Introduction

Classic effects of vitamin D include maintenance of mineral homeostasis by regulation of calcium absorption in the gut and reabsorption by the kidney, and regulation of bone remodeling (1). Among many non-calcemic functions, vitamin D also acts as a necessary cofactor for insulin secretion (2). The main sources of vitamin D are ergocalciferol (D₂)¹ and cholecalciferol, the former normally available in foodstuff and the latter produced in the skin by ultraviolet radiation of 7-dehydrocholesterol. Both of these compounds are hydroxylated in the liver to form 25-hydroxyvitamin D [25(OH)D], which is the major circulating metabolite precursor to the hormonally active form, 1,25-dihydroxyvitamin D [1,25(OH)₂D]. Hydroxylation of 25(OH)D metabolite, stimulated by parathormone (PTH), takes place mainly in the kidney and produces the hormonally active metabolite 1,25(OH)₂D. The 25(OH)D precursor is widely used to assess the efficacy of vitamin D repletion (3) and has a slower clearance rate from the circulatory stream than 1,25(OH)₂D.

Obesity is associated with alterations in the vitamin D endocrine system. The levels of serum intact PTH and 1,25(OH)₂D are elevated in morbidly obese patients, whereas levels of 25(OH)D have been found to be reduced (4, 5, 6, 7, 8, 9, 10, 11). The low levels of 25(OH)D found in obesity have been attributed to several factors such as decreased exposure to sunlight because of limited mobility and negative feedback from elevated 1,25(OH)₂D and PTH levels on hepatic synthesis of 25(OH)D (12). Most likely, obesity-associated vitamin D insufficiency is due to a decreased bioavailability of vitamin D₃ from cutaneous and dietary sources because of its excessive deposition in body fat compartments (7, 13, 14). In healthy subjects, serum 25(OH)D concentration inversely correlates to either fat mass (FM) (14) or BMI (15, 16).

Hypovitaminosis D has been proposed as a risk factor for hypertension (17), metabolic syndrome (16, 18, 19), impaired glucose tolerance, and type 2 diabetes (20, 21, 22, 23, 24). Low serum 25(OH)D has been associated with reduced insulin secretion and sensitivity in 124 healthy subjects (16) and increased concentrations of glucose and insulin during an oral glucose load in 146 elderly men (24).

To test the hypothesis that low levels of 25(OH)D negatively influence insulin sensitivity, we investigated the relation between serum 25(OH)D and insulin sensitivity in morbidly obese women who had undergone biliopancreatic diversion (BPD) and were reevaluated after 5 and 10 years of follow-up. BPD is a type of malabsorptive bariatric surgery (25) that markedly improves insulin sensitivity and lipid metabolism (26, 27) but greatly reduces circulating 25(OH)D and increases PTH levels (28, 29).

Research Methods and Procedures

Study Subjects

The study group included 116 white, non-smoking, morbidly obese women (BMI 40 kg/m²) 20 to 35 years old. The presence of impaired glucose tolerance or type 2 diabetes was evaluated by a standard glucose tolerance test (30). None of the study participants had other relevant endocrine or non-endocrine disease. They were not taking any medications, except that subjects after BPD were prescribed oral supplementation of 525 mg/d sulfate iron, 1 g/d calcium carbonate, 1 tablet/d multivitamins (Supradyn Roche, Milan, Italy), and 400,000 UI intramuscularly (i.m.) D₂ (Ostelin fl, Teofarma, Valle Salimbene, Italy) every 2 weeks. Medical history, physical examinations, electrocardiogram, and blood screening showed that all patients were in good health. None of the subjects had a history of hepatic or renal disorders, and none was taking anticonvulsant medications or corticosteroids. The study was performed during the winter (December through February). The subjects were studied before and 5 and 10 years after BPD.

Body composition was estimated by isotopic dilution (31, 32, 33). Fat free mass (FFM) (in kilograms) was obtained by dividing total body water by 0.73 (33).

The study was approved by the Ethical Committee of the Catholic University. All subjects signed an informed consent document before participation.

Euglycemic-Hyperinsulinemic Clamp (EHC) Procedure

Peripheral insulin sensitivity was measured by the EHC procedure (34). After inserting a cannula in a dorsal hand vein for sampling arterialized venous blood and another in the antecubital fossa of the contralateral arm for infusions, the subjects rested in the supine position for at least 1 hour. They were placed with one hand warmed in a heated air box set at 60 °C to obtain arterialized blood samples. Whole-body glucose uptake (M value in millimoles per kilogram FFM per minute) was determined during a primed constant infusion of insulin (at the rate of 6 pmol/min per kilogram). The fasting plasma glucose concentration was maintained throughout the insulin infusion by means of a variable glucose infusion and blood glucose determinations every 5 minutes. Whole-body peripheral glucose use was calculated during the last 40-minute period of the steady-state insulin infusion.

Surgery

BPD is essentially a malabsorptive surgical procedure (25). It consists of an 60% distal gastric resection with stapled closure of the duodenal stump. The residual volume of the stomach is 300 mL. The small bowel is transected at 250 cm from the ileocecal valve, and its distal end is anastomosed to the remaining stomach. The proximal end of the ileum, comprising the remaining small bowel carrying the biliopancreatic juice and excluded from food transit, is anastomosed in an end-to-side fashion to the bowel 50 cm proximal to the ileocecal valve. The total length of absorbing bowel is brought to 250 cm, the final 50 cm of which, the so-called common channel, represents the site where ingested food and biliopancreatic juices mix.

Analytical Assays

Samples were collected in tubes in an ice bath and frozen immediately at -80 C°. Hormones were all assayed in duplicate. Insulin was measured with a human insulin-specific radioimmunoassay kit (Linco Research Inc, St. Charles, MO) that does not react with proinsulin. 25(OH)D concentration was measured by radioimmunoassay (IDS Immunodiagnostics, IDS Limited, Tyne and Wear, UK) (intra- and interassay coefficients of variation 8.8% and 10.8%, respectively). Serum PTH levels (reference range, 10 to 65 pg/mL) were determined using an intact PTH immunoradiometric assay (Diagnostics Systems Laboratories, Inc., Webster, TX) with a sensitivity of 1.0 pg/mL and intra- and interassay coefficients of variation of 7.1% and 3.5%, respectively.

Statistics

Hypovitaminosis D was defined as a 25(OH)D concentration < 20 ng/mL (3) (to convert to SI units, multiply by 2.496). Data are given as mean SD unless otherwise specified. Differences in continuous variables were estimated by repeated measures ANOVA with multiple-group comparison; Bonferroni's post hoc testing was applied whenever appropriate. p < 0.05 was considered significant. Continuous variables that failed the normality test [25(OH)D, PTH, high-density lipoprotein (HDL)-cholesterol, phosphorum, fasting insulin, and glucose] were logarithmically transformed before analysis. Simple and multiple regression analyses were carried out by standard techniques with 95% confidence limits. SPSS 12.0 for Windows (SPSS Inc, Chicago, IL) was used for statistical analysis.

Results

Analytical Results

Anthropometric data of the study population are summarized in Table 1. Type 2 diabetes was diagnosed in 10 women by oral glucose tolerance test; impaired glucose tolerance was found in five obese patients. Both diseases fully reverted after BPD. Prevalence of hypovitaminosis D was 76% in obese subjects and increased up to 91% after 5 years and 89% after 10 years of follow-up.

Table 1. - Anthropometric characteristics of the study population.

Full table

Serum 25(OH)D concentration decreased from 39.2 22.3 nM (p = 0.0001) to 27.4 16.4 at 5 and 25.1 13.9 nM at 10 years after BPD. Serum PTH levels increased from 50.4 21.3 pg/mL in obese subjects to 82.0 50.5 at 5 years and 93.7 43.8 pg/mL at 10 years (p = 0.0001). Changes of calcium, protein, and albumin concentrations together with other biochemical parameters and insulin-stimulated glucose uptake are reported in Table 2.

Table 2. - Biochemical characteristics of the study population.

Full table

Whole-body glucose uptake (M value) showed a long-lasting amelioration of insulin sensitivity after bariatric surgery. M values increased from 24.3 4.4 in the obese status to 57.3 11.6 and 57.7 8.4 mol/kg_FFM per minute 5 and 10 years, respectively, after BPD (p = 0.0001).

As shown in Table 2, triglycerides and low-density lipoprotein (LDL)-cholesterol significantly fell down after BPD (p = 0.0001), whereas HDL-cholesterol significantly increased (p = 0.0001).

When insulin resistance was defined as the lower 25th percentile and insulin sensitivity as the upper 75th percentile of M values separately in the obese and post-obese status, the prevalence of hypovitaminosis was 76.6% [mean values of serum 25(OH)D 32.1 23.4 nM] in insulin-resistant and 79.3% [mean values of 25(OH) D 40.2 18.6 nM] in insulin-sensitive obese subjects (p = NS) and 83.8% in insulin-resistant and 94.6% in insulin-sensitive subjects after BPD [mean value of 25(OH)D 31.3 16.5 nM vs. 24.2 14.4 nM; p = 0.05].

Predictors of 25(OH)D, PTH, and Whole-Body Glucose Uptake

Linear regression analysis showed a negative correlation between serum 25(OH)D and FM (R² = 0.26; p = 0.0001) both before (Figure 1A) and after (R² = 0.20; p = 0.02) (Figure 1B) BPD.

Figure 1.

Simple scatterplot of the correlation between serum 25(OH)D and FM (kilograms) in obese (A; y = -0.0262x + 4.8323; R² = 0.26; p = 0.0001) and post-BPD women (B; y = -0.0164x + 3.4733; R² = 0.20; p = 0.02). 25(OH)D was logarithmically transformed to normality. () Obese women. () BPD women.

Full figure and legend (22K)

A statistically significant correlation between PTH and M value (R² = 0.16; p = 0.0001) (Figure 2), PTH and FM (R² = 0.19; p = 0.0001) (Figure 3), and PTH and BMI (R² = 0.053; p = 0.01) (Figure 4) was found in obese women.

Figure 2.

Simple scatterplot (y = -0.0275x + 4.522) of the correlation between serum PTH and whole-body glucose uptake (micromolar per kilogram FFM per minute) in obese women (R² = 0.16; p = 0.0001). The skewed variable PTH was logarithmically transformed to normality.

Full figure and legend (27K)

Figure 3.

Simple scatterplot (y = 0.0093x + 3.3953) of the correlation between serum PTH and FM (kilograms) in obese women (R² = 0.11; p = 0.0001). The skewed variable PTH was logarithmically transformed to normality.

Full figure and legend (26K)

Figure 4.

Simple scatterplot (y = 0.010x + 3.37) of the correlation between serum PTH and BMI (kilograms per meter squared) in obese women (R² = 0.053; p = 0.013). The skewed variable PTH was logarithmically transformed to normality.

Full figure and legend (24K)

In a step-wise linear regression (R² = 0.72, p = 0.0001), the most powerful predictors of insulin sensitivity were FM and circulating triglycerides, whereas PTH, 25(OH)D, total cholesterol, both fractions LDL- and HDL-cholesterol, calcium, and phosphorum were not (Table 3).

Table 3. - Multistep regression analysis estimating predictors of insulin sensitivity (M value) in the whole population.

Full table

Discussion

The main findings of the present study are that 25(OH)D serum level is not a major determinant of insulin sensitivity in both morbid obesity and the post-obese status, that hypovitaminosis D and consequent hyperparathyroidism have a large prevalence in post-obese subjects after malabsorptive bariatric surgery, that hypovitaminosis D also has a large prevalence in morbid obesity, and that serum 25(OH)D and PTH levels correlate with FM in the obese condition.

This 10-year observational study provides evidence that hypovitaminosis D does not show any adverse influence on insulin sensitivity in formerly morbidly obese women, at least in a selected population, who lost weight after bariatric surgery. In fact, although serum levels of 25(OH) vitamin D significantly decreased after BPD, insulin sensitivity was reversed to normality. No significant correlation was found between M value and 25(OH)D concentration before and after BPD, whereas powerful predictors of insulin resistance were FM and circulating triglycerides in the whole population and serum PTH in obesity.

The effect of 25(OH)D levels on insulin secretion has been widely proved (24, 35, 36, 37). A clear association between vitamin D levels and insulin sensitivity measured by either the EHC (38, 39, 40) or hyperglycemic clamp (16) technique has been also demonstrated in normal-weight subjects, but no studies have been conducted in morbidly obese subjects.

Concerns about the effective role of hypovitaminosis D on insulin sensitivity have been advanced elsewhere (41). Briefly, latitude-related differences in vitamin D metabolism (42) would be translated into country-related differences in prevalence, unproved at least in the European population (43, 44), of insulin resistance.

The postulated relation between low serum 25(OH)D and reduced insulin sensitivity is a noteworthy hypothesis, which would imply that vitamin D supplementation in obese and diabetic patients might be, as suggested (16), extremely useful in reducing insulin resistance, in lowering the incidence of the metabolic syndrome, and in reverting or reducing glucose intolerance and type 2 diabetes.

In addition, supplementation of calcitriol or its analogue had no effect on insulin-mediated glucose uptake in healthy subjects (39, 45). The main doubt regarding a pivotal influence of low levels of 25(OH)D on insulin sensitivity comes from the evidence that, despite further and significant decrease of 25(OH)D after BPD, obese subjects in our study experienced a full restoration of their insulin sensitivity to normality. It might be argued that the restoration of insulin sensitivity could be related to the large weight loss; nevertheless, some of the subjects in our series, although still obese, had an insulin sensitivity in the range of normality. There is a large body of literature (26, 27, 46, 47, 48, 49, 50, 51) showing that insulin sensitivity is restored to normal in morbidly obese, insulin-resistant subjects within 2 years after BPD. The amelioration of insulin-mediated glucose uptake is likely dependent on reduced FM as well as on lowered circulating and intramuscle triglycerides and free fatty acid (FFA) levels (26, 27, 48, 51, 52). Interestingly, it has been reported that in patients with type 2 diabetes, vitamin D supplementation reduced circulating FFAs (53), and this could be one of the mechanisms by which vitamin D metabolism influences glucose and insulin metabolism. So far, it is conceivable that post-BPD patients would have had enhanced insulin sensitivity if they were replete with vitamin D. In the above-mentioned studies (41, 45), as well as in our study, calcitriol or D₂ has been used. In uremic subjects, intravenous 4-week administration of 1,25(OH)₂D increased insulin sensitivity, measured by the EHC, up to normal values (40). It is possible that 25(OH)D has an effect that is not seen by either calcitriol or D₂.

In the literature, few studies (28, 29, 54) have investigated the effect of malabsorptive bariatric surgery on bone metabolism, 25(OH)D, and PTH levels. To our knowledge, no data are reported concerning the compliance to i.m. D₂ and oral calcium supplementation in subjects who have undergone BPD. In our series, the compliance to i.m. administration of D₂ was poor, but some vitamin D was supplemented through an oral multivitamin complex. In post-BPD subjects, a stimulus on PTH secretion derives not only from the low circulating levels of vitamin D but also from the calcium malabsorption because calcium palmitate is formed in the intestinal lumen and lost with stool. Due to supplementation of calcium and to a hyperproteic diet suggested to our patients to overcome possible protein malabsorption, the serum concentration of total calcium remained in a normal range. However, we are aware that measuring ionized calcium would have provided somewhat better information of calcium deficiency.

Another relevant result of our study is the finding of a high prevalence of hypovitaminosis D in morbidly obese women. Several lines of evidence suggest the role of a good vitamin D status in preventing obesity and diabetes (20, 21, 22, 23, 24, 55, 56, 57, 58).

High levels of PTH and 1,25(OH)₂D stimulate Ca²⁺ influx in human adipocytes. Zemel and colleagues (57) have shown that high levels of intracellular free calcium in the adipocyte stimulate lipogenesis through the activation of fatty acid synthase and inhibit lipolysis, thus leading to the expansion of adipocyte triglyceride stores. Conversely, the reduction of 1,25(OH)₂D levels, obtained by increasing dietary calcium, inhibits adiposity and promotes weight loss (58). High intake of calcium also caused an increase in uncoupling protein-2 expression during energy restriction in mice (58).

In our series, serum 25(OH)D levels varied with total body adiposity in morbidly obese and post-BPD women, confirming previous reports in normal and obese subjects (4, 5, 6, 7, 8, 9, 10, 11, 14). In obese subjects, 25(OH)D decreased by 4.8 nM and PTH increased by 3.4 pg/mL for each kilogram of FM. Adiposity influences volume distribution of 25(OH)D (7, 14, 59), but pharmacokinetic and pharmacodynamic characteristics of vitamin D pathways depend on several other factors, including race, age, sex, dietary intake of dairy products, and exposure to sun rays.

Very recently, Parikh et al. (15) found a significant positive relation between PTH and BMI or total body adiposity in obese subjects that our outcomes fully confirm. The Tromso study (60), a very large epidemiological study, demonstrated that PTH hormone levels correlated well with BMI in the healthy population.

In our series, we also found a positive correlation between serum PTH and insulin resistance, which evokes the old question of the insulin resistance in primary (57, 61) and secondary (45) hyperparathyroidism. It has been demonstrated that insulin sensitivity is not ameliorated by decreasing PTH levels (40, 45). Nevertheless, PTH stimulates intracellular flux of calcium in primary cultures of human adipocytes (56), blunting the lipolytic response to catecholamines through the activation of the phosphodiesterase 3B, the same enzyme that mediates the antilipolytic effect of insulin, and reducing the efficiency of insulin-mediated glucose uptake (56). Moreover, serum PTH seems to interfere with the ability of pancreatic -cells to increase insulin secretion appropriately in response to the glucose stimulus (62).

In conclusion, the prevalence of hypovitaminosis D is elevated in morbidly obese subjects, although 25(OH)D levels do not seem to be the major determinant of their insulin resistance. Furthermore, low levels of 25(OH)D do not necessary imply increased insulin resistance after BPD, a condition where, probably, reduced levels of circulating FFAs and triglycerides and intramyocytic fat depots are more powerful determinants of insulin sensitivity. However, the present data cannot exclude some kind of influence of vitamin D status on glucose and insulin metabolism; thus, further molecular and epidemiological investigations are needed to shed light on this topic.

Notes

¹ Nonstandard abbreviations: D₂, ergocalciferol; 25(OH)D, 25-hydroxyvitamin D; 1,25(OH)₂D, 1,25-dihydroxyvitamin D; PTH, parathormone; FM, fat mass; BPD, biliopancreatic diversion; i.m., intramuscular(ly); FFM, fat free mass; EHC, euglycemic-hyperinsulinemic clamp; HDL, high-density lipoprotein; LDL, low-density lipoprotein; FFA, free fatty acid.

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Acknowledgments

We thank Anna Caprodossi for her technical assistance. There was no funding/outside support for this study.