EMBO/soda-nel huggingface dataset:
Loss of autophagy in hypothalamic POMC neurons impairs lipolysis (2011)
Figure 5

Ageing reduces POMC neuronal autophagy and lipolysis. (A) Immunoblots for Atg7 in MBH from young (3 mo), middle‐aged (12 mo) and aged (22 mo) fed and 6‐h‐fasted (Stv) mice (n=4), and (B) LC3‐II in MBH explants from 3 mo and 22 mo fed mice in presence or absence of lysosomal inhibitors (Inh) (n=11–13). Mean±s.e.m values for (B) steady‐state LC3‐II and (C) LC3‐II flux are shown (n=11–13). (D) Immunoblots for p62 and NBR1 flux in MBH from 3 mo and 22 mo fed mice in presence or absence of Inh (n=11–13). (E) Immunofluorescence for POMC (green) and p62 (red) (n=4), (F) immunoblots for POMC and ACTH (n=4), and (G) immunofluorescence for α‐MSH in MBH from 3 mo and 22 mo mice on RD (n=4). (H) Body wt and (I) eWAT wt of 3‐mo‐ and 22‐mo‐old mice on RD (n=9–13). (J) Serum fatty acids and (K) glycerol from fed or 6‐h‐fasted (Stv) 3 mo and 22 mo mice (n=7–9), and (L,M) after intraperitoneal Iso in 6‐h‐fasted 3 mo and 22 mo mice (n=5–6). (N) Immunoblots for adipose triglyceride lipase (ATGL) in eWAT from 3 mo and 22 mo mice. Values are mean±s.e.m. P values are as compared with controls. *P0.05, **P0.01, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. Yellow arrows indicate merged signal. ACTH, adrenocorticotrophic hormone; Con, control; eWAT, epididymal fat; Iso, isoproterenol; MBH, mediobasal hypothalamic; mo, months; MSH, melanocyte‐stimulating hormone; POMC, proopiomelanocortin; RD, regular chow; wt, weight.
PLIC proteins or ubiquilins regulate autophagy‐dependent cell survival during nutrient starvation (2008)
Figure 1

Ubiquilins protect against starvation‐induced cell death. (A) HeLa cells expressing GFP‐tagged UBQLN1 or UBQLN2 (or GFP for controls) were transferred to starvation medium (PBS) for the indicated time. Where specified, only serum was removed from the culture medium. Viability of transfected cells was assessed by PI staining and analysed by flow cytometry. The graph represents the mean of three experiments±s.e. (B) HeLa cells were depleted of UBQLN1 or UBQLN2 by siRNA transfection; depletion was verified by Western blot. Cells were starved for increasing periods of time, and their viability was analysed as in (A). The graph represents the mean of three experiments±s.e; *P0.05 between control and UBQLN2‐KD at 6 h (paired Student's t‐test). GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GFP, green fluorescent protein; KD, knockdown; PI, propidium iodide; siRNA, short interfering RNA; UBQLN, ubiquilin.
PLIC proteins or ubiquilins regulate autophagy‐dependent cell survival during nutrient starvation (2008)
Figure 2

The ubiquilin‐protective effect against starvation requires autophagy. (A) HeLa cells were transfected with siRNAs for autophagy‐related gene ATG5 (siATG5) or with a non‐silencing siRNA (siC). Depletion of ATG5, which decreased the levels of the ATG5–ATG12 covalent complex, was confirmed by Western blot. Loading control: transferrin receptor (TfR). (B) Control or ATG5‐KD cells transfected with UBQLN1 were starved, and the cell viability was assessed as in Fig 1; the graph represents the mean of three experiments±s.e. (C) HeLa cells were stably transfected with control (shC) or ATG7 (shATG7) shRNA; depletion was verified by Western blot (loading control: GAPDH). (D) Control or ATG7‐KD cells transfected with UBQLN1 were starved for 3 or 6 h and assessed for their viability. The graph represents the mean of three experiments±s.e. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; KD, knockdown; Pl, propidium iodide; shRNA, short hairpin RNA; siRNA, short interfering RNA; UBQLN, ubiquilin.
PLIC proteins or ubiquilins regulate autophagy‐dependent cell survival during nutrient starvation (2008)
Figure 3

Ubiquilins colocalize with autophagosomes. (A) Immunolocalization of transfected Myc‐UBQLN (1 or 2) and GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) was analysed by microscopy. Inset: magnification of UBQLN1 vesicular staining (arrow), which, after acquisition of Z‐stacks, was found in all UBQLN structures analysed. (B) Cells were starved for 2 h, with (top row) or without BAF A1 (bottom row). Cells were stained for endogenous UBQLN2 (magenta) and LC3 (green). (C) Control cells (untransfected, right panel) or cells overexpressing Myc‐UBQLN2 and GFP‐LC3 (left panel) were transferred into starvation medium for 2 h. Localization of Myc‐UBQLN2 and GFP‐LC3 was analysed by immunoelectron microscopy using Myc (arrow) and GFP (arrowhead) antibodies coupled to 10 and 15 nm gold particles, respectively. Double‐membrane vacuoles (autophagosomes) in transfected cells (left panel) show increased staining by both antibodies compared with autophagosomes in untransfected cells (right panel). Scale bar, 0.5 μm. BAF A, bafilomycin A1; GFP, green fluorescent protein; UBQLN, ubiquilin.
PLIC proteins or ubiquilins regulate autophagy‐dependent cell survival during nutrient starvation (2008)
Figure 4

The UBA domain of ubiquilin is required for interaction with autophagosomes and a protective effect against starvation. (A) Generation of Myc‐tagged UBQLN1 deletion mutants. (B) Each mutant was co‐transfected with GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) and the localization of each protein was analysed by microscopy. (C) Starvation‐induced cell death was assessed in cells expressing UBQLN1 or UBQLN1ΔUBA. The graph represents the mean of three experiments±s.e., with *P⩽0.05 (P=0.0017 by one‐way ANOVA test, comparing control, UBQLN1 and UBQLN1ΔUBA). No difference was found between control and UBQLN1ΔUBA at any time point. ANOVA, analysis of variance; GFP, green fluorescent protein; Pl, propidium iodide; UBA, ubiquitin‐associated; UBQLN, ubiquilin.
PLIC proteins or ubiquilins regulate autophagy‐dependent cell survival during nutrient starvation (2008)
Figure 5

Depletion of ubiquilin inhibits the degradation of autophagosomes by lysosomes. (A) Autophagosome subcellular localization during starvation was analysed by microscopy in UBQLN‐ or autophagy‐related gene ATG5‐KD cells expressing GFP‐tagged microtubule associated protein 1 light chain 3 (GFP‐LC3). (B) Autophagosome spatial distribution was quantified by measuring the distance of each LC3 vesicle from the nucleus (in pixels) and by analysing the frequency distribution. (C) Control or UBQLN‐KD cells expressing GFP‐LC3 were starved and stained with the mitochondria dye Mitotracker™. Dual staining of LC3 (green) with Mitotracker (magenta) was analysed by microscopy. (D) Control and UBQLN‐KD cells stained with Mitotracker were starved for 6 h; BAF A was added where indicated. Mitotracker fluorescence was measured by flow cytometry. Unless specified otherwise, values for UBQLN‐KD and control+BAF A were compared with that of controls at the same time points, with ***P0.001, **P0.01 and *P0.05 (paired Student's t‐test). (E) Cells were transfected with pShooter™ together with control or UBQLN (1+2) siRNA. Expression of pShooter was comparable in control and UBQLN‐KD cells (top). Cells were starved for 3 or 6 h, and the intensity of GFP was quantified by flow cytometry (bottom). The fluorescence value obtained with controls (no starvation) was considered as 100%, and the value obtained for the 3‐ and 6‐h time points was normalized to controls. The graph represents the mean±s.e. of four assessments. (F) Cells expressing a chimaeric LC3 fused to both GFP and mCherry were transfected with control or UBQLN siRNA, and starvation was induced for the indicated times. Puncta from 25 cells were scored and analysed by microscopy for their fluorescence colour. The graph represents the mean of three experiments±s.e. The number of mCherry single‐positive puncta in UBQLN‐KD cells was compared with that of controls for each time point; **P0.01 and *P0.05 (paired Student's t‐test). (G) Control or UBQLN‐KD cells were starved for increasing periods. Where indicated, BAF A was added to the starvation medium (+B). Cell lysates were analysed by Western blot for their content in LC3‐II. Loading control: transferrin receptor (TfR). BAF A, bafilomycin A1; GFP, green fluorescent protein; KD, knockdown; siRNA, short interfering RNA; UBQLN, ubiquilin.
DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy (2008)
Figure 1

Functional interaction between DAPK and beclin 1. (A) HEK293 cells were transfected with DAPK ΔCaM or with a control vector (pcDNA3‐luciferase, LUC), together with shRNAs targeting beclin 1 or HcRed, and with GFP–LC3 plasmid. After 72 h, cells were counted and lysates were prepared. (A) The percentage of cells with punctate GFP–LC3 fluorescence per total GFP–LC3‐positive cells was quantified. Data presented are the mean±s.d. from a triplicate of 100 transfected cells. The asterisks denote a significance level of P=0.001. (B) Western blot analysis was performed using the indicated antibodies. (C) Representative GFP–LC3 staining of cells transfected with the control shRNA (HcRed) together with pcDNA3‐luciferase or DAPKΔCaM. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; HEK, human embryonic kidney; shRNA, short hairpin RNA.
DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy (2008)
Figure 2

Beclin 1 is a new substrate of DAPK. (A) Flag‐tagged DAPK (100 ng) was incubated with GST (900 ng) or GST–beclin‐1 (750 ng) in the presence of Ca2+, calmodulin and [γ‐33P]ATP for 30 min or 60 min. Phosphorylated proteins were visualized by X‐ray film exposure, and GST/GST–beclin‐1 levels were visualized by Ponceau S staining. The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples. (B) Flag‐tagged DAPK (60 ng) was incubated with Flag‐tagged beclin 1 (250 ng), which was purified from HEK293T cells, and a kinase assay was performed for 60 min. Where indicated (+LiCl), beclin‐1‐bound beads were first washed stringently in 0.5 M LiCl and 0.5 M KCl. Phosphorylated proteins were visualized by X‐ray film exposure, and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies. DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney.
DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy (2008)
Figure 3

Physical interaction between DAPK and beclin 1. (Aa) Protein extracts from COS7 cells, HEK293T cells, or no extracts were added to bacterially produced GST or GST–beclin‐1. The pulled‐down proteins, as well as the total cell extracts, were blotted with the indicated antibodies. (Ab) Ponceau S staining of GST and GST–beclin‐1 to which the extracts were added. (B) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 or with Flag‐tagged beclin 1 lacking the Bcl‐2‐binding domain (ΔBD) together with Bcl‐XL and HA‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted with DAPK, Bcl‐XL and beclin 1 antibodies. (C) HEK293 cells were transfected with Flag–beclin‐1 or Flag–GFP, and the Flag‐tagged proteins were immunoprecipitated using Flag antibodies, and eluted with an excess of Flag peptides. The blot was reacted with DAPK antibodies or with Flag antibodies at different time exposures. DAPK, death‐associated protein kinase; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin; HEK, human embryonic kidney.
DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy (2008)
Figure 4

DAPK phosphorylates beclin 1 on Thr 119 located within its BH3 domain. (A) A bacterially purified catalytic domain of DAPK was incubated for 15 min at 30°C with increasing concentrations (5–10 nmol) of a peptide corresponding to the BH3 domain of beclin 1 (aa 108–127) and with the same peptide in which Thr 119 was substituted by alanine. An in vitro kinase assay was performed, and the reactions were applied to Whatman filters. Total levels of TCA‐insoluble counts were measured and plotted against substrate concentration. Data are the mean±s.d. of three experiments. (B) A model of the interaction between the BH3 domain of beclin 1, and the hydrophobic pocket of Bcl‐XL. The cyan arrow points to the groove in which phosphorylated Ser 113 can be accommodated. The black arrow points to the site that might be disrupted by phosphorylation of Thr 119. The magenta arrow points to the positively charged groove in which phosphorylated Ser 127 can be accommodated. (C) Flag‐tagged DAPK (60 ng) was incubated with GST–WT beclin 1 or with GST–T119A beclin 1 (1000 ng) in the presence of Ca2+, calmodulin and ATP for 30 min. GST‐beclin 1 levels were visualized by Ponceau S staining, and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody (Western blot). (D) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 with or without ΔCaM DAPK. After 24 h, beclin 1 was immunoprecipitated from cells, using Flag antibodies, and the immunoprecipitates were reacted with phosphoThr 119 antibodies. The Ponceau staining shows equal amounts of immunoprecipitated beclin 1. The cell extract blots were reacted with haemagglutinin antibodies or with beclin 1 antibodies. CaM, calmodulin; DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney; TCA, trichloro‐acetic acid.
DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy (2008)
Figure 5

DAPK promotes the dissociation of beclin 1 from Bcl‐XL. (A) HEK293 cells were transfected with Flag‐tagged beclin 1 and Bcl‐XL with or without haemagglutinin‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. (B) HEK293 cells were co‐transfected with Flag‐tagged T119A or T119E beclin 1 mutants and Bcl‐XL. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. (C) HEK293 cells were transfected with 5 or 10 μg T119A or T119E beclin 1 mutants together with GFP–LC3 plasmid. After 24 h, cells were counted and lysates were prepared. (a) Representative GFP–LC3 staining. (b) The percentage of cells with punctate GFP–LC3 fluorescence per total GFP–LC3‐positive cells was quantified. Data presented are the mean±s.d. from a triplicate experiment with 100 transfected cells. The asterisks denote significance level: *P=0.01; **P=0.005. (c) Western blot analysis was performed using the indicated antibodies. (D) HEK293 cells were transfected with Bcl‐XL, Flag‐tagged beclin 1 (WT) or Flag‐tagged T119A beclin 1 mutant with or without haemagglutinin‐tagged activated DAPK (ΔCaM). Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. Levels of Bcl‐XL were quantified using NIH image software, and the ratio between immunoprecipitated and expressed Bcl‐XL was calculated. (E) Transmission electron micrographs of HEK293 cells 24 h after they were transfected with Bcl‐XL, Flag‐tagged beclin 1 and ΔCaM or pcDNA3‐luciferase as a control. The images in (c) and (d) were taken at higher magnifications of the ΔCaM treatment (see the scale bars). ‘AV’ indicates autophagic vacuoles. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; GST, glutathione S‐transferase; HEK, human embryonic kidney; NIH, National Institutes of Health.
The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells (2009)
Figure 1

DOR moves out of the nucleus in response to the activation of autophagy. (A) The intracellular distribution of DOR is modified by starvation. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal), HBSS (starvation), 50 μM chloroquine or 10 mM 3MA. The intracellular localization of DOR was analysed by immunofluorescence and is shown in red. The nuclei are shown in blue (DAPI staining). Scale bars, 10 μm. (B) Endogenous DOR leaves the nucleus under starvation. C2C12 myoblasts were incubated for 30 min at 37°C with DMEM (basal) or HBSS (starvation). DOR is shown in red and nuclei are in blue (DAPI staining). Scale bars, 5 μm. (C,D) De‐activation of autophagy returns DOR to the nucleus. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal) or HBSS (starvation). Cells were then incubated with normal DMEM for an additional hour (starvation+GM). The intracellular distribution of DOR was determined by immunofluorescence. DOR is shown in red and the nuclei are in blue. Scale bars, 5 μm. Cell lysates were also obtained and western blot assays were performed with specific antibodies. 3MA, 3‐methyladenine; DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; HBSS, Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.
The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells (2009)
Figure 2

DOR localizes to autophagosomes when autophagy is activated. (A) Confocal images of HeLa cells transiently transfected with DOR and GFP‐LC3, and incubated for 1h with DMEM, HBSS (starvation), 2 μM rapamycin or HBSS containing 50 μM chloroquine. Nuclei were labelled with DAPI. Scale bars, 10 μm. (B,C) Confocal images of HeLa cells transiently transfected with DOR or GFP‐LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS. Scale bars, 10 μm. (D) Confocal images of HeLa cells transiently transfected with DOR and LAMP1‐GFP, or with YFP‐LC3 and LAMP1‐GFP. Scale bars, 10 μm. Contrast‐corrected merge RGB pictures and the Z‐projection of Product of the differences from the mean (PDM) images are shown in all panels. Colour scales of PDM images have different maximal values, so the different conditions are not comparable from these projections. Product of the differences from the mean (PDM) values closer to 1 show reliable colocalized pixels. DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; LAMP1, lysosomal‐associated membrane protein 1; LC3, microtubule‐associated protein 1A/1B‐light chain 3.
The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells (2009)
Figure 3

DOR stimulates autophagy. (A) DOR gain‐of‐function accelerates the degradation of proteins of middle–long half‐life. Untreated HeLa cells and those transiently transfected with DOR were incubated in DMEM or HBSS. *Significant effects of starvation, P0.01; τsignificant effects caused by DOR overexpression, P0.01. (B) DOR overexpression enhances autophagosomal formation. LC3‐GFP‐positive vacuoles were counted in 100 transfected HeLa cells (with GFP‐LC3 and TRα1, or GFP‐LC3 and DOR). *Significant effects of starvation, P0.001; τsignificant effects of DOR overexpression, P0.001. (C,D) HeLa cells transiently transfected with empty pCDNA3 or DOR were incubated in DMEM (basal) or in HBSS (starvation). Cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes (diameter, 407±63 nm). Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen transfected cells. *Significant effects of starvation, P0.05; τsignificant effects of DOR overexpression, P0.05. DMEM; Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS; Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.
The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells (2009)
Figure 4

DOR loss‐of‐function reduces autophagy. (A,B) C2C12 myoblasts were previously infected with lentiviruses encoding scrambled RNA, DOR siRNA1 or DOR siRNA2. Endogenous LC3 was detected by immunofluorescence. LC3‐positive puncta were counted in 100 cells per group under basal conditions or starved for 20 min. Scale bars, 10 μm. *Significant effects of starvation, P0.001; τsignificant effects caused by DOR knockdown, P0.001. (C,D) Scramble or DOR siRNA1 C2C12 cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes. Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen cells. *Significant effects of DOR repression, P0.001. (E) Scramble or DOR siRNA1 C2C12 cells were incubated in DMEM with and without 10 mM 3MA. *Significant effects of DOR knockdown, P0.01; τsignificant effects caused by 3MA, P0.01. 3MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; LC3, microtubule‐associated protein 1A/1B‐light chain 3.
The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells (2009)
Figure 5

CG11347 is required for autophagy activation in the fat body of Drosophila. (A) Decrease of mRNA levels in TubG4, UAS‐CG11347‐RNAi animals compared with TubG4 controls. All measurements were normalized to rp49. *Significant change, P0.05. (B) Lysotracker staining of fat bodies. No Lysotracker staining is observed in feeding early third‐instar larvae, whereas Lysotracker‐positive granules (red) accumulate in fat body cells of wandering late third‐instar controls (upper panels, TubG4/+). dDOR knockdown animals (TubG4, UAS‐CG11347‐RNAi T4, lower panels) show decreased staining of Lysotracker‐positive granules in wandering late third‐instar larvae. Nuclei are shown in blue. Scale bars, 50 μm. (C) The number of Lysotracker‐positive puncta per unit area are shown for each genotype (control and RNAi), normalized to the value for control cells processed in parallel. *Significant change, P0.05. (D) Transmission electron microscopy images of fat body cells from wandering late third‐instar larvae. Developmental autophagy results in the accumulation of large autolysosomes (AL) in control (TubG4/+) larvae, whereas only a few autolysosomes are seen in CG11347 RNAi fat body cells of the same age. Control and RNAi fat bodies present lipid droplets (LP) with a ‘striped’ appearance, which is an electron microscopy artefact. Scale bars, 5 nm. dDor, CG11347, a homologue of DOR; DOR, diabetes‐ and obesity‐regulated gene; mRNA, messenger RNA; wt, wild type.
Autophagy in Myf5+ progenitors regulates energy and glucose homeostasis through control of brown fat and skeletal muscle development (2013)
Figure 1

Deleting Atg7 in Myf5+ progenitors disrupts macroautophagy (MA) in brown adipose tissue (BAT) and skeletal muscle (SKM). (A-C) Immunoblots for indicated proteins in BAT, extensor digitorum longus (EDL), epididymal white adipose tissue (eWAT) and heart from 10‐ to 12‐month (mo)‐old control (Con) and knock out (KO) mice. Arrows depict LC3‐I and II. (D) ATG5 and ATG7 mRNA levels in indicated tissues (n=4), and (E) immunoblots for indicated proteins in spleen, liver, lung, kidney, mediobasal hypothalamus (MBH), inguinal white adipose tissue (ingWAT) and perinephric fat (pnWAT) from 10‐ to 12‐mo‐old Con and KO mice. (F) Body weights (wt) of chow diet (RD)‐fed (n=6-29), and (G) high‐fat diet (HFD)‐fed Con and KO mice at indicated ages (n=4-17). (H) Total body fat and lean mass of 4-7 mo RD‐fed Con and KO mice (n=8-12). (I) BAT wt (n=4-7), (J) gastrocnemius (GA) wt (n=5-7), (K) soleus wt (n=4-7), (L) eWAT wt (n=5-7) and (M) visceral (Visc) and subcutaneous (Subc) body fat distribution in 10‐mo‐old Con and KO mice (n=4). Values are mean±s.e., *P0.05, **P0.01, ***P0.001.