Store-Operated Ca2+ Entry Controls Induction of Lipolysis and the Transcriptional Reprogramming to Lipid Metabolism

Abstract

Ca²⁺ signals were reported to control lipid homeostasis, but the Ca²⁺ channels and pathways involved are largely unknown. Store-operated Ca²⁺ entry (SOCE) is a ubiquitous Ca²⁺ influx pathway regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca²⁺ channel ORAI1. We show that SOCE-deficient mice accumulate pathological amounts of lipid droplets in the liver, heart, and skeletal muscle. Cells from patients with loss-of-function mutations in STIM1 or ORAI1 show a similar phenotype, suggesting a cell-intrinsic role for SOCE in the regulation of lipid metabolism. SOCE is crucial to induce mobilization of fatty acids from lipid droplets, lipolysis, and mitochondrial fatty acid oxidation. SOCE regulates cyclic AMP production and the expression of neutral lipases as well as the transcriptional regulators of lipid metabolism, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), and peroxisome proliferator-activated receptor α (PPARα). SOCE-deficient cells upregulate lipophagy, which protects them from lipotoxicity. Our data provide evidence for an important role of SOCE in lipid metabolism.

Keywords: CRAC channel; ORAI1; STIM1; cAMP; calcium; fatty acid oxidation; lipid metabolism; lipolysis; lipophagy; mitochondria.

Figure 1. SOCE regulates lipid balance in cells and tissues

(A–C) Lipid droplet (LD) accumulation in SOCE-deficient mice. EM images of heart (A) and Oil Red O stain of skeletal muscle (B) from wild-type (WT) and Orai1^R93W knock-in mice 5 days postpartum. (C) Oil Red O stain of heart, liver and soleus sections (10x) from 16-week-old Stim1^fl/flStim2^fl/fl UBC-ERT2-Cre mice and Cre-negative littermates 5 days after the first dose of tamoxifen injection. (D) EM image (20,000×) of skeletal muscle from a patient with ORAI1 p.G98R mutation shows LD deposition (arrow). (E) SOCE in fibroblasts from healthy donors (HD, blue) and patients (P, red) with loss-of-function mutations in STIM1 or ORAI1 measured by Ca²⁺ imaging. ER Ca²⁺ stores were depleted with thapsigargin (TG) and SOCE was measured after re-addition of 2 mM Ca²⁺. Traces represent the averages of 3 HDs and 3 Ps (≥ 20 cells per donor analyzed). (F and G) Neutral lipid staining of fibroblasts from HDs and Ps cultured in high glucose medium. Cells were stained with BODIPY 493/503, and analyzed by flow cytometry. Representative histograms (F) and average mean fluorescence intensities (MFI) (G) of BODIPY 493/503 after subtraction of autofluorescence (ΔMFI) in cells from 3 HDs and 3 Ps. ΔMFI values of patients were normalized to the mean ΔMFI in HD cells. (H–I) HD and P fibroblasts were challenged with oleic acid (OA, 0.125 mM, 24 hours) and stained with BODIPY 493/503 (green) to identify neutral lipid stores. (H) Dashed boxes indicate regions shown at higher magnification in the right panels. Nuclei were counterstained with DAPI. (I) Average numbers of LDs per cell (left), percentage of cellular area occupied by LDs (middle) and average LD area (right). All values are mean ± SEM. Statistical analysis was performed using Student’s t-test. * p<0.05.

Figure 2. SOCE is required for mitochondrial function and FAO

(A and B) Mitochondrial volume in fibroblasts from healthy donors (HD, blue) and patients (P) with loss-of-function mutations in STIM1 or ORAI1 (P, red) analyzed with MitoView Green and flow cytometry. Representative histograms (A) and average MFI of MitoView Green after subtraction of autofluorescence (ΔMFI) in HD and P fibroblasts (B). (C) Mitochondrial DNA copy number in fibroblasts from 3 HDs and 3 Ps assessed by real time PCR. (D and E) Relative mRNA levels of transcription factors involved in mitochondrial DNA replication (D) and electron transport chain (ETC) components and uncoupler protein 2 (UCP2) (E) in fibroblasts from 3 HDs and 3 Ps assessed by real time PCR and normalized to the house keeping gene NONO (Non-POU domain-containing octamer-binding protein). (F) Western blot for UCP2 and β-actin in fibroblasts from 3 HDs and 3 Ps. (G and H) Analysis of mitochondrial degradation (mitophagy) by co-staining of fibroblasts from HD and P with MitoTracker (green), LysoTracker DND-99 (red) and DAPI (blue). Images show cells at low (left) and high (right) magnification. Bar graphs show Mander’s coefficients of colocalization of MitoTracker and LysoTracker in cells from 3 HDs and 3 Ps (right). (H–J) Analysis of the mitochondrial membrane potential (MMP) in TMRM-loaded HD (blue) and P (red) fibroblasts by single cell imaging. Averaged traces (H), and averaged basal MMP (I) before and after oligomycin treatment (0 and 1 represent minimum and maximum MMP after de- and hyperpolarization with 1 µM FCCP and 1 µM oligomycin, respectively). (J) Proton pumping rate of the ETC measured as the MMP hyperpolarization rate after oligomycin. (K–M) Mitochondrial superoxide production and ETC electron transport rate. Fibroblasts from 3 HDs (blue) and 3 Ps (red) were loaded with MitoSOX and analyzed by flow cytometry before and after 1 µM oligomycin. Normalized MitoSOX signal (ΔMFI) over time (K), basal MitoSOX signal (ΔMFI) before oligomycin (L) and electron transport rate as the rate of superoxide production after oligomycin (M). (N–P) NADH levels and TCA cycle turnover. The redox state of cells was measured by NADH autofluorescence in fibroblasts from 3 HDs (blue) and 3 Ps (red) by time-lapse microscopy. Relative minimum (0) and maximum (1) NADH fluorescence was determined by treating cells with 1 µM FCCP to deplete NADH and 1 µM antimycin A / 100 nM rotenone to saturate NADH levels. Averaged relative NADH autofluorescence over time on a scale from 0 to 1 (N), basal NADH levels measured as total NADH autofluorescence per cell after background subtraction (O) and TCA cycle turnover (P) calculated as the rate of NADH increase after antimycin A / rotenone. (Q, R) Relative mRNA levels of CPT1B (Q) and ACADVL (R) in fibroblasts after 24h culture in high-glucose or oleic acid (OA) medium followed by 6h starvation. mRNA levels in 3 Ps were normalized to those in 3 HDs. (S) Mitochondrial respiration in fibroblasts cultured in OA medium only (HD, black and P, grey) or with subsequent starvation (HD, blue and P, red) in the presence or absence of the CPT1 inhibitor etomoxir (40 µM). (T) ¹⁴C-OA oxidation measured in cells (HD, black and P, white) cultured in OA alone or OA plus starvation. Data are mean ± SEM and normalized to HD fibroblasts (unless indicated otherwise). Statistical analysis was performed using Student’s t-test. * p<0.05.

Figure 3. SOCE regulates starvation-induced lipolysis

(A) Analysis of lipid droplet (LD) levels in fibroblasts from 3 healthy donors (HD) and 3 patients (P) with loss-of-function mutations in STIM1 or ORAI1. Cells were cultured in high glucose or oleic acid (OA) medium for 12 h without or with subsequent starvation (OA+starv) in low glucose (2 mM) medium for 24 h and stained with BODIPY (open traces) or left unstained (filled gray traces) to measure LD content. Representative histograms (left) and quantification of 3 repeat experiments (right). ΔMFI, mean fluorescence intensity (MFI) _BODIPY - MFI _unstained cells. % LD clearance was calculated as described in Methods. (B–C) Free fatty acid (B) and free glycerol (C) release by fibroblasts from 3 HDs and 3 Ps after 24 h culture in high glucose (11 mM) or OA-containing medium followed by 24 h of starvation in 2 mM glucose in the presence or absence of isoproterenol (10 µM). a.u., arbitrary units. (D–F) Lipolysis in NIH3T3-L1 cells transduced with dominant negative ORAI1-E106Q (red, DN-ORAI1) or empty vector control (blue, empty). (D) SOCE following stimulation with thapsigargin (TG) and re-addition of 1 µM Ca²⁺ Ringer’s solution. (E and F) Free glycerol release by NIH3T3-L1 cells cultured for 24 h in medium containing high glucose (E and F), OA (E) or PA (F) followed by starvation for 6 h in 1 mM glucose in the presence or absence of isoproterenol (10 µM). (G and H) Expression of HSL, ATGL and MGL mRNA (normalized to the housekeeping gene NONO) (G) and protein (H) determined in fibroblasts from 3 HDs and 3 Ps. Data are representative of 3 repeat experiments. (I) HSL phosphorylation on the activating Ser660 residue in NIH3T3-L1 cells transduced with empty vector or DN-ORAI1. Cells were cultured for 30 min in high glucose (24 mM) medium alone or with forskolin (10 µM, Forsk.) + IBMX (1 mM) or with thapsigargin (1 µM, TG), or in medium containing 0.5 mM OA followed by 30 min starvation (OA+starv) in the presence or absence of isoproterenol (10 µM). Representative Western blots (top) and normalized ratios of phospho-HSL to total HSL expression (bottom) from 5 repeat experiments (see Methods for details). (J and K) cAMP levels in NIH3T3-L1 cells (J) cultured as described in (I) and in fibroblasts from 3 HDs and 3 Ps (K) cultured in high glucose (11 mM) RPMI 1640 medium alone (basal) or stimulated for 30 min with 1 µM TG. All values represented as bar graphs in A, B, C, E, F, G, I, J and K were normalized to mean values obtained in HD fibroblasts or empty vector-transduced NIH3T3-L1 cells after culture in glucose medium. All values are mean ± SEM of at least 3 repeat experiments. Statistical analysis was performed using Student’s t-test. * p<0.05.

Figure 4. Increased lipophagy in SOCE-deficient cells

(A) Immunoblot for LC3 in fibroblasts from healthy donors (HD) and patients (P) with loss-of-function mutations in STIM1 or ORAI1 treated or not with lysosomal protease inhibitors (PI). (B) LC3-II steady-state levels (left) and autophagic flux (right) calculated by densitometric analysis of immunoblots. (C and D) Autophagy in HD and P fibroblasts lentivirally transduced with the LC3 autophagy reporter mCherry-GFP-LC3 and stained with DAPI. (C) Images of representative cells. (D) Average number of autophagic vacuoles (AV), autophagosomes (APG) and autolysosomes (AUT) per cell section calculated by imaging >1,200 cells in 9 fields from 3 HDs and 3 Ps in duplicate by high content microscopy. (E and F) Long-lived protein degradation in 3 HD and 3 P fibroblasts. Total protein degradation rates (E) and percentage of lysosomal protein degradation and macroautophagy-dependent degradation (3-methyladenine (3-MA) sensitive) in cells treated without or with 10 mM 3-MA for the duration of the chase. (F). (G) HD and P cells were cultured in oleic acid (OA) medium, transferred to medium without OA for 12 h and stained with BODIPY (green, to detect neutral lipid stores), LC3 antibody (red) and DAPI. Representative images of merged channels. Boxed area at higher magnification (middle) and detail of lipid droplets (right) show colocalized pixels in white. (H and I) Percentages of LC3⁺ lipid droplets (LD) (H) and BODIPY⁺ autophagic vacuoles (AV) (I) calculated from images similar those in (G). (J and K) ATG7 knockdown causes lipotoxicity. (J) ATG7 protein expression in HD and P fibroblasts lentivirally transduced with scrambled shRNA (−) or shRNA targeting ATG7 (+) analyzed by immunoblotting with anti-ATG7 antibody. (K) Viability of cells after culture in 10 mM glucose, 1 mM OA or 0.5 mM palmitic acid (PA) for 24 hours. All values are mean ± SEM. Statistical analysis was performed using Student’s t-test. * p<0.05.

Figure 5. SOCE controls PGC-1α and PPARα expression

(A and B) Relative mRNA levels of PGC-1α and PPARα in fibroblasts from 3 patients (P) with loss-of-function mutations in STIM1 or ORAI1 normalized to 3 healthy donors (HD, in A) and in NIH3T3-L1 cells expressing dominant negative ORAI1-E106Q (DN-ORAI1) normalized to cells expressing an empty vector (B). Cells were cultured in high glucose (−) or oleic acid (OA)-containing medium followed by 6 hours of starvation (OA+starv.). mRNA expression was normalized to the housekeeping genes NONO (Non-POU domain-containing octamer-binding protein, in A) and ACTB (Actin beta, in B). (C–D) Relative mRNA levels of PGC-1α (C) and PPARα (D) in NIH3T3-L1 cells cultured in high glucose medium alone or stimulated with 1 µM thapsigargin (TG) in the presence or absence of calcineurin inhibitor FK506 (1 µM), adenylyl cyclase inhibitor SQ22,536 (100 µM) or CAMK inhibitor KN93 (10 µM) for 6 h. Alternatively, cells were cultured in medium containing 0.5 mM OA followed by starvation for 6 h. mRNA expression was normalized to ACTB. (E) Phosphorylation of CREB on the activating Ser133 residue assessed by Western blotting in NIH3T3-L1 cells transduced with empty vector (−) or DN-ORAI1 (+). Cells were cultured in high glucose with or without 10 mM isoproterenol, 10 µM forskolin (Forsk.) + 1 mM IBMX or 1 µM thapsigargin (TG) for 30 minutes. Alternatively, cells were cultured in 0.5 mM OA followed by starvation for 6 h. (F and G) Relative mRNA levels of PGC-1α, PPARα, STIM1 and STIM2 in the heart (F) and liver (G) of Stim1^fl/flStim2^fl/fl UBC-ERT2-Cre mice and Cre-negative littermates 5 days after the first injection of tamoxifen. mRNA levels were determined by real time PCR and normalized to ACTB. (H and I) Relative mRNA levels of PGC-1α, PPARα, STIM1 and ORAI1 in the heart (H) and liver (I) of WT mice fed a normal diet or fasted for 24 h. mRNA expression was normalized to ACTB (PGC1α, PPARα) or HPRT1 (STIM1, ORAI1). Bar graphs in A, B and F-I show data normalized to mean values obtained from the respective control samples and represent the mean ± SEM from at least 3 repeat experiments. Statistical analysis was performed using Student’s t-test. * p<0.05.