Patients infected with HIV are surviving longer because of antiretroviral therapy (ART). Consequently, there has been an increased awareness of adverse events related to ART, particularly metabolic complications such as insulin resistance [1,2]. The cause of insulin resistance includes patient-level factors, HIV infection, certain protease inhibitors , and thymidine analogs . Insulin resistance contributes to the development of type 2 diabetes mellitus and cardiovascular disease among HIV-infected patients.
Deficiency of vitamin D, the major steroid hormone involved in calcium homeostasis and bone metabolism, has been associated with impaired β-cell function and insulin resistance in humans [5,6]. In HIV-uninfected patients, vitamin D deficiency (25-hydroxyvitamin D <20 ng/ml) is a risk factor for both type 2 diabetes mellitus and metabolic syndrome [7–9]. However, in the setting of HIV infection, the clinical consequences of vitamin D deficiency, which has been reported to have a prevalence ranging from 12 to 86% [10–16], remain unclear. In particular, the association between vitamin D deficiency and type 2 diabetes mellitus and metabolic syndrome among HIV-infected patients is not known.
Our primary objective was to determine whether a low serum concentration of 25-hydroxyvitamin D was associated with type 2 diabetes mellitus among HIV-infected patients. We hypothesized that a low 25-hydroxyvitamin D level was an independent risk factor for type 2 diabetes mellitus in HIV. Our secondary objective was to determine if vitamin D deficiency among HIV-infected patients was associated with the metabolic syndrome, for which the underlying mechanism is considered to be insulin resistance. We hypothesized that individuals with HIV and low serum concentrations of 25-hydroxyvitamin D would be more likely to have metabolic syndrome which has been linked to higher cardiovascular risk, in comparison to individuals with higher serum concentrations of 25 hydroxyvitamin D levels. Confirmation of these associations would identify hypovitaminosis D as a potentially modifiable cardiovascular risk factor among HIV-infected patients.
Study design and participants
We performed a cross-sectional study among participants enrolled in the Modena HIV Metabolic Clinic Cohort, a prospective cohort study initiated in September 2004 to evaluate metabolic alterations among HIV-infected patients followed at the Metabolic Clinic of the University of Modena and Reggio Emilia School of Medicine (Modena, Italy) . HIV-infected patients with metabolic complications are referred to this clinic for further evaluation. Patients have laboratory-confirmed HIV, provide informed consent, and complete a standardized questionnaire that collects demographic, medical, and HIV data at enrollment. Laboratory evaluation at enrollment includes blood for fasting lipids, fasting glucose, 25-hydroxyvitamin D, HIV RNA, CD4 cell count, and hepatitis status.
All patients enrolled in the cohort between 1 January 2005 and 31 January 2008 who had documented 25-hydroxyvitamin D level were eligible for inclusion. For our primary objective, we included patients with a 25-hydroxyvitamin D level and either fasting serum glucose level or documented type 2 diabetes mellitus diagnosis and use of insulin or an oral antidiabetic medication. For our secondary objective, we included patients with a 25-hydroxyvitamin D level and presence or absence of the metabolic syndrome diagnosis. This study was reviewed and approved by the Institutional Review Boards of the University of Modena and University of Pennsylvania.
Main study outcomes
The primary outcome was type 2 diabetes mellitus, defined as a fasting glucose at least 126 mg/dl , a physician's diagnosis of type 2 diabetes mellitus, and/or receipt of an oral antidiabetic agent or insulin.
As a secondary outcome, we evaluated the metabolic syndrome, defined by the presence of any three of the following five characteristics according to the National Cholesterol Education Program's Adult Treatment Panel III report: abdominal obesity based on waist circumference (>102 cm in men and >88 cm in women), triglycerides at least 150 mg/dl, high-density lipoprotein (HDL) below 40 mg/dl for men and 50 mg/dl for women, blood pressure at least 130/85 mmHg, and fasting glucose at least 110 mg/dl .
Demographic (age, sex); clinical [height, body weight, blood pressure, history of type 2 diabetes mellitus (as defined above)]; and HIV data (duration of HIV diagnosis, antiretroviral use and duration) were recorded at patient enrollment.
Blood was drawn from all patients after at least 8 h of fasting for determination of HIV RNA (Abbott RealTime HIV-1 assay; Abbott Laboratories; lower limit of detection: 50 copies/ml); CD4 T-lymphocyte count; hepatitis C virus (HCV) antibody (anti-HCV; Abbott HCV EIA 3.0 enzyme immunoassay; Abbott Laboratories); glucose (Rapidpoint 400 analyzer; Bayer); total cholesterol (Cholesterol/HP; Roche); triglyceride (Triglycerides/GPO; Roche); HDL (HDL-C plus 3rd generation; Roche); and 25-hydroxyvitamin D (DiaSorin 25-hydroxyvitamin D chemiluminescence immunoassay; Stillwater, Minnesota, USA). Vitamin D deficiency was defined as a serum 25-hydroxyvitamin D level less than 20 ng/ml (50 nmol/l). Vitamin D insufficiency was defined as 25-hydroxyvitamin D 30 ng/ml or less and at least 20 ng/ml .
Differences between groups were assessed using Student's t tests for continuous variables and chi-squared tests for categorical variables. Multivariable logistic regression determined unadjusted and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) of type 2 diabetes mellitus and metabolic syndrome between patients with and without low vitamin D. Potential confounding variables evaluated included age, sex, body mass index, vitamin D supplementation, season of vitamin D measurement, thymidine analogs, specific protease inhibitors such as lopinavir, and hepatitis C virus (HCV) co-infection. Backward elimination procedures removed nonsignificant potential confounders from adjusted models. All analyses were performed using STATA 10.0 (College Station, Texas, USA).
Vitamin D status
The mean serum concentration of 25-hydroxyvitamin D was 19 ng/ml (95% CI 18–20 ng/ml). A total of 64% were vitamin D deficient (25-hydroxyvtitamin D <20 ng/ml), and an additional 20% of the patients had vitamin D insufficiency. Among 1405 patients with available information regarding supplementation, 93 (7%) were taking a vitamin D supplement (7500 IUs vitamin D3 weekly).
Association between 25(OH) Vit D levels and type 2 diabetes mellitus
Mean 25-hydroxyvitamin D levels among type 2 diabetes mellitus patients were lower than in individuals without type 2 diabetes mellitus (Table 1). After adjusting for vitamin D supplementation, sex, age, body mass index and HCV co-infection, vitamin D deficiency was associated with type 2 diabetes mellitus (adjusted OR 1.85; 95% CI 1.03–3.32; P = 0.038) (Table 2).
Association between 25-hydroxyvitamin D levels and metabolic syndrome
Overall among 1687 participants, 412 (24%) met the definition of metabolic syndrome. Mean 25-hydroxyvitamin D levels among participants with metabolic syndrome were lower than in those without metabolic syndrome [17 (±12) versus 20 (± 10) ng/ml; P < 0.001]. The association between vitamin D deficiency was not significant after adjusting for age, sex, body mass index, and vitamin D supplementation (Table 2).
Our study identified a high prevalence of vitamin D deficiency among HIV-infected patients. Patients with type 2 diabetes mellitus in this study had lower 25-hydroxyvitamin D levels compared with individuals without type 2 diabetes mellitus, although both groups met criteria for vitamin D deficiency. We also demonstrated that vitamin D deficiency was independently associated with type 2 diabetes mellitus in this population. However, we did not find an association between vitamin D deficiency and metabolic syndrome.
The prevalence of vitamin D deficiency among the HIV-infected patients in this sample was similar to that reported by Garcia Aparicio et al.  among 30 HIV-infected men in Spain (86%). The prevalence of vitamin D deficiency in our population was higher than in several previous studies, which reported prevalence of 12–47% [12–16]. In contrast, a large Italian study of more than 6000 outpatients and inpatients reported a 52% prevalence of vitamin D deficiency . The higher prevalence of vitamin D deficiency in this study may be related to the northern geographic location of Modena (44°N) which may lead to more indoor activities and less sun exposure.
Several cross-sectional studies have shown that low serum 25-hydroxyvitamin D concentration is associated with impaired glucose tolerance in HIV-uninfected patients [7,8]. In the Nurses' Health Study, Pittas et al.  demonstrated an inverse association between vitamin D supplementation and the development of type 2 diabetes mellitus. A combined daily intake of at least 1200 mg of calcium and 800 IUs of vitamin D was associated with a 33% lower risk of type 2 diabetes mellitus. The findings of our study are consistent with those of HIV-uninfected individuals and suggest that the association between vitamin D deficiency and type 2 diabetes mellitus is also present among HIV-infected individuals.
In our study, patients with metabolic syndrome had lower serum 25-hydroxyvitamin D levels. However, we did not find an association between vitamin D deficiency and metabolic syndrome.
The role of HIV and ART in vitamin D deficiency is complex. HIV might affect α-hydroxylation of 25-hydroxyvitamin D in the kidneys to its active form 1, 25-dihydroxyvitamin D . Protease inhibitors and non-nucleoside reverse transcriptase inhibitors utilize the cytochrome p450 enzyme, which is also the site of hydroxylation of vitamin D. Thus, certain antiretrovirals may also predispose to vitamin D deficiency . If individuals with HIV are at a higher risk of developing vitamin D deficiency due to HIV and ART, then the consequences of vitamin D deficiency, such as type 2 diabetes mellitus, may be more prevalent among HIV-infected individuals, as shown by our results.
Additionally, inflammation has been shown to play a key role in the development of type 2 diabetes mellitus and metabolic syndrome. Vitamin D has anti-inflammatory effects among HIV-uninfected individuals, which may explain its protective role in type 2 diabetes mellitus and cardiovascular disease . Vitamin D may lower the risk of type 2 diabetes mellitus and metabolic syndrome in HIV by reducing the heightened inflammation associated with HIV infection .
Our study had several limitations. First, since we used a cross-sectional design, we cannot establish whether the relationship between vitamin D deficiency and type 2 diabetes mellitus is causal. A small prospective study by van Den Bout et al.  evaluated the impact of vitamin D supplementation on insulin sensitivity in 20 HIV-infected patients. At 24 weeks insulin sensitivity was decreased but by 48 weeks insulin sensitivity returned to baseline . Further larger prospective studies are needed to evaluate whether vitamin D deficiency may be a modifiable risk factor for the development of type 2 diabetes mellitus.
Second, our definition of type 2 diabetes mellitus may have resulted in outcome misclassification. Patients with type 2 diabetes mellitus with well controlled fasting blood glucose levels who failed to report antidiabetic medication use may have been misclassified as not having type 2 diabetes mellitus. However, such outcome misclassification would have caused us to underestimate the association between vitamin D deficiency and type 2 diabetes mellitus.
Lastly, participants were from Italy, a homogeneous population, potentially limiting the generalizability of our results.
In summary, we demonstrated that vitamin D deficiency was independently associated with type 2 diabetes mellitus in HIV. Future studies should examine whether vitamin D supplementation can prevent or treat type 2 diabetes mellitus in HIV and possibly reduce complications associated with HIV infection and its treatment.
Z.S. received support from the National Institute of Allergy and Infectious Diseases (T32 AI055435-06) and Penn Center for AIDS Research (P30 AI 045008).
S.S.S. received support from the National Institute of Allergy and Infectious Diseases (K01 AI73729) and the Robert Wood Johnson Foundation under its Physician Faculty Scholar Program. V.L.R. received support from the National Institute of Allergy and Infectious Diseases (K01 AI07001). P.T. received support from the AIDS Clinical Trial Group Grant from the National Institute of Health (AI069467). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Robert Wood Johnson Foundation.
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Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
insulin resistance; metabolic syndrome; type 2 diabetes mellitus; vitamin D deficiency