Insulin-Like Growth Factor-1 Induces Lipid Production in Human SEB-1 Sebocytes Via Sterol Response Element-Binding Protein-1 Terry M. Smith, Zhaoyuan.

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Insulin-Like Growth Factor-1 Induces Lipid Production in Human SEB-1 Sebocytes Via Sterol Response Element-Binding Protein-1 Terry M. Smith, Zhaoyuan Cong, Kathryn L. Gilliland, Gary A. Clawson, Diane M. Thiboutot Journal of Investigative Dermatology Volume 126, Issue 6, Pages 1226-1232 (June 2006) DOI: 10.1038/sj.jid.5700278 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Treatment of SEB-1 sebocytes with adipogenic hormones increases 14C acetate incorporation into non-polar lipids. This increase can be attributed to insulin. (a) SEB-1 sebocytes were treated with methylisobutylxanthine (0.5mm), dexamethasone (0.3μm), and insulin (1.74μm) for 72hours. Cells were then incubated with 14C acetate for 2hours. Lipids were extracted and separated by thin layer chromatography. (b) Insulin alone increases lipogenesis in SEB-1 sebocytes. Treatment with methylisobutylxanthine or dexamethasone alone did not change lipogenesis (not shown). Units are expressed as mean±SE c.p.m./hour/104 cells. Significance was determined by t-test and a P-value of <0.05 in comparison to the vehicle was determined to be significant and denoted with an*. The following abbreviations are used: C=cholesterol; FOH=fatty alcohol; OA=oleic acid; TAG=triglycerides; CO=cholesterol oleate; SQ=squalene. Journal of Investigative Dermatology 2006 126, 1226-1232DOI: (10.1038/sj.jid.5700278) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Treatment of SEB-1 sebocytes with 100nm insulin increases lipogenesis, though to a lesser extent than 1μm insulin or 20ng/ml IGF-1. SEB-1 sebocytes were treated for 24hours with 100nm insulin, 1μm insulin, or 20ng/ml IGF-1. Cells were then incubated with 14C acetate for 2hours. Lipids were extracted and separated by thin layer chromatography. Units are expressed as mean±SE c.p.m./hour/104 cells. Analysis of variance was used to determine statistical significance. Date by treatment interaction was observed for some data points. A P-value of <0.05 in comparison to the vehicle was determined to be significant and denoted with an*, while a P-value of <0.05 compared to 100nm insulin is denoted by an ‡. The following abbreviations are used: C=cholesterol; FOH=fatty alcohol; OA=oleic acid; TAG=triglycerides; CO=cholesterol oleate; SQ=squalene; WE=wax esters. Journal of Investigative Dermatology 2006 126, 1226-1232DOI: (10.1038/sj.jid.5700278) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Both forms of the SREBP-1 protein are increased in SEB-1 sebocytes treated with 20ng/ml IGF-1 or 1μm insulin, but not with 100nm insulin. SEB-1 sebocytes were treated 24hours with insulin or IGF-1. Protein was isolated and a Western blot was performed, probing with antibodies to SREBP-1 and actin. (a) is a representative blot probed for SREBP-1 following 20ng/ml IGF-1 treatment. (b) These data were normalized to actin and quantified. (c) Representative blot probed for SREBP-1 and normalized to actin following insulin treatment: 100nm (lane 2) and 1μm (lane 3). (d) These data are quantified. (b and d) All blots were repeated a minimum of five times, analyzed by densitometry and normalized to actin. Densitometer data were compared to vehicle-treated blots and Student's t-test was performed. A P-value <0.05 was determined to be statistically significant. Journal of Investigative Dermatology 2006 126, 1226-1232DOI: (10.1038/sj.jid.5700278) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Both SREBP-1a and SREBP-1c mRNA transcripts are increased in SEB-1 sebocytes treated with 20ng/ml IGF-1, 100nm insulin, or 1μm insulin. SEB-1 sebocytes were treated with (a) IGF-1 or (b) insulin for 14hours. Following treatment, RNA was isolated and subjected to QPCR to determine abundance of the SREBP-1a and SREBP-1c transcript in comparison to vehicle-treated cells. Data were analyzed using the relative expression software tool program and Student's t-test was performed. A P-value <0.05 was considered statistically significant. Using the relative expression software tool program, it is not possible to generate SD or SE unless the same RT reaction is run repeatedly, thus P-values are used to gauge variation. Journal of Investigative Dermatology 2006 126, 1226-1232DOI: (10.1038/sj.jid.5700278) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The ratio of SREBP-1c:1a mRNA in SEB-1 sebocytes is 0.59:1, while the ratio is 9.8:1 in sebaceous gland. Human sebaceous glands were microdissected. RNA was extracted from the glands and was subjected to QPCR to determine the relative ratio of SREBP-1c:1a. RNA was also extracted from SEB-1 cells to determine the SREBP-1c:1a ratio by QPCR. Data represent the mean ratios of five determinations±SE. Journal of Investigative Dermatology 2006 126, 1226-1232DOI: (10.1038/sj.jid.5700278) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The ratio of insulin receptor isoform A:isoform B mRNA is 5.1:1 in SEB-1 sebocytes. RNA was extracted from SEB-1 sebocytes and QPCR was performed to determine the ratio of insulin receptor isoform A to insulin receptor isoform B. Data represent the mean ratios of five determinations±SE. Journal of Investigative Dermatology 2006 126, 1226-1232DOI: (10.1038/sj.jid.5700278) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions