Deficient chaperone-mediated autophagy in liver leads to metabolic dysregulation - PubMed (original) (raw)
Deficient chaperone-mediated autophagy in liver leads to metabolic dysregulation
Jaime L Schneider et al. Cell Metab. 2014.
Abstract
The activity of chaperone-mediated autophagy (CMA), a catabolic pathway for selective degradation of cytosolic proteins in lysosomes, decreases with age, but the consequences of this functional decline in vivo remain unknown. In this work, we have generated a conditional knockout mouse to selectively block CMA in liver. We have found that blockage of CMA causes hepatic glycogen depletion and hepatosteatosis. The liver phenotype is accompanied by reduced peripheral adiposity, increased energy expenditure, and altered glucose homeostasis. Comparative lysosomal proteomics revealed that key enzymes in carbohydrate and lipid metabolism are normally degraded by CMA and that impairment of their regulated degradation contributes to the metabolic abnormalities observed in CMA-defective animals. These findings highlight the involvement of CMA in regulating hepatic metabolism and suggest that the age-related decline in CMA may have a negative impact on the energetic balance in old organisms.
Copyright © 2014 Elsevier Inc. All rights reserved.
Figures
Figure 1. Liver-specific L2AKO mice are incompetent for CMA and display signs of liver damage and reduced liver function
(A) Schematic of the generation of mice with liver-specific deletion of LAMP-2A. (B) RT-PCR quantification of LAMP-2A and LAMP-1 mRNA levels in livers from Albumin-Cre-L2Af/f (L2AKO) and L2Af/f (Control, Ctr) mice, n=4. (C) Immunoblot for LAMP-2A in liver homogenates from the same animal groups (Alb-Cre mice are shown as an additional control). (D) Immunohistochemistry for LAMP-2A in liver sections (scale bar, 20 μm). (E, F) Immunoblot for the indicated proteins in homogenates (E) and lysosomes (F). (G) Quantification of the fraction of mature Cathepsin D relative to total Cathepsin D detected by immunoblot in isolated liver lysosomes, n=5. (H) β-hexosaminidase activity in liver homogenates (left) and isolated lysosomes (right), n=7; AUF: arbitrary units of fluorescence. (I) Lysosomal membrane stability measured as the percentage of lysosomal β-hexosaminidase activity detectable in the media upon lysosomal incubation, n=4. (J) Immunoblot for the indicated cytosolic proteins in lysosomes isolated from livers of 24h starved mice injected or not with leupeptin (Leup); Pyruvate Carboxylase (Pyr. Carb) is shown as negative control. (K) Immunoblot for the indicated macroautophagy-related proteins in liver homogenates. (L) Serum levels of alanine aminotransferase (ALT), n=4-6. (M) Time for recovery of the righting reflex after zoxazolamine-induced paralysis, n=3. All values are mean + s.e.m. Differences with Ctr were significant for * P < 0.05, ** P < 0.01, and *** P < 0.001. See also Fig. S1.
Figure 2. Altered lipid metabolism and hepatosteatosis in liver-specific L2AKO mice
(A) Representative images of livers from 24h starved control (Ctr) and Albumin-Cre-L2Af/f (L2AKO) mice. (B) Total liver weight (right) or relative to body weight (b.w.) (left), n=7. (C) H&E staining of liver sections from fed, 12h or 48h starved mice. Insets show higher magnification images (scale bar, 10 μm). (D) Left: Oil Red O (ORO) staining of liver sections from fed and 24h starved mice. Insets show higher magnification images (scale bar, 20 μm). Right: quantification of number, area occupied and average area of lipid droplets (LD), n=4. (E) Lipid analysis by thin layer chromatography in livers from fed (top) and 24h starved (bottom) mice. Values are expressed relative to control values, n=4. (F) ORO staining of liver sections from mice after two i.p. injections of isoproterenol to induce peripheral lipolysis. Untreated L2AKO mice liver is shown as reference. Insets show higher magnification images. (G) Levels of triglyceride (TG) and cholesterol (Chol) in livers of the same animals 24h after the two-isoproterenol injections, n=3-4. (H) Serum levels of free fatty acids (FFA) and glycerol before and 15 minutes after isoproterenol injection in mice, n=3-4. (I) Representative images of livers from mice after 16 weeks on a high-fat diet (HFD). (J) H&E and ORO staining of liver sections from the same mice in i. All values are mean + s.e.m. Differences with Ctr were significant for * P < 0.05, ** P < 0.01, and *** P < 0.001. See also Fig. S2.
Figure 3. Increased energy expenditure and reduced peripheral adiposity in liver-specific L2AKO mice
(A) Changes in body weight with age in control (Ctr) and Albumin-Cre-L2Af/f (L2AKO) male mice, n ≥11 (average group size 14.3+0.6 and 13.0+0.4 for Ctr and L2AKO, respectively). (B) Weight loss (in percentage) in the same animals at 4 months of age after 24h (top) or 48h (bottom) starvation, n=11-14. (C) Fat mass (top) and lean (mass) as a percent of body weight (b.w.) in fed or 24h starved mice, n=9-14. (D) Weight of perigonadal white adipose tissue fat in grams (top) or relative to b.w. (bottom) in the same mice as in c, n=3-4. (E) H&E staining sections of perigonadal white adipose tissue (WAT, top) and interscapular brown adipose tissue (BAT, bottom). Right: quantification of the average size of lipid droplets (LD) in the same tissues. (F-H) Average values (left) and 24h time-course (right) of the energy expenditure (EE) during the light and dark cycles or through a 24h cycle (Total) in mice fed a regular chow diet (F; n=8), fed a HFD for 16 weeks (G; n=4), or starved for 24h (H; n=8). (I, J) Average values of the respiratory exchange ratio (RER) during the light and dark cycles and Total in mice fed a regular chow diet (I) or during the first 6h of a 24h starvation period (J), n=8. All values are mean + s.e.m. Differences with Ctr were significant for * P < 0.05, ** P < 0.01, and *** P < 0.001. See also Fig. S3.
Figure 4. Enhanced glucose tolerance and reduced hepatic glycogen storage in liver-specific L2AKO mice
(A) Blood glucose levels in fed and 8h starved control (Ctr) and Albumin-Cre-L2Af/f (L2AKO) mice, n=6-10. (B-C) Glucose (B), insulin (C) and pyruvate tolerance (D) tests after overnight fasting, n=4-8. (E) Glycogen content in livers from normally fed mice, n=4. (F) Periodic acid-Schiff (PAS) staining of liver sections from fed and 24h starved mice (scale bar, 20 μm). (G) Liver lactate content, n=6. (H) Activity of glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase in fed mice livers (n=4). (I, K) Average extracellular acidification rates (ECAR) in primary hepatocytes from mice maintained in serum-supplemented (I) or -deprived (K) media without addition (Basal, top) or after adding 2,4 dinitrophenol (2,4 DNP) to asses inducible ECAR (Maximal, bottom). (n=6). (J) Immunoblot for the indicated enzymes in the same hepatocytes maintained in serum supplemented media. (L) Immunoblot for the indicated glycolytic enzymes in liver homogenates from 24h starved mice. Three individual mice are shown. Right: Densitometric quantification, values are expressed relative to values in Ctr mice, n=3-4. GAPDH, glyceraldehyde 3-phosphate dehydrogenase, Aldo, aldolase A; PK, pyruvate kinase; Pyr Carb., pyruvate carboxylase. All values are mean + s.e.m. Differences with Ctr were significant for * P < 0.05, ** P < 0.01, and *** P < 0.001. See also Fig. S4.
Figure 5. CMA regulates hepatic levels of carbohydrate metabolism enzymes in response to starvation
(A) mRNA levels of the indicated glycolytic enzyme genes in livers from fed or 48h starved control (Ctr) and Albumin-Cre-L2Af/f (L2AKO) mice. Values are expressed relative to Ctr fed mice, n=3. (B) Schematic of the hypothetical changes in levels of proteins in lysosomes isolated from 24h starved Ctr and L2AKO mice injected or not with leupeptin 2h before isolation. Proteins are classified depending on these changes as non-substrates (1), substrates for lysosomal degradation (2) and CMA substrates (3). Right: representative electrophoretic pattern of lysosomes isolated from animals in the indicated conditions and subjected to SDS-PAGE and SyproRuby staining. Arrows indicate examples of proteins in each of the categories indicated in the scheme. (C) Immunoblot for the indicated glycolytic enzymes in the same lysosomal samples as in B. PK, pyruvate kinase; Mdh1, malate dehydrogenase 1, cytoplasmic; Pyr. Carb., pyruvate carboxylase; Eno1, enolase 1; Cath D, cathepsin D. (D) Densitometric quantification of immunoblots as the ones shown in C and in Fig. 1J (for GAPDH). Values are expressed relative to values in untreated Ctr mice (none), n=2-5. (E) Immunoblot for the indicated enzymes of liver homogenates from fed or 24h starved mice injected or not with leupeptin. (F) Rates of lysosomal degradation of the indicated enzymes calculated by densitometric quantification of immunoblots as the ones showin in E, n=2-5. (G) Comparative proteomics of the lysosomes from the four experimental groups described in B. Percentage of proteins classified as lysosomal substrates based on their sensitivity to leupeptin (top) and percentage of CMA substrates based on: their loss of sensitivity to leupeptin in L2AKO lysosomes (middle panel) or the presence of a CMA-targeting motif in their sequence in the group of leupeptin-sensitive proteins (bottom panel), n=3. (H,I) Distribution among cellular biological processes (H) and metabolic processes (I) of proteins classified as CMA substrates in the proteomic analysis. Percentages in each group are shown, n=3. All values are mean+s.e.m. Differences with Ctr (*) or with fed (§) were significant for * § P < 0.05, ** P < 0.01, and *** P < 0.001. See also Fig. S5.
Figure 6. Reduced degradation of glycolytic enzymes leads to enhanced glycolysis in CMA-defective cells
(A) Lactate content in mouse embryonic fibroblasts (right, n=3) from control (Ctr) and L2A-/- (KO) mice. (B) Average extracellular acidification rates (ECAR) in fibroblasts maintained in serum-deprived media without addition (Basal, left) or after adding 2,4 dinitrophenol (2,4 DNP) to asses inducible ECAR. (C) Timecourse of ECAR changes in same cells as in (B) (n=8). (D) Top: Immunoblot for indicated proteins from Ctr fibroblasts transfected with plasmids coding for MYC-tagged GAPDH, Pyruvate Kinase (PK), or Aldolase. Bottom: ECAR values for Ctr fibroblasts overexpressing the indicated glycolytic enzymes individually or in combination, n=4. OE: overexpression. (E,F) Left: Immunoblot for indicated enzymes from fibroblasts transduced with a lentivirus carrying shRNA against GAPDH or PK to knockdown (KD) glycolytic enzymes individually or together (PK+GAP). Right: ECAR values over time and (F) average ECAR values in the transduced cells (n=4). (G) Top: Immunoblot for Myc and GAPDH from total cellular lysates (homogenate) and cytosolic fractions from Ctr fibroblasts transfected with plasmids coding for MYC-tagged wild-type (WT) GAPDH or GAPDH bearing a mutated CMA targeting motif (MT). Bottom: Timecourse of changes of WT and MT GAPDH protein in total and cytosolic cellular fractions. (H) Activity of GAPDH and (I) lactate levels in the same cells at indicated times post-tranfection (n=3). (J) Average ECAR values in the same cells 24h post-transfection (n=4). All values are mean+s.e.m. Differences with Ctr (*) were significant for * P < 0.05 and *** P < 0.001.
Figure 7. Liver enzymes related to lipid metabolism undergo regulated degradation by CMA
(A) Distribution among pathways related to lipid metabolism of all proteins or proteins classified as CMA substrates through the comparative proteomic analysis of lysosomes isolated from 24h starved control (Ctr) and Albumin-Cre-L2Af/f (L2AKO) mice injected or not with leupeptin 2h before isolation, n=3 (B) Folds increase in levels of the indicated proteins related to lipid metabolism in the same lysosomes after leupeptin treatment as determined in the comparative proteomic analysis, n=3. (C) Immunoblot for the indicated enzymes related to lipid metabolism in the same lysosomal samples as in Fig. 5B. GPD, glycerol-3-phosphate dehydrogenase; ACADL, acyl-coenzyme A dehydrogenase long chain; Cyp27A1, cytochrome P450 cholesterol 27 hydroxylase; ACSL1, long chain acetyl-coenzyme A synthetase. Right: quantification of immunoblots shown on left. Values are presented relative to values in untreated groups, n=3-6. (D) Rates of basal and starvation-induced lysosomal degradation in percentage of protein delivered to lysosomes per hour calculated from the differences in the levels of the indicated proteins in lysosomes from animals treated or not with leupeptin 2h before isolation, n=4-6. Representative immunoblots shown in Fig. S5B. (E) Enzymatic activity of GPD in liver homogenates, n=7 (F) Average rates of newly synthesized radiolabeled TG in cells pulsed with 14C-Glycerol. n=3. (G) Rates of TG hydrolysis in cells incubated overnight with 14C-Oleate and chased in serum free media, n=3. (H) Serum triglyceride (TG) measured at the indicated times before and after treatment with lipoprotein lipase inhibitor, Poloxamer 407, in mice starved overnight, n=3-4. (I) Schematic of the enzymes in the indicated carbohydrate and lipid metabolic pathways validated to undergo regulated degradation by CMA in this study and whose levels increase when CMA is compromised in liver. All values are mean+s.e.m. Differences with Ctr (*) or with fed (§) were significant for * § P < 0.05, ** P < 0.01, and *** P < 0.001. See also Fig. S6 and S7.
Comment in
- When autophagy chaperones liver metabolism.
Codogno P, Lotersztajn S. Codogno P, et al. Cell Metab. 2014 Sep 2;20(3):392-3. doi: 10.1016/j.cmet.2014.08.008. Cell Metab. 2014. PMID: 25185945
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