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. 2006 Jan;116(1):125-36.
doi: 10.1172/JCI26040. Epub 2005 Dec 22.

Suppression of oxidative metabolism and mitochondrial biogenesis by the transcriptional corepressor RIP140 in mouse adipocytes

Aimee M Powelka  1 , Asha Seth, Joseph V Virbasius, Evangelos Kiskinis, Sarah M Nicoloro, Adilson Guilherme, Xiaoqing Tang, Juerg Straubhaar, Andrew D Cherniack, Malcolm G Parker, Michael P Czech
Affiliations

Suppression of oxidative metabolism and mitochondrial biogenesis by the transcriptional corepressor RIP140 in mouse adipocytes

Aimee M Powelka et al. J Clin Invest. 2006 Jan.

Abstract

Using an siRNA-based screen, we identified the transcriptional corepressor RIP140 as a negative regulator of insulin-responsive hexose uptake and oxidative metabolism in 3T3-L1 adipocytes. Affymetrix GeneChip profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, TCA cycle, fatty acid oxidation, mitochondrial biogenesis, and oxidative phosphorylation in these cells. Conversely, we show that reexpression of RIP140 in mouse embryonic fibroblasts derived from RIP140-null mice downregulates expression of many of these same genes. Consistent with these microarray data, RIP140 gene silencing in cultured adipocytes increased both conversion of [14C]glucose to CO2 and mitochondrial oxygen consumption. RIP140-null mice, previously reported to resist weight gain on a high-fat diet, are shown here to display enhanced glucose tolerance and enhanced responsiveness to insulin compared with matched wild-type mice upon high-fat feeding. Mechanistically, RIP140 was found to require the nuclear receptor ERRalpha to regulate hexose uptake and mitochondrial proteins SDHB and CoxVb, although it likely acts through other nuclear receptors as well. We conclude that RIP140 is a major suppressor of adipocyte oxidative metabolism and mitochondrial biogenesis, as well as a negative regulator of whole-body glucose tolerance and energy expenditure in mice.

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Figures

Figure 1
Figure 1. An siRNA-mediated screen identifies Nrip1 (RIP140) as a negative regulator of deoxyglucose uptake in 3T3-L1 adipocytes.
(A) Four days after the induction of differentiation, 3T3-L1 adipocytes were transfected with smart pools of siRNA against a panel of genes (GenBank accession numbers are provided in Supplemental Table 1) selected as highly expressed in muscle and 3T3-L1 adipocytes compared with 3T3-L1 fibroblasts. The effect of each knockdown on glucose uptake was determined using a 2-deoxyglucose uptake assay. Arrow indicates insulin-stimulated 2-deoxyglucose uptake in RIP140-knockdown cells. Shown is the average of 2 independent experiments. (B) siRNA-mediated depletion of RIP140 mRNA was confirmed by real-time RT-PCR. The graph represents the mean ± SEM of 3 independent experiments. Scr, scrambled; Q-RT-PCR, quantitative RT-PCR. *P < 0.05 compared with scrambled.
Figure 2
Figure 2. RIP140 depletion enhances GLUT4 expression and hexose uptake in both day-4 and day-8 3T3-L1 adipocytes.
Four (day 4) or eight (day 8) days after the induction of differentiation, 3T3-L1 adipocytes were transfected with scrambled or RIP140 siRNA. Three days later, cell lysates were used for SDS-PAGE to assess GLUT4, GLUT1, and actin expression levels. Intact cells were used to examine the effect of RIP140 depletion on 2-deoxyglucose uptake. The graph shows the mean ± SEM of 5 independent experiments. *P < 0.05 compared with scrambled, by Student’s t test.
Figure 3
Figure 3. Insulin signaling is not significantly affected by RIP140 silencing.
Four days after the induction of differentiation, 3T3-L1 adipocytes were transfected with scrambled or RIP140 siRNA. Three days later, the cells were starved for 2 hours and then stimulated with insulin for 30 minutes. Cell lysates were used for total and phospho-Akt and MAPK blots. The representative blot shows a small but insignificant increase in phospho-serine Akt levels.
Figure 4
Figure 4. RIP140 silencing enhances multiple metabolic pathways in 3T3-L1 adipocytes.
Eight days after the induction of differentiation, 3T3-L1 adipocytes were transfected with scrambled or RIP140 siRNA. Three days later, mRNA was harvested and used for Affymetrix GeneChip analysis. The numbers of genes that were significantly (P < 0.05) increased, decreased, or unchanged in expression within each of the pathways shown are depicted in bar graphs adjacent to the respective pathways. The total number of significantly (P < 0.05) changed probe sets from 3 independent experiments is shown in the upper-right panel. FATP, fatty acid transport protein; TG, triglycerides.
Figure 5
Figure 5. RIP140 reexpression in RIPKO-1 adipocytes results in downregulation of multiple metabolic pathways.
Ten days after the induction of differentiation, RNA was isolated from RIPKO-1 adipocytes and RIPKO-1 adipocytes stably expressing RIP140 using a lentiviral vector and used for Affymetrix GeneChip analysis. The number of genes in the indicated metabolic pathways that were significantly (P < 0.05) upregulated, downregulated, or not changed is shown from left to right in the graphs.
Figure 6
Figure 6. Metabolic and mitochondrial protein expression is enhanced in 3T3-L1 adipocytes depleted of RIP140.
Eight days after the induction of differentiation, 3T3-L1 adipocytes were transfected with scrambled or RIP140 siRNA. After 72 hours, postnuclear supernatants were used for immunoblotting selected proteins in oxidative phosphorylation and fatty acid oxidation pathways. Shown is a representative blot and densitometric quantification of 3 independent experiments (mean ± SEM). All of the changes shown (except those for actin) were significant at P < 0.05 (indicated by asterisk).
Figure 7
Figure 7. RIP140 depletion enhances glycolysis and TCA cycling, but not triglyceride synthesis or de novo free fatty acid synthesis.
Eight days after the induction of differentiation, 3T3-L1 adipocytes were transfected with scrambled or RIP140 siRNA. After 72 hours, cells were starved and glucose metabolism (with continued starvation [basal] or 1 μM insulin stimulation) was measured by [6-14C]-glucose conversion into carbon dioxide (CO2), triglycerides, and fatty acids as described in Methods. Graphs show the mean ± SEM of 3–4 independent experiments. *P < 0.05 compared with scrambled, by Student’s t test.
Figure 8
Figure 8. RIP140 is a negative regulator of cellular respiration in 3T3-L1 adipocytes.
Eight days after the induction of differentiation, 3T3-L1 adipocytes were transfected with scrambled or RIP140 siRNA. (A) After 72 hours, cells were incubated with MitoTracker Red to stain actively respiring mitochondria. Fluorescence microscopy was performed as described in Methods. Shown are representative photographs with identical exposure times. (B) Cells were assayed for respiration using the oxygen biosensor fluorescence assay described in Methods. Basal respiration is indicated on the left, and uncoupled respiration, induced by addition of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 33 μM), is shown on the right. Illustrated are the averages of 3–4 independent experiments. The respiration with or without FCCP of cells transfected with scrambled or RIP140 siRNA are considered statistically different based on the fluorescence intensity at the time when fluorescence of the leading curve was equal to half-maximum for each experiment. RFU, raw fluorescence units.
Figure 9
Figure 9. Absence of RIP140 protects against age- and diet-induced glucose intolerance and enhances insulin responsiveness on a high-fat diet.
Glucose tolerance tests were performed on 3-month-old female mice fed a normal chow diet (A), 8-month-old female mice fed a normal chow diet (B), or 3-month-old female mice fed a high-fat diet (C) for 10 weeks. Values are mean ± SEM; n = 6–12. (D) Insulin tolerance tests were performed on 3-month-old female mice fed a high-fat diet for 9 weeks. Values are mean ± SEM; n = 9.
Figure 10
Figure 10. RIP140 depletion does not require normal PPARγ levels to enhance GLUT4 expression.
Eight days after the induction of differentiation, cells were transfected with scrambled, RIP140, PPARγ, or RIP140 plus PPARγ siRNA. Three days later, cell lysates were used for immunoblotting GLUT4 and actin, while nuclear extracts were used to immunoblot PPARγ and lamin. A 2-deoxyglucose uptake assay was performed with the indicated knockdowns. The graph shows the average and SE of 5 independent experiments. *P < 0.05 compared with scrambled siRNA, by Student’s t test.
Figure 11
Figure 11. RIP140 depletion requires ERRα to enhance GLUT4 expression, deoxyglucose uptake, and mitochondrial protein expression.
Eight days after the induction of differentiation, cells were transfected with scrambled, RIP140, ERRα, or RIP140 plus ERRα siRNA. (A) Three days later, cell lysates were used for immunoblotting GLUT4, SDHB, and actin. A representative blot and the mean ± SEM of 4–5 independent experiments are shown. (B) A 2-deoxyglucose uptake assay was performed with the indicated knockdowns. A representative blot and the mean ± SEM of 3 independent experiments are shown. *P < 0.05 compared with scrambled siRNA, by Student’s t test.
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References

    1. Saltiel A.R., Kahn C.R. Insulin signalling and the regulation of glucose and lipid metabolism. Nature. 2001;414:799–806. doi: 10.1038/414799a. - DOI - PubMed
    1. Czech M.P., Corvera S. Signaling mechanisms that regulate glucose transport. J. Biol. Chem. 1999;274:1865–1868. doi: 10.1074/jbc.274.4.1865. - DOI - PubMed
    1. Watson R.T., Pessin J.E. Subcellular compartmentalization and trafficking of the insulin-responsive glucose transporter, GLUT4. Exp. Cell. Res. 2001;271:75–83. doi: 10.1006/excr.2001.5375. - DOI - PubMed
    1. Minokoshi Y., Kahn C.R., Kahn B.B. Tissue-specific ablation of the GLUT4 glucose transporter or the insulin receptor challenges assumptions about insulin action and glucose homeostasis. J. Biol. Chem. 2003;278:33609–33612. doi: 10.1074/jbc.R300019200. - DOI - PubMed
    1. Cline G.W., et al. Impaired glucose transport as a cause of decreased insulin-stimulated muscle glycogen synthesis in type 2 diabetes. N. Engl. J. Med. 1999;341:240–246. doi: 10.1056/NEJM199907223410404. - DOI - PubMed

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