Research Progress on Lipophagy-Mediated Exercise Intervention in Non-Alcoholic Fatty Liver Disease.

Lipophagy is a cellular pathway targeting the lysosomal degradation of lipid droplets, playing a role in promoting lipid turnover and renewal. Abnormal lipophagy processes can lead to the occurrence and development of non-alcoholic fatty liver disease (NAFLD), characterized by the deposition of lipid droplets (LDs) in the liver. The importance of exercise training in preventing and improving NAFLD has been well-established, but the exact mechanisms remain unclear. Recent research findings suggest that lipophagy may serve as a crucial hub for liver lipid turnover under exercise conditions. Exercise may alleviate hepatic lipid accumulation and mitigate inflammatory responses and fibrosis through lipophagy, thereby improving the onset and progression of NAFLD.

Mitochondria-Associated Membranes as Key Regulators in Cellular Homeostasis and the Potential Impact of Exercise on Insulin Resistance.

The communication between mitochondria and the endoplasmic reticulum (ER) is facilitated by a dynamic membrane structure formed by protein complexes known as mitochondria-associated membranes (MAMs). The structural and functional integrity of MAMs is crucial for insulin signal transduction, relying heavily on their regulation of intracellular calcium homeostasis, lipid homeostasis, mitochondrial quality control, and endoplasmic reticulum stress (ERS). This article reviews recent research findings, suggesting that exercise may promote the remodeling of MAMs structure and function by modulating the expression of molecules associated with their structure and function. This, in turn, restores cellular homeostasis and ultimately contributes to the amelioration of insulin resistance (IR). These insights provide additional possibilities for the study and treatment of insulin resistance-related metabolic disorders such as obesity, diabetes, fatty liver, and atherosclerosis.

FUNDC1: An Emerging Mitochondrial and MAMs Protein for Mitochondrial Quality Control in Heart Diseases.

Heart diseases (HDs) are the leading cause of mortality worldwide, with mitochondrial dysfunction being a significant factor in their development. The recently discovered mitophagy receptor, FUNDC1, plays a critical role in regulating the homeostasis of the Mitochondrial Quality Control (MQC) system and contributing to HDs. The phosphorylation of specific regions of FUNDC1 and varying levels of its expression have been shown to have diverse effects on cardiac injury. This review presents a comprehensive consolidation and summary of the latest evidence regarding the role of FUNDC1 in the MQC system. The review elucidates the association of FUNDC1 with prevalent HDs, such as metabolic cardiomyopathy (MCM), cardiac remodeling/heart failure, and myocardial ischemia-reperfusion (IR) injury. The results indicate that the expression of FUNDC1 is elevated in MCM but reduced in instances of cardiac remodeling, heart failure, and myocardial IR injury, with divergent impacts on mitochondrial function among distinct HDs. Exercise has been identified as a powerful preventive and therapeutic approach for managing HDs. Additionally, it has been suggested that exercise-induced enhancement of cardiac function may be attributed to the AMPK/FUNDC1 pathway.

High-Intensity Interval Training Ameliorates High-Fat Diet-Induced Metabolic Disorders via the Cyclic GMP-AMP Synthase-Stimulator of Interferon Gene Signaling Pathway.

Metabolic diseases are growing in prevalence worldwide. Although the pathogenesis of metabolic diseases remains ambiguous, the correlation between cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) and metabolic diseases has been identified recently. Exercise is an effective intervention protecting against metabolic diseases, however, the role of the cGAS-STING signaling pathway in this process is unclear, and the effect and mechanism of different exercise intensities on metabolic disorders are still unknown. Thus, we explored the association between exercise to ameliorate HFD-induced metabolic disorders and the cGAS-STING signaling pathway and compared the effects of high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT). Male C57BL/6 mice (6-8 weeks old) were fed HFD for 8 weeks to establish a metabolic disease model and were subjected to 8-week MICT or HIIT training. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were used to assess glucose metabolism. Serum triglyceride (TG) and total cholesterol (TC) were measured to evaluate lipid metabolism. Oil red staining was used to observe the lipid droplets in the gastrocnemius muscle. An enzyme-linked immunosorbent assay was used to detect the serum inflammatory factors IL-6 and IFN-ß. The protein expression of the cGAS-STING signaling pathway was detected by the WesTM automatic protein expression analysis system. We reported that HFD induced metabolic disorders with obesity, abnormal glucolipid metabolism, and significant inflammatory responses. Both HIIT and MICT ameliorated the above adverse reactions, but MICT was superior to HIIT in improving glucolipid disorders. Additionally, HIIT significantly increased the expression of STING protein, as well as the phosphorylation of TBKI and the ratio of p-IRF3/IRF3. MICT only increased the expression of STING protein. Our findings suggest that HIIT may alleviate HFD-induced metabolic disorder phenotype through the cGAS-STING signaling pathway. However, the improvement of MICT on metabolic disorder phenotype is less associated with the cGAS-STING pathway, which needs to be further explored.

Codon-optimized FAM132b gene therapy prevents dietary obesity by blockading adrenergic response and insulin action.

BACKGROUND: FAM132b (myonectin) has been identified as a muscle-derived myokine with exercise and has hormone activity in circulation to regulate iron homeostasis and lipid metabolism via unknown receptors. Here, we aim to explore the potential of adeno-associated virus to deliver FAM132b in vivo to develop a gene therapy against obesity. METHODS: Adeno-associated virus AAV9 were engineered to induce overexpression of FAM132b with two mutations, A136T and P159A. Then, AAV9 was delivered into high-fat diet mice through tail vein, and glucose homeostasis and obesity development of mice were observed. Methods of structural biology were used to predict the action site or receptor of the FAM132b mutant. RESULTS: Treatment of high-fat diet-fed mice with AAV9 improved glucose intolerance and insulin resistance, and resulted in reductions in body weight, fat depot, and adipocyte size. Codon-optimized FAM132b (coFAM132b) reduced the glycemic response to epinephrine (EPI) in the whole body and increased the lipolytic response to EPI in adipose tissues. However, FAM132b knockdown by shRNA significantly increased the glycemic response to EPI in vivo and reduced adipocyte response to EPI and adipose tissue browning. Structural analysis predicted that the FAM132b mutant with A136T and P159A may form a weak bond with ß2 adrenergic receptor (ADRB2) and may have more affinity for insulin and insulin-receptor complexes. CONCLUSIONS: Our study underscores the potential of FAM132b gene therapy with codon optimization to treat obesity by modulating the adrenergic response and insulin action. Both structural biological analysis and in vivo experiments suggest that the adrenergic response and insulin action are most likely blockaded by FAM132b mutants.

Transcription factor EB enhances autophagy and ameliorates palmitate-induced insulin resistance at least partly via upregulating AMPK activity in skeletal muscle cells.

This study aimed to elucidate the role of transcription factor EB (TFEB) in protecting C2C12 myotubes against palmitate (PA)-induced insulin resistance (IR) and explored its mechanism associated with autophagy. PA treatment significantly decreased insulin sensitivity in myotubes and downregulated TFEB protein expression. TFEB overexpression significantly reversed the PA-suppressed glucose transporter 4 (GLUT4) protein expression and improved intracellular glucose uptake and consumption, and also alleviated the decrease of autophagy markers induced by PA. The effect of TFEB overexpression on GLUT4 was also abolished by the autophagy inhibitor 3-MA. In addition, AMPKɑ2-DN inhibited or abolished the effects of TFEB overexpression on upregulation of GLUT4 and PA-induced decrease of autophagy marker expressions. Taken together, our data demonstrated that upregulation of TFEB improved PA-induced IR in C2C12 myotubes by enhancing autophagy and upregulating AMPK activity. TFEB, as a critical regulator of glucose homeostasis in skeletal muscle cells, may be a potential therapeutic target for IR and Type 2 diabetes.

A single bout of exhaustive treadmill exercise increased AMPK activation associated with enhanced autophagy in mice skeletal muscle.

Previous studies reported inconsistent findings on autophagy activation in skeletal muscles after acute exercise. In this study, we investigated the effect of a single bout of exhaustive treadmill exercise on AMPK and autophagy activations in mice gastrocnemius muscle in vivo. Male ICR/CD-1 mice were randomly divided into the control and exercise groups. The later was subjected to a single bout of exhaustive treadmill exercise. Changes of AMPK, phosphorylation of AMPKThr172 (pAMPKThr172 ), and autophagy markers including Beclin1, LC3II/LC3I and p62 mRNA and protein expressions in gastrocnemius muscle at different times (0, 6, 12, 24 h) after the exercise were analysed by quantitative real-time PCR and western blot. Our results demonstrated that a single bout of exhaustive treadmill exercise significantly induced AMPK content and AMPK activity at 0, 6 and 12 h after the exercise, and changed the expressions of autophagy markers at different time points in the recovery period, respectively. Moreover, we observed positive correlations between expressions of LC3II/LC3I ratio and pAMPKThr172 or AMPK, and a negative correlation between expressions of p62 and AMPK or pAMPKThr172 . In conclusion, a single bout of exhaustive treadmill exercise in mice caused a prolonged activation of AMPK and improved autophagy in the gastrocnemius muscle. The regulation of autophagic markers were related to enhanced AMPK activity. The findings indicate that acute exercise enhanced AMPK-related autophagy activation may be the underlying molecular mechanism that regulates cellular energy metabolism during exercise.

Exercise-Induced Adipose Tissue Thermogenesis and Browning: How to Explain the Conflicting Findings?

Brown adipose tissue (BAT) has been widely studied in targeting against metabolic diseases such as obesity, type 2 diabetes and insulin resistance due to its role in nutrient metabolism and energy regulation. Whether exercise promotes adipose tissue thermogenesis and browning remains controversial. The results from human and rodent studies contradict each other. In our opinion, fat thermogenesis or browning promoted by exercise should not be a biomarker of health benefits, but an adaptation under the stress between body temperature regulation and energy supply and expenditure of multiple organs. In this review, we discuss some factors that may contribute to conflicting experimental results, such as different thermoneutral zones, gender, training experience and the heterogeneity of fat depots. In addition, we explain that a redox state in cells potentially causes thermogenesis heterogeneity and different oxidation states of UCP1, which has led to the discrepancies noted in previous studies. We describe a network by which exercise orchestrates the browning and thermogenesis of adipose tissue with total energy expenditure through multiple organs (muscle, brain, liver and adipose tissue) and multiple pathways (nerve, endocrine and metabolic products), providing a possible interpretation for the conflicting findings.

Chrono-Aerobic Exercise Optimizes Metabolic State in DB/DB Mice through CLOCK-Mitophagy-Apoptosis.

Although the benefits of aerobic exercise on obesity and type 2 diabetes are well-documented, the pathogenesis of type 2 diabetes and the intervention mechanism of exercise remain ambiguous. The correlation between mitochondrial quality and metabolic diseases has been identified. Disruption of the central or peripheral molecular clock can also induce chronic metabolic diseases. In addition, the interactive effects of the molecular clock and mitochondrial quality have attracted extensive attention in recent years. Exercise and a high-fat diet have been considered external factors that may change the molecular clock and metabolic state. Therefore, we utilized a DB/DB (BSK.Cg-Dock7m +/+ Leprdb/JNju) mouse model to explore the effect of chrono-aerobic exercise on the metabolic state of type 2 diabetic mice and the effect of timing exercise as an external rhythm cue on liver molecular clock-mitochondrial quality. We found that two differently timed exercises reduced the blood glucose and serum cholesterol levels in DB/DB mice, and compared with night exercise (8:00 p.m., the active period of mice), morning exercise (8:00 a.m., the sleeping period of mice) significantly improved the insulin sensitivity in DB/DB mice. In contrast, type 2 diabetes mellitus (T2DM) increased the expression of CLOCK and impaired the mitochondrial quality (mitochondrial networks, OPA1, Fis1, and mitophagy), as well as induced apoptosis. Both morning and night exercise ameliorated impaired mitochondrial quality and apoptosis induced by diabetes. However, compared with morning exercise, night exercise not only decreased the protein expression of CLOCK but also decreased excessive apoptosis. In addition, the expression of CLOCK was negatively correlated with the expression of OPA1 and Fis1. In summary, our research suggests that morning exercise is more beneficial for increasing insulin sensitivity and promoting glucose transport in T2DM, whereas night exercise may improve lipid infiltration and mitochondrial abnormalities through CLOCK-mitophagy-apoptosis in the liver, thereby downregulating glucose and lipid disorders. In addition, CLOCK-OPA1/Fis1-mitophagy might be novel targets for T2DM treatment.

Moderate endurance training reduced hepatic tumourigenesis associated with lower lactate overload compared to high-intensity interval training.

The anti-tumour effects of exercise are still poorly understood. In recent years, high-intensity interval exercise has been recognised as one of the best choices for better health. However, high-intensity interval exercise induces lactate production in muscles and elevates blood lactic acid levels, and the resulting acidic microenvironment may promote tumour progression. Therefore, it is important to compare the anti-tumour effects of different types of exercise. OBJECTIVE: In this study, we aimed to compare the anti-tumour effects of moderate endurance training and high-intensity interval training on diethylnitrosamine (DEN)-induced liver tumours and to explore the underlying mechanisms. METHODS: Three-week-old male C57BL/6 mice were injected intraperitoneally with DEN for 10 weeks to induce hepatocellular carcinoma. DEN-treated mice were grouped and subjected to moderate endurance training (MET) or high-intensity interval training (HIIT) for 18 weeks. We performed real-time PCR to evaluate the mRNA expressions of key enzymes involved in lactate metabolism pathway and western blotting to examine the protein expressions of LDHA, AMPK/P-AMPK, PCK1, and G6Pase in the paracancerous liver tissue. We performed high-performance liquid mass spectrometry (HPLC) to detect lactate in liver. RESULTS: Our results revealed that compared with HIIT, MET decreased hepatic tumour incidence, as HIIT increased blood lactate concentration at rest. Moreover, MET reduced the transcript-level expression of LDH subunit and significantly increased the mRNA levels of COX1 and ND1 in liver. However, no significant changes were observed in liver lactate levels and the expression of LDHA among the groups. In addition, no significant differences in the mRNA levels of critical enzymes involved in the gluconeogenesis pathway in liver were observed among the groups. Additionally, no significant differences were observed in the mRNA levels of MPC2, pdha2, and pdk4 among the groups. CONCLUSIONS: Our findings suggest that MET may be more efficient than HIIT at reducing hepatic tumourigenesis, and that it is associated with improved mitochondrial function in liver and lower lactate load in the circulation at rest.

NLRP3 Inflammasome in Diabetic Cardiomyopathy and Exercise Intervention.

Diabetic cardiomyopathy (DCM), as a common complication of diabetes, is characterized by chronic low-grade inflammation. The NLRP3 inflammasome is a key sensor mediating innate immune and inflammatory responses. However, the mechanisms initiating and promoting NLRP3 inflammasome activation in DCM is largely unexplored. The aim of the present review is to describe the link between NLRP3 inflammasome and DCM, and to provide evidence highlighting the importance of exercise training in DCM intervention. Collectively, this evidence suggests that DCM is an inflammatory disease aggravated by NLRP3 inflammasome-mediated release of IL-1ß and IL-18. In addition, chronic exercise intervention is an effective preventive and therapeutic method to alleviate DCM via modulating the NLRP3 inflammasome.

Research Progress of Mitochondrial Mechanism in NLRP3 Inflammasome Activation and Exercise Regulation of NLRP3 Inflammasome.

NLRP3 is an important pattern recognition receptor in the innate immune system, and its activation induces a large number of pro-inflammatory cytokines, IL-1ß and IL-18 which are involved in the development of various diseases. In recent years, it has been suggested that mitochondria are the platform for NLRP3 inflammasome activation. Additionally, exercise is considered as an important intervention strategy to mediate the innate immune responses. Generally, chronic moderate-intensity endurance training, resistance training and high-intensity interval training inhibit NLRP3 inflammasome activation in response to various pathological factors. In contrast, acute exercise activates NLRP3 inflammasome. However, the mechanisms by which exercise regulates NLRP3 inflammasome activation are largely unclear. Therefore, the mechanism of NLRP3 inflammasome activation is discussed mainly from the perspective of mitochondria in this review. Moreover, the effect and potential mechanism of exercise on NLRP3 inflammasome are explored, hoping to provide new target for relevant research.

Mechanism of tumor cells escaping from immune surveillance of NK cells.

Natural killer (NK) cells play an important role in anti-tumor and anti-infection, and perform their immune surveillance function in various ways. However, no matter what kind of cancer, the functional activity of NK cells in the tumor microenvironment (TME) is suppressed. Understanding the relationship between tumor cells and NK cells is very critical for tumor immunotherapy. This review discusses the mechanism of tumor cells escaping the immune surveillance of NK cells. These include a variety of factors that inhibit the activity of NK cells, an imbalance of activating receptors and inhibiting receptors on NK cells, abnormal binding of receptors and ligands, cross-talk of surrounding cell groups and NK cells in the TME, and other factors that affect NK cell activity. An understanding of these factors is necessary to provide new treatment strategies for tumor immunotherapy.

ER-Mitochondria Contacts and Insulin Resistance Modulation through Exercise Intervention.

The endoplasmic reticulum (ER) makes physical contacts with mitochondria at specific sites, and the hubs between the two organelles are called mitochondria-associated ER membranes (MAMs). MAMs are known to play key roles in biological processes, such as intracellular Ca2+ regulation, lipid trafficking, and metabolism, as well as cell death, etc. Studies demonstrated that dysregulation of MAMs significantly contributed to insulin resistance. Alterations of MAMs' juxtaposition and integrity, impaired expressions of insulin signaling molecules, disruption of Ca2+ homeostasis, and compromised metabolic flexibility are all actively involved in the above processes. In addition, exercise training is considered as an effective stimulus to ameliorate insulin resistance. Although the underlying mechanisms for exercise-induced improvement in insulin resistance are not fully understood, MAMs may be critical for the beneficial effects of exercise.

GASZ and mitofusin-mediated mitochondrial functions are crucial for spermatogenesis.

Nuage is an electron-dense cytoplasmic structure in germ cells that contains ribonucleoproteins and participates in piRNA biosynthesis. Despite the observation that clustered mitochondria are associated with a specific type of nuage called intermitochondrial cement (pi-body), the importance of mitochondrial functions in nuage formation and spermatogenesis is yet to be determined. We show that a germ cell-specific protein GASZ contains a functional mitochondrial targeting signal and is largely localized at mitochondria both endogenously in germ cells and in somatic cells when ectopically expressed. In addition, GASZ interacts with itself at the outer membrane of mitochondria and promotes mitofusion in a mitofusin/MFN-dependent manner. In mice, deletion of the mitochondrial targeting signal reveals that mitochondrial localization of GASZ is essential for nuage formation, mitochondrial clustering, transposon repression, and spermatogenesis. MFN1 deficiency also leads to defects in mitochondrial activity and male infertility. Our data thus reveal a requirement for GASZ and MFN-mediated mitofusion during spermatogenesis.

Acute exercise stress promotes Ref1/Nrf2 signalling and increases mitochondrial antioxidant activity in skeletal muscle.

NEW FINDINGS: What is the central question of this study? How does acute exercise affect the redox effector factor-1 (Ref1) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling and its association with mitochondrial H(2)O(2) production and antioxidant capacity? What is the main finding and its importance? Ref1/Nrf2 signalling in skeletal muscles was activated by acute exercise, and this activation was correlated with increased mitochondrial H(2)O(2) content and antioxidant capacity (reduced glutathione and manganese superoxide dismutase). The finding indicates that the oxidative stress induced by acute exercise in skeletal muscle mitochondria is associated with the upregulation of Ref1/Nrf2 signalling and enhancement of antioxidant defense pathways. This mechanism may play a role in preventing cellular oxidative stress resistance during acute exercise. The molecular mechanism of exercise-induced oxidative stress and adaptive activation of antioxidant responses in skeletal muscle has not been fully elucidated. This study aimed to investigate the effect of acute exercise on redox effector factor-1 (Ref1) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling and associations with mitochondrial H(2)O(2) production and antioxidant mechanisms in skeletal muscles. Groups of male ICR/CD-1 mice were subjected to an acute exercise bout of different durations (45, 90, 120 or 150 min). Muscle tissues (gastrocnemius and quadriceps femoris) were harvested after exercise to measure mitochondrial manganese superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase (CuZnSOD) activities, reduced glutathione (GSH) content and expression of Ref1/Nrf2 genes and Ref1/Nrf2 proteins. The acute exercise increased oxidative stress and activated Ref1/Nrf2 signalling in a time-dependent manner, with a linear correlation between the mitochondrial H(2)O(2) content and Ref1/Nrf2 expressions. The GSH content and MnSOD activity were also significantly increased, but CuZnSOD activity was not significantly affected. The findings indicate that the H(2)O(2) production induced by acute exercise in skeletal muscle mitochondria in the mouse is closely associated with upregulation of the Ref1/Nrf2 signalling pathway and enhancement of antioxidant defense components, including GSH and MnSOD. Activation of Ref1/Nrf2/antioxidant defense pathways may play a role in preventing cellular oxidative stress resistance during acute exercise.

[The cellular mechanisms of exercise adaptation from the perspective of skeletal muscle plasticity].

As highly plastic tissue, skeletal muscle adapts to stressors such as exercise. Reasonable amount of exercise is known to play a role in the prevention of cardiovascular diseases, diabetes and cancer. However, in the field of Exercise Physiology, the debate has been on for a while as to which kind of exercise is more effective in health promotion. In this review, the authors contrast and compare the signaling pathways mediated by different types of exercise, and boldly speculate the intrinsic similarities and discrepancies among them, hoping to enrich the theoretical frames of exercise adaptation, as well as provide effective exercise regimen to the public.

[Mitochondrial quality control: the target for exercise to promote health and prevent disease].

Regular exercise has been known to have many benefits, for example, improving physical performance, promoting health and preventing chronic diseases such as metabolic diseases. As a very important organelles in eukaryotic cells, mitochondria exhibit superb plasticity in response to exercise. Exercise may promote mitochondrial biogenesis and eliminate the dysfunctional mitochondria via mitophagy in order to maintain the normal function of the mitochondrial network. These dynamic changes keep mitochondria in health state and ensure the energy supply for cells. This review summarized the studies on the regulation of mitochondrial quality control by exercise, and provided a reasonable explanation for exercise to promote health and prevent diseases.

PC 18:1/18:1 mediates the anti-inflammatory effects of exercise and remodels tumor microenvironment of hepatocellular carcinoma.

AIM: Phosphatidylcholine (PC) is essential for membrane structural integrity and lipid-dependent signaling pathways, and is an essential component required for cancer cell growth. Using hepatocellular carcinoma (HCC) as a tumor model, this study aims to further screen phospholipid biomarkers of the tumor microenvironment and explore the anti-tumor effects and mechanisms of aerobic exercise. MAIN METHODS: The HCC of C57BL/6J mice was induced by the injection of the carcinogen diethylnitrosamine (DEN). Exercise was performed on an ungraded treadmill for weeks. The inflammation-related markers were detected by ELISA, PCR and immunohistochemistry, hepatic metabolic profile was analyzed by GC/MS, and lipid metabolism profile was further detected by lipid-targeted LC/MS. Cell culture was used to verify the anti-inflammatory effect of PC. KEY FINDINGS: Exercise reduced hepatic inflammation, tumor incidence and volume. Metabolomics analysis showed that palmitic acid is a key metabolic marker for exercise to improve tumor microenvironment. Injection of exogenous palmitic acid following exercise impaired the anti-inflammatory and anti-tumor effects of exercise. Lipid metabolomics analysis further showed that metabolites for exercise were enriched in glycerol phospholipid metabolism, including 14 phosphatidylcholines (PCs), 18 phosphatidylethanolamines (PEs), and 6 triglycerides (TGs). These biomarkers contain different lengths of fatty acid chains and different numbers of unsaturated bonds, respectively. Cell culture verified that PC (18:1/18:1) mediated lipopolysaccharide (LPS)-induced inflammation in HepG2 cell. SIGNIFICANCE: Our results suggest that exercise remodels glycerophospholipid metabolism and reduces hepatic palmitic acid loading and PC (18:1/18:1) level, thereby reconstructing a microenvironment that is hostile to HCC.

Optimization of training for professional rugby union players: investigating the impact of different small-sided games models on GPS-derived performance metrics.

Introduction: Professional rugby union players can improve their performance by engaging in small-sided games (SSGs), which simulate the movement patterns of the game. This study collected metrics related to running performance and mechanical workload and their relative values from both forward and back positions, aiming to explore the impact of different SSGs factors on athlete workload, as well as the workload difference between official games (OGs) and SSGs. Methods: The monitored GPS data were collected from SSGs with different player numbers and pitch sizes (five sessions), SSG rules (5 weeks, four sessions per week), and OGs conducted throughout the year. Additionally, the study compared changes in players' sprinting performance before and after two SSG sessions. Results: Backs had greater workload than forwards. Less space and number of players SSG (4 vs. 4, 660 m2) was conducive to facilitating training for players in acceleration and deceleration. Conversely, larger spaces were associated with improved running performance. However, the introduction of a floater had no significant impact on performance improvement. Additionally, the 7 vs. 4 model (seven players engaged with four opponents) resulted in the greatest workload during medium-hard accelerations (F = 52.76-88.23, p < 0.001, ηp 2 = 0.19-0.28). Japan touch model allowed for more high-speed running training (F = 47.93-243.55, p < 0.001, ηp 2 = 1.52). The workload performed by SSGs can almost cover that of OGs (F = 23.36-454.21, p < 0.05, ηp 2 = 0.03-0.57). In the context of ηp 2, values around 0.01, 0.06 and 0.14 indicate small, medium and large effects respectively. Discussion: However, given the significantly higher workload of SSGs and the slight decrease in sprinting performance, further research is required to examine the training patterns of SSGs. This study provided insight into the impact of player numbers, pitch size, and rules on rugby-specific SSGs. Coaches should optimize SSG setups for enhanced training outcomes, ensuring the long-term development of physical capacity, technical and tactical skills.