AdipoRon enhances healthspan in middle‐aged obese mice: striking alleviation of myosteatosis and muscle degenerative markers

AdipoRon enhances healthspan in middle-aged obese mice: striking alleviation of myosteatosis and muscle degenerative markers Camille M. Selvais 1 *, María A. Davis-López de Carrizosa 1,2 , Maxime Nachit 3 , Romain Versele 1 , Nicolas Dubuisson 1 , Laurence Noel 1 , Justine Gillard 3 , Isabelle A. Leclercq 3 , Sonia M. Brichard 1 & Michel Abou-Samra 1 1 Endocrinology, Diabetes and Nutrition Unit, Institute of Experimental and Clinical Research, UCLouvain, Brussels, Belgium; 2 Department of Physiology, Faculty of Biology, University of Seville, Seville, Spain; 3 Hepato-Gastroenterology Unit, Institute of Experimental and Clinical Research, UCLouvain, Brussels, Belgium Abstract Background Obesity among older adults has increased tremendously. Obesity accelerates ageing and predisposes to age-related conditions and diseases, such as loss of endurance capacity, insulin resistance and features of the metabolic syndrome. Namely, ectopic lipids play a key role in the development of nonalcoholic fatty liver disease (NAFLD) and myosteatosis, two severe burdens of ageing and metabolic diseases. Adiponectin (ApN) is a hormone, mainly secreted by adipocytes, which exerts insulin-sensitizing and fat-burning properties in several tissues including the liver and the muscle. Its overexpression also increases lifespan in mice. In this study, we investigated whether an ApN receptor agonist, AdipoRon (AR), could slow muscle dysfunction, myosteatosis and degenerative muscle markers in middle-aged obese mice. The effects on myosteatosis were compared with those on NAFLD. Methods Three groups of mice were studied up to 62 weeks of age: One group received normal diet (ND), another, high-fat diet (HFD); and the last, HFD combined with AR given orally for almost 1 year. An additional group of young mice under an ND was used. Treadmill tests and micro-computed tomography (CT) were carried out in vivo. Histological, biochemical and molecular analyses were performed on tissues ex vivo. Bodipy staining was used to assess intramyocellular lipid (IMCL) and lipid droplet morphology. Results AR did not markedly alter diet-induced obesity. Yet, this treatment rescued exercise endurance in obese mice (up to 2.4-fold, P<0.05), an event that preceded the improvement of insulin sensitivity. Dorsal muscles and liver densities, measured by CT, were reduced in obese mice (42% and 109%, respectively, P<0.0001), suggesting fatty infiltration. This reduction tended to be attenuated by AR. Accordingly, AR significantly mitigated steatosis and cellular ballooning at liver histology, thereby decreasing the NALFD activity score (30%, P<0.05). AR also strikingly reversed IMCL accumulation either due to ageing in oxidative fibres (types 1/2a, soleus) or to HFD in glycolytic ones (types 2x/2b, extensor digitorum longus) (50% to 85%, P<0.05 or less). Size of subsarcolemmal lipid droplets, known to be associated with adverse metabolic outcomes, was reduced as well. Alleviation of myosteatosis resulted from improved mitochondrial function and lipid oxidation. Meanwhile, AR halved aged-related accumulation of dysfunctional proteins identified as tubular aggregates and cylindrical spirals by electron microscopy (P<0.05). Conclusions Long-term AdipoRon treatment promotes ‘healthy ageing’in obese middle-aged mice by enhancing endurance and protecting skeletal muscle and liver against the adverse metabolic and degenerative effects of ageing and caloric excess. Keywords adiponectin; myosteatosis; intramyocellular lipids; ageing; nonalcoholic fatty liver disease; endurance Received: 13 October 2022 ; Accepted: 16 November 2022 *Correspondence to: Camille Selvais, Endocrinology, Diabetes and Nutrition Unit, Institute of Experimental and Clinical Research, UCLouvain, Brussels, Belgium. Email: [email protected] ORIGINAL ARTICLE © 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Journal of Cachexia, Sarcopenia and Muscle 2023; 14: 464–478 Published online 13 December 2022 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jcsm.13148 Introduction As society ages, the incidence of physical limitations is dramatically increasing. An important cause of physical limitations is the age-related loss of skeletal muscle mass, referred to as sarcopenia. Muscle function is impaired as well, and the endurance capacity declines. 1 Beyond physical performance, muscles also play a crucial role in insulin sensitivity and fuel homeostasis. Muscle disturbances may thus contribute to insulin resistance and metabolic syndrome. 1 Obesity among older adults aged 65 and over has increased noticeably over the last decades on all continents, and its prevalence is reaching 35% in the United States where it is expected to at least double by 2050. 2 Obesity accelerates ageing and predisposes an individual to age-related conditions and diseases. Thus, obesity and ageing go hand in hand with loss of muscle mass, function and endurance capacity, insulin resistance, and features of the metabolic syndrome. 3 Two additional burdens associated with this syndrome involve ectopic lipid deposition in the liver and the skeletal muscle, namely, nonalcoholic fatty liver disease (NAFLD)/steatohepatitis (NASH) and myosteatosis, whose rising prevalence tends to parallel that of obesity and ageing. 4,5 Adiponectin (ApN) is a hormone, mainly secreted by the adipose tissue, which is known to be tightly linked to the metabolic syndrome. ApN exerts insulin-sensitizing, fat-burning and anti-inflammatory actions, thereby effectively counteracting several facets of this syndrome. Liver and muscle are two of its main target tissues. The ApN receptor, AdipoR2 is predominantly expressed in the liver, whereas AdipoR1 is predominantly expressed in the skeletal muscle. In the liver, ApN stimulates fatty-acid oxidation and reduces glucose production. 6 In skeletal muscle, we have shown that ApN does also exert powerful protective effects in mdx mice, a model with a severe muscle disease (Duchenne muscular dystrophy). Thus, ApN reduces muscle inflammation and oxidative stress and enhances the myogenic program, thereby decreasing muscle damage while increasing force/ endurance. 6,7 Importantly, overexpression of ApN in transgenic mice prolongs life span 8 whereas complete deficiency of ApN in knockout mice shortens it. 9 ApN has a complex three-dimensional structure and must be injected to produce its effects. 6 The development of novel molecules that mimic the beneficial effects of ApN is therefore relevant. AdipoRon is an orally active synthetic agonist of ApN receptors, which, based on data in animal models, has been proposed for the treatment of type 2 diabetes and other obesity-related disorders. 10–12 Relevant for translational applications, AdipoRon remains efficient in AdipoR1-humanized mice. 13 We have recently shown that, like ApN, AdipoRon protects the skeletal muscle of mdx mice. 14 However, its potential beneficial properties on ageing muscle have been scarcely addressed. 15 It is also unknown whether this molecule does effectively prolong life span in normal (wild-type) mice. An effect on longevity has been observed in a mouse model with a severe form of genetic diabetes; hence, the marked alleviation of diabetes provoked by AdipoRon was, in this case, a major confounding factor. 10 The overall aim of this work was to explore whether AdipoRon could promote healthy ageing in middle-aged obese mice on a normal genetic background. Mice were rendered obese by chronic excess of caloric intake in order to mimic our western-life style habits and the high prevalence of obesity in the elderly. Some of these mice were concomitantly treated with AdipoRon for approximately 1 year. Herein, we investigated whether an ApN receptor agonist, AdipoRon, could slow down muscle dysfunction, myosteatosis and degenerative muscle markers in middle-aged obese mice. The effects on myosteatosis were compared with those on NAFLD. Methods Experimental design and animals Male C57BL/6J mice were divided into three groups and studied up to 62 weeks of age. They are referred to as old (O) mice (Figure 1A). Two of these groups received a highfat diet (HFD): mice in one HFD group were orally treated with AdipoRon (AR) for approximately 1 year (30 mg/kg/day scaled up to 50 mg/kg/day; O-HFD + AR), while mice in the other group were left untreated (O-HFD). HFD groups were compared with O mice kept on a normal diet (O-ND). An additional group of young (12-week-old) mice under a normal diet (Y-ND) was also studied. Mice were subjected to treadmill exhaustion tests at 32 and 56 weeks of age and to micro-computed tomography (CT) at 59 weeks. Histological, biochemical and molecular analyses were performed on tissues ex vivo. Mice were purchased from Janvier Labs, Genest, France, and AdipoRon from Bio-Techne, Minnesota, USA. Please see the Supporting Information S 1 for detailed experimental procedures. Results Long-term administration of AdipoRon protects against impaired glycaemia, dyslipidaemia and improves endurance The body weight of O-HFD mice steadily increased and was twice that of O-ND animals at the end of study (Figure 1B). In line with others, 10 AdipoRon did not markedly alter obesity and only stabilized the body weight at the end of AdipoRon on myosteatosis and muscle degenerative markers 465 Journal of Cachexia, Sarcopenia and Muscle 2023; 14: 464–478 DOI: 10.1002/jcsm.13148 1353921906009, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcsm.13148 by Universidad De Sevilla, Wiley Online Library on [15/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Figure 1 Chronic administration of AdipoRon improves insulin sensitivity, endurance and enhances computed tomography (CT) scan muscle and liver densities in middle-aged obese mice. (A) Experimental protocol. Four groups of mice were studied. Three groups were studied up to 62 weeks of age and are referred to as old (O) mice. Two of these groups received a high-fat diet (HFD) from 8 weeks. One HFD group was orally treated with AdipoRon (AR), starting from 18 weeks (30 mg/kg/day scaled up to 50 mg/kg/day; O-HFD + AR), while the other one was left untreated (O-HFD). Both groups were compared with O mice kept on a normal diet (O-ND). An additional group of young (12-week-old) mice under a normal diet (Y-ND) was also used for comparison. At the indicated times, mice were submitted to treadmill exhaustion test (T) or micro-CT. (B–D) Evolution of body weight, glycaemia and insulin resistance index during the study. (E) Mice were submitted to an uphill treadmill exhaustion test at different ages, with each time a protocol adapted to mice conditions. Endurance capacity was expressed as work to consider the differences in body weight (kg) over the distance covered (m). (F) Micro-CT evaluation of dorsal muscle and liver density, a decrease in density reflecting fatty infiltration. Data are means ± SEM for 6 Y-ND, and 9–12 mice in the other three groups. Statistical analysis was performed using a mixed-effects analysis (B) or one-way ANOVA followed by Tukey’s test to compare the three groups of O mice (C–F). Comparisons between Y-ND and O-ND were carried out using unpaired two-tailed t-test. *P<0.05, ***P<0.001, ****P<0.0001 versus O-ND mice. † P≤0.07, # P<0.05, ## P<0.01, ### P<0.001 versus O-HFD mice. 466 C.M. Selvais et al. Journal of Cachexia, Sarcopenia and Muscle 2023; 14: 464–478 DOI: 10.1002/jcsm.13148 1353921906009, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcsm.13148 by Universidad De Sevilla, Wiley Online Library on [15/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License the protocol. Likewise, daily energy intake, which was approximately 60% higher under HFD, was not modified by AdipoRon (Figure S1). Yet AdipoRon, when administered at the highest dose (50 mg/kg/day), progressively decreased fasting blood glucose levels, which were moderately elevated in HFD-fed mice, to reach O-ND values (Figure 1C). The insulin resistance index was attenuated as well (Figure 1D). Moreover, AdipoRon decreased plasma cholesterol levels induced by HFD, possibly through increased efflux, without modifying other circulating lipids (Table S1). Plasma ApN levels were decreased in old mice compared with young ones (O-ND vs. Y-ND), and this decrease was further amplified in obese mice when data were normalized by white fat mass (i.e., expressed per ‘secretion unit’ 16 ;Table S1). We next evaluated the effect of AdipoRon on muscle function by using an uphill treadmill exhaustion test. This test was performed twice, with each time a protocol adapted to age and obesity of the mice (Figure 1E, see Supporting information). At 32 weeks, exercise endurance capacity, expressed as work, was reduced in O-HFD mice compared with O-ND ones and was corrected by AdipoRon. At 56 weeks, work of O-HFD was drastically reduced (73% vs. O-ND), while it was partially rescued by AdipoRon. Thus, AdipoRon was able to partially or totally rescue exercise endurance of middle-aged obese mice. This already occurs at the low dose of AdipoRon (30 mg/kg/day), which did affect neither glucose homeostasis nor the body weight (compare Figure 1E left panel vs. Figure 1B,C). Effects of AdipoRon on body composition, and liver and muscle density We measured in vivo the body composition of mice as well as liver and muscle density by micro-CT. Whole body fat mass, which was markedly increased in O-HFD mice, was reduced by 20% under AdipoRon treatment (Figure S1B), in line with the decreased subcutaneous fat measured ex vivo (Table S1). However, AdipoRon did not significantly alter total lean mass (Figure S1C), in agreement with the unchanged dorsal muscle area, an index of muscle mass 17 (Figure S1D) and the unchanged weight of several muscles sampled ex vivo (Table S1). Dorsal muscle density was decreased in HFD mice, suggesting fatty infiltration (Figure 1F,left). However, this decrease tended to be less pronounced under AdipoRon treatment, a result confirmed a posteriori by lipid dosage of dorsal muscle (Figure S1E). Likewise, liver density was reduced in both groups of HFD-mice, but this reduction also tended to be attenuated by AdipoRon (Figure 1F,right). These data suggest therefore that AdipoRon treatment lessens hepatic steatosis and myosteatosis induced by HFD. AdipoRon reduces the severity of NAFLD To further investigate the effects of AdipoRon on the liver, several parameters including steatosis, hepatocellular ballooning and inflammation were quantified on histological sections using the NAFLD activity score (NAS score) (Figure 2A,B). O-ND mice had almost normal liver histology (NAS = 0.4). O-HFD mice were diagnosed with nonalcoholic steatohepatitis (NASH) (i.e., NAS ≥3 with at least 1 point in each sub-score) as they exhibited steatosis (>2), inflammation (>2) and ballooning (>1). By contrast, O-HFD + AR mice did not reach NASH stage: They had lower scores of steatosis (<2), inflammation (<2) and ballooning (<1). Strikingly, only 3 out of 10 O-HFD + AR mice presented ballooning versus 7 out of 8 in untreated HFD ones. The protective effect of AdipoRon on steatosis was corroborated by liver lipid dosage and liver weight (Figure 2C;Table S1). These data indicate that AdipoRon partially protected against liver fatty infiltration and mitigated the severity of NAFLD in diet-induced obese mice. AdipoRon drastically blunts dietor age-induced accumulation of Intramyocellular lipids We next examined the effects of AdipoRon on myosteatosis and more specifically on the excessive accumulation of intramyocellular lipids (IMCLs) that leads to insulin resistance in obesity and type 2 diabetes. 18,19 With this accumulation being fibre type-dependent, we quantified IMCL content and analysed lipid droplet (LD) morphology in the soleus, a slow-twitch oxidative muscle, and in the extensor digitorum longus (EDL), a fast-twitch glycolytic one. We performed fibre typing and LD staining on serial muscle cryosections. The total number of fibres in each muscle was similar between the four groups of mice (data not shown). Fibre typing was carried out by immunofluorescence staining of different MyHCs isoforms (Figure 3A). As expected, the soleus contained a large proportion of slow-oxidative type 1 (blue, ~38%) and 2a fibres (green, ~50%), whereas the EDL was mainly composed of fast-glycolytic type 2b (black, ~65%) and 2x (red, ~17%) fibres (Figure 3A,B). There was also a small proportion of hybrid fibres. Overall, the proportion of fibre types in each muscle did not significantly differ between the four groups of mice (Figure 3B). Thus, AdipoRon did not induce a fibre switch towards a more oxidative phenotype in middle-aged obese mice, unlike in mdx mice. 14 Fibre size, assessed by both cross-sectional area and minimum Feret’s diameter, was also roughly similar in the different groups (not shown). Next, by Bodipy staining, we quantified IMCL content and LD size on a fibre type and subcellular (peripheral vs. central) specific basis (Figures3C and 4). The overall lipid content was higher in the peripheral (subsarcolemmal) region than at the AdipoRon on myosteatosis and muscle degenerative markers 467 Journal of Cachexia, Sarcopenia and Muscle 2023; 14: 464–478 DOI: 10.1002/jcsm.13148 1353921906009, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcsm.13148 by Universidad De Sevilla, Wiley Online Library on [15/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License centre of the myofibre. Peripheral IMCL accumulation has been associated with insulin resistance 18,19 and could also exert a mechanical stress on sarcolemma. As shown on Figure 3C, the plasma membrane presented scalloped edges (arrows) due to the presence of large subsarcolemmal LDs. In Types 1 and 2a fibres of soleus, IMCL content (expressed as the percentage of stained area) was significantly increased in O-ND mice compared with that in the Y-ND ones (both centrally and peripherally) and was not significantly modified by HFD (Figure 4A). However, when compared with HFD-fed mice, AdipoRon reduced total IMCL content in both fibre types. In EDL (type 2x and type 2b fibres), IMCL content of O-ND mice was roughly similar to that of Y-ND mice (except for the periphery in 2x fibres) (Figure 4B). By contrast, this content was significantly increased by HFD in both fibre types (compare O-HFD vs. O-ND mice). AdipoRon drastically reduced HFD-induced IMCL accumulation in both types of fibres and in both regions. Moreover, it tended or reduced LD size in soleus (especially in 2a fibres) and in EDL in both fibre types, both centrally and peripherally (Figure 4C,D). Taken together, these results indicate that AdipoRon strikingly reversed IMCL accumulation either due to ageing in oxidative fibres or to HFD in glycolytic ones. It reduced LD size as well. We then analysed whether AdipoRon enhanced fatty acid oxidation by stimulating the AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α/Ppargc 1 a) axis (Figure S2). 6 The phosphorylated and active form of AMPK (P-AMPK) and protein levels of PGC-1αdoubled under AdipoRon when compared with the other two groups of O-mice. The expression of relevant target genes of this transcriptional co-activator was (or tended to be) increased by AdipoRon, including those involved in fatty acid oxidation or energy dissipation (represented by dark blue boxes, part 1 on Figure S2). Thus, AdipoRon enhanced (or tended to enhance) mRNA abundance of medium-chain acyl-CoA dehydrogenase (Acadm), acyl-CoA oxidase (Acox) and uncoupling protein 3 (Ucp 3 )(Figure 5B). Figure 2 Chronic administration of AdipoRon reduces the severity of nonalcoholic fatty liver disease (NAFLD) in middle-aged obese mice. (A) Representative haematoxylin and eosin-stained liver sections from the different groups of mice. Arrows indicate hepatocellular ballooning, scale bar = 50 μm. (B) Histological NAFLD activity score (NAS) calculated on sections like those shown in (A). (C) Lipid content in liver (biochemical measure). Data are means ± SEM for 6 Y-ND, and 8–10 mice in the other three groups. Unless otherwise specified, statistical analysis was performed using one-way ANOVA followed by Tukey’s test (comparing three groups of O-mice) or by unpaired two-tailed t-test (Y-ND vs. O-N). $$$ P<0.001 versus Y-ND mice. ***P<0.001, ****P<0.0001 versus O-ND mice. # P<0.05 versus O-HFD mice. 468 C.M. Selvais et al. Journal of Cachexia, Sarcopenia and Muscle 2023; 14: 464–478 DOI: 10.1002/jcsm.13148 1353921906009, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcsm.13148 by Universidad De Sevilla, Wiley Online Library on [15/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Figure 3 Effects of AdipoRon on fibre type composition and lipid infiltration in soleus and EDL from middle-aged obese mice. Fibre typing and Bodipy staining were performed on serial muscle cross-sections in an oxidative (soleus) and a glycolytic (EDL) muscle. (A) Fibre typing was carried out by immunofluorescence staining of different myosin heavy chains isoforms (MyHCs). Type 1 fibres were labelled in blue, type 2a in green, 2x in red while 2b were nonlabelled (black). Laminin antibody was used to delineate basal membrane (white). Representative sections for each group are shown. Scale bar = 200 μm. Insets: Higher magnification of immunostaining images (scale bar = 50 μm). (B) Fibre type proportion for each muscle in the 4 groups of mice. Data are means for six mice per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test (comparing three groups of O-mice) or by unpaired two-tailed t-test (Y-ND vs. O-ND). $ P<0.05 versus Y-ND mice. (C) Bodipy staining of lipids (red) on muscle cross-sections to quantify IMCL content on a fibre type specific-basis. Laminin was coloured in cyan. Peripheral (subsarcolemmal, SS) lipid droplets (LDs) are usually more abundant than central ones. Arrows indicate scalloped edges of sarcolemma facing large SS LDs. Representative sections for six mice per group are shown. Scale bar = 20 μm. AdipoRon on myosteatosis and muscle degenerative markers 469 Journal of Cachexia, Sarcopenia and Muscle 2023; 14: 464–478 DOI: 10.1002/jcsm.13148 1353921906009, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcsm.13148 by Universidad De Sevilla, Wiley Online Library on [15/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License AdipoRon improves muscle mitochondrial function PGC-1αalso promotes mitochondrial biogenesis and function via the transcriptional regulation of nuclear transcription factors such as nuclear respiratory factor-1 (NRF-1) (Part 2 on Figure S2). Expression of NRF-1 tended to be increased by AdipoRon (Figure 5C). Moreover, AdipoRon doubled the expression of mitochondrial transcription factor A (Tfam), Figure 4 AdipoRon drastically blunts dietor age-induced accumulation of intramyocellular lipids (IMCL) in middle-aged obese mice. (A) IMCL content of soleus in peripheral and central subcellular regions in type 1 and 2a fibres stained by Bodipy and (B) IMCL content of EDL in subcellular regions in type 2x and 2b fibres. IMCL content in each region was expressed as the percentage of stained area normalized to total fibre area. Symbols for differences among central regions are in dark blue, among peripheral regions in turquoise blue, and those for the total content in black. (C) LD size in peripheral and central subcellular regions in type 1 and 2a fibres from soleus and (D) in type 2x and 2b fibres from EDL. Data are means ± SEM for five mice per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test (comparing three groups of O-mice) or by unpaired two-tailed t-test (Y-ND vs. O-ND). $ P<0.05 versus Y-ND mice. *P<0.05, **P<0.01 versus O-ND mice. # P<0.05, ## P<0.01, ### P<0.001 versus O-HFD mice. 470 C.M. Selvais et al. Journal of Cachexia, Sarcopenia and Muscle 2023; 14: 464–478 DOI: 10.1002/jcsm.13148 1353921906009, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcsm.13148 by Universidad De Sevilla, Wiley Online Library on [15/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License downstream of NRF1, which binds to mitochondrial DNA (mtDNA) and activates mitochondrial biogenesis and function 20 (Figures5C and S2). Myofibre mitochondrial content was assessed by immunodetection of the translocase of outer mitochondrial membrane 20 (TOMM20) in soleus and EDL, in a fibreand subcellular-specific way (Figures6and S2). As described, 21 in soleus, type 2a fibres, which are more oxidative than type 1, have a higher mitochondrial content (Figure 6A,B; compare the percentage of stained area between the 2 fibre types). Likewise, in EDL, type 2x fibres have more mitochondria than 2b, which are more glycolytic and whose mitochondrial content was barely detectable 21 (Figure 6A,B). Mitochondria were usually more abundant in the subsarcolemmal region. In soleus, total mitochondrial content rose under HFD (mainly due to an increase in subsarcolemmal mitochondria) in line with other reports, 22 but this content was not influenced by the treatment. Mitochondria content decreased with age in 2x fibres (Figure 6B). Overall, there was no major change in mitochondrial content induced by AdipoRon in any muscle. Yet, as shown in Figures6C and S3, (immuno)fluorescence co-labelling (TOMM20/Bodipy) revealed physical contacts between LDs and mitochondria, which may facilitate the trafficking of fatty acids released from LDs to mitochondria, Figure 5 AdipoRon increases or tends to increase the expression of genes involved in fatty acid oxidation, mitochondrial biogenesis and function in soleus of middle-aged obese mice. (A) AMPK activity and protein levels of PGC-1α(gastrocnemius), early signalling events of the cascade leading to enhancing effects on mitochondria. (B) mRNA levels of medium-chain acyl-CoA dehydrogenase (Acadm) and acyl-CoA oxidase 1 (Acox 1 ) implicated in fatty acid oxidation and uncoupling protein 3 (Ucp 3 ) in energy dissipation. (C) mRNA levels of nuclear respiratory factor-1 (Nrf 1 ), a target gene of PGC-1αand of mitochondrial transcription factor A (Tfam). mRNA levels were normalized to cyclophilin, and the subsequent ratios presented as relative expression compared with O-ND values. The active phosphorylated form of AMPKα(P-AMPK) and PGC-1αprotein levels were quantified by ELISA, and absorbance data were presented as relative expression compared with O-ND values. Data are means ± SEM for 6 Y-ND, and 8–10 mice in the other three groups (A–C). Statistical analysis was performed by one-way ANOVA followed by Tukey’s test (comparing three groups of O-mice) or by unpaired two-tailed t-test (Y-ND vs. O-ND). $ P<0.05, $$ P<0.01, $$$ P<0.001 versus Y-ND mice. § P= 0.059, *P<0.05, **P<0.01, ****P<0.0001 versus O-ND mice. † P≤0.08, # P<0.05, #### P<0.0001 versus O-HFD mice. AdipoRon on myosteatosis and muscle degenerative markers 471 Journal of Cachexia, Sarcopenia and Muscle 2023; 14: 464–478 DOI: 10.1002/jcsm.13148 1353921906009, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcsm.13148 by Universidad De Sevilla, Wiley Online Library on [15/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Figure 6 Quantification of mitochondrial content in muscle of middle-aged obese mice treated or not with AdipoRon. (A) Mitochondrial content was assessed by immunodetection of the translocase of outer mitochondrial membrane 20 (TOMM20) in soleus and EDL from the four groups of mice on a fibre dependent manner. Representative images for each group are shown. Scale bar = 20 μm. (B) Quantification of mitochondrial content in peripheral (subsarcolemmal) and central (intermyofibrillar) subcellular regions for each fibre type in soleus or EDL. Mitochondrial content in each region was expressed as the percentage of stained area normalized to total fibre area. Data are means ± SEM for 5–6 mice per group. Statistical analysis was performed by one-way ANOVA (comparing three groups of O-mice) or by unpaired two-tailed t-test (Y-ND vs. O-ND). $ P<0.05 versus Y-ND mice. *P<0.05, **P<0.01 versus O-ND mice. Symbols for differences among central regions are in dark green, among peripheral regions in light green, and those for the total content in black. (C) Lipid droplet (LD)-mitochondrion contacts. Left, confocal fluorescence micrographs of soleus from an O-HFD mouse: LDs were stained with Bodipy in red, mitochondria with anti-TOMM20 in green, the edge of the fibre with anti-laminin in cyan and nuclei with DAPI in blue. Some mitochondria co-localized with LDs when channels were merged. Scale bar = 10 μm. Inset: Higher magnification (scale bar = 5 μm). Right, transmission electron micrograph of rectus femoris from an O-HFD + AR mouse illustrating LD-mitochondrion contact. Scale bar = 1 μm(top right) and 0.25 μm (inset, bottom right). 472 C.M. Selvais et al. Journal of Cachexia, Sarcopenia and Muscle 2023; 14: 464–478 DOI: 10.1002/jcsm.13148 1353921906009, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcsm.13148 by Universidad De Sevilla, Wiley Online Library on [15/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License