Correlating Circulating Vitamin D3 with Aspects of the Metabolic Syndrome and Nonalcoholic Fatty Liver Disease in Healthy Female Volunteers

Abstract

Background: Significant inverse correlations between circulating vitamin D3 and the presence and strength of common clinical entities influenced by insulin resistance (IR) have been reported. Among these entities are common maladies such as the metabolic syndrome (MS) and nonalcoholic fatty liver disease (NAFLD). Since hypovitaminosis D3 is considered a common worldwide health disturbance, the consequences emanating from such relationships once soundly established might reasonably be ameliorated by vitamin replacement. Accordingly, a need exists for definitive confirmatory support for the existence of these linkages particularly in ordinary, relatively healthy individuals.

Methodology: Many aspects of MS and NAFLD were examined in healthy females via linear regression analyses to determine significant correlations mainly using vitamin D3 and fasting blood glucose (FBG), the latter a surrogate for IR, as independent variables.

Results: A descending linear regression between vitamin D3 and FBG did not reach full statistical significance, but a negative correlation of vitamin D3 with HbA1C was statistically significant and that with circulating insulin concentrations showed a downward statistical trend. These findings are consistent with previous ones by others suggesting an ameliorating effect of vitamin D3 on IR. Body weight, body mass index (BMI), and body fat correlated statistically significantly with vitamin D3 levels, as did systolic blood pressure. Again, these correlations were negative instead of positive unlike the case when FBG was the independent variable. The significant negative correlation of ALT with vitamin D3 was also consistent with previous reports that higher circulating vitamin D3 favorably influences the extent and intensity of NAFLD.

Conclusions: Employing FBG as a surrogate for IR and ALT for status of NAFLD, linear correlations reasonably suggest that increasing circulating vitamin D3 can favorably influence the initial development of and/or strength of risk factors for MS including NAFLD in relatively fit females.

Keywords:

• Vitamin D3
• metabolic syndrome
• Vitamin D3
• insulin resistance
• insulin resistance
• Vitamin D3
• metabolic syndrome
• Vitamin D3
• Vitamin D3
• nonalcoholic fatty liver disease
• nonalcoholic fatty liver disease
• Vitamin D3

Introduction

It is generally recognized that certain chronic metabolic health disorders primarily connected to poor lifestyle choices in diet and/or exercise are associated with disturbed glucose-insulin metabolism – particularly insulin resistance (IR) [1–6]. Unfortunately, these IR-linked perturbations appear to be gaining in frequency and intensity throughout the world. Hope still exists, however, to favorably modify these maladies by returning insulin sensitivity back toward optimal status. In this regard, numerous studies have made intriguing connections between the level of circulating vitamin D3 and the following IR-involved perturbations: type 2 diabetes mellitus (T2DM) [7–13], the metabolic syndrome (MS) [14–17], as well as obesity [10–12, 18] and nonalcoholic fatty liver disease (NAFLD) [19–25] – to be more exact, the higher the vitamin D3 levels, the healthier the situation regarding the occurrence and strength of the metabolic disturbances. The latter relationship now is particularly unfortunate; because along with the increasing chronic health maladies also mentioned above, hypovitaminosis D3 is prevalent worldwide [7, 19]. Accordingly, definitively proving existence of links between these chronic disturbances and vitamin D3 lack could lead to preventing, ameliorating, and even curing them at least to some extent by simple replacement.

Recently, we examined linear correlations between a variety of surrogates for IR in non-diabetics and the various elements of MS including obesity, and NAFLD [26, 27]. While FBG proved to be the most practical proxy for IR in our hands, we also found significant correlations when providing circulating insulin levels, HbA1C, and the ratio triglyceride/HDL-cholesterol as surrogates for IR in the plethora of correlations [26, 27]. In addition, we employed ALT to determine presence and intensity of NAFLD [24, 25]. In those cases, worsening risk factors for the various entities and their elements were discovered even with supposedly non-pathologic increases in IR and/or hepatic fat accumulation estimated by the use of their surrogates [28, 29]. These happenings have been referred to as “continuum of risks” – a principle purporting that risks for medical perturbations can exist even in the so-called normal ranges of analytic values devoid of diagnostic implications [28].

Hence, the not mutually exclusive aims in the present investigation were twofold: first, to further establish significant links between circulating vitamin D3 levels and the status of IR in relatively healthy females and second, to determine more specifically by comparing a variety of correlations made with vitamin D3 and FBG as the independent variables any potential beneficial effects that vitamin D3 replacement could have on IR-associated medical entities.

Material and methods

Study design

Data for this cross-sectional assessment were obtained from baseline materials gathered from a single earlier study that previously reported on 107 females. When entering the initial clinical investigation, all participants gave written informed consent that complied with the Helsinki Declaration as approved by the RCRC Institutional Review Board. Permission was given to use the obtained data for future research as long as the sources remained unidentified. Some data from this group were used originally in a report examining age-related blood pressure elevation [30]. The intent of the initial study was to assess average female individuals. In doing so, many volunteers were overweight or obese with a group average body mass index (BMI) of 28.7 kg/m² ±1.1 (SEM) (range 16.9 kg/m² to 50.6 kg/m²).¹ The average age was 56.1 years ± 1.2 (SEM)(range 29 years to 80 years). With one exception where a single reading of 132 mg/dl was found, the initial FBG level was below 125 mg/dl in every other case.

In the original enrollment, after reviewing the study with a research technician, volunteers were provided with a copy of the study’s informed consent and were asked to review it with their personal physicians to insure they had no medical conditions that would preclude their participation in the previous particular study. After fasting overnight, participants returned to the research facility and fulfilled much of the test battery described below. The 107 subjects completed a baseline test battery that in most cases included: total-body DEXA measures of body composition, blood pressure and resting heart-rate, blood chemistries, and a complete blood count (CBC) panel. Bloods were drawn at a Lab Corps Service Center of the subject’s choice (www.labcorp.com).

Specific testing

Body composition

In contrast to scale weight readings, which reflect how much not what kind of weight is lost, dual energy x ray absorptiometry (DEXA) measurements were used that provide for assessment of the kind of weight lost—fat mass (FM), fat-free mass (FFM) and bone mineral density (BMD) [31]. Measurements were made using a constant potential energy source at 78 kVp and a K-edge filter (cerium) to achieve a congruent, stable, dual-energy beam with effective energies of 40 and 70 keV. The unit performs a series of transverse scans moving from head to toe at one mm intervals; the scan area was approximately 60 cm × 200 cm. Data were collected for about 120 pixel elements (5 × 10 mm) per transverse. Total body measurements were typically completed in ∼10 minutes with a scan speed of 16 cm/sec or in ∼20 minutes with a scan speed of eight cm/sec. The R-value (ratio of low to high-energy attenuation in soft tissue) ranged from 1.20 to 1.47.

Blood chemistries

After fasting overnight, subjects had venous blood samples drawn at a local drawing station of their choice. Blood samples were collected in EDTA (one mg per milliliter) and were centrifuged within four hours, the plasma was separated, and the plasma sample shipped at 4 °C to a central laboratory.

Analyses

All linear regression statistical analyses were carried out with KaleidaGraph graphing and data analysis, Version 4.1.3, Synergy Software, Reading PA. A p < 0.05 determined by using a table of correlation coefficients (p Value from Pearson (r) Calculator) was considered to be statistically significant.

Results

In Figure 1, correlations between chronological age vs. circulating FBG and vitamin D3 levels are illustrated. FBG correlates significantly positive with age (p < 0.03). In contrast, no statistical significance is found in the correlation of age with vitamin D3 concentrations. In Figure 2, the correlation between vitamin D3 and FBG was negative but not statistically significantly so (r = −0.118, n = 99, p = 0.24). In contrast, the negative correlation between vitamin D3 as the independent variable and ALT as the dependent variable depicted in the same figure proved significant (r = −0.219, n = 105, p < 0.05). Figure 3 reveals a significant negative correlation between vitamin D3 and HbA1C (r = −0.194, n = 105, p < 0.05), while the correlation between vitamin D3 and insulin levels in Fig. 4 displays a trend (r = −0.168, n = 105, p < 0.10 > 0. 05).

Figure 1. Correlations between age (independent variable) and fasting glucose (FBG) and vitamin D3 levels (dependent variables). The rise in FBG levels with age is statistically significant at p < 0.03, whereas aging correlations are not significant for vitamin D3.

Figure 2. Correlations between vitamin D3 (independent variable) and fasting blood glucose (FBG) and ALT levels (dependent variables). The decrease in the ALT levels with increasing circulating vitamin D3 is statistically significant at p < 0.03, whereas, the correlation with FBG does not reach statistical significance.

Figure 3. Correlation between vitamin D3 (independent variable) and HbA1C level (dependent variable). The decrease in the HbA1C levels with increasing circulating vitamin D3 is statistically significant at p < 0.05.

Figure 4. Correlation between vitamin D3 (independent variable) and circulating insulin level (dependent variable). The decrease in the HbA1C levels with increasing circulating vitamin D3 reveals a statistically significant trend at p < 0.10 > 0.05.

In Table 1, when vitamin D3 is the independent variable, linear slopes are predominantly negative whereas, the same slopes with FBG being the independent variable are positive. Accordingly, increases in circulating vitamin D3 concentrations appear to improve overall health by lessening various risk factors, while positive correlations in FBG represent increase in the strength of the risk factors. These divergent correlations occur in body composition (body weight, BMI, body fat, body fat percent); in cardiac dynamics (systolic blood pressure); and in blood chemistries (insulin concentration, HbA1C, and ALT).

Table 1. Vitamin D3 levels compared to circulating glucose levels as independent variables in female subjects. Average age: 56.1 years ± 1.2 (SEM) with a range of 29–80 years.

Parameters	n	Ave ± SEM	Vit D3	FBG
			r Values	r Values
Body Composition
Body Weight (lbs)	106	165.8 ± 3.3	−0.250°	0.230*
BMI (kg/cm^2)	107	28.7 ± 0.6	−0.311°	0.230*
Fat (lbs)	108	73.1 ± 2.4	−0.270°	0.242*
Fat (%)	107	43.1 ± 0.7	−0.214^#	0.237*
Fat Free Mass (lbs)	108	93.8 ± 1.2	−0.142	0.134
BMD (g/cm^2)	106	1.14 ± 0.01	−0.165	0.061
Cardiac Dynamics
Systolic BP (mm Hg)	107	120.3 ± 11	−0.206^#	0.341^°
Diastolic BP (mm Hg)	108	74.5 ± 0.8	−0.059	0.189^#
Heart Rate (beats/min)	108	74.1 ± 1.0	0.057	0.178^$
Blood Chemistries
Fasting Glucose (mg/dl)	99	92.9 ± 0.60	−0.118	——
HbA1C (%)	98	5.71 ± 0.03	−0.194^#	0.566°
Insulin (mIU/ml)	99	4.83 ± 0.38	−0.168^$	0.501°
AST (U/l)	98	21.8 ± 0.7	−0.074	0.141
ALT (U/l)	99	21.4 ± 0.9	−0.219^#	0.306^°
Alk P”ase (U/l)	99	71.4 ± 2.0	−0.119	0.149
Bilirubin (mg/dl)	101	0.49 ± 0 .02	−0.117	−0.106
BUN (mg/dl)	101	14.7 ± 0.4	0.149	0.134
Creatinine (mg/dl)	101	0.84 ± 0.02	0.097	0.016
Blood Count
RBC (10^6/mm^3)	100	4.38 ± 0.03	−0.065	0.074
WBC (10^3/mm^3)	100	6.12 ± 0.16	−0.068	0.137
Neutrophils (10^3/mm^3)	100	3.61 ± 0.13	−0.122	0.156
Platelets ((10^3/mm^3)	100	245.0 ± 5.7	−0.168^$	0.085

BMD = bone mineral density; BP = blood pressure; AST = aspartate aminotransferase (SGOT); ALT = alanine aminotransferase (SGPT); Alk P’ase = alkaline phosphatase; RBC = red blood cells; WBC white blood cells. Minus sign before r value indicates negative slope. ⁰ p < 0.01, * p < 0.02, ^# p < 0.05, ^$p < 0.10 > 0.05. “n” represents correlation number for vitamin D3 as independent variable. Correlations with FBG were made with n of 98-99 subjects.

Discussion

Various chronic metabolic disorders are becoming more ubiquitous and severely harmful in the aging populations worldwide, and much of this is attributed to the development of a health issue referred to as “insulin resistance” (IR) [5, 6, 32–34]. Approximately a century ago after the isolation and purification of insulin by Banting and colleagues, it was the general belief that diabetes, an abnormal increase in circulating glucose, was solely due to a shortage of available insulin [35]. So, the general conviction was that diabetes is curable, because lack of insulin could now be dealt with. Very soon after time wise however, another form of diabetes was discovered when it was observed that an unexpected increase of insulin in the blood stream could be present even in the face of abnormally high circulating glucose concentrations [36]. Ironically, this happening was more common than that described in the original diabetes with low circulating insulin (type 1). This newer, more abundant form attributed greatly to the aforementioned IR was referred to, of course, as type 2 diabetes mellitus (T₂DM). Perhaps as important to the whole health scenario, IR is generally accepted to be a crucial driving force behind the linked components of the metabolic syndrome (MS) including nonalcoholic fatty liver disease (NAFLD) [5, 6, 24, 25].

What exactly is IR? Similar to other endocrine systems such as those involving the thyroid and adrenals, the glucose-insulin system often has a distinctive response to any diminution in hormonal action. In the case of the latter endocrine schema, a tendency exists under certain conditions for peripheral target tissues such as liver, fat and muscle to experience a lessened, apparently sub-optimal response to insulin over time. To compensate, more insulin is produced and released from the pancreas as a “make-up.” While this comeback has some merit and returns circulating glucose more toward normal, the final response is characteristically slightly higher glucose and insulin levels than prior. Unfortunately, a harmful potential may still exist [29].

Similar happenings health-wise sometimes attain a unique nomenclature, namely “trade off”, because they can bring on immediate benefits but may sooner or later cause future harms [37]. As the organ resistance to insulin becomes greater after a while, the back and forth interaction between glucose and insulin can continue to build. Accordingly, both levels of circulating glucose and insulin may rise progressively and be associated with increasing severity of the risk factors involved in T₂DM, as well as MS and NAFLD [29, 34].

To state an old axiom, “an ounce of prevention is worth a pound of cure.” Taking this to heart, the concept of a “continuum or risks” mentioned in the introduction needs a more precise description in order to fully realize the meaningful potentials of an early preventative program [28]. As is the case for blood pressure regulation, the earlier an “IR preventive program” begins, the more successful should be the outcome. For better or worse, our evidence indicates that IR perturbations initiate a blossoming variety of risk factors linked to MS, e.g., T₂DM and obesity as well as NAFLD, even when circulating levels of glucose and insulin fall within the accepted normal ranges but at the higher levels in those ranges [26–28]. Reasonably, the best option is to begin early to overcome IR in order to prevent eventual pathology arising from long-term exposure to any non-optimal phase of the resistance [38, 39].

Besides aging, what else is behind IR that can be used to develop an ideal preventative regimen? Lifestyle choices are very important in this regard. Diets containing too many calories particularly from rapidly absorbed carbohydrates such as table sugars and poor exercise habits gravely influence IR status [40]. However, the public has known about these unhealthful measures for eons, but the mainstream has not been able to effectively comply with the needed remedies. A call for drugs with “insulin sensitizing” capabilities has not come fully to fruition primarily because of ineffectiveness and concomitant adverse reactions – real and potential [34, 41]. Leaving aside drugs, natural safe supplements exist that can afford some measure of insulin sensitization [6]. Unfortunately, however, the potential of natural, safe dietary supplements to produce insulin sensitization has not been fully recognized, especially when it comes to the established medical community. Nevertheless, among the latter possibilities, utilization of vitamin D3 as a preventative may be coming into some well-deserved prominence [7].

New avenues for usefulness of vitamin D3 supplementation are becoming apparent with the passage of time [8]. More and more is being written concerning the healthful benefits of vitamin D3 other than in mineral metabolism. Still, some uncertainty about their useful role has been expressed. Even though the ability of vitamin D3 to positively influence the course of major health disorders such as T2DM, MS, and NAFLD has come under closer scrutiny of late, in the words of George et al., “There is currently insufficient evidence of beneficial effect to recommend vitamin D3 supplementation” [8]. With the above settings in mind, we remembered a previous study where a goodly number of vitamin D3 assays had been carried out in the examination of age-related elevated blood pressure. To be exact, vitamin D3 levels were obtained from over 100 females [30].

The material from this previous study was revisited focusing primarily on the vitamin D3 aspects [30]. We, for the most part, relied upon the effects of vitamin D3 circulatory levels during the period of “continuum of risks” for major IR associated health entities [29]. It has been proposed that many risk factors even of a mild nature may have the ability to take their toll over time and even be responsible for a shorter lifespan [38, 39]. Similar to previous reports, data from Figures 2–4 and Table 1 suggest that higher concentrations of vitamin D3 can lessen the strength of the IR and its associated maladies [42]. Although the negative correlation between vitamin D3 and FBG in Figure 2 did not quite achieve statistical significance in this limited number of volunteers, the association with HbA1C and insulin in Figures 3 and 4 proved respectively statistically significant and trending. So, along with the successful use of FBG in larger previous samples as a surrogate for IR, we believe this justifies its use as an appropriate stand-in for IR in Table 1 [26,27]. In any case, the correlations showing that low levels of vitamin D3 associate with increases in body weight, general fat accumulation, higher systolic blood pressure values, and increasing ALT levels to go along with hepatic fat accumulation strengthen the hypothesis that vitamin D3 consumption may be helpful in ameliorating or even preventing many future chronic disabling aging perturbations [14, 21–23]. Taking the last statement to heart, it is indeed unfortunate that we found in the current study that IR in the form of FBG levels tends to increase during aging but vitamin D3 does not do likewise to help compensate (Figure 1).

Much confusion has been expressed over the effects of vitamin D3 on lifespan – pro and con [43, 44]. Recent studies suggest that improvements of many elements composing the MS over time have the potential to improve lifespan [29, 45]. The fact that vitamin D3 can benefit many aspects of MS favors the hypothesis that this vitamin has the potential to improve prospects for longevity [43, 46]. Considering the entire above material, vitamin D3 can be added to a list of other safe natural dietary supplements that have ability to improve IR status and thus bring about improved overall health either alone or in conjunction with other components [5, 6, 47–49]. Suffice it to say, advising the public to use a daily ‘insulin sensitizer” should be seriously considered by health practitioners.

Disclosure statement

The authors declare that they have no conflict of interest.

Notes

1 The BMI of the average woman in the U.S. is 29.6, which falls into the category of “overweight.”