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

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.