PMC

Adipocyte cilium-associated proteins. Differentiating HWP were immunostained for cilium and for BBS1, BBS10, BBS12, Fzzl1–10, and Smo. Left panels show the cilium, middle panels show the protein localization and right panels the merged pictures. (A) BBS1 is detected throughout the cilium. BBS10 and BBS12 are localized at the basal body (B) and (C), respectively. The Fzzl protein (D) and Smo (E) are also associated to the cilium. (Scale bars, 5 μm.)

Localization of BBS10 and BBS12 proteins in renal epithelial cells and mature adipocytes. (A) Ciliated hRPTEC stained for cilium (acetylated α-tubulin, α-Tub) and for BBS10 or BBS12. Primary cilium and basal body (Left). BBS10 and BBS12 proteins are detected adjacent to the nucleus (Middle) and localized at the basal body (Right, merged pictures). (B) Mature adipocytes stained for the centrioles with γ-tubulin (γ-Tub) and for either BBS10 or BBS12. BBS10 and BBS12 are centriolar proteins. (Scale bars, 5 μm.)

Up-regulation of BBS10 and BBS12 proteins and transient presence of primary cilium in early adipogenesis. (A) BBS10 and BBS12 protein levels during adipogenesis. HWP were cultured to D0 in preadipocyte growth medium. Differentiation was induced, and cellular extracts were immunoblotted at the indicated time points. BBS10 and BBS12 proteins are maximally expressed in early differentiation. Glucose Transporter 4 (GLUT4, 45kDa) served as adipogenic marker and β-tubulin served as loading control. (B) (Left to Right) Proliferating preadipocytes, confluent differentiating preadipocytes, mature adipocytes stained for cilium. Primary cilium is exclusively observed in differentiating preadipocyte. (Scale bars, 5 μm.) (C) Representative scanning electron microscopic pictures of the same 3 stages. Cilium is clearly observable on the differentiating preadipocyte (Middle, arrow) and absent in preadipocyte and mature adipocyte. (Scale bars, 10 μm.) (D) Electron microscopic pictures showing cilium with the centriole at the base (arrow), axonemes inside the cilium. Asterix indicates the intracellular lipid droplets. Right panel shows the two centrioles (arrows). One centriole recruited at the basal body to support the axonemes. (Scale bar, 1 μm.)

Adipogenesis is favored in BBS-deprived adipocytes. (A) Representative immunofluorescence pictures for the detection of the inactive phosphorylated GSK3β (GSK3β-P) and the active unphosphorylated GSK3β in the differentiating preadipocytes following the indicated RNAi treatment. An increase in unphosphorlated GSK3β was observed after inactivation of either BBS10 or BBS12. (Scale bars, 5 μm.) (B) Quantification of the active GSK3β isoform by ELISA. Increases of 20 and 35% in active GSK3β were obtained after BBS10 (lane 2) or BBS12 (lane 3) knock down compared to control RNAi (lane 1). (n = 3; control compared to BBS10: P = 0.007 and control compared to BBS12: P = 0.013). (C) Immunodetection of cytoplasmic and nuclear β-catenin in confluent preadipocytes after 24 h transfection with control, BBS10, and BBS12 RNAi. β-catenin protein was detected at 94kDa. 1, control RNAi transfected; 2, BBS10 RNAi; 3, BBS12 RNAi. Homogenous protein loading was verified (SI Text and Fig. S1D) (D) Immunofluorescence for PPARγ in transfected preadipocyte after 24 h in differentiating medium. Increased PPARγ content was detected in BBS10- and BBS12 RNAi-treated cells compared to controls. (E) A band at 57 kDa was detected by Western blot. 1, control RNAi transfected; 2, BBS10 RNAi; 3, BBS12 RNAi. The Western blot results confirmed the increased PPARγ expression in the adipocytes following the indicated BBS knock down.

Increased fat content in adipocytes derived from BBS patients' fibroblasts. (A) Representative pictures of human dermal fibroblasts with the indicated gene mutation cultured in FGM (SI Text, Methods) to full confluence before adipogenic induction. (Scale bar, 20 μm.) (B) Triglyceride accumulation after 14 days of culture in FDAM was stained with Adipored Assay Reagent. Increased fluorescent-labeled cells were observable in the BBS10- and BBS12-mutated cells (Middle and Right, respectively) compared to control wild-type cells (Left). (Scale bar, 60 μm.) (C) The lipid-derived fluorescence was measured at 572 nm for each condition (CONT for the wild-type healthy fibroblasts, BBS10-, and BBS12-mutated fibroblasts) and was expressed as total measured fluorescence per cell. (n = 3) (control compared to BBS10: P = 0.001 and control compared to BBS12: P < 0.001). (D) Secreted leptin was measured in the culture medium by ELISA and was expressed as the total absorbance at 450 nm per cell. In parallel with the increased intracellular triglyceride content, higher quantities of leptin was detected in the medium from the BBS mutated cells compared to the healthy control (n = 3) (control compared to BBS10: P = 0.05 and control compared to BBS12: P = 0.03).

Impaired cilium formation during adipogenesis upon BBS knock down. (A) Ninety percent confluent preadipocytes were transfected and cultured for 4 days in preadipocytes growth medium. Four days later, immunoblot analysis for BBS10 was done on control transfected RNAi duplexes (medium and low Negative Universal Control Stealth RNAi) (lanes 1 and 2, respectively) and on BBS10 RNAi knockdown confluent preadipocyte cells (BBS10 duplexes 1 and 2) (SI Methods, and lanes 3 and 4, respectively). Significant reduction in BBS10 protein level was obtained. Anti-β-Tubulin was used as loading control. (B) BBS12 was inactivated in a similar way and cell lyzates analyzed by immunoblotting (lanes 1 and 2 were loaded with cellular extracts from medium and low Negative Universal Control Stealth RNAi transfected cells and lanes 3 and 4 corresponded to those from cells transfected with BBS12 duplexes 2 and 3). A significant reduction in BBS12 level was also observed. (C) BBS10 or BBS12 silencing to prevent ciliogenesis. Four days after RNAi-transfection, cilia presence was assessed. Representative pictures of BBS10, BBS12, and control RNAi transfected cells are shown. Arrows indicate the primary cilium. (Scale bar, 40 μm.) (D) The percentage of ciliated cells: that is, (number of cilia/total number of nuclei) × 100% was calculated and plotted. n = 550 for each of 3 independent experiments. Significant reduction in ciliated cells was obtained (control compared to BBS10: P = 0.001 and control compared to BBS12: P = 0.003). (E) Immunolocalization of BBS12 in BBS10-deprived preadipocytes showed that centriolar localization of BBS12 protein was unaffected in the absence of the BBS10. (Scale bar, 5 μm.) (F) In a similar way, BBS10 centriolar localization was also unaffected in BBS12-deprived preadipoctes. (Scale bar, 5 μm.)