Cyclophilin D Deficiency Exacerbates Diet-Induced Obesity and Selectively Enhances Skeletal Muscle Mitochondrial Respiration in Mice

Cyclophilin D Deficiency Exacerbates Diet-Induced Obesity and Selectively Enhances Skeletal Muscle Mitochondrial Respiration in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cyclophilin D Deficiency Exacerbates Diet-Induced Obesity and Selectively Enhances Skeletal Muscle Mitochondrial Respiration in Mice Jazzminn L. Hembree, William A. Abplanalp, John W. Elrod, Paul T. Pfluger This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9472657/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Mitochondrial dysfunction is a hallmark of obesity-associated metabolic disease, with the mitochondrial permeability transition pore serving as a key regulator of cellular energy homeostasis. Cyclophilin D, encoded by Ppif , is the principal regulatory component of this pore and has been implicated in insulin secretion, adipocyte survival, and whole-body energy balance. However, whether Ppif loss produces a consistent metabolic phenotype when challenged with high fat diet (HFD) exposure, and whether systemic metabolic alterations reflect tissue-specific changes in mitochondrial oxidative capacity, remains unresolved. Here, we characterized whole-body Ppif knockout mice longitudinally over 34 weeks under standard (SD) and HFD using body composition analysis, indirect calorimetry, glucose and insulin tolerance testing and tissue-resolved, Seahorse-based mitochondrial respirometry. Under SD, knockout mice displayed elevated fat mass without differences in food intake or energy expenditure. High-fat feeding progressively amplified this adiposity, with knockout mice developing significantly greater body weight and fat mass than wild-type littermates by weeks 28 and 36. Glucose tolerance and insulin sensitivity remained comparable genotype under either diet. Energy expenditure was selectively elevated in high-fat-fed knockout mice, and ANCOVA revealed lean mass as a primary driver, with a significant lean mass-by-genotype interaction. Consistent with that, ADP-stimulated mitochondrial respiration was significantly increased in quadriceps of knockout mice under both diets, without corresponding differences in brown adipose tissue or liver, or changes in mitochondrial content across tissues. These findings establish cyclophilin D as a diet- and tissue-specific regulator of energy homeostasis, informing therapeutic strategies targeting the permeability transition pore or cyclophilin D interactome in metabolic disease. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Obesity and its associated metabolic complications, including type 2 diabetes, dyslipidemia, and cardiovascular disease, represent a growing global health burden that is driven in large part by dysregulated energy homeostasis and adipose tissue expansion. At the cellular level, mitochondria act as key regulators of energy metabolism, and mitochondrial dysfunction is strongly implicated in the development of diet‑induced obesity and insulin resistance [ 9 ][ 19 ]. A key regulator of mitochondrial integrity is the mitochondrial permeability transition pore (mPTP), a high-conductance channel whose aberrant or sustained opening leads to dissipation of the mitochondrial membrane potential, release of pro-apoptotic factors, and disruption of oxidative phosphorylation [ 3 ]. Understanding the molecular mechanisms that govern mPTP activity and their downstream metabolic consequences has therefore become an important area of investigation in the context of metabolic disease. Cyclophilin D (CypD), encoded by the Ppif gene, is a matrix peptidyl-prolyl cis-trans isomerase that serves as a critical facilitator of mPTP opening by directly binding to pore constituents including the adenine nucleotide translocator and components for oxidative stress-induced opening, thereby acting as key modulator of mPTP sensitivity and mitochondrial stress responses [ 1 ][ 6 ]. Beyond its canonical role in cell death regulation, CypD has emerged as a broader regulator of mitochondrial metabolism, with implications for whole-body energy balance and glucose homeostasis [ 17 , 25 ]. However, reports on its metabolic role are highly divergent, with studies describing protection from, susceptibility to, or altered progression of diet‑induced obesity and glucose intolerance in CypD‑deficient mice. These discrepancies highlight the need to clarify how loss of CypD influences whole‑body metabolic regulation. Furthermore, whether alterations in whole-body metabolic parameters in CypD‑deficient mice are mechanistically linked to tissue-specific changes in mitochondrial oxidative capacity has not been fully elucidated. Here, we characterize the metabolic phenotype of whole-body Ppif knockout mice undergoing SD and HFD exposure for 34 weeks, incorporating body composition analysis, comprehensive indirect calorimetry, glucose and insulin tolerance testing, and mitochondrial respiratory function measurements across metabolically distinct tissues. Our findings demonstrate that CypD deficiency exacerbates diet-induced obesity in a dietary context-dependent manner, selectively amplifying adiposity, energy expenditure, and skeletal muscle mitochondrial respiration, thereby establishing a tissue- and diet-specific role for CypD in the regulation of whole-body metabolic homeostasis. Methods Mouse experiments Generation of Ppif knockout mice has been described previously [ 1 ]. All mice were group-housed with a 12hr light, 12hr dark cycle (7am-7pm) at 22°C and maintained on a standard chow diet (Harlan Teklad LM-485) or high-fat diet (Research Diets D12492 (60% fat, New Brunswick, NJ, USA), with ad libitum access to food and water. Fat and lean mass were quantified using NMR technology (EchoMRI, Houston, TX, USA). Energy expenditure, respiratory quotient, food intake, and locomotor activity were assessed using a combined indirect calorimetry system (TSE Systems GmbH, Bad Homburg, Germany) as described previously [ 16 ]. Mice had ad libitum access to food and water and were acclimated to metabolic cages for at least 24 hours followed by the simultaneous measurement over a 72‑hour period. Intraperitoneal glucose and insulin tolerance testing (GTT/ITT) was conducted in mice subjected to 6 hours of fasting and injected intraperitoneally with 1.5 g glucose / kg body weight (20% D-glucose (Sigma) in 0.9% saline) for the GTT, and 1.0 U insulin / kg body weight (Humolog, Lily, Indianapolis, USA) for the ITT. All studies were approved by and performed according to the guidelines of the Institutional Animal Care and Use Committee of the University of Cincinnati, USA. Mitochondrial Isolation and Seahorse Respirometry Mitochondria were isolated from quadriceps muscle, brown adipose tissue (BAT), and liver. Tissue samples were excised and transferred into 2‑ml microcentrifuge tubes containing 1 ml of Isolation Buffer 1 (IB1: 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EGTA, 0.5% fatty‑acid–free BSA). Samples were homogenized and centrifuged at 700 × g for 10 min. The supernatant was collected and centrifuged at 14,000 × g for 10 min, and the resulting pellet was resuspended in fresh IB1 and centrifuged again at 10,000 × g for 10 min. This wash step was repeated using Isolation Buffer 2 (IB2: 210 mM mannitol, 70 mM sucrose, 10 mM MgCl₂, 5 mM potassium diphosphate, 10 mM MOPS, 1 mM EGTA). The final mitochondrial pellet was resuspended in 50–200 µl IB2. Mitochondrial oxygen consumption rate (OCR) was measured using an XF‑24 extracellular flux analyzer (Seahorse Bioscience) following a 10‑min calibration at 37°C. Isolated mitochondria (15 µg per well) were loaded into XF‑24 plates in 500 µl MAS‑1 buffer (70 mM sucrose, 220 mM mannitol, 5 mM KH₂PO₄, 5 mM MgCl₂, 50 mM KCl, 2 mM HEPES, 1 mM EDTA, 0.1% fatty‑acid–free BSA, pH 7.4). Sequential injections of substrates and modulators were used to define respiratory states: State II (5 mM glutamate, 5 mM malate), State III (0.25 mM ADP), State IV₀ (1 µM oligomycin), and maximal uncoupled respiration induced by FCCP (300 nM). OCR traces were used to calculate basal respiration, ADP‑stimulated State III respiration, oligomycin‑inhibited State IV₀ respiration, and FCCP‑stimulated maximal respiration. Respiratory control ratios (RCRs) were determined as State III/State IV₀ OCR to assess mitochondrial coupling efficiency across genotypes and dietary conditions. Citrate synthase activity in mitochondrial isolates from quadriceps, BAT, and liver was quantified spectrophotometrically according to the manufacturer’s instructions (CS0720 kit, Sigma-Aldrich, St. Louis, MO, USA). Statistical Analysis Statistical analyses were performed using GraphPad Prism (V.9 or higher) or SPSS (V31). Longitudinal comparisons of body weight and percentage weight gain, and glucose excursions in the GTT and ITT, were performed using Two-Way RM ANOVA followed by Sidak's multiple comparisons test. AUC values and differences in weight, fat and lean mass, fat and lean mass gain, food intake, respiratory quotient and locomotor activity between genotypes within each dietary condition or light/dark phase were evaluated using two-tailed unpaired t-tests. Energy expenditure values were assessed by Analysis of Co-Variance (ANCOVA), using body weight or lean mass as covariates. Results are shown as means ± SEM. Results Higher weight and fat mass in adult Ppif KO mice on SD To investigate the impact of Ppif -deficiency on the development of adult-onset obesity, we conducted body composition analysis, indirect calorimetry, and glucose homeostasis testing in 20.9-weeks-old Ppif WT and KO mice fed standard diet (SD; week 0; Fig. 1 a). KO mice exhibited significantly higher body weight (Fig. 1 b) and fat mass (Fig. 1 c), while lean mass was comparable between genotypes (Fig. 1 d). Although food intake remained unchanged, locomotor activity during the dark phase was significantly increased in KO mice relative to WT mice (Fig. 1 e,f). Energy expenditure, adjusted for lean mass and assessed by analysis of covariance (ANCOVA), was comparable between genotypes during the light (F(1,13) = 0.948, p = 0.348) and dark (F(1,13) = 1.884, p = 0.193) phases (Fig. 1 g,h). Glucose tolerance, evaluated using a glucose tolerance test (GTT), showed no difference between genotypes, with comparable glucose excursions and area under the curve values (Fig. 1 i). However, during the insulin tolerance test (ITT), blood glucose levels at the 120-minute timepoint were significantly higher in KO mice compared to WT mice (Fig. 1 j), although the overall area under the curve (AUC) values did not reach statistical significance. Together, our data show that Ppif KO mice on SD display modestly increased body weight and fat mass, elevated dark-phase locomotor activity, and a slight impairment in late-phase insulin tolerance at baseline, without noticable differences in food intake, energy expenditure, or overall glucose tolerance. HFD exposure escalates adult-onset obesity in Ppif -deficient mice We next divided Ppif WT and KO mice into two weight-matched cohorts, each receiving either SD or HFD, and monitored body weight and body composition over a period of 34 weeks. Under SD, body weight trajectories and percentage weight gain were comparable between WT and KO mice throughout the 34-week-monitoring (Fig. 2 a,b). Under HFD, KO mice showed significantly greater body weight from weeks 28 to 36 (Fig. 2 c). Although weight gain followed a similar trend, higher variance due to the loss of 3 WT and 2 KO mice over the final 8 weeks of measurement prevented statistical significance in Šídák's multiple comparisons testing despite a significant genotype effect (p = 0.0101) in the 2-Way repeated Measures (RM) ANOVA (Fig. 2 d). After 34 weeks, fat mass gain was greater in HFD-fed KO compared to WT mice (Fig. 2 e), while no significant difference in fat mass gain was observed between genotypes under SD. Lean mass gain was also significantly greater in HFD-fed KO relative to WT mice (Fig. 2 f), with no significant difference between genotypes under SD. Collectively, these results indicate that Ppif deficiency worsens diet-induced obesity, leading to a greater accumulation of body weight, fat mass, and lean mass specifically in response to HFD exposure. Indirect calorimetry does not reveal differences in metabolic homeostasis in SD-fed Ppif -deficient mice To determine whether the initially observed increase in adiposity in Ppif -deficient mice on SD at week 0 was accompanied by alterations in energy homeostasis, we repeated indirect calorimetry monitoring after 16 weeks of SD feeding. Body weights were comparable between genotypes (Fig. 3 a), whereas fat mass was moderately increased in KO compared to WT mice (Fig. 3 b; p < 0.05). Lean mass remained comparable between groups (Fig. 3 c). Food intake patterns were similar between WT and KO mice over 72 continuous hours (Fig. 3 d). Likewise, phase-averaged food intake during the light and dark phases did not differ significantly between genotypes (Fig. 3 e). Respiratory quotient (RQ) monitoring displayed the expected diurnal oscillation (Fig. 3 f) and comparable phase-averaged RQ values during the light and dark phase between WT and KO mice (Fig. 3 g). Similarly, locomotor activity followed a diurnal pattern in both genotypes (Fig. 3 h), and both light and dark phase locomotion were comparable between groups (Fig. 3 i). Energy expenditure (EE) traces over 72 hours demonstrated the expected higher activity during the dark phase in both groups (Fig. 3 j). ANCOVA revealed that lean mass was a significant predictor of the average EE (F(1,13) = 8.880, p = 0.011), and genotype also exerted a significant main effect (F(1,13) = 4.927, p = 0.045), visualized by the regression plot of EE vs lean mass (Fig. 4 k). When adjusted for body weight (F(1,13) = 7.165, p = 0.022), the significant genotype effect was no longer present (Fig. 3 l; F(1,13) = 1.177, p = 0.298). Phase-averaged EE, after adjustment for lean mass, showed a trend toward a genotype effect during the light phase (F(1,13) = 3.661, p = 0.078), and was significantly higher in the dark phase (F(1,13) = 5.733, p = 0.032) in Ppif KO mice (Fig. 3 m,n). In summary, these findings indicate that Ppif KO mice on standard diet at 16 weeks show a modest yet significant increase in fat mass and energy expenditure, but no detectable differences in body weight and lean mass, or food intake, substrate utilization land locomotor activity, compared to WT controls. Ppif -deficient mice are more prone to develop high fat diet-induced obesity After 16 weeks of HFD feeding, Ppif WT and KO mice showed significantly increased body weight and fat mass compared with WT mice (Fig. 4 a,b). Lean mass was comparable across genotypes (Fig. 4 c). Continuous 72-hour food intake monitoring (Fig. 4 d) revealed comparable average food intake during the light and dark phases (Fig. 4 e). Respiratory quotient traces over 72 hours showed comparable oscillations between WT and KO mice (Fig. 4 f); however, the phase-averaged RQ during the dark phase was significantly lower in KO mice (Fig. 4 g), indicating a higher utilization of lipids as fuel substrate. Total locomotor activity over 72 hours showed comparable patterns between groups (Fig. 4 h), and phase-averaged locomotor activity during both light and dark phases was not different between genotypes (Fig. 4 i). However, total energy expenditure (EE) over 72 hours (see Fig. 4 j) was noticeably higher in knockout (KO) mice compared to wild-type (WT) mice. ANCOVA, after adjusting for lean mass, confirmed a significant effect of genotype (F(1, df) = 7.709, p = 0.017), with lean mass being a strong predictor of EE (F(1, df) = 19.597, p < 0.001). Furthermore, a significant Genotype × Lean Mass interaction (F(1, df) = 6.547, p = 0.025) indicated differing EE scaling between genotypes, which was visualized by differing slopes for the linear regression of total EE versus lean mass (Fig. 4 k). Of note, adjustment for body weight in the ANCOVA did not yield significance between genotypes, indicated by largely overlapping regression lines for EE plotted vs body weight (genotype: F(1,12) = 0.280, p = 0.606; body weight: F(1,12) = 12.210; p = 0.004, genotype × body weight: F(1,12) = 0.237, p = 0.635, demonstrated also by largely overlapping regression lines for EE plotted vs body weight (Fig. 4 l). Lean mass-adjusted ANCOVA analyses and regression plots (Fig. 4 m,n) further revealed higher light phase EE (genotype: F(1,12) = 8.020, p = 0.015; lean mass: F(1,12) = 23.906, p < 0.001) and higher dark-phase EE (genotype: F(1,12) = 6.018, p = 0.030; lean mass: F(1,12) = 13.041, p = 0.004) with a significant interaction between genotype and lean mass (light phase EE: F(1,12) = 6.944, p = 0.022; dark-phase EE: F(1,12) = 5.020, p = 0.045). Taken together, these data reveal that HFD-fed Ppif KO mice develop greater obesity than WT mice and exhibit selectively increased dark-phase respiratory quotient and light- and dark-phase energy expenditure without differences in food intake or locomotor activity. Comparable glucose homeostasis in Ppif WT and KO mice on SD and HFD At week 15 of SD exposure, intraperitoneal GTTs revealed comparable glucose excursions and AUC values in WT and KO mice (Fig. 5 a). In HFD-fed mice, both groups had more pronounced and higher glucose excursions compared to those on the SD. Although the AUC for the KO group seemed higher than that of the WT group, there were no statistically significant differences detected between the two genotypes (Fig. 5 b; Two-Way Repeated Measures ANOVA left panel: p = 0.088; t-test right panel: p = 0.12).. ITTs at week 17 of diet exposure in SD-fed mice revealed a comparable decrease in blood glucose following insulin injection, with no significant differences in AUC values between groups (Fig. 5 c). In the HFD-fed ITT, both groups exhibited a blunted glucose-lowering response relative to SD-fed animals, yet AUC values again were not different between WT and KO mice (Fig. 5 d). Collectively, these results indicate that Ppif deficiency did not significantly alter glucose tolerance or insulin sensitivity under either SD or HFD conditions at these timepoints. Elevated State 3 respiration in quadriceps mitochondria from Ppif KO mice To determine if the metabolic phenotype of Ppif knockout (KO) mice was linked to differences in mitochondrial respiratory function or mitochondrial content, we conducted mitochondrial respiration assays and measured citrate synthase activity in isolated mitochondria from the quadriceps, brown adipose tissue (BAT), and liver of wild-type (WT) and KO mice fed SD or HFD for 34 weeks (using four randomly selected mice per group). At the time of sacrifice, the surviving Ppif WT (n = 6) and KO mice (n = 5) on SD had comparable weight, fat and lean mass, respectively. Ppif KO mice on HFD (n = 8) had higher weights (Fig. 2 B), fat mass (KO: 20.74 ± 2.31, WT: 10.45 ± 1.77; p = 0.0041) and lean mass (WT: 22.83 ± 1.08 g, KO: 27.95 ± 1.36 g; p = 0.012) compared to their WT counterparts (n = 6). In quadriceps mitochondria, ADP-stimulated State III respiration was significantly increased in KO mice compared to WT mice under both SD (Fig. 6 a) and HFD (Fig. 6 b), while no additional significant differences were detected at other respiratory states. Respiratory control ratio (RCR) values in quadriceps were not different between genotypes under either dietary condition (Fig. 6 c). In BAT, oxygen consumption rate (OCR) profiles were comparable between WT and KO mice under SD (Fig. 6 d) or HFD (Fig. 6 e), and RCR values were similarly comparable between genotypes under both diets (Fig. 6 f). In liver, OCR profiles under both SD and HFD and RCR values under both dietary conditions were comparable between WT and KO mice (Fig. 6 g-i). Citrate synthase activity was not significantly different between WT and KO mice across all three tissues under SD (Fig. 6 j) or HFD (Fig. 6 k) conditions, indicating comparable mitochondrial content between genotypes. Across all tissues and dietary conditions, liver mitochondria showed the lowest citrate synthase activity, while quadriceps and BAT exhibited higher and relatively similar levels. In summary, these findings indicate that Ppif deficiency selectively increased ADP-stimulated mitochondrial respiration in quadriceps under both dietary conditions, without broadly altering mitochondrial coupling efficiency or mitochondrial content across metabolically active tissues. Discussion This study shows that whole‑body Ppif deficiency exacerbates age-onset obesity when 20-week-old mice are exposed to HFD for up to 36 weeks. Under SD, Ppif ‑deficient mice displayed modestly elevated fat mass early in adulthood, despite unchanged food intake, lean mass, and weight trajectories. Importantly, EE, adjusted for lean mass and analyzed by ANCOVA, was higher in Ppif ‑deficient mice both under SD and HFD, with a significant genotype × lean mass interaction in HFD-fed KO mice, indicating that lean‑mass‑derived tissues, especially skeletal muscle, disproportionately contribute to EE differences. Direct mitochondrial phenotyping supports this interpretation. After 36 weeks of HFD, obese Ppif ‑deficient mice showed selectively increased ADP‑stimulated oxidative phosphorylation in quadriceps, but not in BAT or liver, without changes in mitochondrial abundance or coupling efficiency. These data indicate that CypD loss enhances muscle oxidative responsiveness, while other metabolic tissues remain comparatively unaffected. Together, these findings establish that CypD shapes whole‑body metabolism through diet‑dependent and tissue‑specific effects, particularly within skeletal muscle. CypD, mPTP sensitization, and tissue‑specific metabolic phenotypes CypD regulates the Ca²⁺ and redox sensitivity of the mitochondrial permeability transition pore (mPTP), lowering the threshold for pore opening and thereby influencing cell survival, ROS signaling, and metabolic flexibility [ 1 , 3 , 6 ]. Consequences of CypD deletion are strongly context‑dependent. For instance, CypD is essential for necrotic cell death in Ca²⁺‑overloaded dystrophic muscle [ 13 ], 2008), but dispensable for denervation‑induced atrophy [ 4 ]. Our findings align with this model of conditional mPTP involvement. The elevated ADP‑stimulated respiration in the quadriceps of HFD-fed Ppif ‑deficient mice suggests that reduced mPTP susceptibility enhances mitochondrial stability during sustained nutrient excess. This is consistent with reports that CypD deletion protects mitochondria under metabolic or Ca²⁺ stress [ 17 , 25 ]. However, it should be noted that our study was conducted with isolated mitochondria; in situ assays in permeabilized muscle fibers may differ drastically and are thus warranted, e.g., to exclude an isolation-induced bias by differentially enriching subsarcolemmal vs intermyofibrillar mitochondria. CypD and mitochondrial fuel selection CypD loss‑ and gain‑of‑function have been linked to altered metabolic substrate preference. Proteomic profiling of CypD‑null hearts revealed broad remodeling of the TCA cycle, branched‑chain amino acid catabolism, β‑oxidation, and pyruvate metabolism [ 12 ]. Other studies report that CypD deficiency biases toward greater reliance on carbohydrate metabolism and reduced fatty‑acid oxidation [ 18 , 22 ]. In pancreatic β‑cells, reduced mPTP opening enhances coupling efficiency and insulin secretion [ 21 ], further implicating CypD in the regulation of metabolic substrates. Our long‑term phenotyping aligns with this metabolic shift. Ppif ‑deficient mice accumulated more fat mass on both SD and HFD without consuming more calories, indicating altered metabolic partitioning rather than hyperphagia. Together with the observed increase in skeletal muscle oxidative phosphorylation, without accompanying changes in mitochondrial content, this pattern suggests a preferential use of glucose‑supported respiration, consistent with the AMPK‑driven remodeling described in other CypD‑null models [ 18 ]. Future studies are therefore mandated to integrate longitudinal body‑weight trajectories with tissue‑specific mitochondrial respiration analyses in the same animals, to determine how early divergence in weight gain relates to the distinct respiratory phenotypes we observe across muscle, liver, and adipose tissue. This approach will ultimately help delineate organ‑specific functions of Cyclophilin D in directing fuel utilization and systemic metabolic balance. Tissue‑specificity of CypD effects CypD action varies markedly across tissues. The heart, which expresses high CypD levels, shows strong CypD‑dependent regulation of mPTP activity, Ca²⁺ handling, and susceptibility to heart failure [ 7 ]. In contrast, skeletal muscle exhibits relatively low CypD dependence at rest but stronger involvement during activity‑induced Ca²⁺ flux [ 24 ]. Liver‑specific Ppif deletion increases visceral adiposity and glucose intolerance under HFD, whereas muscle‑specific deletion has minimal effects [ 10 ]. Our respirometry data mirror this tissue‑selective pattern. Only the quadriceps showed increased respiration in Ppif ‑deficient mice, the BAT and liver did not. This skeletal‑muscle‑restricted enhancement of oxidative phosphorylation may explain why EE, adjusted for lean mass, increases in Ppif ‑deficient mice, yet adiposity still rises, as non‑muscle tissues likely exhibit altered survival and storage properties upon Ppif deletion than muscle. CypD also has direct roles in adipocyte biology. High‑fat diet induces adipocyte cell death through a CypD‑dependent intrinsic pathway [ 8 ], and CypD loss protects adipocytes from lipotoxic necrosis [ 5 ]. This protection may favor adipose expansion. Together with our findings, this suggests that increased fat mass in Ppif ‑deficient mice, also under SD feeding, may reflect enhanced adipocyte survival, reduced inflammatory turnover, and greater lipid storage capacity. However, this hypothesis requires further experimental validation. Discrepant reports on obesity susceptibility in Ppif KO mice Previous studies on the metabolism of whole-body CypD knockout mice have produced inconsistent results. Ppif ‑deficient mice either displayed protection from diet‑induced obesity [ 5 ] or a higher propensity for adult‑onset obesity developing under normal SD conditions [ 11 ]. The latter phenotype was further corroborated in liver‑specific Ppif KO mice, which developed increased adiposity and glucose intolerance on HFD, whereas skeletal‑muscle–specific deletion produced no differences in body weight or fat mass [ 10 ]. Our results align more closely with the studies reporting heightened susceptibility to obesity upon loss of CypD. Differences in genetic background, gut microbiome composition, and housing environment remain unavoidable sources of variation across CypD‑knockout studies, including our own, and may interact with genotype in ways that are difficult to fully control. Such cohort‑specific influences likely contribute to the discrepant metabolic fates reported for global or tissue-specific Ppif ‑deficient mice. A clearer understanding of this susceptibility may require deeper analysis of Ppif functions in metabolically active tissues. Such studies should entail adipose‑tissue biology in Ppif ‑deficient mice, including adipocyte size distribution, lipid‑turnover kinetics, macrophage infiltration, and crown‑like structure formation, to determine whether the phenotype reflects cell‑autonomous adipocyte effects, altered adipose immune dynamics [ 8 ], or both. Given the divergent phenotypes of liver‑ and muscle‑specific knockouts, adipocyte‑specific and myeloid‑specific conditional Ppif deletions could help resolve the tissue contributions to whole‑body adiposity. Energy expenditure changes upon Ppif deletion A major discrepancy with earlier reports of reduced obesity susceptibility in Ppif -deficient mice arises from differences in energy expenditure analysis. Devalaraja‑Narashimha et al. [ 5 ] normalized VO₂ to total body weight, a ratio‑based approach now recognized as statistically invalid and prone to misleading interpretations. By contrast, applying ANCOVA with lean mass as the covariate, as recommended by Tschöp et al. [ 23 ], reveals that lean mass is the primary determinant of EE and that genotype modifies the EE–lean‑mass relationship. This methodological distinction likely explains why we observe higher lean‑mass–adjusted EE together with greater adiposity, whereas earlier work suggested protection. Although our calorimetry was performed at discrete timepoints, within-animal, time-aligned studies with longitudinal ANCOVA‑based EE analysis will nonetheless be required to define causal relationships and determine how these effects evolve over time. Such studies may moreover also include housing at thermoneutrality (~ 30°C), to dissociate basal metabolic differences from thermoregulatory demand and to test the temperature dependence of tissue-resolved mitochondrial phenotypes upon Ppif deletion. Last, increased whole‑body VO₂ does not necessarily capture the full spectrum of metabolic costs. Indirect calorimetry quantifies EE exclusively via oxygen‑dependent oxidative metabolism and therefore fails to detect oxygen‑minimal or ATP‑independent energetic processes. Such processes, including NADPH‑driven redox cycling, transsulfuration and glutathione turnover, augmented dNTP and serine/one‑carbon biosynthesis, proteostatic repair, and substrate‑cycling “futility” pathways, are prominent features of mitochondrial stress remodeling, and have been demonstrated across multiple models of mitochondrial dysfunction, where substantial increases in cellular energy demand occur without proportional increases in VO₂ [ 2 , 14 , 15 , 20 ]. Such uncaptured metabolic costs, whether arising early in the phenotype’s development or emerging later once a new adiposity setpoint has been established, combined with potential differences in nutrient absorption, substrate utilization, or lipid‑storage capacity, may help explain why Ppif ‑deficient mice display hypermetabolism yet still accumulate excess adiposity. Because shifts in food intake or energy expenditure can be transient, subtle, or temporally restricted, they may fall outside the specific time window assessed by indirect calorimetry. Likewise, metabolic changes involving resting thermogenesis or muscle‑related energy costs may easily remain undetected because they lie outside the sensitivity range of standard VO₂‑based assays. In this framework, increased obesity is not inconsistent with elevated EE but may instead reflect inefficient or compensatory metabolic rewiring that is not fully quantified by conventional calorimetric measurements. Conclusion Together, our findings position CypD as a diet‑ and tissue‑specific regulator of energy homeostasis, shaping mitochondrial fuel selection, oxidative capacity, adipocyte survival, and whole‑body metabolic adaptability. Rather than acting as a uniformly protective or detrimental factor, CypD emerges as a context‑dependent modulator whose influence on adiposity and mitochondrial function varies with dietary environment, tissue identity, and the energetic demands of individual cell types, consistent with broader models of CypD‑dependent metabolic plasticity. The coexistence of enhanced skeletal‑muscle respiration with greater adiposity in Ppif ‑deficient mice further positions CycD as a potential gatekeeper against hidden energetic sinks in HFD-fed mice and underscores the complex, compensatory nature of mPTP‑linked remodeling across tissues. These insights establish a foundation for precision targeting of the mPTP axis in obesity and metabolic disease, and highlight the need for additional tissue‑specific Ppif knockouts to delineate the distinct contributions of muscle, adipose, and liver to systemic metabolic outcomes. Declarations Research Funding This research received no external funding. Clinical Trial Number Not applicable. This study did not involve a clinical trial and was not registered. Conflict of Interest The authors declare no conflict of interest. Author Contribution J.H. and P.P. conducted the mouse work, W.A. performed mitochondrial respiration assays. J.E. and P.P. conceptualized and designed the studies. P.P. prepared the figures, conducted statistical analyses and wrote the main manuscript text. All authors reviewed the manuscript. Acknowledgement We’d like to thank Zachary Marcus and Nickki Parker for their skilful technical assistance. 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FASEB J 33:11443–11457. 10.1096/fj.201802238R Sergi D, Naumovski N, Heilbronn LK, Abeywardena M, O’Callaghan N, Lionetti L, Luscombe-Marsh N (2019) Mitochondrial (Dys)function and Insulin Resistance: From Pathophysiological Molecular Mechanisms to the Impact of Diet. Front Physiol 10:532. 10.3389/fphys.2019.00532 Southwell N, Primiano G, Nadkarni V, Attarwala N, Beattie E, Miller D, Alam S, Liparulo I, Shurubor YI, Valentino ML, Carelli V, Servidei S, Gross SS, Manfredi G, Chen Q, D’Aurelio M (2023) A coordinated multiorgan metabolic response contributes to human mitochondrial myopathy. EMBO Mol Med 15:e16951. 10.15252/emmm.202216951 Taddeo EP, Alsabeeh N, Baghdasarian S, Wikstrom JD, Ritou E, Sereda S, Erion K, Li J, Stiles L, Abdulla M, Swanson Z, Wilhelm JJ, Bellin MD, Kibbey RG, Liesa M, Shirihai OS (2020) Mitochondrial Proton Leak Regulated by Cyclophilin D Elevates Insulin Secretion in Islets at Nonstimulatory Glucose Levels. Diabetes 69:131–145. 10.2337/db19-0379 Tavecchio M, Lisanti S, Bennett MJ, Languino LR, Altieri DC (2015) Deletion of Cyclophilin D Impairs β-Oxidation and Promotes Glucose Metabolism. Sci Rep 5:15981. 10.1038/srep15981 Tschöp MH, Speakman JR, Arch JRS, Auwerx J, Brüning JC, Chan L, Eckel RH, Farese RV, Galgani JE, Hambly C, Herman MA, Horvath TL, Kahn BB, Kozma SC, Maratos-Flier E, Müller TD, Münzberg H, Pfluger PT, Plum L, Reitman ML, Rahmouni K, Shulman GI, Thomas G, Kahn CR, Ravussin E (2011) A guide to analysis of mouse energy metabolism. Nat Methods 9:57–63. 10.1038/nmeth.1806 Wei-LaPierre L, Ainbinder A, Tylock KM, Dirksen RT (2019) Substrate-dependent and cyclophilin D-independent regulation of mitochondrial flashes in skeletal and cardiac muscle. Arch Biochem Biophys 665:122–131. 10.1016/j.abb.2019.03.003 Zhou S, Yu Q, Zhang L, Jiang Z (2023) Cyclophilin D-mediated Mitochondrial Permeability Transition Regulates Mitochondrial Function. Curr Pharm Des 29:620–629. 10.2174/1381612829666230313111314 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 30 Apr, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers invited by journal 23 Apr, 2026 Editor assigned by journal 21 Apr, 2026 Submission checks completed at journal 21 Apr, 2026 First submitted to journal 20 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9472657","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633584350,"identity":"cf56ce0c-d8e2-455e-9577-b0da5ffbdea1","order_by":0,"name":"Jazzminn L. Hembree","email":"","orcid":"","institution":"Miami University","correspondingAuthor":false,"prefix":"","firstName":"Jazzminn","middleName":"L.","lastName":"Hembree","suffix":""},{"id":633584351,"identity":"fb970f89-51c7-4d54-a7a3-b97b683a1c79","order_by":1,"name":"William A. Abplanalp","email":"","orcid":"","institution":"Cincinnati Children's Hospital Medical Center","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"A.","lastName":"Abplanalp","suffix":""},{"id":633584353,"identity":"67ff05cb-c482-4076-ae8b-cc1a7b5e5c30","order_by":2,"name":"John W. Elrod","email":"","orcid":"","institution":"Lewis Katz School of Medicine at Temple University","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"W.","lastName":"Elrod","suffix":""},{"id":633584354,"identity":"463881ab-f8cc-4e2d-8c6e-0d97f3c5b70f","order_by":3,"name":"Paul T. Pfluger","email":"data:image/png;base64,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","orcid":"","institution":"Helmholtz Zentrum München","correspondingAuthor":true,"prefix":"","firstName":"Paul","middleName":"T.","lastName":"Pfluger","suffix":""}],"badges":[],"createdAt":"2026-04-20 13:08:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9472657/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9472657/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108402733,"identity":"a087bc88-fd3e-4524-8f7a-88499e1de52f","added_by":"auto","created_at":"2026-05-04 09:10:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":417422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental paradigm and metabolic phenotyping of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpif\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e WT and KO mice fed standard diet (SD)\u003c/strong\u003e. \u003cstrong\u003ea) \u003c/strong\u003e\u003cem\u003ePpif\u003c/em\u003e WT and KO mice were monitored for 34 weeks with SD or high fat diet (HFD), indicating the times when glucose tolerance or insulin tolerance tests (GTT/ITT) or combined indirect calorimetry (IC) were performed. Body weight \u003cstrong\u003e(b)\u003c/strong\u003e, fat mass \u003cstrong\u003e(c) \u003c/strong\u003eand lean mass \u003cstrong\u003e(d)\u003c/strong\u003e of SD-fed \u003cem\u003ePpif\u003c/em\u003e WT and KO mice (n=18-20, age 20.9±0.3 weeks). At week 0, subgroups of the SD-fed WT and KO mice (n=8) were subjected to combined indirect calorimetry (n=8) to assess food intake \u003cstrong\u003e(e)\u003c/strong\u003e, locomotor activity \u003cstrong\u003e(f)\u003c/strong\u003e or energy expenditure, statistically assessed by ANCOVA and displayed as linear regression plot of light-phase \u003cstrong\u003e(g)\u003c/strong\u003e and dark-phase \u003cstrong\u003e(h)\u003c/strong\u003eenergy expenditure vs covariate lean mass. Glucose excursions (left panel) and area under the curve values (AUC, right panel after glucose \u003cstrong\u003e(i)\u003c/strong\u003e and insulin \u003cstrong\u003e(j)\u003c/strong\u003e tolerance tests (GTT/ITT, n=8-10). Two-tailed t-tests (b-f,I,j), ANCOVA with covariate lean mass (g,h) and 2-Way RM ANOVA with Šídák's multiple comparisons tests (I,j). Means ± SEM. *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9472657/v1/943b62fd5f570c2366d7f143.png"},{"id":108402731,"identity":"5e41018b-72b3-40ea-9107-33e8df97902c","added_by":"auto","created_at":"2026-05-04 09:10:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":196037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBody weight and body composition trajectories of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpif\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eWT and KO mice fed SD or HFD. \u003c/strong\u003eBody weight \u003cstrong\u003e(a,b)\u003c/strong\u003e, percentage of weight gain \u003cstrong\u003e(c,d)\u003c/strong\u003e and gain of fat \u003cstrong\u003e(e)\u003c/strong\u003e and lean mass \u003cstrong\u003e(f)\u003c/strong\u003e in \u003cem\u003ePpif\u003c/em\u003e WT and KO mice (n=5-10) exposed to SD or HFD for 34 weeks. Two-tailed t-tests (e,f) or 2-Way RM ANOVA with Šídák's multiple comparisons tests (a-d). Means ± SEM. *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9472657/v1/4c3d6f999ab093d4607130ea.png"},{"id":108402730,"identity":"fb3c00ee-3f38-454a-8a8f-4ab74d8ce8c4","added_by":"auto","created_at":"2026-05-04 09:10:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":518870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic phenotype of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpif\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e WT and KO mice fed 16 weeks of SD. \u003c/strong\u003eBody weight \u003cstrong\u003e(a)\u003c/strong\u003e, fat mass \u003cstrong\u003e(b)\u003c/strong\u003e and lean mass \u003cstrong\u003e(c)\u003c/strong\u003e of \u003cem\u003ePpif\u003c/em\u003e WT and KO mice after 16 weeks of SD feeding (n=8). Subsequent combined indirect calorimetry monitoring over 67 hours, averaged for light- and dark-phases, revealed unchanged food intake \u003cstrong\u003e(d,e)\u003c/strong\u003e, respiratory quotients \u003cstrong\u003e(f,g)\u003c/strong\u003e and locomotor activity \u003cstrong\u003e(h,i)\u003c/strong\u003e. Energy expenditure (EE) traces \u003cstrong\u003e(j\u003c/strong\u003e) were subjected to ANCOVA and plotted as average EE vs lean mass \u003cstrong\u003e(k)\u003c/strong\u003e or body weight \u003cstrong\u003e(l)\u003c/strong\u003e, or light- \u003cstrong\u003e(m)\u003c/strong\u003e and dark-phase \u003cstrong\u003e(n)\u003c/strong\u003e EE vs lean mass. Two-tailed t-tests (a,b,c,e,g,i). ANCOVA with covariates lean mass (j,k,m,n) or body weight (j,l). Means ± SEM. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9472657/v1/3b3a0c1fdd9929501df81a6b.png"},{"id":108402736,"identity":"ca88476b-815b-4190-8482-ea35c8260727","added_by":"auto","created_at":"2026-05-04 09:10:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":544128,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic phenotype of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpif\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e WT and KO mice fed 16 weeks of HFD. \u003c/strong\u003eBody weight \u003cstrong\u003e(a)\u003c/strong\u003e, fat mass \u003cstrong\u003e(b)\u003c/strong\u003e, and lean mass \u003cstrong\u003e(c)\u003c/strong\u003e of \u003cem\u003ePpif\u003c/em\u003e WT and KO mice after 16 weeks of high‑fat diet (HFD) feeding (n = 8). Subsequent indirect calorimetry over 72 hours, averaged for light‑ and dark‑phases, revealed unchanged food intake \u003cstrong\u003e(d,e)\u003c/strong\u003e, a significant reduction of dark‑phase respiratory quotient in KO mice \u003cstrong\u003e(f,g)\u003c/strong\u003e, and comparable locomotor activity \u003cstrong\u003e(h,i)\u003c/strong\u003e. Energy expenditure (EE) traces \u003cstrong\u003e(j)\u003c/strong\u003ewere analyzed by ANCOVA and plotted as average EE versus lean mass \u003cstrong\u003e(k)\u003c/strong\u003eor body weight \u003cstrong\u003e(l)\u003c/strong\u003e, as well as lean‑mass–adjusted EE during light \u003cstrong\u003e(m)\u003c/strong\u003e and dark \u003cstrong\u003e(n)\u003c/strong\u003ephases. Two‑tailed t‑tests were used for a,b,c,e,g,i; ANCOVA with covariates lean mass (j,k,m,n) or body weight (j,l). Means ± SEM. *p \u0026lt; 0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9472657/v1/d3166279df4515bca25f2d3e.png"},{"id":108402744,"identity":"48e2ffbc-ceef-4b02-95a9-eab0ed1a9ae3","added_by":"auto","created_at":"2026-05-04 09:10:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlucose tolerance and insulin sensitivity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpif\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e WT and KO mice fed SD and HFD.\u003c/strong\u003e SD and HFD-fed WT and KO mice were subjected to glucose tolerance (\u003cstrong\u003ea,b\u003c/strong\u003e, 15 weeks of diet exposure, n=9-10) or insulin tolerance tests (\u003cstrong\u003ec,d\u003c/strong\u003e, 17 weeks of diet exposure, n=5-8). Two-tailed t-tests and 2-Way RM ANOVA with Šídák's multiple comparisons tests. Means ± SEM.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9472657/v1/892a3cfba492f98cbfedaff6.png"},{"id":108402734,"identity":"b165c382-8d26-4be9-89c7-0b326631bc9f","added_by":"auto","created_at":"2026-05-04 09:10:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":24232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial respiration in skeletal muscle, brown fat and liver in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpif\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e WT and KO mice fed SD or HFD. \u003c/strong\u003eOxygen consumption rates (OCR; \u003cstrong\u003ea,b,d,e,g,h\u003c/strong\u003e), respiratory control ratios (RCR; \u003cstrong\u003ec,f,i\u003c/strong\u003e) and citrate synthase activities (\u003cstrong\u003ej,k\u003c/strong\u003e) in mitochondria isolated from quadriceps \u003cstrong\u003e(a-c)\u003c/strong\u003e, BAT \u003cstrong\u003e(d-f) \u003c/strong\u003eand livers \u003cstrong\u003e(g-i) \u003c/strong\u003eof \u003cem\u003ePpif\u003c/em\u003e WT and KO mice exposed to SD or HFD for 34 weeks. ). Two-tailed t-tests (c,f,I,j,k) and multiple t-tests with Holm-Sidak correction for multiple comparisons (a,b,d,e,g,h). Mice used for mitochondrial isolation \u0026amp; measurement: n=4 per genotype. Means ± SEM. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9472657/v1/8a037b64100653338f4093d1.png"},{"id":108402888,"identity":"52c5feb2-a247-4636-90fb-f1495db82f2d","added_by":"auto","created_at":"2026-05-04 09:11:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1716523,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9472657/v1/630b40ce-253d-414e-a16d-5c6731e94626.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cyclophilin D Deficiency Exacerbates Diet-Induced Obesity and Selectively Enhances Skeletal Muscle Mitochondrial Respiration in Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eObesity and its associated metabolic complications, including type 2 diabetes, dyslipidemia, and cardiovascular disease, represent a growing global health burden that is driven in large part by dysregulated energy homeostasis and adipose tissue expansion. At the cellular level, mitochondria act as key regulators of energy metabolism, and mitochondrial dysfunction is strongly implicated in the development of diet‑induced obesity and insulin resistance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e][\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A key regulator of mitochondrial integrity is the mitochondrial permeability transition pore (mPTP), a high-conductance channel whose aberrant or sustained opening leads to dissipation of the mitochondrial membrane potential, release of pro-apoptotic factors, and disruption of oxidative phosphorylation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Understanding the molecular mechanisms that govern mPTP activity and their downstream metabolic consequences has therefore become an important area of investigation in the context of metabolic disease.\u003c/p\u003e \u003cp\u003eCyclophilin D (CypD), encoded by the \u003cem\u003ePpif\u003c/em\u003e gene, is a matrix peptidyl-prolyl cis-trans isomerase that serves as a critical facilitator of mPTP opening by directly binding to pore constituents including the adenine nucleotide translocator and components for oxidative stress-induced opening, thereby acting as key modulator of mPTP sensitivity and mitochondrial stress responses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Beyond its canonical role in cell death regulation, CypD has emerged as a broader regulator of mitochondrial metabolism, with implications for whole-body energy balance and glucose homeostasis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, reports on its metabolic role are highly divergent, with studies describing protection from, susceptibility to, or altered progression of diet‑induced obesity and glucose intolerance in CypD‑deficient mice. These discrepancies highlight the need to clarify how loss of CypD influences whole‑body metabolic regulation. Furthermore, whether alterations in whole-body metabolic parameters in CypD‑deficient mice are mechanistically linked to tissue-specific changes in mitochondrial oxidative capacity has not been fully elucidated.\u003c/p\u003e \u003cp\u003eHere, we characterize the metabolic phenotype of whole-body \u003cem\u003ePpif\u003c/em\u003e knockout mice undergoing SD and HFD exposure for 34 weeks, incorporating body composition analysis, comprehensive indirect calorimetry, glucose and insulin tolerance testing, and mitochondrial respiratory function measurements across metabolically distinct tissues. Our findings demonstrate that CypD deficiency exacerbates diet-induced obesity in a dietary context-dependent manner, selectively amplifying adiposity, energy expenditure, and skeletal muscle mitochondrial respiration, thereby establishing a tissue- and diet-specific role for CypD in the regulation of whole-body metabolic homeostasis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMouse experiments\u003c/h2\u003e \u003cp\u003eGeneration of \u003cem\u003ePpif\u003c/em\u003e knockout mice has been described previously [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. All mice were group-housed with a 12hr light, 12hr dark cycle (7am-7pm) at 22\u0026deg;C and maintained on a standard chow diet (Harlan Teklad LM-485) or high-fat diet (Research Diets D12492 (60% fat, New Brunswick, NJ, USA), with ad libitum access to food and water.\u003c/p\u003e \u003cp\u003eFat and lean mass were quantified using NMR technology (EchoMRI, Houston, TX, USA). Energy expenditure, respiratory quotient, food intake, and locomotor activity were assessed using a combined indirect calorimetry system (TSE Systems GmbH, Bad Homburg, Germany) as described previously [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Mice had ad libitum access to food and water and were acclimated to metabolic cages for at least 24 hours followed by the simultaneous measurement over a 72‑hour period. Intraperitoneal glucose and insulin tolerance testing (GTT/ITT) was conducted in mice subjected to 6 hours of fasting and injected intraperitoneally with 1.5 g glucose / kg body weight (20% D-glucose (Sigma) in 0.9% saline) for the GTT, and 1.0 U insulin / kg body weight (Humolog, Lily, Indianapolis, USA) for the ITT. All studies were approved by and performed according to the guidelines of the Institutional Animal Care and Use Committee of the University of Cincinnati, USA.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMitochondrial Isolation and Seahorse Respirometry\u003c/h3\u003e\n\u003cp\u003eMitochondria were isolated from quadriceps muscle, brown adipose tissue (BAT), and liver. Tissue samples were excised and transferred into 2‑ml microcentrifuge tubes containing 1 ml of Isolation Buffer 1 (IB1: 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EGTA, 0.5% fatty‑acid\u0026ndash;free BSA). Samples were homogenized and centrifuged at 700 \u0026times; g for 10 min. The supernatant was collected and centrifuged at 14,000 \u0026times; g for 10 min, and the resulting pellet was resuspended in fresh IB1 and centrifuged again at 10,000 \u0026times; g for 10 min. This wash step was repeated using Isolation Buffer 2 (IB2: 210 mM mannitol, 70 mM sucrose, 10 mM MgCl₂, 5 mM potassium diphosphate, 10 mM MOPS, 1 mM EGTA). The final mitochondrial pellet was resuspended in 50\u0026ndash;200 \u0026micro;l IB2.\u003c/p\u003e \u003cp\u003eMitochondrial oxygen consumption rate (OCR) was measured using an XF‑24 extracellular flux analyzer (Seahorse Bioscience) following a 10‑min calibration at 37\u0026deg;C. Isolated mitochondria (15 \u0026micro;g per well) were loaded into XF‑24 plates in 500 \u0026micro;l MAS‑1 buffer (70 mM sucrose, 220 mM mannitol, 5 mM KH₂PO₄, 5 mM MgCl₂, 50 mM KCl, 2 mM HEPES, 1 mM EDTA, 0.1% fatty‑acid\u0026ndash;free BSA, pH 7.4). Sequential injections of substrates and modulators were used to define respiratory states: State II (5 mM glutamate, 5 mM malate), State III (0.25 mM ADP), State IV₀ (1 \u0026micro;M oligomycin), and maximal uncoupled respiration induced by FCCP (300 nM). OCR traces were used to calculate basal respiration, ADP‑stimulated State III respiration, oligomycin‑inhibited State IV₀ respiration, and FCCP‑stimulated maximal respiration. Respiratory control ratios (RCRs) were determined as State III/State IV₀ OCR to assess mitochondrial coupling efficiency across genotypes and dietary conditions.\u003c/p\u003e \u003cp\u003eCitrate synthase activity in mitochondrial isolates from quadriceps, BAT, and liver was quantified spectrophotometrically according to the manufacturer\u0026rsquo;s instructions (CS0720 kit, Sigma-Aldrich, St. Louis, MO, USA).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism (V.9 or higher) or SPSS (V31). Longitudinal comparisons of body weight and percentage weight gain, and glucose excursions in the GTT and ITT, were performed using Two-Way RM ANOVA followed by Sidak's multiple comparisons test. AUC values and differences in weight, fat and lean mass, fat and lean mass gain, food intake, respiratory quotient and locomotor activity between genotypes within each dietary condition or light/dark phase were evaluated using two-tailed unpaired t-tests. Energy expenditure values were assessed by Analysis of Co-Variance (ANCOVA), using body weight or lean mass as covariates. Results are shown as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eHigher weight and fat mass in adult\u003c/b\u003e \u003cb\u003ePpif\u003c/b\u003e \u003cb\u003eKO mice on SD\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the impact of \u003cem\u003ePpif\u003c/em\u003e-deficiency on the development of adult-onset obesity, we conducted body composition analysis, indirect calorimetry, and glucose homeostasis testing in 20.9-weeks-old \u003cem\u003ePpif\u003c/em\u003e WT and KO mice fed standard diet (SD; week 0; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). KO mice exhibited significantly higher body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and fat mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), while lean mass was comparable between genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Although food intake remained unchanged, locomotor activity during the dark phase was significantly increased in KO mice relative to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee,f). Energy expenditure, adjusted for lean mass and assessed by analysis of covariance (ANCOVA), was comparable between genotypes during the light (F(1,13)\u0026thinsp;=\u0026thinsp;0.948, p\u0026thinsp;=\u0026thinsp;0.348) and dark (F(1,13)\u0026thinsp;=\u0026thinsp;1.884, p\u0026thinsp;=\u0026thinsp;0.193) phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg,h). Glucose tolerance, evaluated using a glucose tolerance test (GTT), showed no difference between genotypes, with comparable glucose excursions and area under the curve values (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). However, during the insulin tolerance test (ITT), blood glucose levels at the 120-minute timepoint were significantly higher in KO mice compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej), although the overall area under the curve (AUC) values did not reach statistical significance. Together, our data show that \u003cem\u003ePpif\u003c/em\u003e KO mice on SD display modestly increased body weight and fat mass, elevated dark-phase locomotor activity, and a slight impairment in late-phase insulin tolerance at baseline, without noticable differences in food intake, energy expenditure, or overall glucose tolerance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHFD exposure escalates adult-onset obesity in\u003c/b\u003e \u003cb\u003ePpif\u003c/b\u003e\u003cb\u003e-deficient mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe next divided \u003cem\u003ePpif\u003c/em\u003e WT and KO mice into two weight-matched cohorts, each receiving either SD or HFD, and monitored body weight and body composition over a period of 34 weeks. Under SD, body weight trajectories and percentage weight gain were comparable between WT and KO mice throughout the 34-week-monitoring (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b). Under HFD, KO mice showed significantly greater body weight from weeks 28 to 36 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Although weight gain followed a similar trend, higher variance due to the loss of 3 WT and 2 KO mice over the final 8 weeks of measurement prevented statistical significance in Š\u0026iacute;d\u0026aacute;k's multiple comparisons testing despite a significant genotype effect (p\u0026thinsp;=\u0026thinsp;0.0101) in the 2-Way repeated Measures (RM) ANOVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). After 34 weeks, fat mass gain was greater in HFD-fed KO compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), while no significant difference in fat mass gain was observed between genotypes under SD. Lean mass gain was also significantly greater in HFD-fed KO relative to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), with no significant difference between genotypes under SD. Collectively, these results indicate that \u003cem\u003ePpif\u003c/em\u003e deficiency worsens diet-induced obesity, leading to a greater accumulation of body weight, fat mass, and lean mass specifically in response to HFD exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIndirect calorimetry does not reveal differences in metabolic homeostasis in SD-fed\u003c/b\u003e \u003cb\u003ePpif\u003c/b\u003e\u003cb\u003e-deficient mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether the initially observed increase in adiposity in \u003cem\u003ePpif\u003c/em\u003e-deficient mice on SD at week 0 was accompanied by alterations in energy homeostasis, we repeated indirect calorimetry monitoring after 16 weeks of SD feeding. Body weights were comparable between genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), whereas fat mass was moderately increased in KO compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Lean mass remained comparable between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Food intake patterns were similar between WT and KO mice over 72 continuous hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Likewise, phase-averaged food intake during the light and dark phases did not differ significantly between genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Respiratory quotient (RQ) monitoring displayed the expected diurnal oscillation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef) and comparable phase-averaged RQ values during the light and dark phase between WT and KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Similarly, locomotor activity followed a diurnal pattern in both genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh), and both light and dark phase locomotion were comparable between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). Energy expenditure (EE) traces over 72 hours demonstrated the expected higher activity during the dark phase in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). ANCOVA revealed that lean mass was a significant predictor of the average EE (F(1,13)\u0026thinsp;=\u0026thinsp;8.880, p\u0026thinsp;=\u0026thinsp;0.011), and genotype also exerted a significant main effect (F(1,13)\u0026thinsp;=\u0026thinsp;4.927, p\u0026thinsp;=\u0026thinsp;0.045), visualized by the regression plot of EE vs lean mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). When adjusted for body weight (F(1,13)\u0026thinsp;=\u0026thinsp;7.165, p\u0026thinsp;=\u0026thinsp;0.022), the significant genotype effect was no longer present (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el; F(1,13)\u0026thinsp;=\u0026thinsp;1.177, p\u0026thinsp;=\u0026thinsp;0.298). Phase-averaged EE, after adjustment for lean mass, showed a trend toward a genotype effect during the light phase (F(1,13)\u0026thinsp;=\u0026thinsp;3.661, p\u0026thinsp;=\u0026thinsp;0.078), and was significantly higher in the dark phase (F(1,13)\u0026thinsp;=\u0026thinsp;5.733, p\u0026thinsp;=\u0026thinsp;0.032) in \u003cem\u003ePpif\u003c/em\u003e KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em,n). In summary, these findings indicate that \u003cem\u003ePpif\u003c/em\u003e KO mice on standard diet at 16 weeks show a modest yet significant increase in fat mass and energy expenditure, but no detectable differences in body weight and lean mass, or food intake, substrate utilization land locomotor activity, compared to WT controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePpif\u003c/b\u003e \u003cb\u003e-deficient mice are more prone to develop high fat diet-induced obesity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter 16 weeks of HFD feeding, Ppif WT and KO mice showed significantly increased body weight and fat mass compared with WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). Lean mass was comparable across genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Continuous 72-hour food intake monitoring (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) revealed comparable average food intake during the light and dark phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Respiratory quotient traces over 72 hours showed comparable oscillations between WT and KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef); however, the phase-averaged RQ during the dark phase was significantly lower in KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), indicating a higher utilization of lipids as fuel substrate. Total locomotor activity over 72 hours showed comparable patterns between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), and phase-averaged locomotor activity during both light and dark phases was not different between genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). However, total energy expenditure (EE) over 72 hours (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej) was noticeably higher in knockout (KO) mice compared to wild-type (WT) mice. ANCOVA, after adjusting for lean mass, confirmed a significant effect of genotype (F(1, df)\u0026thinsp;=\u0026thinsp;7.709, p\u0026thinsp;=\u0026thinsp;0.017), with lean mass being a strong predictor of EE (F(1, df)\u0026thinsp;=\u0026thinsp;19.597, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, a significant Genotype \u0026times; Lean Mass interaction (F(1, df)\u0026thinsp;=\u0026thinsp;6.547, p\u0026thinsp;=\u0026thinsp;0.025) indicated differing EE scaling between genotypes, which was visualized by differing slopes for the linear regression of total EE versus lean mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). Of note, adjustment for body weight in the ANCOVA did not yield significance between genotypes, indicated by largely overlapping regression lines for EE plotted vs body weight (genotype: F(1,12)\u0026thinsp;=\u0026thinsp;0.280, p\u0026thinsp;=\u0026thinsp;0.606; body weight: F(1,12)\u0026thinsp;=\u0026thinsp;12.210; p\u0026thinsp;=\u0026thinsp;0.004, genotype \u0026times; body weight: F(1,12)\u0026thinsp;=\u0026thinsp;0.237, p\u0026thinsp;=\u0026thinsp;0.635, demonstrated also by largely overlapping regression lines for EE plotted vs body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el). Lean mass-adjusted ANCOVA analyses and regression plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em,n) further revealed higher light phase EE (genotype: F(1,12)\u0026thinsp;=\u0026thinsp;8.020, p\u0026thinsp;=\u0026thinsp;0.015; lean mass: F(1,12)\u0026thinsp;=\u0026thinsp;23.906, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and higher dark-phase EE (genotype: F(1,12)\u0026thinsp;=\u0026thinsp;6.018, p\u0026thinsp;=\u0026thinsp;0.030; lean mass: F(1,12)\u0026thinsp;=\u0026thinsp;13.041, p\u0026thinsp;=\u0026thinsp;0.004) with a significant interaction between genotype and lean mass (light phase EE: F(1,12)\u0026thinsp;=\u0026thinsp;6.944, p\u0026thinsp;=\u0026thinsp;0.022; dark-phase EE: F(1,12)\u0026thinsp;=\u0026thinsp;5.020, p\u0026thinsp;=\u0026thinsp;0.045). Taken together, these data reveal that HFD-fed Ppif KO mice develop greater obesity than WT mice and exhibit selectively increased dark-phase respiratory quotient and light- and dark-phase energy expenditure without differences in food intake or locomotor activity.\u003c/p\u003e\n\u003ch3\u003eComparable glucose homeostasis in Ppif WT and KO mice on SD and HFD\u003c/h3\u003e\n\u003cp\u003eAt week 15 of SD exposure, intraperitoneal GTTs revealed comparable glucose excursions and AUC values in WT and KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In HFD-fed mice, both groups had more pronounced and higher glucose excursions compared to those on the SD. Although the AUC for the KO group seemed higher than that of the WT group, there were no statistically significant differences detected between the two genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb; Two-Way Repeated Measures ANOVA left panel: p\u0026thinsp;=\u0026thinsp;0.088; t-test right panel: p\u0026thinsp;=\u0026thinsp;0.12).. ITTs at week 17 of diet exposure in SD-fed mice revealed a comparable decrease in blood glucose following insulin injection, with no significant differences in AUC values between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In the HFD-fed ITT, both groups exhibited a blunted glucose-lowering response relative to SD-fed animals, yet AUC values again were not different between WT and KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Collectively, these results indicate that \u003cem\u003ePpif\u003c/em\u003e deficiency did not significantly alter glucose tolerance or insulin sensitivity under either SD or HFD conditions at these timepoints.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eElevated State 3 respiration in quadriceps mitochondria from Ppif KO mice\u003c/h2\u003e \u003cp\u003eTo determine if the metabolic phenotype of Ppif knockout (KO) mice was linked to differences in mitochondrial respiratory function or mitochondrial content, we conducted mitochondrial respiration assays and measured citrate synthase activity in isolated mitochondria from the quadriceps, brown adipose tissue (BAT), and liver of wild-type (WT) and KO mice fed SD or HFD for 34 weeks (using four randomly selected mice per group). At the time of sacrifice, the surviving Ppif WT (n\u0026thinsp;=\u0026thinsp;6) and KO mice (n\u0026thinsp;=\u0026thinsp;5) on SD had comparable weight, fat and lean mass, respectively. \u003cem\u003ePpif\u003c/em\u003e KO mice on HFD (n\u0026thinsp;=\u0026thinsp;8) had higher weights (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), fat mass (KO: 20.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31, WT: 10.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77; p\u0026thinsp;=\u0026thinsp;0.0041) and lean mass (WT: 22.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 g, KO: 27.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 g; p\u0026thinsp;=\u0026thinsp;0.012) compared to their WT counterparts (n\u0026thinsp;=\u0026thinsp;6). In quadriceps mitochondria, ADP-stimulated State III respiration was significantly increased in KO mice compared to WT mice under both SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) and HFD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), while no additional significant differences were detected at other respiratory states. Respiratory control ratio (RCR) values in quadriceps were not different between genotypes under either dietary condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). In BAT, oxygen consumption rate (OCR) profiles were comparable between WT and KO mice under SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed) or HFD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee), and RCR values were similarly comparable between genotypes under both diets (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). In liver, OCR profiles under both SD and HFD and RCR values under both dietary conditions were comparable between WT and KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-i). Citrate synthase activity was not significantly different between WT and KO mice across all three tissues under SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej) or HFD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek) conditions, indicating comparable mitochondrial content between genotypes. Across all tissues and dietary conditions, liver mitochondria showed the lowest citrate synthase activity, while quadriceps and BAT exhibited higher and relatively similar levels. In summary, these findings indicate that \u003cem\u003ePpif\u003c/em\u003e deficiency selectively increased ADP-stimulated mitochondrial respiration in quadriceps under both dietary conditions, without broadly altering mitochondrial coupling efficiency or mitochondrial content across metabolically active tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study shows that whole‑body \u003cem\u003ePpif\u003c/em\u003e deficiency exacerbates age-onset obesity when 20-week-old mice are exposed to HFD for up to 36 weeks. Under SD, \u003cem\u003ePpif\u003c/em\u003e‑deficient mice displayed modestly elevated fat mass early in adulthood, despite unchanged food intake, lean mass, and weight trajectories. Importantly, EE, adjusted for lean mass and analyzed by ANCOVA, was higher in \u003cem\u003ePpif\u003c/em\u003e‑deficient mice both under SD and HFD, with a significant genotype \u0026times; lean mass interaction in HFD-fed KO mice, indicating that lean‑mass‑derived tissues, especially skeletal muscle, disproportionately contribute to EE differences.\u003c/p\u003e \u003cp\u003eDirect mitochondrial phenotyping supports this interpretation. After 36 weeks of HFD, obese \u003cem\u003ePpif\u003c/em\u003e‑deficient mice showed selectively increased ADP‑stimulated oxidative phosphorylation in quadriceps, but not in BAT or liver, without changes in mitochondrial abundance or coupling efficiency. These data indicate that CypD loss enhances muscle oxidative responsiveness, while other metabolic tissues remain comparatively unaffected. Together, these findings establish that CypD shapes whole‑body metabolism through diet‑dependent and tissue‑specific effects, particularly within skeletal muscle.\u003c/p\u003e\n\u003ch3\u003eCypD, mPTP sensitization, and tissue‑specific metabolic phenotypes\u003c/h3\u003e\n\u003cp\u003eCypD regulates the Ca\u0026sup2;⁺ and redox sensitivity of the mitochondrial permeability transition pore (mPTP), lowering the threshold for pore opening and thereby influencing cell survival, ROS signaling, and metabolic flexibility [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Consequences of CypD deletion are strongly context‑dependent. For instance, CypD is essential for necrotic cell death in Ca\u0026sup2;⁺‑overloaded dystrophic muscle [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], 2008), but dispensable for denervation‑induced atrophy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Our findings align with this model of conditional mPTP involvement. The elevated ADP‑stimulated respiration in the quadriceps of HFD-fed \u003cem\u003ePpif\u003c/em\u003e‑deficient mice suggests that reduced mPTP susceptibility enhances mitochondrial stability during sustained nutrient excess. This is consistent with reports that CypD deletion protects mitochondria under metabolic or Ca\u0026sup2;⁺ stress [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, it should be noted that our study was conducted with isolated mitochondria; in situ assays in permeabilized muscle fibers may differ drastically and are thus warranted, e.g., to exclude an isolation-induced bias by differentially enriching subsarcolemmal vs intermyofibrillar mitochondria.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCypD and mitochondrial fuel selection\u003c/h2\u003e \u003cp\u003eCypD loss‑ and gain‑of‑function have been linked to altered metabolic substrate preference. Proteomic profiling of CypD‑null hearts revealed broad remodeling of the TCA cycle, branched‑chain amino acid catabolism, β‑oxidation, and pyruvate metabolism [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Other studies report that CypD deficiency biases toward greater reliance on carbohydrate metabolism and reduced fatty‑acid oxidation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In pancreatic β‑cells, reduced mPTP opening enhances coupling efficiency and insulin secretion [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], further implicating CypD in the regulation of metabolic substrates.\u003c/p\u003e \u003cp\u003eOur long‑term phenotyping aligns with this metabolic shift. \u003cem\u003ePpif\u003c/em\u003e‑deficient mice accumulated more fat mass on both SD and HFD without consuming more calories, indicating altered metabolic partitioning rather than hyperphagia. Together with the observed increase in skeletal muscle oxidative phosphorylation, without accompanying changes in mitochondrial content, this pattern suggests a preferential use of glucose‑supported respiration, consistent with the AMPK‑driven remodeling described in other CypD‑null models [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Future studies are therefore mandated to integrate longitudinal body‑weight trajectories with tissue‑specific mitochondrial respiration analyses in the same animals, to determine how early divergence in weight gain relates to the distinct respiratory phenotypes we observe across muscle, liver, and adipose tissue. This approach will ultimately help delineate organ‑specific functions of Cyclophilin D in directing fuel utilization and systemic metabolic balance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTissue‑specificity of CypD effects\u003c/h2\u003e \u003cp\u003eCypD action varies markedly across tissues. The heart, which expresses high CypD levels, shows strong CypD‑dependent regulation of mPTP activity, Ca\u0026sup2;⁺ handling, and susceptibility to heart failure [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In contrast, skeletal muscle exhibits relatively low CypD dependence at rest but stronger involvement during activity‑induced Ca\u0026sup2;⁺ flux [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Liver‑specific \u003cem\u003ePpif\u003c/em\u003e deletion increases visceral adiposity and glucose intolerance under HFD, whereas muscle‑specific deletion has minimal effects [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur respirometry data mirror this tissue‑selective pattern. Only the quadriceps showed increased respiration in \u003cem\u003ePpif\u003c/em\u003e‑deficient mice, the BAT and liver did not. This skeletal‑muscle‑restricted enhancement of oxidative phosphorylation may explain why EE, adjusted for lean mass, increases in \u003cem\u003ePpif\u003c/em\u003e‑deficient mice, yet adiposity still rises, as non‑muscle tissues likely exhibit altered survival and storage properties upon \u003cem\u003ePpif\u003c/em\u003e deletion than muscle.\u003c/p\u003e \u003cp\u003eCypD also has direct roles in adipocyte biology. High‑fat diet induces adipocyte cell death through a CypD‑dependent intrinsic pathway [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and CypD loss protects adipocytes from lipotoxic necrosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This protection may favor adipose expansion. Together with our findings, this suggests that increased fat mass in \u003cem\u003ePpif\u003c/em\u003e‑deficient mice, also under SD feeding, may reflect enhanced adipocyte survival, reduced inflammatory turnover, and greater lipid storage capacity. However, this hypothesis requires further experimental validation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiscrepant reports on obesity susceptibility in\u003c/b\u003e \u003cb\u003ePpif\u003c/b\u003e \u003cb\u003eKO mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrevious studies on the metabolism of whole-body CypD knockout mice have produced inconsistent results. \u003cem\u003ePpif\u003c/em\u003e‑deficient mice either displayed protection from diet‑induced obesity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] or a higher propensity for adult‑onset obesity developing under normal SD conditions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The latter phenotype was further corroborated in liver‑specific \u003cem\u003ePpif\u003c/em\u003e KO mice, which developed increased adiposity and glucose intolerance on HFD, whereas skeletal‑muscle\u0026ndash;specific deletion produced no differences in body weight or fat mass [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Our results align more closely with the studies reporting heightened susceptibility to obesity upon loss of CypD.\u003c/p\u003e \u003cp\u003eDifferences in genetic background, gut microbiome composition, and housing environment remain unavoidable sources of variation across CypD‑knockout studies, including our own, and may interact with genotype in ways that are difficult to fully control. Such cohort‑specific influences likely contribute to the discrepant metabolic fates reported for global or tissue-specific \u003cem\u003ePpif\u003c/em\u003e‑deficient mice.\u003c/p\u003e \u003cp\u003eA clearer understanding of this susceptibility may require deeper analysis of \u003cem\u003ePpif\u003c/em\u003e functions in metabolically active tissues. Such studies should entail adipose‑tissue biology in \u003cem\u003ePpif\u003c/em\u003e‑deficient mice, including adipocyte size distribution, lipid‑turnover kinetics, macrophage infiltration, and crown‑like structure formation, to determine whether the phenotype reflects cell‑autonomous adipocyte effects, altered adipose immune dynamics [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], or both. Given the divergent phenotypes of liver‑ and muscle‑specific knockouts, adipocyte‑specific and myeloid‑specific conditional \u003cem\u003ePpif\u003c/em\u003e deletions could help resolve the tissue contributions to whole‑body adiposity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnergy expenditure changes upon\u003c/b\u003e \u003cb\u003ePpif\u003c/b\u003e \u003cb\u003edeletion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA major discrepancy with earlier reports of reduced obesity susceptibility in \u003cem\u003ePpif\u003c/em\u003e-deficient mice arises from differences in energy expenditure analysis. Devalaraja‑Narashimha et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] normalized VO₂ to total body weight, a ratio‑based approach now recognized as statistically invalid and prone to misleading interpretations. By contrast, applying ANCOVA with lean mass as the covariate, as recommended by Tsch\u0026ouml;p et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], reveals that lean mass is the primary determinant of EE and that genotype modifies the EE\u0026ndash;lean‑mass relationship. This methodological distinction likely explains why we observe higher lean‑mass\u0026ndash;adjusted EE together with greater adiposity, whereas earlier work suggested protection. Although our calorimetry was performed at discrete timepoints, within-animal, time-aligned studies with longitudinal ANCOVA‑based EE analysis will nonetheless be required to define causal relationships and determine how these effects evolve over time. Such studies may moreover also include housing at thermoneutrality (~\u0026thinsp;30\u0026deg;C), to dissociate basal metabolic differences from thermoregulatory demand and to test the temperature dependence of tissue-resolved mitochondrial phenotypes upon \u003cem\u003ePpif\u003c/em\u003e deletion.\u003c/p\u003e \u003cp\u003eLast, increased whole‑body VO₂ does not necessarily capture the full spectrum of metabolic costs. Indirect calorimetry quantifies EE exclusively via oxygen‑dependent oxidative metabolism and therefore fails to detect oxygen‑minimal or ATP‑independent energetic processes. Such processes, including NADPH‑driven redox cycling, transsulfuration and glutathione turnover, augmented dNTP and serine/one‑carbon biosynthesis, proteostatic repair, and substrate‑cycling \u0026ldquo;futility\u0026rdquo; pathways, are prominent features of mitochondrial stress remodeling, and have been demonstrated across multiple models of mitochondrial dysfunction, where substantial increases in cellular energy demand occur without proportional increases in VO₂ [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Such uncaptured metabolic costs, whether arising early in the phenotype\u0026rsquo;s development or emerging later once a new adiposity setpoint has been established, combined with potential differences in nutrient absorption, substrate utilization, or lipid‑storage capacity, may help explain why \u003cem\u003ePpif\u003c/em\u003e‑deficient mice display hypermetabolism yet still accumulate excess adiposity. Because shifts in food intake or energy expenditure can be transient, subtle, or temporally restricted, they may fall outside the specific time window assessed by indirect calorimetry. Likewise, metabolic changes involving resting thermogenesis or muscle‑related energy costs may easily remain undetected because they lie outside the sensitivity range of standard VO₂‑based assays. In this framework, increased obesity is not inconsistent with elevated EE but may instead reflect inefficient or compensatory metabolic rewiring that is not fully quantified by conventional calorimetric measurements.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTogether, our findings position CypD as a diet‑ and tissue‑specific regulator of energy homeostasis, shaping mitochondrial fuel selection, oxidative capacity, adipocyte survival, and whole‑body metabolic adaptability. Rather than acting as a uniformly protective or detrimental factor, CypD emerges as a context‑dependent modulator whose influence on adiposity and mitochondrial function varies with dietary environment, tissue identity, and the energetic demands of individual cell types, consistent with broader models of CypD‑dependent metabolic plasticity. The coexistence of enhanced skeletal‑muscle respiration with greater adiposity in \u003cem\u003ePpif\u003c/em\u003e‑deficient mice further positions CycD as a potential gatekeeper against hidden energetic sinks in HFD-fed mice and underscores the complex, compensatory nature of mPTP‑linked remodeling across tissues. These insights establish a foundation for precision targeting of the mPTP axis in obesity and metabolic disease, and highlight the need for additional tissue‑specific \u003cem\u003ePpif\u003c/em\u003e knockouts to delineate the distinct contributions of muscle, adipose, and liver to systemic metabolic outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eResearch Funding\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eClinical Trial Number\u003c/strong\u003e \u003cp\u003eNot applicable. This study did not involve a clinical trial and was not registered.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.H. and P.P. conducted the mouse work, W.A. performed mitochondrial respiration assays. J.E. and P.P. conceptualized and designed the studies. P.P. prepared the figures, conducted statistical analyses and wrote the main manuscript text. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe\u0026rsquo;d like to thank Zachary Marcus and Nickki Parker for their skilful technical assistance. A first draft of the manuscript was generated with AI assistance on publicationgod.com, for which we\u0026rsquo;d like to thank its founder, Jens Mittag, for his tokens and kind support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, Brunskill EW, Sayen MR, Gottlieb RA, Dorn GW, Robbins J, Molkentin JD (2005) Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. 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Curr Pharm Des 29:620\u0026ndash;629. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/1381612829666230313111314\u003c/span\u003e\u003cspan address=\"10.2174/1381612829666230313111314\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9472657/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9472657/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMitochondrial dysfunction is a hallmark of obesity-associated metabolic disease, with the mitochondrial permeability transition pore serving as a key regulator of cellular energy homeostasis. Cyclophilin D, encoded by \u003cem\u003ePpif\u003c/em\u003e, is the principal regulatory component of this pore and has been implicated in insulin secretion, adipocyte survival, and whole-body energy balance. However, whether \u003cem\u003ePpif\u003c/em\u003e loss produces a consistent metabolic phenotype when challenged with high fat diet (HFD) exposure, and whether systemic metabolic alterations reflect tissue-specific changes in mitochondrial oxidative capacity, remains unresolved. Here, we characterized whole-body \u003cem\u003ePpif\u003c/em\u003e knockout mice longitudinally over 34 weeks under standard (SD) and HFD using body composition analysis, indirect calorimetry, glucose and insulin tolerance testing and tissue-resolved, Seahorse-based mitochondrial respirometry. Under SD, knockout mice displayed elevated fat mass without differences in food intake or energy expenditure. High-fat feeding progressively amplified this adiposity, with knockout mice developing significantly greater body weight and fat mass than wild-type littermates by weeks 28 and 36. Glucose tolerance and insulin sensitivity remained comparable genotype under either diet. Energy expenditure was selectively elevated in high-fat-fed knockout mice, and ANCOVA revealed lean mass as a primary driver, with a significant lean mass-by-genotype interaction. Consistent with that, ADP-stimulated mitochondrial respiration was significantly increased in quadriceps of knockout mice under both diets, without corresponding differences in brown adipose tissue or liver, or changes in mitochondrial content across tissues. These findings establish cyclophilin D as a diet- and tissue-specific regulator of energy homeostasis, informing therapeutic strategies targeting the permeability transition pore or cyclophilin D interactome in metabolic disease.\u003c/p\u003e","manuscriptTitle":"Cyclophilin D Deficiency Exacerbates Diet-Induced Obesity and Selectively Enhances Skeletal Muscle Mitochondrial Respiration in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 09:10:09","doi":"10.21203/rs.3.rs-9472657/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-30T18:16:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3306677764579946181423788850217851252","date":"2026-04-27T07:31:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337596732158458825063240886424611749985","date":"2026-04-23T12:10:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-23T10:26:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-22T03:31:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-22T03:31:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pflügers Archiv - European Journal of Physiology","date":"2026-04-20T12:56:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c1ae03ba-44e3-4d8b-941d-0871c22d1d61","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-04-30T18:16:11+00:00","index":39,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T09:10:09+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 09:10:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9472657","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9472657","identity":"rs-9472657","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}