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. 2004 Aug 24;101(36):13296–13301. doi: 10.1073/pnas.0405354101

Fig. 3.

Fig. 3.

PLSCR3-/- mice show glucose intolerance and their adipocytes exhibit insulin resistance with diminished expression of IRs. (a) Glucose tolerance testing. WT (triangles), PLSCR3-/- (squares), and combined PLSCR(1&3)-/- (circles) mice were fasted for 18 h before i.p. injection of d-glucose. Blood glucose levels were measured at times indicated. Symbols represent mean ± SD (n = 8) of pooled data for four males and four females of each genotype. Animals were matched at 2 months of age (±2 d). Analysis of these data revealed no obvious or consistent difference in either fasted blood glucose or peak response to i.p. d-glucose in the male versus female animals evaluated (data not shown). These data were from a single experiment but representative of three. (b) Insulin-stimulated uptake of [14C]deoxyglucose. Experiments were performed with adipocytes isolated from epididymal (Upper) and inguinal (Lower) fat pads of 3-month-old WT and PLSCR3-/- mice. Cells were matched for total protein and prestimulated (20 min, 37°C) with insulin (concentrations on abscissa). Net influx of 2-[14C]deoxy-d-glucose was then measured 20 min after addition of tracer. ▴ and •, nonspecific uptake in WT and PLSCR3-/- adipocytes, respectively, measured in the presence of cytocholasin B. See Methods. (c) Western blotting for expression of IR, IRS-1, IRS-3, and GLUT4 in PLSCR3-/- adipocytes. Gonadal adipocytes from WT and PLSCR3-/- mice were matched on the basis of total cellular protein and Western blotted for expression of IR (β-chain), IRS-1, IRS-3, and GLUT4. The gel was also developed for β-actin and lamin (A and C). The relative band intensities by scanning densitometry (Kodak Image Station 440 CF) of these proteins detected in PLSCR3-/- adipocytes (expressed as ratios to that in WT) were: IRS-1 (0.54); IR-β (0.82); IRS-3 (0.63); GLUT4 (0.38); lamin A/C (1.21); β-actin (1.15). Data are representative of results obtained in four independent experiments with different mice. (d) Reduced expression of cell surface IRs. 125I-insulin binding to epididymal adipocytes isolated from 3-month-old WT and PLSCR3-/- mice is shown. Binding was performed at 16°C, with correction for nonspecific binding (see Methods). Cells were matched based on protein content. (Inset) Western blot of PLSCR3 and β-actin in each sample. These data, from a single experiment, are representative of two so performed.