 compared with the control group. (B) plasma leptin levels in HCA-SX-supplemented or nonsupplemented rats after 8 weeks. Plasma leptin levels were measured using ELISA. Data are mean ± SD (n = 7).

Figure 2

Abdominal fat gene expression of leptin, glut-1, and glut-4 following 8 weeks of dietary HCA-SX supplementation. Male Sprague-Dawley rats supplemented with HCA-SX (10 mg/ kg body weight) once every day, 5 times a week for 8 weeks (n = 7). The rats had free access to regular diet and water ad libitum. The control group was gavaged with matching volumes of water and was given regular laboratory diet and water ad libitum. At the end of 8 weeks, rats were killed and abdominal adipose tissue was collected. Real-time RT-PCR was performed from RNA isolated from abdominal fat for quantification of leptin, glut-1, and glut-4 mRNA. Data shown are mean ± SD (n = 3). *p < 005, significantly different compared with the control group.

Figure 3

GeneChip™ data analysis scheme. The illustrated approach was used to identify differentially expressed genes in animals supplemented or not with HCA-SX as described in the legend of Figure 1. Data processing was primarily performed using Microarray Suite v 5.0 (MAS) and Data Mining Tool v 2.0 (DMT) software. Additional data filtration was performed using dChip with following criteria: I) fold change >1.2; II) t-test, p < 0.05; and III) present call in all HCA-SX supplemented animals for upregulated genes, vice versa present call in all baseline control animals for downregulated genes. Details of software and other resources for data analysis have been provided in Materials and Methods. ↑, increase and ↓, decrease in response to HCA-SX supplementation.

Figure 4

Cluster images illustrating genes sensitive to HCA-SX supplementation. Male Sprague-Dawley rats supplemented with HCA-SX (10 mg/kg body weight) once every day, 5 times a week for 8 weeks (n = 7). The rats had free access to regular diet and water ad libitum. The control group was gavaged with matching volume of water and was given regular diet and water ad libitum. At the end of 8 weeks, rats were killed and abdominal adipose tissue was collected. GeneChip microarray analysis was performed on the RNA extracted from the abdominal adipose tissue. For a clear graphic display of HCA-SX-sensitive genes, t-test was performed on data from control animals (4, 5, & 6) and from animals in the HCA-SX-supplemented groups (1, 2, & 3). Each of the six lanes represents data from one animal. The genes that significantly (p < 0.05) changed between the two groups compared were selected and subjected to hierarchial clustering using dChip software as described in Figure 3. Red to green gradation in color represents higher to lower expression signal. (A) Upregulated or (B) downregulated genes in response to HCA-SX supplementation compared with placebo group.

Figure 5

Real-time PCR validation of GeneChip microarray expression analysis. Expression levels of select HCA-SX-sensitive genes were independently determined using real-time PCR. For comparison, the GeneChip expression values were proportionately adjusted to fit to the scale with real-time PCR data. RNA samples used in GeneChip assay were utilized for the real-time PCR analysis. PDGS, prostaglandin D synthase; AldB, aldolase B; LNC2, lipocalin 2.

Figure 6

Mapping of GeneChip expression data onto existing pathways. To obtain insights into the effects of HCA-SX supplementation, results of GeneChip analysis were mapped onto known pathways associated with energy metabolism and obesity. GenMAPP and KEGG were used as the database for existing pathways. Several of the genes that were upregulated following HCA-SX supplemented were found to overlap with the monoamine G-protein-coupled receptors (GPCR) pathway. Genes shown with arrows were upregulated in the HCA-SX-supplemented group compared with the control groups. Gi/Go, Gq/G11, and Gs pathways: GPCRs have been shown to stimulate signaling cascade through a number of linear pathways that are initiated by s, Gi/Go, Gq/11, or Gs proteins.