Resting skeletal muscle PNPLA2 (ATGL) and CPT1B are associated with peak fat oxidation rates in men and women but do not explain observed sex differences

What is the central question of this study? What is the relationship between proteins in skeletal muscle and adipose tissue determined at rest and at peak rates of fat oxidation in men and women? What is the main finding and its importance? The resting contents of proteins in skeletal muscle involved in triglyceride hydrolysis and mitochondrial lipid transport were more strongly associated with peak fat oxidation rates than proteins related to lipid transport or hydrolysis in adipose tissue. Although females displayed higher relative rates of fat oxidation than males, this was not explained by the proteins measured in this study, suggesting that other factors determine sex differences in fat metabolism.


INTRODUCTION
The oxidation of fatty acids as a fuel source continues to be an area of interest for both human health and exercise performance (Goodpaster & Sparks, 2017;Kiens, 2006). Several regulatory sites are likely to contribute to the oxidation of fatty acids in skeletal muscle during exercise; these include: (i) fatty acid delivery to skeletal muscle; (ii) sarcolemmal translocation of fatty acids; (iii) availability of intramyocellular triglycerides (IMTGs); (iv) cytosolic activation (or transportation) of fatty acids; and (v) mitochondrial factors (Jeppesen & Kiens, 2012;Lundsgaard et al., 2018;Sahlin, 2009). The relative importance of these factors is partly dependent on the exercise conditions. For example, fatty acid delivery is a major factor at low-to moderate-intensity exercise [e.g., below 50% of peak oxygen consumption (V O 2 peak )], whereas mitochondrial factors [in particular, the regulation of carnitine palmitoyltransferase 1 (CPT1)] are primarily responsible for the reported decline in fat oxidation rates during highintensity (>70%V O 2 peak ) exercise (Frayn, 2010;Lundsgaard et al., 2018;Sahlin, 2009;Spriet, 2014).
Previous studies have explored associations between the peak fat oxidation (PFO) rate during exercise (a potential marker of the capacity of an individual to oxidize fat) and factors that reside at the level of skeletal muscle (e.g., skeletal muscle fibre composition and capillary density, mitochondrial content and key enzymes involved in β-oxidation or IMTG hydrolysis) (Dandanell et al., 2018;Haufe et al., 2010;Nordby et al., 2006Nordby et al., , 2015Rosenkilde et al., 2015;Shaw et al., 2020;Stisen et al., 2006). However, no study to date has explored such associations within other key tissues that contribute to fatty acid delivery, such as adipose tissue. Thus, there is a need to expand our knowledge on proteins that might contribute to the regulation of PFO.
Lastly, sex differences in concentrations of oestrogen and its actions via oestrogen receptor α might account for the increase in fat oxidation during exercise in females compared with males (Devries, 2016;Oosthuyse & Bosch, 2012). Nevertheless, these inferences derive from a relatively small body of evidence, and further sex-comparison studies would help to improve our understanding of the mechanisms that

New Findings
• What is the central question of this study?
What is the relationship between proteins in skeletal muscle and adipose tissue determined at rest and at peak rates of fat oxidation in men and women?
• What is the main finding and its importance?
The resting contents of proteins in skeletal muscle involved in triglyceride hydrolysis and mitochondrial lipid transport were more strongly associated with peak fat oxidation rates than proteins related to lipid transport or hydrolysis in adipose tissue. Although females displayed higher relative rates of fat oxidation than males, this was not explained by the proteins measured in this study, suggesting that other factors determine sex differences in fat metabolism.
Accordingly, this study has two distinct aims: (i) to explore associations between PFO and the content of key proteins relevant to fatty acid metabolism in adipose tissue and skeletal muscle; and (ii) to assess sex differences in the content of these proteins in males and females when matched, and not matched, for cardiorespiratory fitness, physical activity levels and fat mass index classifications.

Anthropometrics
Upon each arrival in the laboratory, body stature (Stadiometer, Holtain, Pembrokeshire, UK) and body mass (BC-543 Monitor; Tanita, Tokyo, Japan) were measured. At trial C, waist and hip circumference measures (SECA 201, Hamburg, Germany) and a whole-body dual energy X-ray absorptiometry scan (Discovery; Hologic, Bedford, UK) were also performed.

FAT MAX test
Before the FAT MAX test, resting metabolic rate was assessed by indirect calorimetry, and a 10 ml venous blood sample was collected. The  (Achten et al., 2002)]; (ii) FAT MAX (measured values approach ); (iii) peak power output (in watts; power output of the last completed stage, plus the fraction of time in the final non-completed stage, multiplied by the watt increment of that stage); and (iv)V O 2 peak (in millilitres per kilogram per minute).  (Frayn, 1983), assuming urinary nitrogen excretion was negligible].

Habitual lifestyle assessment
Habitual levels of physical activity were assessed using a chest-worn physical activity monitor (Actiheart; Cambridge Neurotechnology, Papworth, UK) that participants wore throughout the 7 days before trial A and throughout the 48 h before trial B to assess pretrial physical activity standardization objectively. A minimum of 4 days were required to determine habitual physical activity levels (excluding n = 1 participant, for whom only 3 days were available). Habitual energy and macronutrient intake were assessed using a self-weighed diet diary (Pro Pocket Scale TOP2KG ; Smart Weigh Scales). Participants kept a written record of their food and fluid intakes for ≥4 days in the week preceding trial A (including ≥1 day at the weekend).
Additionally, the 2 days immediately before trial A were recorded in order that participants could replicate this on the 2 days before trial B.

Adipose tissue and skeletal muscle tissue biopsies
Biopsies were taken from adipose tissue and skeletal muscle in a rested state to determine the content of key proteins involved in sequential regulatory sites of lipid metabolism, such as estrogen signalling (ESR1 (ERα)), plasma membrane lipid transport (FABP pm ), regulation of lipase activity (PLIN1 and ABHD5 (CGI-58)), intracellular lipid hydrolysis (LIPE (HSL) and PNPLA2 (ATGL)), intracellular lipid metabolism (ACSL1) and mitochondrial membrane lipid transport (CPT1B).
Biopsies were taken on the same day (always adipose tissue before muscle). The adipose tissue sample was collected from the

Western blot
Before western blotting, frozen muscle tissue (∼60-100 mg) was freeze-dried and powdered, with visible blood and connective tissue removed. The powdered samples were then mixed in a 1:1 ratio with

Statistical analysis
The relationship between protein content and PFO, expressed in absolute terms (in grams per minute) and relative to FFM (in milligrams per kilogram of FFM per minute), was assessed by bivariate correlation [either a Pearson correlation coefficient (r) and Fisher z 95% confidence intervals (CI), or Spearman's ρ and Fieller et al.
The maximum sample size for correlation analysis with the content of proteins in skeletal muscle and adipose tissue was n = 28 and n = 32, respectively. To explore potential sex differences in exercise metabolic data, habitual physical activity levels, blood analytes and the content of proteins in skeletal muscle and adipose tissue, either Data are presented as the mean (SD) unless otherwise stated below. Abbreviations: BMI, body mass index; FAT MAX , exercise intensity at which peak fat oxidation rate occurs; FFM, fat-free mass; HR, heart rate; PFO, peak fat oxidation;V O 2 peak , peak oxygen consumption. † Derived from trial C. ‡ Average of trials A and B using dual energy X-ray absorptiometry body fat percentage from trial C. § Median, Mann-Whitney U test on medians.
for parametric or non-parametric data, respectively. Sex differences in the content of these proteins were explored in the whole sample Data are presented as the mean ± SD for parametric data or the median and range or mean rank for non-parametric data. Statistical significance was accepted at P ≤ 0.05, and sensitivity analyses were performed as appropriate.  Data are presented as the mean (SD) unless otherwise stated below. Abbreviations: BMI, body mass index; FAT MAX , exercise intensity at which peak fat oxidation rate occurs; FFM, fat-free mass; HR, heart rate; PFO, peak fat oxidation;V O 2 peak , peak oxygen consumption. † Derived from trial C. ‡ Average of trials A and B using dual energy X-ray absorptiometry body fat percentage from trial C. § Median, Mann-Whitney U test on medians.

Aim 1: Relationship between the content of key proteins involved in fatty acid
*P < 0.05 **P < 0.001 and ***P < 0.001 male versus female.
LIPE (HSL) in adipose tissue, but these were not apparent when PFO was expressed relative to FFM (in milligrams per kilogram of FFM per minute; Table 4). No significant correlations were evident between PFO (expressed in absolute rates or relative to FFM) and the content of ABHD5 (CGI-58), PNPLA2 (ATGL) or ACSL1 or ex vivo basal lipolysis rates in adipose tissue (Table 4; all P-values >0.05).
Significant positive correlations were found between PFO expressed in both absolute terms and relative to FFM and the content of PNPLA2 (ATGL) and CPT1B in skeletal muscle (Figure 1a-d). The protein content of skeletal muscle FABP 3 (FABPpm) was positively correlated with absolute PFO (

Sexual dimorphism in participant characteristics
Sex differences in participant characteristics for the whole sample and for matched adipose tissue and muscle subgroups are highlighted in Tables 1-3. No sex differences in the subgroups were detected when PFO was expressed in absolute terms (P > 0.05; Figure 2a), but in the whole sample males had a higher absolute PFO compared with females (P ≤ 0.01). Alternatively, females had a higher PFO when expressed relative to FFM compared with males in both the adipose tissue and TA B L E 4 Correlations between peak fat oxidation and the content of key fatty acid metabolism proteins in skeletal muscle and adipose tissue (quantified by western blots)

DISCUSSION
In this study, we investigated associations between the abundance of proteins within adipose tissue and skeletal muscle with PFO rates during exercise and explored sexual dimorphism in the content of these proteins. We found that the content of proteins involved in processes relating to intramyocellular triglyceride hydrolysis (PNPLA2 (ATGL)) and mitochondrial fatty acid transport (CPT1B) exhibited moderate and consistent positive correlations with PFO rate expressed in absolute terms and relative to FFM. In contrast, the protein contents of ESR1 (ERα), PLIN1 and LIPE (HSL) in adipose tissue exhibited significant correlations with PFO only when expressed in absolute rates. This suggests that a greater capacity to oxidize fatty acids during exercise has a stronger association with the abundance of proteins in skeletal muscle rather than adipose tissue. Additionally, we showed that although females had a greater rate of PFO (relative to FFM) and basal ex vivo adipose tissue lipolysis rates compared with wellmatched males, sexual dimorphism was not evident in the total content of any of the measured proteins in skeletal muscle or adipose tissue.
Collectively, these pilot findings help to provide support and insight into the proteins that are likely to be involved in the capacity of an individual to oxidize fat during exercise.

Peak fat oxidation and the content of skeletal muscle and adipose tissue proteins
Intramuscular factors, in particular aspects revolving around mitochondria, are suggested to be the major rate-limiting site for fat oxidation during moderate-to high-intensity exercise (Jeppesen

F I G U R E 1
The relationship between peak fat oxidation (PFO) and the content of proteins involved in lipid metabolism in skeletal muscle: (a) skeletal muscle adipose triglyceride lipase (PNPLA2 (ATGL); n = 28) and (b) skeletal muscle carnitine palmitoyltransferase 1B (CPT1B; n = 28), with PFO expressed as an absolute rate (in grams per minute); (c) skeletal muscle adipose triglyceride lipase (PNPLA2 (ATGL); n = 28) and (d) skeletal muscle carnitine palmitoyltransferase 1B (CPT1B; n = 28), with PFO expressed relative to fat-free mass (in milligrams per kilogram of FFM per minute). The Pearson correlation coefficient with 95% confidence intervals is reported and a simple linear regression line plotted for all data. RI, relative intensity & Kiens, 2012;Lundsgaard et al., 2018;Sahlin, 2009). The prevalent mechanism is believed to be via reduced free carnitine availability during intensive exercise, which inhibits the carnitine-palmitate transferase 1 (CPT1) reaction and the rate of entry of long-chain fatty acids into mitochondria (Lundsgaard et al., 2018;Stephens, 2018;Wall et al., 2011). Consistent with this, we found a moderate correlation between skeletal muscle CPT1B content and PFO rates.
Other mitochondrial factors that have previously been associated with PFO, although possibly moderated by training status and cardiorespiratory fitness, are as follows: (i) mitochondrial volume density and content (Dandanell et al., 2018); (ii) markers of mitochondrial capacity (oxidative phosphorylation complexes II-V) (Shaw et al., 2020;Stisen et al., 2006); and (iii) the activity and content of key enzymes in β-oxidation [β-hydroxyacyl CoA dehydrogenase (βHAD)] (Nordby et al., 2015;Rosenkilde et al., 2015;Shaw et al., 2020). Overall, this suggests that the ability to transport fatty acids into the mitochondria alongside the oxidative capacity of skeletal muscle are likely to be important factors that contribute to the capacity of an individual to oxidize fat.
The regulation of PFO is also attributable, in part, to factors involved in the mobilization of IMTG stores. The moderate relationship found between PFO and the content of PNPLA2 (ATGL) in skeletal muscle aligns neatly with a recent study that found positive correlations between PFO and the total abundance of skeletal muscle LIPE (HSL), PNPLA2 (ATGL) and perilipin 5 in trained and untrained males (Shaw et al., 2020). These findings are consistent with the suggestion that during low-to moderate-intensity exercise, when PFO typically occurs, IMTG stores are an important fuel source for energy production (Romijn et al., 1993;van Loon et al., 2003;Watt & Cheng, 2017). Interestingly, however, no associations have been reported between PFO and the content of IMTG or glycogen in trained and untrained subjects (Dandanell et al., 2018;Haufe et al., 2010;Shaw et al., 2020;Stisen et al., 2006), albeit participants were assessed either after a 4 h fast (Shaw et al., 2020) or in a fed state (Haufe et al., 2010;Stisen et al., 2006). Thus, it could be that factors involved in the mobilization of IMTG, rather than the content per se, contribute to the regulation of PFO.
A number of other factors identified in the molecular regulation of skeletal muscle fatty acid oxidation during exercise were also assessed in this study; these included: (i) proteins, enzymes and receptors involved in the mobilization of fatty acids from adipose tissue (i.e., PLIN1, ABHD5 (CGI-58), PNPLA2 (ATGL), LIPE (HSL) and ESR1 (ERα)); (ii) sarcolemmal and cytosolic transport of skeletal muscle fatty acids (by FABP 3 (FABPpm) and ACSL1, respectively); and (iii) the content of ESR1 (ERα) in skeletal muscle (a mediating factor proposed to upregulate fatty acid oxidation in skeletal muscle). However, despite this physiological rationale, no consistent correlations between these molecular factors and rates of PFO were found here ( Table 4). Given that this pilot study is the first to explore many of the above aspects F I G U R E 2 Peak fat oxidation rates expressed as absolute (a) and relative to total body fat-free mass (b) in females and males in the whole sample and in adipose and muscle subgroups matched for cardiorespiratory fitness, physical activity levels and fat mass index classifications. n = 36 (15 females), n = 14 (seven females) and n = 12 (six females) for the whole sample, matched adipose tissue subgroup and matched muscle subgroup, respectively. (c) Peak fat oxidation rates normalized for leg fat-free mass are also shown from the larger cohort associated with this study . Data are reported as the median and interquartile range for absolute peak fat oxidation rates owing to non-normality evident in the skeletal muscle subgroup, whereas the mean ± SD is reported for relative peak fat oxidation rates alongside the content of certain proteins proving difficult to quantify (namely PLIN1, ABHD5 (CGI-58) and ERa), we suggest that further studies that combine alternative techniques to provide an insight into the localization of these proteins (e.g., through immunohistochemistry) should be conducted. Moreover, factors not explored in the present study, such as capillary density and muscle fibre type composition, have also been shown to be correlated with PFO in young men (Dandanell et al., 2018;Shaw et al., 2020). The influence that skeletal muscle fibre type composition has on PFO could be of particular importance, given that the majority of skeletal muscle proteins assessed in the present study are more highly expressed in type I versus type II muscle fibres.
Taken collectively, the research to date suggests that the regulation of PFO is likely to be multifactorial, whereby the upregulation of several mechanisms is likely to be responsible for the increased capacity of an individual to oxidize fat during exercise.

Sexual dimorphism in fat utilization during exercise
The present study provides new insights alongside confirmatory work on whether sexual dimorphism exists in the total content of key proteins involved in fatty acid metabolism. In sex-comparison studies it is often recommended to match males and females for training status (e.g., maximum oxygen uptake expressed relative to FFM) (Lundsgaard et al., 2017;Skelly & Gibala, 2019;Tarnopolsky, 2008). As such, we performed sex-comparison analyses on the whole sample and in subgroups of males and females matched for cardiorespiratory fitness, habitual physical activity levels and fat mass index classifications.
Additionally, given the sexual dimorphism in body composition, the expression of PFO relative to FFM is proposed to be most appropriate when comparing males and females (Amaro-Gahete et al., 2018;Maunder et al., 2018). Aligned with the general consensus, female participants in this study had a higher PFO when expressed relative to FFM compared with well-matched males in both subgroup analyses (Amaro-Gahete, Sanchez-Delgado & Ruiz, 2018;Maunder et al., 2018).
Given that the increased FFM in males can often be driven by a higher upper body FFM, this could result in inappropriate normalization of fat oxidation rates to total body FFM during cycling exercise, where the lower limbs are the primary contributors to fat oxidation. However, even when normalizing for leg FFM, females displayed a higher PFO rate than males. This provides confirmation of sex differences in fat oxidation, whereby females display a greater capacity for fat oxidation in a fasted state.
At the level of adipose tissue, ex vivo basal lipolysis rates were higher in females compared with males. This might contribute to the greater PFO reported here in females and corresponds to whole-body data in basal rested conditions (Mittendorfer et al., 2001), albeit not to all ex vivo findings (Lundgren et al., 2008). Nevertheless, this suggests that there might be inherent sexual dimorphism within adipose tissue, independent from important in vivo regulators of adipose tissue metabolism, such as humoral factors and/or blood flow (Lafontan & Langin, 2009;Thompson et al., 2012). Moreover, females generally have greater whole-body lipolysis rates during exercise compared with males (indicated by circulating glycerol concentrations) (Carter et al., 2001;Henderson et al., 2007;Mittendorfer et al., 2002), although the source of increased systemic glycerol concentrations in women (i.e., adipose tissue and/or IMTG lipolysis) remains unclear (Devries, 2016). Furthermore, the present study suggests that sex differences F I G U R E 3 Sex comparisons, from the whole sample, of the protein content of oestrogen receptor alpha (Erα; a); perilipin-1 (PLIN1; b); comparative gene identification-58 (ABHD5 (CGI-58); c); adipose triglyceride lipase (PNPLA2 (ATGL); d); hormone-sensitive lipase (LIPE (HSL); e); long chain acyl-CoA synthase 1 (ACSL1; f); ex vivo basal lipolysis rates (g); and representative immunoblot for each protein of interest (h). Symbols with the same colour represent the same participant, in order to highlight participants who displayed the highest values for any given protein and their respective values across other proteins. All data are presented as the median and interquartile range (Mann-Whitney U test on medians), except for ABHD5 (CGI-58) and ex vivo basal lipolysis rates, which are shown as the mean ± SD (Student's unpaired t test) in PFO and fuel metabolism appear to be independent of some important factors involved in the regulation of fatty acid mobilization from adipose tissue (i.e., PLIN1, ABHD5 (CGI-58), LIPE (HSL) and PNPLA2 (ATGL)). Given that this study is, to our knowledge, the first to explore sexual dimorphism in the content of lipolytic gatekeeper proteins (PLIN1 and ABHD5 (CGI-58)) and lipases (PNPLA2 (ATGL) and LIPE (HSL)) in adipose tissue, future studies should provide clarification alongside exploration of alternative factors, such as adipose tissue blood flow and the regulation of different adipose tissue depots.
No sex differences were found in the protein content of skeletal muscle PNPLA2 (ATGL) (involved in IMTG mobilization) nor in factors involved in cytosolic (ACSL1) or mitochondrial transportation (CPT1B) of fatty acids. Moreover, the content of FABP 3 (FABPpm) (a sarcolemma fat transporter) and the content of ESR1 (ERα) in skeletal muscle and adipose tissue do not appear to explain sexual dimorphism in fat oxidation during exercise. Given that these findings in skeletal muscle are largely confirmatory of prior research (Berthon et al., 1998;Costill et al., 1979;Lundsgaard et al., 2017;Miotto et al., 2018;Wiik et al., 2009), future sex-comparison studies should look to explore alternative regulatory factors to those studied here and previously, such as the localization of proteins, the content of FATP1/4 and the specific regulation of lipases involved in IMTG mobilization.
There are several considerations that should be reflected upon when interpreting the findings from the present study. Firstly, care should be applied when extending these findings to the precise molecular regulation of the capacity of an individual to oxidize fat during exercise, given that the total contents of several proteins were measured in basal, rested conditions and a number of days after the measurement of fat oxidation. Exercise evokes a large physiological stimulus at both the whole-body and tissue levels (Hawley et al., 2014), where the importance of molecular mechanisms in regulating PFO might differ, and the time gap between tissue sampling and measurement of fat oxidation could mean that some associations were missed. However, in a subsidiary analysis of individuals with the smallest time gap between biopsies and measurement of fat oxidation, no new correlations were revealed. Secondly, the cross-sectional design prevents causality from being inferred, that is, that a greater content of skeletal muscle CPT1B or PNPLA2 (ATGL) leads to a higher PFO. Furthermore, the heterogeneity of the subject population recruited might have impacted the associations reported, particularly given that training status might F I G U R E 4 Sex comparisons, from the whole sample, of the skeletal muscle protein content of oestrogen receptor alpha (Erα; a); fatty acid binding protein plasma membrane (FABP 3 (FABPpm); b); adipose triglyceride lipase (PNPLA2 (ATGL); c); long chain acyl-CoA synthase 1 (ACSL1; d); carnitine palmitoyltransferase 1B (CPT1B; e) and a representative immunoblot for each protein of interest (f). Symbols with the same colour represent the same participant, in order to highlight participants who displayed the highest values for any given protein and their respective values across other proteins. Data for CPT1B and PNPLA2 (ATGL) are presented as the mean rank (Mann-Whitney U test); data for ESR1 (ERα) are presented as the median and interquartile range (Mann-Whitney U test on median); and data for FABP 3 (FABPpm) and ACSL1 are the mean ± SD (Student's unpaired t test) moderate the molecular regulation of fatty acid oxidation (Dandanell et al., 2018;Kiens et al., 1993;Shaw et al., 2020). The quantification of ABHD5 (CGI-58) and PLIN1 was surprisingly difficult given their abundant expression in adipose tissue, where western blot analyses on 3T3-L3 adipocytes confirmed the identification of these bands for quantification.
Finally, the relatively small sample size (especially in some of the subgroup analyses) has the potential to lead to an increased false-negative rate, and the study might be underpowered for some outcomes. Nevertheless, the sex differences in means/medians were small, and thus even if these were underpowered to detect differences statistically, the magnitude of these differences is likely to be small.
To overcome the limitations of the present study, in future studies it might be preferable to take skeletal muscle and adipose tissue biopsies immediately after participants have been exercising at their FAT MAX intensity, in order to assess the activation status of proteins involved in lipid metabolism and of mitochondrial enzymes. In addition, the use of stable isotope methods would allow for greater tissue-specific insight into lipid metabolism in vivo by enabling characterization of adipose tissue lipolysis rates and the contribution of fat oxidation from plasma and muscle-based sources.

Conclusion
In the present study, we assessed associations between PFO and the resting content of key proteins involved in fatty acid metabolism in adipose tissue and skeletal muscle. The data demonstrate that factors involved in regulation of IMTG lipolysis (PNPLA2 (ATGL)) and fatty acid entry into the mitochondria (CPT1) were consistently associated with PFO, whereas no such relationships were observed for proteins measured in adipose tissue. This suggests that skeletal muscle has a more important role than adipose tissue in the capacity of an individual to oxidize fat during exercise. Although females consistently displayed increased basal adipose tissue lipolysis rates, no sex differences were found in the content of any of the measured proteins involved in lipid F I G U R E 5 Sex comparisons of the protein content of oestrogen receptor alpha (Erα; a); perilipin-1 (PLIN1; b); comparative gene identification-58 (ABHD5 (CGI-58); c); adipose triglyceride lipase (PNPLA2 (ATGL); d); hormone-sensitive lipase (LIPE (HSL); e); long chain acyl-CoA synthase 1 (ACSL1; f); and ex vivo basal lipolysis rates (g), in the subgroup matched for cardiorespiratory fitness, physical activity levels and fat mass index classifications. Symbols with the same colour represent the same participant, in order to highlight participants who displayed the highest values for any given protein and their respective values across other proteins. All data are presented as the median and interquartile range (Mann-Whitney U test on medians), except for ABHD5 (CGI-58), which are the mean ± SD (Student's unpaired t test). n = 7 females and n = 7 males, except for PLIN1 (n = 7 females, n = 5 males), ABHD5 (CGI-58) (n = 5 females, n = 5 males) and ex vivo basal lipolysis rates (n = 7 females, n = 4 males) metabolism within adipose tissue or skeletal muscle. This suggests that higher relative rates of fat oxidation during exercise in females than in males are unlikely to be attributable to a higher abundance of key proteins within adipose tissue or skeletal muscle determined in the resting state.

ACKNOWLEDGEMENTS
All participants are greatly thanked for graciously volunteering their time and effort to partake in the study. We would like to thank Andrea ; adipose triglyceride lipase (PNPLA2 (ATGL); c); long chain acyl-CoA synthase 1 (ACSL1; d); and carnitine palmitoyltransferase 1B (CPT1B; e) in the subgroup matched for cardiorespiratory fitness, physical activity levels and fat mass index classifications. Symbols with the same colour represent the same participant, in order to highlight participants who displayed the highest values for any given protein and their respective values across other proteins. All data are presented as the median and interquartile range (Mann-Whitney U test on medians), except for FABP 3 (FABPpm), which are the mean ± SD (Student's unpaired t test). n = 6 females and n = 6 males, except for ESR1 (ERα) (n = 3 females, n = 5 males) authors qualify for authorship, and all those who qualify for authorship are listed.

DATA AVAILABILITY STATEMENT
The data underpinning this work is available from the University of