Lean Mass Gain from a Moringa-Enriched Protein Diet Confers Reno-Protective Effects and Modulates Gut Microbiota and sACE2 Activity

Lean Mass Gain from a Moringa-Enriched Protein Diet Confers Reno-Protective Effects and Modulates Gut Microbiota and sACE2 Activity | 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 Lean Mass Gain from a Moringa-Enriched Protein Diet Confers Reno-Protective Effects and Modulates Gut Microbiota and sACE2 Activity Pradeep B Patil, Parimala A, Srinivas M, Tulja B, Sreenivasa J. Rao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8275989/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Obesity and protein-energy malnutrition represent dual nutritional challenges, with high-protein diets often prescribed to restore lean mass but simultaneously risking renal overload. Moringa oleifera (MOL), a nutrient-rich plant with antioxidant and prebiotic properties, may offer a dietary strategy to improve muscle metabolism, microbial balance, and renal protection, particularly when combined with exercise. This study evaluated the effects of 2% and 4% MOL in 20% protein diet and 8% MOL in 40% protein diet, with or without forced swimming, and study includes three experimental phases in NIN/Sprague Dawley, Wistar/NIN, and obese mutant rats ( Ob/ob and Gr/ob ). In Phase I (20% protein diet), MOL improved neuromuscular performance: grip strength increased by 21–26% compared with 7.6% in controls (p < 0.0001). Gastrocnemius glycogen decreased by 15–35% (p < 0.0001), reflecting greater glycogen utilization. Morphometry revealed fiber hypertrophy, with short fibers enlarging by 87% in Group 5 (p < 0.0001) in gastrocnemius muscles. Phase II demonstrated microbiota remodeling, with Lactobacillus and Bifidobacteria increasing by 08–26% and the Firmicutes/Bacteroides ratio decreasing by up to 11%. Circulating sACE2 declined by 7–52% with MOL, contrasting with elevations in Ob/ob rats (+ 64%). Phase III, involving 40% protein diets, revealed renal stress with elevated NAGase (~ 45 ng/mL) and β2-microglobulin (+ 11%); these were attenuated by MOL with fiber (− 49%). Collectively, MOL enhanced muscle performance, promoted microbial homeostasis, lowered sACE2, and provided renoprotection under high-protein load, particularly when combined with exercise. These findings highlight MOL as a functional food candidate for obesity management and metabolic resilience. Nutrition & Dietetics Animal Science Lean Mass High protein diet Moringa oleifera leaves sACE2 NAGase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In India, rapid demographic and epidemiological transitions driven by economic growth have accelerated the rise in abdominal obesity, largely due to sedentary lifestyles and reduced physical activity[ 1 ] with steep increase and sharp regional variation[ 2 ] reaching up to abdominal obesity affected 56% of women and 49% of men in India[ 1 ]. Obesity has emerged as one of the foremost health crises of the twenty-first century, contributing to type 2 diabetes, cardiovascular disease, and renal impairment. At the same time, many low- and middle-income countries face an ongoing burden of protein-energy malnutrition (PEM), which compromises growth, immunity, and metabolic efficiency[ 3 ]. This coexistence of overnutrition and undernutrition usually termed the “double burden of malnutrition” which complicates dietary recommendations and underscores the need for nutrient-dense interventions capable of supporting lean mass [ 4 ]without imposing organ stress[ 5 ]. High-protein diets are often advocated for weight management and muscle maintenance. However, excessive protein intake is associated with glomerular hyperfiltration and tubular damage, raising concern about renal safety in both obese and malnourished individuals[ 6 ]. Moringa oleifera: a multifunctional food source Moringa oleifera leaves (MOL), commonly known as the “drumstick tree,” is native to South Asia found to be a uniquely nutrient-dense food. Beyond its nutritional profile, MOL exhibits anti-inflammatory, antioxidant, and metabolic modulatory properties. The choice of MOL is also supported by their suitability and cost-effectiveness as a plant-based protein alternative, free from pesticides and heavy metals, and readily available throughout the year. Their cultivation and utilization require minimal investment and labor, making them a practical option from soil to fork. Animal studies demonstrate that MOL extracts lower serum cholesterol, improve glucose tolerance, and ameliorate oxidative stress markers [ 7 , 8 ]. Importantly, MOL also contains soluble fibers and phytochemicals with prebiotic potential, suggesting it could favorably shift gut microbiota composition[ 9 – 11 ]. Given its affordability and cultural acceptance in South Asia, MOL represents an attractive candidate for addressing both obesity and malnutrition. Gut microbiota as a metabolic regulator The gut microbiome plays a critical role in nutrient absorption, immune function, and host metabolism[ 12 , 13 ]. Dysbiosis is an imbalance in microbial communities which is strongly associated with obesity and metabolic syndrome. Specifically, an elevated Firmicutes/Bacteroides ratio and reduced probiotic taxa such as Lactobacillus and Bifidobacteria have been reported in obese humans and rodents [ 14 – 17 ]. Interventions that lower this ratio and enrich probiotics improve insulin sensitivity and reduce systemic inflammation. Exercise also remodels the microbiota, increasing diversity and favoring beneficial genera[ 18 – 20 ]. Given MOL’s prebiotic activity in various models[ 21 – 23 ], a combined diet–exercise intervention could exert synergistic effects on microbial balance. Skeletal muscle as a target of nutritional intervention Skeletal muscle, comprising up to 40% of body mass, is a key determinant of energy expenditure and metabolic health. In obesity, muscles accumulate intramyocellular lipid (IMCL), which correlates with insulin resistance[ 24 , 25 ]. Exercise reduces IMCL and promotes mitochondrial biogenesis, increasing oxidative enzyme activity and fiber hypertrophy[ 26 , 27 ]. Glycogen utilization during exercise further reflects metabolic efficiency. Nutritional supplementation with antioxidant-rich plant products can amplify these adaptations, protecting muscle from oxidative damage and enhancing endurance capacity. MOL extract has already been shown to attenuate chemical-induced muscle atrophy and improve exercise performance in rodent models[ 28 ]. Renal stress and high-protein diets High-protein diets, while effective in increasing lean body mass, raise concerns about long-term kidney health. Increased nitrogen load and glomerular pressure can cause microstructural injury, reflected by early biomarkers such as N-acetyl-β-D-glucosaminidase (NAGase) and β2-microglobulin[ 29 ]. Plant-derived bioactives that attenuate oxidative and inflammatory stress within the kidney are therefore of great relevance. MOL’s nephroprotective effects have been documented in models of chemically induced kidney damage [ 30 – 32 ], but its capacity to offset protein-induced renal stress has not been systematically evaluated. sACE2 as a metabolic and immunological node ACE2 exists as tissue ACE2 and serum ACE2 (sACE2). Tissue ACE2 is expressed in lung alveolar cells, intestine, heart, kidney, and endothelium, where it regulates the renin–angiotensin system (RAS) by degrading Ang II into Ang (1–7), promoting vasodilation and protection against inflammation and fibrosis. sACE2 originates from shedding of tissue ACE2 by ADAM17/TACE and reflects both tissue expression and inflammatory activity. Thus, tissue ACE2 mediates local effects, while sACE2 serves mainly as a biomarker of tissue stress or systemic disease reflecting its paradoxical role [ 33 , 34 ]. However, it has been observed that ACE2 regulation differs by context. Tissue ACE2 increases physiologically with estrogen, exercise, and ARBs, and pathologically in diabetes, obesity, and heart failure, while it decreases in ARDS, viral infections (via SARS-CoV-2 internalization), aging, and kidney disease. sACE2 rises with inflammation and oxidative stress due to increased shedding, but decreases when tissue expression is globally reduced, as in advanced fibrosis[ 35 ]. Therefore, sACE2 was considered a parameter in this study. ACE2 links with diseases are strongly supported in the literature. In COVID-19, tissue ACE2 is reduced after viral binding, driving Ang II–mediated lung injury and cytokine storm, while sACE2 rises and correlates with severity[ 36 ]. In sepsis, sACE2 increases via ADAM17 activation. In metabolic disease, tissue ACE2 is upregulated in adipose, pancreas, and kidney as compensation, while sACE2 is persistently elevated, marking higher cardiovascular risk. Hypertension therapy with ARBs increases tissue ACE2, while liver diseases (cirrhosis, NAFLD) also show high sACE2[ 37 , 38 ]. Dynamics vary with time. In acute and chronic injury, tissue ACE2 is downregulated through overutilization acting as transient molecular disease [ 39 ] or upregulated depends through body ability to fight back through protective mechanism [ 40 ] by increasing ACE2 however sACE2 rises due to increased stress [ 41 ], predicting poor outcomes. Drugs such as ARBs, statins, and SGLT2 inhibitors favorably modulate ACE2. Clinically, sACE2 is a prognostic biomarker, and tissue ACE2 is a therapeutic target, with recombinant soluble ACE2 being tested as a viral decoy. Aerobic exercise reduces ACE2 expression in several organs, suggesting a protective effect[ 42 – 44 ]. Whether MOL influences ACE2 regulation in obesity and exercise remains unknown, but such an effect would be highly relevant to cardiometabolic health. In summary, tissue ACE2 protects but can be hijacked by viruses, whereas sACE2 reflects injury and prognosis. Low tissue ACE2 with high sACE2 indicates severe disease, while high tissue ACE2 (without viral exploitation) is protective in cardiovascular, renal, and metabolic conditions. Study rationale and novelty Although MOL is recognized for its nutritional and medicinal value, its systemic role across multiple endpoints like muscle remodeling, glycogen use, gut microbiota balance, renal stress under high-protein load, and sACE2 modulation has not been studied in an integrated framework. Previous studies tend to isolate single outcomes, limiting translational relevance. By combining MOL supplementation with exercise in both lean and obese rat models across three experimental phases, we aimed to create a high-fidelity picture of how MOL functions as a nutraceutical (Fig. 1a). We hypothesized that dietary MOL supplementation would enhance neuromuscular performance while simultaneously promoting favorable remodeling of the gut microbiota. Under high-protein feeding conditions, we anticipated that supplementation would alleviate renal stress, reflected by reductions in circulating biomarkers of kidney injury. To further address the practical question of whether plant-based high-protein diets differ from animal-based sources in their renal impact, we evaluated whether MOL and dietary fiber could provide protection against protein-induced kidney damage. We also hypothesized that supplementation might influence systemic sACE2 levels, which are elevated in obesity, thereby contributing to metabolic regulation. Finally, we proposed that structured exercise would act synergistically with MOL supplementation, amplifying beneficial effects across muscular, microbial, renal, and systemic endpoints. Objectives and Hypotheses This study examined whether MOL supplementation, alone or with exercise, improves muscle performance, modulates gut microbiota, reduces renal stress from high-protein diets, and regulates circulating sACE2 levels. We hypothesized that MOL would enhance lean mass, promote microbial balance, protect renal function, and lower sACE2, with exercise amplifying these benefits. Materials and Methods All experimental procedures were carried out at the National Institute of Nutrition (NIN), Hyderabad, in compliance with the guidelines of the CCSEA (Animal Ethics Approval: ICMR-NIN/IAEC-IV/02/001/2020 and ICMR-NIN/IAEC/2021-1/005). Male rats were selected to maintain uniformity and avoid confounding effects of hormonal variations. Three distinct cohorts were employed across the study (Fig. 1b-1d). Each group consisted six male animals of 3 months old rat. In all phase’s animals were randomized based on body weight. In Phase I, NIN/Sprague Dawley rats (Fig. 1e-left) were used to investigate the impact of isocaloric and isonitrogenous diet (20% protein) MOL supplementation (2% and 4%) with or without forced exercise (30 minutes swimming) on muscle physiology and metabolism. Phase II utilized Wistar/NIN rats (Fig. 1e-right) along with obese mutant strains including Ob/ob and Gr/ob , as well as their lean counterparts with standard pelleted diet (20% protein), to evaluate impact of exercise (30 minutes swimming) on sACE2 levels of obese mutants. Phase III was designed on Wistar/NIN male to address the effects of high-protein diets (40%) on renal stress and to test whether MOL supplementation (8%) with or without high fiber (8%) could provide protection under these conditions. Animals were housed in polypropylene cages with autoclaved bedding under controlled temperature (22 ± 2°C) and humidity (50–70 percent) with a reversed 12:12 h light–dark cycle. They were acclimatized for one week before the initiation of interventions and were provided autoclaved water and standard pelleted diet and customized AIN-93M diet ad libitum . The diets were formulated in the NIN animal diet section (Fig. 2a-2b). The ingredients included wheat flour, roasted Bengal gram, casein, skimmed milk powder, groundnut oil, sucrose, vitamin and mineral mixtures, cellulose, and dried MOL powder prepared from sun-dried leaves pulverized into fine powder. In phase I, the MOL intervention was designed to provide either 2 percent or 4 percent supplementation by weight. In Phase I and II, the protein content was maintained at 20 percent, while in Phase III the diet was modified to provide 40 percent protein with or without 8% MOL. To evaluate the effects of fiber and MOL interaction, Phase III diets incorporated either 4 percent or 8 percent fiber. All diets were isocaloric and isonitrogenous, with proximate analysis confirming macronutrient content. The diet preparation followed a standard procedure [ 45 ] where it was pelletized, sterilized using autoclaving, and stored under refrigeration (Cold room) until use, and all batches were confirmed to be free of microbial contamination. The exercise intervention consisted of forced swimming (Fig. 1f), selected as a model for endurance-type activity[ 45 ]. Animals were trained to swim in stainless steel tanks filled with water (Fig. 1h) maintained at 30 ± 5°C. Swimming was performed for 30 minutes for 6 days/week for six weeks. To minimize variability, all exercise groups were subjected to swimming sessions simultaneously, with animals identified by tail markings under red light illumination during the dark phase. A reversed light–dark cycle was maintained to align the intervention with the anabolic window of opportunity and to prevent stress or depressive effects associated with electric stimulation for exercise and circadian disruption. This duration was selected to provide consistent aerobic challenge without inducing exhaustion. Following exercise, animals were towel-dried to prevent hypothermia and returned to their home cages. As shown in our earlier publication, MOL supplementation has improved feed intake and serum protein levels throughout the intervention, where body weight and feed intake were recorded weekly to monitor physiological adaptation[ 45 ]. At initial week and 6th week of study phase, neuromuscular assessments were performed, including measurement of grip strength (Fig. 1g-left) using GSM-01 LI Model (Orchid Scientific and Innovation India Pvt. Ltd, India) and locomotor performance using actimeter (Fig. 1g-right). Grip strength was measured with a digital grip strength meter, where the rat was allowed to grasp a horizontal bar using forelimbs and gently pulled backward by the tail until grip release. The maximum force was recorded, and three attempts were averaged per animal. Locomotor activity was recorded using automated actimetry chambers to capture exploratory and repetitive activity. Body composition was evaluated by both total body electrical conductivity (TOBEC) [ 46 ]and dual-energy X-ray absorptiometry (DEXA). TOBEC (EM-SCAN model 3000) measurements provided estimates of fat-free mass, lean body mass, total body water, and fat percentage, while DEXA scans were performed (Hologic Discovery A QDR system) to assess bone mineral content and bone mineral density. At the end of the intervention, blood samples were collected from the retro-orbital plexus under light isoflurane anesthesia. Serum was separated and stored at − 80°C until further analysis. Serum biochemistry was performed using the COBAS C-311 analyzer (Roche, Switzerland) to measure lactate dehydrogenase (LDH) and serum lactate levels. The biochemical assays were performed as per kit protocols provided by respective companies. It included measurement of gastrocnemius glycogen concentration (E-BC-K073-S, Elabscience, China) using a colorimetric assay kit, and determination of sACE2 (LS-F10723, LSBio, USA), β2-microglobulin (E-EL-R1085, Elabscience, China), and N-acetyl-β-D-glucosaminidase (NAGase; NBP2-81174, Novus Biologicals, USA) using ELISA reader (BioTek Synergy H1, USA). These markers were chosen based on their physiological relevance: sACE2 as an indicator of metabolic and cardiovascular stress, β2-microglobulin as a marker of glomerular function, and NAGase as a sensitive indicator of proximal tubular injury. Organ weights were recorded at necropsy. Liver, kidney, spleen, heart, lungs, and brain were excised, cleaned of connective tissue, blotted dry, and weighed. Skeletal muscles including gastrocnemius and soleus were dissected, weighed, and measured for length before being processed for histological analysis. The soleus and gastrocnemius muscles were selected for analysis because they represent distinct fiber-type compositions and metabolic profiles. The soleus is predominantly composed of slow-twitch oxidative fibers, making it highly fatigue-resistant and suited for sustained, endurance-type activity, whereas the gastrocnemius contains a mixture of fast- and slow-twitch fibers, contributing both to powerful, propulsive movements and glycolytic metabolism. During swimming, both muscles are actively recruited, with the soleus supporting continuous low-intensity contractions and the gastrocnemius generating bursts of propulsive force. Examining these muscles therefore provides complementary insights into exercise-induced adaptations across oxidative and glycolytic pathways, as well as muscle-specific responses to swimming training. Histological evaluations were conducted on muscle tissue using hematoxylin and eosin staining for general morphology, Oil Red O staining for lipid deposition, and succinate dehydrogenase activity staining for oxidative capacity. Sections were imaged and quantified using ImageJ software. Morphometric parameters included fiber diameter along both long and short axes, cross-sectional area, and connective tissue thickness. At least 5 sections/animal were measured to ensure statistical validity. These analyses provided an integrated view of structural and metabolic adaptations within skeletal muscle. Gut microbiota was evaluated using quantitative polymerase chain reaction (qPCR) targeting bacterial 16S ribosomal RNA genes as per our earlier published procedure [ 47 ]. DNA was extracted from caecal fecal pellets using the QIAamp DNA Stool Mini Kit (Qiagen, Germany), and species-specific primers were used to quantify Lactobacillus , Bifido, Firmicutes, Bacteroides, and total bacteria. Relative abundances were calculated and used to derive the Firmicutes to Bacteroides ratio and the Firmicutes to Bifido bacteria ratio, both of which are considered key indicators of metabolic dysbiosis. Statistical analyses were conducted using GraphPad Prism version 8.0. Data are presented as mean ± standard deviation. One-way or two-way ANOVA was applied as appropriate, followed by Tukey’s or Sidak’s post hoc tests. A p-value < 0.05 was considered statistically significant. Results Muscle Function and Morphology MOL supplementation markedly enhanced neuromuscular performance (grip strength) in NIN/Sprague Dawley rats (Fig. 2c). While the Normal Control group showed only a 7% increase in grip strength over six weeks, MOL-fed groups demonstrated much higher gains, ranging from + 21.2% to + 26.2%, with the largest improvements seen in the Group 5 (p < 0.0001; Fig. 2c-left). Exercise potentiated the diet effect, with all MOL + exercise groups outperforming either treatment alone. Locomotor activity assessed through actimeter readings (Fig. 2c-right) revealed progressive improvement with MOL supplementation and exercise. Although no significant differences were observed at baseline, by the sixth week, the 4% MOL with Forced Exercise (FE) group exhibited the highest activity count (1456 ± 111), significantly surpassing the normal control (1190 ± 50; p < 0.001). Control has minimal change (-1.5%) whereas MOL group has shown dose dependent trend increasing significantly from baseline in MOL groups without exercise (1.4%-8.4%) and MOL with forced exercise (7.5%-19.1%). Energy handling paralleled these changes observed in from frozen tissue samples of dissected gastrocnemius muscles (Fig. 2d). Gastrocnemius glycogen stores fell by 15–35% relative to control, indicating greater utilization during activity (p < 0.0001; Fig. 2d). Histological stains confirmed these functional findings: Oil Red O staining (Fig. 2e) showed fewer and smaller lipid droplets in MOL-fed groups, particularly in the MOL + exercise cohort, while succinate dehydrogenase staining (Fig. 2f) intensity increased, signifying improved oxidative metabolism. Morphometric analysis (Fig. 3a-3f) supported hypertrophic remodeling. Gastrocnemius mass rose by 5–17% across supplemented groups (Fig. 3a), while soleus mass remained near baseline. Both muscle lengths (Fig. 3b), no such change was observed however, muscle fiber diameter (Fig. 3c) increased significantly in both soleus (+ 1%-21%) and gastrocnemius (+ 7–21%). Connective tissue thickness slightly declined in both muscles, suggesting healthier tissue remodeling (Fig. 3d). The long-axis fiber hypertrophy (+ 18%-49) and short-axis hypertrophy reaching up to + 87% in the Group 5 (p < 0.0001) in gastrocnemius but no such change was observed with soleus (Fig. 3e-3f). Body Composition Whole-body composition reflected these muscle-specific adaptations. Fat-free mass increased most prominently in MOL + exercise groups (+ 13–15%), while lean body mass improved slightly upto + 6%. Fat percentage and total fat declined slightly, with the greatest reductions observed in 2% MOL alone (− 10% fat, − 15% total fat; Fig. 5a–b). Skeletal indices showed modest improvements compared to the normal control (Group 1). At the end of the study, bone mineral content (BMC) and bone mineral density (BMD) increased by approximately 7% (Fig. 4d left) and 5% (Fig. 4d right), respectively, in the combined treatment group (Group 5). Across all five Groups, BMC improved from baseline by 8–16%, and BMD increased by 2–7%, whereas Group 5 demonstrated an 15% and 11% improvement in BMC and BMD respectively. Organ Weights and Microbiota Systemic organ remodeling showed distinct trends (Fig. 5a-5f). Spleen mass declined consistently (− 21.7 to − 35.6% across groups), indicating reduced inflammatory load. Liver and lung weights were lower in supplemented groups (− 3.5 to − 9%), while kidney mass showed a mild rise/decline. Brain weight remained stable, indicating no adverse central effects. Gut microbiota analysis revealed robust diet- and exercise-driven restructuring (Fig. 5g-5s). Lactobacillus increased by 8–13%, and Bifidobacteria by 9–26% in supplemented groups, while the Firmicutes/Bacteroides ratio fell by up to 11%. The Firmicutes/ Bifidobacteria ratio declined even more strongly (− 5% to − 13.7%). These changes oppose the dysbiosis signature observed in obese mutants (Fig. 5t), where Bifidobacteria dropped (− 16% in Gr/ob; −39% in Ob/ob) and Bacteroides rose (+ 12% in Ob/ob). Thus, MOL reversed obesity-associated microbiota shifts while enhancing probiotic abundance in lean rats. Circulating sACE2 sACE2 responses were striking (Fig. 6a-6c). In lean Sprague Dawley rats, circulating sACE2 fell progressively with MOL supplementation: −7% with 2% MOL, − 22% with 4% MOL, and − 52% in Group 5 (p < 0.001). In obese mutants, however, sACE2 was elevated (+ 64% in Ob/ob; +38% in Ob/ob lean; +9% in Gr/ob), highlighting obesity-driven maladaptation. Exercise reduced sACE2 modestly across all strains (− 6 to − 15%; Fig. 6c), suggesting a protective effect that was amplified by MOL. The directionality was consistent: MOL and exercise lowered sACE2, counteracting the obesity-associated rise. Phase III – Renal Stress under High-Protein Diets Renal biomarkers differentiated tubular and glomerular responses. NAGase levels were uniformly elevated (~ 42–45 ng/mL) in all 40% protein groups, confirming that tubular stress is unavoidable under protein overload and resistant to dietary intervention (Fig. 6d). In contrast, β2-microglobulin (B2M) showed divergent trends (Fig. 6e). The high-protein control diet (HPD-1) group (with 8% fiber but no MOL) produced an 11% rise in B2M, showing that fiber alone failed to protect glomeruli. By comparison, the addition of MOL to fiber diets strongly reduced B2M: −49% with HPD-2 (4% fiber + 8% MOL) group, and − 37% with HPD-3 (8% fiber + 8% MOL) group (both p < 0.01–0.0001 vs high-protein control). These data demonstrate that MOL provides clear glomerular renoprotection in a fiber-containing context, although tubular injury persists. Across phases, MOL supplementation consistently enhanced muscle strength, glycogen utilization, and hypertrophy, while reorganizing the gut microbiota toward a healthier profile and downregulating sACE2 in lean but not obese mutants. Under high-protein feeding, MOL provided robust protection against glomerular stress but not tubular stress, underscoring the specificity of its nephroprotective action. The convergence of muscular, microbial, renal, and systemic endpoints supports the role of MOL as a functional food for obesity-linked metabolic resilience. Discussion The present findings build upon our earlier report where MOL supplementation improved serum protein levels by 14–19% and reduced serum urea, AST, ALT, and ALP, demonstrating clear hepatic and renal protection[ 45 ]. In the current extended study, these biochemical improvements are complemented by functional outcomes such as increased grip strength and muscle fiber hypertrophy, alongside enhanced glycogen utilization, which mechanistically explain the protein-sparing effects seen earlier[ 45 ]. Similarly, the weight gain observed with 4% MOL in the previous study is corroborated here by improved lean mass and reduced fat deposition, suggesting a consistent anabolic and body-composition benefit. The nephroprotective trend seen earlier through stable creatinine [ 45 ] is further validated in this study through reductions in tubular (NAGase) and glomerular (β2-microglobulin) damage markers, particularly under high-protein stress, confirming the resilience of renal physiology. Importantly, while earlier work was limited to biochemical endpoints, the current data also demonstrate gut microbiota remodeling and sACE2 downregulation, which not only extend the hepatoprotective and nephroprotective roles but also position MOL as a systemic modulator of metabolic and immune health. The present study demonstrates that dietary supplementation with MOL, particularly when combined with endurance-type exercise, yields systemic benefits across muscle performance, body composition, gut microbial ecology, renal resilience, and circulating sACE2 regulation. By conducting experiments across three phases, we show that MOL exerts context-dependent effects, amplifying exercise-induced adaptations, correcting obesity-associated dysbiosis, and mitigating renal stress induced by high-protein diets. The neuromuscular data from Phase I highlight a striking improvement in grip strength and muscle hypertrophy. While controls improved by only 7 percent over six weeks, MOL-fed rats exhibited increases of 21–28 percent, with the highest gains in those also undergoing exercise. The increased actimeter scores in the 4% MOL + FE group indicate that MOL supplementation augments exercise-induced improvements in locomotor activity, possibly through enhanced glycogen utilization and muscle fiber remodeling. This aligns with the observed rise in grip strength and oxidative enzyme activity reported in the main study. The absence of significant change in lower-dose groups suggests a threshold effect requiring both adequate MOL concentration and physical stimulus to induce measurable behavioral outcomes. The results collectively imply that MOL enhances neuromuscular efficiency and endurance, validating its potential as a functional dietary supplement for improving metabolic resilience and physical activity levels. This effect parallels the observed increase in oxidative staining and reduction in lipid deposition, suggesting that MOL enhances the adaptive responses usually attributed to endurance training. Glycogen depletion in MOL-fed groups, particularly under exercise, indicates greater glycogen mobilization and oxidative turnover. These findings are consistent with previous evidence that endurance training increases mitochondrial biogenesis and oxidative enzyme activity in skeletal muscle[ 48 ]. Furthermore, reduced intramyocellular lipid content aligns with improvements in metabolic efficiency, as lipid accumulation is linked to insulin resistance in sedentary muscle[ 49 ]. Importantly, the pattern of short fiber hypertrophy, which increased by as much as 87 percent in the 4 percent MOL plus Exercise group, indicates that MOL potentiates exercise-induced remodeling of glycolytic and oxidative fibers. This resonates with prior work showing that MOL extracts can attenuate drug-induced muscle atrophy and improve exercise capacity in rodent models[ 50 , 51 ]. Evidence also suggest MOL improves bone health[ 52 ], however as our study was for short duration, slight difference was noted. Gut microbiota data from Phase II provide a complementary dimension to these muscular adaptations. MOL supplementation enriched Lactobacillus and Bifidobacteria by up to 26 percent, while lowering the Firmicutes/Bacteroides ratio by as much as 11 percent. These microbial changes are widely interpreted as signatures of improved metabolic health, as obesity is associated with a higher Firmicutes/Bacteroides ratio and reduced probiotic representation[ 53 , 54 ]. Exercise itself is known to remodel the microbiota, increasing diversity and abundance of taxa linked to short-chain fatty acid production and improved metabolic balance. The combined effect of MOL and exercise, therefore, likely reflects a convergence of prebiotic and activity-induced remodeling. The contrast with obese mutants is notable. Ob/ob rats showed a 39 percent decline in Bifidobacteria and a 13 percent increase in Bacteroides, consistent with dysbiosis associated with obesity. That these shifts were absent in MOL-fed lean groups underscores the corrective capacity of MOL to stabilize microbial composition. Phase II also revealed important changes in sACE2, a molecule at the intersection of metabolism and immunity. Circulating sACE2 declined progressively with MOL supplementation, reaching a 52 percent reduction in the 4 percent MOL plus Exercise group. Conversely, obese mutants exhibited a 64 percent elevation in sACE2, consistent with findings that obesity and diabetes upregulate sACE2 expression. Exercise alone has been shown to lower sACE2 in muscle, heart, and kidney, helping to rebalance the renin–angiotensin system[ 55 – 57 ]. The additive effect of MOL with exercise suggests that phytochemicals may reinforce this protective remodeling. The interpretation of sACE2 modulation is complex because circulating sACE2 can be both a marker of metabolic stress and a mediator of viral entry. Nevertheless, the observed decline in MOL plus Exercise groups, when coupled with improvements in grip strength, fiber hypertrophy, and microbial balance, strongly suggests that sACE2 reduction here reflects systemic metabolic improvement rather than loss of protective angiotensin 1–7 signaling. Phase III extended the findings to renal stress under high-protein diets. Consistent with prior evidence that high protein intake elevates glomerular pressure and tubular injury markers [ 58 , 59 ], rats on a 40 percent protein diet showed a rise in NAGase to approximately 45 ng/mL and an 11 percent increase in β2-microglobulin. Importantly, these changes were mitigated when MOL was combined with fiber, leading to a 49 percent reduction in β2-microglobulin compared with the high-protein control. This highlights a synergistic effect between MOL’s antioxidant and anti-inflammatory bio-actives and the protective role of dietary fiber in reducing tubular reabsorption stress. Prior studies confirm that MOL extracts reduce oxidative and inflammatory injury in models of nephrotoxicity[ 60 , 61 ]. The present findings extend this evidence by demonstrating a protective effect even in the context of protein overload, which has greater translational relevance for individuals adopting high-protein diets for resolving obesity issues or athletic purposes. In the high-protein phase, all groups exhibited elevated NAGase levels (~ 42–45 ng/mL), confirming that tubular injury is an unavoidable outcome of protein overload, irrespective of fiber or MOL supplementation. Thus, tubular stress appears resistant to dietary modulation under these conditions. However, glomerular injury, reflected by β2-microglobulin, showed a more nuanced response. The high-protein control diet, despite already containing 8% fiber, led to an 11% rise in β2-microglobulin, indicating that fiber alone failed to protect glomerular strain. By contrast, the combination of MOL with fiber reduced β2-microglobulin substantially, by 37–49% relative to the high-protein control, demonstrating a strong renoprotective effect. These findings emphasize that fiber does not independently safeguard renal function; instead, MOL is the critical protective factor, with fiber acting as a supportive matrix that enhances the bio-efficacy of MOL’s phytochemicals. A detailed study on fiber type and content is needed to understand its impact. This synergy may involve slower protein digestion, improved nitrogen handling, or enhanced microbial fermentation, but it is clear that MOL drives the protective effect. Taken together, these results point to a coherent systems-level model. In skeletal muscle, MOL amplifies exercise-induced improvements in glycogen turnover, oxidative metabolism, and hypertrophy. In the gut, MOL restores microbial balance, enriching probiotic taxa and lowering obesity-associated ratios. In circulation, MOL downregulates sACE2, reversing obesity-associated elevations, while in the kidney it prevents protein-induced stress when fiber is present. The cross-organ convergence strengthens the case for MOL as a functional food that supports both anabolic and protective pathways. Several limitations warrant mention. The study was conducted exclusively in male rats, and sex-specific differences in microbiota, sACE2, and renal adaptation remain unexplored. Sample sizes were modest, and although differences reached statistical significance, power analysis should guide future work. Control exercise group was intentionally avoided in the MOL group as we assessed its impact in obese study (phase II) for sACE2. Microbiota assessments were limited to selected taxa; metagenomics and metabolomics would better characterize microbial function. Likewise, renal assessments were restricted to serum markers; inclusion of other urinary markers and histopathological scoring would improve sensitivity. Despite these limitations, the consistency of effects across independent endpoints and phases provides compelling evidence of MOL’s beneficial role. High-protein diets (40% HPD), whether combined with MOL or dietary fiber, did not prevent tubular damage, as reflected by elevated NAGase levels; however, this injury appears to be sublethal or reversible to certain extent[ 62 , 63 ]. More strikingly, glomerular injury showed a differential response: β2-microglobulin (B2M) levels were reduced by 37–49% in the 8% MOL-supplemented HPD group compared to the normal control, indicating a clear renoprotective effect. In contrast, B2M increased by 11% in the 40% HPD group without MOL, a pattern that suggests glomerular damage is non-reversible once it reaches to threshold level[ 64 ] in the absence of supplementation. These findings underscore the importance of MOL in mitigating renal stress associated with high-protein feeding. The translational implications are considerable. In populations experiencing rising obesity, MOL supplementation could serve as a low-cost dietary adjunct to structured exercise programs, enhancing muscle strength and metabolic resilience. For individuals relying on high-protein diets for weight control or athletic performance, the combination of MOL and dietary fiber may lower renal risks. Additionally, the modulation of sACE2 observed in this context suggests cardiometabolic protection, with possible relevance to viral susceptibility pathways. Future investigations should explore dose–response dynamics, long-term safety, and the potential for human applications through rigorously designed clinical trials. Conclusion This study demonstrates that dietary supplementation with MOL, particularly at 4 percent when combined with endurance exercise, exerts comprehensive benefits across neuromuscular, metabolic, microbial, and renal domains. Grip strength and gastrocnemius hypertrophy improved substantially, accompanied by enhanced glycogen utilization and reduced intramuscular lipid deposition. Gut microbiota composition shifted toward a healthier profile, with increases in Lactobacillus and Bifidobacteria and reductions in Firmicutes/Bacteroides ratios, while obese mutants displayed dysbiosis that MOL counteracted. Circulating sACE2, elevated in obese rats, declined markedly in MOL-fed groups, suggesting improved systemic balance. High-protein diets induced renal stress, yet MOL with fiber reduced β2-microglobulin by nearly half, indicating protective synergy. These multi-system effects highlight MOL as a potent functional food candidate capable of bridging the nutritional paradox of obesity and protein-energy malnutrition. Future research should extend these findings to human cohorts, integrating long-term outcomes, sex differences, and mechanistic omics approaches to fully realize its translational potential. Declarations Conflict of Interest: Nil Acknowledgement: The authors would like to thanks Director ICMR-NIV and ICMR-NIN for providing support conducting and publishing this study (20-NINAF03 and 21-NINAF01). Mr. Subash Tatikayala has provided necessary assistance in the animal experimentation. Authors contribution: PBP - Conduct of the study, Study designing, Data collection, Funding Acquisition, Ethics Approval, Supervision and guidance in the project, Data Curation, Formal Analysis, Writing – Original Draft, Writing – Review & Editing PA –Animal Study, Sample collection, Histology, Data collection, TOBEC, DEXA, blood collection and Biochemical Staining SM – Sectioning and Blood collection TB and KR- Serum Biochemistry, ELISA sample pooling and reading SJR – MOL and diet sample analysis SD – Diet formulation and preparation, Review of manuscript VVP- Gut microbe experiments and analysis References Sengupta R et al (2025) Sedentary work and expanding waistlines: a cross-sectional study on occupational roles and abdominal obesity in India. BMC Public Health J 25(1):748 Singh A et al (2025) Trends and determinants of obesity among ever-married women aged 15–49 in India: insights from National Family Health Surveys (NFHS 1998–2021). 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Cardiovasc Pathol 23(5):298–305 Meyer TW, Anderson S, Brenner BM (1983) Dietary protein intake and progressive glomerular sclerosis: the role of capillary hypertension and hyperperfusion in the progression of renal disease. Ann Intern Med 98(5 Pt 2):832–838 Martin WF, Armstrong LE, Rodriguez NR (2005) Dietary protein intake and renal function. Nutr Metabolism 2(1):25 Karthivashan G et al (2016) The modulatory effect of Moringa oleifera leaf extract on endogenous antioxidant systems and inflammatory markers in an acetaminophen-induced nephrotoxic mice model. J PeerJ 4:e2127 Abdel-Daim MM et al (2020) Ethanolic extract of Moringa oleifera leaves influences NF-κB signaling pathway to restore kidney tissue from cobalt-mediated oxidative injury and inflammation in rats. J Nutrients 12(4):1031 Edelstein CL, Ling H, Schrier RW (1997) The nature of renal cell injury. Kidney Int 51(5):1341–1351 Zhang PL, Liu ML (2025) From acute tubular injury to tubular repair and chronic kidney diseases - KIM-1 as a promising biomarker for predicting renal tubular pathology. Curr Res Physiol 8:100152 Nangaku M (2004) Mechanisms of tubulointerstitial injury in the kidney: final common pathways to end-stage renal failure. Intern Med 43(1):9–17 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted 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. 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Activity\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn India, rapid demographic and epidemiological transitions driven by economic growth have accelerated the rise in abdominal obesity, largely due to sedentary lifestyles and reduced physical activity[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] with steep increase and sharp regional variation[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] reaching up to abdominal obesity affected 56% of women and 49% of men in India[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Obesity has emerged as one of the foremost health crises of the twenty-first century, contributing to type 2 diabetes, cardiovascular disease, and renal impairment. At the same time, many low- and middle-income countries face an ongoing burden of protein-energy malnutrition (PEM), which compromises growth, immunity, and metabolic efficiency[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis coexistence of overnutrition and undernutrition usually termed the \u0026ldquo;double burden of malnutrition\u0026rdquo; which complicates dietary recommendations and underscores the need for nutrient-dense interventions capable of supporting lean mass [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]without imposing organ stress[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. High-protein diets are often advocated for weight management and muscle maintenance. However, excessive protein intake is associated with glomerular hyperfiltration and tubular damage, raising concern about renal safety in both obese and malnourished individuals[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eMoringa\u003c/b\u003e \u003cb\u003eoleifera: a multifunctional food source\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eMoringa oleifera\u003c/em\u003e leaves (MOL), commonly known as the \u0026ldquo;drumstick tree,\u0026rdquo; is native to South Asia found to be a uniquely nutrient-dense food. Beyond its nutritional profile, MOL exhibits anti-inflammatory, antioxidant, and metabolic modulatory properties. The choice of MOL is also supported by their suitability and cost-effectiveness as a plant-based protein alternative, free from pesticides and heavy metals, and readily available throughout the year.\u003c/p\u003e\u003cp\u003eTheir cultivation and utilization require minimal investment and labor, making them a practical option from soil to fork. Animal studies demonstrate that MOL extracts lower serum cholesterol, improve glucose tolerance, and ameliorate oxidative stress markers [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Importantly, MOL also contains soluble fibers and phytochemicals with prebiotic potential, suggesting it could favorably shift gut microbiota composition[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Given its affordability and cultural acceptance in South Asia, MOL represents an attractive candidate for addressing both obesity and malnutrition.\u003c/p\u003e\n\u003ch3\u003eGut microbiota as a metabolic regulator\u003c/h3\u003e\n\u003cp\u003eThe gut microbiome plays a critical role in nutrient absorption, immune function, and host metabolism[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Dysbiosis is an imbalance in microbial communities which is strongly associated with obesity and metabolic syndrome. Specifically, an elevated \u003cem\u003eFirmicutes/Bacteroides\u003c/em\u003e ratio and reduced probiotic taxa such as \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacteria\u003c/em\u003e have been reported in obese humans and rodents [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Interventions that lower this ratio and enrich probiotics improve insulin sensitivity and reduce systemic inflammation. Exercise also remodels the microbiota, increasing diversity and favoring beneficial genera[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Given MOL\u0026rsquo;s prebiotic activity in various models[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], a combined diet\u0026ndash;exercise intervention could exert synergistic effects on microbial balance.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSkeletal muscle as a target of nutritional intervention\u003c/h2\u003e\u003cp\u003eSkeletal muscle, comprising up to 40% of body mass, is a key determinant of energy expenditure and metabolic health. In obesity, muscles accumulate intramyocellular lipid (IMCL), which correlates with insulin resistance[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Exercise reduces IMCL and promotes mitochondrial biogenesis, increasing oxidative enzyme activity and fiber hypertrophy[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Glycogen utilization during exercise further reflects metabolic efficiency. Nutritional supplementation with antioxidant-rich plant products can amplify these adaptations, protecting muscle from oxidative damage and enhancing endurance capacity. MOL extract has already been shown to attenuate chemical-induced muscle atrophy and improve exercise performance in rodent models[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRenal stress and high-protein diets\u003c/h3\u003e\n\u003cp\u003eHigh-protein diets, while effective in increasing lean body mass, raise concerns about long-term kidney health. Increased nitrogen load and glomerular pressure can cause microstructural injury, reflected by early biomarkers such as N-acetyl-β-D-glucosaminidase (NAGase) and β2-microglobulin[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Plant-derived bioactives that attenuate oxidative and inflammatory stress within the kidney are therefore of great relevance. MOL\u0026rsquo;s nephroprotective effects have been documented in models of chemically induced kidney damage [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], but its capacity to offset protein-induced renal stress has not been systematically evaluated.\u003c/p\u003e\n\u003ch3\u003esACE2 as a metabolic and immunological node\u003c/h3\u003e\n\u003cp\u003eACE2 exists as tissue ACE2 and serum ACE2 (sACE2). Tissue ACE2 is expressed in lung alveolar cells, intestine, heart, kidney, and endothelium, where it regulates the renin\u0026ndash;angiotensin system (RAS) by degrading Ang II into Ang (1\u0026ndash;7), promoting vasodilation and protection against inflammation and fibrosis. sACE2 originates from shedding of tissue ACE2 by ADAM17/TACE and reflects both tissue expression and inflammatory activity. Thus, tissue ACE2 mediates local effects, while sACE2 serves mainly as a biomarker of tissue stress or systemic disease reflecting its paradoxical role [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, it has been observed that ACE2 regulation differs by context. Tissue ACE2 increases physiologically with estrogen, exercise, and ARBs, and pathologically in diabetes, obesity, and heart failure, while it decreases in ARDS, viral infections (via SARS-CoV-2 internalization), aging, and kidney disease. sACE2 rises with inflammation and oxidative stress due to increased shedding, but decreases when tissue expression is globally reduced, as in advanced fibrosis[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, sACE2 was considered a parameter in this study.\u003c/p\u003e\u003cp\u003eACE2 links with diseases are strongly supported in the literature. In COVID-19, tissue ACE2 is reduced after viral binding, driving Ang II\u0026ndash;mediated lung injury and cytokine storm, while sACE2 rises and correlates with severity[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In sepsis, sACE2 increases \u003cem\u003evia\u003c/em\u003e ADAM17 activation. In metabolic disease, tissue ACE2 is upregulated in adipose, pancreas, and kidney as compensation, while sACE2 is persistently elevated, marking higher cardiovascular risk. Hypertension therapy with ARBs increases tissue ACE2, while liver diseases (cirrhosis, NAFLD) also show high sACE2[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDynamics vary with time. In acute and chronic injury, tissue ACE2 is downregulated through overutilization acting as transient molecular disease [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] or upregulated depends through body ability to fight back through protective mechanism [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] by increasing ACE2 however sACE2 rises due to increased stress [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], predicting poor outcomes. Drugs such as ARBs, statins, and SGLT2 inhibitors favorably modulate ACE2. Clinically, sACE2 is a prognostic biomarker, and tissue ACE2 is a therapeutic target, with recombinant soluble ACE2 being tested as a viral decoy. Aerobic exercise reduces ACE2 expression in several organs, suggesting a protective effect[\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Whether MOL influences ACE2 regulation in obesity and exercise remains unknown, but such an effect would be highly relevant to cardiometabolic health.\u003c/p\u003e\u003cp\u003eIn summary, tissue ACE2 protects but can be hijacked by viruses, whereas sACE2 reflects injury and prognosis. Low tissue ACE2 with high sACE2 indicates severe disease, while high tissue ACE2 (without viral exploitation) is protective in cardiovascular, renal, and metabolic conditions.\u003c/p\u003e\n\u003ch3\u003eStudy rationale and novelty\u003c/h3\u003e\n\u003cp\u003eAlthough MOL is recognized for its nutritional and medicinal value, its systemic role across multiple endpoints like muscle remodeling, glycogen use, gut microbiota balance, renal stress under high-protein load, and sACE2 modulation has not been studied in an integrated framework. Previous studies tend to isolate single outcomes, limiting translational relevance. By combining MOL supplementation with exercise in both lean and obese rat models across three experimental phases, we aimed to create a high-fidelity picture of how MOL functions as a nutraceutical (Fig.\u0026nbsp;1a).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWe hypothesized that dietary MOL supplementation would enhance neuromuscular performance while simultaneously promoting favorable remodeling of the gut microbiota. Under high-protein feeding conditions, we anticipated that supplementation would alleviate renal stress, reflected by reductions in circulating biomarkers of kidney injury. To further address the practical question of whether plant-based high-protein diets differ from animal-based sources in their renal impact, we evaluated whether MOL and dietary fiber could provide protection against protein-induced kidney damage. We also hypothesized that supplementation might influence systemic sACE2 levels, which are elevated in obesity, thereby contributing to metabolic regulation. Finally, we proposed that structured exercise would act synergistically with MOL supplementation, amplifying beneficial effects across muscular, microbial, renal, and systemic endpoints.\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003eObjectives and Hypotheses\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003eThis study examined whether MOL supplementation, alone or with exercise, improves muscle performance, modulates gut microbiota, reduces renal stress from high-protein diets, and regulates circulating sACE2 levels. We hypothesized that MOL would enhance lean mass, promote microbial balance, protect renal function, and lower sACE2, with exercise amplifying these benefits.\u003c/b\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e All experimental procedures were carried out at the National Institute of Nutrition (NIN), Hyderabad, in compliance with the guidelines of the CCSEA (Animal Ethics Approval: ICMR-NIN/IAEC-IV/02/001/2020 and ICMR-NIN/IAEC/2021-1/005). Male rats were selected to maintain uniformity and avoid confounding effects of hormonal variations. Three distinct cohorts were employed across the study (Fig.\u0026nbsp;1b-1d). Each group consisted six male animals of 3 months old rat. In all phase\u0026rsquo;s animals were randomized based on body weight. In Phase I, NIN/Sprague Dawley rats (Fig.\u0026nbsp;1e-left) were used to investigate the impact of isocaloric and isonitrogenous diet (20% protein) MOL supplementation (2% and 4%) with or without forced exercise (30 minutes swimming) on muscle physiology and metabolism. Phase II utilized Wistar/NIN rats (Fig.\u0026nbsp;1e-right) along with obese mutant strains including \u003cem\u003eOb/ob\u003c/em\u003e and \u003cem\u003eGr/ob\u003c/em\u003e, as well as their lean counterparts with standard pelleted diet (20% protein), to evaluate impact of exercise (30 minutes swimming) on sACE2 levels of obese mutants. Phase III was designed on Wistar/NIN male to address the effects of high-protein diets (40%) on renal stress and to test whether MOL supplementation (8%) with or without high fiber (8%) could provide protection under these conditions. Animals were housed in polypropylene cages with autoclaved bedding under controlled temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and humidity (50\u0026ndash;70 percent) with a reversed 12:12 h light\u0026ndash;dark cycle. They were acclimatized for one week before the initiation of interventions and were provided autoclaved water and standard pelleted diet and customized AIN-93M diet \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe diets were formulated in the NIN animal diet section (Fig.\u0026nbsp;2a-2b). The ingredients included wheat flour, roasted Bengal gram, casein, skimmed milk powder, groundnut oil, sucrose, vitamin and mineral mixtures, cellulose, and dried MOL powder prepared from sun-dried leaves pulverized into fine powder. In phase I, the MOL intervention was designed to provide either 2 percent or 4 percent supplementation by weight. In Phase I and II, the protein content was maintained at 20 percent, while in Phase III the diet was modified to provide 40 percent protein with or without 8% MOL. To evaluate the effects of fiber and MOL interaction, Phase III diets incorporated either 4 percent or 8 percent fiber. All diets were isocaloric and isonitrogenous, with proximate analysis confirming macronutrient content. The diet preparation followed a standard procedure [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] where it was pelletized, sterilized using autoclaving, and stored under refrigeration (Cold room) until use, and all batches were confirmed to be free of microbial contamination.\u003c/p\u003e\u003cp\u003eThe exercise intervention consisted of forced swimming (Fig.\u0026nbsp;1f), selected as a model for endurance-type activity[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Animals were trained to swim in stainless steel tanks filled with water (Fig.\u0026nbsp;1h) maintained at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C. Swimming was performed for 30 minutes for 6 days/week for six weeks. To minimize variability, all exercise groups were subjected to swimming sessions simultaneously, with animals identified by tail markings under red light illumination during the dark phase. A reversed light\u0026ndash;dark cycle was maintained to align the intervention with the anabolic window of opportunity and to prevent stress or depressive effects associated with electric stimulation for exercise and circadian disruption. This duration was selected to provide consistent aerobic challenge without inducing exhaustion. Following exercise, animals were towel-dried to prevent hypothermia and returned to their home cages. As shown in our earlier publication, MOL supplementation has improved feed intake and serum protein levels throughout the intervention, where body weight and feed intake were recorded weekly to monitor physiological adaptation[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAt initial week and 6th week of study phase, neuromuscular assessments were performed, including measurement of grip strength (Fig.\u0026nbsp;1g-left) using GSM-01 LI Model (Orchid Scientific and Innovation India Pvt. Ltd, India) and locomotor performance using actimeter (Fig.\u0026nbsp;1g-right). Grip strength was measured with a digital grip strength meter, where the rat was allowed to grasp a horizontal bar using forelimbs and gently pulled backward by the tail until grip release. The maximum force was recorded, and three attempts were averaged per animal. Locomotor activity was recorded using automated actimetry chambers to capture exploratory and repetitive activity. Body composition was evaluated by both total body electrical conductivity (TOBEC) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]and dual-energy X-ray absorptiometry (DEXA). TOBEC (EM-SCAN model 3000) measurements provided estimates of fat-free mass, lean body mass, total body water, and fat percentage, while DEXA scans were performed (Hologic Discovery A QDR system) to assess bone mineral content and bone mineral density.\u003c/p\u003e\u003cp\u003eAt the end of the intervention, blood samples were collected from the retro-orbital plexus under light isoflurane anesthesia. Serum was separated and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further analysis. Serum biochemistry was performed using the COBAS C-311 analyzer (Roche, Switzerland) to measure lactate dehydrogenase (LDH) and serum lactate levels. The biochemical assays were performed as per kit protocols provided by respective companies. It included measurement of gastrocnemius glycogen concentration (E-BC-K073-S, Elabscience, China) using a colorimetric assay kit, and determination of sACE2 (LS-F10723, LSBio, USA), β2-microglobulin (E-EL-R1085, Elabscience, China), and N-acetyl-β-D-glucosaminidase (NAGase; NBP2-81174, Novus Biologicals, USA) using ELISA reader (BioTek Synergy H1, USA). These markers were chosen based on their physiological relevance: sACE2 as an indicator of metabolic and cardiovascular stress, β2-microglobulin as a marker of glomerular function, and NAGase as a sensitive indicator of proximal tubular injury.\u003c/p\u003e\u003cp\u003eOrgan weights were recorded at necropsy. Liver, kidney, spleen, heart, lungs, and brain were excised, cleaned of connective tissue, blotted dry, and weighed. Skeletal muscles including gastrocnemius and soleus were dissected, weighed, and measured for length before being processed for histological analysis. The soleus and gastrocnemius muscles were selected for analysis because they represent distinct fiber-type compositions and metabolic profiles. The soleus is predominantly composed of slow-twitch oxidative fibers, making it highly fatigue-resistant and suited for sustained, endurance-type activity, whereas the gastrocnemius contains a mixture of fast- and slow-twitch fibers, contributing both to powerful, propulsive movements and glycolytic metabolism. During swimming, both muscles are actively recruited, with the soleus supporting continuous low-intensity contractions and the gastrocnemius generating bursts of propulsive force. Examining these muscles therefore provides complementary insights into exercise-induced adaptations across oxidative and glycolytic pathways, as well as muscle-specific responses to swimming training.\u003c/p\u003e\u003cp\u003eHistological evaluations were conducted on muscle tissue using hematoxylin and eosin staining for general morphology, Oil Red O staining for lipid deposition, and succinate dehydrogenase activity staining for oxidative capacity. Sections were imaged and quantified using ImageJ software. Morphometric parameters included fiber diameter along both long and short axes, cross-sectional area, and connective tissue thickness. At least 5 sections/animal were measured to ensure statistical validity. These analyses provided an integrated view of structural and metabolic adaptations within skeletal muscle.\u003c/p\u003e\u003cp\u003eGut microbiota was evaluated using quantitative polymerase chain reaction (qPCR) targeting bacterial 16S ribosomal RNA genes as per our earlier published procedure [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. DNA was extracted from caecal fecal pellets using the QIAamp DNA Stool Mini Kit (Qiagen, Germany), and species-specific primers were used to quantify \u003cem\u003eLactobacillus\u003c/em\u003e, Bifido, Firmicutes, Bacteroides, and total bacteria. Relative abundances were calculated and used to derive the Firmicutes to Bacteroides ratio and the Firmicutes to Bifido bacteria ratio, both of which are considered key indicators of metabolic dysbiosis.\u003c/p\u003e\u003cp\u003eStatistical analyses were conducted using GraphPad Prism \u003cem\u003eversion\u003c/em\u003e 8.0. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way or two-way ANOVA was applied as appropriate, followed by \u003cem\u003eTukey\u0026rsquo;s\u003c/em\u003e or \u003cem\u003eSidak\u0026rsquo;s\u003c/em\u003e post hoc tests. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eMuscle Function and Morphology\u003c/h2\u003e\u003cp\u003eMOL supplementation markedly enhanced neuromuscular performance (grip strength) in NIN/Sprague Dawley rats (Fig.\u0026nbsp;2c). While the Normal Control group showed only a 7% increase in grip strength over six weeks, MOL-fed groups demonstrated much higher gains, ranging from +\u0026thinsp;21.2% to +\u0026thinsp;26.2%, with the largest improvements seen in the Group 5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;2c-left). Exercise potentiated the diet effect, with all MOL\u0026thinsp;+\u0026thinsp;exercise groups outperforming either treatment alone.\u003c/p\u003e\u003cp\u003eLocomotor activity assessed through actimeter readings (Fig.\u0026nbsp;2c-right) revealed progressive improvement with MOL supplementation and exercise. Although no significant differences were observed at baseline, by the sixth week, the 4% MOL with Forced Exercise (FE) group exhibited the highest activity count (1456\u0026thinsp;\u0026plusmn;\u0026thinsp;111), significantly surpassing the normal control (1190\u0026thinsp;\u0026plusmn;\u0026thinsp;50; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Control has minimal change (-1.5%) whereas MOL group has shown dose dependent trend increasing significantly from baseline in MOL groups without exercise (1.4%-8.4%) and MOL with forced exercise (7.5%-19.1%).\u003c/p\u003e\u003cp\u003eEnergy handling paralleled these changes observed in from frozen tissue samples of dissected gastrocnemius muscles (Fig.\u0026nbsp;2d). Gastrocnemius glycogen stores fell by 15\u0026ndash;35% relative to control, indicating greater utilization during activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;2d). Histological stains confirmed these functional findings: Oil Red O staining (Fig.\u0026nbsp;2e) showed fewer and smaller lipid droplets in MOL-fed groups, particularly in the MOL\u0026thinsp;+\u0026thinsp;exercise cohort, while succinate dehydrogenase staining (Fig.\u0026nbsp;2f) intensity increased, signifying improved oxidative metabolism.\u003c/p\u003e\u003cp\u003eMorphometric analysis (Fig.\u0026nbsp;3a-3f) supported hypertrophic remodeling. Gastrocnemius mass rose by 5\u0026ndash;17% across supplemented groups (Fig.\u0026nbsp;3a), while soleus mass remained near baseline. Both muscle lengths (Fig.\u0026nbsp;3b), no such change was observed however, muscle fiber diameter (Fig.\u0026nbsp;3c) increased significantly in both soleus (+\u0026thinsp;1%-21%) and gastrocnemius (+\u0026thinsp;7\u0026ndash;21%). Connective tissue thickness slightly declined in both muscles, suggesting healthier tissue remodeling (Fig.\u0026nbsp;3d). The long-axis fiber hypertrophy (+\u0026thinsp;18%-49) and short-axis hypertrophy reaching up to +\u0026thinsp;87% in the Group 5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in gastrocnemius but no such change was observed with soleus (Fig.\u0026nbsp;3e-3f).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eBody Composition\u003c/h2\u003e\u003cp\u003eWhole-body composition reflected these muscle-specific adaptations. Fat-free mass increased most prominently in MOL\u0026thinsp;+\u0026thinsp;exercise groups (+\u0026thinsp;13\u0026ndash;15%), while lean body mass improved slightly upto\u0026thinsp;+\u0026thinsp;6%. Fat percentage and total fat declined slightly, with the greatest reductions observed in 2% MOL alone (\u0026minus;\u0026thinsp;10% fat, \u0026minus;\u0026thinsp;15% total fat; Fig.\u0026nbsp;5a\u0026ndash;b). Skeletal indices showed modest improvements compared to the normal control (Group 1). At the end of the study, bone mineral content (BMC) and bone mineral density (BMD) increased by approximately 7% (Fig.\u0026nbsp;4d left) and 5% (Fig.\u0026nbsp;4d right), respectively, in the combined treatment group (Group 5). Across all five Groups, BMC improved from baseline by 8\u0026ndash;16%, and BMD increased by 2\u0026ndash;7%, whereas Group 5 demonstrated an 15% and 11% improvement in BMC and BMD respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eOrgan Weights and Microbiota\u003c/h2\u003e\u003cp\u003eSystemic organ remodeling showed distinct trends (Fig.\u0026nbsp;5a-5f). Spleen mass declined consistently (\u0026minus;\u0026thinsp;21.7 to \u0026minus;\u0026thinsp;35.6% across groups), indicating reduced inflammatory load. Liver and lung weights were lower in supplemented groups (\u0026minus;\u0026thinsp;3.5 to \u0026minus;\u0026thinsp;9%), while kidney mass showed a mild rise/decline. Brain weight remained stable, indicating no adverse central effects.\u003c/p\u003e\u003cp\u003eGut microbiota analysis revealed robust diet- and exercise-driven restructuring (Fig.\u0026nbsp;5g-5s). \u003cem\u003eLactobacillus\u003c/em\u003e increased by 8\u0026ndash;13%, and \u003cem\u003eBifidobacteria\u003c/em\u003e by 9\u0026ndash;26% in supplemented groups, while the \u003cem\u003eFirmicutes/Bacteroides\u003c/em\u003e ratio fell by up to 11%. The Firmicutes/\u003cem\u003eBifidobacteria\u003c/em\u003e ratio declined even more strongly (\u0026minus;\u0026thinsp;5% to \u0026minus;\u0026thinsp;13.7%). These changes oppose the dysbiosis signature observed in obese mutants (Fig.\u0026nbsp;5t), where \u003cem\u003eBifidobacteria\u003c/em\u003e dropped (\u0026minus;\u0026thinsp;16% in Gr/ob; \u0026minus;39% in Ob/ob) and Bacteroides rose (+\u0026thinsp;12% in Ob/ob). Thus, MOL reversed obesity-associated microbiota shifts while enhancing probiotic abundance in lean rats.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCirculating sACE2\u003c/h2\u003e\u003cp\u003esACE2 responses were striking (Fig.\u0026nbsp;6a-6c). In lean Sprague Dawley rats, circulating sACE2 fell progressively with MOL supplementation: \u0026minus;7% with 2% MOL, \u0026minus;\u0026thinsp;22% with 4% MOL, and \u0026minus;\u0026thinsp;52% in Group 5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In obese mutants, however, sACE2 was elevated (+\u0026thinsp;64% in Ob/ob; +38% in Ob/ob lean; +9% in Gr/ob), highlighting obesity-driven maladaptation. Exercise reduced sACE2 modestly across all strains (\u0026minus;\u0026thinsp;6 to \u0026minus;\u0026thinsp;15%; Fig.\u0026nbsp;6c), suggesting a protective effect that was amplified by MOL. The directionality was consistent: MOL and exercise lowered sACE2, counteracting the obesity-associated rise.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003ePhase III \u0026ndash; Renal Stress under High-Protein Diets\u003c/h2\u003e\u003cp\u003eRenal biomarkers differentiated tubular and glomerular responses. NAGase levels were uniformly elevated (~\u0026thinsp;42\u0026ndash;45 ng/mL) in all 40% protein groups, confirming that tubular stress is unavoidable under protein overload and resistant to dietary intervention (Fig.\u0026nbsp;6d). In contrast, β2-microglobulin (B2M) showed divergent trends (Fig.\u0026nbsp;6e). The high-protein control diet (HPD-1) group (with 8% fiber but no MOL) produced an 11% rise in B2M, showing that fiber alone failed to protect glomeruli. By comparison, the addition of MOL to fiber diets strongly reduced B2M: \u0026minus;49% with HPD-2 (4% fiber\u0026thinsp;+\u0026thinsp;8% MOL) group, and \u0026minus;\u0026thinsp;37% with HPD-3 (8% fiber\u0026thinsp;+\u0026thinsp;8% MOL) group (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u0026ndash;0.0001 vs high-protein control). These data demonstrate that MOL provides clear glomerular renoprotection in a fiber-containing context, although tubular injury persists.\u003c/p\u003e\u003cp\u003eAcross phases, MOL supplementation consistently enhanced muscle strength, glycogen utilization, and hypertrophy, while reorganizing the gut microbiota toward a healthier profile and downregulating sACE2 in lean but not obese mutants. Under high-protein feeding, MOL provided robust protection against glomerular stress but not tubular stress, underscoring the specificity of its nephroprotective action. The convergence of muscular, microbial, renal, and systemic endpoints supports the role of MOL as a functional food for obesity-linked metabolic resilience.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present findings build upon our earlier report where MOL supplementation improved serum protein levels by 14\u0026ndash;19% and reduced serum urea, AST, ALT, and ALP, demonstrating clear hepatic and renal protection[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In the current extended study, these biochemical improvements are complemented by functional outcomes such as increased grip strength and muscle fiber hypertrophy, alongside enhanced glycogen utilization, which mechanistically explain the protein-sparing effects seen earlier[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Similarly, the weight gain observed with 4% MOL in the previous study is corroborated here by improved lean mass and reduced fat deposition, suggesting a consistent anabolic and body-composition benefit. The nephroprotective trend seen earlier through stable creatinine [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] is further validated in this study through reductions in tubular (NAGase) and glomerular (β2-microglobulin) damage markers, particularly under high-protein stress, confirming the resilience of renal physiology. Importantly, while earlier work was limited to biochemical endpoints, the current data also demonstrate gut microbiota remodeling and sACE2 downregulation, which not only extend the hepatoprotective and nephroprotective roles but also position MOL as a systemic modulator of metabolic and immune health.\u003c/p\u003e\u003cp\u003eThe present study demonstrates that dietary supplementation with MOL, particularly when combined with endurance-type exercise, yields systemic benefits across muscle performance, body composition, gut microbial ecology, renal resilience, and circulating sACE2 regulation. By conducting experiments across three phases, we show that MOL exerts context-dependent effects, amplifying exercise-induced adaptations, correcting obesity-associated dysbiosis, and mitigating renal stress induced by high-protein diets.\u003c/p\u003e\u003cp\u003eThe neuromuscular data from Phase I highlight a striking improvement in grip strength and muscle hypertrophy. While controls improved by only 7 percent over six weeks, MOL-fed rats exhibited increases of 21\u0026ndash;28 percent, with the highest gains in those also undergoing exercise. The increased actimeter scores in the 4% MOL\u0026thinsp;+\u0026thinsp;FE group indicate that MOL supplementation augments exercise-induced improvements in locomotor activity, possibly through enhanced glycogen utilization and muscle fiber remodeling. This aligns with the observed rise in grip strength and oxidative enzyme activity reported in the main study. The absence of significant change in lower-dose groups suggests a threshold effect requiring both adequate MOL concentration and physical stimulus to induce measurable behavioral outcomes. The results collectively imply that MOL enhances neuromuscular efficiency and endurance, validating its potential as a functional dietary supplement for improving metabolic resilience and physical activity levels.\u003c/p\u003e\u003cp\u003eThis effect parallels the observed increase in oxidative staining and reduction in lipid deposition, suggesting that MOL enhances the adaptive responses usually attributed to endurance training. Glycogen depletion in MOL-fed groups, particularly under exercise, indicates greater glycogen mobilization and oxidative turnover. These findings are consistent with previous evidence that endurance training increases mitochondrial biogenesis and oxidative enzyme activity in skeletal muscle[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Furthermore, reduced intramyocellular lipid content aligns with improvements in metabolic efficiency, as lipid accumulation is linked to insulin resistance in sedentary muscle[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Importantly, the pattern of short fiber hypertrophy, which increased by as much as 87 percent in the 4 percent MOL plus Exercise group, indicates that MOL potentiates exercise-induced remodeling of glycolytic and oxidative fibers. This resonates with prior work showing that MOL extracts can attenuate drug-induced muscle atrophy and improve exercise capacity in rodent models[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Evidence also suggest MOL improves bone health[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], however as our study was for short duration, slight difference was noted.\u003c/p\u003e\u003cp\u003eGut microbiota data from Phase II provide a complementary dimension to these muscular adaptations. MOL supplementation enriched \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacteria\u003c/em\u003e by up to 26 percent, while lowering the \u003cem\u003eFirmicutes/Bacteroides\u003c/em\u003e ratio by as much as 11 percent. These microbial changes are widely interpreted as signatures of improved metabolic health, as obesity is associated with a higher \u003cem\u003eFirmicutes/Bacteroides\u003c/em\u003e ratio and reduced probiotic representation[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Exercise itself is known to remodel the microbiota, increasing diversity and abundance of taxa linked to short-chain fatty acid production and improved metabolic balance. The combined effect of MOL and exercise, therefore, likely reflects a convergence of prebiotic and activity-induced remodeling. The contrast with obese mutants is notable. \u003cem\u003eOb/ob\u003c/em\u003e rats showed a 39 percent decline in \u003cem\u003eBifidobacteria\u003c/em\u003e and a 13 percent increase in Bacteroides, consistent with dysbiosis associated with obesity. That these shifts were absent in MOL-fed lean groups underscores the corrective capacity of MOL to stabilize microbial composition.\u003c/p\u003e\u003cp\u003ePhase II also revealed important changes in sACE2, a molecule at the intersection of metabolism and immunity. Circulating sACE2 declined progressively with MOL supplementation, reaching a 52 percent reduction in the 4 percent MOL plus Exercise group. Conversely, obese mutants exhibited a 64 percent elevation in sACE2, consistent with findings that obesity and diabetes upregulate sACE2 expression. Exercise alone has been shown to lower sACE2 in muscle, heart, and kidney, helping to rebalance the renin\u0026ndash;angiotensin system[\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The additive effect of MOL with exercise suggests that phytochemicals may reinforce this protective remodeling. The interpretation of sACE2 modulation is complex because circulating sACE2 can be both a marker of metabolic stress and a mediator of viral entry. Nevertheless, the observed decline in MOL plus Exercise groups, when coupled with improvements in grip strength, fiber hypertrophy, and microbial balance, strongly suggests that sACE2 reduction here reflects systemic metabolic improvement rather than loss of protective angiotensin 1\u0026ndash;7 signaling.\u003c/p\u003e\u003cp\u003ePhase III extended the findings to renal stress under high-protein diets. Consistent with prior evidence that high protein intake elevates glomerular pressure and tubular injury markers [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], rats on a 40 percent protein diet showed a rise in NAGase to approximately 45 ng/mL and an 11 percent increase in β2-microglobulin. Importantly, these changes were mitigated when MOL was combined with fiber, leading to a 49 percent reduction in β2-microglobulin compared with the high-protein control. This highlights a synergistic effect between MOL\u0026rsquo;s antioxidant and anti-inflammatory bio-actives and the protective role of dietary fiber in reducing tubular reabsorption stress. Prior studies confirm that MOL extracts reduce oxidative and inflammatory injury in models of nephrotoxicity[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The present findings extend this evidence by demonstrating a protective effect even in the context of protein overload, which has greater translational relevance for individuals adopting high-protein diets for resolving obesity issues or athletic purposes.\u003c/p\u003e\u003cp\u003eIn the high-protein phase, all groups exhibited elevated NAGase levels (~\u0026thinsp;42\u0026ndash;45 ng/mL), confirming that tubular injury is an unavoidable outcome of protein overload, irrespective of fiber or MOL supplementation. Thus, tubular stress appears resistant to dietary modulation under these conditions. However, glomerular injury, reflected by β2-microglobulin, showed a more nuanced response. The high-protein control diet, despite already containing 8% fiber, led to an 11% rise in β2-microglobulin, indicating that fiber alone failed to protect glomerular strain. By contrast, the combination of MOL with fiber reduced β2-microglobulin substantially, by 37\u0026ndash;49% relative to the high-protein control, demonstrating a strong renoprotective effect. These findings emphasize that fiber does not independently safeguard renal function; instead, MOL is the critical protective factor, with fiber acting as a supportive matrix that enhances the bio-efficacy of MOL\u0026rsquo;s phytochemicals. A detailed study on fiber type and content is needed to understand its impact. This synergy may involve slower protein digestion, improved nitrogen handling, or enhanced microbial fermentation, but it is clear that MOL drives the protective effect.\u003c/p\u003e\u003cp\u003eTaken together, these results point to a coherent systems-level model. In skeletal muscle, MOL amplifies exercise-induced improvements in glycogen turnover, oxidative metabolism, and hypertrophy. In the gut, MOL restores microbial balance, enriching probiotic taxa and lowering obesity-associated ratios. In circulation, MOL downregulates sACE2, reversing obesity-associated elevations, while in the kidney it prevents protein-induced stress when fiber is present. The cross-organ convergence strengthens the case for MOL as a functional food that supports both anabolic and protective pathways.\u003c/p\u003e\u003cp\u003eSeveral limitations warrant mention. The study was conducted exclusively in male rats, and sex-specific differences in microbiota, sACE2, and renal adaptation remain unexplored. Sample sizes were modest, and although differences reached statistical significance, power analysis should guide future work. Control exercise group was intentionally avoided in the MOL group as we assessed its impact in obese study (phase II) for sACE2. Microbiota assessments were limited to selected taxa; metagenomics and metabolomics would better characterize microbial function. Likewise, renal assessments were restricted to serum markers; inclusion of other urinary markers and histopathological scoring would improve sensitivity. Despite these limitations, the consistency of effects across independent endpoints and phases provides compelling evidence of MOL\u0026rsquo;s beneficial role.\u003c/p\u003e\u003cp\u003eHigh-protein diets (40% HPD), whether combined with MOL or dietary fiber, did not prevent tubular damage, as reflected by elevated NAGase levels; however, this injury appears to be sublethal or reversible to certain extent[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. More strikingly, glomerular injury showed a differential response: β2-microglobulin (B2M) levels were reduced by 37\u0026ndash;49% in the 8% MOL-supplemented HPD group compared to the normal control, indicating a clear renoprotective effect. In contrast, B2M increased by 11% in the 40% HPD group without MOL, a pattern that suggests glomerular damage is non-reversible once it reaches to threshold level[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] in the absence of supplementation. These findings underscore the importance of MOL in mitigating renal stress associated with high-protein feeding. The translational implications are considerable. In populations experiencing rising obesity, MOL supplementation could serve as a low-cost dietary adjunct to structured exercise programs, enhancing muscle strength and metabolic resilience. For individuals relying on high-protein diets for weight control or athletic performance, the combination of MOL and dietary fiber may lower renal risks. Additionally, the modulation of sACE2 observed in this context suggests cardiometabolic protection, with possible relevance to viral susceptibility pathways. Future investigations should explore dose\u0026ndash;response dynamics, long-term safety, and the potential for human applications through rigorously designed clinical trials.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that dietary supplementation with MOL, particularly at 4 percent when combined with endurance exercise, exerts comprehensive benefits across neuromuscular, metabolic, microbial, and renal domains. Grip strength and gastrocnemius hypertrophy improved substantially, accompanied by enhanced glycogen utilization and reduced intramuscular lipid deposition. Gut microbiota composition shifted toward a healthier profile, with increases in \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacteria\u003c/em\u003e and reductions in \u003cem\u003eFirmicutes/Bacteroides\u003c/em\u003e ratios, while obese mutants displayed dysbiosis that MOL counteracted. Circulating sACE2, elevated in obese rats, declined markedly in MOL-fed groups, suggesting improved systemic balance. High-protein diets induced renal stress, yet MOL with fiber reduced β2-microglobulin by nearly half, indicating protective synergy. These multi-system effects highlight MOL as a potent functional food candidate capable of bridging the nutritional paradox of obesity and protein-energy malnutrition. Future research should extend these findings to human cohorts, integrating long-term outcomes, sex differences, and mechanistic omics approaches to fully realize its translational potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNil\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thanks Director ICMR-NIV and ICMR-NIN for providing support conducting and publishing this study (20-NINAF03 and 21-NINAF01). Mr. Subash Tatikayala has provided necessary assistance in the animal experimentation.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eAuthors contribution:\u003c/h3\u003e\n\u003cul\u003e\n \u003cli\u003ePBP - Conduct of the study, Study designing, Data collection, Funding Acquisition, Ethics Approval, Supervision and guidance in the project, Data Curation, Formal Analysis, Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review \u0026amp; Editing\u003c/li\u003e\n \u003cli\u003ePA \u0026ndash;Animal Study, Sample collection, Histology, Data collection, TOBEC, DEXA, blood collection and Biochemical Staining\u003c/li\u003e\n \u003cli\u003eSM \u0026ndash; Sectioning and Blood collection\u003c/li\u003e\n \u003cli\u003eTB and KR- Serum Biochemistry, ELISA sample pooling and reading\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSJR \u0026ndash; MOL and diet sample analysis\u003c/li\u003e\n \u003cli\u003eSD \u0026ndash; Diet formulation and preparation, Review of manuscript\u003c/li\u003e\n \u003cli\u003eVVP- Gut microbe experiments and analysis\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSengupta R et al (2025) Sedentary work and expanding waistlines: a cross-sectional study on occupational roles and abdominal obesity in India. 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J Front Nutr 9:1018212\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbenavoli L et al (2019) \u003cem\u003eGut microbiota and obesity: a role for probiotics.\u003c/em\u003e 11(11): p. 2690\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShao Z et al (2019) Soluble angiotensin converting enzyme 2 levels in chronic heart failure is associated with decreased exercise capacity and increased oxidative stress-mediated endothelial dysfunction. Translational Res 212:80\u0026ndash;88\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSousa Cunha T et al (2016) \u003cem\u003eExercise and Renin Angiotensin System\u003c/em\u003e, in \u003cem\u003eNew Aspects of the Renin Angiotensin System in Cardiovascular and Renal Diseases\u003c/em\u003e. Bentham Science\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGu Q et al (2014) Contribution of renin\u0026ndash;angiotensin system to exercise-induced attenuation of aortic remodeling and improvement of endothelial function in spontaneously hypertensive rats. Cardiovasc Pathol 23(5):298\u0026ndash;305\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeyer TW, Anderson S, Brenner BM (1983) Dietary protein intake and progressive glomerular sclerosis: the role of capillary hypertension and hyperperfusion in the progression of renal disease. Ann Intern Med 98(5 Pt 2):832\u0026ndash;838\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartin WF, Armstrong LE, Rodriguez NR (2005) Dietary protein intake and renal function. 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Curr Res Physiol 8:100152\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNangaku M (2004) Mechanisms of tubulointerstitial injury in the kidney: final common pathways to end-stage renal failure. Intern Med 43(1):9\u0026ndash;17\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"fbbbc0ca-67f2-489a-b62b-ae1768e7025e","identifier":"10.13039/501100001411","name":"Indian Council of Medical Research","awardNumber":"20-NINAF03 and 21-NINAF01","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"National Institute of Nutrition","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lean Mass, High protein diet, Moringa oleifera leaves, sACE2, NAGase","lastPublishedDoi":"10.21203/rs.3.rs-8275989/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8275989/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eObesity and protein-energy malnutrition represent dual nutritional challenges, with high-protein diets often prescribed to restore lean mass but simultaneously risking renal overload. \u003cem\u003eMoringa oleifera\u003c/em\u003e (MOL), a nutrient-rich plant with antioxidant and prebiotic properties, may offer a dietary strategy to improve muscle metabolism, microbial balance, and renal protection, particularly when combined with exercise.\u003c/p\u003e\u003cp\u003eThis study evaluated the effects of 2% and 4% MOL in 20% protein diet and 8% MOL in 40% protein diet, with or without forced swimming, and study includes three experimental phases in NIN/Sprague Dawley, Wistar/NIN, and obese mutant rats (\u003cem\u003eOb/ob\u003c/em\u003e and \u003cem\u003eGr/ob\u003c/em\u003e). In Phase I (20% protein diet), MOL improved neuromuscular performance: grip strength increased by 21\u0026ndash;26% compared with 7.6% in controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Gastrocnemius glycogen decreased by 15\u0026ndash;35% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), reflecting greater glycogen utilization. Morphometry revealed fiber hypertrophy, with short fibers enlarging by 87% in \u003cem\u003eGroup 5\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in gastrocnemius muscles. Phase II demonstrated microbiota remodeling, with \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacteria\u003c/em\u003e increasing by 08\u0026ndash;26% and the \u003cem\u003eFirmicutes/Bacteroides\u003c/em\u003e ratio decreasing by up to 11%. Circulating sACE2 declined by 7\u0026ndash;52% with MOL, contrasting with elevations in \u003cem\u003eOb/ob\u003c/em\u003e rats (+\u0026thinsp;64%). Phase III, involving 40% protein diets, revealed renal stress with elevated NAGase (~\u0026thinsp;45 ng/mL) and β2-microglobulin (+\u0026thinsp;11%); these were attenuated by MOL with fiber (\u0026minus;\u0026thinsp;49%).\u003c/p\u003e\u003cp\u003eCollectively, MOL enhanced muscle performance, promoted microbial homeostasis, lowered sACE2, and provided renoprotection under high-protein load, particularly when combined with exercise. These findings highlight MOL as a functional food candidate for obesity management and metabolic resilience.\u003c/p\u003e","manuscriptTitle":"Lean Mass Gain from a Moringa-Enriched Protein Diet Confers Reno-Protective Effects and Modulates Gut Microbiota and sACE2 Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 04:57:33","doi":"10.21203/rs.3.rs-8275989/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eaa69493-7897-403e-9431-f61b2178ff06","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59069029,"name":"Nutrition \u0026 Dietetics"},{"id":59069030,"name":"Animal Science"}],"tags":[],"updatedAt":"2025-12-05T04:57:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-05 04:57:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8275989","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8275989","identity":"rs-8275989","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}