Production of Leptin in E. coli and Its Effect on Glucose and Acetate Transport and Expression of Uncoupling Protein-2 Gene in Adipose Tissues of Korean Cattle (Hanwoo)

K S Kim,M G Baik

doi:10.5713/ajas.2004.1062

Abstract

Leptin has a major role in the regulation of food intake and energy homeostasis. In addition, leptin participates in many physiological functions including regulation of lipid metabolism. Bovine recombinant leptin protein was produced in E. coli cells in order to understand function of leptin in the regulation of lipid metabolism. The leptin expression vector was constructed in pGEX-4T-3 vector and transformed into E. coli BL21 cells. Expression of the GST-leptin fusion protein was induced with IPTG. The fusion protein was purified using glutathione sepharose 4B batch method, and the recombinant leptin was eluted after thrombin protease digestion. The effect of leptin on glucose transport was examined in the differentiated adipocytes of 3T3-L1 cells. Leptin had no effect on basal and insulin-stimulated glucose transport in 3T3-L1 cells (p>0.05). Effect of recombinant leptin on glucose and acetate transport was examined in adipose tissues of Korean cattle (Hanwoo). Insulin stimulated glucose transport in both intramuscular and subcutaneous adipose tissues (p 0.05). Insulin stimulated acetate transport in bovine adipose tissues (p 0.05). Northern and RT-PCR analyses showed that mRNA levels of uncoupling protein-2 were increased by leptin treatment in 3T3-L1 cells without statistical difference (p>0.05). In conclusion, bovine recombinant leptin did not affect glucose and acetate transport in both 3T3-L1 adipocytes and bovine adipose tissues, while it stimulates UCP-2 mRNA expression in 3T3-L1 cells. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 8 : 1062-1068)

Highlights

Lipid metabolism is regulated by many hormones, and leptin is the most recently discovered among them (Zhang et al, 1994)
The profound importance of leptin is demonstrated by the fact that mice which are homozygous for a mutation in the ob gene (Pelleymounter et al, 1995) or in its receptor (Lee et al, 1996), demonstrate massive obesity, a reduced basal metabolic rate, hyperglycemia, hyperinsulinemia, and reduced fertility
Bovine recombinant leptin was produced in E. coli and purified, and effects of leptin on glucose or acetate transport were examined in 3T3-L1 adipocytes and in adipose tissues of Korean cattle

Summary

INTRODUCTION

Lipid metabolism is regulated by many hormones, and leptin is the most recently discovered among them (Zhang et al, 1994). Administered leptin has been shown to increase insulin-stimulated glucose utilization and to favor the expression of uncoupling proteins (UCPs). EFFECT OF LEPTIN ON GLUCOSE AND ACETATE TRANSPORT level of obese mRNA and its response to fasting are different among several bovine adipose tissues (Kim et al., 2000). The expression of the obese gene in intramuscular adipose tissues was very low and did not respond to fasting This result may suggest that increasing intramuscular fat instead of subcutaneous fat would have a favorable effect in that a lower leptin level could result in greater food intake and further marbling. Bovine recombinant leptin was produced in E. coli and purified, and effects of leptin on glucose or acetate transport were examined in 3T3-L1 adipocytes and in adipose tissues of Korean cattle. The effect of leptin on expression UCP-2 gene was examined by northern and RTPCR analyses in 3T3-L1 cells

MATERIALS AND METHODS

RESULTS AND DISCUSSION

A Control GST