Decreased Alu methylation in type 2 diabetes mellitus patients increases HbA1c levels

Abstract Introduction Alu hypomethylation is a common epigenetic process that promotes genomic instability with aging phenotypes, which leads to type 2 diabetes mellitus (type 2 DM). Previously, our results showed significantly decreased Alu methylation levels in type 2 DM patients. In this study, we aimed to investigate the longitudinal changes in Alu methylation levels in these patients. Results We observed significantly decreased Alu methylation levels in type 2 DM patients compared with normal (p = 0.0462). Moreover, our findings demonstrated changes in Alu hypomethylation over a follow‐up period within the same individuals (p < 0.0001). A reduction in Alu methylation was found in patients with increasing HbA1c levels (p = 0.0013) and directly correlated with increased HbA1c levels in type 2 DM patients (r = −0.2273, p = 0.0387). Conclusions Alu methylation in type 2 DM patients progressively decreases with increasing HbA1c levels. This observation suggests a potential association between Alu hypomethylation and the underlying molecular mechanisms of elevated blood glucose. Furthermore, monitoring Alu methylation levels may serve as a valuable biomarker for assessing the clinical outcomes of type 2 DM.

accumulated DNA damage, [36][37][38] leading to genomic instability and cellular senescence. 39,40Therefore, it is plausible to consider that Alu hypomethylation-induced DNA damage is one of the processes contributing to genomic instability in type 2 DM.
Presently, blood tests are used to diagnose type 2 DM by measuring fasting blood sugar (FBS) and hemoglobin A1C (HbA1c) levels, [41][42][43] and guidelines gauge good diabetes care primarily based on HbA1c levels. 44,45Although it is an effective diagnostic test for type 2 DM, 46 it is mainly used to detect the disease in patients who already exhibit symptoms.Therefore, blood test results for type 2 DM diagnosis should be confirmed with other measurements before patients display DM symptoms, such as Alu methylation levels.
In this study, we employed ALU-Combined Bisulfite Restriction Analysis (COBRA) to assess Alu methylation in all samples (Fig- ure S1).[49] In our previous study, we categorized the Alu methylation pattern in our samples, which included control, pre-DM, and type 2 DM patients.Our findings revealed lower Alu methylation levels in type 2 DM patients compared to the general population. 50Here, we observed Alu methylation levels during 4 years of follow-up in normal, pre-DM, and type 2 DM groups.
We hypothesized that the extent of Alu hypomethylation would undergo changes within the same individuals over the follow-up period.The aim of this study was to determine whether Alu methylation could serve as a highly specific novel biomarker for more accurate screening and monitoring of the progression of type 2 DM in the future.

| Participants
We conducted this study with a total of 203 samples.The participants' blood glucose levels were monitored using HbA1c, and they were divided into three groups: normal ( 56

| DNA extraction and bisulfite DNA modification
DNA was extracted from the buffy coat using proteinase K digestion and phenol-chloroform extraction protocols.The denatured genomic DNA was then incubated at 37°C for 10 min in 0.22 M NaOH, followed by incubation with 30 μL of 10 mM hydroquinone and 520 μL of 3 M sodium-bisulfite at 50°C for 16-20 h.Subsequently, the DNA was purified and treated with 0.33 M NaOH for 3 min at 25°C, after which it was ethanol precipitated, washed with 70% ethanol, and finally resuspended in 20 μL of H 2 O. 50

| ALU-Combined bisulfite restriction analysis (COBRA)
To detect methylated levels at thousands of CpG loci, a set of conserved primers for each IRS (interspersed repetitive sequence) was used.The following components were used to determine the methylation level of Alu in the samples: 1X PCR buffer (Qiagen, Germany), 0.2 mM deoxynucleotide triphosphate (dNTP) (Promega, USA), 1 mM magnesium chloride (Qiagen, Germany), 25 U of HotStarTaq DNA Polymerase (Qiagen, Germany), and a primer pair at a concentration of 0.3 μM.The primer sequences used for Alu amplification were as follows: ALU-Forward (5′-GGYGY GGT GGT TTA YGT TTGTAA-3′) of TasI (Thermo Scientific, USA), 5X NEB3 buffer (New England Biolabs, USA), and 1 μg/μL bovine serum albumin (BSA) (New England Biolabs, USA).Each digestion reaction was incubated overnight at 65°C, followed by separation on an 8% acrylamide gel and staining with SYBR (Lonza, USA).The intensity of the Alu methylation band was observed and measured using a phosphoimager and Image-Quant software (GE Healthcare, UK). 50

| Methylation analysis
The COBRA results were categorized into the following four groups based on the methylation status of the two CpG dinucleotides:

| Statistical analyses
The average and distributions of characteristic data for all the samples were analyzed and presented as the mean ± SD and median.
Differences in blood glucose levels between groups in matched cases were determined using t-tests, with a significance threshold set at a p value of 0.05.To explore the relationship between two continuous variables, Spearman's correlation coefficient was utilized.

| Alu methylation levels in patients with type 2 DM
In this study, a total of 203 samples were categorized into three groups based on their HbA1c levels; there were 56 normal control subjects, 64 pre-DM patients, and 83 type 2 DM patients (Table 1).
The levels of Alu methylation in the normal group were compared to those in the pre-DM and type 2 DM groups.Our analysis revealed that type 2 DM patients exhibited significantly lower Alu methylation levels than normal control subjects (p = 0.0462) (Figure 1A).To investigate the potential influence of sex differences on Alu methylation levels, we further examined and compared the methylation levels between males and females within each group.Our findings indicated that there were no significant differences in Alu methylation levels between males and females across all three groups (Figure 1B).

| Alu methylation levels during case follow-up
The results also showed significantly different levels of Alu methylation for 4 years of follow-up in all the samples (Figure 2A) (p < 0.0001).Additionally, when we classified the samples based on the HbA1c indicator, the Alu methylation levels in the normal, pre-DM, and type 2 DM groups were significantly decreased (Figure 2B) (p = 0.0028, p = 0.0309, and p = 0.0231, respectively).

| DISCUSS ION
Previously, our data showed that Alu methylation in patients with type 2 DM was lower than that in the general population.Furthermore, the results demonstrated a direct correlation between Alu hypomethylation levels and high blood glucose in type 2 DM. 50reover, this association is similar to the correlation between Alu hypomethylation and lower bone mass or hypertension patients. 27,51erefore, the aim of this study was to determine the correlation between changes in Alu methylation levels in individual patients, particularly those who had increased HbA1c levels, and the progression of type 2 DM.Interestingly, our results revealed that Alu methylation levels were consistently reduced in the same individuals, with significantly decreased levels observed in the normal, pre-DM, and type 2 DM groups during the follow-up.Alu hypomethylation might represent an epigenetic alteration that plays a key role in the molecular pathogenesis of DM.
Our results showed significantly decreased Alu methylation in all of the samples in the increased HbA1c group during case follow-up.We divided the samples into two subgroups based on the changes in HbA1c levels during the case follow-up: those with decreased or equal HbA1c levels (good control) and those with increased HbA1c levels (bad control).[57] The association between Alu methylation and increased HbA1c levels in type 2 DM patients might be linked to inflammatory disorders.
As a result, our findings suggest that managing diabetes, along with acknowledging the connection between heightened hyperglycemia and inflammatory complications in special populations such as elderly patients with type 2 DM and heart conditions, necessitates specific attention. 65,66cumulating evidence suggests that oxidative stress can directly damage DNA, leading to the formation of DNA adducts and strand breaks. 67,68The presence of DNA damage triggers p53 activation as a protective response. 69,70Moreover, p53 can directly induce Our data suggest that decreased Alu methylation was more strongly associated with the poorly controlled blood glucose group, indicating a possible link between Alu hypomethylation and inadequate hyperglycemia management in type 2 DM.Importantly, previous studies have demonstrated that Alu methylation plays a role in preventing the accumulation of endogenous DNA damage. 30Therefore, Alu hypomethylation, which is associated with hyperglycemia, might induce DNA damage and potentially contribute to insulin resistance, reduced glucose uptake, elevated blood sugar levels, high HbA1c levels, and the development of type 2 DM.

Furthermore, we observed an inverse relationship between
ΔAlu hypomethylation and ΔHbA1c in both normal control subjects and type 2 DM patients.These findings indicate that changes in blood glucose levels corresponded to changes in Alu hypomethylation, suggesting that decreased Alu methylation is associated with HbA1c levels.These findings are similar to those of HbA1c, which is an effective biomarker. 77The amount of Alu methylation is inversely associated with the degree of aging, and the degree of aging phenotype is directly associated with the severity of type 2 DM.
Thus, Alu methylation levels could provide an overall picture of the average disease severity over a long period when detecting type samples), pre-DM (64 samples), and type 2 DM (83 samples).The pre-DM and type 2 DM patients were admitted to Thailand's Tambon Health Promoting Hospital.The age range of the patients was 31 to 85 years.The participants were recruited for the study and provided written informed consent.Access to participant information that could identify individuals was maintained during data collection.All methods were performed in accordance with the relevant guidelines and regulations.Approval for the study was obtained on September 28, 2015, from the Ethics Clearance Committee on Human Rights Related to Research Involving Human Subjects at Walailak University in Nakorn Sri Thammarat, Thailand, in accordance with the Declaration of Helsinki.The study has been reviewed and approved by the Thai Clinical Trials Registry Committee with the identification number TCTR20220324001.
and ALU-Reverse (5′-CTAAC TTT TTA TAT TTT TAA TAA AAA CRA AATTTCACCA-3′), where R represents A and G, and Y represents C and T. The PCR program for Alu amplification consisted of an initial denaturation step at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 45 s, annealing at 57°C for 45 s, extension at 72°C for 45 s, and a final extension step at 72°C for 7 min.After the Alu PCR products were obtained, COBRA was performed.The COBRA reaction included 2 U of TaqI (Thermo Scientific, USA), 2 U hypermethylation at both CpGs ( m C m C); hypomethylation at CpGs ( u C u C); partial methylation of the m C u C; and partial methylation of the u C m C. To calculate the Alu methylation levels for each group, the intensity of COBRA-digested Alu products was measured using a Typhoon FLA 7000 biomolecular imager (GE Healthcare, UK).The band intensities of five Alu products with sizes of 133, 90, 75, 58, and 43 bp were used in the Alu methylation analysis.The following formula was employed: A = 133/133, B = 58/58, C = 75/75, D = 90/90, E = 43/43, and F = 32/32.The Alu methylation levels were calculated using the following formulas: Alu methylation level percentage (% m C) = 100 × (E + B)/(2A + E + B + C + D); percentage of m C m C loci (% m C m C) = 100 × F/(A + C + D + F); percentage of u C m C loci (% u C m C) = 100 × C/(A + C + D + F); percentage of m C u C loci (% m C u C) = 100 × D/(A + C + D + F); and percentage of u C u C loci (% u C u C) = 100 × A/(A+ C + D + F).These formulas allow for the calculation of Alu methylation levels based on the band intensities obtained from the COBRA analysis.

Furthermore, we further
divided the three groups (normal, pre-DM, and type 2 DM) into two subgroups based on changes in HbA1c levels during the case follow-up.The first subgroup represented individuals with decreased or equal HbA1c levels, and the second subgroup included those with increased HbA1c levels.Our results demonstrated a significant decrease in Alu methylation levels in the subgroup with increased HbA1c levels during the case follow-up, encompassing all the samples, normal, pre-DM, and type 2 DM groups (Figure 3A-D) (p < 0.0001, p = 0.0078, p = 0.0230, and p = 0.0013, respectively).
We observed the association between different levels of Alu methylation (ΔAlu methylation) and different levels of HbA1c (ΔHbA1c) in the four-year follow-up in all samples, normal, pre-DM, and type 2 DM patients (Figure4A-D, respectively).The results showed a significant inverse correlation between ΔAlu methylation and ΔHbA1c in all samples and those from the normal and type 2 DM patients (Figure 4A,B,D) (r = −0.1581,p = 0.0242, r = −0.3068,p = 0.0214 and r = −0.2273,p = 0.0387, respectively).

F I G U R E 1 76 F I G U R E 3 F I G U R E 4
The Alu methylation levels in the samples were analyzed based on the HbA1c levels.(A) The percentage of each Alu methylation level in normal, pre-DM, and type 2 DM samples.(B) The Alu methylation levels in males and females.The results are presented as box plots, where the boxes represent the interquartile ranges (25th to 75th percentile), the median lines represent the 50th percentile, and the whiskers represent the minimum and maximum values *p < 0.05 (t test) (Mann-Whitney test).F I G U R E 2 Alu methylation levels over a follow-up period in the same individuals from all the samples (A).(B) The percentage distribution of each follow-up level in the normal, pre-DM, and type 2 DM samples.*p < 0.05, **p < 0.001 (Wilcoxon signed-rank test).tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), and this effect has been found to inhibit insulin signaling and hinder glucose uptake.Consequently, increased DNA damage-inducing p53 activity can lead to a decrease in IRS1 expression, alterations in IRS1 phosphorylation, and disruption of downstream insulin signaling pathways, ultimately contributing to insulin resistance. 71-74Therefore, prolonged hyperglycemia causes an imbalance in oxidative production and suppression, which induces p53 and contributes to impaired insulin signaling, compromised glucose metabolism, and the pathogenesis of type 2 DM. 75,Alu methylation levels of follow-up samples with a cutoff point determined by the change in HbA1c levels in all samples (A), normal (B), pre-DM (C), and type 2 DM groups (D).*p < 0.05, **p < 0.001 (Wilcoxon signed-rank test).Correlation between ΔAlu methylation levels and ΔHbA1c for all samples (A) and the normal (B), pre-DM (C), and type 2 DM groups (D).Spearman's correlation coefficients (r) with p values are indicated (*p < 0.05).

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DM.In practical applications, it might be necessary to increase the sample size to generate a standardized graph demonstrating the decrease in Alu methylation in each age group, comparing individuals with diabetes to those without diabetes.Consequently, Alu methylation could potentially serve as a highly specific and novel biomarker for more accurate prediction and follow-up of type 2 DM patients.CON CLUS IONS Our results not only showed significant Alu hypomethylation in type 2 DM but also demonstrated a decrease in Alu hypomethylation levels during the follow-up of type 2 DM patients, particularly those who had increased HbA1c levels.This suggests that Alu hypomethylation might contribute to DNA damage and the subsequent decline in cellular function in type 2 DM.Therefore, Alu methylation could serve as a promising biomarker for monitoring type 2 DM and might facilitate the development of improved therapeutic approaches for prevention and treatment in the future.AUTH O R CO NTR I B UTI O N S JT isolated DNA from plasma samples and processed the data.JT performed the Bisulfite DNA modification.JT and AM discuss the results part.JT and AM wrote the first draft of the manuscript and approved the final manuscript.