RNAi screens reveal novel metabolic regulators: RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP) 27

Abstract

Adipose tissue modulates whole body metabolism and insulin sensitivity by controlling circulating lipid levels and producing molecules that can regulate fatty acid metabolism in such tissues as muscle and liver. We have developed RNA interference (RNAi) screens to identify genes in cultured adipocytes that regulate insulin signalling and key metabolic pathways. These short interfering RNA (siRNA)-based screens identified the transcriptional corepressor receptor interacting protein 140 (RIP140) (J Clin Invest 116: 125, 2006) and the mitogen-activated protein kinase (MAP4k4) (Proc Natl Acad Sci USA 103: 2087, 2006) as negative regulators of insulin-responsive hexose uptake and oxidative metabolism. Gene expression profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, tricarboxylic acid cycle, fatty acid oxidation, mitochondrial biogenesis and oxidative phosphorylation. RIP140-null mice resist weight gain on a high-fat diet and display enhanced glucose tolerance. MAP4k4 depletion in adipocytes increases many of the RIP140-sensitive genes, increases adipogenesis and mediates some actions of tumour necrosis factor-alpha (TNF-alpha). Remarkably, another hit in our RNAi screens was fat specific protein 27 (FSP27), a highly expressed isoform of Cidea. We discovered that FSP27 unexpectedly associates specifically with lipid droplets and regulates fat storage. We conclude that RIP140, MAP4k4 and the novel lipid droplet protein FSP27 are powerful regulators of adipose tissue metabolism and are potential therapeutic targets for controlling metabolic disease. The discovery of these novel proteins validates the power of RNAi screening for discovery of new therapeutic approaches to type 2 diabetes and obesity.

Figure 1

Domain structure of receptor interacting protein 140 (RIP140). Interaction of the RIP140 protein with nuclear receptors is mediated by nine LXXLL motifs capable of interacting with the ligand binding domain of the receptor. In turn, via four independent repressor domains (RD1-4) RIP140 can recruit histone deacetylases as well as C-terminal binding protein (CtBP) whose activities repress transcription from the promoter bound by the nuclear receptor.

Figure 2

Function of receptor interacting protein 140 (RIP140) in adipocytes. Via interaction with nuclear receptors (here depicted as a heterodimer such as PPAR-RXR) bound to specific recognition sites in promoters, RIP140 recruits transcription repressor activities [histone deacetylases (HDAC) and C-terminal binding protein (CtBP)] to promoter complexes thereby inhibiting transcription. Major categories of genes repressed by RIP140 function in adipocytes include those in the glycolysis, tricarboxylic acid (TCA) cycle, fatty acid oxidation and oxidative phosphorylation pathways. Inset charts depict numbers of genes whose expression is changed upon RIP140 depletion: upregulated (red bars); unchanged (white bars); downregulated (green bar).

Figure 3

(a) Protein kinases related to Saccharomyces cerevisiae Sterile 20. Depicted are the two families, p21-activated protein kinases (PAK) and the germinal centre protein kinases (GCK), the subfamilies, PAK I and II and GCK-I to -VIII. Members from each one of the subfamilies are also shown. Depicted in red is MAP4K4, a member of the GCK-IV subfamily. (b) Schematic diagram depicting predicted structural motifs of mouse MAP4K4. Within the MAP4K4 sequence shown is the N-terminal catalytic domain in the hatched area, the coiled-coil region in grey and the Nck-binding motif in black, followed by the C-terminal CNH domain. The two proline-rich motifs that match consensus SH3 binding motifs and mediate the association of MAP4K4 with Nck are shown (amino acids 574–616).

Figure 4

(a) Model for the increase in MAP4k4 expression via TNF-α signalling. TNF-α activates the catalytic activity of MAP4k4, but in addition elevates its expression. Our data are consistent with the following hypothesis. Treatment of 3T3-L1 adipocytes with TNF-α stimulates TNFR1 and causes enhanced activation of JNK1/2 and p38 SAP kinase. In turn, activated JNK1/2 and p38 SAP kinase cause increased phosphorylation and activation of c-Jun and ATF2. Increased activation of c-Jun and ATF2 leads to increased MAP4k4 transcription, thus increasing MAP4k4 expression. This increase in MAP4k4 expression then negatively regulates PPAR-γ expression and adipogenesis in 3T3-L1 adipocytes (adapted from Tesz et al. 2007). (b) MAP4k4 is required for TNF-α signalling in adipocytes and skeletal muscle. Model for MAP4k4 downregulation of GLUT4-mediated glucose transport in muscle and adipose cells and the effect of this kinase in insulin receptor signalling in skeletal muscle. Increased levels of circulating fatty acids lead to lipid overload and impairment of insulin-stimulated glucose transport through GLUT4 in muscle. PPAR-γ activity promotes fatty acid storage as triglycerides (TG) in adipocytes. TNF-α signals through MAP4K4 and downregulates PPAR-γ expression and TG biosynthesis in adipocytes. MAP4K4 may also mediate in part the actions of TNF-α on insulin-stimulated glucose transport through GLUT4 in adipocytes as well as in muscle cells.

Figure 5

siRNA-based screen identifies FSP27 as a negative regulator of 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes. (a) Four days after the induction of differentiation, 3T3-L1 adipocytes were transfected with pools of siRNA against the panel of genes shown (GenBank accession numbers will be provided upon request). The effect of each knockdown on 2-DOG uptake was determined by using a 2-DOG uptake assay. Shown is the average of two independent experiments. siRNA was purchased from Dharmacon (Chicago, IL, USA). Pools of siRNA consisting of a mix of four individual oligonucleotides against each gene were used for the screen. (b) Dose-dependent insulin-stimulated 2-DOG uptake in 3T3-L1 adipocytes. Cells on their fourth day of differentiation were transfected with scrambled siRNA or siRNA against, Akt, Pten or FSP27 and assayed for 2-DOG uptake after 72 h. Results are representative of at least six independent experiments each performed in triplicate. (c) Graphic representation of FSP27 and Cidea showing N-terminal CIDE-N domain and COOH-terminal CIDE-C domain. (d) Real-time PCR analysis of mRNA levels of FSP27 in mouse inguinal and axillary subcutaneous white adipose tissue, epididymal white adipose tissue, interscapular brown adipose tissue, liver, muscle, kidney, heart, lung, brain and testis. These tissues or organs were excised from 6-week-old C57Bl/6J male mice that were housed on a 12-h light/dark schedule and had free excess to water and food. After procurement, the samples were immediately stored flash frozen and stored at −80 °C. All procedures were carried according to the guidelines of the University of Massachusetts Medical School Institutional Animal Care and Use Committee (UMMS-IACUC). 36B4 gene was used as a reference gene for quantitative analysis.

Figure 6

The expression of FSP27 is greatly upregulated during differentiation of 3T3-L1 adipocytes. (a) mRNA levels of FSP27 in 3T3-L1 adipocytes at different days of differentiation. Data were generated using Affymetrix GeneChip microarrays. (b) Fold changes, indicating the difference in expression of PPAR-γ and FSP27, from day 0 to day 6 during adipogenesis and after transfecting adipocytes (day 4) with scrambled siRNA or siRNA against PPAR-γ (P < 0.0001). (c) Real-time PCR analysis of mRNA levels of various adipogenic markers in 3T3-L1 adipocytes transfected with scrambled siRNA or siRNA against FSP27 on day 4 of their differentiation. Results are an average of three or more independent experiments. (d) Working model and hypothesis. We hypothesize that FSP27 is a lipid droplet protein that localizes to lipid droplets in adipocytes. Its presence on lipid droplets inhibits basal lipolysis, thereby promoting net triglyceride storage. Hypothetically, FSP27 depletion in our RNAi screen may have increased expression of GLUT4 through the release of PPAR-γ ligand(s) from lipid droplets, with the resultant increase in adipogenesis and adipocyte-specific genes (see text for details).