Diabetes mellitus (DM), a chronic disease, was the third leading cause of death in Indonesia, with a percentage of 6.7% after stroke (21.1%) and coronary heart disease (12.9%). The DM prevalence in Indonesia has increased substantially from 6.9% in 2013 to 8.5% in 20181. Other data has estimated that approximately 30% of Indonesia's population (30 million people) with diabetes remains undiagnosed. The diabetics in Indonesia were estimated could reach 30 million people in 2030 if lifestyles including unhealthy diet, obesity, lack of physical activity, alcohol consumption, and smoking are not a concern2. In line with this report, The International Diabetes Federation (IDF) found that people with diabetes in Indonesia have increased precipitously in the last ten years from 2021. Without proper management, people with diabetes will jump to a staggering 28.57 million in 2045, or 47% greater than 19.47 million in 20213.

Type 2 diabetes mellitus (T2DM) is the most common in adults and accounts for 90% of all diabetes cases. In past years, T2DM typically develops in adulthood. However, in recent years, it has been increasingly seen in children and adolescents partially due to lifestyle, including rising obesity rates, unhealthy diet, lack of physical inactivity, alcohol consumption, and smoking4. The Basic Health Research of Indonesia (Riset Kesehatan Dasar, RISKESDAS) 2018 reports that T2DM prevalence in the Daerah Istimewa Yogyakarta (DIY) Province was second among provinces in Indonesia5. About 74,668 DIY people have been diagnosed with diabetes, but only 55,190 patients have received standard health services or the equivalent of 73.9%6.

Several studies have suggested that poor smoking behavior is associated with chronic complications of T2DM compared to non-smokers7,8. Another study has reported that smoking can increase glycohemoglobin (HbA1c) blood levels9. This HbA1c value can accurately reflect glucose control 2-3 months ago. HbA1c levels are normal if <5.7%, prediabetes 5.7 to 6.4%, and diabetes ≥6.5%10. Nicotine, the main compound in cigarettes, was considered most responsible for increasing blood sugar levels due to insulin resistance11.

Nicotine is primarily metabolized by the CYP2A6 enzyme to cotinine and excreted in the urine12. The CYP2A6 enzyme encoded by the CYP2A6 gene is a polymorphic gene. The active allele gene is CYP2A6*1, and the inactive is CYP2A6*4, CYP2A6*7, and CYP2A6*9. Due to their genotype, a person with having CYP2A6*4, CYP2A6*7, and CYP2A6*9 allele genes is associated with a slow metabolizer or poor metabolizer13. Furthermore, according to Liu et al.14, reduced metabolism function CYP2A6 in smokers appears to be associated with a higher risk of T2DM.

Our preliminary study15 revealed a high-frequency CYP2A6*4 allele gene among smokers and non-smokers in Javanese Indonesian. We have also reported that smoking can increase diabetes risk factors. Prediabetes was developing in smokers who had smoked for at least 25 years with 25 cigarettes per day16. Furthermore, in our recent study on diabetic patients, both smokers and non-smokers, high-frequency CYP2A6*4, the inactive allele gene of CYP2A6, was detected17. In high frequency, the other inactive alleles, CYP2A6*7 and CY2A6*9, have also been found among Javanese Indonesian smokers13. So, in this research, we study the association of the CYP2A6*4, CYP2A6*7, and CYP2A6*9 on glycohemoglobin levels in Indonesian Javanese smokers.

Materials

A Norudia® N HbA1c Immunoassay Method using the Architect 600 instrument, calibrated using Diabetes Control and Complications Trial (DCCT) standards with a coefficient of variation <2.5% was used to analyze total HbA1c in the Clinical Pathology Laboratory, Bethesda Hospital, Yogyakarta. Genomic DNA was extracted using a DNA Mini Kit from Bioron GmbH (Germany). The CYP2A6*4, *7, and *9 allele genes were analyzed using the Polymerase Chain Reaction (PCR) method. The forward and reverse primers used in this study were 5' CCT CAT CAC ACA CAA CTT CCT C 3' and 5' TGC AGG TAC TGG GTG CTT GGT AG 3' for CYP2A6*4; 5’-CTC CCA GTC ACC TAA GGA CAC-3' and 5’-AAA ATG GGC ATG AAC GCC C-3' for CYP2A6*7; as well as 5’-GAT TCC TCT CCC CTG GAA C-3' and 5’-GGC TGG GGT GGT TTG CCT TTC-3' for CYP2A6*9.

The PCR mixture contained 12.5 μL Promega Go Taq Green Master Mix, 1.25 μL forward primer, 1.25 μL reverse primer, 5.0 μL genomic DNA, and 5.0 μL nuclease-free water in a final volume of 25 μL. This mixture was run using a PCR machine (Thermal cycler Perkin Elmer 2400) to amplify the genomic DNA. The PCR conditions used are shown in Table I.

TableI. PCR condition used.

Methods

Research subject

It is an observational study using a cross-sectional design to analyze the CYP2A6 polymorphism among Javanese Indonesian Smokers associated with glycohemoglobin blood levels, the main predictor for diabetes disease. Participants were enrolled between July and August 2022. They live in Yogyakarta, as indicated by their identity card. A preliminary survey was conducted to find respondents who smoked using a self-reported smoking questionnaire adopted from the Fagerström Test for Nicotine Dependence (FTND) questionnaire18. The participants had to meet the study's inclusion criteria: active smokers who had smoked for at least ten years, Javanese Indonesians with at least three grandparents of Javanese descent due to their self-reported, male, aged 20-50 years, weight between 46 to 75 kg, with a varying height between 150-170 cm. This study excluded the participant who had an infection at the blood sampling and was taking an anticoagulant. All participants had agreed to participate in this study indicated by signing the informed consent. The study was approved by the Ethics Commission for General Medicine Research, Universitas Duta Wacana, Yogyakarta (No. 1413/C.16/FK/2022).

Blood sample collection

Three mL of blood was sampled from a cubital vein in all participants who had met the inclusion and exclusion criteria. Blood samples were collected in a vacutainer containing EDTA (1.8 mg/mL blood) and immediately stored in the refrigerator at 4°C.

PCR analysis

The PCR products were analyzed using electrophoresis with 1.5% agarose and evaluated using a UV transilluminator. Expressed PCR products are documented using a Polaroid camera.

Data analysis

To describe the study population and evaluate data, we used Microsoft Excel 2019. All values are displayed as the mean ±SD or number (%). We assumed p <0.05 indicated significant differences. Using a box plot diagram, we also described the distribution of HbA1c levels among the subjects based on their CYP2A6 allele gene. The chi-square test was used to analyze the relationship between CYP2A6 polymorphism and HbA1c levels.

A total of 106 participants were participating in this study. There are three groups of test subjects, based on the number of cigarettes per day (CPD) they smoked: light smokers (CPD: 1-10), intermediate smokers (CPD: 11-20), and heavy smokers (CPD: 21-30)19. All the respondents were smoking a white filter cigarette containing 12 mg of nicotine/cigarette. Table II below shows the respondent characteristics participating in this study. Based on Table II, 88.7% of the respondents are light and intermediate smokers, while 11.3% are heavy smokers. The Ministry of Health of the Republic of Indonesia has reported that the average CPD by Indonesian adults was 13 cigarettes or the equivalent of one pack6. Some of the respondents started smoking at the age of under ten years. Several factors influence smoking behavior among children and adolescents, including easy access to cigarettes, family and peer environment, and cigarette promotion/advertising20. All respondents had smoked for at least ten years, indicating that they had been exposed to nicotine for a long time.

TableII. Respondent characteristics.

Several studies have proven that cigarette dependence can trigger the occurrence of T2DM21,22. Compared to non-smokers, active smokers have a 76% higher risk of developing T2DM23,24. Nicotine in cigarette smoke was responsible for the development of T2DM in smokers25-27. Nicotine in cigarettes has caused insulin resistance and reduced insulin secretion28. Xie et al.29 has revealed that nicotine exposure in the long term will decrease insulin secretion through the activation of nAChRs present in pancreatic cells. Furthermore, Xie et al.29 also mentioned that nicotine exposure for a short period (24 hours) will inhibit insulin release from the pancreas. Other studies have shown that nicotine exposure can cause pancreatic cell dysfunction and increased cell apoptosis30,31. Eventually, it will cause an increase in blood glucose levels and the T2DM risk factor in smokers7.

Our study assesses the T2DM risk factor in smokers using the HbA1c blood level. Several studies have used the HbA1c parameter to control blood glucose levels8,16,23,32. Indonesian Endocrinology Society (Perkumpulan Endokrinologi Indonesia, PERKENI) stated that people with HbA1c levels <6.5 have a normal glucose level. People with HbA1c levels between 5.7% and 6.4% have prediabetes and a higher chance of getting diabetes. The diabetes condition is established if the HbA1c levels are higher than 6.5%33. Akkuzulu et al.23 has reported a positive correlation between nicotine dependence and HbA1c levels in smokers. Several other previous studies have also revealed that compared to non-smokers, smokers have higher HbA1c levels and a 30-40% higher risk of T2DM8,34. Somm et al.31 has revealed that nicotine administration in low doses will increase HbA1c levels by 8.8%, and at high doses, after being given nicotine for two days, increase HbA1c levels by 34.5%.

Figure 1 describes the distribution of HbA1c levels among the respondents. According to Figure 1, 16.04% of the respondents participating in this study had diabetes, and 13.16% were pre-diabetic. They are mainly distributed among intermediate and heavy smokers with smoking for more than 20 years. It is in line with our previous study that prediabetes among Javanese smokers will occur at a minimum CPD of 20 cigarettes with a minimum smoking duration of 25 years. Meanwhile, diabetes will occur at a minimum CPD of 20 cigarettes with a minimum smoking duration of 29 years16. Therefore, it is possible for respondents whose HbA1c levels <5.7 will still develop T2DM if they continue to smoke. Diabetes was an underdiagnosed disease. Approximately 30% of diabetics are often unaware of their condition, resulting in 25% of people with diabetes being diagnosed with microvascular complications. The average delay from onset to diagnosis is about seven years35. This study has also supported the report issued by RISKESDAS 2018, that only about 25% of diabetics in Indonesia know that they have diabetes36.

Figure1. The HbA1c distribution among participants.

In addition, another factor that can increase the T2DM risk in a smoker is the CYP2A6 polymorphism. The three CYP2A6 inactive allele genes have been identified in this study: CYP2A6*4, *7, and *9. The CYP2A6*4, a whole gene deletion, due to the unequal crossover junction with CYP2A7. CYP2A6*7 occurred due to the Single Nucleotide Polymorphism (SNPs) in the 8454th nucleotide base sequence (T>C). The CYP2A6*9 allele formed due to the SNPs in the TATA box in the CYP2A6 promoter region at the -48T>G point37. These three allele genes will decrease the CYP2A6 enzyme activity, either intermediate, slow, or poor metabolizer. Smokers with slow or poor metabolizers are more susceptible to suffering T2DM than fast metabolizers38.

Table III shows that the CYP2A6*4, CYP2A6*7, and CYP2A6*9 allele frequency were 50.9%, 4.3%, and 3.8%, respectively. It is consistent with our previous studies15,16, where the CYP2A6*4 allele frequency in Javanese was high. These allele genes will decrease the CYP2A6 enzyme activity. Several studies39-41 have revealed that smokers with the inactive allele would slowly metabolize the nicotine compared to the active allele. Consequently, the nicotine blood level becomes higher, and the CPD and nicotine dependence become lower. Based on the three allele genes, Peamkrasatam et al.42 and Malaiyandi et al.43 classified the CYP2A6 phenotype into four groups: fast metabolizer (CYP2A6*1/*1), intermediate metabolizer (CYP2A6*1/*4; CYP2A6*1/*7, CYP2A6*1/*9), slow metabolizer (CYP2A6*4/*7; CYP2A6*4/*9, CYP2A6*7/*9), and poor metabolizer (CYP2A6*4/*4). As shown in Table III, only four (3.8%) smokers are fast metabolizers, and most smokers are intermediate metabolizers (74.5%), while the rest are slow and poor metabolizers (21.7%).

TableIII. CYP2A6 genotype and allele frequency among respondents.

Figure 2 describes the distribution of HbA1c levels among the respondent based on their phenotype. Figure 2 shows that all participants with fast metabolizers and most intermediate metabolizers had HbA1c levels <5.7. There are only 10 participants with intermediate metabolizers had HbA1c >5.7. In the slow metabolizer, two people have HbA1c values <5.7, and the rest have >5.7. On the other hand, all participants with poor metabolizers have HbA1c levels >5.7, indicating that they have diabetes condition. It is in line with another study44 that heavy smokers with slow and poor metabolizers would have a high risk of developing T2DM compared to light smokers with fast and intermediate metabolizers. Furthermore, we used a chi-square test to analyze the effect of the inactive alleles on the HbA1c levels among the participants.

Figure2. HbA1c levels distribution among the test subjects according to their genotype. FM: fast metabolizers; SM: slow metabolizers; IM: intermediate metabolizers; PM: poor metabolizers.

As shown in Table IV, due to its p-value (0.000 <0.005) and χ2 (54.6) with df=1, it is known that CYP2A6 polymorphism could have affected the HbA1c levels among the participants. The homozygous and heterozygous *4, *7, and *9 among smokers would increase the risk of HbA1c levels in smokers. CYP2A6 enzyme encoded by CYP2A6 is the enzyme corresponding to nicotine inactivation. The inactive metabolites of nicotine excreted in the urine are cotinine and trans-3-hydroxycotinine45. Therefore, heavy smokers with slow or poor metabolizers tend to have higher nicotine plasma levels than light smokers with fast or intermediate metabolizers. Several studies have revealed that smokers may increase the risk of T2DM, indicated by an increase in HbA1c levels. It is due to pancreatic β cell dysfunction and insulin resistance34,46.

TableIV. The relationship between CYP2A6 polymorphism to HbA1c values among participants.

CYP2A6 is also known as the enzyme responsible for nitrosamine metabolic activation, the pre-carcinogen compound in tobacco smoke, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosonornicotine (NNN), N′-nitrosoanabasine (NAB), and N′-nitrosoanatabine (NAT)47. Therefore, smokers with slow or poor metabolizers could reduce the hepatic first-pass clearance of tobacco nitrosamines, resulting in greater exposure to other organs, such as the pancreas, due to its higher systemic levels48. The increased exposure of nitrosamine in pancreatic islet cells could lead it’s the metabolic activation by other cytochrome P450 enzymes (CYPs), including CYP2E147, resulting in inflammation and apoptosis of pancreatic cells, which is furthermore might decrease insulin secretion and the increased risk of developing T2DM49.

According to Bergman et al.50, insulin sensitivity will recover in a smoker who has quit smoking; therefore, to prevent diabetes, a smoker must stop smoking. It is also supported by other studies51-53 on preventing T2DM among smokers through smoking cessation strategies. Several studies54-56 have also shown that smokers who have inactive alleles tend to quit smoking more easily. Therefore, to increase efforts to reduce the prevalence of diabetes in Indonesia, cooperation from various parties is needed to reduce cigarette consumption in Indonesia. RISKESDAS in Indonesia has reported that the number of smokers over 15 years of age was 33.8%, of which 62.9% were male and 4.8% were female57. In addition, The Southeast Asia Tobacco Control Alliance (SEATCA) in The Tobacco Control Atlas has reported that the number of smokers in Indonesia was 65.19 million, placing Indonesia as the highest number in Southeast Asia58. Therefore, based on our study, we suggest promoting smoking cessation campaigns is the best effort to reduce cigarette consumption and diseases related to cigarettes, such as T2DM, stroke, and coronary heart disease.

Quite a few limitations of our study are: this was a cross-sectional study, the causal association between CYA2A6 polymorphism and HbA1c levels should be interpreted carefully; we used self-report surveys to collect the data regarding smoking behavior, thus it might have been caused bias data; the other inactive allele of CYP2A6 might be reduced CYP2A6 activity resulting in the alteration of phenotype, primarily on fast and intermediate metabolizers; and some confounding factor, including obesity, physical activity, and dietary factors have not fully accounted in our analysis.

In conclusion, this study reveals that the heterozygote CYP2A6 alleles, including *4, *7, and *9, corresponding to slow and poor metabolizers, may worsen HbA1c levels among Javanese Indonesian smokers. Furthermore, due to our result, it may be crucial for the government to encourage smoking cessation programs in Indonesia, which are trusted to prevent various health problems, especially diseases related to smoking behavior, including T2DM, stroke, and coronary heart disease.

This study was supported by the Institute of Research and Community Services Universitas Sanata Dharma (No. 017 Penel./LPPMUSD/IV/2022).

Conceptualization: Christine Patramurti

Data curation: Christine Patramurti

Formal analysis: Christine Patramurti

Funding acquisition: Christine Patramurti

Investigation: Dita Maria Virginia

Methodology: Dita Maria Virginia

Project administration: Dita Maria Virginia

Resources: Christine Patramurti

Software: -

Supervision: Christine Patramurti

Validation: Christine Patramurti

Visualization: Dita Maria Virginia

Writing - original draft: Christine Patramurti

Writing - review & editing: Dita Maria Virginia

None.

The authors declare no conflict of interest.

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