Type 2 diabetes, sex and metabolic dysfunction-associated steatotic liver disease

Table 1 Epidemiological evidence that the association between steatotic liver disease and risk of dysglycemia and type 2 diabetes is modulated by sex

Method	Findings	Conclusion	Risk of bias	Ref.
This retrospective cohort study included 13596 men and 6037 women to examine the association between SLD, sex, and T2D in non-obese Japanese adults undergoing health check-ups. Baseline BMI was grouped into six categories, and SLD was diagnosed by abdominal ultrasonography	During the follow-up, 738 men and 138 women developed T2D. Men with a BMI of 20-22.9 kg/m2 and SLD had a higher risk, while women in this group did not. Among men, higher BMI without SLD did not raise the risk of diabetes, but women with a BMI of 23-27.4 kg/m2 had an increased risk regardless of SLD status	Sex influences the relationship between liver biomarkers and the development of T2D. SLD is associated with an increased risk of T2D in non-obese Asian males, but not in females	Low	Narisada et al[59]
This analysis used cross-sectional data from 1554 participants in the Maastricht study. T2D was determined by OGTT, and IHL content was measured via 3T Dixon MRI. Mediation analysis evaluated whether serum SHBG mediates the link between IHL content and T2D	IHL content was linked to T2D in both women (OR = 1.08, 95%CI: 1.04-1.14) and men (OR = 1.12, 95%CI: 1.08-1.17), and serum SHBG significantly mediated this association. Serum SHBG contributed more in women (OR = 1.04, 95%CI: 1.02-1.07; 50.9% mediated, 95%CI: 26.7-81.3) than men (OR = 1.02, 95%CI: 1.01-1.03; 17.2% mediated, 95%CI: 9.6-27.6). Repeating analyses with T2D proxies and covariate adjustment yielded similar results	Serum SHBG mediated the association between IHL content and T2D in both sexes, but its contribution was stronger in women than in men	Low	Simons et al[37]
Of 1309 screened individuals, 748 without diabetes were enrolled. Over 15 years, correlations between metabolic markers (including APN) and T2D onset were assessed in this group	In multivariate LRA, the independent predictors of incident T2D differed by sex: In men, only eGFR and SLD were significantly related to the onset of T2D. In women, APN was the only significant risk factor	Intervention strategies to prevent T2D vary between men and women	Low	Yoshimoto et al[60]
PPG was determined as the AUC for glucose during OGTT using the trapezoidal rule among 794 middle-aged Soweto cohort participants. PC analysis clustered sex hormones, liver enzymes, and cardiometabolic factors by sex. Multivariable linear regression assessed how much variance in PPG was explained by PCs and T2D PRSs, adjusting for relevant covariates in men and women	In men, the PCs’ cluster of sex hormones, liver enzymes, and cardiometabolic factors explained 10.6% of the variance in PPG, with PC1 (peripheral fat), PC2 (liver enzymes and steroid hormones), and PC3 (lipids and peripheral fat) contributing significantly to PPG. In women, PC factors of sex hormones, cardiometabolic factors, and liver enzymes explained a similar amount of the variance in PPG (10.8%), with PC1 (central fat) and PC2 (lipids and liver enzymes) contributing significantly to PPG	Variations in PPG responses to an OGTT among individuals may be influenced by factors such as body fat distribution, serum lipid profiles, liver enzyme levels, and steroid hormone concentrations, with differing effects observed between men and women	Low	Masango et al[61]
The PREVADIAB2 study assessed metabolic changes over 5 years in people initially without T2D. Researchers used hierarchical clustering on 953 participants based on IGI, fasting insulin secretion rate, HOMA-IR, and insulin clearance; 488 had their lipid profiles analyzed by LC/MS-QTOF	Four clusters (LS, PGS, ID, IR) with distinct dysglycemia risk were found. Although metabolic features were similar for both sexes, threshold differences led to sex-specific lipid profiles. Women had higher dihydroceramide and SM levels, while men showed a greater ceramide-to-SM ratio. All clusters except LS showed unique lipid signatures linked to metabolic dysfunction, with clear sex-based differences	Distinct insulin-associated metabolic characteristics and sex delineate phenotypes with unique lipidomic profiles, highlighting the importance of considering prediabetes within a comprehensive metabolic framework that extends beyond glycaemic measures	Low	Pina et al[62]

T2D: Type 2 diabetes; SLD: Steatotic liver disease; OGTT: Oral glucose tolerance test; BMI: Body mass index; IHL: Intrahepatic lipids; MRI: Magnetic resonance imaging; SHBG: Sex hormone-binding globulin; APN: Serum adiponectin; AUC: Area under the curve; PPG: Postprandial glucose; PC: Principal component; PRSs: Polygenic risk scores; IGI: Insulinogenic index; HOMA-IR: Homeostasis model assessment of insulin resistance; LC: Liquid chromatography; QTOF: Quadrupole time-of-flight; MS: Mass spectrometry; OR: Odds ratio; CI: Confidence interval; LRA: Logistic regression analysis; eGFR: Estimated glomerular filtration rate; LS: Liver sensitive; PGS: Pancreas glucose sensitive; ID: Insulin deficient; IR: Insulin resistant; SM: Sphingomyelin(s).

Table 2 Sex disparities in the pathophysiology across states of impaired glucose tolerance and type 2 diabetes in mice and rats

Method	Findings	Comment	Ref.
This study examined protein malnutrition in rats fed a LP or NP diet from weeks 3 to 6, after which all rats received the NP diet for the remainder of the experiment	Fasting glucose levels and GTT results significantly deteriorated from 12 weeks to 48 weeks in all rats, with the most pronounced changes observed in LP males	Male rats are particularly susceptible to impaired glucose tolerance due to protein malnutrition	Crace et al[63]
The effects of obesity and sex on hepatic insulin metabolism were evaluated in the SHR/Mcc-cp rat model	CLR decreased by 58% in obese females and 68% in obese males vs lean controls. In lean animals, males had 46% lower CLR; in obese animals, 59% lower than in females. Obesity led to a 50% drop in insulin-receptor binding in hepatocytes. Male sex reduced insulin binding by about 34% in both groups, mainly due to fewer cell surface receptors. Receptor-mediated insulin degradation was 40% less in obese animals compared to lean animals, and 27% lower in males than in females	Male sex and obesity are independently and additively linked to reduced hepatic insulin clearance and fewer cell surface insulin receptors, resulting in proportionally lower insulin compartmentalization and degradation	Hennes et al[64]
Adult male Wistar albino rats were orchidectomized and given daily doses of testosterone, estradiol, or both for 15 days starting 11 days after surgery. Plasma levels of glucose, insulin, testosterone, and estradiol were measured after treatment. The gastrocnemius muscle, adipose tissue, and liver were isolated to assess Akt phosphorylation, GLUT2/4 expression, glucose uptake, and the activities of glycogenic and glycogenolytic enzymes	Castration raised blood glucose and inhibited serum insulin, Akt phosphorylation, GLUT4 expression, glucose uptake, glycogen content and synthase activity, while increasing GLUT2 expression and glycogen phosphorylase activity. Testosterone alone or plus estradiol restored normal levels for all parameters. Estradiol alone increased Akt phosphorylation but did not affect other measures	Deficiency of sex steroids results in impaired glucose uptake in skeletal muscle and adipose tissue, associated with reduced Akt phosphorylation and decreased expression of GLUT4 on the plasma membrane	Muthusamy et al[65]
To identify protective factors in females, hepatic molecular profiles of non-diabetic obese fZDF rats were compared to mZDF or HF-fZDF rats	HFDs in fZDF rats caused greater weight gain, elevated glucose and insulin, lower insulin sensitivity, and prediabetes. mZDF rats were diabetic, with low insulin, high glucose, poor insulin sensitivity, and impaired glucose tolerance. Diet changes shifted female transcript and metabolite profiles toward a male pattern with reduced lipogenesis, increased FA oxidation, and higher oxidative stress. Males showed reduced GSH production capacity, while females had increased GSH turnover	Overall, the data support the hypothesis that anabolic pathways such as lipogenesis and lipid export may restrict FA oxidation and mitigate oxidative stress in female rats. Along with an enhanced capacity for GSH production, these hepatic sex differences could play a role in the sex-specific progression of T2D in ZDF rats	Gustavsson et al[31]
Obesity was induced, and food intake and weights were monitored weekly in age-matched mice	Hepatic EST induction is common in T2D. In female mice, loss of EST improved metabolic function in various T2D models. However, this benefit disappeared in ovariectomized mice. In males, EST ablation worsened diabetes due to reduced β-cell mass and impaired insulin secretion, with increased inflammation in WAT	The findings indicate that EST plays a critical, sex-specific role in energy metabolism and T2D pathogenesis	Gao et al[66]
A replacement vector was generated to delete two exons covering the entire Id2 coding region, with successful deletion confirmed by qRT-PCR. The resulting 2.2 kb mutant protein from neo-exon3 lacks functional domains, as exon 3 consists only of 3’ UTR sequence. Id2 WT and Id2-/- mice were bred on mixed backgrounds and received standard chow and antibiotic-treated water ad libitum. Wild-type controls were always Id2+/+ littermates	Male Id2-/- mice ate more food per body mass but gained less weight, while female Id2-/- mice had smaller adipocytes, indicating sex-specific effects on adipogenesis. Male Id2-/- mice also showed improved glucose tolerance and insulin sensitivity, especially with age. FDG-PET scans showed higher glucose uptake in their skeletal muscle and brown fat, suggesting increased metabolism and thermogenesis. These mice also had reduced intramuscular triacylglycerol and diacylglycerol, which may explain their enhanced insulin sensitivity	The findings enhance current knowledge regarding the development of sex-specific obesity and diabetes, with relevance to shift work personnel at risk of metabolic disorders	Mathew et al[67]
To evaluate the impact of hepatic STS overexpression on metabolic functions in mouse models of obesity and T2D through sex-specific mechanisms, STS transgenic mice were exposed to an HFD	STS liver expression increased in HFD and ob/ob models of obesity and T2D, as well as during fasting. Overexpressing STS in transgenic mice reduced weight, improved insulin sensitivity, and lowered liver fat and inflammation. In females, benefits were linked to the conversion of estrogen sulfates to active estrogens, which was lost after ovariectomy. In males, advantages were due to decreased inflammation and higher energy expenditure, even after castration. Treatment with estrone sulfate also improved metabolic health in both models	These findings reveal a previously unidentified role of STS in energy metabolism and T2D	Jiang et al[33]
C57/BL6J mice were bred and housed on either a CD or HFD from conception through weaning. The offspring then received either CD or HFD between 6 and 20 weeks of age. At 20 weeks, those exposed to maternal HFD showed glucose intolerance and insulin resistance	At 20 weeks, maternal HFD induced glucose intolerance and insulin resistance in offspring. Hepatic triacylglycerol levels, adipose tissue mass, and inflammatory markers were elevated following maternal HFD exposure. In male offspring, insulin secretion, islet area, insulin content, and PDX-1 mRNA expression decreased; conversely, these parameters increased in females. Islet oxidative stress was heightened in males, while remaining unchanged in females. Plasma estradiol concentrations were lower in males and further diminished by HFD, indicating that female offspring may be protected against insulin deficiency due to reduced oxidative stress	Maternal HFD caused insulin resistance and impaired pancreatic β-cell function in adult offspring, with notable sex-specific differences. These effects included adipose inflammation and liver steatosis. The differing β-cell outcomes may relate to increased oxidative stress and lower estradiol levels in males	Yokomizo et al[68]
An experimental model of streptozotocin-induced GD was used to assess the protein expression of LXRα (NR1H3) and LXRβ (NR1H2)	LXR expression in CO varied by tissue and receptor, with sex differences only seen for hypothalamic LXRβ at 35 days and 9 months. Most groups showed differences between CO and DO at 1 day, but these mostly disappeared except for male hypothalamic LXRβ. Glucose tolerance correlated negatively with LXRβ in CO, not DO; however, in male DO animals, this correlation was positive, as seen in intolerant subjects	Data indicate that GD affects hypothalamic LXR expression differently in male and female offspring	Kruse et al[69]
The effects of sex hormones on hepatic fetuin expression were evaluated in a rat model subjected to sex hormone administration. Results were corroborated by complementary in vitro investigations	E2 and DHT exerted contrasting influences on body weight among male and female rats. The expression of Ft-A and Ft-B in rat livers demonstrated sex-specific patterns and was subject to regulation by hormone receptors. In vitro experiments utilizing E2 or DHT corroborated the in vivo results, while antagonist assays confirmed that these effects were mediated through sex hormone receptors	Sex hormones modulate the sex-specific expression of hepatic fetuins via direct engagement with their respective hormone receptors	Kim et al[27]
Selected candidate genes were validated using real-time PCR analysis performed on hepatic tissues from both obese and lean rats	A total of 103 sex-different genes were found, mainly linked to chemical response, lipid metabolism, and organic substance response. Male-specific genes are related to liver metabolism, detoxification, and secretion, while most female-specific genes are involved in lipid metabolism or glycolysis	The data underscore the importance of considering sex- and diabetes-related variations during pre-clinical evaluation of drugs metabolized and secreted by the liver	Babelova et al[70]
This study used untargeted metabolomics to assess water-soluble metabolites in C57BL/6J male and female mice fed five different diets (Japanese, ketogenic, Mediterranean, American, and standard chow) for 7 months. Metabolite levels were measured in the liver, muscle, and fat, focusing on sex, diet, and their interaction	ANOVA shows liver tissue is most metabolically adaptable to diet changes, compared to adipose and skeletal muscle. The ketogenic diet was clearly distinct for both sexes by partial least-squares discriminant analysis. Most affected pathways, identified through pathway analysis, involve liver amino acid metabolism. Dietary patterns also influenced skeletal muscle amino acid profiles. Adipose tissue showed minimal metabolite changes, indicating stability despite dietary shifts	The ketogenic diet had the strongest physiological impact, especially in female mice. Metabolomics showed that diet alters metabolites differently by tissue, with the liver being the most responsive	Wells et al[71]
Hepatic pathology was compared between male and female SHROB rats, with key biochemical and molecular signaling pathways related to hyperinsulinemia and hyperlipidaemia assessed. The expression of 45 lipid biosynthesis and metabolism genes in rat livers was quantified using qPCR and Western blot	All SHROB rats exhibited hepatic steatosis, accompanied by increased expression of SREBP1, SREBP2, ACC1, and FASN proteins. In male rats, liver tissue showed greater induction of Pparg, Ppara, Slc2a4, Atox1, Skp1, Angptl3, and Pnpla3 mRNA levels. Conversely, female SHROB rat livers demonstrated consistently higher concentrations of phosphorylated JNK and AMPK, along with elevated CD36 expression	In SHROB rats, increased de novo lipogenesis primarily caused hepatic steatosis, with males and females showing different severity due to sex-specific differences in fatty acid transport and esterification	Dong et al[72]
Metabolic changes in the heart, liver, and kidneys of male and female mice, from healthy to diabetic states, were analyzed using 1H NMR metabolomics to identify sex-specific mechanisms in diabetes and its complications	Male mice experienced more pronounced metabolic disorders during diabetes progression than females. The kidneys were most affected, followed by the liver and heart. The altered metabolites chiefly involved energy, amino acid, choline, and nucleotide metabolism	The findings of this study indicate that the progression of DKD varies according to sex	Zhang et al[73]
Rats developed T2D after 3 weeks of a high-fat diet and daily 2-hour immobilization stress followed by streptozotocin treatment on day 15, and a 3-week course of LA	FBG increased in HFD-fed male rats, but not females. CS further raised FBS in HFD-fed rats; CS alone had no effect. HOMA-IR followed the same pattern as FBS. Serum corticosterone rose notably only in HFD-fed males exposed to CS. Pancreatic NF-κB levels were higher in HFD-fed males with CS exposure compared to controls, with no sex difference. LA at 10 mg/kg significantly lowered FBS, serum insulin, HOMA-IR, and corticosterone in HFD-fed males with CS, tended to reduce pancreatic NF-κB, and significantly improved liver MMP	Male rats are vulnerable to CS- and HFD-induced T2D, and LA can prevent T2D by reducing insulin resistance and corticosterone levels and also by increasing MMP in the liver	Shin et al[53]
The effect of diets on glycerol metabolism in mice and the influence of sex and GLP-1RA agonist treatment were investigated in female and male C57BL/6JRj mice fed either a CD or HFD for 12 or 24 weeks, with liraglutide administered to a subset of female mice	After 12 weeks of HFD, females gained less weight than males. Only females showed increased AQP7 in adipose tissue, while only males had higher glycerol kinase and larger adipocytes. At 24 weeks, weight gain and adipose changes became similar for both sexes. HFD caused notable hepatic steatosis in males but did not affect AQP9 levels in the liver. Liraglutide generally reduced HFD effects on glycerol metabolism. Overall, there was no coordinated increase in glycerol channels in adipose and liver tissues following HFD	The impact of a HFD on glycerol metabolism exhibits sex-specific differences in mice. An elevated abundance of AQP7 in female adipose tissue may underlie their comparatively attenuated response to HFD exposure	Iena et al[74]
This study examines the effects of exogenous SHBG on metabolically impaired hepatocytes, focusing on ER stress and lipid metabolism, using palmitate-treated HepG2 cells and post-mortem liver tissue samples cultured with 50 nM and 100 nM SHBG	SHBG protects against the development and progression of endoplasmic reticulum stress. It leads to reduced expression of IRE1α, ATF6, CHOP, and BIP. Additionally, SHBG regulates lipolytic gene expression in ex vivo settings	This research elucidates the cellular and molecular processes through which SHBG influences hepatocyte metabolism	Kornicka-Garbowska et al[75]
Young 3-month-old and middle-aged 12-month-old male and female mice were subjected to ad libitum or TRF of a Western diet to assess the metabolic impact	TRF confers metabolic benefits that are independent of age but dependent on sex. TRF protects against fatty liver and glucose intolerance in both males and females, whereas reductions in body weight are observed exclusively in males	These findings emphasize the notion that both timing and biological sex can significantly influence the effectiveness of treatments in preventing diet-induced metabolic disorders	Chaix et al[76]
This research analyzed the effects of a HFD on glucose regulation and T2D risk in Swiss Webster male and female subjects across the preweaning, peripubertal, and post pubertal developmental stages	In males, lifelong HFDs resulted in the highest T2D rates, but switching to chow after puberty lowered risk more than switching at weaning. The timing of HFD affected liver steatosis more than duration. Females avoided hyperglycemia with any HFD pattern, though postpubertal HFD caused notable liver and fat changes without impacting glucose tolerance. Most females had peri-insulitis, which did not affect glucose regulation	The data indicate distinct phases of glucose dysregulation induced by a high-fat diet in Swiss Webster mice, with notable sex disparities	Glavas et al[77]
The study investigated the role of Negr1 in preserving systemic metabolism, including glucose homeostasis, using male and female Negr1-/- mice receiving a standard or HFD	After 6 weeks of HFD Negr1-/- mice had higher glycaemic levels. HFD induced glucose tolerance variations only in male mice; Negr1-/- male mice displayed altered glucose tolerance, accompanied with upregulated BCAA circulating levels. Negr1-/- mice are biased towards gluconeogenesis, FA synthesis, and higher protein catabolism, all of which are amplified by HFD. Negr1 deficiency impairs the efficiency of energy storage, and reduced food intake could attempt to compensate for the metabolic challenge present in the Negr1-/- males during HFD feeding	Male mice exposed to HFD are protected from developing glucose intolerance, liver steatosis, and excessive weight gain by the presence of functional Negr1	Kaare et al[78]
For 8 weeks, weight, food and water intake, and blood glucose were measured in mice to assess sex differences in T2D	Water intake and fasting blood glucose did not differ between db/db female and male mice, but glomerular injury and hepatic fibrosis showed sex-specific differences	Experimental design should account for sex differences in both male and female animals	Gao et al[79]
Ay mice underwent a seven-day course of FGF-21 administration, followed by an assessment of metabolic parameters and gene expression profiles in multiple tissues	Placebo-treated females vs males exhibited more obesity, reduced insulin levels and liver fat content, higher expression of insulin signaling and inflammatory genes. FGF-21 increased food intake without affecting body weight and the expression of genes for fat catabolism and insulin action in both sexes. Only in males, FGF-21 reduced high insulin and liver fat, while boosting muscle Cpt1 and Irs1 expression	FGF-21 demonstrates a more pronounced beneficial effect on metabolic disorders associated with melanocortin obesity in male animals	Makarova et al[80]
18FDG-PET was used to evaluate baseline differences in whole-body glucose uptake between young male and female mice maintained on standard chow and HFDs	Sex and diet each affect glucose uptake in organs. Brown fat and heart showed the most FDG, with young females having 47% more brown fat uptake than males, and males having 49% more skeletal muscle uptake. HFDs lowered FDG uptake in brown fat, muscle, and heart, but increased it in the brain for both sexes	Glucose homeostasis regulation differs based on contextual factors and organ systems, highlighting the necessity to examine sex-specific outcomes and mechanisms with respect to T2D, obesity, and MetS	Gandhi et al[52]
Sex differences in hepatic histology were evaluated in TSOD and db/db mice, both being genetic models of NAFLD. Male and female mice from each strain received a standard diet and water, and groups of six were sacrificed at 3 months and 9 months for serum, pathology, and molecular analyses	At 3 months, male mice of both strains showed significantly higher serum AST and ALT levels (in TSOD mice), as well as greater steatotic and fibrotic areas (in db/db and both strains, respectively) compared to females; these sex differences were not observed at 9 months	Age-related sex differences in serum liver enzymes, as well as hepatic steatosis and fibrosis in TSOD and db/db mice, were consistent with patterns observed in human NAFLD	Dungubat et al[40]
Streptozotocin-induced diabetic rats received eugenol treatment at doses of 12 mg/kg and 24 mg/kg for four weeks	Streptozotocin raised serum glucose, cholesterol, triglycerides, LDL, liver enzymes, oxidative stress markers, pancreas damage, COX-2 expression, ovarian cysts, and anovulation, while lowering insulin, HDL, antioxidant activity, sex hormones, reproductive hormones, and PPAR-α. Eugenol improved diabetes indicators by enhancing lipid profile, antioxidant status, hormone levels, liver function, COX-2 and PPAR-α expression, and pancreas health, but did not affect ovarian cysts or follicle development	Eugenol may be useful for ameliorating some adverse features of diabetes regardless of sex	Kokabiyan et al[81]
Elderly Sprague-Dawley rats were divided into four groups: Control (STD diet), HFHSD diet, HFHSD plus metformin (HFHSD + M), and HFHSD plus liraglutide (HFHSD + L). Antidiabetic treatment began 5 weeks after diet initiation and continued for 13 weeks	Contrary to expectations, animals fed a HFHSD did not exhibit weight gain; however, they experienced notable metabolic alterations. Both antidiabetic interventions demonstrated sex-specific effects, yet neither was effective in preventing the development of prediabetes or diabetes	Liraglutide conferred beneficial effects on hepatic and skeletal muscle tissue in males, while in females it was associated with insulin resistance	Ivić et al[82]
This study started from the assumption that chronic CYP1A1/CYP1A2 activation may play a role in HFD-induced metabolic dysfunction in mice, and deletion of these enzymes could be protective. Male and female global CYP1A1/CYP1A2 KO and Cyp WT mice aged 29 weeks to 31 weeks were fed either a 45% HFD or standard chow for 14 weeks	Cyp KO females demonstrated partial protection against HFD-induced glucose intolerance, and chow-fed Cyp KO females exhibited reduced plasma insulin levels as well as diminished insulin secretion from isolated islets compared to Cyp WT females. Additionally, HFD-induced hyperinsulinemia developed later in Cyp KO males relative to Cyp WT males. Elevated expression of CYP1A1 and other stress-related genes was observed in Cyp WT male islets following HFD feeding, but not in Cyp KO islets, suggesting that CYP1A1 plays a role in mediating islet stress responses. Across both sexes, liver pathology, adiposity, and adipose tissue inflammation were predominantly influenced by dietary factors rather than genotype	This study reveals a sex-specific role for CYP1A1/CYP1A2 in regulating systemic metabolism under HFD conditions. Deleting CYP1A1/CYP1A2 partially protects female mice from HFD-induced glucose intolerance and lowers insulin on a chow diet, while in males, it delays HFD-induced hyperinsulinemia and reduces islet stress. The finding that these genotype effects are limited to islets, indicates a distinct function for islet CYP1A1/CYP1A2 in metabolic stress response	Ching et al[47]
Both male and female DNAJB3 KO and WT mice were administered high-sucrose, HFDs, or LFDs for 12 weeks. Body weight, food intake, glucose tolerance, and energy expenditure were evaluated; blood, adipose, and liver tissues were collected for histological examination and gene expression analyses	HF-fed KO females had greater body and fat mass, reduced glucose clearance, and lower energy expenditure than other groups. In males, both genotypes were affected by the HFD. HF-KO females had increased leptin, IL-6, and insulin, while HF-KO males showed higher leptin and resistin. DNAJB3 deficiency altered inflammatory and glucose transporter gene expression in adipose tissue and pancreas, indicating impaired glucose metabolism	This study highlights the important role of DNAJB3 in the regulation of glucose and metabolism, with a notable emphasis on its impact among female individuals	Nejat et al[48]

LP: Low protein; NP: Normal protein; Akt: Protein kinase B; GLUT: Glucose transporter; fZDF: Female Zucker diabetic fatty rat; mZDF: Male Zucker diabetic fatty rat; HF-fZDF: High-fat-fed female Zucker diabetic fatty rat; qRT-PCR: Quantitative real-time polymerase chain reaction; UTR: Untranslated regions; WT: Wild-type; T2D: Type 2 diabetes; STS: Steroid sulfatase; HFD: High-fat diet; CD: Control diet; GD: Gestational diabetes; LXRs: Liver X receptors; SHROB: Spontaneously hypertensive obese rats; LA: Linalyl acetate; NMR: Nuclear magnetic resonance; GLP-1RA: Glucagon-like peptide-1 receptor agonist; SHBG: Sex hormone-binding globulin; ER: Endoplasmic reticulum; TRF: Time-restricted feeding; FGF-21: Fibroblast growth factor 21; FDG-PET: Fluorodeoxyglucose proton emission tomography; TSOD: Tsumura Suzuki obese diabetes; NAFLD: Nonalcoholic fatty liver disease; STD: Standard diet; HFHSD: High-fat and high-sucrose diet; M: Metformin; L: Liraglutide; Cyp: Cytochrome P; KO: Knockout; GTT: Glucose tolerance test; CLR: Insulin clearance rate; FA: Fatty acid; GSH: Glutathione; EST: Estrogen sulfotransferase; WAT: White adipose tissue; PDX-1: Pancreatic and duodenal homeobox factor-1; mRNA: Messenger RNA; CO: Control; DO: Diabetic; DHT: Dihydrotestosterone; E2: 17β-estradiol; Ft-A: Fetuin A; Ft-B: Fetuin B; SREBP: Sterol regulatory element-binding protein; ANOVA: Analysis of variance; ACC1: Acetyl-CoA-carboxylase 1; JNK: C-Jun N-terminal kinase; AMPK: Adenosine 5’-monophosphate-activated protein kinase; CD36: Cluster of differentiation 36; FBG: Fasting blood glucose; CS: Chronic stress; HOMA-IR: Homeostatic model assessment-insulin resistance; NF-κB: Nuclear factor kappa-B; MMP: Mitochondrial membrane potential; IRE1α: Inositol-requiring enzyme 1; ATF6: Activating transcription factor 6; CHOP: DNA damage-inducible transcript 3; BIP: Immunoglobulin heavy chain-binding protein; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BCAA: Branched-chain amino acids; COX-2: Cyclooxygenase-2; LDL: Low-density lipoprotein; HDL: High-density lipoprotein; PPAR: Peroxisome proliferator-activated receptor; qPCR: Quantitative polymerase chain reaction; IL: Interleukin; HF: High fat; MetS: Metabolic syndrome.

Table 3 Evidence that sex-specific associations link metabolic dysfunction, liver health and cardiovascular risk in humans

Method	Findings	Conclusion	Risk of bias	Ref.
Of 158 women (47 normal and 111 with IGT) and 148 men (74 normal and 74 with IGT) were enrolled. They underwent a hyperinsulinemic normoglycemic clamp to determine M3, besides liver enzymes (ALT, AST, and GGT), metabolic and anthropometric parameters	Significant bivariate correlations were found between clamp measured M3 and all three liver enzymes in both sexes. After adjustment for possible metabolic confounders, correlations ceased in the male population alone. Feature selection analysis showed that ALT is an important attribute for M3 among women alone. MRA confirmed that BMI (P < 0.0001) and ALT (P = 0.00991) significantly and independently predicted clamp measured muscle glucose uptake in women [R (2) = 0.5259], while in men serum fasting insulin (P = 0.0210) and leptin levels (P = 0.0294) but none of the liver enzymes were confirmed as significant independent predictors of M3 [R (2) = 0.4989]	The existing sex-specific association between insulin sensitivity, MRFs, and ALT might explain the sex difference in the predictive role of ALT elevation for CVD. Accordingly, ALT may be used as a simple biomarker of IR in women	Moderate	Buday et al[83]
A cohort with T2D (n = 64, 30 male/34 female) and a sample of healthy subjects (n = 25, 13 male/12 female) were enrolled. Intraorgan and visceral fat were quantified by MR and VLDL1-TG export by intralipid infusion techniques	Intrahepatic and intrapancreatic TG content was elevated among those with T2D, irrespective of sex. In non-diabetic subjects, fat levels in both organs were significantly lower in women than men [1.0% (0.9%-1.7%) vs 4.5% (1.9%-8.0%), P = 0.005, and 4.7% ± 0.4% vs 7.6% ± 0.5%, P < 0.0001, respectively]. T2D women had higher hepatic VLDL1-TG production rate and plasma VLDL1-TG than healthy women [559.3 ± 32.9 mg/kg/day vs 403.2 ± 45.7 mg/kg/day, P = 0.01, and 0.45 (0.26-0.77) mmol/L vs 0.25 (0.13-0.33) mmol/L, P = 0.02], whereas there were no differences in men [548.8 ± 39.8 mg/kg/day vs 506.7 ± 29.2 mg/kg/day, P = 0.34, and 0.72 (0.53-1.15) mmol/L vs 0.50 (0.32-0.68) mmol/L, P = 0.26]. Weight loss decreased intraorgan fat content and VLDL1-TG production rates regardless of sex, and these changes were accompanied by similar rates of T2D remission (65.4% vs 71.0%) and CVD risk reduction (59.8% vs 41.5%) in women and men, respectively	In T2D, women have liver and pancreas fat accumulation like men, associated with raised hepatic VLDL1-TG production rates. Dynamics of TG turnover differ between sexes in T2D and after weight loss. Collectively, these changes may explain the raised CVR among women with T2D	High	Jesuthasan et al[84]
This cross-sectional observational study enrolled n = 16126 adult participants from the KNHANES. Absolute and body size-adjusted HGS indices were evaluated	Prevalence of T2D in all, younger, and older groups were 131%, 4.2%, and 21.4%, respectively. Proportions of cardiometabolic diseases were all higher in those with than without T2D in sex-stratified age groups, whereas obesity and MASLD were higher in younger T2D, and HTN was higher in older T2D in both sexes. Adjusted HGS significantly correlated with cardiometabolic parameters, and thus, high ORs for T2D in low tertiles of adjusted HGS were shown in all groups, whereas high ORs for T2D in low tertiles of absolute HGS were observed only in older men	Obesity and MASLD were more prevalent in younger T2D, while HTN was more prevalent in older T2D in both sexes. Low adjusted HGS was associated with higher T2D risk in all groups, whereas low absolute HGS was associated with higher T2D risk in older men, indicating that adjusted HGS might be useful in screening, especially for younger or obese individuals with T2D	Low	Bae et al[85]
The study comprised 332 hospitalized patients. The following data on leading CVD and risks related to CV drug administration were collected: Age, hyperlipidemia, T2D, CKD, CLD, HF, HTN, MI, and S. The amount of the CV drugs administered during hospitalization was expressed as the sex-specific DDD/100 BD	During hospitalization in the ICU, women were less likely to be treated with statins than male patients (30.1 DDD/100 BD vs 57.5 DDD/100 BD, P < 0.05). There was no difference between sexes regarding the use of antihypertensive drugs. Women were less likely to be treated with ASA (30.4 DDD/100 BD vs 36.9 DDD/100 BD, P < 0.05)	This study identifies sex differences in CV drug administration, and it is possible that these discrepancies mirror differences in primary care	Moderate	Drakul et al[86]
HTG was prospectively assessed among 2331 individuals using 1H MRS magnetic resonance spectroscopy, and plasma concentrations of TG, T-chol, LDL-chol, HDL-chol, and UA	The 95th percentile for HTG in lean non-AI individuals was 1.85%. Plasma insulin, TG, T-chol, LDL-chol, and UA concentrations were increased, and HDL-chol was decreased in individuals with HTG content > 1.85% and ≤ 5.56% compared to those individuals with HTG content ≤ 1.85%, and these altered parameters were associated with increased IR. Lean non-AI women had lower mean HTG than lean non-AI men, but both lean AI men and women showed a 40%-100% increase in HTG compared to their non-AI counterparts, with this rise linked to higher CMRFs	HTG concentrations > 1.85% (the 95th percentile of HTG in lean non-AI individuals) were associated with IR and CVR factors. Premenopausal women were protected from these changes, while young, lean AI men and women showed increased HTG levels and related CMRFs	Low	Petersen et al[87]
Of 5027 men were enrolled in this population-based study conducted in China. Low eGFR was defined as 60 mL/minute/1.73 m2	After adjusting for age, smoking, metabolic factors, and testosterone, through increasing quartiles of SHBG, a significantly positive association between SHBG quartiles and eGFR was detected in men (Q1 vs Q4, β = -2.53, 95%CI: -3.89 to -1.17, P < 0.001). Compared to the highest quartile of SHBG, SHBG in the lowest quartile was associated with 96% higher odds of low eGFR (OR = 1.96, 95%CI: 1.10 to 3.48) in the fully adjusted model. At stratified analyses, the associations between a 1-SD increase in serum SHBG and the prevalence of low eGFR were significant in men aged ≥ 60 years old, WC, diabetes, HTN, dyslipidemia, and MASLD	Reduced serum SHBG levels were independently associated with decreased eGFR and an increased prevalence of low eGFR among Chinese men, after adjusting for demographic characteristics, lifestyle factors, MRFs, and testosteronemia	Low	Zhang et al[88]
This cross-sectional study included data from 33216 randomly selected Spanish adult workers submitted to occupational medical check-ups. Sociodemographic, anthropometric, and clinical parameters were recorded, and MASLD was identified with FLI ≥ 60. MetS was diagnosed according to the IDF criteria, and CVR was determined with the REGICOR-Framingham equation	The global prevalence of MASLD was 19.1%, 27.9% (95%CI: 23.3%-28.5%) for men and 6.8% (95%CI: 6.4%-7.3%) for women, and increasing across age intervals. Compared to women, men had worse cardiometabolic and anthropometric profiles. At MVA, SLD was strongly associated with age, HDL-chol, social class, prediabetes, diabetes, preHTN, HTN, and smoking in both sexes. The association between diabetes, HTN, and MASLD was stronger in women than in men	Compared to women, men had a higher prevalence of MASLD, a worse cardiometabolic profile and a higher CVR. Nevertheless, MASLD was more strongly associated with T2D or HTN in women than in men, suggesting that metabolic dysfunction poses a more severe threat to liver health in women than in men	Low	Fresneda et al[89]
This retrospective longitudinal study of 92997 patients seen between January 2017 to January 2019 used electronic medical records to abstract data back to April 1997	Globally, 59323 individual without known CLD received an ALT test. Age, ethnicity, and metabolic factors were associated with higher odds of ALT testing (P < 0.01). At MVA, female sex (OR = 2.7), Latinx ethnicity (OR = 2.6), API race (OR = 1.3), overweight/obesity (OR = 1.8, OR = 2.6), and dyslipidemia (OR = 1.3) were associated with abnormal ALT (P ≤ 0.001)	Among individuals without any known CLD, women, Latinx, API, and persons with excess BMI had greater risks of abnormal ALT	Low	Kim and Khalili[90]
Two-hundred and twelve T2D patients, equally assigned to oral or subcutaneous semaglutide (n = 106 per group), were enrolled. The primary endpoint was the change in HbA1c. Secondary endpoints included variations in anthropometric and metabolic parameters.	A total of 208 patients completed the study. In men, sc semaglutide significantly reduced Δ weight (P = 0.010), Δ HbA1c (P = 0.037), and Δ LDL-chol (P = 0.038) compared to oral semaglutide. In women, sc semaglutide caused a significant decrease in Δ HSI (P = 0.024) and also led to a greater Δ GOT reduction (P = 0.035) than in men	This real-world study indicates that sc semaglutide offers superior metabolic benefits compared to the oral formulation, especially among male patients	High	Piccione et al[91]
This retrospective study included 6107 subjects submitted to annual health check-ups. CMI was calculated by multiplying the ratio of TG and HDL-chol (TG/HDL-C) by WHtR	MAFLD prevalence rose with higher CMI quartiles in both sexes. Higher CMI independently increased MAFLD risk (per SD: OR = 2.72 for males, OR = 3.26 for females). Those in the highest CMI quartile had the greatest odds of MAFLD (males: OR = 15.82; females: OR = 22.60), with significant trends (P < 0.001). CMI showed a non-linear association with MAFLD and had the largest AUC among obesity indexes for identifying MAFLD (males: 0.796; females: 0.853). FLD in males (AUC = 0.796, 95%CI: 0.782-0.810) and females (AUC = 0.853, 95%CI: 0.834-0.872)	CMI served as an effective marker for MAFLD screening in Chinese adults. The diagnostic value of CMI for MAFLD was higher in women than in men	Low	Gu et al[92]
A total of 7950 Taiwan Biobank participants were enrolled; 6478 had anthropometric, biochemical, and hematologic data, and 4185 underwent abdominal sonography for MASLD assessment	High PCSK9 levels were linked to older age, female sex, adverse cardiometabolic factors, and changes in blood markers. In women, associations with platelet count were stronger. Increasing PCSK9 quartiles correlated with higher odds of IR, MetS, DM, and MASLD, particularly for women. Elevated PCSK9 predicted higher risks of all-cause, non-cardiovascular, and cancer mortality, mainly affecting women	Elevated PCSK9 levels are linked to higher risks of IR, MetS, DM, MASLD, and mortality, reflecting poorer metabolic health among Taiwanese individuals. These effects are more pronounced in women, emphasizing the need for sex-specific risk assessment in metabolic disorders	Low	Yeh et al[56]

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: Gamma glutamyl transferase; IGT: Impaired glucose tolerance; VLDL: Very low density lipoproteins; T2D: Type 2 diabetes; TG: Triglycerides; MR: Magnetic resonance; CVD: Cardiovascular disease; HGS: Hand-grip strength; CKD: Chronic kidney disease; CLD: Chronic liver disease; CV: Cardiovascular; HF: Heart failure; HTN: Arterial hypertension; MI: Myocardial infarction; S: Stroke; DDD: Defined daily dose; BD: Bed-days; HTG: Hepatic triglycerides; MRS: Magnetic resonance spectroscopy; T-chol: Total cholesterol; LDL: Low-density lipoprotein; HDL: High-density lipoprotein; chol: Cholesterol; UA: Uric acid; eGFR: Estimated glomerular filtration rate; MASLD: Metabolic dysfunction-associated steatotic liver disease; FLI: Fatty liver index; MetS: Metabolic syndrome; IDF: International diabetes federation; CVR: Cardiovascular risk; HbA1c: Glycated hemoglobin; CMI: Cardiometabolic index; WHtR: Waist-to-height ratio; MRA: Magnetic resonance angiography; BMI: Body mass index; OR: Odds ratio; CI: Confidence interval; ICU: Intensive care unit; ASA: Acetylsalicylic acid; AI: Asian Indians; CMRFs: Cardiometabolic risk factors; IR: Insulin resistance; KNHANES: Korea National Health and Nutrition Examination Survey; SHBG: Sex hormone-binding globulin; WC: Waist circumference; MVA: Multivariate analysis; SLD: Steatotic liver disease; API: Asian/pacific islander; HIS: Hepatic steatosis index; GOT: Glutamic oxalacetic transaminase; MAFLD: Metabolic dysfunction-associated fatty liver disease; AUC: Area under the curve; FLD: Fatty liver disease; pre-HTN: Prehypertension; PCSK9: Proprotein convertase subtilisin/kexin type 9; DM: Diabetes mellitus; MRFs: Metabolic risk factors; M3: Muscle glucose uptake; Q: Quartile; sc: Subcutaneous.