Novel assessment of post-prandial metabolism reveals sex-specific metabolic flexibility and lipid remodeling following volumetric muscle loss

Abstract

Localized skeletal muscle injury (e.g., volumetric muscle loss; VML) can disrupt diurnal metabolic flexibility. However, it remains unclear how the evaluation of whole-body metabolism and physical activity following a prandial stimuli may reveal differences in metabolic flexibility between injured and uninjured states. This study aimed to develop a novel tool to examine whole-body metabolic impairments following VML injury, with consideration of lipid-related mechanisms. Adult C57Bl/6J mice (n=50; equal males and females) were randomized to a methodology development cohort; to undergo VML injury, intramuscular glycerol injection, or remain injury naïve controls. The developed tool using an i.p. glucose injection and indirect calorimetry was used to characterize the dynamic nature of whole-body metabolism. Whole-body metabolism, in vivo muscle function, and markers of lipid and glycemic regulation were evaluated 6 weeks following injury. Females, regardless of injury, exhibited greater daily energy expenditure alongside increases in activity. Females exhibit lower whole-body lipid oxidation during the inactive period, despite higher in active period, suggesting more coordinated substrate utilization. Biologic sex differences in post-prandial substrate utilization, reveal males fail to suppress whole-body lipid oxidation; exhibiting marked impairments in post-prandial metabolic flexibility. VML injury increases protein expression of perilipin 2 and SIRT1 in the muscle remaining. While inducing sex-specific changes, with ATGL expression markedly increased and perilipin 5 expression is reduced in females. The remaining muscle following VML accumulates neutral lipids and perilipin 1-positive adipocytes. This work highlights sex-specific mechanisms of metabolic disruption following traumatic skeletal muscle injuries, such as VML.

Graphical Abstract

New & Noteworthy:

We developed a novel, freely ambulatory pre-clinical tool to assess dynamic post-prandial metabolic flexibility. Utilizing this tool, we identified sex differences in whole-body energy expenditure and substrate utilization. The use of a single intramuscular glycerol injection model is not sufficient as a comparative model for evaluating chronic ectopic lipid accumulation and metabolic disruptions. In the context of volumetric muscle loss injury, disruptions in metabolic flexibility occur alongside alterations in lipid-handling and ectopic lipid accumulation.

Introduction

Metabolic flexibility is characterized by the ability to efficiently shift between fat and carbohydrate oxidation when faced with various physiological stimuli, and changes in energy supply and/or demand (e.g., physical activity and/or meals) (1). Skeletal muscle is a key contributor to metabolic flexibility, as it plays a critical role in glycemic regulation and triglyceride clearance, with fuel selection shifting in response to varying physical activity intensities. Evaluation of impairments in whole-body metabolism can be understood by examining pathologies that compromise skeletal muscle capacity. Skeletal muscle capacity is broadly defined by the plasticity of skeletal muscle and its ability to adapt to physiological cues, which is foundational for metabolic flexibility. Metabolic inflexibility, in contrast, is marked by impaired substrate (i.e., carbohydrate and fat) switching and is commonly associated with insulin resistance (2), ectopic fat accumulation (3, 4), and mitochondrial dysfunction (5, 6). The loss of cellular metabolic flexibility compounds to impair whole-body metabolism, and is associated with cardiometabolic disease (e.g., type 2 diabetes) development. Recent investigations of whole-body skeletal muscle-related conditions, such as sarcopenia (7) and cachexia (8) reveal disruptions in systemic and cellular metabolic flexibility. Similarly, instances of prolonged skeletal muscle disuse (i.e., physical inactivity or bedrest), which may occur following injury, disrupt mitochondrial respiration and subsequent substrate utilization (9, 10). It currently remains unclear how a localized skeletal muscle injury (e.g., volumetric muscle loss – VML), which exhibits dynamic and temporal fluctuations in energy metabolism (11, 12) responds to a physiological stimulus that encompasses whole-body metabolic flexibility. Understanding metabolic flexibility in the context of a localized skeletal muscle injury could provide insight into the intersection between injury, repair and regeneration, and chronic cardiometabolic disease risk.

Pre-clinical evaluation of whole-body metabolic flexibility has focused on substrate utilization across a 24-hr period with emphasis on diurnal rhythms between 12-hr photoperiods. Disruption in the oscillations between diurnal periods is a hallmark of metabolic disease and often emerges before overt disease develops, marking an early indicator of impaired metabolic regulation (13, 14). Further, evaluation of post-prandial absorptive and post-absorptive states provide insight into glucose disposal, a timely insulin response, and substrate preference. During times of metabolic inflexibility, persistent lipid oxidation occurs despite a transition in energetic demand (e.g., post-prandially), indicating a failure to appropriately adjust substrate preference in response to physiological cues. Clinical evaluation of whole-body metabolic flexibility post-prandially can be accomplished with whole-room calorimetry measurements, while measurements at rest are commonly assessed using a metabolic cart and ventilated hood or mask-based systems (i.e., indirect calorimetry). However, it is notable that simple indirect calorimetry alone is unable to capture the dynamic metabolic response that occurs post-prandially; limiting the ability to reveal early impairments in metabolic flexibility often associated with disease risk. Assessment of pre-clinical metabolic flexibility in vivo has been limited to diurnal and non-ambulatory post-prandial evaluation (i.e., hyperinsulinemic-euglycemic clamp; HIEC) (15) and novel pre-clinical tools to evaluate post-prandial metabolic flexibility in a free ambulatory environment are needed. Developing such tools is critical to enable translational research that bridges mechanistic understanding with clinical metabolic phenotypes, ultimately to improve the assessment and treatment of metabolic disorders.

Localized skeletal muscle injuries, including VML, have the capacity to perturb whole-body metabolism, despite cellular disruption confined to the remaining injured muscle. An injury model such as an intramuscular cardiotoxin injection, which is marked by impaired tissue remodeling and persistent inflammation, is likely to disrupt overall energy homeostasis and thus, whole-body metabolism (16). Immediate functional and structural mitochondrial impairments characterize the pathophysiology of VML injuries (17, 18) and subsequent disruptions to whole-body metabolism following VML, including limitations in diurnal substrate preference, become apparent at chronic time points (e.g., ~6 weeks) (11, 19). Suggesting that continued disruption in β-oxidation following VML could result in ectopic fat accumulation and alter lipid signaling (e.g., perilipins, lipases), further driving whole-body metabolic dysfunction. However, ectopic fat accumulation in skeletal muscle following VML has only recently been recognized (20, 21), though it has been noted anecdotally without quantification (22-24). An injury model utilizing an intramuscular glycerol injection (25-27), which induces ectopic fat accumulation, presents an additional model by which disruption to cellular metabolism leads to delayed tissue repair and regeneration. However, characterization of the intramuscular glycerol injection model has been limited to early, site-specific effects on muscle regeneration, failing to capture its broader influence on whole-body metabolic consequences; providing a novel comparative model to VML injury. The demonstrated ectopic fat accumulation following intramuscular glycerol injection can aid in framing VML pathology as part of a broader skeletal muscle injury continuum. It is posited that a localized skeletal muscle pathology with impairments in cellular metabolism may subsequently disrupt systemic metabolic flexibility. Thus, the primary aim of this study was to interrogate potential mechanisms driving whole-body metabolic disruptions after localized skeletal muscle injuries, such as VML and intramuscular glycerol injection, with particular interest in the role of lipids. Whole-body metabolism (e.g., energy expenditure, substrate oxidation rates) were evaluated alongside circulating (e.g., free fatty acids) and localized (e.g., perilipin 2) lipid metabolism-related factors. This work further aimed to develop a pre-clinical tool to evaluate post-prandial whole-body metabolism.

Methods and Materials

Ethical approval

All protocols were approved by the Institutional Animal Care and Use Committee at the University of Minnesota (2107-39253A) in compliance with the Animal Welfare Act, the Implementing Animal Welfare Regulations and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals.

Experimental design

Adult C57Bl/6J (n=50; equal numbers male and female) mice were purchased from Jackson Laboratory (#000664, Bar Harbor, ME, USA; RRID:IMSR_JAX:000664) at 11 weeks of age. Mice were provided with at least 1 week acclimation prior to initiating any part of the study. Mice were given ad libitum access to chow and water and were housed on a 12h light-dark cycle, with weekly evaluation of bodyweight for the duration of the study.

An initial study was designed to assist in the development of methodology to quantify metabolic flexibility in response to a prandial stimulus. A subset of control naïve mice (n=8; equal numbers male and female) were used in a repeated measures cross-over study over 6 weeks (Fig. 1A), beginning at 11 weeks of age. Evaluation of post-prandial, whole-body metabolism occurred following glucose injections at 12-, 14-, and 16-weeks of age and saline injections at 13-and 17-weeks of age. Mice were euthanized with pentobarbital (> 100mg/kg; s.q.) at 18-weeks of age.

Figure 1.

A) Schematic of cross over study design and B) average longitudinal body weights by weeks of age between male and female mice. C) Fluctuations in RER plotted across ~240 minutes, collected every 5 minutes following saline or glucose injection averaged across all mice within each group. D) RER area under the curve (AUC) following injection (main effect stimuli p=0.918; sex p=0.869; interaction p=0.829). E) Average RER 60-mins prior to injection (main effect stimuli p=0.202; sex p=0.809; interaction p=0.176). F) Peak RER following injection (main effect stimuli p<0.001; sex p=0.459; interaction p=0.936). G) Nadir RER following injection (main effect stimuli p=0.360; sex p=0.594; interaction p=0.044). H) Delta RER (Δ RER) from injection to peak RER following injection (main effect stimuli p=0.004; sex p=0.666; interaction p=0.793). I) Δ RER from peak to nadir RER following injection (main effect stimuli p=0.003; sex p=0.334; interaction p=0.273). J) Fluctuations in RER for all individual mice plotted across ~240 minutes, collected every 5 minutes following saline or glucose injection. All scales are the same with the dashed line representing the injection point (see C). Red data points represent the peak and nadir, respectively (see F and G). Dots represent an individual animal. Data analyzed by two-way ANOVA; statistically significant Main Effects are noted.

In a second study, male and female mice at ~12 weeks of age were randomly assigned to a VML injury to the posterior compartment of the hindlimb or intramuscular glycerol injection to the gastrocnemius muscle. An additional experimental group remained injury naïve and were age- and sex-matched controls (n=10/group; equal numbers male and female), a subset of mice (n=4; equal numbers male and female) within the same experimental groups were designated for histological analysis. Glucose uptake was evaluated at 17 weeks of age. Diurnal and post-prandial whole-body metabolism was assessed at 18 weeks of age. Terminally, at 6-weeks following injury (~18 weeks of age), in vivo muscle function was assessed and gastrocnemius muscles, gonadal adipose tissue and blood were collected and saved for later analyses. Mice were euthanized with pentobarbital (> 100mg/kg; s.q.).

Skeletal muscle injuries by intramuscular glycerol injection and volumetric muscle loss

In a subset of mice, intramuscular glycerol injections were performed in the gastrocnemius muscle of the hindlimb, unilaterally on the left side. A pre-procedural analgesic of buprenorphine SR (1mg/kg, s.q.) was administered ~2 hours prior to the procedure for analgesia and mice were anesthetized by isoflurane inhalation (~2.0%) during the procedures. Using a 50μl Gastight Neuros Syringe (Hamilton Cat# 65460-16), three 25μl injections of 50% (v/v) glycerol in sterile saline (Teknova Cat# G1798) were administered into the gastrocnemius muscle – one at the mid-belly and two at proximal sites – totaling 75 μl. In a separate subset of mice, a full thickness VML injury was created surgically to the posterior compartment of the hindlimb, including the gastrocnemius, soleus, and plantaris muscle. Mice received buprenorphine SR (1mg/kg, s.q.) approximately ~2 hours prior to surgery for analgesia. Mice were anesthetized by isoflurane inhalation (~2.0%) and prepped under aseptic surgical conditions. As previously described (28) a posterior-lateral incision was created to expose the gastrocnemius muscle. After blunt dissection to isolate the posterior muscle compartment, a metal plate was inserted between the tibia and the deep aspect of the soleus. A 4-mm punch biopsy was performed on the middle third of the muscle compartment with any bleeding stopped with light pressure. Tissue removed was weighed and recorded (overall 20.1±3.2mg; males 21.4±3.8mg; females 18.9±2.2mg). The skin incision was closed with 6-0 suture. Animals were monitored throughout acute recovery and twice daily for 72-hrs following both procedures.

Evaluation of whole-body metabolism and metabolic flexibility

Whole-body metabolism and metabolic flexibility were assessed using Comprehensive Laboratory Animal Monitoring System (CLAMS; Columbus Instruments) and the data examination tool (CI-LINK, v1.11.10; Columbus Instruments, Columbus, OH, USA). Mice were acclimated for ~24hrs, then diurnal and post-prandial data was collected over the subsequent 48hrs. Whole-body metabolic data was collected as previously described (11) with a modification in the frequency of data collection points. Specifically, metabolic data was collected in 5-min intervals over a 24-hr period, with a 45s reference period and 15s measurement period, resulting in cage sampling every ~5min. Post-prandial data collection was standardized to time of day and performed at the mid-point between active and inactive photoperiods. The respiratory exchange ratio (RER) was calculated as the volume ratio of O₂ and CO_2. Calorific value (CV) (29) was calculated for use in quantification of energy expenditure as the product of CV and VO₂, which is reported in kilocalorie/hr (i.e., kcal/hr). Carbohydrate and lipid oxidation rates were calculated as recommended by the manufacturer (CLAMS; Columbus Instruments). Infrared X, Y, and Z axis beam breaks on the CLAMS were used as previously described (11) to calculate ambulatory distance in meters. A normalizing factor was calculated for beam breaks and body weight using the following equation: 0.757kcal/1000 beam breaks/g body weight. Next, the energy cost of physical activity (PAEE) was calculated as the sum of X and Y beam breaks * normalizing factor * g body weight (30). All data processing was done using MATLAB (version R2023a, MathWorks, Natick, MA, USA); RER, energy expenditure, carbohydrate and lipid oxidation rates, ambulatory distance, and PAEE averages over 24-hr and 12-hr active and inactive periods, and to evaluate diurnal metabolic flexibility.

The tool to assess metabolic flexibility in response to a prandial stimulus included an administered injection of either D-glucose (Sigma G7201; 2mg/g, i.p.) in sterile saline or sterile saline. Injection volumes were scaled by bodyweight with a range between 20-35ml. Following injection, mice were immediately placed in the CLAMS and monitored for 180-mins, without access to food. The two data points (~5-mins) immediately following the opening of the cage for injections were excluded from analysis to allow the chambers to return to normal pressure. MATLAB was used to calculate RER, with specific quantification of pre-injection, peak, and nadir RER. Pre-injection RER was calculated as an average of 11 data bins (~1hr) prior to the injection. Peak and nadir RER were determined as the first peak RER value and lowest RER value following injection, respectively. Delta outcomes (i.e., Δ RER injection to peak) were calculated as the difference between various post-prandial timepoints. Area under the curve for post-prandial outcomes was determined using the trapezoidal rule to approximate the integral of the curve over time.

Glucose tolerance test

Glucose tolerance testing was performed after ad libitum access to food. Baseline blood glucose was obtained from the lateral tail vein, nicked with a 20-G needle, with a glucometer (Freestyle Lite, Abbott). Following injection of D-glucose (Sigma #G7021; 2 mg/g, i.p.) in sterile saline, glucose measurements were obtained at 15-, 30-, 45-, 60-, 90-, and 120-mins following injection. Mice were continuously monitored, and any additional bleeding was stopped with light pressure. Following testing, mice were returned to home cages with ad libitum access to food and water.

In vivo muscle function

Muscle function of the posterior hindlimb compartment was determined 6-week following injury as previously described (17, 28). Briefly, mice were anesthetized with isoflurane (1-2%) and the left knee and hip were stabilized with a knee clamp at 90°. The left foot was attached to a footplate of a dual-mode muscle lever system (300C-LR; Aurora Scientific, Aurora, Ontario, Canada). The common peroneal nerve was visualized and severed to avoid stimulation of the anterior compartment muscles. Platinum-Iridium percutaneous needle electrodes were placed across the sciatic nerve for nerve stimulation. Isometric torque was measured across a range of frequencies (5-250hz), with 120 seconds between each frequency tested. Peak isometric torque was achieved between 150 and 250 Hz and is expressed as mN·m per kg body weight. The frequency relationship was graphed using a four-parameter logistic curve. Average rates of contraction and relaxation were measured at the frequency in which peak isometric torque was achieved.

Histological analyses

In a subset of mice (n=12), the whole gastrocnemius was placed in OCT prior to freezing in isopentenyl cooled by liquid nitrogen, before being stored at −80°C until histological processing. Systematic 10 μm thick cross-sections were obtained from 5 standardized regions across the length of the whole muscle using a previously established technique (21, 31). Each of the five muscle sections were stained with Masson’s Trichrome (Abcam ab150686) for histological analysis of total myofiber number. Oil Red O (ORO; Sigma-Aldrich Cat# O0625) was dissolved in triethyl phosphate at 1.4% w/v, and volumed to concentration of 36%. Muscle sections were fixed in 4% PFA for 10 minutes and rinsed with 85% propylene glycol. Slides were incubated in ORO solution for 15 minutes at room temperature, rinsed twice with propylene glycol and distilled water, respectively. Next, slides were counterstained with hematoxylin for 1 minute. All sections were cover-slipped with either DPX Mounting Media (Electron Microscopy Sciences Cat#1351) or Fluoro-Gel (Electron Microscopy Science Cat# 17985) depending on the stain. Brightfield images were acquired on the TissueScope LE slide scanner (Huron Digital Pathology, St. Jacobs, ON, Canada) using a 20X objective (0.75 NA, 0.5 μm/pixel resolution). Myofibers were counted in a semi-automated manner in FIJI (32), briefly, the Masson’s Trichome-stained image was split using color deconvolution. Next, the thresholding tool was used to identify each individual myofiber and subsequently counted using the particle analysis function. Outstanding myofibers not identified by the thresholding tool and as necessary were quantified using the multipoint tool. Both automated and manual counts were summated for total myofiber number. For the ORO stained slides, the total area of the muscle was determined by outlining the perimeter using the polygon selection tool. Next, the image was split using color deconvolution and thresholding (range 0-115) tools. Then the area of ORO was calculated by measuring ORO-positive area compared to total area of each muscle in FIJI.

Gastrocnemius muscle sections were also stained for perilipin 1 (Cell Signaling Cat# 9349S; RRID:AB_10829911; 1:1000) and DAPI (Thermo Fisher Scientific Cat# D21490; 10 μg/ml) to visualize myonuclei. Briefly, sections were fixed with 4% PFA before being washed with 1x PBS and incubated in the primary antibody overnight at 4°C. The host- and isotype-specific cross-absorbed Alexa Fluor 647 anti-rabbit IgG (Thermo Fisher Scientific Cat# A-21245; RRID_AB_2535813; 2mg/ml) was used to detect anti-perilipin 1 with a 60 min incubation at room temperature. A subsequent 10 min incubation with DAPI was completed at room temperature. All sections were airdried and cover slipped with ProLong^™ Diamond Antifade Mountant (Thermo Fisher Scientific Cat# P36970). Sections were visualized on a Nikon AXR confocal microscope using galvano scanner and a Plan Flour 4x PhL DL objective (0.13 NA). Images (3891x3891 pixels, 2.14 μm/px) were captured using the large image acquisition add-on within Nikon NIS-Elements AR which automated stitching of smaller images to capture on images of the whole muscle section for analysis. Using FIJI (32), the percent area of positively stained tissue expressing perilipin 1 was quantified as previously described (21).

Biochemical analyses

At the 6-week terminal time point, the gastrocnemius muscle was harvested and cut into thirds, including the proximal, middle, and distal regions with the middle portion containing the VML defect and one of the intramuscular glycerol injections. The middle portion of the muscle was used for biochemical analyses. The gastrocnemius muscle was homogenized in a phosphate buffer with a protease inhibitor (Thermo Scientific Cat#78443) at a ratio of 1:5 (mg/μl) and 1:100 (mg/ μl), respectively. Total muscle protein content was analyzed using the Protein A280 setting on a NanoDrop One spectrophotometer (Thermo Scientific) in triplicate and averaged. Gonadal adipose tissue compartments were visualized following a midline incision and opening of the peritoneum. Both gonadal adipose tissue compartments were harvested, weighed and saved for biochemical analyses. Gonadal adipose tissue was homogenized using the Minute^™ Adipose Tissue Fractionation Kit for Adipose Tissues (Invent Biotechnologies, Inc. Cat# AT-023) and protein content was analyzed using a Pierce^™ BCA Protein Assay (Thermo Scientific Cat#23225). Whole blood was collected terminally, allowed to clot at room temperature, and centrifuged at 2,000 rpm for 15 minutes at 4°C. Following centrifuge, serum was collected and stored until analysis.

Immunoblot analyses were performed on skeletal muscle and gonadal adipose tissue by loading and separating 25-50 μg of protein by 4-15% Criterion TGX Stain-Free Gel. Proteins were transferred onto a low-fluorescence PVDF membrane and immunoblotted with primary antibodies to detect protein levels. The following antibodies were used to detect protein expression in the middle portion of the gastrocnemius muscle: perilipin 5 (Proteintech Cat#26951-1-AP; RRID:AB_2880699; 1000 μl/ml), perilipin 2 (Proteintech Cat#15294-1-AP; RRID:AB_2878122; 1000 μl/ml), adipose triglyceride lipase (Cell Signaling Cat#2138; RRID:AB_2167955; 129 μg/ml), SIRT1 (Abcam Cat#ab110304; RRID:AB_10864359; 1 mg/ml). Gonadal adipose tissue was probed for the following antibodies: adiponectin (Cell Signaling Technology Cat# 2789; RRID:AB_2221630; 10 μg/ml). Primary antibodies were detected using host- and isotype-specific horseradish peroxidase conjugated secondary antibodies as follows: anti-mouse IgG HRP (Cell Signaling Technology Cat #7076; RRID_330924), or anti-rabbit IgG HRP (Cell Signaling Technology Cat #7074; RRID_2099233, 1:1000). Immunoblots were blocked and incubated with primary and secondary antibodies with 5% milk in 0.1% Tween TBS or EveryBlot Blocking Buffer (Bio-Rad Laboratories Cat #13010020) and imaged with stain-free chemiluminescence using a ChemiDoc MP System (Bio-Rad Laboratories, Hercules, CA, USA). Total protein was quantified in each lane. Bands of interest were identified by the manufacturer-suggested molecular weight, and the intensity of each band was normalized to the total protein in the respective lane. Naïve, within sex mice were used as a comparison to which all experimental samples were normalized to during analysis.

Serum free fatty acid (FFA) and triglyceride concentrations were assessed in duplicate using Free Fatty Acid Assay (Abcam Cat#65341) and Triglyceride Assay (Abcam Cat#Ab65336) Kits, respectively. The ratio between FFA and triglycerides was calculated to serve as an indicator of lipolytic activity (33), in that a lower ratio may indicate suppressed lipolysis and reduced FFA release from adipose tissue. Terminal blood glucose was assessed using a glucometer at the time of harvest. Serum insulin (Crystal Chem Cat#90080) and leptin (R&D Systems; #MOB00B) were evaluated using enzyme-linked immunosorbent assay (ELISA) according to manufacturers’ protocols. Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) was calculated with terminal blood glucose and serum insulin values using the well-established formula (34): [glucose (mmol/L) x insulin (μU/mL) / 22.5].

Statistical analyses

All data was analyzed using JMP software (version 17.0, SAS Institute, Cary, NC, USA). For all mice differences in weight between males and females were evaluated using an independent t-test. For initial method development study, two-way ANOVAs (stimuli x sex) with Tukey’s HSD post hoc evaluated differences between post-prandial metabolic flexibility outcomes (e.g., peak RER) across groups. Paired t-tests were conducted temporally within each prandial stimulus. Following experimental injuries, two-way ANOVAs (injury x sex) with Tukey’s HSD post hoc evaluated differences in masses, whole-body metabolism (e.g., 24-hr energy expenditure), function (i.e., maximal isometric torque), and biochemical outcomes (e.g., triglycerides, perilipin 2) across groups. To account for the potential influence of physical activity on whole-body metabolism, PAEE was included as a covariate in all relevant models. Statistical significance of PAEE was evaluated and its contribution to model variance was reported when p≤0.05. A three-way ANOVA was used to evaluate the fitted force frequency curve (injury x sex x frequency, repeated). Two-way ANOVAs were used to assess myofiber number, ORO-positive area, and perilipin 1 across muscle regions (injury x whole muscle region) with Tukey’s HSD post hoc to evaluate interactions. Significance was set at p≤0.05.

Results

Development of a tool to assess post-prandial metabolic flexibility

A repeated measures cross-over design (Fig. 1A) was used to confirm if an i.p. glucose injection could mimic a whole-body, post-prandial metabolic response, thereby modeling a clinical assessment of metabolic function. Glucose and saline injections volumes were normalized to bodyweight, given that males exhibited significantly greater bodyweight than females (Fig. 1B; p<0.0001). Paired t-tests were conducted within each stimulus to confirm there were no significant effects of time (p≥0.052); then data for the glucose and saline injections were pooled within time points. Respiratory exchange ratio (RER) was captured in the 60-mins prior to the injection and for 180-mins following (Fig. 1C & J). Neither sex nor prandial stimuli impacted RER post-injection (Fig. 1D; p≥0.829). Importantly, there were no differences in pre-injection RER prior to injecting either glucose or saline (Fig. 1E; p≥0.176), confirming comparable baseline metabolic states. Peak RER was greatest following the glucose injections (Fig. 1F; main effect p<0.001) with no differences in nadir RER between sexes or prandial stimuli (Fig. 1G; interaction p=0.044). Lack of differences in nadir RER confirm the return to a post-absorptive metabolic state, regardless of sex or prandial stimuli. Glucose injection resulted in greater delta RER (Δ RER) from injection to peak (Fig. 1H; main effect p=0.004) and peak to nadir (Fig. 1I; main effect p=0.003). These results indicate that an i.p. glucose injection effectively stimulates the metabolic shift in RER characteristic of the fasting to fed transition, suggesting its potential use as a pre-clinical model to evaluate post-prandial metabolic flexibility.

Assessment of whole-body metabolic flexibility following a localized skeletal muscle injury

Next, a study was designed to characterize the effect of a localized skeletal muscle injury on diurnal and post-prandial metabolic flexibility, while employing the tool developed in the repeated measures cross-over study. Mice were randomized into VML-injured, intramuscular glycerol injection or remained age- and sex-matched naïve controls. Mice recovered promptly from the VML surgery and glycerol injection procedure without complication. Whole-body metabolic flexibility was assessed 5 weeks following intramuscular glycerol injection or VML injury across a 24-hr period and collapsed into 12-hr active and inactive diurnal periods. Regardless of injury, females had greater ambulatory distance across a 24-hour period, with over two times the distance accumulated compared to males (Fig. 2A; main effect p<0.0001). Sex differences in ambulation were independent of diurnal period, with females exhibiting greater ambulation in both active and inactive periods (Fig. 2A; main effect p≤0.033). Similarly, females presented with greater PAEE across 24-hr, inactive, and active periods, with ~69% greater total PAEE across 24-hrs (Fig. 2B; main effects p≤0.002).

Figure 2.

A) Ambulatory distance averages in meters over 24-hr (main effect injury p=0.586; sex p<0.0001; interaction p=0.880) and 12-hr inactive (main effect injury p=0.306; sex p=0.033; interaction p=0.707) and active periods (main effect injury p=0.813; sex p<0.0001; interaction p=0.795). B) Activity-associated energy expenditure (PAEE) averages over 24-hrs (main effect injury p=0.719; sex p<0.0001; interaction p=0.942) and 12-hr inactive (main effect injury p=0.609; sex p=0.002; interaction p=0.788) and active periods (main effect injury p=0.884; sex p<0.0001; interaction p=0.998). C) Energy expenditure averaged over 24-hrs (main effect injury p=0.048; sex p<0.0001; interaction p=0.928). D) RER averaged over 24-hrs (main effect injury p=0.243; sex p=0.404; interaction p=0.631). E) Carbohydrate oxidation averaged over 24-hrs (main effect injury p=0.152; sex p=0.122; interaction p=0.841). F) Lipid oxidation averaged over 24-hrs (main effect injury p=0.481; sex p=0.407; interaction p=0.190). G) Fluctuations in energy expenditure across 24-hrs in all mice with averages of the 12-hr inactive (main effect injury p=0.185; sex p<0.0001; interaction p=0.928) and active periods (main effect injury p=0.015; sex p<0.0001; interaction p=0.928). H) Fluctuations in RER across 24-hrs in all mice with averages of the 12-hr inactive (main effect injury p=0.295; sex p=0.148; interaction p=0.439) and active period (main effect injury p=0.323; sex p=0.025; interaction p=0.853; activity p=0.006). I) Fluctuations in lipid oxidation across 24-hrs in all mice with averages of the 12-hr inactive (main effect injury p=0.664; sex p=0.026; interaction p=0.125) and active periods (main effect injury p=0.274; sex p=0.004; interaction p=0.731; activity p=0.004). J) Fluctuations in carbohydrate oxidation across 24-hrs in all mice with averages of the 12-hr inactive (main effect injury p=0.271; sex p=0.844; interaction p=0.865) and active periods (main effect injury p=0.129; sex p=0.029; interaction p=0.796). Shading across all graphs indicates the inactive period. Bar graph data is presented as mean ± SD. Dots represent an individual animal. Data analyzed by two-way ANOVA; statistically significant Main Effects are noted. Note legend for all groups at top of figure.

Sex-specific differences in whole-body energy expenditure were observed, with females expending ~30% greater total energy expenditure across 24-hrs compared to males, as determined by metabolic rate (kilocalories per hour) (Fig. 2C; main effect p<0.0001). This was independent of diurnal period, in that females exhibited ~34% and ~25% higher energy expenditure across active and inactive periods, respectively (Fig. 2G; main effects p<0.0001). Neither injury nor sex influenced 24-hr RER (Fig. 2D; p≥0.243), however, active period RER was greater in males (Fig. 2H; main effect p=0.025) and PAEE significantly contributed to the observed sex-specific differences (PAEE as covariate p=0.006). This was reflected in whole-body substrate usage, in that males utilized ~19% greater carbohydrates in the active period compared to females (Fig. 2E&J; main effect p=0.029) with concomitant decreases in active period lipid oxidation (Fig. 2F&I; main effect p=0.044). Inactive period lipid oxidation was a primary outcome influenced by PAEE (Fig. 2I; PAEE as covariate p=0.004). Interestingly, energy expenditure was also influenced by injury, in that VML-injured mice exhibited greater energy expenditure compared to glycerol-injured mice across 24-hrs and specifically impacted the active period (Fig. 2C&G; main effects p≤0.048). Together, these results demonstrate sex-specific differences in energy expenditure and substrate utilization, independent of injury. However, energy expenditure may be impacted by a localized skeletal muscle injury, specifically in that greater total energy expenditure occurs following VML injury despite a lack of change in ambulation and activity.

Using the tool developed, post-prandial metabolic flexibility was assessed by observing whole-body RER across ~240-mins (Fig. 3A). Neither injury nor sex impacted post-prandial RER across 180-mins (Fig. 3B; p≥0.134). Post-prandial ambulation was greatest in females, regardless of injury, with females covering ~51% greater ambulatory distance than males over the ~180-mins (Fig. 3C, main effect p=0.006). Females following VML injury exhibited the greatest pre-injection RER, as measured as the average RER 60-mins prior to injection (Fig. 3D; interaction p=0.046). Neither injury nor sex contributed to peak RER (Fig. 3E; p≥0.423) or nadir RER (Fig. 3F; p≥0.075); however, activity-associated energy expenditure contributed significantly to nadir RER (PAEE as covariate p=0.019). No significant effects of injury or sex on the magnitude of change in RER between injection to peak (Fig. 3G; p≥0.741) and peak to nadir (Fig. 3H; p≥0.418) were identified. Whole-body carbohydrate and lipid oxidation rates were examined across 180-mins following glucose injection (Figs. 3I&L). Despite lack of sex differences in post-injection carbohydrate oxidation (Figs. 3J; p≥0.171), males exhibited ~21% greater lipid oxidation across 180-mins (Figs. 3K; main effect p=0.019). These results highlight that sex-based differences in post-prandial metabolism may be driven by differences in ambulation and whole-body lipid oxidation. Further, persistent lipid oxidation in males may suggest a failure to appropriately switch substrate usage during changes in substrate availability, indicating poorer post-prandial metabolic flexibility.

Figure 3.

A) Fluctuations in RER plotted across ~240 minutes, collected every 5 minutes following glucose injection averaged across all mice within a group. B) RER area under the curve following injection (main effect injury p=0.134; sex p=0.765; interaction p=0.850). C) Ambulatory distance in meters across 180-mins following injection (main effect injury p=0.351; sex p=0.006, interaction p=0.363). D) Average of RER for 60-minutes prior to injection (main effect injury p=0.200; sex p=0.319; interaction p=0.046). E) Peak RER following injection (main effect injury p=0.423; sex p=0.504; interaction p=0.792). F) Nadir RER following injection (main effect injury p=0.075; sex p=0.757; interaction p=0.530). G) Δ RER from injection to peak RER following injection (main effect injury p=0.741; sex p=0.834; interaction p=0.679). H) Δ RER from peak to nadir RER following injection (main effect injury p=0.904; sex p=0.609; interaction p=0.418). I) Fluctuations in whole-body carbohydrate oxidation plotted across ~180 minutes, collected every 5 minutes following glucose injection. J) Carbohydrate oxidation area under the curve following injection (main effect injury p=0.163; sex p=0.171; interaction p=0.560). K) Lipid oxidation area under the curve following injection (main effect injury p=0.165; sex p=0.019; interaction p=0.124). L) Fluctuations in whole-body lipid oxidation plotted across ~180 minutes, collected every 5 minutes following glucose injection. Bar graph data is presented as mean ± SD. Dots represent an individual animal. Data analyzed by two-way ANOVA; statistically significant Main Effects are noted.

Glucose uptake is impaired in the context of metabolic disease with poorer uptake and greater HOMA-IR indicative of insulin resistance. Blood glucose was evaluated across 120-mins following i.p. glucose injection (Fig. 4A), in which females demonstrated improved glucose tolerance regardless of injury (Fig. 4B; main effect p<0.0001). Terminally, blood glucose and serum insulin failed to differ between injury or sex (Fig. 4C&D; p≥0.134). As a result, calculated HOMA-IR was not statistically different across groups (Fig. 4E; p≥0.078).

Figure 4.

A) Average blood glucose across 120-mins following i.p. glucose injection for all mice. B) Average blood glucose area under the curve from A (main effect injury p=0.846; sex p<0.0001; interaction p=0.745). C) Average terminal blood glucose concentration (main effect injury p=0.575; sex p=0.283; interaction p=0.134). D) Average terminal serum insulin concentration (main effect injury p=0.689; sex p=0.212; interaction p=0.774). E) HOMA-IR based on terminal blood glucose and serum insulin concentrations (main effect injury p=0.411; sex p=0.759; interaction p=0.078). Data is presented as mean ± SD. Dots represent an individual animal. Data analyzed by two-way ANOVA; statistically significant Main Effects are noted.

Assessment of injury-induced changes in adipose and skeletal muscle

As expected, male mice had greater initial and terminal body mass compared to females, regardless of injury (Table 1). Terminally, gonadal adipose tissue normalized to total body weight was greatest in Naïve males and glycerol-injected mice of both sexes (interaction p=0.015). Both male and female mice subjected to VML injury had reduced gonadal adipose tissue depots and the remaining gastrocnemius muscle mass compared to their sex-matched counterparts, despite having similar total body mass. At 6-weeks post-injury, the ratio between left (injured) and right (uninjured) gastrocnemius muscle mass remained significantly reduced following VML (main effect of injury p<0.0001).

Table 1. Body, muscle, and adipose depot masses.

	Male			Female			Two-way ANOVA p-value
	Injury Naïve	VML	Glycerol	Injury Naïve	VML	Glycerol	Main Effect Injury	Main Effect Sex	Interaction
	n=7	n=7	n=7	n=7	n=7	n=7
Initial Body Mass (g)	29. 5 ± 2.7	26.7 ± 2.5	27.3 ± 2.2	19.6 ± 0.7	20.4 ± 1.5	20.4 ± 1.8	0.395	<0.0001	0.056
Terminal Body Mass (g)	31.9 ± 2.8	29.1 ± 2.9	29.4 ± 2.7	21.3 ± 0.6	22.4 ± 2.5	22.4 ± 1.9	0.603	<0.0001	0.075
Gonadal Adipose Tissue Mass/Body Mass (mg/g)	15.0 ± 2.8*	6.5 ± 2.6‡	14.2 ± 4.2*	7.1 ± 1.1‡	7.6 ± 1.4‡	13.4 ± 5.2§	--	--	0.015
Left (injured) Gastrocnemius Mass (mg)	178.1 ± 15.6*§	108.6 ± 31.5‡	181.4 ± 16.8*§	123.4 ± 4.3‡	91.6 ± 18.6*‡	131.1 ± 5.4‡	--	--	0.016
L:R Gastrocnemius Mass	1.04 ± 0.06a	0.68 ± 0.17	1.17 ± 0.11a	1.02 ± 0.06a	0.75 ± 0.14	1.05 ± 0.06a	<0.0001	0.743	0.162

Data mean ± SD

Significant main effect of injury different than ^a VML

Significant interaction different than * Naïve Females, ‡ Naïve Males, § VML Males

Terminally, in vivo isometric torque of the posterior compartment was measured across a range of stimulation frequencies to evaluate the force frequency relationship. Across all stimulation frequencies, VML injury resulted in significant deficits in torque compared to other experimental groups (Fig. 5A; repeated effect p<0.0001). More specifically, maximal isometric torque was lowest following VML injury with no influence of biologic sex (Fig. 5B; main effect p<0.0001). Twitch torque was impaired by ~60% following VML when compared to other experimental groups (Table 2; main effect p<0.0001). Males exhibited greater average rates of contraction compared to females (main effect p=0.001), while VML-injured mice had a ~59% and ~51% decrease in average rate of contraction compared to Naïve and glycerol-injured mice, respectively (main effect p<0.0001), indicating a slower rate of force development. Average rates of relaxation were lowest following VML, regardless of sex, with greater than ~67% deficit compared to other experimental groups (interaction p=0.014). Time to peak torque during maximal isometric contraction was influenced by sex, in that females exhibited greater times to reach maximal force output (main effect sex p=0.017). Within group sex differences were only observed in time to half relaxation (i.e., ½ RT), specifically between Naïve males and females (interaction p=0.014). Taken together, these confirm that VML injury leads to persistent deficits in muscle and adipose depot masses, accompanied by marked impairments in contractile function and slowed muscle kinetics, irrespective of sex.

Figure 5.

A) Isometric torque normalized to total body weight displayed across 5-200hz stimulation frequencies (main effect injury p<0.0001; sex p=0.150; interaction p=0.338; stimulation frequency repeated measure p<0.0001). B) Maximal isometric torque of the posterior compartment normalized to total body weight (main effect injury p<0.0001; sex p=0.359; interaction p=0.925). Data is presented as mean ± SD. Dots represent an individual animal; males are closed circles and females are open circles. Data analyzed by two-way ANOVA or two-way repeated measures ANOVA; statistically significant Main Effects are noted.

Table 2. Contractile properties.

	Male			Female			Two-way ANOVA p-value
	Injury Naïve	VML	Glycerol	Injury Naïve	VML	Glycerol	Main Effect Injury	Main Effect Sex	Interaction
	n=5	n=5	n=6	n=7	n=6	n=6
Isometric twitch torque normalized to body mass (mN·m/kg)	103.7 ± 17.2a	40.9 ± 20.7	113.9 ± 29.4a	113.5 ± 24.1a	47.2 ± 15.6	94.1 ± 21.0 a	<0.0001	0.871	0.222
Time to Peak – Twitch (ms)	23.2 ± 2.3	22.7 ± 1.0	25.1 ± 1.4	25.8 ± 0.1	23.8 ± 2.5	23.3 ± 2.2	--	--	0.018
+dP/dT (mN·m/s)	300.0 ± 87.9a	105.7 ± 39.1	234.1 ± 75.3a	177.6 ± 45.5a	84.6 ± 41.0	150.0 ± 37.2a	<0.0001	0.001	0.119
−dP/dT (mN·m/s)	−557.2 ± 169.5	−135.9 ± 57.5*‡#†	−430.2 ± 55.3	−312.7 ± 78.4‡#	−122.8 ± 61.0*‡#†	−364.9 ± 68.6‡#	--	--	0.011
Time to Peak – Maximum (ms)	192.5 ± 60.4	193.8 ± 49.0	190.6 ± 57.4	233.2 ± 2.3	206.4 ± 28.1	243.5 ± 16.1	0.602	0.017	0.499
½ RT (ms)	37.7 ± 4.9	44.2 ± 5.5	42.6 ± 3.5	48.1 ± 0.8‡	44.3 ± 4.4	41.0 ± 3.3	--	--	0.014

Data mean ± SD

Significant main effect of injury different than ^a VML

Significant interaction different than * Naïve Females, ‡ Naïve Males, § VML Males, # Glycerol Males, † Glycerol Females

Histological evaluation of lipids in the remaining muscle

A systematic histological evaluation of five sections per muscle was performed to characterize neutral lipids and lipid droplet-associated proteins across the whole gastrocnemius muscle (Fig. 6A, C&E) in a subset of mice. Across the entire muscle, VML injury resulted in a ~20% deficit in total myofiber number compared to intramuscular glycerol injury (Fig. 6B; main effect injury p=0.012). As expected, there are regional differences across muscles sections, with the total myofiber number greater in the middle cross-sections of the gastrocnemius muscle, with nearly 2-fold greater myofibers than the distal and proximal sections (main effect whole muscle region p<0.0001). Neutral lipids species, as evaluated by ORO, were greatest in VML-injured muscles with a ~91% increase in total ORO-positive area compared to Naïve (Fig. 6D; main effect injury p<0.0001). Interestingly, glycerol-injured muscle failed to accumulate neutral lipid species at this time point (6-weeks post-injection), with a ~65% decrease in accumulation compared to VML injury (main effect injury p<0.0001). Regional differences in neutral lipid accumulation also emerged; the proximal whole muscle region displayed greater accumulation compared to mid and mid-distal whole muscle regions (main effect whole muscle region p=0.003). The lipid-droplet associated protein perilipin 1, which is commonly associated with mature adipocytes, was also assessed across the whole gastrocnemius muscle. VML injury resulted in the most perilipin 1-positive area across the whole muscle with more than 3-fold increases over other experimental groups (Fig. 6F; main effect injury p<0.0001). Across the whole gastrocnemius muscle, VML injury exhibited a loss of total myofiber number and robust lipid accumulation, suggesting a shift toward adipogenic remodeling that may impair functional recovery.

Figure 6.

A) Representative images of the mid whole muscle region with Masson’s Trichome. B) Total myofiber number across systematic whole muscle regions (main effect injury p=0.012, whole muscle region p<0.0001; interaction p=0.365). C) Representative images of the mid whole muscle region with ORO. D) ORO-positive area across systematic whole muscle regions (main effect injury p=<0.0001; whole muscle region p=0.003; interaction p=0.340). E) Representative images of the mid whole muscle region with perilipin 1 (green) and DAPI (blue). F) Perilipin 1-positive area across systematic whole muscle regions (main effect injury p<0.0001; whole muscle region p=0.176; interaction p=0.300). Data is presented as mean ± SD. Dots represent an individual animal; males are closed circles and females are open circles. Data analyzed by two-way ANOVA; statistically significant Main Effects are noted.

Markers of lipid mobilization and metabolic health

Circulating triglycerides and FFA were assessed to examine metabolic factors contributing to sex-based differences in whole-body substrate utilization. Next, gonadal adipose depots were analyzed for adiponectin protein expression, a systemic marker of overall metabolic health. Though injury and sex did not influence circulating triglycerides (Fig. 7A; p≥0.215), FFA concentrations were greatest in males post-VML, showing a trend towards significance (Fig. 7B; interaction p=0.051). Similarly, FFA to triglyceride ratios failed to reach statistical significance between injuries; however, males trended towards larger ratios than females (Fig. 7C; main effect sex p=0.055). Adiponectin protein expression within adipose was higher in females, compared to males (Fig. 7D; main effect sex p=0.030). These findings suggest that males may exhibit greater lipid mobilization and reduced overall metabolic health, which may be contributing to sex-based differences in whole-body substrate metabolism.

Figure 7.

A) Average terminal serum triglyceride concentration (main effect injury p=0.961; sex p=0.215; interaction p=0.995). B) Average terminal serum free fatty acid concentration (main effect injury p=0.239; sex p=0.438; interaction p=0.051). C) Average ratio between terminal serum free fatty acid and triglyceride concentrations (main effect injury p=0.675; sex p=0.055; interaction p=0.488). D) Average adiponectin protein expression in adipose depot (main effect injury p=0.392; sex p=0.030; interaction p=0.274). E) Representative image of a stain-free blot for total lane protein quantification and F) cropped chemiluminescence bands for quantification of protein expression. G) Average SIRT1 protein expression in remaining muscle (main effect injury p=0.002; sex p=0.042; interaction p=0.068). H) Average perilipin 2 protein expression in remaining muscle (main effect injury p=0.002; sex p=0.825; interaction 0.349). I) Average perilipin 5 protein expression in remaining muscle (main effect injury p=0.213; sex p=0.114; interaction p=0.008). J) Average adipose triglyceride lipase (ATGL) protein expression in the remaining muscle (main effect injury p<0.0001; sex p=0.025; interaction p=0.004). Data is presented as mean ± SD. Dots represent an individual animal. Dashed line represents normalized Naïve expression. Data analyzed by two-way ANOVA; statistically significant Main Effects are noted; statistically significant Interactions are noted by: * different than VML females.

Next, the expression of lipid droplet regulatory proteins in the remaining muscle was evaluated (Figs. 7E&F). Primarily responsible for activating transcription factors that promote mitochondrial biogenesis and oxidative metabolism, VML-injured mice exhibited the greatest SIRT1 expression with 2-fold and 1.6-fold increases in expression over Naïve and glycerol-injured mice, respectively (Fig. 7G; main effect p=0.002). Lipid droplet-associated proteins perilipin 2 and 5 were also influenced by VML injury with some sex-specific differences. Perilipin 2 expression was greatest following VML with ~30% increase compared to Naïve (Fig. 7H; main effect p=0.002). Sex influenced perilipin 5 expression specifically post-VML, with VML females presenting 2.4-fold lesser expression compared to VML males (Fig. 7I; interaction p=0.008). Most associated with facilitating the initiation of lipolysis, ATGL expression was highest in VML females with 3-fold greater expression than their injury-matched counterparts (Fig. 7J; interaction p=0.004). Taken together, VML injury induces a coordinated shift in lipid droplet regulation, marked by increases in lipid-handling proteins (e.g., perilipin 2) with notable sex-specific differences in perilipin 5 and ATGL expression.

Discussion

This study aimed to investigate mechanisms of whole-body metabolic disruption following VML injury, with particular focus on lipid regulation. This included the development of a novel non-invasive pre-clinical tool for assessing post-prandial whole-body metabolism. There are four key conclusions based on the results. First, the usage of an i.p. glucose injection in combination with indirect calorimetry can successfully model a whole-body metabolic transition from pre- to post-prandial states. Second, pronounced sex differences in whole-body metabolism and substrate utilization reflect fundamental differences in energy regulation between male and female C57Bl/6J mice. Third, biologic sex influences metabolic proteins that impact tissue remodeling and lipid accumulation in the muscle remaining, adding complexity to the pathophysiology of VML injury. And fourth, the use of a single intramuscular glycerol injection does not provide a sufficiently robust comparative model to investigate chronic lipid accumulation and metabolic disruption in C57Bl/6J mice.

Evaluating metabolic flexibility can characterize aspects of overall metabolic health and assist in the identification and prevention of cardiometabolic disease. While investigating post-prandial metabolic flexibility clinically can yield valuable insights into glycemic regulation and substrate utilization (35, 36), there is currently a scarcity of pre-clinical tools to assess the whole-body RER response to a prandial stimulus. To the best of our knowledge, this is the first pre-clinical tool to combine indirect calorimetry and standardized glucose injections in freely moving rodents, to assess metabolic flexibility non-invasively. This tool demonstrated that an i.p. saline injection does not induce fluctuations in RER that would be indicative of a prandial stimulus. In contrast, an i.p. glucose injection induced a ~7% change or fluctuation in RER over the 180-minute test. The i.p. glucose injection also resulted in a return to a post-absorptive RER by the end of the test period. Individual RER traces for each mouse were included to highlight inherent variability that exists between animals in this assay. Use of this tool should be adequately powered to ensure biological differences can be identified, with consideration of how physical activity (i.e., ambulatory activity) may impact observed outcomes.

Both clinically and pre-clinically, the use of hyperinsulinemic-euglycemic clamps (or HIEC) is a well-documented method to assess whole-body metabolic flexibility (15). The HIEC approach is used to induce a prandial state and assess post-prandial responses, however, the moderate restriction of activity with this technique may limit non-exercise activity thermogenesis (NEAT), which contributes 15-50% of total energy expenditure and may affect substrate oxidation measurements (37). Physical activity is known to promote glucose disposal and supports a return to a post-absorptive RER, thus the inclusion of activity in the assessment of metabolic flexibility is warranted. Though NEAT was not directly assessed in this study, calculation and inclusion of activity-associated energy expenditure (i.e., PAEE) (30) was considered. Herein, PAEE contributed to post-prandial nadir RER and active period lipid oxidation, confirming that physical activity influences substrate preference and usage. Metabolic assessments that preserve activity are essential to accurately capture whole-body metabolic flexibility in a physiologically relevant context. The HIEC method solely assesses glycemic control and the responsiveness of skeletal muscle to insulin and glucose stimuli. However, it doesn’t account for the coordinated suppression of alternative pathways, such as lipid oxidation, and misses the broader capacity for efficient substrate switching that characterizes metabolic flexibility. These limitations have been recognized by others (38-40), and some have attempted to address them using mixed-meal challenges within a whole-room calorimeter clinically (36, 41, 42). Pre-clinical assessments of metabolic flexibility using the HIEC approach requires considerable technical expertise and the procedure is highly invasive, both of which often preclude its routine use; these limitations have been widely acknowledged (43, 44). Ultimately, the novel pre-clinical tool developed assesses the post-prandial state outside of labor-intensive, non-ambulatory techniques, and enables translational research that bridges mechanistic understanding.

The impact of localized skeletal muscle injuries, such as VML, on whole-body metabolism has only recently begun to be investigated. Most studies evaluating VML injury across the field have been conducted exclusively in males; and work evaluating any aspects of whole-body metabolism have been in males only (11, 12, 19, 45, 46). This presents a significant shortcoming for advancing a sex-specific understanding of metabolic flexibility and whole-body metabolic adaptations to injury. The initial single-sex study in males reported temporal shifts in RER following VML, with increases in lipid oxidation at 2- and 6-weeks post-injury (11). Subsequent studies documented alterations in lipid oxidation at similar timepoints, although, findings were inconsistent with prior work (11) indicating blunted lipid oxidation compared to naïve controls (19, 45). In this work, daily RER and lipid oxidation remain largely unaffected by the VML injury. However, females exhibit a greater reliance on lipid oxidation in the active period, suggesting inherent differences in diurnal substrate usage and possibly biologic sex-specific metabolic responses post-VML.

Differences in whole-body metabolism and substrate utilization between biologic sexes has been increasingly recognized in pre-clinical studies, in that females display enhanced metabolic flexibility relative to males across stimuli (47-52). Whereas most of these studies have focused on the cellular mechanisms of metabolic flexibility (51, 52), only a few have examined whole-body metabolism (47-50). In response to dietary challenges, such as a high-fat diet, females increase whole-body energy expenditure and mitochondrial respiration in both adipose tissue and skeletal muscle (50-52); notably, these adaptations are evident both acutely and with prolonged high-fat diet exposure. Exercise stimuli have been used as an additional approach to assess metabolic flexibility, providing insight into sex-based differences in exercise capacity and substrate utilization. Females, independent of body weight and skeletal muscle mass, have greater times-to-exhaustion during treadmill tests compared to males (47-49). In terms of substrate utilization, females exhibit greater free fatty acid availability and a reliance on lipid substrates during exercise, suggesting a greater ability to mobilize endogenous lipid stores when faced with a physiological stimulus such as exercise (48, 53). Here, there were no differences in post-prandial RER outcomes between males and females; however, females exhibited lower lipid oxidation following the i.p. glucose injection, though mechanisms confirming suppression of lipid oxidation have yet to be determined. While this contrasts with the lipid reliance during exercise (47, 49), it reflects an appropriate substrate shift when glucose is acutely elevated following a prandial stimulus, indicating metabolic flexibility. Though ambulatory activity following glucose tolerance test was not evaluated, slightly lower blood glucose in females may be due to increased activity and thus, increased glucose uptake. Females, irrespective of injury, expend ~30% greater total energy and 1.7-fold more calories per hour as determined with PAEE. Further evaluation of diurnal substrate oxidation revealed females oxidize lipids at a greater rate and present with a lower active period RER, despite greater PAEE. This aligns with the idea posited by others, that substrate utilization is influenced by changes in mitochondrial bioenergetics across a diurnal period rather than by activity alone, though this has been documented solely in male mice (54-56). How loading or gait, in addition to physical activity, could contribute to VML-induced or sex-specific changes in whole-body metabolism is not yet clear, and it is possible even with similar ambulatory activity the metabolic costs of loading could be different. Future work is needed to understand this complex relationship. Males herein present with fluctuations in whole-body substrate preferences across photoperiods, prioritizing carbohydrate oxidation in the active period and lipid oxidation in the inactive period. Although these substrate preferences broadly align with what is considered favorable to metabolic flexibility, recent work has urged the field to look beyond conventional photoperiod frameworks when assessing rodent metabolic physiology. Focusing solely on differences between active and inactive periods may overlook important episodic or ultradian shifts in energy expenditure and substrate utilization within shorter timescales (30, 57). These findings highlight that while photoperiod substrate shifts provide a useful framework, they may not capture the full complexity of metabolic regulation. Further justifying the deployment of translational tools, such as the one developed, to assess additional layers of metabolic flexibility that occur independent of diurnal photoperiods.

Impairments in oxidative pathophysiology have been well documented following VML injury, with particular focus on mitochondrial bioenergetics and associated pathways. Recently, a few studies have begun recognizing sex-specific responses following VML, with growing evidence spanning mitochondrial (51) and qualitative adaptations (20, 58). Lipid accumulation has been introduced as an additional local sequela of VML injury (24, 59). In the present study, lipid accumulation was observed across systematic whole-muscle regions post-VML, inclusive of mature adipocytes (i.e., perilipin 1-positive) and neutral lipid species (i.e., oil red o-positive). Though due to the evaluation of only a subset of samples, sex could not be analyzed due to limited sample size. Recent evidence of lipid accumulation following VML has been indirectly quantified and evaluated distinctly at the VML defect (20, 21). The direct evaluation of mature adipocytes throughout the muscle demonstrates that lipid accumulation is not restricted to the VML defect but also occurs more broadly as a result of this pathology. While the effect of sex on post-VML lipid deposition remains unresolved, in other work, females appear to accumulate more lipids (60), and accumulation may be influenced by ovarian hormone loss (61). Further work should explore how sex and hormonal factors influence lipid deposition and its relationship to tissue remodeling following VML. Sex-specific observations in lipid-related protein expression and signaling may support two proposed mechanisms that could contribute to lipid accumulation following VML. First in males, increases in perilipin 5 and SIRT1 expression may reflect compensatory lipid droplet-mitochondrial coupling; however, limited mitochondrial flux (18) and accumulation of triglycerides and aclycarnitine (19) could promote pathological lipid droplet expansion. Second in females, robust ATGL expression suggests dynamic lipid turnover, while reduced perilipin 5 expression may serve to limit excessive fatty acid influx into mitochondria (62). Thus, limiting the accumulation of lipotoxic intermediates and resulting in smaller, metabolically efficient lipid droplets. Knowing that many of these lipid-related mechanisms can be influenced by physical activity, it is notable to acknowledge that sex-specific differences in expression following VML may be attributed to differential physical activity. The ratio between serum free fatty acid and triglycerides, though not significant, trended (p=0.055) lower in females, suggesting greater re-esterification, reduced systemic lipolysis, and supporting lipid metabolism efficiency. Further studies should explore these metabolic byproducts within the local tissue to further test this hypothesis. Prior work suggests that females may exhibit distinct regenerative responses following localized skeletal muscle injury (e.g., cardiotoxin injection) (63, 64). Estrogen-dependent mechanisms are also known to contribute to the activation of satellite cells (65), reductions in the inflammatory response (63), and functional regeneration of the muscle (64). Females also have higher rates of reinnervation (~7 weeks post injury), which should support long-term muscle repair (58). Together, these sex-specific differences in lipid handling may influence efficient repair and regeneration, highlighting the need to consider biologic sex in strategies to improve tissue remodeling following VML.

An intramuscular glycerol injection has been increasingly utilized as a localized skeletal muscle injury model to induce ectopic fat accumulation in pre-clinical studies (26, 27, 60) as well as for a model to understand aspects of skeletal muscle function and regeneration (66-68). Due to the relationship between ectopic fat accumulation and metabolic dysfunction (69), an intramuscular glycerol injection was used as a comparative model to VML injury to evaluate lipid deposition and its functional and metabolic consequences. One hypothesis is that ectopic fat accumulation impairs muscle function by physically displacing existing contractile tissue and hindering the formation of early de novo myofibers during regeneration. Early changes to myofiber morphology include immediate reductions in myofiber area, which can recover by 2-3 weeks post-injection (26), though this is influenced by rodent strain and sex (60). To note, most glycerol studies to date perform injections into the tibialis anterior muscle (27, 60, 68) or rotator cuff (70-72) and to the best of our knowledge none have explored the effects in the gastrocnemius muscle used in this study. Use of the gastrocnemius here is a notable strength, given its critical high duty cycle during load-bearing and its heterogeneity in myosin heavy chain isoform composition. The glycerol injection failed to induce changes in myofiber number or lipid accumulation in the muscle 6 weeks post-injection, whether assessed by mature adipocytes or neutral lipid species. This may be due to the glycerol injection model mostly being used acutely (~3-4 weeks) and primarily as a model to understand functional regeneration of the muscle. Within 3 weeks of the glycerol injection, when normalized by physiological cross-sectional area, muscles display reductions in vitro at twitch and tetanic force (73). Although correlations with lipid volume suggest an influence of ectopic fat accumulation across muscle contractile parameters. There were no differences in maximal in vivo isometric torque between Naïve and glycerol-injected muscles at 6 weeks post-injection. Lack of ectopic fat accumulation is a noted limitation of a single injection time point for a chronic study, and future studies should consider if repeat injections (74) are needed to address experimental aims.

This work emphasizes the importance of evaluating the intersection of VML injury and cardiometabolic disease risk, while considering biologic sex. By enabling non-invasive assessments of post-prandial metabolism, this approach provides a foundation for capturing whole-body fuel utilization outside of the technical and biological constraints of current methodology. Using this tool, we demonstrate that whole-body and cellular metabolic responses are influenced by a localized skeletal muscle injury, such as VML, but are also fundamentally shaped by biologic sex. These insights could inform sex-specific strategies to preserve metabolic flexibility and mitigate metabolic disruptions observed following VML.

Data availability:

The datasets used and/or analyzed during the current study are primarily presented in the current manuscript and are available from the corresponding author on request.