Adipokines such as leptin and adiponectin, specifically and highly expressed in human adipose cells, play an important role in the regulation of energy balance, insulin sensitivity, and atherogenesis 1–3. Low circulating adiponectin and high leptin levels are significantly correlated with endothelial dysfunction, increased intima media thickness, and progression of coronary artery calcification, independently of other cardiovascular risk factors 4–7. However, adverse atherogenic effects of leptin and beneficial effects of adiponectin could paradoxically disappear in people with advanced atherosclerosis 8,9. Therefore, the plasma leptin/adiponectin ratio (LAR) has been proposed as a preferential marker of atherosclerosis susceptibility compared with leptin and adiponectin alone 10,11.
Hp 2-2 diabetic patients represent a high-risk group for atherosclerotic disease. It has been demonstrated that the Hp 2-2 genotype is associated with a two-fold to five-fold increased risk for microvascular complications as well as with event rates for stroke, nonfatal myocardial infarction, heart failure, and total and cardiovascular mortality in diabetic patients 12–15. As vitamin D has antiatherogenic effects, we suggest that an inexpensive and safe intervention such as vitamin D treatment would provide cardiovascular protection to Hp 2-2 diabetic patients with high risk for cardiovascular disease.
The present study was designed to evaluate the long-term impact of high doses of vitamin D on LAR in diabetic individuals with different haptoglobin phenotypes.
Materials and methods
The study group consisted of 47 white patients with type 2 diabetes mellitus. Patients with a history of unstable angina, myocardial infarction (MI), stroke, or major surgery within 6 months preceding the study were excluded. Patients with unbalanced endocrine disease or any disease that might affect the absorption of medications were excluded, as were patients with plasma creatinine more than 3 mg/dl, elevation of liver enzymes to more than twice the upper normal limit, and calcium metabolism abnormalities.
Study participants were randomly assigned to two groups: group 1 received an oral supplementation of cholecalciferol (vitamin D3) daily at a dose of 1000 U/day and group 2 received placebo capsules containing microcrystalline cellulose. In addition, each group was divided into two subgroups by Hp phenotype: subgroup 1 included diabetic patients homozygous for allele 2 (the Hp 2-2 group) and subgroup 2 included patients heterozygous at the Hp locus, Hp 2-1, and patients homozygous for allele 1, Hp 1-1 (the non-Hp 2-2 group).
The patients were stabilized for 3 months before the study. This was intended to stabilize their condition and treatment in an effort to minimize treatment changes during the study. Patients unbalanced during the 3-month period were not included in the study. Concomitant medications such as oral glucose-lowering agents, insulin, inhibitors of the renin–angiotensin system, and statins did not change in dose during the study.
The study was approved by the Institutional Review Board, and all patients signed an informed consent form before participation.
Blood sampling for complete biochemical and metabolic parameters, including fasting glucose, lipid profile, HbA1C, insulin, CRP, 25-OH vitamin D, leptin, and adiponectin, was performed. Plasma leptin was analyzed using Luminex xMAP technology [Linco Research Inc., St. Charles, Missouri, USA; Lincoplex panel B (HADK-2-61K-B)]. Adiponectin was determined by a commercial sandwich enzyme immunoassay technique (R&D Systems, Minneapolis, Minnesota, USA) (catalog number DRP300). Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated by the following formula: fasting plasma insulin (mU/ml)×fasting plasma glucose (mg/dl)/405. Haptoglobin phenotyping was performed by polyacrylamide gel electrophoresis according to established methods 16.
Analysis of data was carried out using SPSS 11.0 statistical analysis software (SPSS Inc., Chicago, Illinois, USA). For continuous variables, descriptive statistics were calculated and are reported as mean±SD. Normality of distribution of continuous variables was assessed using the Kolmogorov–Smirnov test (cutoff at P<0.01). Categorical variables such as sex and concomitant illnesses were described using frequency distributions and are presented as frequency (%). The t-test for independent samples was used to compare continuous variables by treatment group. All tests are two-sided and considered significant at a P-value less than 0.05.
Clinical and demographic characteristics of the four study groups are presented in Table 1. As can be seen, all groups were similar with respect to age, sex, BMI, and the presence of cardiovascular risk factors. Concomitant medications were similarly distributed in all groups at the initiation and termination of the study. Vitamin D therapy was generally well tolerated; one patient from the active treatment group withdrew because of diarrhea and the other because of weakness. In the control group, two patients discontinued follow-up because of prolonged hospitalization for respiratory infection and elective hospitalization for cholecystectomy. Two patients, both women, discontinued follow-up because of fracture (hip and radial). The reason for the rest of the dropouts was loss to follow-up.
Changes in hemodynamic and metabolic parameters in patients treated with vitamin D supplementation
Table 2 shows hemodynamic and metabolic parameters of patients treated with vitamin D supplementation for 12 months. The heart rate and systolic, diastolic, and mean arterial pressure did not change significantly during the 12-month treatment period in either subgroup. During the study, 25-OH vitamin D level marginally increased in subgroup 1 (P=0.094) and significantly increased in subgroup 2 (P=0.007). LAR decreased significantly from 3.6±2.5 to 2.3±1.5% in patients with the Hp 2-2 phenotype (P=0.039). LAR tended to decrease in non-Hp 2-2 diabetic patients; however, this decrease did not reach statistical significance (P=0.185). As shown in Table 2, glucose homeostasis parameters such as fasting glucose, HbA1C, and HOMA-IR did not change during the study in either subgroup.
Changes in hemodynamic and metabolic parameters in the placebo group
As shown in Table 3, the heart rate and blood pressure level did not change significantly during the 12-month treatment period. No change was detected in adiponectin, leptin, and LAR in either subgroup of diabetic patients. 25-OH Vitamin D levels did not change during the treatment period. Glucose homeostasis parameters did not change during the study in either subgroup receiving placebo.
The present randomized, placebo-controlled study demonstrates that 1-year treatment with high doses of vitamin D was associated with a significant decrease in LAR in diabetic patients with the Hp 2-2 phenotype.
Although the beneficial effects of the activated form of vitamin D, 1,25(OH)2D3, on insulin sensitivity have been observed in animal models, results from human studies have been varied 17–19. Some authors have reported improvements in parameters of insulin secretion and/or resistance, whereas others have not observed any changes. Recently, it was suggested that abnormal glucose tolerance is a proinflammatory state causing changes in adipokine levels probably through the action of proinflammatory cytokines such as TNF-α and IL-1 20. Therefore, the anti-inflammatory action of vitamin D may modulate circulating adiponectin and leptin, which are two important adipokines with opposite effects on insulin sensitivity and inflammation.
Adiponectin and leptin emerged as an important link between obesity, insulin resistance, and atherosclerotic vascular disease. It was shown that low plasma adiponectin levels are significantly correlated with endothelial dysfunction, increased intima media thickness, and progression of coronary artery calcification independently of other cardiovascular risk factors 5,6. Leptin is also involved in insulin sensitivity, angiogenesis, vascular and endothelial function, and myocyte proliferation 7,21. Recently, plasma LAR has been proposed as a preferential marker of atherosclerosis compared with leptin and adiponectin alone 22,23.
Consistent with previous reports that have observed beneficial effects of the antioxidant therapy in Hp 2-2 diabetic patients 12, the present study demonstrates that vitamin D supplementation may have provided cardiovascular benefit in high-risk diabetic patients such as Hp 2-2 phenotype patients who have a two-fold to five-fold increased risk for cardiovascular disease as compared with diabetic individuals without the Hp 2-2 genotype. Hp is an antioxidant protein that binds free hemoglobin with very high affinity and stability. Hemoglobin binding inhibits hydroxyl free radical formation and prevents the development of oxidative tissue damage that contributes to vascular complications in diabetic patients 24. Furthermore, Hp may influence the development of vascular complications by modulating immune and inflammatory reactions 25. Vitamin D has anti-inflammatory and immunomodulatory effects and, as such, may have an important role in providing protection against hemoglobin-driven oxidative tissue damage. The anti-inflammatory action of vitamin D may modulate the effect of proinflammatory cytokines on circulating adiponectin and leptin 26. Moreover, because the vitamin D receptor has been identified in preadipocytes, it is likely that 1,25-dihydroxyvitamin D may regulate the adipokine gene expression and modulate an adipose tissue inflammation. Consistent with previous reports, in the present study, we did not observe significant changes in glucose homeostasis parameters, suggesting that vitamin D mediates its beneficial effects through glycemic control-independent mechanisms.
Our study has several limitations. Our study included a small number of participants; larger studies are required to establish the beneficial vascular effect of vitamin D supplementation as well as its clinical impact on cardiovascular outcomes in the high-risk subgroups with type 2 diabetes mellitus.
Long-term treatment with high doses of vitamin D was associated with a decrease in LAR in diabetic patients with the Hp 2-2 phenotype. The findings of the present study justify future studies investigating the effects of vitamin D supplementation on cardiovascular outcomes in different subgroups of type 2 diabetic patients.
Conflicts of interest
There are no conflicts of interest.
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