Methods
Patients and control subjects
This 52-week observational monocentric study was conducted at the University Clinic for Diabetes, Endocrinology and Metabolic Diseases Vuk Vrhovac, Zagreb, Croatia. The study included 185 (70 male, 115 female) patients diagnosed with type 2 diabetes aged 20–80 years with inadequate glycemic control [HbA1c level from 7 to 11% (53–97 mmol/mol)] on a retrospective documented treatment with premix insulin analogues and 156 (52 male, 104 female) healthy control subjects, sampled during their routine laboratory check-ups. All subjects were Caucasians of Croatian origin. Patients treated with antipsychotic medications, those who had clinically significant gastroparesis, an end stage renal disease, severe chronic pancreatitis, a severe liver dysfunction with portal hypertension or cirrhosis, an inflammatory bowel disease (Crohn’s disease, ulcerative colitis), unregulated hypothyroidism or hyperthyroidism, a known malignant disease, who underwent bariatric surgery, or with a history of drug or alcohol abuse were not included in this study.
After selection and randomization of patients with type 2 diabetes, premixed insulin analogues were replaced with three doses of insulin aspart applied before main meals, and one dose of insulin detemir at bedtime, and followed for 52 weeks. Administration of metformin if not contraindicated was proceeded. A dose adjustment of insulin detemir and insulin aspart was performed according to glucose profile based on self-monitoring measurement and HbA1c level.
All recruited patients went through a comprehensive educational program, and became familiar with meal planning, exercising (compatible with their physical condition), glucose self-monitoring on regular basis four times per day, as well as with insulin dose adjustment according to American Diabetes Association (ADA) guidelines [
35]. Preinclusion data was obtained retrospectively from medical records.
Clinical measurements
All examinations were performed in the morning after an overnight fasting period by the same research nurses and physician-at the baseline visit on both, T2DM patients and healthy controls, and after 52 weeks only on T2DM patients. Body weight was measured using a balanced-beam scale and was expressed in kilograms (kg). Height was measured using a wall-mounted stadiometer and expressed in centimeters (cm). Body mass index (BMI) was calculated based on these measures as kilograms per square meter (kg/m2). Blood pressure was measured on the right arm after a resting period of 10 min in a sitting position with a mercury sphygmomanometer and expressed in millimeters of mercury (mmHg). Venous blood samples were collected for determination of biochemistry, lipid profile status and HbA1c, both on baseline and after a 52 week treatment period. Blood samples for DNA isolation and DBH and COMT gene polymorphism genotyping were taken at the end of the 52 week study period.
HbA1c was measured spectrophotometrically by turbidimetric immuno inhibition (Olympus AU600 Beckman Coulter, USA). Glucose, cholesterol and triglycerides in serum were measured by an enzymatic colorimetric method.
Written informed consent was obtained from all participants, after explaining the aims and procedures of the study, under guidelines approved by Ethics committee of the University of Zagreb School of Medicine and Clinical Hospital Merkur Zagreb. All studies have been carried out with the full cooperation of participants, adequate understanding, and have therefore been performed in accordance with the ethical standards of the Declaration of Helsinki.
Molecular genetic analyses
DNA was isolated from whole blood using DNeasy Blood and Tissue Kit (Qiagen, Chatsworth, CA) according to manufacturer’s instructions. DNA extraction and genotyping were performed at Department for Functional Genomics, Center for Translational and Clinical Research, University of Zagreb School of Medicine, Croatia and at the Laboratory for Molecular Neuropsychiatry, Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia. COMT Val108/158Met (rs4680) and DBH-1021C/T (rs1611115) polymorphisms were determined by ABI Prism 7300 Real time PCR System apparatus (Applied Biosystems, Foster city, California, USA), according to the procedures described by Applied Biosystems. The primers and probes were purchased from Applied Biosystems as TaqMan® Drug Metabolism Genotyping Assay (C_25746809_50 for COMT) or TaqMan® SNP Genotyping Assay (C_2535786_10 for DBH). All genotyping procedures were done blindly to clinical data. As a quality control for genotyping analyses, 5% of all samples were genotyped again.
Statistical analysis
Baseline data was reported using descriptive statistics. The results, expressed as means (x) ± standard deviation (SD) or medians, were evaluated with Sigma Stat 3.5 (Jandell Scientific Corp. San Raphael, California, USA) using one-way and repeated measures analysis of variance (ANOVA) and t test, or with Kruskal–Wallis ANOVA on ranks, Mann–Whitney test, and Wilcoxon Signed Rank Test, when the normality of the data failed. The Hardy–Weinberg analysis was used to test the equilibrium of the population. The differences in the genotype frequencies were evaluated using the Chi square test. The level of significance was set to p value less than 0.05.
Discussion
This study revealed that A carriers (i.e. the combined AA and AG genotype) of the COMT Val108/158Met polymorphism achieved significantly better HbA1c values after 52 weeks of treatment, compared to patients with T2DM carrying GG genotype. Although we expected to detect the association of COMT Val108/158Met and/or DBH-1021C/T polymorphism with the effectiveness of insulin detemir in achieving glucose control and body weight control, our results did not confirm any other significant association with BMI, body weight or fasting glucose values in patients with T2DM.
In our study, COMT Val108/158Met or DBH-1021C/T polymorphisms were not associated with T2DM. These data do not agree with a significant association found between COMT Val108/158Met and T2DM [
36]; however this association was detected in the Asian population, which could have additional confounding factors. In contrast to our results, in a large Caucasian population COMT Val108/158Met was associated with T2DM [
29], while DBH-1021C/T was significantly associated with T2DM and other clinical phenotypes responsive to peripheral sympathetic tone in a tissue-specific manner [
37], implying that present study lacked the statistical power or the needed sample size to detect these associations. However, this was not the main goal of the study, since we evaluated the possible association between COMT Val108/158Met and DBH-1021C/T polymorphisms and detemir-induced control of glucose control and body weight.
Subjects diagnosed with T2DM have a two- to fourfold higher chance of developing a serious cardiovascular outcome compared to those without diabetes [
38]. Weight gain is one of the major problems associated with insulin therapy. The vast majority of patients with T2DM are resistant to insulin and have associated significant cardiovascular risk factors. Hyperglycemia is considered as a principal cause of diabetic complications. Elevated blood glucose levels in patients with diabetes increases the rate of glycation, a nonenzymatic process of reducing sugars with free amino groups of proteins, lipids and amino acids [
39]. Glycated substances can be further modified in compounds called advanced glycation end products (AGE) which can trigger inflammatory reactions leading to atherosclerosis, kidney tissue damage, damage to small vessels in the eye and other major complications of diabetes acids [
39]. Glycation is a process whose significance has recently been revealed also in many other diseases, including neurodegeneration [
40]. Although at lowered blood glucose levels the sugars will be released from the amino groups, it is argued that most of the risk factors can be successfully controlled but the contribution of decreasing hyperglycemia is lower than expected [
41,
42]. Increment of 1% in HbA1c increased the risk of cardiovascular disease mortality by 53% in type 1 diabetic patients, but only by 7.5% in patients with T2DM [
41,
42]. Based on those data, it can hardly be expected that lowering of HbA1c of 1–2% alone is sufficient to significantly decrease the mortality risk in people with T2DM [
41,
43]. On the other hand, weight loss represents one of the main goals of therapy in overweight patients with T2DM [
44]. Clinical studies demonstrate that therapeutic benefit rises with increasing weight loss, but even losses as low as 0.45–4 kg have positive effects on metabolic control, cardiovascular risk factors and mortality rates [
44]. In the present study, at the end of the 52 week of treatment, the main cardiovascular risk factors were significantly reduced for patients with T2DM. There was a significant decline in mean HbA1c value and in mean fasting plasma glucose value, which corresponds to the fundamental function of insulin. We did not observe weight gain which could be expected due to insulin therapy. These data agree with the previous known weight sparing effect of insulin detemir in comparison to other basal insulins [
33,
45]. Zafar et al. [
45] showed a dose-dependent weight gain of patients treated with insulin detemir. Since our patients had adjusted doses of detemir and aspart insulin, according to the glucose profile, it was not possible to detect a correlation between weight gain and insulin detemir dose. However, our results revealed that the group with the smallest BMI (BMI < 27) at baseline needed significantly lower doses of insulin detemir than other groups to obtain adequate glycemic control.
Some of the known cardiovascular risk factors, such as obesity and hypertension, are in part genetically determined, but the entire array of specific genes remains unidentified [
46]. Our results showed different patterns of weight change and differences in achieving adequate glucose control in patients treated for 52 weeks with insulin detemir. Since COMT Val108/158Met and/or DBH-1021C/T polymorphisms are implicated in cardiovascular, sympathetic, and endocrine pathways [
37,
47], we expected that treatment induced differences in weight change and glucose control might be associated with these polymorphisms. In line with other data that failed to show an association of COMT polymorphisms with weight, BMI, or obesity risk [
48,
49], no significant association between COMT Val108/158Met or DBH-1021C/T genotypes and the change in body weight was detected. Still, our data showed that patients with TT genotype of the DBH-1021C/T or with AA genotype of the COMT Val108/158Met achieved a slight BMI decline, since there was a trend that did not reach the level of statistical significance. These results are partly consistent with previous reports showing an association between GG genotype with a fat-BMI [
29], and with a slight decrease in percentage of body fat in AA carriers [
49]. Although an association of COMT Val108/158Met genotypes with abdominal obesity and high blood pressure was found in Swedish men, connecting AA genotype with a higher risk of abdominal obesity, they failed to find a significant correlation to BMI [
27]. These results only underline the ambiguous impact of COMT polymorphism on obesity.
On the other hand, our results showed that patients with best glycemic response were predominantly COMT Val108/158Met A carriers (i.e. carriers of the combined AA and AG genotypes). They achieved significantly better HbA1c values after the 52-week treatment compared to patients carrying the GG genotype, pointing to the fact that presence of one or two A allele of the COMT Val108/158Met could be associated with a better response to insulin detemir therapy. This finding differs from the data from a recent study that reported an association of the G allele with lower values of HbA1c [
50], but is partly consistent with the results from a male obesity study in Denmark in which the GG genotype was associated with impaired glucose tolerance and high fat BMI [
29,
50].
In our study, and in line with previous data [
51], DBH-1021C/T polymorphism was not associated with changes in BMI values, body weight, fasting plasma glucose, or HbA1c in T2DM patients, or with BMI, body weight and fasting glucose values in healthy controls. A preclinical study showed that DBH deficient mice exhibit hyperinsulinemia, lower plasma glucose levels, and insulin resistance [
52]. However, in our study there was no association between fasting glucose levels and DBH-1021C/T and/or COMT Val108/158Met polymorphisms.
Estrogen regulates COMT activity [
53], and women have lower COMT enzymatic activity than men and genotype effect was more pronounced in males than in females [
25]. In agreement with our previous results including 1058 healthy Caucasian subjects [
54], a lack of gender dependent differences in the COMT Val108/158Met genotype frequency was detected in healthy controls, or in patients with T2DM (present study); and genotype effect was similar in both sexes. The differences with previous study [
25], might be explained by the different diagnoses, different number and different ethnicities of subjects included.
In line with well-known effects of insulin detemir as a well-tolerated and effective long acting insulin [
55], our data confirmed its highly beneficial weight sparing effect, especially in overweight patients, which is consistent with previous studies [
34,
45], showing that treatment with insulin detemir was associated with less pronounced weight gain.
In conclusion, our results revealed that the presence of one or two A allele of the COMT Val108/158Met was associated with improved glycemic response, since the carriers of the combined AA and AG genotypes achieved significantly better HbA1c values after the 52-week insulin detemir treatment compared to patients carrying the GG genotype. These data suggest a protective effect of the COMT Val108/158Met A allele and a better response in A carriers to insulin detemir therapy. As far as we are aware, this is the first study to reveal a lack of significant association between DBH-1021C/T and effectiveness of insulin detemir in achieving glucose control as well as body weight control.
Authors’ contributions
TB, FB, NP and TO designed the study concept and planned the experiments. TB, AS and LSD selected and clinically classified patients and controls, and collected clinical data. MNP, KGJ and AB conducted the experiments. AB analysed and interpreted patient data. TB and AB were major contributors in writing the manuscript with support from FB and NP. FB, NP and TO were involved in critical revision of the manuscript. All authors provided critical feedback and helped shape the research, analysis and manuscript. All authors read and approved the final manuscript.