Loss of mitochondrial protease ClpP protects mice from diet‐induced obesity and insulin resistance
2018; Springer Nature; Volume: 19; Issue: 3 Linguagem: Inglês
10.15252/embr.201745009
ISSN1469-3178
AutoresShylesh Bhaskaran, Gavin Pharaoh, Rojina Ranjit, Ashley R. Murphy, Satoshi Matsuzaki, Binoj C. Nair, Brittany Forbes, Suzana Gispert, Georg Auburger, Kenneth M. Humphries, Michael Kinter, Timothy M. Griffin, Sathyaseelan S. Deepa,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoArticle2 February 2018free access Transparent process Loss of mitochondrial protease ClpP protects mice from diet-induced obesity and insulin resistance Shylesh Bhaskaran Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Gavin Pharaoh Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Rojina Ranjit Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Ashley Murphy Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Satoshi Matsuzaki Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Binoj C Nair The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Brittany Forbes Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Suzana Gispert Experimental Neurology, Goethe University Medical School, Frankfurt am Main, Germany Search for more papers by this author Georg Auburger Experimental Neurology, Goethe University Medical School, Frankfurt am Main, Germany Search for more papers by this author Kenneth M Humphries Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Michael Kinter Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Timothy M Griffin Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Sathyaseelan S Deepa Corresponding Author [email protected] orcid.org/0000-0002-3669-4820 Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Shylesh Bhaskaran Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Gavin Pharaoh Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Rojina Ranjit Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Ashley Murphy Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Satoshi Matsuzaki Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Binoj C Nair The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Brittany Forbes Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Suzana Gispert Experimental Neurology, Goethe University Medical School, Frankfurt am Main, Germany Search for more papers by this author Georg Auburger Experimental Neurology, Goethe University Medical School, Frankfurt am Main, Germany Search for more papers by this author Kenneth M Humphries Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Michael Kinter Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Timothy M Griffin Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Sathyaseelan S Deepa Corresponding Author [email protected] orcid.org/0000-0002-3669-4820 Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Search for more papers by this author Author Information Shylesh Bhaskaran1, Gavin Pharaoh1,2, Rojina Ranjit1, Ashley Murphy1, Satoshi Matsuzaki1, Binoj C Nair3, Brittany Forbes1, Suzana Gispert4, Georg Auburger4, Kenneth M Humphries1, Michael Kinter1, Timothy M Griffin1 and Sathyaseelan S Deepa *,1,† 1Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA 2Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA 3The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4Experimental Neurology, Goethe University Medical School, Frankfurt am Main, Germany †Present address: Department of Geriatric Medicine and the Reynolds Oklahoma Center on Aging, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA *Corresponding author. Tel: +1 405 271 2633; E-mail: [email protected] EMBO Rep (2018)19:e45009https://doi.org/10.15252/embr.201745009 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Caseinolytic peptidase P (ClpP) is a mammalian quality control protease that is proposed to play an important role in the initiation of the mitochondrial unfolded protein response (UPRmt), a retrograde signaling response that helps to maintain mitochondrial protein homeostasis. Mitochondrial dysfunction is associated with the development of metabolic disorders, and to understand the effect of a defective UPRmt on metabolism, ClpP knockout (ClpP−/−) mice were analyzed. ClpP−/− mice fed ad libitum have reduced adiposity and paradoxically improved insulin sensitivity. Absence of ClpP increased whole-body energy expenditure and markers of mitochondrial biogenesis are selectively up-regulated in the white adipose tissue (WAT) of ClpP−/− mice. When challenged with a metabolic stress such as high-fat diet, despite similar caloric intake, ClpP−/− mice are protected from diet-induced obesity, glucose intolerance, insulin resistance, and hepatic steatosis. Our results show that absence of ClpP triggers compensatory responses in mice and suggest that ClpP might be dispensable for mammalian UPRmt initiation. Thus, we made an unexpected finding that deficiency of ClpP in mice is metabolically beneficial. Synopsis Mitochondrial matrix protease ClpP is proposed to play an important role in the initiation of mammalian mitochondrial unfolded protein response (UPRmt). This study reveals that ClpP deficiency in mice has paradoxical beneficial effects on metabolism and ClpP might be dispensable for mammalian UPRmt initiation. ClpP−/− mice have reduced adiposity and improved insulin sensitivity. Mitochondrial biogenesis markers and respiration are selectively up-regulated in white adipose tissue of ClpP−/− mice. ClpP−/− mice are protected from diet-induced obesity, glucose intolerance, and insulin resistance. Introduction Mitochondria are critical for the normal function of eukaryotic cells through production of ATP, maintenance of calcium homeostasis, and regulation of programmed cell death, and are also the major site for fatty acid oxidation 1. The mitochondrion has its own quality control system consisting of proteases and chaperones that helps to maintain protein homeostasis that in turn preserves mitochondrial integrity 2. The QC proteases present in the outer mitochondrial membrane (ubiquitin–proteasome system), inner mitochondrial membrane (PARL, OMA1, YME1L1, AFG3L2, and paraplegin), the intermembrane space (HtrA2), and mitochondrial matrix (Lon and ClpXP) help to maintain mitochondrial proteostasis through degradation of misfolded or damaged proteins 3. Failure of the QC system has been linked to various neurological diseases, aging, and metabolic disorders 45. For example, muscle-specific knockdown of PARL impairs insulin signaling and deficiency of OMA1 causes obesity and defective thermogenesis in mice 67. Caseinolytic peptidase P (ClpP) is a highly conserved QC protease from bacteria to humans. ClpP lacks ATPase activity and multimerizes with the mitochondrial chaperone and ATPase, ClpX to form the functional protease ClpXP. In Caenorhabditis elegans, ClpP plays a critical role in the activation of UPRmt, a retrograde signaling response that induces the expression of mitochondrial chaperones that helps to maintain mitochondrial proteostasis. The peptides generated through the proteolytic cleavage of unfolded proteins by ClpP initiate the UPRmt response in C. elegans 89. In mammalian cells, accumulation of unfolded proteins in mitochondrial matrix results in the transcriptional up-regulation of mitochondrial chaperones and ClpP 1011. While the role of ClpP in mammalian UPRmt is not clear, a potential involvement of ClpP in mitochondrial peptide release, similar to C. elegans, has been suggested 12. In addition to its proposed role in UPRmt, ClpP is involved in other mitochondrial functions; for example, knockdown of ClpP in muscle cells causes mitochondrial dysfunction and reduces cell proliferation 13, and ClpP is also involved in the regulation of mitochondrial protein synthesis through mitochondrial ribosome assembly 14. Recessive mutations in CLPP cause Perrault syndrome in humans, characterized by sensorineural deafness and ovarian failure 15. Acquired obesity in humans is associated with an impaired UPRmt response in subcutaneous WAT (sWAT) suggesting a possible relationship between metabolic stress and UPRmt 16. Because ClpP is proposed to play an important role in UPRmt, we analyzed mice deficient in ClpP (ClpP−/− mice) to understand the role of UPRmt-mediated proteostasis in metabolism. ClpP−/− mice recapitulate the phenotypes of Perrault syndrome in humans and are characterized by mild mitochondrial dysfunction, up-regulation of mitochondrial chaperones, and accumulation of ClpX and mtDNA in various tissues 17. We hypothesized that a defective UPRmt response and mitochondrial dysfunction due to ClpP deficiency will cause insulin resistance in ClpP−/− mice. On the contrary, ClpP−/− mice fed ad libitum showed improved insulin sensitivity, reduced adiposity, and elevated mitochondrial respiration in WAT. When challenged with a metabolic stress such as high-fat diet (HFD), ClpP−/− mice are protected from diet-induced obesity, glucose intolerance, insulin resistance, and hepatic steatosis. Our findings suggest that compensatory responses due to ClpP deficiency could contribute to the paradoxical beneficial metabolic effects in ClpP−/− mice. Results ClpP−/− mice have reduced adiposity and white adipocytes from ClpP−/− mice exhibit increased respiration Gispert et al 17 reported that ClpP−/− mice have reduced gain in body weight compared to wild-type (WT) littermates. In our cohort, at 5 months of age ClpP−/− male mice have 28% reduction in body weight compared to WT littermates or heterozygous ClpP+/− mice, despite increased food consumption by ClpP−/− mice (34% more compared to WT) (Fig 1A and B). ClpP−/− mice have 64% reduction in fat mass and 24% reduction in lean mass (Appendix Fig S1A). After normalizing to body weight, fat mass in ClpP−/− mice showed 38% reduction compared to WT, whereas lean mass showed a tendency to increase but did not reach statistical significance (Fig 1C). Thus, reduced body weight in ClpP−/− mice is attributable to reduced fat mass. Body weight, food intake, fat mass, and lean mass of ClpP+/− mice were similar to WT littermates (Fig 1A–C). ClpP−/− female mice (5-month-old) also showed a reduction in body weight and fat mass, comparable to male mice (Appendix Fig S1B and C). H&E staining of gonadal white adipose tissue (gWAT) showed smaller adipocytes (37% reduction in adipocyte area) in ClpP−/− mice compared to adipocytes in WT mice (Fig 1D). Transcript levels of adipocyte differentiation factors peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and adipocyte protein 2 (aP2) in gWAT were similar in ClpP−/− and WT mice, suggesting that adipocyte differentiation is not impaired in ClpP−/− mice (Appendix Fig S1D). Figure 1. ClpP−/− mice have reduced adiposity and elevated respiration in WAT Body weights of WT, ClpP+/−, and ClpP−/− male mice fed ad libitum at 5 months of age (n = 8–10). Food consumption of WT, ClpP+/−, and ClpP−/− mice fed ad libitum, normalized to body weight, at 5 months of age (n = 8–10). Fat mass and lean mass in WT, ClpP+/−, and ClpP−/− mice, assessed by quantitative magnetic resonance imaging and normalized to body weight age (n = 8–10). H&E staining of gWAT sections of WT and ClpP−/− mice (left panel, scale 100 μm, magnification 20×) and quantification of the average adipocyte area of gWAT sections (right panel) age (n = 3). Oil red O staining of differentiated adipocytes from WT, ClpP+/−, and ClpP−/− mice (left panel, scale 100 μm, magnification 20×). Quantification of total oil red O extracted from differentiated adipocytes (right panel) (n = 3). Data shown are mean ± SEM from three independent experiments. Graphical representation of cellular bioenergetics in differentiated primary adipocyte cultures from WT, ClpP+/−, and ClpP−/− mice was measured using the Seahorse Bioscience XF24 Extracellular Flux Analyzer mitostress assay, normalized to protein concentration per well. Data represent mean ± SEM from three independent experiments. Immunostaining of differentiated adipocytes from WT and ClpP−/− mice using Tom20 antibody (left panel, scale 100 μm, magnification 20×) and quantification of fluorescent intensity (right panel) (n = 3). Electron micrographs of gWAT from WT and ClpP−/− mice (n = 3). Scale 500 nm. Magnification 5,000×. Data information: (A–G) Bars represent mean ± SEM (ANOVA, *: WT vs. ClpP−/−; #: ClpP+/− vs. ClpP−/−; ^: WT vs. ClpP+/−; */#/^P < 0.05). WT: white bars, ClpP+/−: gray bars, ClpP−/−: black bars. Download figure Download PowerPoint Differentiated adipocytes from ClpP−/− mice accumulated 50% less triglycerides than adipocytes from WT or ClpP+/− mice (Fig 1E). Basal respiration (30% increase), ATP-linked respiration (93% increase), maximal respiration (127% increase), and spare respiratory capacity (1,319% increase) were increased in ClpP−/− mice compared to WT or ClpP+/− adipocytes (Fig 1F). Levels of non-mitochondrial respiration and proton leak were similar in WT, ClpP+/−, and ClpP−/− adipocytes. For ClpP+/− adipocytes, only basal respiration showed a 17% increase compared to WT adipocytes, whereas other parameters were comparable to WT mice. Similarly, in vitro knockdown of ClpP (90% reduction in ClpP protein) in 3T3-L1 cells also increased respiration, compared to control cells (Appendix Fig S1E and F). Thus, ClpP deficiency increased respiration in adipocytes both in vitro and in vivo. An increased number of mitochondria per cell or the same number of highly active mitochondria per cell can contribute to high mitochondrial respiration. Immunostaining of differentiated adipocytes using an antibody for mitochondrial outer membrane protein Tom20 showed more intense staining in ClpP−/− adipocytes than WT adipocytes, suggesting there is an increase in mitochondrial content in ClpP−/− adipocytes (Fig 1G). In agreement with this, mitochondrial content in gWAT of ClpP−/− mice was higher (and the mitochondria appeared larger) than WT mice, as assessed by electron microscopy (Fig 1H). Thus, elevated mitochondrial content could contribute to elevated respiration and reduced fat mass in ClpP−/− mice. Markers of mitochondrial biogenesis, mitochondrial chaperones, and mitochondrial fission/fusion regulator OPA1 are elevated in gWAT of ClpP−/− mice Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key transcription factor that regulates mitochondrial biogenesis. In gWAT of ClpP−/− mice, expression of PGC-1α is up-regulated 3.3-fold compared to WT mice (Fig 2A). Protein expression of transcription factor A, mitochondrial (Tfam) (mitochondrial DNA transcription factor, 3.3-fold), and voltage-dependent anion channel (VDAC) (a highly conserved outer mitochondrial membrane protein, 2.8-fold) is also elevated in ClpP−/− mice of gWAT (Fig 2A). Protein expression of PGC-1α and Tfam was comparable in gWAT of ClpP+/− mice and WT mice; however, expression of VDAC was twofold higher in ClpP+/− mice compared to WT mice (Fig 2A). We also found a significant increase in the protein expression of electron transport chain (ETC) subunits: ATP Synthase, H+ Transporting, Mitochondrial F1 Complex, Alpha Subunit 1 (ATP5a1, 2.4-fold); ATP5ab (2.5-fold); Succinate Dehydrogenase Complex Flavoprotein Subunit A (SDHA, 3.4-fold); SDHAB (2.4-fold); SDHC (2.2-fold); and Ubiquinol-Cytochrome C Reductase Core Protein II (UQCRC2, 2.6-fold) in gWAT of ClpP−/− mice, compared to WT mice (Fig 2B and Table EV1). However, expression of ETC subunits in WAT of ClpP+/− mice was similar to WT mice (Fig 2B). In addition, protein expression of citrate synthase was increased twofold (Appendix Fig S2A and Table EV1) and mitochondrial DNA (mtDNA) content was increased fourfold in the WAT of ClpP−/− mice (Fig 2C). Taken together, mitochondrial biogenesis markers are increased in gWAT of ClpP−/− mice suggesting an increase in mitochondrial mass, compared to WT or ClpP+/− mice. PGC-1α expression is also elevated (2.7-fold) in subcutaneous WAT (sWAT) of ClpP−/− mice, but not in brown adipose tissue (BAT), heart, or skeletal muscle (Fig 2D). We also looked for a potential signal that might drive PGC-1α expression in gWAT. Increased levels of reactive oxygen species (ROS) are linked to activation of PGC-1α expression in skeletal muscle 181920. In humans, oxidative stress induced by short-term exercise increases PGC-1a expression in skeletal muscle 21. Based on this, we also tested whether ROS levels are altered in gWAT of ClpP−/− mice. Levels of 4-Hydroxynonenal (4-HNE) were used as a marker of oxidative stress 22 and Western blotting of gWAT showed increased 4-HNE levels (1.3-fold) in ClpP−/− mice compared to WT mice, suggesting increased oxidative stress in gWAT (Fig 2E). Elevated levels of H2O2 are shown to induce PGC-1α expression through activation of AMPK in skeletal muscle 18. Assessing AMP-activated protein kinase (AMPK) activation in gWAT of ClpP−/− mice showed that the ratio of phospho-AMPK/AMPK is increased in ClpP−/− mice compared to WT mice, suggesting increased AMPK activation (Fig 2F). Thus, increase in ROS and AMPK activation could contribute to the increased expression of PGC-1α in gWAT of ClpP−/− mice. Figure 2. Markers of mitochondrial biogenesis, mitochondrial chaperones, and mitochondrial fission/fusion regulator OPA1 are elevated in gWAT of ClpP−/− mice Left panel: immunoblots of gWAT extracts from WT, ClpP+/−, and ClpP−/− mice for PGC-1α, Tfam, VDAC, β-actin, and ClpP (n = 5). Right panel: graphical representation of quantified blots normalized to β-actin. Quantification of protein levels of electron transport chain (ETC) subunits ATP5a1, ATP5ab, SDHA, SDHAB, SDHC, and UQCRC2 in gWAT of WT, ClpP+/−, and ClpP−/− mice obtained by mass spectrometry (n = 5). Quantification of mtDNA/nDNA content in gWAT from WT and ClpP−/− mice (n = 6–8). Top panels: immunoblots of sWAT, BAT, heart, and muscle extracts from WT and ClpP−/− mice for PGC-1α and β-tubulin (Western blot shows representative examples for n = 4, and quantification is based on n = 6). Bottom panels: graphical representation of quantified blots normalized to β-tubulin. Left panel: immunoblots of gWAT extracts (30 μg/lane) from WT and ClpP−/− mice for 4-HNE. Right panel: Quantification of band intensity of the entire line represented graphically (n = 5). Top panel: immunoblots of gWAT extracts from WT and ClpP−/− mice for P-AMPK and AMPK (n = 5). Right panel: graphical representation of the ratio of P-AMPK to AMPK. Left panel: immunoblots of gWAT extracts from WT, ClpP+/−, and ClpP−/− mice for Lon, Hsp60, Hsp40, Hsp10, ClpX, and ClpP (n = 5). Right panel: graphical representation of quantified blots normalized to β-actin. Western blots showing protein expression of OPA1 isoforms and β-actin in gWAT, sWAT, BAT, muscle and heart of WT, and ClpP−/− mice (top panel) (Western blot shows representative examples for n = 2, and quantification is based on n = 5). Quantification of the blots normalized to β-actin is shown as bar graphs (bottom panel). Data information: Bars represent mean ± SEM (ANOVA, *: WT vs. ClpP−/−; #: ClpP+/− vs. ClpP−/−; ^: WT vs. ClpP+/−; */#/^P < 0.05). WT: white bars, ClpP+/−: gray bars, ClpP−/−: black bars. Download figure Download PowerPoint Loss of ClpP was reported to induce the expression of mitochondrial chaperones and the mitochondrial protease Lon in testis, heart, liver, and brain of ClpP−/− mice 17. In gWAT of ClpP−/− mice, protein expression of Lon protease (2.1-fold) and mitochondrial chaperones [Heat shock protein 60 (Hsp60, 3.5-fold), Hsp40 (2.6-fold), Hsp10 (2.5-fold), and ClpX (1.9-fold)] was significantly elevated compared to WT mice (Fig 2G). However, this increase in the expression of mitochondrial chaperones in gWAT could be attributed to the increase in mitochondrial number. In ClpP+/− mice, protein levels of Lon, Hsp10, and ClpX were similar to WT mice, whereas protein expression of Hsp60 (2.2-fold) and Hsp40 (2.2-fold) was significantly elevated (Fig 2G). Similarly, sWAT of ClpP−/− mice showed a strong induction of Lon and mitochondrial chaperones and BAT showed increased expression of Lon protease and Hsp40 compared to WT mice (Appendix Fig S2B). Previously, we found that knockdown of ClpP (70% reduction compared control cells) in C2C12 muscle cells increased the expression of mitochondrial fission protein dynamin-related protein1, Drp1 13. In gWAT of ClpP−/− mice, we did not see any change in the expression of fusion protein mitofusin 2 (Mfn2) or fission proteins Drp1 or Fis1 (data not shown). However, protein expression of mitochondrial fission–fusion regulator Optic Atrophy 1 (OPA1, total) was increased in the gWAT (17%) and sWAT (114%) of ClpP−/− mice (Fig 2H). OPA1 exists in multiple long forms (L-OPA1) and short forms (S-OPA1) and processing of OPA1 to L-OPA1 and S-OPA1 balances mitochondrial fission/fusion and the antibody we used detected five different isoforms, as previously reported 2324. In BAT and skeletal muscle of ClpP−/− mice, total OPA1 was similar to WT mice, whereas in heart, total OPA1 was reduced by 34% (Fig 2H). Thus, PGC-1α and OPA1 showed a tissue-specific difference in their expression and WAT depots showed higher expression, compared to other tissues. Pink1 and Parkin are known initiators of mitophagy 25. Therefore, we quantified the expression of mitophagy markers PINK1 and Parkin in gWAT to test whether activation of mitophagy contributes to the recovery of healthy mitochondria in the absence of ClpP. Surprisingly, expression of PINK1 and Parkin is decreased in gWAT of ClpP−/− mice, suggesting that mitophagy is not activated in ClpP−/− mice (Appendix Fig S2C). Absence of ClpP increases whole-body energy expenditure and mitochondrial uncoupling and alters expression of metabolic enzymes in gWAT of ClpP−/− mice ClpP−/− mice exhibited 37 and 12% increases in oxygen consumption during dark and light phases, respectively, when normalized to total body mass (Fig 3A). When normalized to lean body mass, ClpP−/− mice showed a 15% increase in oxygen consumption during dark phase and this difference was not statistically significant (Fig 3B). Similarly, energy expenditure (EE) normalized to total body mass was 40 and 34% higher for ClpP−/− mice during dark and light phases, respectively (Fig 3C). Normalizing EE to lean body mass also reduced the increased EE in ClpP−/− mice to 10%, which did not reach statistical significance (Fig 3D). Furthermore, WT and ClpP−/− mice had a similar respiratory exchange ratio (RER) and cage activity levels (Fig 3E and F). Metabolically, ClpP+/− mice were similar to WT mice except for significantly reduced RER during the light phase (Fig 3E). Thus, the finding that higher oxygen consumption and EE rates of ClpP−/− mice were reduced when normalized to lean rather than total body mass is consistent with increased adipose tissue metabolism in ClpP−/− mice. Figure 3. Absence of ClpP increases whole-body energy expenditure and mitochondrial uncoupling and alters expression of metabolic enzymes in gWATMetabolic cage data of WT, ClpP+/−, and ClpP−/− mice. A–F. Oxygen consumption rate (OCR) normalized to body weight (A), OCR normalized to lean body mass (B), EE normalized to body weight (C), EE normalized to lean body mass (D), RER (E), and cage activity (F) (n = 6). G. Transcript levels of UCP1 and UCP2 in gWAT and sWAT of WT and ClpP−/− mice (n = 6–8). H. Heatmaps showing changes in the expression of protein in fatty acid metabolism (first panel), glucose metabolism (second panel), TCA cycle, electron transport chain (ETC), and other mitochondrial proteins (third panel) and stress response (detoxification/antioxidant enzymes, chaperones, heat shock proteins, and proteases) (forth panel) in gWAT of WT, ClpP+/−, and ClpP−/− mice (n = 5). Average value of WT was used to normalize values of ClpP+/− and ClpP−/− mice. The darker the red indicates the greater the increase in expression, and the darker the blue indicates the greater the decrease in expression. Data information: (A–F) Circles indicate values of individual mice. (A–G) Bars represent mean ± SEM (ANOVA, *: WT vs. ClpP−/−; #: ClpP+/− vs. ClpP−/−; ^: WT vs. ClpP+/−; */#/^P < 0.05). WT: white circles/bars, ClpP+/−: gray circles/bars, ClpP−/−: black circles/bars. Download figure Download PowerPoint Transcript level of uncoupling protein 1 (UCP1) was significantly increased in sWAT, not in gWAT, of ClpP−/− mice compared to WT mice (Fig 3G). UCP2 was significantly elevated in gWAT and sWAT of ClpP−/− mice compared to WT mice (Fig 3G), whereas UCP3 levels were similar in WT and ClpP−/− mice WAT depots (data not shown). Beige fat or “brown-like” fat present in WAT is known to increase energy expenditure 2627. Therefore, we tested markers of browning/beiging in sWAT and found that in addition to UCP1, transcript levels of PGC-1α (11.6-fold), cell death-inducing DFFA-like effector A (CIDEA, 11.4-fold), and cytochrome c oxidase subunit 8b (Cox8b, 15.8-fold) were elevated in sWAT of ClpP−/− mice; however, levels of PR/SET Domain 16 (Prdm16) were similar in ClpP−/− and WT mice (Appendix Fig S3A). Western blotting to assess protein expression of UCP1 in BAT showed no significant change in UCP1 expression (Appendix Fig S3B). Thus, increased uncoupling in WAT depots as well as “browning” of sWAT could contribute to increased energy expenditure in ClpP−/− mice. A targeted quantitative proteomic approach was employed for a detailed study of the changes in protein expression of mitochondrial metabolic enzymes in gWAT of WT, ClpP+/−, and ClpP−/− mice. Protein expression of mitochondrial fatty acid oxidation enzymes, and enzymes/proteins involved glucose metabolism, tricarboxylic acid (TCA) cycle, and ETC, and antioxidants are altered in gWAT of ClpP−/− mice, compared to WT mice (Fig 3H and Table EV1). Thus, absence of ClpP altered expression of metabolic enzymes in gWAT, and the increase in mitochondrial biogenesis might partly contribute to this increase. ClpP−/− mice have improved insulin sensitivity Mitochondrial dysfunction is associated with the development of insulin resistance 28. To understand the effect of ClpP deficiency on glucose metabolism, glucose clearance was measured by glucose tolerance test (GTT) and was found to be similar in WT, ClpP+/−, and ClpP−/− mice (Fig 4A). However, ClpP−/− mice exhibited improved insulin sensitivity compared to WT or ClpP+/− mice when subjected to insulin tolerance test (ITT) (Fig 4B). Improved insulin sensitivity suggests enhanced insulin-stimulated Akt activation to enable faster glucose uptake. Consistent with this, insulin-stimulated Akt phosphorylation was significantly elevated in skeletal muscle (45%), liver (62%), and gWAT (27%) of ClpP−/− mice compared to WT mice (Fig 4C). Circulating level of insulin and glucose was reduced by 68 and 44%, respectively, in ClpP−/− mice compared to WT mice, further supporting improved insulin sensitivity in ClpP−/− mice (Fig 4D and E). Circulating triglycerides were also significantly reduced (33%) in ClpP−/− mice, whereas free fatty acid levels were similar in ClpP−/− mice and WT mice (Fig 4F and G). Surprisingly, circulating level of the insulin-sensitizing adipokine, adiponectin was significantly lower (23%) in ClpP−/− mice compared to WT (Fig 4H). Figure 4. ClpP−/− mice exhibit improved insulin sensitivity A, B. Glucose tolerance test (A) and insulin tolerance test (B) of WT, ClpP+/−, and ClpP−/− mice fed ad libitum (n = 6–8). C. Western blots showing expression P-Akt (T308), Akt, β-actin, and ClpP in WT and ClpP−/− mice muscle (first panel), liver (second panel), and gWAT (third panel) injected with PBS (−Ins) or insulin (+Ins) (top panels) (n = 3–4). Quantification of P-Akt/Akt is shown in bottom panels. D–H. Levels of circulating insulin (D), glucose (E), triglyceride (F), free fatty acids
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