Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers - PubMed (original) (raw)

Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers

Hwan-Woo Park et al. Nat Commun. 2014.

Abstract

Autophagy deregulation during obesity contributes to the pathogenesis of diverse metabolic disorders. However, without understanding the molecular mechanism of obesity interference in autophagy, development of therapeutic strategies for correcting such defects in obese individuals is challenging. Here we show that a chronic increase of the cytosolic calcium concentration in hepatocytes during obesity and lipotoxicity attenuates autophagic flux by preventing the fusion between autophagosomes and lysosomes. As a pharmacological approach to restore cytosolic calcium homeostasis in vivo, we administered the clinically approved calcium channel blocker verapamil to obese mice. Such treatment successfully increases autophagosome-lysosome fusion in liver, preventing accumulation of protein inclusions and lipid droplets and suppressing inflammation and insulin resistance. As calcium channel blockers have been safely used in clinics for the treatment of hypertension for more than 30 years, our results suggest they may be a safe therapeutic option for restoring autophagic flux and treating metabolic pathologies in obese patients.

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Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Saturated fatty acids induce protein inclusions and arrest autophagy

(a–f) HepG2 cells were treated with BSA (Con), 500 μM palmitic acid (PA) or 100 nM rapamycin (Rap) for 9 hr and subjected to following analyses. (a, c–f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c, d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f) (n = 3). (b) Cells were subjected to serial protein extraction (solubility fractionation) with indicated concentration of Triton X-100 (TX100) or sodium dodecyl sulfate (SDS) and analyzed by immunoblotting with indicated antibodies. (g, h) HepG2 cells stably transduced with mCherry (mCh)-GFP-LC3-expressing retroviruses were treated with Con, Rap or PA for 9 hr and examined under a live confocal microscope (g). Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out. Number of autolysosomes was quantified (h) (n = 7). Scale bars, 5 μm. All data are shown as mean ± s.e.m. ***P < 0.001 (Student’s t test). Molecular weight markers are indicated in kDa.

Figure 2

Figure 2. Calcium channel blockers suppress saturated fatty acid-induced protein inclusion formation

HepG2 cells were treated with BSA (Con or (−)), PA (500 μM), PA + verapamil (Ver, 50 μM) or PA + nicardipine (Nic, 100 μM) for 9 hr. (a–d) After each treatment, cells were loaded with a calcium indicator X-Rhod-1-AM (a, b) or Fura-2-AM (c, d). Calcium levels were visualized by laser confocal microscopy (a) or by dual fluorescent microscopy (c, 340/380 nm ratio image) and quantified (b, d; n = 8 and 30, respectively). (e–h) Cells with indicated treatments were subjected to solubility fractionation. 1% Triton X-100-insoluble fractions were dissolved in 2% SDS, analyzed by immunoblotting (e, f) and quantified (g, h) (n = 3). (i–l) Cells with indicated treatments were subjected to immunostaining with indicated antibodies (i, k). DNA was stained with DAPI (blue). Amount of aggregated proteins was quantified (j) (n = 10). Co-localization between LAMP1 and LC3 was quantified (l) (n = 4). Boxed areas in fluorescence images are magnified in right-most panels (i, k). Scale bars, 10 μm (a), 20 μm (c), 5 μm (i, k). All data are shown as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test). Molecular weight markers are indicated in kDa.

Figure 3

Figure 3. Verapamil relieves hepatosteatosis of obese mice

4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg kg−1 body weight, i.p., n = 3) for 10 days. LFD-kept mice (n = 5) of same age were used as a negative control. (a) Body weight was daily monitored during injection period. (b) Daily food consumption was measured during injection period. (c) Livers were harvested from indicated mice and photographed. (d, e) Total liver mass (d) and total epididymal white adipose tissue (eWAT) mass (e) were measured from indicated mice. (f–h) Relative mRNA expression was analyzed from eWAT of indicated mice through quantitative RT-PCR. (i, j) Liver sections were analyzed by hematoxylin and eosin (H&E, upper panels) and Oil Red O (ORO, lower panels) staining (i). ORO densities were quantified (j). (k, l) Calcium-induced CaMKII autophosphorylation in livers was analyzed by immunoblotting (k) and quantified (l). (m) Primary hepatocytes from 2 month-old C57BL/6 mice kept on LFD were treated with BSA (Con), PA (500 μM), PA + verapamil (Ver, 50 μM) or PA + nicardipine (Nic, 100 μM) for 12 hr. After each treatment, cells were loaded with a calcium indicator Fura-2-AM. Calcium levels were visualized by dual fluorescent microscopy at 340/380 nm, and ratio of fluorescence intensities of Fura-2-AM at 340 nm over 380 nm was quantified (n = 17). Scale bars, 1 cm (c), 200 μm (i). All data are shown as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test). Molecular weight markers are indicated in kDa.

Figure 4

Figure 4. Verapamil suppresses obesity-induced accumulation of protein aggregates

4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg kg−1 body weight, i.p., n = 3) for 10 days. LFD-kept mice (n = 5) of same age were used as a negative control. (a–d) Livers were subjected to solubility fractionation. 1% Triton X-100 soluble (a) and insoluble (b) fractions were analyzed by immunoblotting (a, b) and quantified (c, d). (e, f) Paraffin sections of indicated livers were subjected to p62 immunostaining and hematoxylin counterstaining (e). p62-positive areas were quantified (f). Scale bar, 200 μm (e). All data are shown as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test). Molecular weight markers are indicated in kDa.

Figure 5

Figure 5. Verapamil restores lysosomal degradation of autophagosomes in liver of obese mice

4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg/Kg body weight, i.p., n = 3) for 10 days. LFD-kept mice (n = 5) of same age were used as a negative control. (a, b) Levels of LC3-II from 1% Triton X-100 insoluble fraction of livers were analyzed by immunoblotting (a) and quantified (b). (c, d) Frozen sections of indicated livers were subjected to LC3/LAMP1 immunostaining and DAPI counterstaining (c). Co-localization between LC3 and LAMP1 was quantified (d). Boxed areas in fluorescence images are magnified in right-most panels (c). Scale bars, 5 μm (c). All data are shown as mean ± s.e.m. ***P < 0.001 (Student’s t test). Molecular weight markers are indicated in kDa.

Figure 6

Figure 6. Verapamil reduces liver inflammation and improves metabolic homeostasis

4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg kg−1 body weight, i.p., n = 3) for 10 days. LFD-kept mice of same age (n = 5) were used as a negative control. (a, b) Liver sections were subjected to F4/80 immunostaining, which visualizes macrophage infiltration and hematoxylin counterstaining (a). F4/80-positive areas were quantified (b). (c–f) Glucose tolerance tests (GTT, c, d) and insulin tolerance tests (ITT, e, f) were conducted using indicated mice (c, e). Area-under-the-curve (AUC) was quantified from GTT and ITT data (d, f). (g) Serum insulin levels were measured from indicated mice before (Basal) and 10 min after (Glucose-stimulated) glucose injection (n = 4). (h, i) Pancreas sections were analyzed by hematoxylin and eosin (H&E) staining (h). Islet areas were quantified (i) (n = 4). Scale bar, 200 μm (a), 100 μm (h). All data are shown as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

Figure 7

Figure 7. Effects of verapamil on blood pressure and cardiac functionality of obese mice

(a–g) 4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg/Kg body weight, i.p. n = 4) for 10 days. Systolic blood pressure (a), left ventricular (LV) wall mass (b), diastolic LV volume (c), systolic LV volume (d), ejection fraction (e, % EF), stroke volume (f, SV), and cardiac output (g, CO) were analyzed by tail-cuff method (a) or echocardiography (b–g). All data are shown as mean ± s.e.m. *P < 0.05 (Student’s t test).

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