Title

Comprehensive Review on Diabetes Associated Cardiovascular Complications - The Vitamin D Perspective

Running Title

Diabetes Associated Cardiovascular Complications - Vitamin D Perspective

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Comprehensive Review on Diabetes Associated Cardiovascular Complications - The Vitamin D Perspective

Durgarao Y1, *Poornima A Manjrekar1, Prabha Adhikari2, Chakrapani M3, Rukmini MSs1,

1Department of Biochemistry, Kasturba Medical College, Manipal Academy of Higher Education, Mangaluru, Karnataka, India. 2Department of Internal Medicine, Yenepoya University, Mangalore, Karnataka, India. 3Department of Internal Medicine, Kasturba Medical College, Manipal Academy of Higher Education, Mangaluru, Karnataka, India.

Corresponding AddressDr Poornima A Manjrekar,Professor, Department of BiochemistryKasturba Medical College, Manipal Academy of Higher Education, Mangaluru, Karnataka, India. Mobile no. +91 944903390Email: drpamanjrekar@gmail.com

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Abstract

Vitamin D, a steroid hormone is primarily known for its role in calcium and bone mineral homeostasis. Over the

years, vitamin D has been implicated in various non-skeletal diseases. The extraskeletal phenomenon can be

attributed to the presence of vitamin D receptors (VDRs) in almost all cells and identification of 1-α hydroxylase in

extrarenal tissues. The vitamin D deficiency (VDD) pandemic was globally reported with increasing evidence and

paralleled the prevalence of diabetes, obesity and cardiovascular diseases (CVDs). A dependent link was proposed

between hypovitaminosis D glycemic status, insulin resistance and also the other major factors associated with type

2 diabetes leading to CVDs. Insulin resistance plays a central role in both type 2 diabetes and insulin resistance

syndrome. These 2 disorders are associated with distinct etiologies including hypertension, atherogenic

dyslipidemia, and significant vascular abnormalities that could lead to endothelial dysfunction. Evidence from

randomised clinical trials and meta-analysis, however, yielded conflicting results. This review summarizes the role

of vitamin D in the regulation of glucose homeostasis with an emphasis on insulin resistance, blood pressure,

dyslipidaemia, endothelial dysfunction and related cardiovascular diseases and also underline the plausible

mechanisms for all the documented effects.

Keywords: Vitamin D, Diabetes, insulin resistance, dyslipidemia, hypertension, endothelial dysfunction and

cardiovascular diseases

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1 Introduction

Diabetes-associated cardiovascular complications are typically characterized by atherosclerosis and endothelial

dysfunction [1]. Type 2 diabetes mellitus (T2DM) confers a two- to the threefold higher risk of cardiovascular

disease (CVD) [2]. In diabetes individuals, 65-75 per cent of deaths are due to CVDs [3,4]. Among the 50%

world’s population with heart diseases, almost 25% would be Indians in the next 15 years [5]. It has been reported

that Type 2 diabetes mellitus (T2DM) and impaired glucose tolerance confer a three to eight-fold increase in

cardiovascular disease (CVD) risk [2]. T2DM associated abnormal lipid profile contributes to atherosclerosis which

alone is responsible for almost 75-80% of mortality [3]. More than 80% of deaths among diabetes individuals are

due to CVDs and two-thirds of them are because of coronary artery disease (CAD). This clearly suggests that the

“epidemic of T2DM is followed by an epidemic of diabetes-related cardiovascular disease (CVD)” [4]. In T2DM

individuals, hypovitaminosis D predict future macrovascular events [5].

Susceptibility to a metabolic syndrome characterized by impaired glucose tolerance, dyslipidemia, obesity,

hypertension, insulin resistance and T2DM are the known causes for the higher incidence of CVDs in Indians [6].

Diabetes individuals are 8 times more predisposed to heart and blood vessel diseases and 70–80% of mortality

among them is attributed to cardiovascular diseases. T2DM individuals are at risk forto an increased incidence and

accelerated development of atherosclerotic vascular lesions. Compared to non-diabetes individuals, cardiovascular

diseases such as myocardial infarction and stroke are 2 to 4 times more prevalent in them [7].

The effect of tight glycemic control on cardiovascular risk reduction in T2DM individuals is debatable, with many

clinical trials providing conflicting results [8-11]. The United Kingdom Prospective Diabetes Study (UKPDS)

revealed that the early intensive treatment of hyperglycemia within the first five years of T2DM lead to long-term

cardiovascular benefit, compared to patients in the conventional treatment group. On the contrary, intensive

glycemic control regimen results from three large clinical trials namely the Veterans Affairs Diabetes Trial (VADT),

Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation

(ADVANCE) and Action to Control Cardiovascular Risk in Diabetes (ACCORD) trials showed no cardiovascular

benefit. The difference in the outcomes was partly attributed to clinical characteristics of the population involved,

duration of diabetes and type of intensive intervention. The results ofEvidence from these trials proved proved that

hyperglycemia alone can is not the sole cause coronary artery disease (CAD), for CVD risk, but plays its role with

other risk in the merging of multiple factors that predispose T2DM individuals to [12]CAD. [12] Tthisus

underlinesning the role of non-glycemic risk factors in the prevention or treatment of CAD in them T2DM patients.

The same view has been recommended by both the American Diabetes Association and the American Heart

Association [13]. Hence, assessing and controlling the non-glycemic risk factors such as hypertension, dyslipidemia,

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obesity and nutrient deficiencies is vital in the prevention and management of cardio-metabolic risk in T2DM

individuals.

In recent years, the role of vitamin D, an essential fat-soluble nutrient in the regulation of non-skeletal disorders is

gaining prominence. This review is aimed at discussing vitamin D deficiency (VDD), a pandemic worldwide and its

association with diabetes-related cardiovascular diseases.

2 Vitamin D Deficiency (VDD) – surprisingly common

Vitamin D deficiency (VDD) is defined as a 25-hydroxyvitamin D [25 (OH)D] level of less than 20 ng/mL (50 nmol

/ liter) [14] insufficiency between 20-30ng/ml and sufficient when the levels are > 30ng/ml [15-17]. It has been

estimated that 1 billion people worldwide have VDD or insufficiency [14]. As cited in the literature, VDD is highly

prevalent in varying degrees among the Indian population As per the literature, there exists widespread prevalence to

varying degrees (50- 90%) of VDD in Indian population along with low dietary calcium intake [18-20]. According

to International Osteoporosis Foundation, in North India, 84% of pregnant women, 96% of neonates, 91% of healthy

school girls and 78% of the healthy hospital staff were found to have VDD [21]. On the other hand, in Ssouth India,

40% of males and 70% of females were found to be Vit D deficient [22,23].

Exposure to sunlight - UV-B radiation (λ=290-315 nm) is the limiting factor for the dermal synthesis of vitamin D.

Even at optimal sunlight exposure, dark-skinned individuals (high melanin) synthesize less Vit D compared to the

whites. Decreased bioavailability mainly occurs due to malabsorption syndromes and obesity (sequestration of Vit D

in adipose tissue results in low plasma concentration). Increased catabolism occurs through the induction of hepatic

cytochrome p450 enzymes. Certain classes of drugs like anti-epileptics, anti-retrovirals, immunosuppressants

increase hepatic degradation of Vit D. Decreased conversion of 25(OH)D to 1,25(OH)D by the renal 1-alpha-

hydroxylase due to renal dysfunction and excess urinary loss of vitamin D binding protein in chronic kidney disease

limiting the renal 1-alpha-hydroxylase activity and excess urinary loss of vitamin D binding protein. Thyroid

disorders namely hyperthyroidism and primary hyperparathyroidism enhance the metabolism of Vit D [23]. Besides

all the above-mentioned factors, with increasing age, the ability of skin to convert 7-dehydrocholesterol to vitamin

D decreases with increasing age thus predisposing individuals to VDD associated skeletal and non-skeletal diseases

[24,25].

3 Literature search methodology

For this narrative review, MEDLINE and PUBMED databases were primarily explored. Search terms used were

vitamin D (Vit D), prediabetes, obesity, inflammation, hyperglycemia, insulin resistance (IR), type 2 diabetes

(T2DM), dyslipidemia, endothelial dysfunction and cardiovascular diseases. Additional articles relevant to the

context were identified in google scholar and Scopus search engines for citation. The included studies were of

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various designs namely, cross-sectional, prospective randomized control trials, systematic reviews and meta-

analysis. We excluded studies with small sample size and those conducted in children, gestational diabetes and type

1 diabetes subjects.

4 Vitamin D and diabetes – an emphasis on insulin resistance (IR)

VDD is more prevalent among T2DM patients [26] and was found to be significantly higher compared to controls

[27,28]. Vitamin D has been suggested to play a its role in T2DM pathogenesis [29]. Insulin resistance (IR) is a

characteristic feature of T2DM and its attenuation may decrease the incidence of T2DM associated cardiovascular

disease [30]. In IR the insulin released by pancreatic β cells is not able to sensitize the target cells such as hepatic,

skeletal muscle and adipose tissues leading to hyperinsulinemia coupled with hyperglycemia. Hyperinsulinemia is

associated with glucose intolerance, obesity, hypertension and dyslipidemia [31] which together constitute the

“metabolic syndrome” [32] or Insulin resistance syndrome.

4.1 Pieces of evidence from in vitro and animal studies

Pancreatic β-cells possess vitamin D receptors and vitamin D-dependent calcium-binding proteins. Both intracellular

Ca2+ levels and 1,25-dihydroxy vitamin D have an influence on insulin secretion. Vit D has been shown to regulate β

cell calcium flux in vitro and in mouse models [33]. On the other hand, intracellular response to insulin can also be

affected by the changes in intracellular Ca2+ levels [34]. Hence vitamin D may be involved in the β-cell secretory

activity and in the modulation of tissue response to insulin. In vitro, culture studies by Zhou et al. [35] revealed the

development of insulin resistance and atrophic changes on myotubes incubated with free fatty acids (FFAs).

1,25(OH)2D3 antagonized the actions of FFAs leading to an amelioration of insulin-mediated glucose uptake in a

time and dose-dependent manner coupled with a complete prevention of atrophy. Additionally, in skeletal muscle

1,25(OH)2D also facilitated glucose uptake [36]. Experimental studies on insulin-secreting (INS-1E) β-cell-derived

cell line revealed that 1,25(OH)2D improves insulin secretion and FFA-induced insulin resistance in skeletal muscle

through PPAR-δ activation [37].

4.2 Data from Clinical studies

Epidemiological studies stated that VDD and insulin resistance are interlinked. This was further reinforced by the

identification of vitamin D response elements (VDRE) in the promoter region of the human insulin receptor gene

[38]. Few studies demonstrated an inverse relation between serum Vit D levels and HbA1C and also the functional

role of Vit D on glucose tolerance through the modulation of insulin secretion and insulin sensitivity [39,29].

Emerging evidence proposed a role for vitamin D in T2DM pathogenesis [40]. However, studies conducted on the

association of vitamin D with IR and particularly β - cell dysfunction have yielded conflicting results [41–46].

Retnakaran et al. [47] found a significant positive association between vitamin D and β-cell function, using

validated measures of β-cell function. In a study conducted on individuals at risk for T2DM, Kayaniyil et al. [48]

reported an independent association between serum 25(OH)D levels, insulin sensitivity and β-cell function.

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Cross-sectional studies provided conflicting results few studies demonstrated no association between serum 25-

OHD levels and indices of insulin resistance [49,50] whereas othersand some reported varying degrees of

association [42,45,46,48,51,52,53-56,]. After adjustment forwith adiposity, the associations were not significant in

some studies [45,51,52]. However, these studies were limited by their heterogeneous population with a varied

degree of glucose levels [49,51,54,57] and secondary hyperparathyroidism [50]. Hence it is inappropriate to draw

definite conclusions from these studies and there is a need for further studies in defined cohorts. Evidence from

earlier studies demonstrated that T2DM individuals with VDD are more likely to have elevated HbA1c compared to

the Vit D sufficient counterparts [58,59-61]. Jorde and Figenschau [62] study demonstrated that T2DM individuals

with normal serum 25(OH) D levels when supplemented with Vit D for six months, did not show a significant

difference in HbA1c levels from baseline. Similar findings were reported by another study [63]. A significant

negative association was reported between serum 25(OH) D and HbA1c concentration, suggesting that VDD is

linked to poor glycemic control [27]. This was consistent with a recent study that demonstrated an inverse

relationship between VDD and elevated HbA1c levels in T2DM subjects [64]. In a study conducted by

Ghavamzadeh et al. [65], aAlthough there was no difference in post-intervention and baseline mean HbA1c levels,

Ghavamzadeh et al. [65] found a significant reduction in HbA1c levels of vitamin D supplemented group had

significantly low HbA1c levels compared to placebo at the post-intervention stage. This was suggestive of vitamin

D3 effect on diabetes control.

Recently published systematic reviews regarding the effect of vitamin D supplementation on glycemic control in

T2DM individuals revealed no benefits [66-68]. The observed effect could be attributed to the heterogeneity across

studies, duration of treatment and the baseline 25(OH)D status of the population included. Furthermore, Lee et al.

[69] found the favourable effects of vitamin D particularly on the glycemic profile (fasting glucose) in studies that

corrected VDD among their participants indicating that normalization of Vit D levels in Vit D deficient individuals

might be necessary to detect the beneficial outcomes of vitamin D therapy. According to Al-Sofiani et al. [70]

study, replenishment of VDD in T2DM individuals had resulted in significant improvement in β-cell activity

(HOMA-%B) without affecting HbA1c and insulin resistance.

Vitamin D3 sSupplementation in of large doses given of vitamin D3 to Vit D deficient T2DM individuals subjects

did not alter insulin sensitivity or insulin secretion [71]. These results were in acceptance with other studies that

studied the effect of Vit D administration on glucose metabolism in different patient population. Wagner et al. [72]

found no effects of 30,000 IU/week in subjects with prediabetes or non-pharmacologically managed T2DM subjects.

Contrary to this in the Calcium and Vitamin D for Diabetes Mellitus study, vitamin D supplementation to

individuals with prediabetes and normal vitamin D status resulted in improvement of insulin secretion [73]. A recent

review also highlighted the beneficial effects of vitamin D supplementation on fasting glucose in poorly controlled

diabetes [66].

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In T2DM subjects with VDD (20 ng/mL), Sugden et al. [74] reported contradictory results in the secondary outcome

of their study. Firstly there was no change in HbA1c or HOMA-IR between the groups. However, subgroup analysis

revealed a significant decrease in HOMA-IR in the individuals whose serum 25-OHD levels had increased. Talaei et

al. [75] demonstrated an inverse relation between fasting blood glucose values and serum 25(OH)D level. According

to them, vitamin D levels of 40–60 ng/ml (100–150 nmol/l) had a significant effect on insulin resistance (IR). This

could be due to the appearance of extra-skeletal effects which are evident at such higher concentrations. The

Medical Research Council Ely Study has demonstrated a prospective correlation between serum 25(OH)D levels

and IR. After 10 years, baseline serum 25(OH)D levels were inversely related with fasting glucose, 2 hours plasma

glucose on oral glucose tolerance test, fasting plasma insulin levels and HOMA-IR [44].

The probable mechanism(s) through which vitamin D potentially regulates glucose metabolism, decreases insulin

resistance (IR) and thus can be beneficial to T2DM individuals include: 1) direct stimulation of insulin secretion

through the Vit D receptor on β-cells of pancreas, 2) attenuation of systemic inflammation and consequent

improvement in IR and 3) improvement of peripheral insulin resistance through the modulation of cytosolic calcium

concentration (indirect) and also via vitamin D receptors in muscles and liver [35,76,77,]. It is evident from earlier

studies that Vit D can also elicit rapid responses in target cells via its interaction with certain Vit D receptors in the

plasma membrane and with 1,25(OH)2 D3 Membrane Associated Rapid Response Steroid Binding Protein (1,25D3 -

MARRS). The rapid response to Vit D is of prime importance in diabetes management due to its involvement in the

modulation of insulin secretion [78-81]. The relationship between VDD and T2DM is well documented. This link is

mediated both by direct and indirect effects of Vit D on insulin secretion, insulin sensitivity, and systemic

inflammation [82]. Vit D also decreases the accumulation of advanced glycated end products (AGEs) [83] which are

involved in the development of IR [84] and its associated macrovascular complications.

4.3 Vitamin D Deficiency and Type 2 Diabetes - Blame the Genes

Circulating 25(OH)D levels were found to be influenced by the allelic variations in 7-dehydrocholesterol-reductase

(DHCR7), vitD-25-hydroxylase and CYP2R1 enzymes [85-87]. Both DHCR7 and CYP2R1 genes are concerned

with endogenous production and liver conversion of 25(OH)D respectively. Genetic variations in DHCR7

(endogenous production of 25(OH)D) but not CYP2R1 might partially be associated with an increased risk of

T2DM [88]. The probable explanations for the phenomenon are Llow Vit D level through decreased endogenous

production (from exposure to sunlight) is linked to high T2DM risk but low vitamin D level via hepatic conversion

from all sources is not. Concomitantly, CYP2R1 genetic variants were preferentially associated with high

concentrations of plasma 25(OH)D hence might poorly predict plasma 25(OH)D levels [86,89,90]. Genetic

alterations in the VDR gene has been suggested to play a role in the T2DM pathogenesis. TaqI, FokI, ApaI and

BsmI are the four common allelic variants of VDR gene identified [91]. In a study conducted on healthy Asian

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Bangladeshi population of London who had a greater prevalence of VDD and at risk of T2DM, lower insulin

secretion has been linked with ApaI polymorphism (homozygosis for the allele) [92]. In an unknown diabetic cohort

of older adults, ApaI polymorphism had a correlation with fasting plasma glucose and glucose intolerance. Speer et

al [93] demonstrated that obese diabetes individuals with the BB genotype of the BsmI allele in the VDR gene had

elevated postprandial serum C-peptide levels indicative as an indicator of its role in T2DM. It has been stated that

TaqI polymorphism is a determinant of insulin secretion in Vit D insufficient individuals [94].

4.4 Inflammation, IR and T2DM Related Cardiovascular Disease - Vit D the Connecting Link

Autier et al. [95] in their systematic review suggested that VDD is a surrogate marker of systemic inflammation

rather than a cause of it. It has been postulated that IR is linked to chronic low-grade inflammation [96].

Inflammation links obesity to IR through the blockade of insulin receptor signalling cascade [97,98] In T2DM

subjects, chronic low-grade systemic inflammation plays a vital role inon the onset and disease progression as well

as the development of micro and macrovascular dysfunction [99,100]. Further, a strong relationship has been

designated between systemic inflammation and CVD risk, independent of existing risk factors [101,102]. Vit D has

been shown to inactivate inflammatory cytokines associated with IR [82].

T2DM is characterized by abnormalities in systemic inflammatory markers like tumour necrosis factor-α (TNF-α),

interleukin-6 (IL-6), plasminogen activator inhibitor-I and C-reactive protein [65]. TNF-α can directly interfere with

the insulin-signalling pathway triggering IR via various mechanisms [103]. In adipocytes / skeletal muscle cells,

TNF-α may increase serine phosphorylation of insulin receptor, insulin receptor substrate-1 (IRS-1) and perhaps

other proteins involved in intracellular insulin-signalling. Such IRS-1 has been known to prevent insulin receptor

tyrosine kinase activity, which in turn leads to impaired downstream signalling [104]. Vitamin D has been reported

to downregulate the synthesis of many cytokines such as TNF-α, TNF-β, IL-2, and interferon-γ [105,106].

5 Vitamin D: an adjunct antihypertensive?

Vitamin D deficiency is characterised by elevated parathyroid hormone (PTH) levels thus associated with high

blood pressure (BP) and myocardial hypertrophy [107]. Moreover, studies have highlighted the interplay between

vitamin D, PTH and aldosterone free of the renin-angiotensin-aldosterone system (RAAS) in the cardiovascular

damage [108,109].

In a meta-analysis (comprised of 11 prospective studies published between 2005 and 2012) conducted by Kunutsor

et al. [110], they reported a significant inverse relationship between risk of incident hypertension and baseline serum

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vitamin D levels with no heterogeneity. When evaluating dose-response, the authors found that for every 10 ng/mL

increase of 25(OH) D there was a 12% reduction in hypertension risk. In contrast, studies conducted in various

cohorts to evaluate the effect of vitamin D on BP yielded conflicting results [111-113]. The probable mechanisms

put forward to explain the role of vitamin D in the regulation of BP are - a direct vascular effect which is evident by

the presence of VDRs in endothelial cells and occurrence of 1α -hydroxylase activity in both vascular and

endothelial smooth muscle cells [114]. Vit D can improve endothelial function, modulate smooth muscle function

and decrease vasculotoxic PTH [115]. Vitamin D mediated negative regulation of RAAS may lower BP and thus

provide cardiovascular protection [116].

Table 1 Major Studies on Vitamin D and Hypertension

Authors Study design and Sample Size

Intervention Major findings

NHANES III [117] Cross sectional(n = 12644)

- Participants in higher Vit D category had lower SBP and DBP compared to those in the lowest Vit D quartile.

Health Professionals study [118]

Prospective cohort(n = 613)

- Vit D deficientinsufficient (≤ 15.2 ng/mL) mensubjects had a higher incidence of HTN than the Vit D sufficient people (≥ 30 ng/mL).

Nurses' Health Study [118] Prospective cohort (n = 1198) of women

- Same as above.

Women's Health Initiative [119]

Double-blind RCT(n = 36282) postmenopausal women

Vit D 400 IU + 1000 mg Ca2+/day

No significant reduction in either SBP or DBP or risk of HTN after 7 years follow up.

Pfeifer's et al. [120] Prospective cohort(n = 148)

Vit D3 800 IU +1200 mg Ca2+/day or 1200 mg Ca2+/day alone x 8 weeks

Vit D3+ Ca2+ supplementation to elderly women significantly reduced SBP than calcium alone.

Witham et al. [121] Meta-analysis Vit D (D2 and D3) and UV-B radiation

Vit D caused a small but significant decline in DBP in HTNhypertensive subjects.D2 produced greater fall in BP than D3

Beveridge LA et al. [122] Systematic review(n = 4541)

Vit D (D2 and D3) for minimum 4 weeks

Vit D supplementation is ineffective as an antihypertensive.

RCT – Randomised controlled trial, SBP - Systolic Blood Pressure, DBP - Diastolic Blood Pressure, HTN – Hypertension, NHANES – National Health and Nutrition Examination Survey.

6 Vitamin D deficiency and diabetes associated dyslipidemia – the compatriots

The high prevalence of coronary artery disease among South Asians is ascribed mainly to dyslipidemia characterised

by elevated triglyceride (TG), apoB, Lp(a) levels, borderline high low-density lipoprotein cholesterol (LDL-C), low

levels of high-density lipoprotein cholesterol (HDL-C) and apoA1 [123]. Results of INTERHEART Study

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demonstrated nearly 10 mg/dL lower mean LDL-C levels in Asians compared to non-Asians. Majority of Asians had

LDLC ≤100 mg/dL with a slightly lower HDL-C level compared to that of non-Asians [124]. In South Asians,

apoA1 fluctuations predicted cardiac risk better than HDL-C. ApoB/apoA1 ratio had the strongest association with

the risk of acute myocardial infarction [125]. Among Bangladeshi immigrants of United Kingdom, VDD showed a

positive association with decreased apoA1 levels that is independent of anthropometric, glycemic, dietary and

lifestyle associated risk factors for T2DM and ischemic heart disease [126]. Lower apoA1 levels, in turn, lead to low

HDL levels thus augmenting CAD risk [127].

Diabetes-associated dyslipidemia predisposes individuals to CAD in an abnormally aggressive manner. In T2DM

individuals, altered effects of insulin on hepatic lipoprotein synthesis and release, skeletal muscle and adipose tissue

metabolism, lipoprotein lipase (LPL) and cholesterol ester transfer protein activity lead to post-prandial lipemia,

hypertriglyceridemia, and low high-density lipoprotein cholesterol (HDL-C) [128]. Along with these abnormalities,

there exists an attended increase in atherogenic small dense low-density lipoprotein (sd-LDL) levels, which are not

easily reversed even with tight glycemic control [129]. The imbalance between sd-LDL (elevated) and HDL

(decreased) play a crucial role in atherosclerosis development [130,131]. Besides this, diminished HDL function due

to oxidative damage to apolipoprotein A-1 and decreased HDL bound paraoxonase-1 activity encountered in the

diabetic environment also contribute to CAD [132,133]. Diabetes-associated dyslipidemia is also manifested by low

LPL activity and high TG [134]. Earlier studies showed that VDD individuals are at cumulative risk for

dyslipidemia [135,136]. In the cells, Vit D can induce the expression of There is evidence about the LPL expression

inducing the effect of Vit D in cells [137]. In summaryAltogether 25(OH)D levels were directly associated with LPL

and LPL had an inverse relation with IR and T2DM [138]. On the other hand, the atherogenicity of Lp(a) is

primarily driven by the effective binding of oxidized phospholipids to the, by the expression of adhesion molecules

[139]. Whereas, the prothrombotic effect is due to the similarity of apo(a) to plasminogen. This enhances Lp(a)

deposition in the arterial wall resulting in increased cardiovascular risk [140,141]. The European Atherosclerosis

Society (EAS) 2010 guidelines suggested a target level of < 50 mg/dL for Lp(a) as a feature of global cardiovascular

risk [142].

Data from the majority of cross-sectional studies reported a positive association between serum 25(OH)D and HDL

levels [143-144]. On the contrary serum 25(OH)D levels were inversely related to triglyceridesTGs, TC/HDL,

Triacylglycerol and LDL/HDL ratios [145]. Interventional studies conducted among T2DM patients [62,146,147,]

and other individuals [56,148,149] yielded conflicting results. Querfeld U et al. [150] demonstrated the inhibitory

effect of Vit D and PTH on LPL activity which is probably mediated through regulation of intracellular calcium that

in turn may contribute to the alterations in lipoprotein metabolism. Lind L et al. [151] reported a significant negative

association between 25(OH)D levels insulin, LPL activity in both skeletal muscle and adipose tissues. In the same

study Vit D also demonstrated inverse relation with BP, TG and VLDL. Few major studies on vitamin D association

with lipid profile are mentioned in the below ttable 2.

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Table 2 Major Clinical Studies on the Association of Vitamin D and DyslipidaemiaDyslipidemia

Authors Study design and Sample size

Intervention Major findings

Gannage -Yared MH et al. [152]

Cross sectional (n=381)

- 25(OH)D is inversely correlated with BMI, SBP, WC, FBS, Insulin, HOMA and positively correlated with adiponectin and HDL. After adjustment for with sex and BMI 25(OH)D is an independent predictor of FBS, SBP as well as LDL and SBP in males

Carbone LD et al. [153]

RCT (n = 51) adults suberythemal doses of whole-body irradiation twice weekly x 12 weeks

25(OH)D had a significant positive association with Apo-A and Lp-A1 and negative association with LDL/HDL ratio. Significant differences were evident in Vit D insufficient category only.

Aguirre P et al.[154]

Double-blind RCT on postmenopausal T2DM women (n = 104)

Vit D 4000 IU daily vs placebo x 6 months

Vit D administered group showed low serum TG levels with no effect on other lipid fractions

Saedisomeolia A et al. [155]

Cross sectional (n=108) on T2DM patients

- Reported higher TC and LDL with relatively low HDL levels in VDD T2DM individuals compared withto their normal Vit D normal individuals. But 25(OH) D had a significant negative association with TGs only.

Chaudhuri JR et al. [156]

Prospective cohort (n = 150) asymptomatic individuals

- Demonstrated an inverse and direct correlations between 25(OH) D levels and serum TC, LDL, TG, CRP levels and HDL. Association persisted even after multiple logistic regression.

Garry John and colleagues [127]

Cross-sectional (n=170) British Bangladeshi non- diabetes adults

- 25(OH)D showed a positive relationship with fasting apo A-I levels independent of glycemia, anthropometric and other lifestyle risk factors for T2DM and IHD. VDD individuals are likely to have a high risk of IHD independent of T2DM risk.

Women's Health Initiative [157]

Prospective cohort (n = 1259) postmenopausal women

Ca2+ 1 gm and Vit D 400 IU per day supplementation

Ca2+ and Vit D supplementation for 5 years is not associated with any lipid changes.

Zittermann A et al. [158]

Healthy overweight VDD individuals (n =200)

Vit D 83µg/day x 12 months

Independent of weight loss, BMI and sex Vit D supplementation produced a significant reduction in PTH (-26.5%), TGs (-13.5%) and , TNF-α (-10.2%) alongnd TG (16%) along with an increase in LDL (+5.4 8%) in LDL compared to placebo.

RCT – Randomised Control Trial, BMI - Body Mass Index, WC - Waist Circumference, FBS - Fasting Blood Sugar, HOMA - Homeostasis model assessment, TC - Total Cholesterol, TGs - Triglycerides, HDL - High Density Lipoprotein, LDL - Low Density Lipoprotein, SBP - Systolic Blood Pressure, IHD - Ischemic Heart Disease, PTH - Parathyroid hormone, TNF-α – Tumor Necrosis Factor-alpha.

Vitamin D has been linked to various cardiometabolic risk factors such as dyslipidemia, insulin resistance, diabetes,

systemic inflammation and obesity [14,159]. The mechanisms responsible for dyslipidemia in hypovitaminosis D

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are not well documented. Vitamin D through its immuno-modulatory / anti-inflammatory actions may attenuate the

proinflammatory cytokines and can thus affect serum lipid profile [160,161]. 1,25(OH)2D mediated activation of

PPAR-δ [162] lead to decreased FFAs due to improved oxidation of fat and fatty acids consumption by skeletal

muscle [163]. Vit D supplementation could also hasten fatty acid synthesis along with inhibition of lipolysis in

adipocytes [164,165] thus attenuating the rise in serum FFAs.

Besides this, the favourable effects of vitamin D on lipid profile can be attributed to photo metabolism of 7-

dehydrocholesterol. Squalene present in the dermis, when exposed to sunlight, gets converted to 7-

dehydrocholesterol, vitamin D and its metabolites. Inappropriate sunlight triggers the formation of cholesterol

through the alternative pathway [166]. The favourable effects of Vit D onpathways put forward for vitamin D

mediated reduction in serum triglycerides are attributed to the, elevation of in serum Ca2+ levels which could

decrease hepatic triglyceride formation and secretion [167]. Vit D by inhibitingInhibition of serum PTH levels, Vit

D may augment peripheral removal of triglycerides. Vitamin D by attenuating insulin resistance [167] may regulate

the VLDL (expression of VLDL receptors in certain cells) and triglyceride levels [168,169].

7 Diabetes-associated endothelial dysfunction (ED) - the role of Vit D

Plasma of dDiabetes individuals’ plasma is rich in glucose and fatty acids with their metabolites affecting coronary

vasculature thus leading to vascular dysfunction probably via reactive oxygen species generation and inflammation

[170]. Moreover, these abnormalities may trigger endothelial injury, an impending event in atherogenesis [171], or

plaque rupture which can lead to the acute coronary syndrome. T2DM attendant Insulin resistance induces a

selective deficiency in the PI3K/ Akt pathway and suppresses normal nitric oxide (NO) synthesis [172]. During

hyperglycemia or hyperinsulinemia insulin over activates mitogen-activated protein kinase (MAPK) pathway which

results in endothelin-1 [173] expression and cellular proliferation leading to endothelial dysfunction. This further

worsens hyperinsulinaemia and atherogenesis [173,174]. Hyperglycemia increases cyclooxygenase-2 expression,

[175] liberation of arachidonic acid and prostanoid synthesis, which cumulatively modifies vasomotor tone. [176]

Endothelial dysfunction (ED), an underlying vascular pathology observed in T2DM patients is a predictor of

cardiovascular events [177,178]. Endothelial cell damage results in leukocyte adhesion, migration and loss of

endothelial anti-aggregatory properties leading to platelet aggregation and existence of vasoconstrictive substances

[179].

The primary mechanisms for T2DM associated ED include attenuation of endothelial NO synthase (eNOS)

expression, eNOS uncoupling (conversion to superoxide anions producing form) due to oxidative stress and

increased NO breakdown [74]. The alternative pathways were, reduced endothelial prostacyclin secretion, altered

endothelium-dependent hyperpolarizing factors mediated responses, impaired fibrinolytic ability, increased

procoagulant activity, overproduction of growth factors and extracellular matrix proteins, increased endothelial

permeability and increased oxidative stress [174,180]. All these mechanisms also take part in the pathogenesis of the

13

atherosclerotic cardiovascular disease [181,182] Elevated advanced glycated end products impair the function of and

promote apoptosis in endothelial progenitor cells (EPCs) via increased oxidative stress [183].

In T2DM patients, serum 25(OH)D levels were inversely correlated with monocyte adhesion to endothelial cells. 1,

25(OH)2D3 also inhibited endoplasmic reticular stress, [184] and promoted M1-predominant phenotype of

monocytes with lesser endothelial adhesion [185]. VDD status may also indirectly cause ED via its effects on other

cardiovascular risk factors, including glycemic control, lipid profile and blood pressure. Uncontrolled blood glucose

related to VDD might also contribute to endothelial dysfunction. According to Yiu et al. [186] study, both Vit D

and HbA1c levels were independent predictors of flow-mediated dilation. They suggested that VDD in T2DM

patients wasis associated with ED and decreased EPC counts independent of traditional cardiovascular risk factors.

8 Obesity and metabolic syndrome: the D - dimension

Metabolic syndrome (MetS) is typically characterized by intra-abdominal obesity (visceral fat accumulation) a

feature that enhances insulin resistance (IR) by way of adipokines secretion and excess free fatty acids accumulation

[187,188]. Obesity is associated with low serum 25(OH)D [189-190], IR [191-193], MetS [194], inflammation [195]

and T2DM [196,197] all of which are risk factors for CVD. Sequestration of Vit D in body fat (adipose tissue) could

lead to its decreased bioavailability. Thus obesity can synergistically modulate the association between vitamin D,

the risk of IR and T2DM [198]. Obesity is prevalent in 70% of T2DM patients. There exist s evidence of vitamin D

role in the differentiation of brown and white adipose tissue as well as adipose tissue development [199].

Esteghamati et al. [200] reported a significant association between IR and Mets. They also established a direct

relation between HOMA-IR values and MetS components. The cut-offs reported by them to define MetS in

nondiabetes and diabetes individuals were 1.775 and 4 respectively. Evidence from cross-sectional and Mendelian

randomization studies demonstrated a link between low plasma 25(OH)D levels, obesity and components of MetS

[201,202]. Data from randomized interventional studies established obesity as a predisposing factor for diabetes.

[203,204] Previous studies also showed an association between a set of genotypes linked to high BMI, T2DM risk

[205,206] and low serum Vit D levels [90]. Other than hypertension, predominant components of MetS like

elevated glucose, TGs, high waist circumference and low HDL-C are inversely related to serum Vit D status [207].

14

Table 3 Clinical Studies on Metabolic Syndrome, Obesity and Vitamin D

Authors Study design and Sample Size

Major findings

NHANES III 1988-1994) [208]

Cross-sectional(n= 8421 men, non-pregnant women)

Obese individuals with VDD are predisposed to a higher incidence of MetS and IR.

NHANES(2003-2004) [209]

Cross-sectional(n = 834 men, 820 women free of diabetes) age > 20 years

25(OH)D had an inverse relation with MetS independent of age, sex, lifestyle, race/ethnicity, PTH and Ca2+ intake. PTH is directly linked with MetS in older men.

NHANES(2001-2006) [210]

Cross-sectional(n = 12900) age > 20 years

Insufficient 25(OH)D and abdominal obesity synergistically influence IR risk. The magnitude of IR for obese individuals with VDD and Vit D sufficiency was 32.13 and 19.97 folds respectively.

Maki KC et al. [211] Cross-sectional(n = 257 men and women) age > 18 years

25(OH)D was inversely related to MetS prevalence and the components of MetS namely, HDL-C, TG, WC, BMI.

British Birth Cohort [212] Prospective cohort(n = 6810) age 45 years

25(OH)D was inversely associated with MetS. A negative association between vitD and IGF-1 was found only in VDD individuals.

Durmaz et al [213] Cross-sectional(n = 170 men=24 women=146)

Obese individuals had higher VDD and it may further increase T2DM risk.

Afzal and colleagues [88] Mendelian randomization study

(n = 96423) white Danish population

Genetic variants for low 25(OH) D synthesis could have a higher T2DM risk and low Vit D levels seem to mediate 3-4% of the effect on BMI and risk of T2DM.

Rotterdam study [207] Cohort (n = 3240) with no history of diabetes

Elderly people with higher 25(OH)D levels had lower MetS prevalence and experienced beneficial effects on HDL-C, TGs, WC, and serum glucose.

VDD – vitamin D deficiency, MetS – Metabolic syndrome, IR – Insulin resistance, PTH – parathyroid hormone, BMI - Body mass index, WC – waist circumference, TGs – triglycerides, HDL-C – High-density lipoprotein cholesterol, T2DM – type 2 diabetes

9 The D-bate on vitamin D dosage – Institute of Medicine versus The American Association of Clinical

Endocrinologists recommendations

Institute of Medicine (IOM), USA [214] defined VDD as 25(OH)D levels < 20 ng/mL or 50 nmol/L (For conversion

nmol/l divided by 2.5 = value in ng/mL). Garland and colleagues suggested that the cut off for VDD recommended

was too low to make full use of the preventive health benefits and the risk of various diseases like cancer,

15

autoimmune, inflammatory disorders, etc. Therefore the cut off level should be set at 30 ng/mL [215]. Two meta-

analyses [215,216] suggested that maintenance of serum 25(OH)D between 36 and 70 ng/mL was associated with

reduced risk of mortality due to cardiovascular disease and cancer. Supplementation of 2000IU to 4000IU Vit D 3 per

day will increase serum 25(OH)D levels above 30 ng/mL [217,218]. For individuals of 9 years and older IOM

recommended a daily tolerable upper intake level for vitamin D of 4000 IU. Moreover, obese individuals, children

and adults on medications such as glucocorticoids, anti-retrovirals and anticonvulsants need at least two to three-fold

higher vitamin D to meet their daily requirement [217-223]. The American Association of Clinical Endocrinologists

(AACE) guidelines (1500-2000 IU/day) for the prevention of VDD are applicable to adults of all racial groups >18

years. There is no difference in recommendations for whites and blacks to prevent or treat VDD (217). For every

100 IU of Vit D intake serum 25(OH)D levels increase by about 1 ng/mL [224].

101 Conclusion

In type 2 diabetes individuals cardiovascular diseases (CVDs) are the commonest cause of mortality. Considering

the beneficial effects of vitamin D on various risk factors of CVDs such as, hyperglycemia, dyslipidemia,

endothelial dysfunction and blood pressure, maintenance of optimum vitamin D levels (40 – 45 ng/mL) may have a

positive influence on T2DM individuals subjects in context to glycemic profile improvement and cardiovascular

disease (CVD) prevention. Nevertheless, the conflicting evidence regarding the positive effects of Vit D

supplementation can be attributed to the form of Vit D administered (D2 or D3) disparity in the dosage,

duration/allocation of treatment (single or multiple doses) and poor adherence. This provides an impetus to conduct

larger studies considering specific markers of vitamin D that ardeficiency (“Calcium-Vitamin D-Parathyroid

hormone” endocrinal axis) and its relation to e associated with T2DM associated and CVD.

Acknowledgements: Nil

Conflict of Interest: The authors declare no conflict of interest.

16

Figure 1: Schematic Representation of Hypovitaminosis D and its Role in Diabetes

Associated Cardiovascular Diseases

17

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