Chaperone-mediated autophagy regulates adipocyte differentiation

Abstract

Adipogenesis is a tightly orchestrated multistep process wherein preadipocytes differentiate into adipocytes. The most studied aspect of adipogenesis is its transcriptional regulation through timely expression and silencing of a vast number of genes. However, whether turnover of key regulatory proteins per se controls adipogenesis remains largely understudied. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal protein degradation that, in response to diverse cues, remodels the proteome for regulatory purposes. We report here the activation of CMA during adipocyte differentiation and show that CMA regulates adipogenesis at different steps through timely degradation of key regulatory signaling proteins and transcription factors that dictate proliferation, energetic adaptation, and signaling changes required for adipogenesis.

Fig. 1.. CMA activity increases during adipocyte differentiation.

(A) mRNA levels of indicated genes from 3T3L1 cells during adipocyte differentiation. n = 6 independent experiments (i.e.). (B) Representative immunoblots of indicated proteins from 3T3L1 cells during adipocyte differentiation (left) and quantification expressed as arbitrary densitometric units (ADU) relative to day 0 (right). n = 4 i.e. (C) Representative images of 3T3L1 cells stably expressing KFERQ-PS-Dendra2 reporter at the indicated times during adipocyte differentiation. Insets show boxed area at higher magnification. Nuclei are highlighted with 4′,6-diamidino-2-phenylindole (DAPI). Full-field images are shown in fig. S1A. Right: Quantification of CMA activity as number (left) or total area (right) of fluorescent puncta per cell. n = 6 i.e. with 40 cells each. (D) Immunostaining for PreF1 of eWAT from KFERQ-Dendra-mice maintained on regular diet (RD) or high-fat diet for 7 days (7d HFD). Left: Representative full-field images. Insets show higher-magnification images of preadipocytes and perinuclear region of adipocytes. Right: quantification of percentage of PreF1⁺ cells displaying fluorescent puncta (top) and average number of fluorescent puncta per cell (bottom). Nuclei are highlighted with DAPI. n = 4 mice. Values are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ns P > 0.05 using one-way analysis of variance (ANOVA) (A to C), unpaired t test (D, top), and two-way ANOVA (D, bottom).

Fig. 2.. Blocking CMA impairs adipocyte differentiation.

(A) Immunoblot of 3T3L1 cells control (CTR) or stably knocked down for L2A [L2A(−)] and densitometric quantification. n = 5 i.e. (B) Phase contrast images at the indicated differentiation days. Green fluorescent protein (GFP) shows transduction efficiency. (C) Percentage of differentiated cells at day 8 from (B). n = 10 i.e. (D) Immunoblot of the indicated proteins and densitometric quantification during adipocyte differentiation. n = 3 − 6 i.e. (E) DPH, PLIN1, and PLIN2 staining at day 9 of adipocyte differentiation. (F and G) Quantification of area occupied by LD (F) and PLIN1 intensity (G) from (E). n = 4 (F) and 3 (G) i.e. (H) % eWAT/body weight for WT and L2AKO mice. n = 10 mice. (I and J) Hematoxylin and eosin (H&E) staining (I) and size distribution of cells (J) of eWAT. Zoom: Higher magnification. n = 3 mice. (K) eWAT immunostaining for Mac2 and caveolin1 and quantification of number of macrophages. Arrowheads: Macrophages (Mac2⁺ cells). Nuclei are highlighted with Hoechst. n = 4 mice. (L and M) mRNA expression of indicated genes (L) and H&E staining (left) and quantification of crown-like structures (CLS; arrowheads) (right) (M) in eWAT from WT and L2AKO mice on HFD. n = 4 mice. (N) Staining for BODIPY 493/503 (to stain LD) and quantification of LD area in in vitro differentiated primary preadipocytes from WT and L2AKO mice eWAT at day 9. Nuclei are highlighted with DAPI. Full-field images are in fig. S1K. n = 4 mice with 15 fields each. All insets are boxed areas at higher magnification. Values are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 using two-way ANOVA (D and J) or unpaired t test (the rest of the panels).

Fig. 3.. CMA is required for preadipocyte commitment to differentiation.

(A) mRNA expression of indicated genes of 3T3L1 cells control (CTR) or stably knocked down for lamp2a [L2A(−)] at indicated times of adipocyte differentiation. n = 3 to 10 i.e. (B and C) mRNA expression of indicated preadipocyte (B) and adipocyte (C) genes of eWAT from WT and L2AKO mice. n = 5 mice. (D) Flow cytometry for WT and L2AKO eWAT adipose progenitor cells showing percentage of Lin⁻ PDGFR⁺ adipose progenitors. n = 3 mice. (E) Left: Immunostaining for PreF1 and caveolin1 of eWAT from WT and L2AKO mice. Arrowheads: PreF1⁺ cells. Right: Quantification of percentage of PreF1⁺ cells. Nuclei are highlighted with Hoechst. n = 4 mice. Values are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ns P > 0.05 using two-way ANOVA (A) and unpaired t test (B to E).

Fig. 4.. CMA blockage deregulates functional pathways involved in adipocyte differentiation.

(A) Differentially expressed genes in CTR and L2A(−) 3T3L1 cells, comparing days 0 and 2. Venn diagrams depict the up-regulated (top) and down-regulated (bottom) genes in the two cell lines. (B) Heatmap and hierarchical clustering analysis between CTR and L2A(−) cells at days 0 and 2 of differentiation. (C) Venn diagram of genes increased basally in L2A(−) cells and those that fail to decrease in L2A(−) with differentiation when compared to CTR. (D and E) Gene ontology enrichment analysis of genes with constitutively (basal) increased expression (D) or that fail to decrease with differentiation (E) in L2A(−) cells when compared to CTR. (F and G) Pathway enrichment analysis of genes with constitutively (basal) increased expression (F) or that fail to decrease expression with differentiation (G) in L2A(−) cells when compared to CTR. (H to K) Predicted upstream activators of genes with constitutively (basal) increased expression (H) or that fail to decrease expression with differentiation (J) in L2A(−) cells when compared to CTR. Hierarchical arrangement places Myc on top of upstream regulators constitutively activated in L2A(−) and TGFβ on top of upstream regulators that fail to decrease during differentiation [see detailed maps in fig. S4 (D and E)]. Percentage of proteins bearing CMA motifs (KFERQ-like motifs) among the proteins shown in (H) and (J) are shown in (I) and (K), respectively. All gene ontology terms are significant for P < 0.001.

Fig. 5.. Lack of CMA in preadipocytes leads to sustained proliferation and glycolysis during differentiation.

(A) Cell cycle analysis of 3T3L1 cells control (CTR) or stably knocked down for lamp2a [L2A(−)] at days 0 and 1 of differentiation. Left: Representative histograms. Right: Quantification of phases of cell cycle. n = 3 i.e. (B) mRNA expression of indicated gene of eWAT. n = 5 mice. (C) Left: Immunostaining of eWAT for Ki67 and quantification of percentage of Ki67⁺ cells (arrowheads). Insets: Higher magnification. n = 4 mice. (D) Immunoblot of CTR and L2A(−) 3T3L1 cells at indicated times of adipocyte differentiation (left) and quantification of protein levels (right). n = 4 i.e. (E to G) Immunostaining for Myc (E) and quantification of Myc levels in cytosol (F) and nuclei (G) in 3T3L1 cells at day 0 untreated or treated with inhibitors of lysosomal proteolysis (Lys Inh). Nuclei are highlighted with DAPI. n = 3 i.e. (H to J) Extracellular acidification rates (ECAR) (H), energy phenotype (I) and glycolysis, glycolytic capacity, and glycolytic reserve (J) in 3T3L1 cells CTR or L2A(−) at days 0 to 4 of differentiation. n = 4 i.e. (K) Immunoblot and quantification of 3T3L1 cells at indicated times of differentiation. n = 9 i.e. (L) Immunoblots and lysosomal degradation rates for indicated proteins in 3T3L1 cells untreated or treated with Lys Inh at indicated times of differentiation. LC3 is a positive control for Lys Inh. n = 7 i.e. Values are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ns P > 0.05 using two-way ANOVA (A, D, F, J, and L) and unpaired t test (B, C, G, and K).

Fig. 6.. Dysregulated TGFβ signaling is responsible for the adipogenesis defect in CMA-deficient preadipocytes.

(A) mRNA expression of indicated gene of eWAT from WT and L2AKO mice. n = 8 mice. (B) Trichrome A staining of eWAT and quantification of percentage of Trichrome⁺ area. n = 5 mice. (C) mRNA expression of indicated genes in eWAT. n = 5 mice. (D) Trichrome A staining of eWAT from WT and L2AKO mice on HFD and quantification of percentage of Trichrome⁺ area. n = 4 mice. (E) Representative immunoblots for indicated proteins from eWAT of WT and L2AKO mice and quantification. n = 10 mice. (F) Representative immunoblots for indicated proteins and quantification of CTR and L2A(−) 3T3L1 cells at indicated times of adipocyte differentiation. n = 4 i.e. (G) Representative immunoblots and quantification of SMAD2/3 lysosomal degradation rates in CTR and L2A(−) 3T3L1 cells untreated or treated with Lys Inh at indicated times of adipocyte differentiation. n = 7 i.e. (H to J) Immunostaining of SMAD2/3 (H) and quantification of SMAD2/3 levels in cytosol (I) and nuclei (J) in 3T3L1 cells at day 0 untreated or treated with Lys Inh. Image shows color mapping by gradient intensity. n = 3 − 4 i.e. (K) DPH staining and quantification of percentage of differentiated cells in 3T3L1 cells at day 7 untreated or treated with TGFβ inhibitor for 12 hours at day 1.5. n = 4 i.e. Values are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 using unpaired t test (A to E and J), two-way ANOVA (F, G, and I), and one-way ANOVA (K).