Prevalence of GCKR rs1260326 Variant in Subjects with Obesity Associated NAFLD and T2DM: A Case-Control Study in South Punjab, Pakistan

The glucokinase regulatory protein (GCKR) regulates glycogen metabolism and insulin secretion, and the GCKR rs1260326 is a putative single nucleotide polymorphism (SNP) associated with metabolic disorders including nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). This study was conducted to investigate the genetic association of the GCKR rs1260326 in NAFLD and T2DM in our population. NAFLD (n = 103), T2DM (n = 100), and control (n = 100) samples were collected and genotyped for GCKR rs1260326 by tetra-arm PCR. The genetic variant GCKR rs1260326 was significantly linked with NAFLD and T2DM, while the GCKR rs1260326 was significantly associated with the progression of obesity only in NAFLD subjects. The frequency of the C allele (mutant) was higher in both NAFLD (f = 0.69) and T2DM (f = 0.66) subjects as compared to healthy controls of NAFLD (0.52) and T2DM (f = 0.32). The frequency of the C allele was also positively linked with the progression of obesity in both diseases. The frequency of the C allele was 0.66, 0.67, and 0.74 in NAFLD normal weight, overweight, and obese subjects, respectively, while the frequency of the C allele was 0.60, 0.60, and 0.74 in T2DM in normal weight, overweight, and obese subjects, respectively. Homozygous mutant (CC) was 53% in both NAFLD and T2DM subjects, while heterozygous mutant (CT) was 15.53% in NAFLD and 22% in T2DM subjects. Wild-type allele (TT) was 31.06% in NAFLD and 25% in T2DM subjects. In conclusion, the GCKR rs1260326 is a highly prevalent SNP in NAFLD and T2DM subjects, which possibly contributed to obesity, insulin resistance, and metabolic disorders in our population.


Introduction
Glucokinase regulatory protein, encoded by GCKR, is an inhibitory protein of glucokinase (GCK) and is expressed in the liver and β-cells of Islets.GCK is a glucose phosphorylating enzyme which is important in glucose-stimulated insulin release [1].Te GCKR is a highly pleiotropic gene on chromosome 2p23.3with 19 exons encoding 625 amino acids.Terefore, the GCKR gene plays a pivotal role in various metabolic and biochemical pathways.Tus, the rare exonic mutations in GCKR are associated with several metabolic dysfunctions including elevated triglycerides and glucose levels [2].GCKR inhibits GCK by forming an inactive heterodimer in an allosteric manner concerning glucose levels [3].Te activity of GCKR is increased by fructose-6-phosphate and antagonized by fructose-1-phosphate [4].GCKR is a vital candidate protein related to glycogen metabolism and insulin secretion.However, genetic variations in this gene can lead to irregular blood glucose levels and decreased glucose responsiveness to develop insulin resistance, a condition called type 2 diabetes mellitus (T2DM).T2DM is a multifactorial metabolic disorder characterized by high plasma glucose levels resulting in insulin resistance or impaired β-cell function [5,6].Te GCKR rs1260326 genetic variant is functionally relevant comprising of an amino acid substitution as C to T coding for a proline-to-leucine at position 446 (P446L) (Supplementary Figure 1) [7].A missense variant in the GCKR rs1260326 is associated with abnormal fasting glucose levels and a higher risk of T2DM development [8].Similarly, genome-wide association studies (GWASs) also reported that functional variants of the GCKR including rs1260326 are associated with low plasma insulin and fasting plasma glucose, as well as higher fasting and postprandial serum triglyceride levels [9,10].
GCKR-mediated GCK inhibition maintains normal glucose and insulin levels, which play a pivotal role in hepatic de novo lipogenesis (DNL), a metabolic pathway that involves in the synthesis of fatty acids from carbohydrates when in excess.However, genetic polymorphism in the GCKR rs1260326 is associated with impaired hepatic lipid metabolism and secretion of very low density lipoprotein (VLDL), which can lead to hyperlipidemia and hepatic fat accumulation, and is signifcantly associated with the development of nonalcoholic fatty liver disease (NAFLD) in obese children and adolescents [7].Terefore, an impaired cross-talk of GCKR and GCK along with elevated insulin and glucose levels promotes the DNL and subsequent hepatic steatosis, which leads to another metabolic condition called NAFLD [11].NAFLD is a complex disease that results from multiple factors including environmental factors (western or high-fat diet), obesity, epigenetic factors, and genetic factors including polymorphism in diseasesusceptible genes.Te genetic factors associated with NAFLD and its underlying mechanisms are not fully understood.Te diet-induced obesity is considered a primary event in the onset and development of NAFLD [11].However, many studies also reported the role of single nucleotide polymorphism within the GCKR gene, in the development of NAFLD even independent of a high-fat diet, BMI, or the age of the subjects [12][13][14][15].Furthermore, a previous study also reported a signifcant association of the GCKR rs1260326 with high fasting glycemia in the young Mexican population, without a signifcant association with obesity [16].Tus, multiple previous studies have reported a signifcant genetic association between GCKR rs1260326 and NAFLD with and without obesity [12][13][14][15].Previously, NAFLD was considered a part of metabolic syndrome (MS) and associated with obesity and T2DM [17].Meta-analysis and genome-wide association studies (GWASs) have also shown a signifcant association of the GCKR rs1260326 with NAFLD in many populations worldwide including Iran [18], Swedish [19], Japanese [20], Danish [21], Pakistan [22], and China [23].In Pakistan, prevalence of T2DM and NAFLD is 17.1% and 14%, respectively, while the prevalence of NAFLD in subjects with T2DM is 32-72% [24].In Punjab, Pakistan, prevalence of T2DM in males and females is 12.14% and 9.83%, respectively [25].Meta-analysis and GWAS analysis indicate that the GCKR rs1260326 has possibly impaired the hepatic lipid metabolism and signifcantly contributes to the development of obesity and obesity-associated metabolic disorder including NAFLD and T2DM.Tus, this study was conducted to evaluate the genetic association and contribution of GCKR rs1260326 to induce obesity in NAFLD and T2DM subjects in a local population of South Punjab.

Study Subject and Ethical
Approval.In this study, a total of three hundred and three (n � 303) subjects were included (cases � 203 and controls � 100).Te blood samples were collected from nonalcoholic fatty liver disease (NAFLD) (n � 103) and type 2 diabetes mellitus (T2DM) subjects (n � 100).Age-and sex-matched healthy control (HC) subjects (n � 100) were also collected for both diseases (n � 50 each).All samples were collected from Nishtar Hospital, Multan, Pakistan, under the signed Memorandum of Understanding (MoU) between Bahauddin Zakariya University and Nishtar Medical University, Multan, after ethical approval by the Institutional Review Board (IRB) of BZU (case number IMBB-334b).Informed written consent was also obtained from all the participants of the study as per the Declaration of Helsinki from all enrolled subjects.Samples were collected and transported to the molecular pathology laboratory of the Institute of Molecular Biology and Biotechnology, B.Z University, Multan, and stored at 4 °C for further analysis.
Inclusion criteria for the NAFLD group were based on the confrmation of fatty liver duly confrmed with ultrasound irrespective to age, sex, and BMI.All fatty liver subjects sufering from hepatic cirrhosis, hepatocellular carcinoma, anaemia (bone marrow transplant), and alcohol consumption were excluded from this group.All T2DM subjects included in this study were adults (age >35) without insulin dependency, while subjects sufering from type 1 diabetes, gestational diabetes, maturity-onset diabetes, anaemia, chronic kidney disease, cardiovascular diseases, and alcohol consumption were excluded from this group.Te subjects with a high-fat dietary intake were also excluded from both studies.

Physical and Biochemical Analysis for Sample
Preparation.Te demographic data included age, marital status, gender, weight, height, smoking, alcohol consumption family history, systolic blood pressure (BP), and diastolic blood pressure (BP).Body mass index (BMI) and fat content (FC) parameters were calculated using BMI calculator as previously described [18] and presented in Table 1.Te biochemical parameters related to NAFLD including ALT (alanine aminotransferase), AST (aspartate aminotransferase), total bilirubin, and alkaline phosphatase were analyzed in disease and control subjects.Similarly, FBG (fasting blood glucose), RBG (random blood glucose), and Hb1Ac (haemoglobin A1c) were analyzed in T2DM subjects and related control subjects as previously described [26].

Statistical Analysis.
Te chi-square test was performed using IBM SPSS Statistics V.23 software (IBM, Chicago, USA) for statistical signifcance between case (NAFLD and T2DM) and control subjects.Te Hardy-Weinberg equilibrium was used to compare the genotype and allelic frequencies.Data were presented as mean ± SD or the number of cases.Binary logistic regression was also performed along with the Hosmer-Lemeshow goodness-of-ft test to fnd associations.P values (P ≤ * , ≤ * * , and ≤ * * * ) were considered signifcant.

Demographic Data of NAFLD and T2DM Subjects.
Demographic and clinical parameters have been analyzed for all subjects and presented (Tables 1 and 2).Age, disease history, hypertension, duration, BMI, fat content, and blood pressure were signifcantly associated with NAFLD.Gender, family history, exercise, BMI, fat content, FBGL, RBGL, and Hb1Ac were also signifcantly associated with T2DM.Furthermore, the GCKR rs1260326 genetic variant was also signifcantly associated with the development of NAFLD and T2DM.Most of the other risk factors were commonly associated with both disease subjects except gender, age, disease history, smoking, blood pressure, and RBGL (Supplementary Table 2).

Genotypic and Allelic
Frequency of the GCKR rs1260326 in NAFLD Subjects.Te distribution of GCKR rs1260326 genotype has been shown for case-control groups in NAFLD subjects (Table 3).Allelic and genotypic frequencies of normal (BMI <25), overweight (BMI 25-to <30), and obese (BMI >30) NAFLD subjects were calculated and compared with the control subjects.Te frequency of the C allele in obese NAFLD was 74.19%, while the C allele frequency was 52% in control subjects.Te frequency of the T allele (wild type) was 25.81% in NAFLD obese subjects, whereas the T allele frequency was 48% in healthy controls.Te allelic frequency of the mutant allele (CC) was higher in NAFLD subjects than that of the wild type allele (TT) (Table 3).Furthermore, the frequency of mutant C allele also increased with the progression of obesity in NAFLD subjects.Te liver damage markers including ALT (61.20 ± 110.41) and AST (61.20 ± 110.41) were signifcantly elevated in NAFLD normal weight subjects, while ALT and AST were in the normal range in Journal of Obesity overweight and obese subjects.However, bilirubin and alkaline phosphatase levels were normal range in all NAFLD subjects (Supplementary Table 3).

Genotypic and Allelic
Frequency of the GCKR rs1260326 in T2DM Subjects.Te distribution of GCKR rs1260326 variant has been shown for case-control groups in T2DM subjects (Table 4).Allelic and genotypic frequencies of normal (BMI <25), overweight (BMI 25-to <30), and obese (BMI >30) T2DM subjects were calculated and compared with the control subjects.Te frequency of the C allele (mutant type) in obese T2DM was 74.07%, whereas the T allele frequency was 32% in healthy controls.Te frequency of the T allele (wild type) was 25.93% in obese T2DM subjects, whereas the T allele frequency was 68% in control subjects.Te allelic frequency of the mutant allele (CC) was higher in T2DM subjects than that of wild type allele (TT).Te frequency of the mutant C allele also increased with the progression of obesity in T2DM subjects.

Association of the GCKR rs1260326 with Obesity in
NAFLD and T2DM.Allelic and genotypic frequencies of the mutant allele (CC) in all NAFLD and T2DM subjects were 68.93% and 65.5%, respectively, as compared to wild type allele (TT) in NAFLD (31.06%) and T2DM (34.5%) control subjects (Table 5).Te genetic variant GCKR rs1260326 was signifcantly associated with both diseases (NAFLD and T2DM), and mutant type (CC) was the most prevalent genotype as compared to wild type (TT) in both diseases, especially in obese patients.Binary logistic regression has shown a signifcant association of the GCKR rs1260326 variant with obesity in NAFLD as compared to normalweight subjects.However, all other combinations analysis did not show statistical signifcance (Table 6).

Discussion
Te GCKR rs1260326, substitutes proline to leucine at position 446 of amino acid (P446L), is a genetic risk factor associated with obesity-associated nonalcoholic fatty liver (NAFLD) and type 2 diabetes mellitus (T2DM) in diferent population [9,13,30].Tis study reports an association of the GCKR rs1260326 genetic variant with the development of NAFLD and T2DM in the obese subjects, in the local population of South Punjab, Pakistan.In this population, the mutant risk allele (C) of GCKR rs1260326 was highly prevalent in NAFLD (74.19%) and T2DM (74.07%) obese subjects as compared to wild type allele (T).While in some studies, the frequency of C allele was 42.6% in NAFLD [13] and T2DM subjects [15].A genome-wide association study (GWAS) in Malaysia has reported a signifcant association of the GCKR rs1260326 with the development of hepatic steatosis, NAFLD, and nonalcoholic steatohepatitis (NASH) [31].Single nucleotide polymorphism of the GCKR rs1260326 alters the adiponectin function and infuences the lipogenesis pathway to increase susceptibility and severity of NAFLD and hepatic fbrosis [13].In line with this, the mutant C risk allele of the GCKR rs1260326 has been reported genetic risk factor associated with NAFLD in obese subjects [30].Obesity usually coexists with metabolic disorders including NAFLD and T2DM.Visceral obesity and high BMI were high risk factors, especially in both metabolic disorders.Previous studies indicated that the prevalence of obesity usually surpassed 90% of the subjects in NAFLD [8].Te development of obesity was also signifcantly linked with the GCKR rs1260326 mutant allele (C) especially in NAFLD subjects, while the frequency of mutant allele (C) was considerably higher in T2DM obese subjects but did not show a statistical signifcance in comparison to normal weight subjects.Tus, the GCKR rs1260326 was a genetic 4 Journal of Obesity risk factor and signifcantly linked to NAFLD and T2DM in the local population, which can contribute to the development of obesity and insulin resistance.Similarly, the GCKR rs1260326 was also linked with hepatic fat metabolism and fat contents in T2DM [15].Serval risk factors have also been linked to the development of obesity and NAFLD [32].High-fat dietary uptake was considered a major contributory factor in the development of NAFLD.Similarly, this study also reports several other demographic risk factors associated with the development of NAFLD and T2DM without high-fat dietary uptake.In this study, the risk factors including age, disease history, diabetes, hypertension, duration, blood pressure, ALT, AST, and AP have shown a signifcant association with NAFLD development.

Journal of Obesity
Te association of GCKR rs1260326 has been repeatedly reported especially in NAFLD with a considerable contribution to liver damage and hepatic fbrosis [33].TeGCKR rs1260326 was associated with a higher chance of developing NAFLD and NASH with severe fbrosis [34] and increased hepatic de novo lipogenesis (DNL) in obese youngsters [35].In this study, we also reported a signifcant elevation of liver damage markers (AST, ALT, and AP) in normal-weight NAFLD subjects; however, the liver damage markers were in the normal range with the progression of obesity in overweight and obese NAFLD subjects.Higher liver damage in normal-weight NAFLD subjects as compared to the overweight and obese NAFLD subjects was indicating a protective role of fat storage until overloading of fat in hepatocytes and infammation.Te persistent fat storage in hepatocyte possibly leads to loss of hepatocyte function and promotes the infltration of infammatory cells to contribute to hepatic steatohepatitis, NASH, and further liver damage [11].A previous study indicated that proliferator-activated receptor c (PPARc) mediated adipogenesis and browning of    6 Journal of Obesity fat protected liver damage in NAFLD and improved insulin sensitivity [36].Tis partially explained a possible mechanism of moderate liver damage in obese NAFLD subjects as compared to normal weight NAFLD subjects.However, circulatory markers also dropped with the progression of chronic liver disease, for example, AST and ALT levels could not rule out advance stages of chronic liver diseases [37].Tis can be another explanation for low levels of liver damage markers with the advance stage of NAFLD in this study.
In this study, BMI, body fat, FBGL, RBGL, and Hb1Ac were signifcant risk factors associated with the development of T2DM.Similarly, the genetic polymorphisms in diferent genes such as CAMK2, IGF1, IRS1, GCKR, PPARG, GCK1, and KCTD1 are found associated with insulin resistance and T2DM [38,39].Similarly, genetic variations in the GCKR expression can also regulate glucose metabolism and insulin sensitivity in obesity-associated metabolic disorders including NAFLD and T2DM [5,6].Te P446L substitution afects the ability of GCKR negative regulation of glucokinase in response to fructose-6-phosphate and increased the activation of hepatic glucose uptake, resulting in reduced circulating insulin and fasting glucose [40].Other studies reported elevated insulin secretion and low plasma glucose levels associated with the GCKR rs1260326 in Danish diabetic populations [10,41].Similar to the previous studies, this study also reported a signifcant association of the GCKR rs1260326 in the mutant (CC) variants with T2DM subjects without a signifcant diference in HbA1c, fast, or random glucose levels as compared to the wild-type variants.However, obesity (body fat content and BMI) was considerably higher in the mutant-type T2DM subjects.Similar to previous studies [34], this study also indicated that the GCKR rs1260326 possibly contributed to the pathology of T2DM and insulin resistance through lipogenesis or obesityassociated pathways.
Similarly, the GCKR variant rs1260326 has been correlated with metabolic traits, such as higher levels of triglycerides and a higher incidence of dyslipidemia, without the onset of metabolic syndrome [3] and T2DM [9].Tis indicated the possibility of two separate biological mechanisms which contributed to the development of metabolic disorders including metabolic syndrome, T2DM, and NAFLD.

Conclusion
Te GCKR rs1260326 genetic variant contributed to the impaired hepatic lipid and glucose metabolism and promoted the development of metabolic disorders including NAFLD and T2DM.Te GCKR rs1260326 mutant allele (CC) was considerably higher in subjects with obesity and insulin resistance independent of the high fat and glucose uptake.Tis study highlighted the importance of additional lipid metabolic pathways regulated by glucose metabolism and signifcantly contributed to the development of NAFLD and T2DM.Tis study indicated that the GCKR gene is also a novel therapeutic target in the treatment of obesity and obesity-associated metabolic disorders.Te GCKR rs1260326 variant has potential research and clinical implications regarding to its impact on metabolism of glucose and lipid.
Future direction aspects of the GCKR rs1260326 include investigating the role in developing targeted therapies and personalized medicine as well as potential biomarker for the risk assessment of the disease.However, further studies are still required to better understand the underlying mechanisms and concluding the interaction with other environmental and genetic factors involved in lipid metabolism in glucokinase regulator (GCKR) defcient subjects.

Figure 1 :
Figure1: Tetra-arms PCR and gel electrophoresis of GCKR rs1260326 variant.Tetra-arms PCR was optimized and analyzed by gel electrophoresis.DNA was extracted from all samples and amplifed with GCKR rs1260326 specifc primers.Tis fgure shows 1 to 10 representative samples from NAFLD subjects.Te 541 bp amplicon was considered enteral control for all PCR reactions.Te 338 bp amplicon was identifed as a homozygous mutant C/C, while 240 bp was considered a homozygous wild type T/T.Tetra-arm primer amplifed 338 bp in mutant variants and 240 bp in wild-variants and both amplicons in heterozygous variants.Amplicon length was identifed with a 100 bp DNA ladder (L).

Table 2 :
Te demographic data of T2DM and control subjects.

Table 3 :
Genotype and allelic frequencies of GCKR rs1260326 in obese NAFLD vs control.

Table 5 :
Genotype and allelic frequencies of GCKR in NAFLD and T2DM subjects.

Table 6 :
Association of GCKR rs1260326 with obesity in NAFLD and T2DM subjects.