The inverse association between fasting blood glucose and the occurrence of gallbladder cancer in type 2 diabetes mellitus patients: a case–control study

This study aimed to explore the correlation between diabetes mellitus (DM) and gallbladder cancer (GBC) in an epidemiological setting. The study summarized the clinical and laboratory data of 2210 GBC Chinese patients in the authors’ hospital. A total of 17 influencing factors for GBC, including gender, body mass index (BMI), fasting blood glucose (FBG), fasting insulin (FINS), the homeostasis model assessment of insulin resistance (HOMA-IR), retinol-binding protein 4 (RBP4), and lipid indexes were analyzed using unconditional logistic regression analysis. Based on the results of univariate logistic regression, the risk of GBC was significantly and positively correlated with serum triglyceride, low-density lipoprotein, FINS, HOMA-IR, being female, BMI, DM, non-alcoholic fatty liver disease, and gallbladder stone disease (GSD), and significantly negatively correlated with high-density lipoprotein and FBG concentrations in serum, as well as hypertension. According to multivariate analysis, FINS was significantly positively associated with GBC risk, while DM showed an insignificant negative association; FBG was also not important. The most significant independent factor of GBC risk in patients with DM was HOMA-IR. Fasting blood glucose levels showed a significant negative relationship with GBC in patients with DM. In addition, this study indicated a significantly negative association between serum RBP levels and GBC. The findings of the current study revealed that the efficient treatment of insulin resistance is an important approach for decreasing GBC risk, as opposed to lowering blood sugar only, particularly in patients with DM. Interestingly, FBG may have had an inverse association with the development of GBC in patients with type 2 DM. Of note, the study found that a dramatic initial drop in RBP may help predict the occurrence of GBC.


Introduction
Evidence gathered to date appears to confirm that type 2 diabetes mellitus (DM) results in a consistent increase in the risk of cancers, including those of the liver and colorectum, the pancreas, biliary tract, and esophagus in men, and breast and endometrium cancers in women (Gallagher and LeRoith 2015). A recent systematic review indicated that, compared with non-diabetic individuals, both men and women with type 2 DM had an increased risk of developing gallbladder cancer (GBC) (Gu et al. 2016). However, the relevant association with the risk of developing gallbladder cancer remains unclear.
Individuals with insulin resistance (IR) are at an increased risk of developing numerous medical problems, including Bin-Wu Sheng and Jian-Qin Zhang have contributed equally to this work. obesity, type 2 DM, metabolic syndrome (MetS), and particular cancers (Jeong and Lee 2012;Pischon and Nimptsch 2016). Several prospective epidemiological studies showed that excessive weight along with low levels of physical inactivity was a key determining factor in the development of IR with associated hyperglycemia and hyperinsulinemia, which would further promote tumor development involving several biological pathways, e.g., chronic low-grade inflammation, glucose toxicity, advanced glycosylated end-product metabolism, and the adenosine monophosphate kinase pathway (Becker et al. 2009). Moreover, a population-based study suggested that MetS and IR had roles in the etiology of biliary tract cancers and biliary stones (Shebl et al. 2011). However, recent epidemiological data support the hypothesis that obesity may offer protection against some cancers; for example, there appears to be some association with a reduced likelihood of premenopausal breast cancer, a lower incidence rate and mortality for small cell lung and head/ neck cancers (the World Cancer Research Fund/American Institute for Cancer Research, and the International Agency for Research on Cancer Working Group), as well as a better survival rate for those with metastatic colorectal and nonsmall cell lung cancers and renal cell carcinoma (Trestini et al. 2018;Avgerinos et al. 2019). The current authors' existing study (Lai and Lau 2008) additionally highlighted that an elevated weight was inversely associated with gallbladder stone disease (GBD) in patients with hypercholesterolemia, which implied the existence of an unknown link between GBD and GBC. Furthermore, the potential risk factors underlying the associations between the above factors, particularly IR concerning obesity, DM, MetS, and cancers, have not been fully clarified.
With its typically late manifestation and poor prognosis, GBC is the most prevalent biliary tract tumor type (Lai and Lau 2008). A new epidemiological inquiry by the Global Health Observatory (Huang et al. 2021a) revealed that morbidity from GBC varied between countries and differed according to the frequency of potential risk factors, including excess weight/obesity, hyperlipidemia, lifestyle, and DM, several of which are interactively related. A study (Liu et al. 2016) conducted in recent years demonstrated a close relationship between obesity and GBC, in which the former was found to disturb the metabolism of lipids and endogenous hormones, affect the motility of the gallbladder, and increase incidences of gallstones; accordingly, these impacts serve as fundamental contributors to GBC.
The rates of IR, type 2 DM, hypertension, dyslipidemia, and other factors increase in obese patients, and in type 2 DM-linked hyperinsulinemia, insulin receptors or insulin growth factor (IGF) may mediate target cells to directly or indirectly promote cancer through the mechanisms of proliferation, anti-apoptosis, angiogenesis, and lymphogenesis (Avgerinos et al. 2019). Disrupted blood glucose control contributes to the emergence of cancer, and the link between genomic damage and blood glucose control is clear and strong in patients with type 2 DM (Grindel et al. 2017) and may, for example, impair gallbladder emptying (Pazzi et al. 2000). Thus, an important research aim is to prevent the occurrence of GBC and reduce its mortality rate by clarifying its epidemiological mechanisms related to IR, including hyperglycemia and hyperinsulinemia. In the current casecontrol investigation, the authors aimed to dissect the role of common risk factors (e.g., IR) on the etiology of GBC by comparing clinical and laboratory data.

Participant selection and data eligibility
The current authors retrospectively considered the anthropometric and laboratory parameters (e.g., blood lipids, fasting blood glucose [FBG], and fasting insulin [FINS]) of 2210 study participants who were enrolled from a total of 3524 Chinese GBC patients aged 30-80 years in the First Affiliated Hospital of Medical School, Xi'an Jiaotong University, Xi'an, China, from Jan 2009 to Dec 2020 (see Fig. 1). Included criteria: (1) age 30 to 80 years old; (2) BMI ≥ 18.5 Kg/m2; (3) GBC was verified by pathology; (4) underwent radical cholecystectomy in our hospital. 1259 patients were excluded because: (1) without cholecystectomy and not verified by pathology; (2) complicated with metastasis of other organs; and (3) lack of any of the data.
The study compared these participants to 2210 matched subjects without cancer among 10,500 individuals who attended check-ups in the same healthcare facility each year during the same period after excluding those who: (1) consumed more than 20 g/day of alcohol during the past 12 months (for both men and women) by the diagnostic standard of non-alcoholic fatty liver and the characteristics of Chinese men's drinking habits (Sheng et al. 2020); (2) BMI <18.5 Kg/m 2 ; (3) any missing data including age, gender, height, weight, systolic pressure (SBP), diastolic pressure (DBP), serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-c), high-density lipoprotein (HDL-c), fasting insulin, fasting blood glucose (FBG), retinol-binding protein (RBP), or ultrasonographic examination for gallbladder stones and NAFLD; 4) matching failure in any of the following items: age, ethnicity, occupation, and drinking.
The final control data were selected from the mean value of multiple check-up results over 5 years to reduce the selection bias caused by differences in observation times, while the data in the GBC group ranged from the time of the initial diagnosis. All participants in the two groups were matched by age, ethnicity, occupation, and alcohol consumption habits. This study complied with the institutional ethics committee requirements of the above-noted hospital (no. XJTU1AF2020LSK-160).

Data questionnaires and determining the clinical parameters
Participants were interviewed using questions about ethnicity, identification number, occupation, medication history, DM, hypertension, surgery, hepatobiliary disease, and the presentation of any types of cancer (including family histories). Participant identities were confirmed using their identity cards, and their height and weight were recorded during the medical examination or in-hospital by a full-time nurse. All of the study points for each GBC patient were completed by the surgeons in charge during their stay in the hospital and stored in the hospital's information system. For the control participants, a screening panel, including common tumor marks, transabdominal ultrasonography, and measurements pertaining to the study items were completed, and the data were stored in the authors' medical information management system.
Antecubital vein blood samples were collected from all of the participants following a 10-h fast, and the serum was immediately assayed for total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), FBG, retinolbinding protein (RBP), and FINS. The Glycerophosphate oxidation-peroxidase-4-aminoantipyrine-phenol (GPO-PAP) and Cholesterol oxidase-peroxidase-4-aminoantipyrine-phenol (COD-PAP) methods were applied to the TG and TC analyses, respectively. Homogenous methods were used in the LDL-c HDL-c analyses, and the glucose oxidase approach was implemented for measuring FBG. An immunoturbidimetry assay and a radioimmunoassay were used to measure the concentration of blood RBP and FINS, respectively. All assays were conducted as blinded procedures and carried out in the same central laboratory following the relevant manufacturer's protocols. Blood pressure readings were recorded three times after sitting down while relaxed or after resting for 30 min using an RBP 900 automatic blood pressure measuring instrument (Shenzhen Reycome Science and Technology Ltd., China); the lowest measured values were used in the analyses. By adopting an Fig. 1 The flow chart for identifying the study participants.
Step 1 shows that 3524 Chinese gallbladder cancer (GBC) patients, at the time of preliminary diagnosis and prior to surgery in our hospital, were selected from January 2009 to December 2020; 10,500 healthy controls underwent annual medical examinations in the same healthcare center for more than 5 years.
Step 2 shows that 2265 patients with GSD and 3308 healthy controls were included following the process denoted in step 1.
Step 3 shows the 2210 GBC patients and 3308 healthy controls, aged 30-80 years, respectively, after matching according to age, ethnicity, and alcohol consumption HW-900Y ultrasonic wave height and weight scale (Jiangsu Hengfeng Weighting, China), the authors measured the height and weight of all the patients included in the study BMI = weight(Kg)/height 2 m 2 ].

Type-B ultrasonic-based diagnosis of gallbladder stone disease and non-alcoholic fatty liver disease
Ultrasonic color Doppler (Toshiba, SSA-510A, Japan) was used to examine volunteers for gallbladder and hepatic diseases. After fasting and placement in the supine position, each patient's liver, gallbladder, pancreas, and spleen were examined in turn. Both gallstones and non-alcoholic fatty liver disease (NAFLD) were diagnosed (as noted in the authors' existing publication (Sheng et al. 2020)).

Determining diabetes, hypertension, and insulin resistance
Diabetes diagnoses were completed by a professional endocrinologist according to the latest diagnostic criteria used in China; accordingly, the most recent diabetes patients were diagnosed according to the guidelines for type 2 DM prevention and treatment in China 2020 (Chinese Elderly Type 2 Diabetes P and Treatment of Clinical Guidelines Writing G Geriatric E, Metabolism Branch of Chinese Geriatric S, Geriatric E, Metabolism Branch of Chinese Geriatric Health Care S 2022). Hypertension was diagnosed by a cardiovascular specialist who followed guidelines on the pharmacological treatment of hypertension in adults published by the World Health Organization; those with systolic blood pressure (SBP) ≥140 mmHg or diastolic blood pressure (DBP) ≥90 mmHg were diagnosed as having hypertension, as advised in the most recent edition of these guidelines (Al-Makki et al. 2022). Establishing the homeostasis model assessment-insulin resistance (HOMA-IR) method incorporated fasting glucose and insulin according to the following formula: A HOMA-IR ≥2.69 was defined as IR noted in epidemiological survey data (involving assessment data from a Chinese population of 13,045 individuals collected by the China Diabetes Prevention and Control Cooperative Group) in China in 2014 (Roa et al. 2006).

Statistical analysis
All of the data underwent statistical analysis using the SPSS Statistics 19.0 software program (SPSS Inc., Chicago, IL, USA), and a P value <0.05 was considered significant. Data were presented as mean ± standard deviation (SD), as these were continuous and tended towards a normal distribution, and because the study comprised a sufficient number of participants. Continuous and categorical variables were presented as values and frequencies (%), respectively. The variables from the two groups were compared using a Pearson's chi-square test (categorical variables) or a Student's t test (continuous variables). The relationships between gender, BMI, RBP, FBG, FINS, blood lipids, NAFLD, hypertension, diabetes, and GBC risk were determined via multivariate logistic regression models and stratified according to the involvement/exclusion of IR.

General characteristics and serum indexes of the gallbladder cancer and control groups
The differences in the general characteristics and serum indexes of the GBC and control groups are outlined in Table 1. The table shows that 2210 of the 2265 GBC patients and 2210 of the 3808 healthy control patients were aged 30-80 years, and there were 1421 (64.3%) and 1034 (46.8%) females in the GBC and control groups, respectively. A higher risk of GBC was detected for females (odds ratio [OR] = 2.048, 95% confidence interval CI 1.816-2.311). In the GSD group, there were significantly higher BMI, FINS, TG, LDL-c, and HOMA-IR and lower SBP, DBP, HDL-c, and FBG levels compared with the control group; there were also significantly higher incidences of DM, NAFLD, GSD, and IR in GSD patients (P < 0.05, Table 1).

Relationships between the studied indexes and the risk rate of gallbladder cancer
The factors related to the risk of GBC development, based on univariate and multivariate logistic regression, are listed in Table 2.
There was a significantly positive correlation between serum TG, LDL-c, FINS, HOMA-IR levels, being female, BMI, DM, NAFLD, GSD, and the risk of GBC (P < 0.05), and a significantly negative correlation between serum RBP, HDL-c, FBG levels, SBP, DBP, HBP, and the risk of GBC (all values, P < 0.001), based on univariate logistic regression. When including HOMA-IR in multivariate logistic regression, the authors found no significant correlation between BMI, HBP, and the risk of GSD (all values, P > 0.05), while these factors had a significantly negative association with GBC risk in patients with DM and NAFLD (all values, P < 0.05). However, after including FINS and FBG in the multivariate analysis, FINS showed a positive association with GBC risk (P < 0.001), while the association with DM and FBG levels was non-significant (P > 0.05).
To investigate the possible causes of the above results, the authors analyzed data using univariate and multivariate logistic regression analyses (as shown in Tables 3, 4 and 5).
The difference between the GBC and control groups was studied by stratifying the patients according to IR. Univariate logistic regression calculated no significant positive correlation between BMI, GSD, NAFLD, and GBC risk (all values, P > 0.05), and similar results were observed for serum TG, LDL-c, HDL-c, RBP, FINS, FBG, HOMA-IR, DBP, SBP, and HBP vs. the overall data, while the opposite result was observed for DM. Multivariate logistic regression, including HOMA-IR, indicated that the risk of GBC had a positive correlation with serum TG, LDL-c, and HOMA-IR levels (P < 0.01) and a significantly negative correlation with serum HDL-c, RBP levels, and DM (all values, P < 0.001). Significant positive relationships between GBC risk and serum TG, LDL-c, and FINS levels, and negative relationships between GBC risk and serum RBP and HDL-c were observed when the authors included FINS and FBG in the multivariate logistic regression analysis; no significant correlation with being female, FBG, or DM was observed (Table 3).
In the non-IR patients, similar results were obtained for gender, BMI, GSD, NAFLD, HBP, DBP, SBP, serum TG, LDL-c, HDL-c, RBP, HOMA-IR, FINS, and FBG vs. the overall data included in univariate logistic regression, while Table1 Demographic and clinical data of the patients before and after matching GBC gallbladder cancer, BMI body mass index, TG triglyceride, TC total cholesterol, HDL-c high-density lipoprotein cholesterol, LDL-c lowdensity lipoprotein cholesterol, NAFLD non-alcoholic fatty liver disease, SBP systolic blood pressure, DBP diastolic blood pressure, FBG fasting blood glucose, FINS fasting blood insulin, RBP Retinol binding protein, HOMA-IR Homeostasis Model Assessment of IR, IR insulin resistance, HBP high blood pressure, DM diabetes mellitus *means χ 2 for making a difference with student test the association between DM and GBC risk was inverted (OR = 0.03, 95% CI 0.01-0.095) vs. the overall data (OR = 1. 252, 95% CI 1.037-1.512). In the multivariate analysis, the inclusion of HOMA-IR showed that the relationships between GBC risk and serum TG, LDL-c levels, HOMA-IR levels, GSD, and being female were significantly positive; however, those for serum RBP, HDL-c levels, DM, and NAFLD were significantly negative (P < 0.05); when considering FBG and FINS, the outcomes were similar to the above results, and elevated FINS levels predicted a high GBC risk, while elevated FBG levels lowered the risk of GBC (P < 0.05, Table 4). Data for the patients with DM were analyzed considering HOMA-IR (concerning IR) or FBG (Table 5). The results of univariate logistic regression showed significant positive correlations between serum TG, LDL-c, FINS levels, being female, HOMA-IR, GSD, and the risk of GBC (P < 0.05), while the latter had a significantly negative correlation with serum RBP, HDL-c, FBG levels, SBP, DBP, and BMI (P < 0.05). The inclusion of HOMA-IR in the multivariate logistic regression showed no significant correlation between serum HDL-c levels, BMI, GSD, SBP, DBP, or GBC risk (P > 0.05, respectively), while the other indexes had similar relationships to GBC risk as revealed by univariate logistic regression. There were significant positive correlations between being female, serum TG, LDL-c, HOMA-IR, and GBC risk, and significant negation between serum RBP and GBC risk, based on multivariate analysis that included HOMA-IR. When considering FBG and excluding FINS levels in the analysis, a negative correlation was observed between FBG and GBC (OR = 0.843, P = 0.014); similar outcomes were revealed for being female, serum TG, LDLc, and RBP, while negative correlations were found for SBP and serum HDL-c (all values, P < 0.05).

Discussion
In this clinical case-control study of patients, matched according to age, ethnicity, occupation, and alcohol consumption habits in northwestern China, the authors identified HOMA-IR levels to be a risk factor for GBC; GSD and being female (Wernberg and Lucarelli 2014), as well as patients with type 2 DM, had an increased risk of developing GBC, based on a recent study (Gu et al. 2016). Interestingly, the current authors found that controlling HOMA-IR or insulin levels, rather than FBG levels, could more effectively lower the GBC risk related to DM, and there was no significantly higher morbidity from GBC in IR patients with GSD or NAFLD, including females. The multivariate logistic regression that considered HOMA-IR stratified by IR showed a significantly negative correlation between DM and GBC risk; after including FINS and FBG levels, the significant association between DM and GBC risk, as well as FBG levels, was absent. In contrast, in the relationships between the risk of GBC and FINS, HOMA-IR was significantly positive. Moreover, HOMA-IR was the most significant independent risk factor for GBC in the non-IR and total patient groups. The results tended to further indicate that hyperinsulinemia or insulin resistance played key roles in increasing GBC risk, and that poor blood glucose control could also increase the risk of developing GBC, even in non-IR patients.
On the one hand, the above results are supported by a recent study in which insulin was observed to reduce the levels of IGF-binding protein (IGFBP)-1 and IGFBP-2 in the circulation process, resulting in a circulating IGF increase, where IGF and insulin could stimulate target cells to move toward malignant transformation (Pollak 2012). On the other hand, to the current authors' knowledge, the goal of diabetes treatment is to correct metabolic disorders, improve clinical symptoms (e.g., polyuria and increased eating and drinking), and delay the occurrence of diabetic complications by controlling blood sugar concentration. In China, the most common hypoglycemic drugs are insulin secretagogues and metformin. The former may induce hyperinsulinemia after long-term oral administration, particularly in IR patients. The latter, metformin hydrochloride, a first-line hypoglycemic drug, induced porcelain gallbladder in C57Bl/6 mice while reducing the risk of gallstone formation (Dorvash et al. 2018) and may reduce blood glucose by promoting the uptake of glucose by peripheral tissue, thus inhibiting gluconeogenesis and delaying the absorption of glucose in the small intestines, reducing hepatic glycogen output and increasing the use of sugar by inducing its anaerobic fermentation in the small intestine, leading to increased triglyceride liver synthesis.
Triglyceride synthesis in cells is heavily reliant on the glycerol and fatty acids produced by glucose metabolism; glycerol is derived from the glycolysis-produced dihydroxyacetone phosphate, and fatty acids are synthesized by acetyl coenzyme A, a product of the oxidative decomposition of sugar (Smelt 2010). Briefly, a high-carbohydrate diet or sugar accumulation cause fatty liver or hypertriglyceridemia (Asgharpour et al. 2016), which supports the notion that inappropriately controlled blood glucose could promote GBC risk with hypertriglyceridemia and/or IR. As this study showed, high TG levels were the most significant factors related to GSC risk, particularly in IR patients (OR = 96.437). Hirasawa et al. (2013) analyzed the association between endometrial cancer and the risk of MetS in a case-control study and verified that hypertriglyceridemia was significantly frequent in endometrial cancer patients. Retrospective analysis conducted by Wuermli et al. (Hirasawa et al. 2013) of the anthropometric and laboratory parameters of 504 prostate patients and 565 age-matched patients with benign prostatic hyperplasia highlighted a relationship between hypertriglyceridemia and a greater risk of prostate cancer. Although few reports have clarified the mechanisms that were applied to derive their correlations, the current authors propose that hypertriglyceridemia is closely associated with IR, and chronic hyperinsulinemia in the IR state could stimulate very-low-density lipoprotein (VLDL)-triglyceride synthesis (Smelt 2010). Reportedly, the VLDL receptor participates in the pathogenesis of GBC by its mitogen-activated protein kinase-based regulation of the expression of fibroblast growth factor receptor signaling pathway components (Zhou et al. 2014). Moreover, hypertriglyceridemia has been established as closely related to NAFLD, which may progress into non-alcoholic steatohepatitis, triggering the development of hepatocellular carcinoma (Asgharpour et al. 2016). The present study demonstrated that NAFLD may not be a predictive factor of GBC risk in IR or patients with DM, possibly because it is linked to glucose metabolism disorder.
Other studies have suggested the association between cancer advancement or risk and cholesterol-related parameters to be either positive, negative, or neutral (Boretti 2020). A recent study of Bangladeshi women revealed total cholesterol and BMI to be independent predictors of breast cancer (Chowdhury et al. 2021), while the results of the current study showed that BMI and TC may not be independent predictors of GBC after matching for age, ethnicity, occupation, and alcohol consumption habits. Although an early Table 4 Univariate and multivariate analysis of gallbladder cancer risk in the patients with non-insulin resistance BMI body mass index, TG triglyceride, TC total cholesterol, HDL-c high-density lipoprotein cholesterol, LDL-c low-density lipoprotein cholesterol, NAFLD non-alcoholic fatty liver disease, SBP systolic blood pressure, DBP diastolic blood pressure, FBG fasting blood glucose, FINS fasting blood insulin, RBP Retinol binding protein, HOMA-IR homeostasis model assessment of IR, IR insulin resistance, HBP high blood pressure, DM diabetes mellitus *eliminated for the definition of HOMA-IR in multivariate regression -eliminated for no statistical significance in univariate regression /eliminated for its association with HBP case report demonstrated that the GBC cells of hypercholesterolemia patients could secrete a substance that stimulated LDL receptor activity as a result of reducing serum cholesterol (Ueyama et al. 1990), few research efforts have clarified the cellular mechanisms involved. The synthesis of a molecule of cholesterol requires 18 molecules of acetyl coenzyme A (CoA), 36 molecules of ATP, and 16 molecules of nicotinamide adenine dinucleotide phosphate (NADPH); most of the acetyl CoA and ATP are derived from the aerobic oxidation of sugar in mitochondria, and NADPH is obtained from the pentose phosphate pathway metabolism of sugar in the cytosol (Schade et al. 2020). However, it may be difficult to complete this process in tumor tissue because its pattern of glucose metabolism is anaerobic glycolysis. Furthermore, LDL-c was an independent risk factor for GBC in the results of the current study, including for patients stratified by IR/ non-IR. This was likely because blood LDL is a product of VLDL breakdown, which is the primary form of endogenous triglyceride transport (Smelt 2010). Similarly, anthropometric and laboratory parameters in non-IR patients revealed that abnormal lipid metabolism was closely related to GBC, which may result from a disorder of fatty acid-binding protein regulation (Lin et al. 2021).
Most notably, this study revealed that indexes related to glucose metabolism (including TG, FINS, LDL-c, RBP, and HOMA-IR levels) had significant correlations with GBC in patients with DM. The multivariate analysis further demonstrated that HOMA-IR was the most significant factor for predicting GBC risk (OR = 5.756), while FBG levels had a negative association with GBC (OR = 0.869). These outcomes prompted the authors to consider that hyperinsulinemia, a common phenomenon in DM, directly or indirectly regulated the activity of IGF-1, thereby contributing to the proliferation and inhibition of apoptosis in GBC cells (Jing et al. 2020). Moreover, increasingly, research is focusing on the roles of the insulin/IGF system and dyslipidemia in cancer types among women (Kang et al. 2018). This indicates that blood glucose disorder, not only hyperglycemia, plays a key role in carcinogenesis. Furthermore, the authors considered interventions that employed medical nutritional therapy to treat obesity, IR, and DM as being superior to other measures, including bariatric surgery, particularly as this bypass procedure may be related to remnant gastric cancer from residual Helicobacter pylori infections in the stomach (Ohira et al. 2016). Rather, it is important to treat diabetes by adjusting diet and increasing aerobic exercise to control  (Hemmingsen et al. 2017;Hu et al. 2001;Lauterbach and Wunderlich 2017); drugs are the second most effective means for controlling diabetes, and anti-IR drugs should be the first choice, as these may have further benefits for tumor prevention.
Notably, serum retinol-binding protein 4 (RBP-4) was found to be primarily positively associated with type 2 DM and obesity (Olsen and Blomhoff 2020). Huang et al. (Huang et al. 2021b) demonstrated that retinoic acid 6, RBP-4's only known specific membrane receptor, was expressed in betacells and mediated the inhibitory effect of RBP-4 on insulin synthesis through the Janus kinase 2/STAT1/ISL-1 pathway; the current study findings showed that serum RBP levels had a significant negative association with GBC, including for patients with IR, although the OR was low (0.922-0.964).
To the authors' knowledge, RBP-4 is the main retinol transporter in plasma. Therefore, the serum RBP levels that the current authors studied may reflect accurate RBP-4 levels (Blaner 1989). Moreover, among the several secreted bioactive signaling molecules of adipose and liver tissue, RBP-4 has been associated with systemic insulin resistance, dyslipidemia, type 2 diabetes, and other metabolic diseases (Nono Nankam and Bluher 2021). A recent study revealed that RBP-4, which links obesity and cancer, was one of the pathogenic mechanisms of cancers (including prostate cancer) that are related to obesity (Uehara et al. 2013), breast cancer (Jiao et al. 2016), and pancreatic cancer (El-Mesallamy et al. 2012). However, Sobotka et al. (2017) found low levels of RBP-4 at the time of renal cell carcinoma diagnosis to be associated with poorer prognosis after surgery, while Lorkova et al. (2012) verified a decrease in RBP-4 levels in the sera of patients with ovarian cancer using two independent methods. The observed RBP-4 decrease in the research results of the current authors may advocate RBP-4 as a potential diagnostic or prognostic biomarker of tumors with high malignancy and poor prognosis (e.g., GBC).

Study strengths and limitations
The current study reviewed more than 2000 GBC cases during the preceding 12 years that were matched for occupation, age, ethnicity, and alcohol consumption habits, thereby strengthening the study's overall validity. However, the research nonetheless has several limitations. First, case-control studies typically all have limitations in the form of recall bias (the ability of patients to remember their exposure and habits), uncertainties regarding the links between cause and effect, and sampling bias. Second, this study does not present a cohesive explanation for the negative correlations between HBP, SBP, DBP, and GBC. Third, the current authors opted to define IR using HOMA-IR within a large dataset, based on an epidemiological inquiry in China; the reason for this was because the reference values for indirect indicators of an IR diagnosis may be affected by the study population members' age, gender, and ethnicity and the laboratory methods used for the determination of glucose and insulin concentrations (Placzkowska et al. 2020). Finally, errors related to cancer family histories may have occurred due to the typically small family units in China and because of Chinese personality characteristics. Accordingly, the authors did not complete accurate data related to a family history of cancer and matching, as these statistics may have been biased.

Conclusions
Overall, the findings presented in this study supported the verification that IR, being female, and GBD were closely related to the predictive risk of developing GBC for four levels of patient stratification (total, IR, non-IR, and DM). The authors found that the correct anti-insulin treatment was more important for decreasing the GBC risk than lowering blood sugar, particularly for patients with DM. Interestingly, there may be an inverse association between FBG and the occurrence of GBC in patients with type 2 DM. It was initially found that a sharp drop in RBP may be a predictor of GBC, and the observed RBP-4 decrease was advocated as a potential diagnostic or prognostic biomarker of tumors with high malignancy and poor prognosis, including GBC. Despite these novel new findings, due to the relatively small sample, as well as regional and ethnic limitations of this study, its results must be confirmed by a large number of prospective studies and multi-center validation, as well as further research employing animal experiments.

Conflict of interest
The authors declare that they have no competing interests.
Ethics approval This study was conducted with approval from the Ethics Committee of First Affiliated Hospital of Xi'an Jiaotong University (No. XJTU1AF2020LSK-160).

Consent to participate
This study was conducted in accordance with the declaration of Helsinki. Written informed consent was obtained from all participants.