Insulin responsiveness of the glucose transporter 4 in 3T3- L1 cells depends on the presence of sortilin
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Glucose homeostasis is vital to mammalian functioning and depends on the ability of skeletal muscle and adipose cells to translocate the GLUT4 glucose transporter to the plasma membrane in response to insulin stimulation. The GLUT4 transporter traffics in insulin-responsive vesicles (IRVs) that also incorporate sortilin as a major protein constituent. Another protein, AS160 (Akt substrate of 160kD, a Rab GTPase) is thought to maintain the intracellular localization of the IRVs in the absence of insulin. Previous studies have shown that sortilin is induced upon differentiation of pre-adipocytes to adipocytes and plays the key role in the formation of the IRVs. We have found that ectopic expression of GLUT4 in undifferentiated pre-adipocytes does not lead to its marked translocation to the plasma membrane upon insulin stimulation. On the contrary, sortilin expressed in undifferentiated pre-adipocytes translocated to the plasma membrane in response to insulin. Moreover, upon co-expression with GLUT4, sortilin dramatically increased insulin responsiveness of GLUT4 to the levels observed in fully differentiated adipocytes. Thus, sortilin may represent the key component of the IRVs that is responsible not only for vesicle formation, but for their insulin responsiveness as well. Our experiments also show that undifferentiated pre-adipocytes express little AS160 and lack a mechanism for the full intracellular retention of GLUT4. The latter can be achieved by ectopic expression of AS160, despite its limited co-localization with GLUT4 or sortilin. Mutant AS160 that cannot be phosphorylated by Akt does not allow GLUT4 to translocate to the plasma membrane in response to insulin but does provide basal retention of GLUT4, showing that AS160 is a major component of basal retention. Because free fatty acids are elevated in obesity and associated with insulin resistance, we studied the effects of palmitate on insulin signaling. Palmitate decreased phosphorylation of the insulin-signaling molecule Akt and attenuated the ability of adipocytes to take up glucose in response to insulin. We have also shown that adipocytes responded to palmitate by forming stress granules throughout the cytoplasm. As circulating levels of fatty acids are generally increased in obesity, this observation may explain the negative effect of obesity on protein expression.
Thesis (Ph.D.)--Boston University by Dana Buckler-Peña