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Low Vitamin D Levels Correlate With the Proinflammatory State in Type 1 Diabetic Subjects With and Without Microvascular Complications

Sridevi Devaraj PhD, DABCC, FACB, CCRP, Jung-Mi Yun PhD, Catherine R. Duncan-Staley, Ishwarlal Jialal MD, PhD
DOI: http://dx.doi.org/10.1309/AJCPJGZQX42BIAXL 429-433 First published online: 1 March 2011

Abstract

Epidemiologic studies link vitamin D deficiency to onset of type 1 diabetes mellitus (T1DM). T1DM exhibits increased inflammation, which is pronounced with microvascular complications (T1DM-MV). However, there are a paucity of data on vitamin D in T1DM-MV in relation to biomarkers of inflammation, and this formed the aim of the study.

Healthy control subjects (n = 36), patients with T1DM (n = 24), and patients with T1DM-MV (n =26) were recruited. Serum vitamin D levels, monocyte toll-like receptor (TLR) 2 and TLR4 expression and nuclear factor-κB (NFκB) activity were assessed.

Patients with T1DM and T1DM-MV were significantly vitamin D deficient compared with control subjects (P < .01). There was a significant negative correlation between vitamin D levels and high-sensitivity C-reactive protein, NFκB activity, and TLR4 expression (P < .05). Preincubation with vitamin D significantly decreased lipopolysaccharide-activated TLR4 expression and cytokine levels in monocytes (P < .05).

Low vitamin D levels may contribute to increased inflammation in T1DM. Future studies will elucidate the immunomodulatory effects of vitamin D in decreasing vascular risk in this population.

Key Words:
  • Vitamin D
  • Diabetes
  • Inflammation

Epidemiologic studies suggest a link between vitamin D deficiency in early life and the later onset of type 1 diabetes (T1DM).13 T1DM is associated with an increased risk of vascular complications, and patients with T1DM with proteinuria and/or retinopathy have a significantly increased risk of fatal coronary artery disease.4 Studies have explored the relationship of vitamin D concentrations with these complications; however, many of these have not been studied in persons with diabetes.

Wang et al5 studied participants of the Framingham Offspring study (n = 1,739) who did not have cardiovascular disease at the time of enrollment. Levels of 25-hydroxyvitamin D (25-OH vitamin D) of less than 15 ng/mL (37 nmol/L) were associated with a greater risk for cardiovascular events after adjustment for cardiovascular risk factors and renal function (1.62; 95% confidence interval [CI], 1.11–2.36; P = .01). A recent meta-analysis of 18 randomized, controlled trials for vitamin D noted that intake of ordinary supplemental doses of vitamin D (300–2,000 IU) was associated with a reduced risk of mortality (relative risk, 0.93; 95% CI, 0.87–0.99).6 Chonchol and Scragg7 studied the relationships between serum levels of 25-OH vitamin D, insulin resistance, and kidney function in National Health and Nutrition Examination Survey (NHANES) III participants (n = 14,679). The level of 25-OH vitamin D was significantly lower in people with severely decreased glomerular filtration rate when compared with people with normal kidney function. In addition, people in this study with higher levels of 25-OH vitamin D had decreased insulin resistance (homeostasis model of assessment-insulin resistance). Svoren et al8 demonstrated significant vitamin D deficiency in youth (age <18 years) with type 1 diabetes, while Bierschenk et al9 demonstrated low levels of vitamin D in healthy control subjects and people with type 1 diabetes in Florida, with no significant differences between the groups.

Vitamin D has also been shown to have several immunomodulatory effects in vitro such as decreasing C-reactive protein (CRP) levels and abrogation of nuclear factor-κB (NFκB) activity, cytokines, and toll-like receptor (TLR) 4 expression.10 We and others have demonstrated that patients with T1DM exhibit increased inflammation as evidenced by increased plasma CRP levels and increased monocyte activity and increased TLR2 and TLR4 expression, and these are more pronounced in T1DM with microvascular complications (T1DM-MV).1113 However, there are a paucity of data examining the role of vitamin D in T1DM with complications and also examining its relationship with biomarkers of inflammation, and this formed the basis of this study. Herein we report on decreased levels of vitamin D in T1DM with and without complications compared with levels in healthy control subjects.

Materials and Methods

We recruited 24 patients with T1DM and 26 with T1DM-MV (onset <20 years and receiving insulin therapy since diagnosis; present age ≥18 years; and duration of diabetes ≥1 year) from the Diabetes and Pediatric Clinics at University of California Davis Medical Center, Sacramento, and by advertisements in the local newspaper. Microvascular complications included retinopathy and nephropathy. None of the patients were receiving metformin (Glucophage, Bristol-Myers Squibb, Princeton, NJ) and/or the thiazolidinediones, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, or statins, as described previously.11 Other exclusion criteria were as follows: mean glycated hemoglobin (HbA1c) during the last year more than 10%; inflammatory disorders, eg, rheumatoid arthritis; macrovascular complications such as stroke or myocardial infarction, abnormal liver, renal, or thyroid function; malabsorption; steroid therapy; smoking, abnormal CBC, and alcohol consumption of more than 1 oz/d; consumption of fish oil capsules (>1 g/d); and “chronic” high-intensity exercisers.

Healthy control subjects (n = 36), older than 18 years, were included if they had a normal CBC; no family history of diabetes or other chronic diseases; normal kidney, liver, and thyroid function; and a fasting plasma glucose level less than 100 mg/dL (5.6 mmol/L). Healthy control subjects and patients with T1DM and T1DM-MV were matched for age (within 10 years), sex, and race. Exclusion criteria were as described previously.11 Informed consent was obtained from participants in the study, which was approved by the institutional review board at the University of California Davis. After a history and physical examination, fasting blood samples (30 mL) were obtained.

High-sensitivity (hs) CRP levels in serum were assayed using the Beckman LxPro autoanalyzer (Beckman Coulter, Brea, CA). Total calcium and phosphate levels were also assessed in the clinical laboratory. Vitamin D levels were measured using the LIAISON 25-OH Vitamin D TOTAL assay (DiaSorin, Saluggia, Italy), which is an antibody-based vitamin D chemiluminescent assay that measures total 25-OH vitamin D, including D2 and D3 metabolites. Interassay and intra-assay coefficients of variation of the vitamin D assay were less than 8.5%.

Mononuclear cells were isolated from fasting heparinized blood by Ficoll Hypaque centrifugation, followed by magnetic separation of monocytes using the depletion technique (Miltenyi Biotech, Auburn, CA), as described previously.11 NFκB p65 activity was examined and expressed per milligram of cell protein, as described previously.1113 TLR2 and TLR4 expression was examined by flow cytometry, as described previously.13

To examine the relationship between vitamin D and TLRs, we incubated monocytes with 1,25-dihydroxyvitamin D3 (0.1 μmol/L) for 24 hours and then activated the cells with lipopolysaccharide (LPS; 100 ng/mL) and examined TLR4 expression by flow cytometry and cytokine analyses by multiplex array.

Statistical analyses were performed using SAS software (SAS Institute, Cary, NC). Data are expressed as mean ± SD for parametric data and as median and interquartile range for nonparametric data. Following analyses of variance, parametric data were analyzed using paired, 2-tailed t tests and nonparametric data using Wilcoxon signed rank tests. The level of significance was set at a P value less than .05. The Spearman rank correlation was computed to assess association between variables.

Results

Baseline subject characteristics are given in Table 1. There were no significant differences in age, body mass index, and male/female ratio in the control, T1DM, and T1DM-MV groups. In addition, there were no significant differences in the lipid profile, including low- and high-density lipoprotein cholesterol (data not shown). As expected, levels of glucose and HbA1c were significantly higher in the T1DM and T1DM-MV groups compared with the control group. Also, levels of hs-CRP were significantly increased in the T1DM and T1DM-MV groups compared with the control group, and this was more pronounced in the T1DM-MV group as reported previously11 (Table 1).

Monocyte surface expression of TLR2 and TLR4 was significantly up-regulated in the T1DM-MV group compared with the T1DM and control groups, as reported previously13 (Table 1). Furthermore, downstream signaling of TLR, ie, NFκB activity, was significantly increased in resting monocytes from the T1DM-MV group compared with T1DM and control groups (P < .005).13

View this table:
Table 1

The novel data we report here are the levels of vitamin D in T1DM with and without microvascular complications compared with control subjects. As shown in Figure 1, patients with T1DM and T1DM with complications were significantly vitamin D deficient compared with healthy control subjects (P < .01). Although decreased compared with the control group, the levels of vitamin D were not significantly decreased in the T1DM-MV group compared with the T1DM group. While 17% (6/36) of control subjects were also deficient and had values less than 20 ng/mL (50 nmol/L), 29% (7/24) and 35% (9/26) of the T1DM and T1DM-MV groups, respectively, had vitamin D values less than 20 ng/mL (50 nmol/L). There were no significant changes in total calcium or phosphate levels in patients with T1DM compared with control subjects (data not shown).

Because vitamin D seems to have immunomodulatory activity and T1DM has been shown to be a proinflammatory state, we examined correlations between low vitamin D levels and increased biomarkers of inflammation in T1DM, ie, hs-CRP, mononuclear NFκB, and monocyte TLR expression. As shown in Table 2, there was a significant negative correlation between vitamin D levels and hs-CRP (r = −0.67; P < .01); NFκB activity (r = −0.49; P < .05); and TLR4 expression (r = −0.37; P < .05); there was no significant correlation with TLR2 expression.

To examine the relationship between vitamin D and TLRs, we incubated monocytes with 1,25-dihydroxyvitamin D3 (0.1 μmol/L) for 24 hours and then activated cells with LPS and examined TLR4 expression and cytokine release. As shown in Table 3, preincubation with vitamin D significantly decreased LPS-activated TLR4 expression and cytokine levels in these monocytes (P < .05).

Figure 1

Serum vitamin D levels in 36 healthy control subjects, 24 patients with type 1 diabetes mellitus (T1DM), and 26 patients with T1DM with microvascular complications (MV). Data are expressed as median and interquartile range. * P < .05 compared with healthy control subjects. Values are given in conventional units; to convert to Système International units (nmol/L), multiply by 2.496. OH, hydroxy.

View this table:
Table 2

Discussion

T1DM is associated with increased microvascular complications. Inflammation has a crucial role in vascular complications. T1DM is a proinflammatory state characterized by increased levels of circulating biomarkers of inflammation such as hs-CRP. We have previously shown increased systemic and cellular inflammation in T1DM-MV compared with T1DM1113 as evidenced by increased hs-CRP, increased monocyte cytokines, increased monocytic expression of TLR2 and TLR4, and their downstream signaling such as increased NFκB activity. Several lines of evidence from epidemiologic studies and in vitro studies indicate that T1DM is associated with decreased vitamin D levels79 and that low vitamin D levels may contribute to increased risk of microvascular and macrovascular complications. However, there are no studies examining the vitamin D level in T1DM-MV compared with age- and sex-matched T1DM cases and its contribution to the accentuated proinflammatory state of T1DM. In this report, we provide novel data on significantly decreased levels of vitamin D in T1DM compared with control subjects and significant negative associations with biomarkers of inflammation such as hs-CRP, NFκB activity, and TLR4 expression.

While the relationship of vitamin D to complications has not been studied in T1DM, this association has been studied in nondiabetic subjects and reported. In the Framingham Offspring study,5 participants did not have cardiovascular disease at the time of enrollment. At baseline, the mean 25-OH vitamin D level was 19.7 ng/mL (49 nmol/L), and 37% of persons had levels less than 15 ng/mL. At a mean follow-up of 5.4 years, 120 participants had experienced a cardiovascular event. Levels of 25-OH vitamin D of less than 15 ng/mL (37 nmol/L) were associated with a greater risk for cardiovascular events after adjustment for age and sex, other cardiovascular risk factors, and renal function (relative risk, 1.62; 95% CI, 1.11–2.36; P = .01). When models were created to examine the following categories of vitamin D concentrations (≥15, 10–15, and <10 ng/mL [37, 25–37, and 25 nmol/L]), the hazards ratio demonstrated a linear trend (1.00, 1.53, and 1.80, P = .01) for increased cardiovascular risk. The authors suggested that correction of the vitamin D deficiency might be important for the prevention of cardiovascular disease. A recent meta-analysis of 18 randomized, controlled trials for vitamin D noted that intake of ordinary supplemental doses of vitamin D (300–2,000 IU) was associated with a reduced risk of mortality (relative risk, 0.93; 95% CI, 0.87–0.99).6 In the NHANES III cohort,7 the level of 25-OH vitamin D was significantly lower in people with a severely decreased glomerular filtration rate compared with people with normal kidney function. All of the aforementioned studies indicate that low vitamin D levels are associated with increased cardiovascular risk.

View this table:
Table 3

In the present study, we showed significantly decreased levels of vitamin D in T1DM with and without microvascular complications. However, despite lower values in T1DM-MV, these were not significantly lower compared with T1DM, and this relationship needs to be confirmed in larger studies. Previously, European studies have reported decreased 25-OH vitamin D and 1,25-dihydroxyvitamin D3 in patients with newly diagnosed T1DM and in type 2 diabetes mellitus.1,2,6 A recent study failed to show the association of decreased vitamin D in patients with T1DM in Florida; however, despite the sun-rich environment, all subjects, including healthy control subjects, were vitamin D insufficient, confounding analyses of differences between control subjects and patients with T1DM.9

Vitamin D has been reported to be immunomodulatory.10 In this study, we report in T1DM cases a significant negative association between vitamin D levels and hs-CRP, TLR4 expression, and NFκB activity. Previously, negative correlations between hs-CRP and 25-OH vitamin D levels have been reported in some studies of kidney transplant and obese subjects.14 Also, 1,25-dihydroxyvitamin D3 in vitro has been shown to decrease LPS-induced cytokine release and NFκB activity from monocytes.15,16 In addition, in monocytes, vitamin D treatment (1,25-dihydroxyvitamin D3) significantly reduces the response/immune activation of cells in response to TLR2 and TLR4 agonists.17 Since T1DM and T1DM-MV have increased monocyte TLR2 and TLR4 expression and increased NFκB activity, we examined relationships with low vitamin D status. The significant negative correlation between vitamin D levels and biomarkers of inflammation shown in this study indicate that low vitamin D levels may contribute to the increased inflammation in T1DM. These data are supported by our in vitro studies on the efficacy of vitamin D in decreasing TLR4 and cytokine release from monocytes. Future studies will provide mechanistic insights into this association, and clinical trials using vitamin D in T1DM will be important to elucidate the immunomodulatory effects of vitamin D in decreasing vascular risk in this population.

CME/SAM

Upon completion of this activity you will be able to:

  • list findings that provide evidence that type 1 diabetes is a proinflammatory state.

  • understand vitamin D levels in type 1 diabetes.

  • list specific immunomodulatory actions of vitamin D.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.

The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Questions appear on p 478. Exam is located at www.ascp.org/ajcpcme.

Acknowledgments

We thank Manpreet Kaur for technical assistance.

Footnotes

  • Supported by grants K24AT00596 and DK69801 from the National Institutes of Health, Bethesda, MD.

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