Dapagliflozin and Aerobic Exercise Synergistically Attenuate Hepatic Steatosis via Complementary Regulation of Lipogenesis and Fatty Acid Oxidation in Type 2 Diabetic Rats

Dapagliflozin and Aerobic Exercise Synergistically Attenuate Hepatic Steatosis via Complementary Regulation of Lipogenesis and Fatty Acid Oxidation in Type 2 Diabetic Rats | 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 Dapagliflozin and Aerobic Exercise Synergistically Attenuate Hepatic Steatosis via Complementary Regulation of Lipogenesis and Fatty Acid Oxidation in Type 2 Diabetic Rats Longxiang Zhou, Xinghan Du, Liangzhi Zhang, Xudong Yang, Jun Shen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8972569/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Background Pharmacological interventions and regular exercise are widely regarded as core strategies for improving type 2 diabetes mellitus (T2DM) accompanied by non-alcoholic fatty liver disease (NAFLD), as supported by previous metabolic and clinical research. Nevertheless, the potential synergistic impact of combination therapy involving dapagliflozin (Dapa) and physical exercise on hepatic lipid metabolism—particularly its mechanism in regulating the balance between lipogenesis and lipolysis—remains incompletely understood and a definitive consensus has not yet been established. Therefore, the present study was designed to elucidate the effects of Dapa, administered either as monotherapy or in combination with aerobic exercise, on hepatic lipid deposition and its potential underlying mechanisms. Methods A total of 24 four-week-old male Sprague-Dawley (SD) rats were randomly assigned to four groups (n = 6 per group) following successfully model establishment. T2DM was induced in using a high-fat diet combined with streptozotocin administration (30 mg/kg, intraperitoneally), a protocol commonly employed in metabolic disease research. Dapagliflozin was administered daily by gavage to the treatment group. The combination group received both dapagliflozin treatment and a progressive treadmill running program designed to represent aerobic exercise intervention. Hepatic lipid deposition was quantified using Oil Red O staining, whereas Western blot analysis was conducted to determine the expression of key proteins involved in lipid metabolic regulation. Results Both dapagliflozin monotherapy and the combined intervention with aerobic exercise significantly attenuated hepatic steatosis and were associated with improvement in insulin resistance, as reflected by HOMA-IR. Although no additive improvement in HOMA-IR was observed with the combined therapy, a more pronounced reduction in hepatic lipid accumulation was detected. Moreover, the findings suggest that dapagliflozin monotherapy primarily acted through inhibition of hepatic de novo lipogenesis. In contrast, the combined intervention appeared to exert additional effects through enhanced fatty-acid catabolism, thereby contributing to a greater reduction in hepatic lipid content. Conclusion (1) Dapagliflozin suppresses hepatic de novo lipogenesis; (2) Aerobic exercise preferentially enhances lipolysis, thereby producing a complementary therapeutic effect when combined with Dapa; and (3) The combined intervention, through a dual mechanism characterized by suppressing of lipid synthesis and promotion of lipid breakdown, results in an additive metabolic benefit. Aerobic Exercise Dapagliflozin Type 2 Diabetes Mellitus Liver Lipid Accumulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Background Type 2 diabetes mellitus (T2DM) is a prevalent chronic metabolic disorder characterized by impaired glucose regulation. Its global incidence and mortality have continued to rise over the past decades, rendering it a major public health challenge with significant implications for global health[ 1 ]. A substantial body of evidence indicates that patients with T2DM frequently present with non-alcoholic fatty liver disease (NAFLD)[ 2 ]. NAFLD has emerged as one of the leading causes of chronic liver disease worldwide, and its prevalence is increasing rapidly in parallel with the global epidemic of metabolic disorders , [ 3 , 4 ]. As a spectrum of metabolic dysfunction-associated liver diseases, NAFLD encompasses simple hepatic steatosis, non-alcoholic steatohepatitis (NASH), and progressive complications including liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC)[ 5 ]. Epidemiological studies consistently demonstrate close, bidirectional, association between T2DM and NAFLD: individuals with NAFLD have approximately twice the risk of developing T2DM compared with the general population, and the incidence of T2DM increases with the severity of NAFLD histopathology[ 6 , 7 ]. The liver is a critical organ in the maintenance of systemic metabolic homeostasis[ 8 ]. It regulates lipid metabolism through coordinated control of fatty acid uptake and release, de novo lipogenesis (DNL), and fatty acid oxidation (FAO)[ 9 ]. Disruption of these pathways promotes hepatic lipid accumulation and triggers fibrotic and inflammatory signaling cascades, thereby accelerating the progression of liver pathology[ 10 ]. Pharmacotherapy remains the cornerstone of T2DM management. Among available agents, sodium-glucose cotransporter-2 (SGLT2) inhibitors are widely used for their glucose-lowering efficacy. These drugs reduce circulating blood glucose by inhibiting SGLT2 activity in the renal proximal tubule, thereby promoting urinary glucose excretion[ 11 , 12 ]. Recent studies demonstrates that SGLT2 inhibitors not only improve glycemic control but also alleviate hepatic steatosis in both clinical and experimental settings[ 13 – 16 ]. The underlying mechanisms involve at least two primary pathways: First, by increasing urinary glucose excretion and thereby lowering blood glucose and insulin levels, SGLT2 inhibitors improve insulin resistance and reduces the stimulation of hepatic lipogenesis[ 13 ]. Second, inhibition of SGLT2 in pancreatic alpha cells elevate glucagon secretion[ 17 ], which activates hepatic peroxisome proliferator-activated receptor (PPAR) signaling, promotes gluconeogenesis, and enhances hepatic FAO[ 18 ]. These changes collectively drive a metabolic shift from carbohydrates to fatty acids, thereby reducing hepatic fat content[ 19 , 20 ]. Nevertheless, whether individual SGLT2 inhibitors act through both pathways simultaneously remains controversial. Some reports support concurrent regulation of lipogenesis and lipolysis[ 21 , 22 ]; whereas others describe predominant inhibition of lipogenesis[ 19 , 23 ] or a primary promotion of FAO[ 24 ]. These discrepancies are particularly evident for dapagliflozin (Dapa), where debate persists regarding its effects on FAO-related genes and proteins expression. Exercise is a fundamental non-pharmacological strategy for preventing and managing metabolic diseases. Well-designed exercise programs not only attenuate the progression of T2DM but also reduce the risk of its complications. Aerobic exercise (AE), in particular, has been shown to decrease visceral adiposity[ 25 ]. The principal mechanism by which exercise attenuates hepatic fat accumulation is thought to involves activation of hepatic AMP-activated protein kinase (AMPK), which orchestrates downstream changes in the expression and activity of lipid metabolism-related genes and proteins[ 26 , 27 ]. Both AE and pharmacological therapies are therefore recognized as effective interventions for improving T2DM outcomes and reducing hepatic fat steatosis[ 12 , 28 ]. Given these insights, understanding the combined metabolic effects of SGLT2 inhibitors and AE is critical for the development of optimized therapeutic strategies. Current evidence on their interaction, however, remains limited and inconclusive. Some studies report synergistic effects of combined SGLT2 inhibition and AT[ 29 , 30 ] whereas others suggest neutral or even antagonistic interactions[ 31 , 32 ]. To address these uncertainties, the present study aims to investigate the effects of Dapa monotherapy and Dapa combined with AE on hepatic lipid accumulation in a rat model of T2DM induced by a high-fat diet (HFD) and streptozotocin (STZ). Furthermore, this study seeks to elucidate the underlying mechanisms by examining the independent and combined impacts of these interventions on key processes including DNL, FAO, and hepatic lipid uptake and export. 2 Materials and Methods 2.1 Experimental Animals Forty male Sprague-Dawley rats (4 weeks old; body weight 200 ± 20 g) were obtained from the laboratory Animal Center of the Zhejiang Academy of Medical Sciences (Hangzhou, China). Animal were housed in the Laboratory Animal Facility of Zhejiang Normal University (ZJNU) under specific pathogen-free (SPF) conditions with a 12-h light/12-h dark cycle and maintained at a constant temperature and humidity. Two rats were housed per cage with free access to standard chow and water. After a 1-week of acclimatization period, the animals were randomly assigned to either a normal diet (ND) group (n = 10) or a high-fat diet (HFD) group (n = 30). Type 2 diabetes mellitus(T2DM) was induced in the HFD group by two intraperitoneal injections of streptozotocin(STZ) according to established protocols[ 33 – 35 ]. Specifically, rats were fed an HFD for four weeks, followed by an intraperitoneal injection of STZ (30 mg/kg, dissolved in 0.1 M citrate buffer, pH 4.5), and then maintained on the HFD for an additional four weeks. This procedure was repeated once. Rats with fasting blood glucose levels ≥ 14 mmol/L, measured using the OneTouch Ultra™ system(Life Scan, USA), were considered diabetic[ 33 ]. All animal procedures conformed to the guidelines for laboratory animal care and were approved by the Animal Welfare and Ethics Committee of ZJNU (Approval No. ZSDW2022015). At week 13, T2DM rats were further randomized into three experimental groups (n = 6 per group), while ND rats served as non-diabetic controls. After six weeks of treatment, the final grouping was as follows: (1) Control group (CON), ND rats receiving no intervention. (2) Disease group (DM), T2DM rats administered 0.9% NaCl solution by gavage. (3) Drug treatment group (DMDa), T2DM rat gavage with dapagliflozin (Dapa) at 10 mg/kg/day. (4) Drug-combined exercise group (DMDa + AE), T2DM rat receiving Dapa combined with aerobic exercise. 2.2 Aerobic Exercise Protocol Aerobic exercise was performed on a motorized treadmill. Before the formal intervention, rats completed three 30-min adaptation sessions at speeds of 0–10 m/min. The training regimen consisted of non-inclined treadmill running for 60 min/day, five days per week, for six consecutive weeks. Each session included a 5-min warm-up at 10 m/min, and 50 min main exercise period at 15–20 m/min, and a 5-min cool-down at 10 m/min, corresponding to approximately 70%–75% of maximal oxygen consumption (VO 2 max)[ 36 ]. 2.3 Liver Tissue Extraction At the end of intervention period, rats were fasted for 12 h and deeply anesthetized with urethane (2 g/kg, intraperitoneally). Adequate anesthesia was confirmed by the absence of pedal withdrawal and corneal reflexes prior to tissue collection. Whole livers were rapidly excised, blotted to remove surface moisture, and weighed using analytical balance (Sartorius Balances, Gottingen, Germany). For biochemical and protein analysis, liver samples were rinsed in ice-cold saline, rapidly frozen in liquid nitrogen, and stored at -80°C (Thermo Fisher Scientific, Waltham, USA). For histological staining, liver tissues were embedded in optimal cutting temperature (OCT) compound (Sakura, Tokyo, Japan), Frozen in isopentane (Macklin, C14950843, Shanghai, China), and stored at -80°C. Cryosections of 10 µm thickness were prepared using a cryostat (Leica Microsystems, Nuss Loch, Germany). 2.4 Oil Red O Staining Hepatic lipid accumulation was assessed using Oil Red O staining (Beyotime, Shanghai, China) following the manufacturer’s instructions. Briefly, frozen liver sections were equilibrated in 85% isopropanol for 20s, stained with Oil Red O solution for 30 min, and washed with distilled water. Sections were counterstained with hematoxylin for 2 min, rinsed with tap water, and mounted with glycerol gelatin for microscopic evaluation. 2.5 Biochemical Analyses Triglyceride (TG) concentrations in liver tissue and plasma were measured using a TG assay kit (Nanjing Jian Cheng Bioengineering Institute, China). Plasma glucose levels were determined using a glucose oxidase assay kit (Nanjing Jiacheng), and serum insulin levels were quantified with a high-sensitive rat insulin ELISA kit (Sangon Biotech, China). Insulin resistance was calculated using the homeostatic model assessment (HOMA-IR) as follows: HOMA-IR = (fasting plasma insulin, in µIU/mL) × (fasting blood glucose, in mg/dL)) / 405. 2.6 Western Blot Analysis Total liver protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific, USA) containing protease and phosphatase inhibitors. Equal amounts of protein resolved by 6–10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane (Bio-Rad, USA). Membranes were blocked with 5% skimmed milk for 2 h and incubated overnight at 4°C with the following primary antibodies: AMPKα(ABclonal, #A12718), p-AMPK(Thr172; ABclonal, #AP1441), CD36(#A5792), ATGL(#A5126), MTTP(#A14028), CPT1A(#A22899), PPARα(# A25296), ACOX1(#A21217), ACC(#A23129), FAS(#A0223). After washing with TBST, membranes were incubated for 2 h at room temperature with HRP-conjugated goat anti-rabbit secondary antibody (ABclonal, AS014). Protein bands were visualized using enhanced chemiluminescence (Thermo Fisher Scientific, 34578) and quantified by densitometry with Quantity One software v4.6.2(Bio-Rad, USA). β-Actin served as a loading control. 2.7 Statistical Analysis All data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using IBM SPSS Statistics 20(IBM. Chicago, IL, USA). Group differences were assessed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons. Graphs were generated using Fiji ImageJ and GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA). A two tailed p < 0.05 was considered statistically significant. 3 Results 3.1 Effects of Dapa Monotherapy and Combined with AE on Body Weight and Body Composition T2DM rats exhibited marked reductions in body weight loss and persistent hyperglycemia compared with control (CON) group ( p < 0.01, p < 0.001, respectively; Fig. 2 A, B). After six weeks of intervention, both fasting body weight and caloric intake were significantly higher in the DMDa group relative to the untreated DM group ( p < 0.05; Fig. 2 C, D), indicating that dapagliflozin attenuated diabetes-related weight loss. No significant differences were observed between the DMDa + AE group and the other groups. Analysis of epididymal white adipose tissue (eWAT) revealed a significant reduction in eWAT mass in both the DMDa and DMDa + AE groups compared with the DM group ( p < 0.01, p < 0.0001, respectively; Fig. 2 F). 3.2 Effects on Metabolic Parameters Relative to the CON group, DM rats showed pronounced exhibited hyperglycemia, hypoinsulinemia, and insulin resistance. DMDa and DMDa + AE treatments significantly lowered fasting blood glucose and elevated serum insulin compared with the DM group ( p < 0.001 for both; Fig. 3 A, B). Moreover, the DMDa + AE group demonstrated further reduction in fasting glucose and additional increase in insulin relative to DMDa alone ( p < 0.05 and p < 0.001, respectively). HOMA-IR was significantly improved in both treatment groups compared with DM ( p < 0.001, p < 0.0001; Fig. 3 C), although combination therapy conferred no additional benefit over dapagliflozin monotherapy. 3.3 Effects on Hepatic Lipid Accumulation Liver wet weight was significantly higher in the DM group than in the CON group ( p < 0.05), whereas both DMDa and DMDa + AE markedly reduced liver weight ( p < 0.001; Fig. 4 A). Consistent with these findings, Oil Red O staining demonstrated a significant decrease in hepatic lipid droplet accumulation in both treatment groups compared with DM ( p < 0.001; Fig. 4 D, E). Notably, DMDa + AE achieved a further reduction in lipid droplets compared with DMDa alone ( p < 0.05). Plasma and hepatic triglyceride (TG) concentrations were similarly reduced by both interventions (Fig. 4 B, C), indicating that dapagliflozin, either alone or combined with aerobic exercise, effectively attenuates hepatic steatosis, with the combined regimen exhibiting an additive histological effect. 3.4 Effects on Hepatic De Novo Lipogenesis Western blot analysis revealed that the ratio of phosphorylated AMPK (p-AMPK, Thr172) to total AMPK was significantly elevated in both DMDa and DMDa + AE groups compared with DM( p < 0.05, p < 0.001; Fig. 5 B), whereas total AMPK expression did not differ among groups (Fig. 5 C). These results indicate that both interventions activate hepatic AMPK signaling. Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), downstream targets of AMPK and key enzymes in de novo lipogenesis, were significantly downregulated in both treatment groups relative to DM ( p < 0.0001; Fig. 5 D, E). No further reductions were observed with combination therapy. FAS expression remained significantly higher in DM group compared with CON group ( p < 0.001; Fig. 5 E). 3.5 Effects on Hepatic Lipolysis Dapa monotherapy exerted no significant effects on hepatic TG hydrolysis or fatty acid (FA) oxidation. In contrast, the combination of Dapa and AE significantly upregulated proteins involved in hepatic lipolysis. Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for TG hydrolysis, was significantly elevated in the DMDa + AE group compared with both CON and DM groups ( p < 0.05 and p < 0.01, respectively; Fig. 6 B). Carnitine palmitoyl transferase 1A (CPT1A), a key enzyme mediating mitochondrial FA transport, was also significantly increased in the DMDa + AE group compared with CON, DM, and DMDa groups ( p < 0.01; Fig. 6 C). Although peroxisome proliferator-activated receptor-α(PPARα), a principal regulator of mitochondrial β-oxidation, displayed an upward trend in both treatment groups, only the DMDa + AE group reached statistical significance ( p < 0.05; Fig. 6 D). Similarly, acyl-CoA oxidase 1(ACOX1), the rate-limiting enzyme of peroxisomal β-oxidation, was significantly upregulated exclusively in the DMDa + AE group ( p < 0.001; Fig. 6 E). Collectively, these findings demonstrate that while dapagliflozin alone does not significantly promote hepatic lipolysis, its combination with AE markedly enhances hepatic fat catabolism and mitigates lipid accumulation. . 3.6 Effects on Hepatic Fatty Acid Uptake and Export Hepatic lipid metabolism is also influenced by fatty acid uptake and very low-density lipoprotein (VLDL) export. In DM group, microsomal triglyceride transfer protein (MTTP), a critical mediator of VLDL assembly, tended to increase compared with the CON ( p < 0.05; Fig. 7 C). However, neither Dapa monotherapy nor its combination therapy significantly altered MTTP expression. Expression of CD36, a fatty acid translocase that facilitates circulating fatty acid uptake, was elevated in the DM group relative to CON ( p < 0.001; Fig. 7 B). Both DMDa and DMDa + AE significantly reduced CD36 expression, with no additional effect of combined intervention. 4 Discussion This study aimed to systematically evaluate the effects of Dapa monotherapy and its combination with AE on hepatic lipid accumulation and the underlying mechanisms in T2DM rats. The results demonstrated that 20-week-old T2DM rats developed significant hepatic steatosis and IR. The principal can be summarized as follows: (1) Six-week of Dapa monotherapy significantly ameliorated hepatic steatosis and IR in T2DM rats, an effect mechanistically linked to enhanced phosphorylation of AMPK at Thr172 and subsequent suppression of key DNL-related proteins; (2) Although Dapa effectively suppressed DNL, it did not substantially upregulate hepatic lipolysis-related proteins; (3) Most notably, compared with Dapa alone, the combined intervention with AE exerted a marked additive effect in alleviating hepatic lipid accumulation, as demonstrated by both metabolomic and histopathological assessments. Further analysis revealed that this additive benefit was primarily attributable to the robust upregulation of hepatic lipolytic proteins by the combined intervention, whereas no further additive suppression of lipogenesis was observed; (4) Dapa monotherapy downregulated hepatic fatty acid transporter CD36, but did not significantly affect MTTP, while the combined regimen failed to further augment these effects. Effects of Dapagliflozin Monotherapy and Combined AE Therapy on Body Weight and Body Composition in Rats In this model, T2DM rats exhibited a progressive decline in body weight accompanied by a notable increase in hepatic mass. This paradoxical phenotype is closely associated with IR and hyperglycemia, which are hallmarks of T2DM[ 37 ]. IR may drive a compensatory upregulation of FAO, leading to increased influx of FFAs into the liver and providing substrate for DNL[ 38 , 39 ]. In parallel, compensatory hyperinsulinemia[ 40 , 41 ]and hyperglycemia[ 42 , 43 ]—both consequences of IR—directly stimulate DNL, thereby exacerbating hepatic triglyceride accumulation. Consistent with this well-established pathogenic framework, the T2DM rats in our study developed significant IR and hyperglycemia. Both Dapa monotherapy and its combination with AE reduced hepatic mass, likely reflecting improvement in IR and glycemic control, as reported in earlier studies on Dapa[ 44 , 45 ], AE alone[ 46 ], and their combined application[ 29 ]. Interestingly, although the combined regimen produced a pronounced additive effect in reducing fasting blood glucose, no similar additive effect was observed in reducing hepatic mass, likely because both monotherapy and combined intervention had already restored hepatic mass to near-normal levels. Regulatory Role of AMPK in Hepatic Lipid Metabolism AMPK activity is typically suppressed in T2DM, contributing to disrupted energy homeostasis[ 47 ]. Numerous studies have shown that SGLT2 inhibitors ameliorate hepatic lipid metabolism by promoting AMPK(Thr172) phosphorylation [ 21 , 24 , 48 – 53 ]. The present findings are consistent with this evidence. Moreover, AE is a well-established activator of AMPK[ 54 – 56 ].However, prior investigations into combined intervention of Dapa and AE have demonstrated tissue-specific heterogeneity. For example, at the skeletal muscle level, the combined regimen showed no significant effect on AMPK phosphorylation, suggesting that exercise-induced AMPK activation is tissue dependent and produces divergent downstream effects across organs[ 57 ]. This study demonstrates that although combined intervention significantly activates AMPK phosphorylation in the liver, it does not yield results surpassing those of Dapa monotherapy. This discrepancy may be related to additive effects at the hepatic lipid metabolism level, whereas no such correlation was observed in skeletal muscle. Future studies should directly compare the differential regulation of the AMPK signaling pathway and its downstream lipid metabolism targets in the liver and skeletal muscle by combined interventions. In light of this study, two possibilities warrant consideration: (1) the combined Dapa and AE intervention may not synergistically amplify AMPK phosphorylation; (2) the AE protocol employed (6 weeks, 5 days/week, 60 min/day, at ∼70–75% of VO₂max) may have lacked sufficient intensity or duration to produce a synergistic effect. Future studies directly comparing different exercise modalities, such as high-intensity interval training (HIIT) versus moderate-intensity continuous training (MICT), will be valuable for clarifying these mechanisms. Effects of SGLT2 Inhibitors on Molecular Mechanisms of Hepatic Lipid Metabolism The literature reports heterogeneous findings regarding the molecular mechanisms by which SGLT2 inhibitors regulate hepatic lipid metabolism[ 15 , 16 , 18 , 19 , 21 , 22 , 24 , 48 , 50 – 53 , 58 – 68 ]. Such variability may reflect compound-specific pharmacological properties or differences in animal models and experimental designs. The present study provides novel evidence that Dapa predominantly suppresses DNL without significantly enhancing FAO-related proteins expression, a profile more consistent with empagliflozin[ 19 , 48 , 66 ] than with canagliflozin[ 50 – 52 , 61 – 65 ]. The focus of this study is the controversy surrounding FAO and its associated protein expression changes. Under T2DM conditions, hepatic FAO is modulated by insulin signaling, with IR typically inducing compensatory FAO upregulation[ 38 ]. Several studies have suggested that SGLT2 inhibitors, including Dapa, may enhance hepatic FAO[ 13 , 21 , 22 ]. However, our results showed that no significant changes in FAO-related proteins expression after Dapa intervention, diverging from certain published findings. One plausible explanation is that as IR improves under Dapa treatment, the compensatory drive to upregulate FAO is attenuated, thereby offsetting any potential drug-induced enhancement. In addition, the effect of Dapa on FAO may be inherently modest or context-dependent, influenced by factors such as the disease model, treatment duration, or exercise intensity. The precise mechanisms—including whether Dapa directly augments FAO and how this relates to IR improvement and actual FAO flux—remain unresolved and merit further investigation. Dapagliflozin Ameliorates Hyperglycemia and Hepatic Lipid Deposition Primarily Through Inhibition of De Novo Lipogenesis The central conclusion of this study is that combined Dapa and AE intervention produced a significant additive effect in alleviating hepatic lipid accumulation, as confirmed by metabolomic and histopathological analyses. While AMPK activation alone does not fully account for this effect, molecular analyses revealed downstream regulatory mechanisms of hepatic lipid metabolism that explain the additive benefit. Hepatic steatosis is primarily driven by an imbalance between lipogenesis and lipolysis[ 10 ]. Regarding lipogenesis, Dapa monotherapy already suppressed DNL substantially, and the combined intervention did not further reduce it. This may reflect two key points: (1) both Dapa and exercise suppress DNL through the ACC/FAS axis, but Dapa alone markedly reduced ACC and FAS protein expression (by 49% and 59%, respectively, relative to the disease group), approaching the lower physiological limit of regulation. The AMPK phosphorylation results in this study further support this interpretation; and (2) Dapa appears to act predominantly via transcriptional repression of ACC/FAS[ 15 , 16 ], whereas exercise may primarily act by inhibiting enzymatic activity through ACC phosphorylation[ 69 ]. When protein expression is already at low level, further suppression through phosphorylation alone becomes unlikely. Future research should include an exercise-only group and directly assess ACC phosphorylation to confirm these hypotheses. Aerobic Exercise Provides Complementary Benefits Through Promotion of Lipolysis and Fatty Acid Oxidation Combined intervention additive effect appears to derive mainly from enhanced lipolysis, as the combined intervention significantly upregulated proteins associated with TG hydrolysis and FAO. This interpretation is consistent with the findings by Tanaka[ 29 ], who reported that wheel running combined with canagliflozin shifted whole-body substrate utilization toward lipids. Given that Dapa’s pharmacological profile more closely resembles that of empagliflozin than canagliflozin, the observed protein-level changes warrant further comparative investigation. ATGL serves as the rate-limiting enzyme in hepatic TG hydrolysis and determining substrate availability for FAO[ 70 – 72 ]. Previous studies have shown that certain SGLT2 inhibitors, such as tofogliflozin and empagliflozin, significantly increase ATGL expression[ 58 , 73 ], whereas Dapa alone does not substantially affect ATGL or its downstream effector HSL[ 74 ]. Consistent with these findings, our study indicates that AE compensates for Dapa’s limited effect on TG hydrolysis by promoting ATGL expression[ 75 ]. Similarly, the combined intervention also enhanced expression of FAO-related proteins, including PPARα, CPT1A, and ACOX1, which aligns with previous evidence that AT enhances FAO pathway [ 26 , 76 ]. Another potential mechanism involves Lipophagy, a selective form of autophagy critical for intracellular lipid droplet mobilization and energy balance[ 77 ]. Previous studies have shown that both Dapa[ 22 ] and AE[ 78 ] can activate Lipophagy through the AMPK/SIRT1 pathway, thereby improving hepatic lipid metabolism. Although our study did not provide direct evidence of additive effects on Lipophagy, further investigation is warranted to determine whether combined interventions exert superior regulatory effects on this pathway. Collectively, these findings suggest that AE enhances hepatic fat-oxidative capacity through mechanisms that are either distinct from or synergistic with those of Dapa. This synergistic effect, not observed under Dapa monotherapy, highlights the therapeutic potential of integrating SGLT2 inhibitors with exercise interventions. Differential Regulation of Fat Uptake and Export To ensure comprehensiveness, this study also examined key proteins involved in hepatic fatty acid uptake and export. Dapa monotherapy significantly downregulated CD36 expression, consistent with prior reports, suggesting a reduction in hepatic fatty acid uptake[ 79 , 80 ]. However, the combined intervention did not further reduce CD36 expression. Regarding MTTP, neither Dapa alone nor its combination with AE significantly altered expression, indicating that lipid export is unlikely to represent a principal mechanism in this model. Future studies should investigate whether longer-term interventions affect other VLDL-associated proteins, such as ApoB100, and their possible contributions to hepatic lipid regulation. 5 Conclusion In summary, Dapa monotherapy robustly ameliorates hepatic steatosis and IR in T2DM rats by activating AMPK and suppressing DNL. Importantly, the combination of Dapa and AE produces a significant additive effect on hepatic lipid reduction, primarily through AE produced a significant additive effect on hepatic lipid reduction, primarily through AT induced enhancement of lipolysis and FAO rather than further suppression of DNL. These findings highlight the therapeutic potential of integrating SGLT2 inhibitors with structured exercise for the management of T2DM-associated hepatic steatosis. Abbreviations ACC Acetyl–CoA carboxylase ACOX1 Acyl–CoA oxidase 1 AE Aerobic exercise AMPK Adenosine monophosphate–activated protein kinase ANOVA Analysis of variance AT Aerobic training ATGL Adipose triglyceride lipase β Actin–Beta–actin CD36 Cluster of differentiation 36 (fatty acid translocase) CON Control group CPT1A Carnitine palmitoyltransferase 1A Dapa Dapagliflozin DM Diabetes mellitus group DMDa Dapagliflozin–treated diabetic group DMDa + AE Dapagliflozin combined with aerobic exercise group DNL De novo lipogenesis ELISA Enzyme–linked immunosorbent assay eWAT Epididymal white adipose tissue FA Fatty acid FAO Fatty acid oxidation FAS Fatty acid synthase FFAs Free fatty acids HCC Hepatocellular carcinoma HFD High–fat diet HIIT High–intensity interval training HOMA IR–Homeostatic model assessment of insulin resistance HRP Horseradish peroxidase IR Insulin resistance MICT Moderate–intensity continuous training MTTP Microsomal triglyceride transfer protein NAFLD Non–alcoholic fatty liver disease NASH Non–alcoholic steatohepatitis ND Normal diet OCT Optimal cutting temperature compound PPARα Peroxisome proliferator–activated receptor alpha RIPA Radioimmunoprecipitation assay buffer SD rats Sprague–Dawley rats SDS PAGE–Sodium dodecyl sulfate–polyacrylamide gel electrophoresis SEM Standard error of the mean SGLT2 Sodium–glucose cotransporter–2 SPF Specific pathogen–free STZ Streptozotocin TBST Tris–buffered saline with Tween 20 TG Triglyceride Declarations Ethics approval and consent to participate The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Animal Welfare and Ethics Committee of Zhejiang Normal University (ZSDW2022015). Consent for publication Not applicable. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding This study was supported in part by grants from Science and Technology Bureau of Jinhua City (Grant No. 2023–4–003) ,China; Fundamental Research Funds for the Central Universities (Grant No. 2412025QD044), China Authors’ contributions Design: All authors participated in the design experiment, with HLQ, LJ and as the main leaders. Data collection: LZZ, XDY,SJ and Wei Li completed the work. Analysis: LXZ and XHD completed the data statistical work, while other authors checked. Writing manuscript: LXZ, XHD and HLQ completed the main contents, with other authors participating in revision and verification. Acknowledgements Longxiang Zhou 1,2† and Xinghan Du 1† contributed equally to this work. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8972569","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":602518720,"identity":"2af073b2-d87e-4748-9bbd-b57ec830412b","order_by":0,"name":"Longxiang Zhou","email":"","orcid":"","institution":"Northeast Normal University","correspondingAuthor":false,"prefix":"","firstName":"Longxiang","middleName":"","lastName":"Zhou","suffix":""},{"id":602518722,"identity":"f0ac9b25-3b7d-47fa-91eb-8ba4f59cddd0","order_by":1,"name":"Xinghan Du","email":"","orcid":"","institution":"Northeast Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xinghan","middleName":"","lastName":"Du","suffix":""},{"id":602518724,"identity":"e7eb3fea-6572-411c-9d3c-9658bfb365c2","order_by":2,"name":"Liangzhi Zhang","email":"","orcid":"","institution":"Shanghai University of Sport","correspondingAuthor":false,"prefix":"","firstName":"Liangzhi","middleName":"","lastName":"Zhang","suffix":""},{"id":602518726,"identity":"54c105e1-4cd7-41f9-9d7f-2bddf33544b2","order_by":3,"name":"Xudong Yang","email":"","orcid":"","institution":"Chungnam National University","correspondingAuthor":false,"prefix":"","firstName":"Xudong","middleName":"","lastName":"Yang","suffix":""},{"id":602518730,"identity":"ade2d1f5-c407-48fc-bbe3-6f374ad45ab0","order_by":4,"name":"Jun Shen","email":"","orcid":"","institution":"Zhejiang Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Shen","suffix":""},{"id":602518732,"identity":"de52dbdc-8ed7-4833-97e0-e8fbe89fc775","order_by":5,"name":"Wei Li","email":"","orcid":"","institution":"Zhejiang Normal University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Li","suffix":""},{"id":602518733,"identity":"e599071e-f9df-4136-b935-df6c38ceb74d","order_by":6,"name":"Lei Ji","email":"","orcid":"","institution":"Zhejiang Normal University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Ji","suffix":""},{"id":602518735,"identity":"bd8e6b2f-9bab-42ed-8786-a014d0084f16","order_by":7,"name":"Helong Quan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYJACZgYGCxB1AMpPIEqLBJBigyklXguPAXFa+G4kP3tcUCEhZ86/5tuDjzsOM/Cz5xjg1SJ5I83ceMYZCWPLGW+3G848c5hBsucNfi0GNxLMpHnbJBI33Di7Dcg4DBQhYIvBjfRv0rz/QFrOPJP+C9RiT1hLDtCWBqCW8z1s0owgWyQI+eXMmzJpnmMSxgY32Mwke9vSeSTOPCvAq4XvePo2aZ4aGzmD84efSfxss5bjb0/egFcLwwEYQyIBTPHgV46ihf8AbkWjYBSMglEwsgEARoVHB3CYGZEAAAAASUVORK5CYII=","orcid":"","institution":"Northeast Normal University","correspondingAuthor":true,"prefix":"","firstName":"Helong","middleName":"","lastName":"Quan","suffix":""}],"badges":[],"createdAt":"2026-02-26 02:53:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8972569/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8972569/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104785653,"identity":"5d269190-e91b-41dd-9f63-515f49005e3e","added_by":"auto","created_at":"2026-03-17 08:12:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82873,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental model modeling and intervention process\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8972569/v1/d1d5a30c366ee8c13bacd9d6.png"},{"id":104784291,"identity":"443a4c03-408f-4ff2-9eb1-e1e930a126c9","added_by":"auto","created_at":"2026-03-17 08:06:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":154708,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of dapagliflozin (Dapa) monotherapy or combined with aerobic exercise (AE) on body weight and adiposity in T2DM rats. (A) Body weight curves over 20 weeks. (B) Fasting blood glucose at week 14. (C) Fasting body weight at week 20. (D–E) Caloric intake during the 6-week intervention and corresponding area under the curve (AUC). (F) Ratio of epididymal white adipose tissue(eWAT) to body weight. Data are mean ± SEM; n = 6 per group. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8972569/v1/3d81180b3bc33bb8f93e273d.png"},{"id":104785844,"identity":"a17d25b4-1d5c-40ac-aa5a-421725d57ab8","added_by":"auto","created_at":"2026-03-17 08:13:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64640,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Dapa monotherapy or combined with AE on metabolic parameters in T2DM rats. (A) Fasting blood glucose. (B) Serum insulin levels. (C) HOMA-IR index. Data are mean ± SEM; n = 6 per group. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8972569/v1/663aee8344f7429e4f831fae.png"},{"id":104785693,"identity":"bf0aa4a6-e382-4071-b224-a239f510e386","added_by":"auto","created_at":"2026-03-17 08:12:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":619915,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Dapa monotherapy or combined with AE on hepatic lipid accumulation in T2DM rats. (A) Liver wet weight. (B) Serum triglyceride (TG) levels. (C) Hepatic TG content. (D–E) ORO staining and quantification of lipid area percentage. Scale bar: 100 µm; magnification: 400×. Four random fields per sample were analyzed. Data are mean ± SEM; n = 6 per group. \u0026nbsp;*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8972569/v1/8833de2c2da8af419475f088.png"},{"id":104785796,"identity":"712b49d3-7705-4734-af30-7066af1921e5","added_by":"auto","created_at":"2026-03-17 08:13:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":179225,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Dapa monotherapy or combined with AE on hepatic de novo lipogenesis in T2DM rats. (A) Representative Western blot of p-AMPKα, AMPKα, ACC, FAS, and β-actin (loading control). (B–E) Quantification of p-AMPKα/AMPK ratio, AMPKα, ACC, and FAS protein levels. Data are mean ± SEM; n = 6 per group. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8972569/v1/89eb1d7652f90378fffda192.png"},{"id":104785614,"identity":"64737b9c-7847-4cf5-989c-3c264b7db193","added_by":"auto","created_at":"2026-03-17 08:12:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":184403,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Dapa monotherapy or combined with AE on hepatic lipolysis in T2DM rats. (A) Representative Western blot of CPT1A, PPARα, ACOX1, ATGL, and β-actin (loading control). (B–E) Quantification of CPT1A, PPARα, ACOX1, and ATGL protein levels. Data are mean ± SEM; n = 6 per group. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8972569/v1/e9618caf23e6a236c5550862.png"},{"id":104785647,"identity":"ea5157d9-c561-4019-b639-b25ddef62e1c","added_by":"auto","created_at":"2026-03-17 08:12:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":121972,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Dapa monotherapy or combined with AE on hepatic fatty acid uptake and export in T2DM rats. (A) Representative Western blot of CD36, MTTP, and β-actin (loading control). (B–C) Quantification of CD36 and MTTP protein levels. Data are mean ± SEM; n = 6 per group. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8972569/v1/1c311c31554013ede64e6e8d.png"},{"id":104835534,"identity":"8ba1d312-90e0-4557-b825-af2d4eb677ad","added_by":"auto","created_at":"2026-03-17 17:45:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2635739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8972569/v1/2a150fd9-3005-46ee-ab5e-80ccb3d38f4d.pdf"},{"id":104785845,"identity":"e1ecd084-51f9-458d-8718-bf99844fa445","added_by":"auto","created_at":"2026-03-17 08:13:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2556289,"visible":true,"origin":"","legend":"","description":"","filename":"WBrawdata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8972569/v1/2623873ddfc9198d9b47e2d1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dapagliflozin and Aerobic Exercise Synergistically Attenuate Hepatic Steatosis via Complementary Regulation of Lipogenesis and Fatty Acid Oxidation in Type 2 Diabetic Rats","fulltext":[{"header":"1 Background","content":"\u003cp\u003eType 2 diabetes mellitus (T2DM) is a prevalent chronic metabolic disorder characterized by impaired glucose regulation. Its global incidence and mortality have continued to rise over the past decades, rendering it a major public health challenge with significant implications for global health[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A substantial body of evidence indicates that patients with T2DM frequently present with non-alcoholic fatty liver disease (NAFLD)[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. NAFLD has emerged as one of the leading causes of chronic liver disease worldwide, and its prevalence is increasing rapidly in parallel with the global epidemic of metabolic disorders\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As a spectrum of metabolic dysfunction-associated liver diseases, NAFLD encompasses simple hepatic steatosis, non-alcoholic steatohepatitis (NASH), and progressive complications including liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Epidemiological studies consistently demonstrate close, bidirectional, association between T2DM and NAFLD: individuals with NAFLD have approximately twice the risk of developing T2DM compared with the general population, and the incidence of T2DM increases with the severity of NAFLD histopathology[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe liver is a critical organ in the maintenance of systemic metabolic homeostasis[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It regulates lipid metabolism through coordinated control of fatty acid uptake and release, de novo lipogenesis (DNL), and fatty acid oxidation (FAO)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Disruption of these pathways promotes hepatic lipid accumulation and triggers fibrotic and inflammatory signaling cascades, thereby accelerating the progression of liver pathology[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePharmacotherapy remains the cornerstone of T2DM management. Among available agents, sodium-glucose cotransporter-2 (SGLT2) inhibitors are widely used for their glucose-lowering efficacy. These drugs reduce circulating blood glucose by inhibiting SGLT2 activity in the renal proximal tubule, thereby promoting urinary glucose excretion[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Recent studies demonstrates that SGLT2 inhibitors not only improve glycemic control but also alleviate hepatic steatosis in both clinical and experimental settings[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The underlying mechanisms involve at least two primary pathways: First, by increasing urinary glucose excretion and thereby lowering blood glucose and insulin levels, SGLT2 inhibitors improve insulin resistance and reduces the stimulation of hepatic lipogenesis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Second, inhibition of SGLT2 in pancreatic alpha cells elevate glucagon secretion[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], which activates hepatic peroxisome proliferator-activated receptor (PPAR) signaling, promotes gluconeogenesis, and enhances hepatic FAO[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These changes collectively drive a metabolic shift from carbohydrates to fatty acids, thereby reducing hepatic fat content[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Nevertheless, whether individual SGLT2 inhibitors act through both pathways simultaneously remains controversial. Some reports support concurrent regulation of lipogenesis and lipolysis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]; whereas others describe predominant inhibition of lipogenesis[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] or a primary promotion of FAO[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These discrepancies are particularly evident for dapagliflozin (Dapa), where debate persists regarding its effects on FAO-related genes and proteins expression.\u003c/p\u003e \u003cp\u003eExercise is a fundamental non-pharmacological strategy for preventing and managing metabolic diseases. Well-designed exercise programs not only attenuate the progression of T2DM but also reduce the risk of its complications. Aerobic exercise (AE), in particular, has been shown to decrease visceral adiposity[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The principal mechanism by which exercise attenuates hepatic fat accumulation is thought to involves activation of hepatic AMP-activated protein kinase (AMPK), which orchestrates downstream changes in the expression and activity of lipid metabolism-related genes and proteins[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Both AE and pharmacological therapies are therefore recognized as effective interventions for improving T2DM outcomes and reducing hepatic fat steatosis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven these insights, understanding the combined metabolic effects of SGLT2 inhibitors and AE is critical for the development of optimized therapeutic strategies. Current evidence on their interaction, however, remains limited and inconclusive. Some studies report synergistic effects of combined SGLT2 inhibition and AT[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] whereas others suggest neutral or even antagonistic interactions[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To address these uncertainties, the present study aims to investigate the effects of Dapa monotherapy and Dapa combined with AE on hepatic lipid accumulation in a rat model of T2DM induced by a high-fat diet (HFD) and streptozotocin (STZ). Furthermore, this study seeks to elucidate the underlying mechanisms by examining the independent and combined impacts of these interventions on key processes including DNL, FAO, and hepatic lipid uptake and export.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental Animals\u003c/h2\u003e \u003cp\u003eForty male Sprague-Dawley rats (4 weeks old; body weight 200\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were obtained from the laboratory Animal Center of the Zhejiang Academy of Medical Sciences (Hangzhou, China). Animal were housed in the Laboratory Animal Facility of Zhejiang Normal University (ZJNU) under specific pathogen-free (SPF) conditions with a 12-h light/12-h dark cycle and maintained at a constant temperature and humidity. Two rats were housed per cage with free access to standard chow and water. After a 1-week of acclimatization period, the animals were randomly assigned to either a normal diet (ND) group (n\u0026thinsp;=\u0026thinsp;10) or a high-fat diet (HFD) group (n\u0026thinsp;=\u0026thinsp;30). Type 2 diabetes mellitus(T2DM) was induced in the HFD group by two intraperitoneal injections of streptozotocin(STZ) according to established protocols[\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Specifically, rats were fed an HFD for four weeks, followed by an intraperitoneal injection of STZ (30 mg/kg, dissolved in 0.1 M citrate buffer, pH 4.5), and then maintained on the HFD for an additional four weeks. This procedure was repeated once. Rats with fasting blood glucose levels\u0026thinsp;\u0026ge;\u0026thinsp;14 mmol/L, measured using the OneTouch Ultra\u0026trade; system(Life Scan, USA), were considered diabetic[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. All animal procedures conformed to the guidelines for laboratory animal care and were approved by the Animal Welfare and Ethics Committee of ZJNU (Approval No. ZSDW2022015).\u003c/p\u003e \u003cp\u003eAt week 13, T2DM rats were further randomized into three experimental groups (n\u0026thinsp;=\u0026thinsp;6 per group), while ND rats served as non-diabetic controls. After six weeks of treatment, the final grouping was as follows: (1) Control group (CON), ND rats receiving no intervention. (2) Disease group (DM), T2DM rats administered 0.9% NaCl solution by gavage. (3) Drug treatment group (DMDa), T2DM rat gavage with dapagliflozin (Dapa) at 10 mg/kg/day. (4) Drug-combined exercise group (DMDa\u0026thinsp;+\u0026thinsp;AE), T2DM rat receiving Dapa combined with aerobic exercise.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Aerobic Exercise Protocol\u003c/h2\u003e \u003cp\u003eAerobic exercise was performed on a motorized treadmill. Before the formal intervention, rats completed three 30-min adaptation sessions at speeds of 0\u0026ndash;10 m/min. The training regimen consisted of non-inclined treadmill running for 60 min/day, five days per week, for six consecutive weeks. Each session included a 5-min warm-up at 10 m/min, and 50 min main exercise period at 15\u0026ndash;20 m/min, and a 5-min cool-down at 10 m/min, corresponding to approximately 70%\u0026ndash;75% of maximal oxygen consumption (VO\u003csub\u003e2\u003c/sub\u003emax)[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Liver Tissue Extraction\u003c/h2\u003e \u003cp\u003eAt the end of intervention period, rats were fasted for 12 h and deeply anesthetized with urethane (2 g/kg, intraperitoneally). Adequate anesthesia was confirmed by the absence of pedal withdrawal and corneal reflexes prior to tissue collection. Whole livers were rapidly excised, blotted to remove surface moisture, and weighed using analytical balance (Sartorius Balances, Gottingen, Germany). For biochemical and protein analysis, liver samples were rinsed in ice-cold saline, rapidly frozen in liquid nitrogen, and stored at -80\u0026deg;C (Thermo Fisher Scientific, Waltham, USA). For histological staining, liver tissues were embedded in optimal cutting temperature (OCT) compound (Sakura, Tokyo, Japan), Frozen in isopentane (Macklin, C14950843, Shanghai, China), and stored at -80\u0026deg;C. Cryosections of 10 \u0026micro;m thickness were prepared using a cryostat (Leica Microsystems, Nuss Loch, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Oil Red O Staining\u003c/h2\u003e \u003cp\u003eHepatic lipid accumulation was assessed using Oil Red O staining (Beyotime, Shanghai, China) following the manufacturer\u0026rsquo;s instructions. Briefly, frozen liver sections were equilibrated in 85% isopropanol for 20s, stained with Oil Red O solution for 30 min, and washed with distilled water. Sections were counterstained with hematoxylin for 2 min, rinsed with tap water, and mounted with glycerol gelatin for microscopic evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Biochemical Analyses\u003c/h2\u003e \u003cp\u003eTriglyceride (TG) concentrations in liver tissue and plasma were measured using a TG assay kit (Nanjing Jian Cheng Bioengineering Institute, China). Plasma glucose levels were determined using a glucose oxidase assay kit (Nanjing Jiacheng), and serum insulin levels were quantified with a high-sensitive rat insulin ELISA kit (Sangon Biotech, China). Insulin resistance was calculated using the homeostatic model assessment (HOMA-IR) as follows: HOMA-IR = (fasting plasma insulin, in \u0026micro;IU/mL) \u0026times; (fasting blood glucose, in mg/dL)) / 405.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western Blot Analysis\u003c/h2\u003e \u003cp\u003eTotal liver protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific, USA) containing protease and phosphatase inhibitors. Equal amounts of protein resolved by 6\u0026ndash;10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane (Bio-Rad, USA). Membranes were blocked with 5% skimmed milk for 2 h and incubated overnight at 4\u0026deg;C with the following primary antibodies: AMPKα(ABclonal, #A12718), p-AMPK(Thr172; ABclonal, #AP1441), CD36(#A5792), ATGL(#A5126), MTTP(#A14028), CPT1A(#A22899), PPARα(# A25296), ACOX1(#A21217), ACC(#A23129), FAS(#A0223). After washing with TBST, membranes were incubated for 2 h at room temperature with HRP-conjugated goat anti-rabbit secondary antibody (ABclonal, AS014). Protein bands were visualized using enhanced chemiluminescence (Thermo Fisher Scientific, 34578) and quantified by densitometry with Quantity One software v4.6.2(Bio-Rad, USA). β-Actin served as a loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were performed using IBM SPSS Statistics 20(IBM. Chicago, IL, USA). Group differences were assessed by one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons. Graphs were generated using Fiji ImageJ and GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA). A two tailed \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effects of Dapa Monotherapy and Combined with AE on Body Weight and Body Composition\u003c/h2\u003e \u003cp\u003eT2DM rats exhibited marked reductions in body weight loss and persistent hyperglycemia compared with control (CON) group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). After six weeks of intervention, both fasting body weight and caloric intake were significantly higher in the DMDa group relative to the untreated DM group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D), indicating that dapagliflozin attenuated diabetes-related weight loss. No significant differences were observed between the DMDa\u0026thinsp;+\u0026thinsp;AE group and the other groups. Analysis of epididymal white adipose tissue (eWAT) revealed a significant reduction in eWAT mass in both the DMDa and DMDa\u0026thinsp;+\u0026thinsp;AE groups compared with the DM group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effects on Metabolic Parameters\u003c/h2\u003e \u003cp\u003eRelative to the CON group, DM rats showed pronounced exhibited hyperglycemia, hypoinsulinemia, and insulin resistance. DMDa and DMDa\u0026thinsp;+\u0026thinsp;AE treatments significantly lowered fasting blood glucose and elevated serum insulin compared with the DM group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for both; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Moreover, the DMDa\u0026thinsp;+\u0026thinsp;AE group demonstrated further reduction in fasting glucose and additional increase in insulin relative to DMDa alone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). HOMA-IR was significantly improved in both treatment groups compared with DM (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), although combination therapy conferred no additional benefit over dapagliflozin monotherapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects on Hepatic Lipid Accumulation\u003c/h2\u003e \u003cp\u003eLiver wet weight was significantly higher in the DM group than in the CON group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas both DMDa and DMDa\u0026thinsp;+\u0026thinsp;AE markedly reduced liver weight (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Consistent with these findings, Oil Red O staining demonstrated a significant decrease in hepatic lipid droplet accumulation in both treatment groups compared with DM (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). Notably, DMDa\u0026thinsp;+\u0026thinsp;AE achieved a further reduction in lipid droplets compared with DMDa alone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Plasma and hepatic triglyceride (TG) concentrations were similarly reduced by both interventions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C), indicating that dapagliflozin, either alone or combined with aerobic exercise, effectively attenuates hepatic steatosis, with the combined regimen exhibiting an additive histological effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effects on Hepatic De Novo Lipogenesis\u003c/h2\u003e \u003cp\u003eWestern blot analysis revealed that the ratio of phosphorylated AMPK (p-AMPK, Thr172) to total AMPK was significantly elevated in both DMDa and DMDa\u0026thinsp;+\u0026thinsp;AE groups compared with DM(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), whereas total AMPK expression did not differ among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results indicate that both interventions activate hepatic AMPK signaling.\u003c/p\u003e \u003cp\u003eAcetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), downstream targets of AMPK and key enzymes in de novo lipogenesis, were significantly downregulated in both treatment groups relative to DM (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). No further reductions were observed with combination therapy. FAS expression remained significantly higher in DM group compared with CON group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effects on Hepatic Lipolysis\u003c/h2\u003e \u003cp\u003eDapa monotherapy exerted no significant effects on hepatic TG hydrolysis or fatty acid (FA) oxidation. In contrast, the combination of Dapa and AE significantly upregulated proteins involved in hepatic lipolysis. Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for TG hydrolysis, was significantly elevated in the DMDa\u0026thinsp;+\u0026thinsp;AE group compared with both CON and DM groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Carnitine palmitoyl transferase 1A (CPT1A), a key enzyme mediating mitochondrial FA transport, was also significantly increased in the DMDa\u0026thinsp;+\u0026thinsp;AE group compared with CON, DM, and DMDa groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Although peroxisome proliferator-activated receptor-α(PPARα), a principal regulator of mitochondrial β-oxidation, displayed an upward trend in both treatment groups, only the DMDa\u0026thinsp;+\u0026thinsp;AE group reached statistical significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Similarly, acyl-CoA oxidase 1(ACOX1), the rate-limiting enzyme of peroxisomal β-oxidation, was significantly upregulated exclusively in the DMDa\u0026thinsp;+\u0026thinsp;AE group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Collectively, these findings demonstrate that while dapagliflozin alone does not significantly promote hepatic lipolysis, its combination with AE markedly enhances hepatic fat catabolism and mitigates lipid accumulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effects on Hepatic Fatty Acid Uptake and Export\u003c/h2\u003e \u003cp\u003eHepatic lipid metabolism is also influenced by fatty acid uptake and very low-density lipoprotein (VLDL) export. In DM group, microsomal triglyceride transfer protein (MTTP), a critical mediator of VLDL assembly, tended to increase compared with the CON (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). However, neither Dapa monotherapy nor its combination therapy significantly altered MTTP expression.\u003c/p\u003e \u003cp\u003eExpression of CD36, a fatty acid translocase that facilitates circulating fatty acid uptake, was elevated in the DM group relative to CON (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Both DMDa and DMDa\u0026thinsp;+\u0026thinsp;AE significantly reduced CD36 expression, with no additional effect of combined intervention.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThis study aimed to systematically evaluate the effects of Dapa monotherapy and its combination with AE on hepatic lipid accumulation and the underlying mechanisms in T2DM rats. The results demonstrated that 20-week-old T2DM rats developed significant hepatic steatosis and IR. The principal can be summarized as follows: (1) Six-week of Dapa monotherapy significantly ameliorated hepatic steatosis and IR in T2DM rats, an effect mechanistically linked to enhanced phosphorylation of AMPK at Thr172 and subsequent suppression of key DNL-related proteins; (2) Although Dapa effectively suppressed DNL, it did not substantially upregulate hepatic lipolysis-related proteins; (3) Most notably, compared with Dapa alone, the combined intervention with AE exerted a marked additive effect in alleviating hepatic lipid accumulation, as demonstrated by both metabolomic and histopathological assessments. Further analysis revealed that this additive benefit was primarily attributable to the robust upregulation of hepatic lipolytic proteins by the combined intervention, whereas no further additive suppression of lipogenesis was observed; (4) Dapa monotherapy downregulated hepatic fatty acid transporter CD36, but did not significantly affect MTTP, while the combined regimen failed to further augment these effects.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of Dapagliflozin Monotherapy and Combined AE Therapy on Body Weight and Body Composition in Rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this model, T2DM rats exhibited a progressive decline in body weight accompanied by a notable increase in hepatic mass. This paradoxical phenotype is closely associated with IR and hyperglycemia, which are hallmarks of T2DM[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. IR may drive a compensatory upregulation of FAO, leading to increased influx of FFAs into the liver and providing substrate for DNL[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In parallel, compensatory hyperinsulinemia[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]and hyperglycemia[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u0026mdash;both consequences of IR\u0026mdash;directly stimulate DNL, thereby exacerbating hepatic triglyceride accumulation. Consistent with this well-established pathogenic framework, the T2DM rats in our study developed significant IR and hyperglycemia. Both Dapa monotherapy and its combination with AE reduced hepatic mass, likely reflecting improvement in IR and glycemic control, as reported in earlier studies on Dapa[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], AE alone[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and their combined application[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Interestingly, although the combined regimen produced a pronounced additive effect in reducing fasting blood glucose, no similar additive effect was observed in reducing hepatic mass, likely because both monotherapy and combined intervention had already restored hepatic mass to near-normal levels.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRegulatory Role of AMPK in Hepatic Lipid Metabolism\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAMPK activity is typically suppressed in T2DM, contributing to disrupted energy homeostasis[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Numerous studies have shown that SGLT2 inhibitors ameliorate hepatic lipid metabolism by promoting AMPK(Thr172) phosphorylation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR49 CR50 CR51 CR52\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The present findings are consistent with this evidence. Moreover, AE is a well-established activator of AMPK[\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].However, prior investigations into combined intervention of Dapa and AE have demonstrated tissue-specific heterogeneity. For example, at the skeletal muscle level, the combined regimen showed no significant effect on AMPK phosphorylation, suggesting that exercise-induced AMPK activation is tissue dependent and produces divergent downstream effects across organs[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. This study demonstrates that although combined intervention significantly activates AMPK phosphorylation in the liver, it does not yield results surpassing those of Dapa monotherapy. This discrepancy may be related to additive effects at the hepatic lipid metabolism level, whereas no such correlation was observed in skeletal muscle. Future studies should directly compare the differential regulation of the AMPK signaling pathway and its downstream lipid metabolism targets in the liver and skeletal muscle by combined interventions. In light of this study, two possibilities warrant consideration: (1) the combined Dapa and AE intervention may not synergistically amplify AMPK phosphorylation; (2) the AE protocol employed (6 weeks, 5 days/week, 60 min/day, at \u0026sim;70\u0026ndash;75% of VO₂max) may have lacked sufficient intensity or duration to produce a synergistic effect. Future studies directly comparing different exercise modalities, such as high-intensity interval training (HIIT) versus moderate-intensity continuous training (MICT), will be valuable for clarifying these mechanisms.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of SGLT2 Inhibitors on Molecular Mechanisms of Hepatic Lipid Metabolism\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe literature reports heterogeneous findings regarding the molecular mechanisms by which SGLT2 inhibitors regulate hepatic lipid metabolism[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan additionalcitationids=\"CR59 CR60 CR61 CR62 CR63 CR64 CR65 CR66 CR67\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Such variability may reflect compound-specific pharmacological properties or differences in animal models and experimental designs. The present study provides novel evidence that Dapa predominantly suppresses DNL without significantly enhancing FAO-related proteins expression, a profile more consistent with empagliflozin[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] than with canagliflozin[\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan additionalcitationids=\"CR62 CR63 CR64\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The focus of this study is the controversy surrounding FAO and its associated protein expression changes. Under T2DM conditions, hepatic FAO is modulated by insulin signaling, with IR typically inducing compensatory FAO upregulation[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Several studies have suggested that SGLT2 inhibitors, including Dapa, may enhance hepatic FAO[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, our results showed that no significant changes in FAO-related proteins expression after Dapa intervention, diverging from certain published findings. One plausible explanation is that as IR improves under Dapa treatment, the compensatory drive to upregulate FAO is attenuated, thereby offsetting any potential drug-induced enhancement. In addition, the effect of Dapa on FAO may be inherently modest or context-dependent, influenced by factors such as the disease model, treatment duration, or exercise intensity. The precise mechanisms\u0026mdash;including whether Dapa directly augments FAO and how this relates to IR improvement and actual FAO flux\u0026mdash;remain unresolved and merit further investigation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDapagliflozin Ameliorates Hyperglycemia and Hepatic Lipid Deposition Primarily Through Inhibition of De Novo Lipogenesis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe central conclusion of this study is that combined Dapa and AE intervention produced a significant additive effect in alleviating hepatic lipid accumulation, as confirmed by metabolomic and histopathological analyses. While AMPK activation alone does not fully account for this effect, molecular analyses revealed downstream regulatory mechanisms of hepatic lipid metabolism that explain the additive benefit. Hepatic steatosis is primarily driven by an imbalance between lipogenesis and lipolysis[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding lipogenesis, Dapa monotherapy already suppressed DNL substantially, and the combined intervention did not further reduce it. This may reflect two key points: (1) both Dapa and exercise suppress DNL through the ACC/FAS axis, but Dapa alone markedly reduced ACC and FAS protein expression (by 49% and 59%, respectively, relative to the disease group), approaching the lower physiological limit of regulation. The AMPK phosphorylation results in this study further support this interpretation; and (2) Dapa appears to act predominantly via transcriptional repression of ACC/FAS[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], whereas exercise may primarily act by inhibiting enzymatic activity through ACC phosphorylation[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. When protein expression is already at low level, further suppression through phosphorylation alone becomes unlikely. Future research should include an exercise-only group and directly assess ACC phosphorylation to confirm these hypotheses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAerobic Exercise Provides Complementary Benefits Through Promotion of Lipolysis and Fatty Acid Oxidation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCombined intervention additive effect appears to derive mainly from enhanced lipolysis, as the combined intervention significantly upregulated proteins associated with TG hydrolysis and FAO. This interpretation is consistent with the findings by Tanaka[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], who reported that wheel running combined with canagliflozin shifted whole-body substrate utilization toward lipids. Given that Dapa\u0026rsquo;s pharmacological profile more closely resembles that of empagliflozin than canagliflozin, the observed protein-level changes warrant further comparative investigation. ATGL serves as the rate-limiting enzyme in hepatic TG hydrolysis and determining substrate availability for FAO[\u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Previous studies have shown that certain SGLT2 inhibitors, such as tofogliflozin and empagliflozin, significantly increase ATGL expression[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], whereas Dapa alone does not substantially affect ATGL or its downstream effector HSL[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Consistent with these findings, our study indicates that AE compensates for Dapa\u0026rsquo;s limited effect on TG hydrolysis by promoting ATGL expression[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Similarly, the combined intervention also enhanced expression of FAO-related proteins, including PPARα, CPT1A, and ACOX1, which aligns with previous evidence that AT enhances FAO pathway [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother potential mechanism involves Lipophagy, a selective form of autophagy critical for intracellular lipid droplet mobilization and energy balance[\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Previous studies have shown that both Dapa[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and AE[\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] can activate Lipophagy through the AMPK/SIRT1 pathway, thereby improving hepatic lipid metabolism. Although our study did not provide direct evidence of additive effects on Lipophagy, further investigation is warranted to determine whether combined interventions exert superior regulatory effects on this pathway.\u003c/p\u003e \u003cp\u003eCollectively, these findings suggest that AE enhances hepatic fat-oxidative capacity through mechanisms that are either distinct from or synergistic with those of Dapa. This synergistic effect, not observed under Dapa monotherapy, highlights the therapeutic potential of integrating SGLT2 inhibitors with exercise interventions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferential Regulation of Fat Uptake and Export\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo ensure comprehensiveness, this study also examined key proteins involved in hepatic fatty acid uptake and export. Dapa monotherapy significantly downregulated CD36 expression, consistent with prior reports, suggesting a reduction in hepatic fatty acid uptake[\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. However, the combined intervention did not further reduce CD36 expression. Regarding MTTP, neither Dapa alone nor its combination with AE significantly altered expression, indicating that lipid export is unlikely to represent a principal mechanism in this model. Future studies should investigate whether longer-term interventions affect other VLDL-associated proteins, such as ApoB100, and their possible contributions to hepatic lipid regulation.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn summary, Dapa monotherapy robustly ameliorates hepatic steatosis and IR in T2DM rats by activating AMPK and suppressing DNL. Importantly, the combination of Dapa and AE produces a significant additive effect on hepatic lipid reduction, primarily through AE produced a significant additive effect on hepatic lipid reduction, primarily through AT induced enhancement of lipolysis and FAO rather than further suppression of DNL. These findings highlight the therapeutic potential of integrating SGLT2 inhibitors with structured exercise for the management of T2DM-associated hepatic steatosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcetyl\u0026ndash;CoA carboxylase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACOX1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcyl\u0026ndash;CoA oxidase 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAerobic exercise\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMPK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdenosine monophosphate\u0026ndash;activated protein kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnalysis of variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAerobic training\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATGL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdipose triglyceride lipase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eβ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eActin\u0026ndash;Beta\u0026ndash;actin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCD36\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCluster of differentiation 36 (fatty acid translocase)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCON\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPT1A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarnitine palmitoyltransferase 1A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDapa\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDapagliflozin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDiabetes mellitus group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMDa\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDapagliflozin\u0026ndash;treated diabetic group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMDa\u0026thinsp;+\u0026thinsp;AE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDapagliflozin combined with aerobic exercise group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDe novo lipogenesis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme\u0026ndash;linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eeWAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpididymal white adipose tissue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFatty acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFAO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFatty acid oxidation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFAS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFatty acid synthase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFFAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFree fatty acids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHFD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh\u0026ndash;fat diet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHIIT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh\u0026ndash;intensity interval training\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHOMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIR\u0026ndash;Homeostatic model assessment of insulin resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHorseradish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInsulin resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMICT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eModerate\u0026ndash;intensity continuous training\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMTTP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMicrosomal triglyceride transfer protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAFLD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon\u0026ndash;alcoholic fatty liver disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNASH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon\u0026ndash;alcoholic steatohepatitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eND\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNormal diet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptimal cutting temperature compound\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPARα\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeroxisome proliferator\u0026ndash;activated receptor alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRIPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRadioimmunoprecipitation assay buffer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD rats\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSprague\u0026ndash;Dawley rats\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePAGE\u0026ndash;Sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard error of the mean\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSGLT2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium\u0026ndash;glucose cotransporter\u0026ndash;2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSpecific pathogen\u0026ndash;free\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTZ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStreptozotocin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTBST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTris\u0026ndash;buffered saline with Tween 20\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTriglyceride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Animal Welfare and Ethics Committee of Zhejiang Normal University (ZSDW2022015). \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNot applicable.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe authors declare no competing interests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThis study was supported in part by grants from Science and Technology Bureau of Jinhua City (Grant No. 2023–4–003) ,China; Fundamental Research Funds for the Central Universities (Grant No. 2412025QD044), China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDesign: All authors participated in the design experiment, with HLQ, LJ and as the main leaders. Data collection: LZZ, XDY,SJ and Wei Li completed the work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalysis: LXZ and XHD completed the data statistical work, while other authors checked.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting manuscript: LXZ, XHD and HLQ completed the main contents, with other authors participating in revision and verification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLongxiang Zhou\u003csup\u003e1,2†\u003c/sup\u003e and Xinghan Du\u003csup\u003e1†\u003c/sup\u003e \u003cstrong\u003econtributed equally to this work. The study was conducted with financial support from Lei Ji and\u0026nbsp;\u003c/strong\u003eHelong Quan\u003cstrong\u003e. We thank\u0026nbsp;\u003c/strong\u003eLiangzhi Zhangand Xudong Yang\u003cstrong\u003e\u0026nbsp;for their help in the animal feeding and intervention process.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, et al. IDF Diabetes Atlas: Global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045. Diabetes Res Clin Pract. 2022;183:109119. https://doi.org/10.1016/j.diabres.2021.109119\u003c/li\u003e\n \u003cli\u003eFu Y, Zhou Y, Shen L, Li X, Zhang H, Cui Y, et al. Diagnostic and therapeutic strategies for non-alcoholic fatty liver disease. Front Pharmacol. 2022; 13:973366. https://doi.org/10.3389/fphar.2022.973366.\u003c/li\u003e\n \u003cli\u003eWhalley S, Puvanachandra P, Desai A, Kennedy H. Hepatology outpatient service provision in secondary care: a study of liver disease incidence and resource costs. 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Diabetologia. 2023;66(4):754\u0026ndash;767. https://doi.org/10.1007/s00125-022-05851-x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"lipids-in-health-and-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lhad","sideBox":"Learn more about [Lipids in Health and Disease](http://lipidworld.biomedcentral.com/)","snPcode":"12944","submissionUrl":"https://submission.nature.com/new-submission/12944/3","title":"Lipids in Health and Disease","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aerobic Exercise, Dapagliflozin, Type 2 Diabetes Mellitus, Liver, Lipid Accumulation","lastPublishedDoi":"10.21203/rs.3.rs-8972569/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8972569/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePharmacological interventions and regular exercise are widely regarded as core strategies for improving type 2 diabetes mellitus (T2DM) accompanied by non-alcoholic fatty liver disease (NAFLD), as supported by previous metabolic and clinical research. Nevertheless, the potential synergistic impact of combination therapy involving dapagliflozin (Dapa) and physical exercise on hepatic lipid metabolism\u0026mdash;particularly its mechanism in regulating the balance between lipogenesis and lipolysis\u0026mdash;remains incompletely understood and a definitive consensus has not yet been established. Therefore, the present study was designed to elucidate the effects of Dapa, administered either as monotherapy or in combination with aerobic exercise, on hepatic lipid deposition and its potential underlying mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 24 four-week-old male Sprague-Dawley (SD) rats were randomly assigned to four groups (n\u0026thinsp;=\u0026thinsp;6 per group) following successfully model establishment. T2DM was induced in using a high-fat diet combined with streptozotocin administration (30 mg/kg, intraperitoneally), a protocol commonly employed in metabolic disease research. Dapagliflozin was administered daily by gavage to the treatment group. The combination group received both dapagliflozin treatment and a progressive treadmill running program designed to represent aerobic exercise intervention. Hepatic lipid deposition was quantified using Oil Red O staining, whereas Western blot analysis was conducted to determine the expression of key proteins involved in lipid metabolic regulation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBoth dapagliflozin monotherapy and the combined intervention with aerobic exercise significantly attenuated hepatic steatosis and were associated with improvement in insulin resistance, as reflected by HOMA-IR. Although no additive improvement in HOMA-IR was observed with the combined therapy, a more pronounced reduction in hepatic lipid accumulation was detected. Moreover, the findings suggest that dapagliflozin monotherapy primarily acted through inhibition of hepatic de novo lipogenesis. In contrast, the combined intervention appeared to exert additional effects through enhanced fatty-acid catabolism, thereby contributing to a greater reduction in hepatic lipid content.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e(1) Dapagliflozin suppresses hepatic de novo lipogenesis; (2) Aerobic exercise preferentially enhances lipolysis, thereby producing a complementary therapeutic effect when combined with Dapa; and (3) The combined intervention, through a dual mechanism characterized by suppressing of lipid synthesis and promotion of lipid breakdown, results in an additive metabolic benefit.\u003c/p\u003e","manuscriptTitle":"Dapagliflozin and Aerobic Exercise Synergistically Attenuate Hepatic Steatosis via Complementary Regulation of Lipogenesis and Fatty Acid Oxidation in Type 2 Diabetic Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 12:59:38","doi":"10.21203/rs.3.rs-8972569/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-22T18:55:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-20T18:35:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-08T06:43:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208725443726001071223103380753631652409","date":"2026-03-06T05:00:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304851352865007534167083061642622884270","date":"2026-03-06T02:00:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-06T01:18:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-04T12:11:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-04T01:12:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Lipids in Health and Disease","date":"2026-03-03T14:30:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"lipids-in-health-and-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lhad","sideBox":"Learn more about [Lipids in Health and Disease](http://lipidworld.biomedcentral.com/)","snPcode":"12944","submissionUrl":"https://submission.nature.com/new-submission/12944/3","title":"Lipids in Health and Disease","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"28c67b17-5c6f-455e-8019-517a5a7b77e3","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-22T19:09:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 12:59:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8972569","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8972569","identity":"rs-8972569","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}