Rosiglitazone Modulates Insulin-Induced Plasma Membrane Area Changes in Single 3T3-L1 Adipocytes

Abstract

In this study we hypothesized that rosiglitazone, an antidiabetic high-affinity agonist for the peroxisome proliferator-activated receptor gamma, affects the plasma membrane (PM) turnover in single 3T3-L1 adipocytes. To study the PM turnover, the patch-clamp electrophysiological method was used to measure changes in membrane capacitance (Cm), a parameter linearly related to the PM area. Microscopy results show that the presence of rosiglitazone in the differentiating medium significantly increased the differentiation of 3T3-L1 adipocytes in cell culture, based on oil red O-stained area (11.4 +/- 1.2%) vs. controls (3.1 +/- 0.5%). Moreover, rosiglitazone treatment significantly reduced the size of single 3T3-L1 adipocytes; their average radius of 21.1 +/- 1.1 microm in controls was reduced to 17.5 +/- 0.5 microm in rosiglitazone-treated cells. Consistent with this, insulin application increased the rate of Cm increase to 2.34 +/- 0.10%/min, which was significantly different from controls (0.12 +/- 0.08%/min). However, pretreatment of cells with rosiglitazone prior to the treatment with insulin resulted in an attenuated rate of Cm increase. These data support the involvement of insulin in the modulation of membrane area and show that treatment by rosiglitazone reduced the insulin-mediated membrane area increase in 3T3-L1 adipocytes.

Rosiglitazone balances insulin-induced exo- and endocytosis in single 3T3-L1 adipocytes

“…Furthermore, adipocyte differentiation leads to the enhanced expression of adipocyte-specific genes, such as GLUT4 and insulin receptor substrate-1 (IRS-1), which are important components of the insulin receptor signaltransduction pathway (Ntambi and Young-Chuel, 2000;Okuno et al, 1998;White and Kahn, 1994;Wu et al, 1998). It was shown that the treatment of 3T3-L1 adipocytes with Rosi during differentiation results in the accumulation of GLUT4 at the plasma membrane surface (Martinez et al, 2010), which is consistent with the concept that TZDs regulate membrane dynamics (Velebit et al, 2008). However, the mechanism by which Rosi affects the increased density of GLUT4 at the plasma membrane remains unclear.…”

“…However, the mechanism by which Rosi affects the increased density of GLUT4 at the plasma membrane remains unclear. It has been shown that insulin induces an increase in net changes of plasma membrane surface area of single 3T3-L1 cells by increasing the rate of exocytosis, via phosphatidylinositol 3-kinase, as in the case of insulin-induced GLUT4 translocation (Velebit et al, 2008), consistent with insulin-induced surface area measurements in adipocytes (Chowdhury et al, 2002(Chowdhury et al, , 2005. Interestingly, the pretreatment of 3T3-L1 cells with Rosi significantly attenuated the insulin-induced increase in membrane area (Velebit et al, 2008), which could be due to a simultaneous insulininduced increase in exo-and endocytosis of Rosi-pretreated cells.…”

“…It has been shown that insulin induces an increase in net changes of plasma membrane surface area of single 3T3-L1 cells by increasing the rate of exocytosis, via phosphatidylinositol 3-kinase, as in the case of insulin-induced GLUT4 translocation (Velebit et al, 2008), consistent with insulin-induced surface area measurements in adipocytes (Chowdhury et al, 2002(Chowdhury et al, , 2005. Interestingly, the pretreatment of 3T3-L1 cells with Rosi significantly attenuated the insulin-induced increase in membrane area (Velebit et al, 2008), which could be due to a simultaneous insulininduced increase in exo-and endocytosis of Rosi-pretreated cells. To directly address this, we here employed quantitative confocal microscopy and the styryl fluorescent dye FM1-43, a membrane area marker (Cochilla et al, 1999) to monitor cumulative exocytosis as well as cumulative endocytosis of single 3T3-L1 adipocytes pretreated with Rosi and stimulated with insulin.…”

“…This appears to be associated with a plasma membrane area increase (Chowdhury et al, 2005;Velebit et al, 2008). When insulin is removed, GLUT4 is rapidly cleared from the plasma membrane back into intracellular compartments (Muretta et al, 2008).…”

“…At cellular level the insulin sensitizing effects of TZDs are mediated through M a n u s c r i p t 4 the peroxisome proliferator-activated receptor γ (PPARγ) (Bhatia and Viswanathan, 2006) which is highly abundant in adipose tissue and to a lesser extent in skeletal muscle and liver (Auwerx, 1999;Olefsky, 2000). A representative of TZD, rosiglitazone (Rosi), is a potent agonist of PPARγ and improves the differentiation of 3T3-L1 cells into adipocytes (Rangwala and Lazar, 2004;Velebit et al, 2008). Furthermore, adipocyte differentiation leads to the enhanced expression of adipocyte-specific genes, such as GLUT4 and insulin receptor substrate-1 (IRS-1), which are important components of the insulin receptor signaltransduction pathway (Ntambi and Young-Chuel, 2000;Okuno et al, 1998;White and Kahn, 1994;Wu et al, 1998).…”