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. 2007 Apr 13;355(3):745-50.
doi: 10.1016/j.bbrc.2007.02.025. Epub 2007 Feb 12.

PACSIN3 overexpression increases adipocyte glucose transport through GLUT1

William Roach  1 , Markus Plomann
Affiliations

PACSIN3 overexpression increases adipocyte glucose transport through GLUT1

William Roach et al. Biochem Biophys Res Commun. .

Abstract

PACSIN family members regulate intracellular vesicle trafficking via their ability to regulate cytoskeletal rearrangement. These processes are known to be involved in trafficking of GLUT1 and GLUT4 in adipocytes. In this study, PACSIN3 was observed to be the only PACSIN isoform that increases in expression during 3T3-L1 adipocyte differentiation. Overexpression of PACSIN3 in 3T3-L1 adipocytes caused an elevation of glucose uptake. Subcellular fractionation revealed that PACSIN3 overexpression elevated GLUT1 plasma membrane localization without effecting GLUT4 distribution. In agreement with this result, examination of GLUT exofacial presentation at the cell surface by photoaffinity labeling revealed significantly increased GLUT1, but not GLUT4, after overexpression of PACSIN3. These results establish a role for PACSIN3 in regulating glucose uptake in adipocytes via its preferential participation in GLUT1 trafficking. They are consistent with the proposal, which is supported by a recent study, that GLUT1, but not GLUT4, is predominantly endocytosed via the coated pit pathway in unstimulated 3T3-L1 adipocytes.

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Figures

Fig. 1
Fig. 1. Expression of PACSIN isoforms during 3T3-L1 differentiation
3T3-L1 cells were isolated as fibroblasts (F) or following incubation in differentiation media for 0, 2, 4, 6, 8 or 10 days. SDS samples (25 μg) were immunoblotted for the stated proteins. Brain homogenate was included as a positive control (+) for PACSIN1. Blots are representative of two independent experiments.
Fig. 2
Fig. 2. Overexpression of PACSIN3 in 3T3-L1 adipocytes promotes glucose uptake
(A) Schematic diagram of the PACSIN3 constructs expressed through adenoviral expression. (B) 3T3-L1 adipocytes at day 4–5 after differentiation were infected with equal viral titers of beta-galactosidase (βgal), mycWTPACSIN3 (WT), mycΔC354PACSIN3 (ΔC354), mycΔC329PACSIN3 (ΔC329) or mycΔN244PACSIN3 (ΔN244) constructs; 3 days post-infection, cells were incubated with or without 1 μM insulin for 30 min, followed by the measurement of 2-deoxy-glucose uptake. *, p < 0.05 and **, p < 0.01 compared to the corresponding βgal control by Student T-test. Values are the mean ± standard error from three independent experiments.
Fig. 3
Fig. 3. PACSIN3 overexpression increases GLUT1 and transferrin receptor in the plasma membrane
3T3-L1 adipocytes were infected with equal titers of βGal, WT or ΔN244 viruses. Three days post-infection, cells were either left untreated (−) or were stimulated with 1 μM insulin (+) for 30 min and fractionated as described in Materials and Methods; purified plasma membrane fractions were isolated. Protein was then separated by SDS-PAGE. For (A) GLUT1 and (B) GLUT4, 0.5 μg of protein was loaded per lane. For (C) transferrin receptor (Tfr), and the (D) Na+K+ATPase, 25 μg of protein was loaded per lane. Representative immunoblots are depicted. The values of the relative intensities on the immunoblots have been normalized to the value for the basal βgal control. *, p < 0.05 compared to the corresponding βgal control as determined by Student T-test . Values represent mean ± standard error from two to five experiments.
Fig. 4
Fig. 4. PACSIN3 overexpression promotes GLUT1 but not GLUT4 exofacial presentation at the plasma membrane
3T3-L1 adipocytes were infected with equal titers of βgal, WT or ΔN244 viruses. Three days post-infection, cells were treated with or without 1 μM insulin for 30 min and then UV irradiated for 1 min in the presence of 40 μM bio-ATB-BGPA as described in Materials and Methods. Equal volumes of each sample were separated by SDS-PAGE and immunoblotted with (A) anti-GLUT1 and (B) anti-GLUT4 antibodies. Immunoblots were quantified and relative labeling was calculated by setting basal βgal values as 1.0, Values within respective GLUT species were then calculated relative to the βgal basal signal. **, p < 0.01 compared to the corresponding βgal control. Values represent mean ± standard error from three independent experiments.
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