Sex-specific adipose tissue’s dynamic role in metabolic and inflammatory response following peripheral nerve injury

Summary Epidemiological data and research highlight increased neuropathy and chronic pain prevalence among females, spanning metabolic and normometabolic contexts, including murine models. Prior findings demonstrated diverse immune and neuroimmune responses between genders in neuropathic pain (NeP), alongside distinct protein expression in sciatic nerves. This study unveils adipose tissue’s (AT) role in sex-specific NeP responses after peripheral nerve injury. Metabolic assessments, metabolomics, energy expenditure evaluations, AT proteomic analyses, and adipokine mobilization depict distinct AT reactions to nerve damage. Females exhibit altered lipolysis, fatty acid oxidation, heightened energy expenditure, and augmented steroids secretion affecting glucose and insulin metabolism. Conversely, male neuropathy prompts glycolysis, reduced energy expenditure, and lowered unsaturated fatty acid levels. Males’ AT promotes regenerative molecules, oxidative stress defense, and stimulates peroxisome proliferator-activated receptors (PPAR-γ) and adiponectin. This study underscores AT’s pivotal role in regulating gender-specific inflammatory and metabolic responses to nerve injuries, shedding light on female NeP susceptibility determinants.

Since SCs are the main players in the first days of Wallerian degeneration (WD) and are highly sensitive to metabolic alterations, 16 we investigated some metabolic parameters (glycemia, insulin, glucagon and triglycerides serum levels) after peripheral nerve injury in male and female CD1 mice subjected to chronic constriction injury 17 (CCI), which induces WD and allodynia in the ipsilateral hindpaw ( Figures 1A and 1B) (all statistical analyses are reported in figures legend).
We found sex-differences in baseline (BL) condition and time-and sex-dependent changes in metabolic rates after the induction of NeP (in vivo time-points: BL, 6h, 24h, day 3 -D3 and D7; ex vivo: BL, 6h, D7). Neither body weight nor food intake were affected by neuropathy ( Figure S1).
Sex-dependent differences in glycemia and insulin levels were observed in BL condition and after CCI. After CCI, a reduction in glucose levels in all time-points was observed in males, while in females after an initial decrease at 24h a significant enhancement in comparison with BL condition was measured at D7. An early (6h) upregulation of insulin was found in both sexes, restoring later to BL value at D7 in males while maintaining a bit higher level in females. Although no sex differences were observed in BL condition, glucagon and triglycerides (TGs) were modulated by sex and by the induction of CCI. Upon CCI and NeP development, glucagon was significantly decreased in females at 6h and D7, while in males it was first upregulated (6h) and finally downregulated (D7). Likewise, serum TGs changes were sex-dependent and strongly dependent on the effects induced by the CCI: TGs were upregulated at 24h and downregulated at D3 in male mice, while they were always downregulated in females.
(D) s-PLDA and Loading plot based on whole blood amino acid (AA) and acylcarnitine (AC) profiling of female and male mince before (NAIVE) and after (D7) injury. Histograms show levels of AAs and ACCs expressed as mmol/L N = 10-12/group (*)p < 0.05; (**)p < 0.01; (***)p < 0.001 vs. female; ( B )p < 0,05; ( BB )p < 0,01; ( BBB ) p < 0.001 vs. BL. (E) Indirect calorimetry analysis. The left panel shows energy expenditure (EE) kcal/h/kg in male and female mice at BL and D7 after CCI. One-way ANOVA significant main effect for sex/condition (F 3,504 = 25.36; p < 0.0001) and Tukey-Kramer test post-hoc analysis. The middle panel shows resting energy expenditure (REE) kcal/h/kg at BL and D7 after CCI. One-way ANOVA significant main effect for sex/condition (F 3,238 = 46.62; p < 0.0001) and Tukey-Kramer test post-hoc analysis. The right panel shows respiratory exchange ratio (RER) VCO2/VO2 in male and female mice at BL and D7 after CCI. One-way ANOVA significant main effect for sex/condition (F 3,504 = 882.9; p < 0.0001) and Tukey-Kramer test post-hoc analysis; (**) p < 0.01; (***)p < 0.001. iScience Article Sex-differences are recognized as a crucial factor in energy metabolism and homeostasis of amino acids and fatty acids. 19 We previously showed 15 that nerve injury induced several metabolic alterations in adult male mice, suggesting an increase in fatty acids oxidation to cope with the rise of energy need. Thus, targeted profiling of blood amino acids (AAs) and acylcarnitines (ACCs) was performed (Tables S1-S3) to highlight sex differences in the metabolic response to nerve insult. Blood AAs and ACCs profiling were analyzed in male and female mice, showing a good ability to cluster mice before and after injury ( Figure 1D).
Fatty acids and ACCs mobilization mirror the energy fuel required and, to better understand whole-body energy metabolism and sexdependent differences before and after CCI, we performed an indirect calorimetry (IC) analysis, thus assessing in vivo energy expenditure (EE), resting EE (i.e., in lack of motor activity) (REE) and substrate oxidation (respiratory exchange ratio, RER) ( Figure 1E).
The measure of EE, after 48-h continuous recording in BL condition, revealed sex-associated differences in energy metabolism. Female mice showed higher levels of both EE and REE at BL while exhibited lower RER.
Moreover, as compared to BL, peripheral nerve injury at D7 induced a decrease of EE and REE in both sexes. However, the reduction of EE and REE was more pronounced in male than in female mice. As for the RER, female mice showed a lower level at BL that was further decreased after CCI. By contrast, the RER level was increased at D7 in male mice.
From these data emerge that EE and REE are decreased by peripheral nerve damage in both sexes but also that whole-body EE and REE resulted always higher in female animals. The main fuel use appears different between males and females, with an increase of mixed protein/ fatty acids oxidation in female mice followed by a switch toward a prevalent increase of fatty acids oxidation after injury. Thus, response to nerve damage can change glucose and fat metabolism, and, in females, enhance EE, in which energy supply seems to rely mainly on the increase of protein and lipid oxidation. To exclude other effects affecting energy metabolism, we evaluated the basal body temperature (BT) and adaptive thermoregulation after exposure to cold temperature. As reported ( Figure S3), no significant differences were revealed.

Role of adipose tissue in orchestrating metabolic response to neuropathy
The secretory activity of adipose tissue (AT), and its involvement in the inter-organ communication has been repeatedly recognized in the last years. 20 AT is an endocrine organ that is able to regulate glucose, lipids and energy homeostasis, through the secretion of signaling mediators such as adipokines (e.g., leptin and adiponectin), metabolites, fatty acids and steroids. The most part of these investigations were focused on the role of AT in metabolic dysfunction such as in insulin resistance (IR), obesity and diabetes; nothing is known about its potential pathophysiological role in the onset and development of NeP in normo-metabolic subjects.
We previously 21 showed that leptin was strongly upregulated in sciatic nerve from female mice subjected to peripheral nerve injury. ATreleased leptin acts as pleiotropic hormone 22 in energy metabolism, being involved in the regulation of several homeostatic functions.
To understand whether white adipose tissue (WAT) has an active role in the peripheral neurodegeneration, inflammatory processes, and in sex-dependent metabolic alterations, we performed an analysis of WAT proteomics (Proteomics Data are available via ProteomeXchange with identifier PXD030391). Hence, we made a comparative analysis of the differentially expressed proteins (DEPs) in WAT before (BL) and seven days (D7) after sciatic nerve injury by means of LC-MS based shotgun proteomics. 23,24 This experiment allowed us the overall quantification of 525 proteins in the four groups of mice (F_BL; F_D7; M_BL; M_D7). From 525 proteins we filtered out 291 DEPs with a maximum fold change (MFC) of the protein expression levels that was set as MFC R1.5 in statistically significant observation (ANOVA p value %0.05) (Table S3); details on analysis are reported in supplementary material.
A heatmap representation (Figure 2A) of the dataset based on protein recurrence illustrates the different expression regulation between females and males, which results of particular relevance at D7 post-injury.
DEPs protein dataset was used to perform gene ontology enrichment analysis with WebGestalt toolkit 25 (https://www.webgestalt.org/) and pathway enrichment analysis on the Reactome database of pathways and reaction (https://reactome.org/). 26 Figure S4 shows the enrichment of the complete dataset among the gene ontology categories Biological Process, Cellular Component, and Molecular Function. Interestingly, in the Biological Process bar graph, it is shown that the majority of the protein of this dataset is included iScience Article in the subcategories biological regulation and metabolic process. More in detail, in Figure 2B, a directed acyclic graph (DAG), shows that different small molecules, metabolic process and oxidative reduction process are enriched in the collected dataset (Table S4).
To further study the involvement of WAT in the metabolic changes after nerve injury, including the GO overrepresentation analysis, we compared males and females at baseline (BL) and seven days after injury (D7), and finally matched males and females at D7. In this last comparison, male mice were considered as the reference group since they showed a complete functional recovery, not observed in female mice (Table S5 resume DEPs relative to each comparison). Volcano scatterplots in Figure S5 allows a quick visual identification of proteins with large fold changes that are also statistically significant in each comparison.
From Reactome Pathway database expression analysis emerges that both in males and females, the proteins upregulated at D7 group are involved in pathways such as glycolysis, gluconeogenesis, creatine metabolism or metabolism of carbohydrates that, being essential metabolic pathways, are able to match the energy requirement (e.g., ATP production and metabolite precursor) for the biosynthesis of small molecules and macromolecules ( Figure 2C).
These pathways are also enriched from DEPs in females versus males at D7 after CCI, as well as in other pathways involved in insulin effect on the increase of the small molecules formed during glycolysis and pathways involved in the insulin growth factor (IGF) and Insulin-like growth factor-binding proteins (IGFBPs). A list of the Top 20 ranking pathways enriched for each analysis is shown in Table S6.
Furthermore, if we compare males and females after CCI or females before and after CCI, we observe that WAT of female mice changed after nerve injury with the production of sex hormones and steroids, as revealed by the high presence of proteins such as Steroid receptor RNA activator 1, 20-alpha-hydroxysteroid-dehydrogenase (Akr1c18), estradiol17beta-dehydrogenase5 (Akr1c6) and 3beta-hydroxysteroiddehydrogenase (hsd3b1 and hsd3b6) (pink letter in Tables S5 and S6), while males utilized antioxidant, tissue repair, protection and suppressors of endoplasmic reticulum (ER) stress proteins (i.e., Alpha-1-Microglobulin/Bikunin Precursor -AMBP-, Serpina 1day, 1a, 1b) (blue letter in Tables S5 and S6).
To validate the idea that WAT is involved in the response to peripheral nerve damage, we measured the expression of Peroxisome proliferator-activated receptor gamma (PPARg), widely diffused in AT where regulates fatty acid storage, glucose metabolism 27 and the levels of adiponectin, which is an adipokine involved in the regulation of insulin signaling and inflammatory processes 28 ( Figure 2D). The analysis of adiponectin and PPARg revealed that adiponectin was upregulated after nerve damage only in males with a parallel increase of PPARg expression. Of note, adiponectin can influence insulin, glucagon, glucose metabolism, TGs and free-fatty acids concentrations, while PPARg can change accordingly to medium chain ACCs, being involved in antioxidant activity as well as in peripheral nerve injury and diabetic neuropathy.

DISCUSSION
In the last years, 29 AT-derived signaling have been recognized to control and influence other organs and participate to inter-organ communication. AT can be regarded as an endocrine organ regulating glucose and lipid metabolism as well as energy homeostasis throughout the secretion of multiple adipokines such as leptin, fatty acids and steroids. 20,22,[28][29][30] In spite of this evidence and the literature focused on metabolic disorders in which AT is recognized to play a primary role in the activation of inflammatory events and neurological disorders, there is no knowledge available regarding the potential pro-active role of AT in NeP development. In seeking for an explanation about why females are more pain sensitive and susceptible to NeP, recent studies 3,6,21 have focused on neuroinflammatory, inflammatory and immune response in female and male mice after peripheral nerve injury, suggesting a specific influence of sex-hormones.
Despite the leading hypothesis for the higher female susceptibility to pain takes mostly into account the different production of endogenous sex hormones, 6,7,31 the literature is still controversial. Indeed, we previously shown 10 a decrease in both sexes of 17-b-estradiol levels after nerve injury, while estrogen receptors were upregulated after sciatic nerve lesion. Moreover, several experimental investigations, [32][33][34][35][36] including ours, 5,10 demonstrated that exogenous estrogen prevents neuropathy and pain chronicization and that estrus cycle does not interfere with NeP development. Thus, estrogens appear to protect against chronic neuropathy even though estrogens secretion in females do not seem to confer a higher protection than observed in males, especially considering the major vulnerability to chronic pain observed in the female sex. However, not only ovaries and adrenal glands but also adipocytes are responsible for production of sex hormones in females, and their synthesis from adipocytes can affect whole metabolism, as shown for insulin metabolism. 30,37 On one hand, sex-steroids appear as a possible cause of the sexual dimorphic response to NeP development and could account for the different neuroinflammatory, inflammatory and immune response found between males and females. On the other hand, no evidence emerged linking the direct action of these hormones to the mechanisms underlying the neuropathy onset in order to account for the processes responsible of the sex-dependent neuropathic response.
Sex steroids are synthesized from lipids and one source is the AT, which is an endocrine organ having an active role in the control of inflammatory processes and a master regulator of metabolism.
Starting from the idea that neurological damage causes important metabolic changes and a high increase of energy requirements, 15,38 we asked if AT could be involved in these mechanisms in a sex-dependent manner.
Here, we provide evidence for sex-associated metabolic alterations in response to NeP. Moreover, we demonstrated that neuropathy in female mice produced changes in lipolysis and fatty acids oxidation (FAO), as well as an enhancement of whole-body energy expenditure and higher secretion of sex hormones from AT, affecting glucose and insulin metabolism. These data could explain the early anti-inflammatory response and the different immunological profile found in females. By contrast, males showed marked alteration of glycolysis and a decrease of systemic energy expenditure and unsaturated fatty-acids levels. In males, the AT response elicited the secretion of factors involved in regenerative process and in oxidative stress, as well as in the stimulation of peroxisome proliferator activated receptors (PPARs) gamma subtype (PPAR-g) and adiponectin, which are important for pain management and inflammation. 39,40 From this analysis emerges that nerve injury elicited in female mice selective changes of the AT sex-hormones (as showed by AT proteomics), revealing a strong up-regulation of enzymes that catalyze for different steroids, together with leptin upregulation as previously demonstrated, 6 while no changes were detected for adiponectin and PPAR-g expression.
It is therefore possible that AT-dependent secretion of leptin and AT sex hormones composition in female mice subjected to nerve injury, might explain not only the inhibition of inflammatory process and immunomodulation but also partly account for the metabolic changes triggered by peripheral neuropathy. It should be reminded that sex steroids affect carbohydrates and liver metabolism, 41 increasing glycemia and insulin release but decreasing muscle sensitivity to insulin. 42 Being part of the carnitine pool, the ACCs are mitochondria-and peroxisomes-derived intermediate oxidative metabolites consisting in fatty acids (C2-C26) esterified to a carnitine moiety to facilitate their carrying across the mitochondrial membrane. 43,44 For this reason, circulating ACCs can be considered indirect markers of mitochondrial function and fatty acid oxidation (FAO) that can mirror FAO defects or metabolic disorders. 45 Seven days after nerve injury short-chain ACCs, such as C5, were found reduced in female mice, while circulating medium-chain C12 and long-chain C14:1 and C14 C18 were found increased.
In the last years, several studies have helped to disclose additional mechanisms underlying IR in which IR incidence has been linked to the increase of catabolism of branched-chain amino acids (BCAA) and to the accumulation of intermediary metabolites of FAO that may interfere with insulin sensitivity, and in particular with increased plasma levels of C3 and C5 ACCs. 46,47 Interestingly, plasma insulin levels were found reduced at D7 in female mice as compared to male mice that underwent to the same experimental procedure of peripheral nerve injury. Moreover, reduced plasma levels of C5 ACCs in female mice might be indicative of energy production relying for the most part on the increase of lipid oxidation, as further corroborated by the decrease at D7 of whole-body RER (VCO2/VO2), thus revealing an increased ratio of fat-to-carbohydrate utilization. The parallel increase of medium-chain C12 and long-chain C14:1 and C14 ACCs may further suggest the presence of incomplete fatty-acid b-oxidation and therefore increase of energy production/expenditure. Within this context, a decrease of circulating TGs was observed at D7 after nerve injury in female mice that may suggest an increase of FAO. As for the increase of wholebody lipid oxidation (i.e., RER reduction), the IC analysis also disclosed an increase of energy expenditure (EE = Kcal/Kg) in female mice as compared to male mice at D7 after nerve lesion.
This complex metabolic mechanism, having in AT a key player, could explain some early events observed in female mice subjected to experimental neuropathy (i.e., macrophages/microglia and myelin proteins strongly influenced by insulin, glucose metabolism and steroids), in which AT-mediated response appear to interfere with the physiological progression of WD and facilitate the development of chronic NeP, thus underlying a sex-dependent impact of AT on metabolic and inflammatory response after nerve injury.
In male mice subjected to peripheral nerve injury, we observed some AT-dependent changes that activate the glycolytic and pyruvate pathway (as shown by proteomics analysis), as well as an upregulation of both PPAR-g and adiponectin in AT. PPAR-g activation is reported to normalize insulin level 48 as well as to exert a direct action on macrophages. 49  iScience Article nonesterified fatty acids, decreasing glucose plasma levels, TGs and concentration of free fatty acids. 51,52 Low adiponectin levels can contribute to diabetic neuropathy, 53 and the lack of adiponectin exacerbates thermal nociception in mice. 54 Our data show a marked sexual dimorphism in PPAR-g expression and higher adiponectin activation that might account for the lower allodynic response found in male mice, which exhibited a progressive decrease of sensitivity to mechanical stimulation up to a complete remission from neuropathy. For both sexes the metabolic changes observed can be, at least in part, explained as consequence of AT mobilization in response to nerve injury. After peripheral nerve lesion, male mice showed a rapid (6h) insulin and glucagon increase, together with the decrease of glycemia levels. Moreover, TGs levels followed a biphasic trend in which the initial increase (24h) was followed by a subsequent decrease (D3).
A rapid increase of insulin may have beneficial effects on macrophages activation through the upregulation of PPAR-g 49 that promotes an anti-inflammatory phenotype as well as the stimulation of phagocytic activity taking advantage of low glucose levels. 55 Finally, insulin exerts a neurotrophic action (through also IRS1) on sciatic nerve stimulating remyelination processes 56,57 while glucose levels are essential for the phenotype of SCs. 58,59 By contrast with female mice, both medium-and long-chain AACs were found decreased after nerve lesion (D7) in male mice. The differing profile of medium-chain AACs plasma levels is indicative of an opposite use of the main oxidative substrate after nerve lesion among the two sexes. Indeed, while RER was decreased in female mice with regard to baseline and was lower than RER scores showed by male mice after nerve lesion, RER was conversely increased in male mice in comparison to baseline. An increase of RER is indicative of a decreasing rate of lipid oxidation as main fuel and, therefore, indicative of a decreased ratio of fat-to-carbohydrate utilization. An increase of insulin sensitivity has been found associated with a decrease of long-chain AACs after glucose loading, while no decrease was detected in diabetic rats. 60 Moreover, the proteomic analysis of AT composition discloses the upregulation of glycolysis and pyruvate pathways observed after CCI in male mice. In line with a decrease of medium-and long-chain AACs, an increase of glucose uptake by AT may be viewed as an index of insulin sensitivity. 61 In other words, an upregulation of the glycolysis pathway in AT appears to be a downstream effect of the insulin-dependent glucose uptake in AT upon glucose transporter 4 (GLUT4) translocation. Both increase of glycolysis and expression of PPAR-g after nerve injury in male mice should facilitate fat storage in adipocytes and energy sparing as suggested by the decrease of energy expenditure found at D7, which was reduced as compared to both baseline heat (Kcal/Kg) and heat emitted at the same time point by female animals.
Our data strongly suggests that AT can play a pivotal role in inter-organ communication and in the regulation of metabolic response after nerve injury, since peripheral nerves are highly sensitive to glucose, lipids and hormonal changes.
In summary, we demonstrated that peripheral nerve injury can trigger a metabolic stress that induces AT activation which plays a fundamental role in the orchestration of the sex-dependent response to NeP. These data may help to find a missing piece in the puzzle of pain gender gap. A possible new scenario for NeP therapy might be introduced by considering AT and AT-associated targets as new players for the development of gender-specific medicine.

Limitations of the study
This study aims to uncover novel contributors to the generation of gender-based disparities in neuropathy and the initiation, progression, and recovery of pain. This endeavor seeks to consolidate insights into the mechanisms underpinning the heightened susceptibility and prevalence of neuropathy in females. Our findings unveil AT's dynamic engagement in a sex-dependent manner within the metabolic response following nerve injury and NeP induction. This active involvement leads to alterations in metabolic rates and the release of both pro-and anti-inflammatory agents that modulate various stages of WD. These revelations introduce a prospective therapeutic landscape for addressing peripheral neural damage, albeit necessitating further exploration to identify potential targets. Given the utilization of an animal model in this research, clinical investigations are imperative to validate these gender-based disparities in AT's proactive role in neuropathy onset and perpetuation. Moreover, comprehensive exploration is warranted to elucidate the triggers initiating the AT's responsive behaviors.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
We, the authors, declare no conflict of interest.

INCLUSION AND DIVERSITY
We support inclusive, diverse, and equitable conduct of research.