Control of adipose tissue expandability in response to high fat diet by the insulin-like growth factor-binding protein-4 - PubMed (original) (raw)

Control of adipose tissue expandability in response to high fat diet by the insulin-like growth factor-binding protein-4

Olga Gealekman et al. J Biol Chem. 2014.

Abstract

Adipose tissue expansion requires growth and proliferation of adipocytes and the concomitant expansion of their stromovascular network. We have used an ex vivo angiogenesis assay to study the mechanisms involved in adipose tissue expansion. In this assay, adipose tissue fragments placed under pro-angiogenic conditions form sprouts composed of endothelial, perivascular, and other proliferative cells. We find that sprouting was directly stimulated by insulin and was enhanced by prior treatment of mice with the insulin sensitizer rosiglitazone. Moreover, basal and insulin-stimulated sprouting increased progressively over 30 weeks of high fat diet feeding, correlating with tissue expansion during this period. cDNA microarrays analyzed to identify genes correlating with insulin-stimulated sprouting surprisingly revealed only four positively correlating (Fads3, Tmsb10, Depdc6, and Rasl12) and four negatively correlating (Asph, IGFbp4, Ppm1b, and Adcyap1r1) genes. Among the proteins encoded by these genes, IGFbp4, which suppresses IGF-1 signaling, has been previously implicated in angiogenesis, suggesting a role for IGF-1 in adipose tissue expandability. Indeed, IGF-1 potently stimulated sprouting, and the presence of activated IGF-1 receptors in the vasculature was revealed by immunostaining. Recombinant IGFbp4 blocked the effects of insulin and IGF-1 on mouse adipose tissue sprouting and also suppressed sprouting from human subcutaneous adipose tissue. These results reveal an important role of IGF-1/IGFbp4 signaling in post-developmental adipose tissue expansion.

Keywords: Adipocyte; Angiogenesis; Endothelial Cell; Insulin; Insulin-like Growth Factor (IGF); Obesity; Vascular Biology.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.

FIGURE 1.

Time course of epididymal fat pad expansion in response to ND or HFD feeding. Body weight (A), epididymal fat pad weight (B), and mean adipocyte size (C) are given at the indicated time points after initiation of diets in 5-week-old mice. Points represent the mean and S.E. of eight mice per condition. Statistical significance was assessed by two-tailed paired t tests for each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Comparison of the size distribution of adipocytes at 3 weeks (D), 7 weeks (E), and 11 weeks (F) of HFD feeding compared with ND is shown. G, size distribution of adipocytes at 3, 7, and 11 weeks of ND or HFD feeding. Arrows point to appearance of peaks in lower and higher size bins after 11 weeks of HFD.

FIGURE 2.

FIGURE 2.

Effect of insulin on sprouting from adipose tissue of ND-fed mice. A, representative images of adipose tissue explants embedded in Matrigel and incubated in the absence (top) and in the presence (bottom) of insulin, visualized at day 14 post-embedding. B, effect of insulin on the percent of explants displaying capillary sprouts after 14 days in culture. C, effects of insulin on the average number of capillary sprouts formed by explants that displayed sprouting. The value assigned to no added insulin was 5 n

m

, which is contributed by 10% FBS in the growth medium. Statistical significance of the difference between no added insulin and insulin was assessed by two-tailed paired t tests. *, p < 0.05; **, p < 0.01, n = 4. Effects of 2-week rosiglitazone (Rosi) treatment initiated at 28 weeks of ND on the percent of explants displaying capillary sprouts (D) and the number of sprouts per explant (E). *, p < 0.05; **, p < 0.01 compared with no added insulin; #, p < 0.05 versus no rosiglitazone, n = 3.

FIGURE 3.

FIGURE 3.

Effect of HFD and rosiglitazone treatment on basal and insulin-stimulated sprout formation. A, representative images of adipose tissue explants from mice fed HFD for 18 weeks, embedded in Matrigel, and incubated in the absence (top) and presence (bottom) of 170 n

m

insulin, visualized at day 14 post-embedding. B, effect of insulin and HFD on the percent of explants displaying capillary sprouts after 14 days in culture. C, effects of insulin on the average number of capillary sprouts formed by explants that displayed sprouting. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with no added insulin. #, p < 0.05; ##, p < 0.01; ####, p < 0.0001 compared with 3-week value. Effects of 2-week rosiglitazone (Rosi) treatment initiated at 28 weeks of HFD on the percent of explants displaying capillary sprouts (D) and the number of sprouts per explant (E). *, p < 0.05; ****, p < 0.0001 compared with no insulin; #, p < 0.05 compared with no rosiglitazone. Data represent the means and S.E. from four mice. Statistical significance was assessed by two-tailed paired t tests.

FIGURE 4.

FIGURE 4.

Time course of changes in capillary density in response to HFD. A, representative images of anti-CD31 immunohistochemistry of epididymal adipose tissue from ND- (top) or HFD (bottom)-fed mice at 7 (left panels) or 25 (right panels) weeks after diet initiation. B, mean and S.E. of capillary lumens per adipocyte assessed by immunohistochemistry. Values were derived from analysis of five fields per section, from two independent sections per mouse derived from four independent mice. Statistical significance was assessed by two-tailed paired t tests. *, p < 0.05; **, p < 0.01.

FIGURE 5.

FIGURE 5.

Effect of HFD on adipose tissue capillary network integrity. A, maximal intensity projections of 25 image stacks acquired at 10-μm intervals (250-μm thickness) of IB4-stained fragments of adipose tissue from mice fed ND or HFD (top or bottom panels, respectively) for 5, 15-, or 30 weeks (left, middle, and right panels, respectively). B, superimposition of images in A over corresponding Bodipy-stained images. C, optical section through a fragment of adipose tissue from 18-week HFD-fed mouse double-stained with IB4 (left panel) and antibody to F4/80 (middle panel). Right panel is the overlap of IB4 and F4/80. Arrows point to examples of a single cells positive for both IB4 and F4/80.

FIGURE 6.

FIGURE 6.

Effects of HFD and rosiglitazone on insulin-stimulated sprout formation. Sprouting potential ((number of sprouts/explant)(% of explants sprouting)) of explants derived from mice fed ND or HFD for 30 weeks and treated without or with rosiglitazone (Rosi) for 2 weeks starting at 28 weeks of diet feeding. Statistical significance was assessed by two-tailed paired t tests. **, p < 0.01; ****, p < 0.0001 compared with ND; #, p < 0.05 compared with HFD.

FIGURE 7.

FIGURE 7.

Expression levels and effects of IGFbp4 on sprout formation. Expression levels of IGFbp4 (A), Emr-1 (B), and CD68 (C) mRNAs in adipose tissue from mice maintained on ND or HFD for the indicated length of time. Statistical significance of the difference between ND and HFD was assessed by two-tailed paired t tests **, p < 0.01; ***, p < 0.005. D, representative Western blots from whole tissue extracts from mice on ND or HFD for the weeks indicated probed with antibodies to IGFbp4 or actin. E, quantification of data in D, showing mean and S.E. of four independent experiments. F, sprouting potential ((number of sprouts/explant)(% of explants sprouting)) of explants derived from mice maintained on ND or HFD for 25 weeks and cultured in the presence or absence of insulin and 0.5 μg/ml IGFbp4 as indicated. Statistical significance was assessed by two-tailed paired t tests as indicated. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.0001. G, production of IGF-1 by explants. The supernatant from 20 wells per experiment from four independent experiments was pooled and analyzed for IGF-1 content. Statistical significance was assessed by one-way analysis of variance and the Krustal-Wallis test with Dunn's correction for multiple comparisons *, p < 0.05; ***, p < 0.005.

FIGURE 8.

FIGURE 8.

Effect of IGF-1 on sprout formation. A, effects of insulin, IGF-1 and IGF-2, and of IGFbp4 on the percent of explants displaying capillary sprouts after 14 days in culture. Statistical significance of the difference from no treatment was calculated by one-way analysis of variance corrected for multiple comparisons (Holm-Sidak). **, p < 0.005; ****, p < 0.0001. B, Western blotting for IGF-1 receptor (top panel) and insulin receptor (bottom panel) β subunits in tissue from 8-week ND- and HFD-fed mice. a indicates lane with extract from isolated adipocytes. C, whole mount staining of adipose tissue after 24 h of incubation in the absence (top panels) or presence (bottom panels) of 150 n

m

insulin. Arrows highlight regions of coincidence between phosphorylated receptors and IB4 staining. Arrowhead points to cell outside the vascular wall. D, model for interaction between high fat diet, insulin, and IGFbp4 to enhance adipose tissue growth.

FIGURE 9.

FIGURE 9.

Effect of IGFbp4 on human adipose tissue sprout formation. A, explant from human subcutaneous adipose tissue embedded in Matrigel and visualized after 5 (left) and 10 (right) days in culture. Average number of sprouts per explant from subcutaneous human adipose tissue explants plotted as a function of donor's fasting serum insulin (B) or circulating triglyceride concentration (C). D, effect of IGFbp4 on the average number of capillaries formed per explant. In each case, ∼30 explants were cultured in the presence or absence of IGFbp4, and sprouting was assessed after 14 days. ***, p < 0.001. A p value of 0.037 was obtained in a one-tailed, paired Student's t test comparing the mean of three individuals without or with IGFbp4.

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