Genistein and metformin regulate glycerol kinase and the enzymes of glycerol 3-phosphate shuttle in a differential manner in myocytes, hepatocytes and adipocytes.

Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by ∼45 %, while metformin reduced cGPDH and mGPDH activities by ∼32 % and âˆ¼43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited His-GK activity (Ki 19.12 µM), while metformin non-competitively inhibited His-cGPDH (Ki 75.52 µM) and mGPDH (Ki 54.70 µM) activities. siRNAs transfection showed ∼50 % and âˆ¼35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.

Purification and characterization of human glycerol 3-phosphate dehydrogenases (mitochondrial and cytosolic) by NAD+/NADH redox method.

Glycerol 3-phosphate (G3P) shuttle is composed of mGPDH and cGPDH and serves as the interface between carbohydrate- and lipid-metabolism. Recently, these metabolic enzymes have been implicated in type II diabetes mellitus but the detailed kinetic parameters and crystal structure of human mGPDH is unknown, though fewer studies on cGPDH are available. To characterize these enzymes, the human mGPDH and cGPDH genes were optimized and cloned into the pET-SUMO vector and pET-24a(+) vector, respectively, and over-expressed in Escherichia coli BL21 (DE3). However, SUMO-mGPDH was expressed as inclusion bodies. Hence, various culture parameters, solubilizing agents and expression vectors were used to solubilize the protein but they did not produce functional SUMO-mGPDH. Over-expression of SUMO-mGPDH along with molecular chaperone (pG-KJE8) produced a functional SUMO-mGPDH. The functional SUMO-mGPDH was purified and characterized using NAD+/NADH redox method. cGPDH was also over-expressed and purified for its characterization. DLS analysis and CD spectra of the purified proteins were performed. The mGPDH was a monomeric enzyme with MW of ∼74 kDa and displayed optimal activity in the Tris-HCl buffer (pH 7.4); while, cGPDH was a homodimer with a monomeric MW of ∼37 kDa and showed optimal activity in imidazole buffer (pH 8.0). The Kmapp was 0.475 mM for G3P, and 0.734 mM for DHAP. These methods may be used to characterize these enzymes to understand their role in metabolic disorders.

Expression and characterisation of human glycerol kinase: the role of solubilising agents and molecular chaperones.

BACKGROUND: Glycerol kinase (GK; EC 2.7.1.30) facilitates the entry of glycerol into pathways of glucose and triglyceride metabolism and may play a potential role in Type 2 diabetes mellitus (T2DM). However, the detailed regulatory mechanisms and structure of the human GK are unknown. METHODS: The human GK gene was cloned into the pET-24a(+) vector and over-expressed in Escherichia coli BL21 (DE3). Since the protein was expressed as inclusion bodies (IBs), various culture parameters and solubilising agents were used but they did not produce bioactive His-GK; however, co-expression of His-GK with molecular chaperones, specifically pKJE7, achieved expression of bioactive His-GK. The overexpressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics. RESULTS: The overexpressed bioactive His-GK was purified apparently to homogeneity (∼295-fold) and characterised. The native His-GK was a dimer with a monomeric molecular weight of ∼55 kDa. Optimal enzyme activity was observed in TEA buffer (50 mM) at 7.5 pH. K+ (40 mM) and Mg2+ (2.0 mM) emerged as prefered metal ions for His-GK activity with specific activity 0.780 U/mg protein. The purified His-GK obeyed standard Michaelis-Menten kinetics with Km value of 5.022 µM (R2=0.927) for its substrate glycerol; whereas, that for ATP and PEP was 0.767 mM (R2=0.928) and 0.223 mM (R2=0.967), respectively. Other optimal parameters for the substrate and co-factors were also determined. CONCLUSION: The present study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.