New Insights into Cytosolic Glucose Levels during Differentiation of 3T3-L1 Fibroblasts into Adipocytes

Abstract

Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. In adipocytes, glucose utilization is considered very rapid, meaning that every glucose molecule entering the cytoplasm is quickly phosphorylated. Thus, the cytosolic free glucose concentration is considered to be negligible; however, it was never measured directly. In the present study, we monitored cytosolic glucose dynamics in 3T3-L1 fibroblasts and adipocytes by expressing a fluorescence resonance energy transfer (FRET)-based glucose nanosensor: fluorescent indicator protein FLIPglu-600. Specifically, we monitored cytosolic glucose responses by varying transmembrane glucose concentration gradient. The changes in cytosolic glucose concentration were detected in only 56% of 3T3-L1 fibroblasts and in 14% of 3T3-L1 adipocytes. In adipocytes, the resting cytosolic glucose concentration was reduced in comparison with the one recorded in fibroblasts. Membrane permeabilization increased cytosolic glucose concentration in adipocytes, and glycolytic inhibitor iodoacetate failed to increase cytosolic glucose concentration, indicating low adipocyte permeability for glucose at rest. We also examined the effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration in adipocytes by a factor of 3.6; however, we recorded no effect on delta ratio (⌬R) in fibroblasts. Adrenaline increased cytosolic glucose concentration in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls (p < 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose.Adipocytes utilize glucose to supply energy needs for cellular activities and to promote synthesis and storage of lipids in the cell. Glucose utilization is regulated by the supply of intracellular glucose-6-phosphate. This, in turn, depends on the transport of glucose across the cell membrane and its phosphorylation in the presence of hexokinase and ATP. Adipocytes contain two isoforms of a family of proteins that facilitate transport of glucose across the plasma membrane: GLUT1, the constitutive glucose transporter located mainly at the plasma membrane, and GLUT4, the insulin-sensitive glucose transporter (1, 2). The effect of insulin to increase glucose uptake is exerted by stimulating the translocation of GLUT4 from an intracellular pool to the plasma membrane and thus increasing the permeability of the plasma membrane to glucose (3, 4). However, the accompanying changes in dynamics of intracellular glucose concentration in a single adipocyte are not known as, until recently, it was not possible to measure this parameter directly. To image and quantify changes in cytosolic glucose, Fehr et al. (6) constructed a...

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