Metabolic Flux Analysis in Insulin-Stimulated Adipocytes Form Temporal Patterns of Intracellular Metabolome Data

The hormone insulin is secreted from beta-cells in the pancreas, and plays a great role for maintaining blood glucose homeostasis. In adipocytes, insulin activates glucose uptake and utilization, and lipid biosynthesis. Abnormalities of blood insulin level are widely known to be related to type-II diabetes mellitus, thus deep understanding of adipocyte metabolism by insulin stimulation has been desired. However, how metabolic fluxes change in adipocytes by insulin is poorly understood. In this study, we estimated metabolic fluxes in insulin-stimulated differentiated 3T3-L1 adipocytes from intracellular metabolome data.

Materials and Methods

3T3-L1 adipocyte cell lines were cultured, and the media were swapped with those including U-¹³C glucose and insulin. Cell samples were obtained at 1-60 min after insulin stimulation (100 nM), and intracellular metabolite concentrations and their mass isotopomer abundances were measured by CE-MS. Control experiments without insulin addition were also examined. Measured experimental metabolome data seemed to consist of two phases, the early transient and the late sustained phase, after insulin stimulation. Then we estimated time average fluxes within each phase by a modified metabolic flux analysis approach, considering temporal patterns of metabolite concentrations and mass isotopomer fractions. The optimal time dividing two experimental phases were also estimated.

Results

¹³C -tracer experiments for adipocyte cell lines and the modified metabolic flux analysis approach exhibited different characteristics in the estimated metabolic fluxes between the early and the late experimental phase, and between with or without insulin stimulation. Regarding experiments with insulin, large absolute fluxes from glucose to triacylglycerol through upstream of glycolysis were estimated in the early phase, which decreased by more than 80% in the late phase. In contrast, less than 30% of absolute fluxes in the early phase decreased in glycogen biosynthesis pathway in the late phase. Regarding experiments without insulin stimulation, overall absolute fluxes were estimated to be much smaller than those without insulin, but a flux ratio at the branching point of glucose-6-phosphate and triose-phosphate were similar between with or without insulin stimulation.

In summary, in adipocytes, insulin stimulation lead to a large increase in fluxes of glucose uptake, upstream of glycolysis, and triacylglycerol biosynthesis pathway, whereas increased fluxes in glycogen biosynthesis pathway were relatively small. In future, we will develop more sophisticated method to estimate dynamics of metabolic fluxes. Moreover, we will integrate the fluxome, metabolome, and phosphoproteome data which we’ve already obtained, for comprehensive understanding of insulin action on adipocyte cells.