Diabetes mellitus (DM) is a highly prevalent metabolic disease characterized by uncontrolled elevation of blood glucose levels, which leads to multiple complications, morbidity, and mortality ^[1][2][3][1,2,3]. DM is predominantly categorized as type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) ^[2][3][2,3]. T2DM is the most common type of DM, and its global prevalence is steadily increasing ^[3][4][3,4]. Pre-diabetes mellitus (pre-DM) is a metabolic state that is closely associated with T2DM, in which plasma glucose levels lie above the range of normoglycemia but below diabetes thresholds ^[5][6][7][5,6,7]. Indeed, pre-DM has been termed “Intermediate hyperglycemia” by the World Health Organization and “High Risk State of Developing Diabetes” by the International Expert Committee congregated by the American Diabetes Association [8]. Globally, the number of patients with pre-DM is increasing rapidly, and the prevalence of pre-DM is expected to increase to 470 million or higher by 2030 [8]. Individuals with pre-DM have a high risk of developing T2DM ^[6][8][9][6,8,9]. In total, 5–10% of patients who progress to DM per year present with pre-DM-related complications ^[6][7][6,7]. In pre-DM, abnormalities such as insulin resistance (IR) and β-cell dysfunction persist simultaneously prior to detectable changes in glucose levels [8]. Observational evidence supports an association of pre-DM with early stages of nephropathy, diabetic retinopathy, small fiber neuropathy, and the risk of macrovascular disease ^[8][9][8,9].

Obesity is a prime risk factor for the progression of pre-DM to T2DM ^[10][11][10,11]. Epidemiological studies have indicated that an increase in dietary fat consumption promotes obesity [12]. However, obesity alone is insufficient to explain disease progression, as only a small proportion of obese individuals have T2DM. In this regard, genetic factors likely contribute to the progression of pre-DM to T2DM. Relevant animal models of various progressive stages of T2DM development (i.e., from insulin-resistant pre-DM to T2DM) may facilitate the elucidation of associated genetic signatures in insulin-sensitive tissues and reveal the molecular mechanisms underlying disease progression [13].

To induce pre-DM, various animal models, such as high-fat/high-calorie diet-fed rodents and high-fat diet (HFD)-fed streptozotocin (STZ)-injected canine models, have been established [14]. The progressive stages of T2DM in HFD models are classified as 6 weeks (pre-DM), 12 weeks (T2DM), and 20 weeks (late-stage T2DM) [13]. As the progression of pre-DM results in T2DM, detailed investigations of pre-DM and associated biomarkers may attenuate the risk of progression to T2DM.

Biomarkers generally represent features that indicate pathogenic processes, normal biological processes, and/or pharmacological reactions to clinical treatment. HbA1c is a well-established, traditional biomarker. MicroRNAs and several proteomic and cytokine markers are emerging biomarkers, referred to as novel biomarkers [3].

Given that the development of T2DM is preceded by pre-DM, which is often undiagnosed [15], investigations of pre-DM and associated biomarkers may help to delay or prevent progression to T2DM.

2. Pre-Diabetic Biomarkers Identification in Progression of Diabetes Mellitus

T2DM is a serious chronic metabolic disease that has become a major global health issue. T2DM develops gradually and is preceded by the pre-DM stage, which is often undiagnosed ^[15][16][15,22]. T2DM and pre-DM are components of metabolic disorders, which significantly overlap [6]. However, studies on pre-DM and the associated biomarkers are limited. In this study, we identified major pre-DM and T2DM biomarkers in an HFD-induced pre-DM mouse model compared to standard chow diet-fed control mice. Mice fed an HFD are well established as an experimentally induced pre-DM animal model [14]. The progression of T2DM occurs in stages of 6 weeks (pre-DM stage), 12 weeks (T2DM stage), and 20 weeks (late-stage T2DM) [13]. Obesity is a major contributing factor to the progression from pre-DM to T2DM ^[10][11][10,11]. In this regard, HFD-fed mice exhibited significant body weight gain compared to control mice, which may have been due to an increase in fat mass and concomitant decrease in lean mass. β-cell dysfunction has been associated with increased serum TG levels in pre-DM [5]. In this study, serum levels of lipids, such as TG and cholesterol, increased from the pre-DM to T2DM stages. Previous studies have highlighted an association of T2DM with increased TG levels ^[17][23]. Increased TG levels indicate pre-DM and DM disease, as it is an indirect sign of insulin resistance, which is also responsible for NAFLD development and its progression to non-alcoholic steatohepatitis (NASH) ^[18][24]. Moreover, increased lipogenic gene expression may promote TG synthesis ^[19][25]. HFD-induced pre-DM mice exhibited significantly elevated mRNA and protein levels of lipogenic genes in the liver compared to control mice. Moreover, serum glucose and insulin levels were increased in pre-DM mice. Both hyperglycemia and hyperinsulinemia aggravate IR and stimulate hepatic lipogenesis ^[20][26]. A recent study demonstrated that an increase in serum insulin and glucose levels may underpin elevated serum TG and cholesterol levels ^[21][27]. Collectively, these data demonstrate that an increase in serum glucose and insulin levels elevate lipogenic gene expression and serum lipid levels, indicating an increased risk of developing T2DM. IR in the liver is the predominant contributor to metabolic syndrome, which includes disorders such as T2DM ^[20][26]. IR and T2DM are associated with elevated ALT and AST enzyme levels ^[22][28]. Indeed, serum levels of the liver enzymes, ALT and AST, were significantly increased in HFD-induced pre-DM mice, implying early signs of hepatic dysfunction. A human study reported that increased levels of liver enzymes are associated with an increased risk of T2DM ^[22][28]. Moreover, T2DM is characterized by low-grade inflammation ^[23][29]. Increased serum transaminase levels are also explained by the low-grade inflammation present in DM, which is one of the main pathophysiological mechanisms underlying NASH development ^[24][30]. Low-grade inflammation, caused by pro-inflammatory cytokines, is also implicated in the development of endothelial dysfunction, which can occur in pre-DM patients, thus highlighting the importance of detecting pre-DM as quickly as possible, as complications begin to develop in this phase ^[25][31]. Inflammation plays a key role in the development of IR in individuals with obesity. In this study, the expression of pro-inflammatory genes, such as Mcp1, Tnfα, and Il-1β, was increased in HFD-fed mice. MCP-1 links obesity to IR, and plasma levels of MCP-1 are increased in T2DM ^[26][32]. TNFα, which is associated with obesity, has been linked to the onset of IR and T2DM ^[22][27][28,33]. Previous studies have reported increased levels of TNFα in serum samples of individuals with pre-DM ^[28][34]. Additionally, IL-1β is a macrophage-induced immune system modulator that decreases βcell activity and insulin sensitivity ^[29][35]. Overall, these findings suggest that elevated levels of liver enzymes and inflammation lead to the progression of T2DM. To identify pre-DM and T2DM biomarker genes, semi-quantitative assessment of 97 cytokines in serum samples from control and HFD-induced pre-DM mice was performed. Of these cytokines, 13 were selected for subsequent analysis of mRNA expression in the liver samples. mRNA expression of several genes was elevated in the pre-DM and T2DM stages, respectively. In this study, HFD-fed mice showed significantly increased mRNA and protein levels of Fcgr2b, Lgals1, Vcam1, Igfbp5, and Gas6 at the pre-DM stage compared to those in control mice. FCGR2B is a member of the immune receptor IgG Fc gamma receptor family. A recent study reported the role of the FcgR family in the development of T2DM ^[30][36]. Similarly, increased levels of LGALS1 in the subcutaneous adipose tissue of individuals with T2DM have been observed ^[31][37]. Studies have reported elevated Vcam1 and Igfbp5 expression in association with high glucose levels ^[32][33][38,39]. GAS6 is a vitamin K-dependent cytokine for receptor tyrosine kinases such as AXL. However, the association among GAS6, IR, and T2DM remains controversial ^[34][40]. The elevated mRNA and protein levels of these genes at the pre-DM stage in HFD-fed mice compared to those in control mice highlight their potential utility as novel pre-DM biomarkers. Genes with elevated mRNA expression at the T2DM stage in HFD-induced pre-DM mice may act as novel T2DM biomarkers. The expression of known biomarkers of T2DM (i.e., cytokine Il-12b) was significantly upregulated after 8 weeks. A recent study on patients with chronic plaque psoriasis revealed that variation of IL-12b influences T2DM ^[35][41]. Moreover, Il-12, consisting of an Il-12b allele, has been previously shown to promote the development of T1DM in nonobese diabetic mice ^[36][37][42,43]. Several studies have reported an association of Il-12b with T1DM ^[34][40]. Similarly, HFD-induced pre-DM mice exhibited increased mRNA expression of Tnfrsf1b and Tnfrsf12a at the T2DM stage. Previous studies have demonstrated an association of TNFRSF1B genotype with clinical neuropathy in T2DM ^[38][44]. However, Tabassum et al. reported a lack of association between TNFRSF1B and T2DM in patients from North India ^[23][39][29,45]. TNFRSF12A (also known as fibroblast growth factor-inducible 14) has been associated with an increased risk of T2DM in women ^[40][46]. Furthermore, the expression of TNFRSF18, a glucocorticoid-induced tumor necrosis factor receptor (GITR), is upregulated during inflammation. In this study, we observed increased mRNA expression of Tnfrsf18 at the T2DM stage. However, this result is in contrast with previous findings, suggesting a protective role of GITR in the development of T2DM ^[41][47]. mRNA expression of Il-21 was upregulated after 8 weeks in HFD-fed mice. Studies on IL-21 receptor-deficient mice have reported complete termination of T1DM development ^[42][48]. Furthermore, mRNA expression of Axl was increased at the T2DM stage in HFD-induced pre-DM mice. These findings agree with previous reports of increased expression of Axl and Gas6 in STZ-induced diabetic rats ^[43][49]. IGFBPs are associated with IR and diabetic complications ^[44][50]. In this study, increased expression of Igfbp6 was observed after 8 weeks in HFD-fed mice. However, a study by Bergman et al. on STZ-induced diabetic rats reported downregulated Igfbp6 expression in the eye ^[45][51]. In contrast to the findings of Bergman et al., Lu et al. reported that serum levels of IGFBP6 were increased in T1DM patients ^[44][50]. Although IL-21, AXL, and IGFBP6 are strongly implicated in the progression of DM, increased expression of Il-21, Axl, and Igfbp6 in T2DM has not been reported to date. The chemokine (C-X-C motif) ligand (CXCL) family plays a vital role in inflammation ^[46][52]. mRNA expression of Cxcl11 was increased in the T2DM stage in HFD-induced pre-DM mice. A recent study reported a close association between CXCL11 and inflammation in the progression of IR in obese individuals ^[47][53]. Collectively, this model exhibited increased mRNA expression of Il-12b, Tnfrsf1b, Tnfrsf12a, Tnfrsf18, Il-21, Axl, Igfbp6, and Cxcl11 at the T2DM stage, suggesting their potential role as T2DM biomarkers.