Vitamin D deficiency impairs glucose-stimulated insulin secretion and increases insulin resistance by reducing PPAR-γ expression in nonobese Type 2 diabetic rats

    Vitamin D deficiency impairs glucose-stimulated insulin secretion and increases insulin resistance by reducing PPAR-γ expression in non-obese type 2 diabetic rats Sunmin Park, Da Sol Kim, Suna Kang PII: DOI: Reference:

S0955-2863(15)00249-1 doi: 10.1016/j.jnutbio.2015.09.013 JNB 7459

To appear in:

The Journal of Nutritional Biochemistry

Received date: Revised date: Accepted date:

21 April 2015 3 September 2015 15 September 2015

Please cite this article as: Park Sunmin, Kim Da Sol, Kang Suna, Vitamin D deficiency impairs glucose-stimulated insulin secretion and increases insulin resistance by reducing PPAR-γ expression in non-obese type 2 diabetic rats, The Journal of Nutritional Biochemistry (2015), doi: 10.1016/j.jnutbio.2015.09.013

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ACCEPTED MANUSCRIPT Vitamin D deficiency impairs glucose-stimulated insulin secretion and increases insulin

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resistance by reducing PPAR-γ expression in non-obese type 2 diabetic rats

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Sunmin Park, Da Sol Kim, Suna Kang

Dept. of Food & Nutrition, Obesity/Diabetes Research Institutes, College of Natural Science,

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Hoseo University, Asan, Korea

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Running Head: Vitamin D and glucose metabolism

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Correspondence author: Sunmin Park, Ph.D Dept. of Food and Nutrition, Hoseo University 165 Sechul-Ri Baebang-Myun Asan-Si Chungnam-Do, 336-795, Korea Tel: 82-41-540-5633 Fax: 82-41-548-0670 E-mail: [email protected] 1

ACCEPTED MANUSCRIPT Abstract Human studies have provided relatively strong associations of poor vitamin D status

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with type 2 diabetes, but do not explain the nature of the association. Here, we explored

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the physiological pathways that may explain how vitamin D status modulates energy, lipid and glucose metabolisms in non-obese type 2 diabetic rats. Goto-Kakizaki (GK) rats were fed

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high fat diets containing 25 (VD-low), 1,000 (VD-normal), or 10,000 (VD-high)

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cholecalciferol IU/kg diet for 8 weeks. Energy expenditure, insulin resistance, insulin secretory capacity, and lipid metabolism were measured. Serum 25-OH-D levels, an index of vitamin D status, increased dose-dependently with dietary vitamin D. VD-low resulted in less fat oxidation without a significant difference in energy expenditure, and less lean body mass

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in the abdomen and legs comparison to the VD-normal group. In comparison to VD-low, VD-normal had lower serum triglycerides and intracellular fat accumulation in the liver and

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skeletal muscles which was associated with downregulation of the mRNA expressions of

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sterol regulatory element binding protein-1c and fatty acid synthase, and upregulation of gene expressions of peroxisome proliferator-activated receptors (PPAR)-α and carnitine

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palmitoyltransferase-1. In euglycemic hyperinsulinemic clamp, whole body and hepatic insulin resistance was exacerbated in the VD-low group but not in the VD-normal group, possibly through decreasing hepatic insulin signaling and PPAR-γ expression in the adipocytes. In 3T3-L1 adipocytes 1,25-(OH)2-D (10 nM) increased triglyceride accumulation by elevating PPAR-γ expression and treatment with a PPAR-γ antagonist blocked the triglyceride deposition induced by 1,25-(OH)2-D treatment. VD-low impaired glucosestimulated insulin secretion in hyperglycemic clamp and decreased β-cell mass by decreasing β-cell proliferation. In conclusion, vitamin D deficiency resulted in the dysregulation of 2

ACCEPTED MANUSCRIPT glucose metabolism in GK rats by simultaneously increasing insulin resistance by

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decreasing adipose PPAR-γ expression, and deteriorating β-cell function and mass.

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Key words: vitamin D, insulin resistance, insulin secretion, energy expenditure, lean body

Introduction

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1.

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mass, PPAR-γ

Vitamin D plays an important role in bone metabolism through the classical pathway that regulates serum calcium and phosphorous levels, and its deficiency results in bone diseases such as rickets in children and osteomalacia in adults [1]. However, in addition to organs

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associated with bone metabolism such as bone, kidney and intestines; vitamin D receptors (VDR) are also expressed in many other tissues, including pancreatic β-cells, vascular

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endothelial cells, neurons, immune cells, osteoblasts, and myocytes [2]. These observations

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suggest that vitamin D may modulate metabolic actions in energy, glucose, and/or lipid metabolism. Growing evidence from human observational studies indicates that vitamin D

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deficiency is positively associated with metabolic diseases such as musculoskeletal, cardiovascular, neoplastic diseases, and type 2 diabetes [2]. However, the mechanisms by which vitamin D exerts these effects remains unknown. A possible mechanism is as follows: VDR is a nuclear receptor that dimerizes with retinoid X receptors (RXRs) in adipose tissues like peroxisome proliferator-activated receptor (PPAR)-γ [3]. Vitamin D enhances PPAR-γ expression during adipogenesis and vitamin D and PPAR-γ may participate in cross-talk to conduct the VD-mediated action [4]. However, this is still controversial due to the differences in dosage and species in previous studies [5,6]. Since PPAR-γ is also involved in glucose 3

ACCEPTED MANUSCRIPT metabolism and inflammation [7], vitamin D may modulate these metabolisms by modulating PPAR-γ signaling.

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Vitamin D status is reportedly associated with energy balance and body composition in

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humans. Obesity is inversely associated with serum 25-hydroxy cholecalciferol (25-OH-D) levels in humans [8,9]. Moreover, vitamin D deficiency was associated with loss of skeletal

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muscle mass and function independent of obesity in both a cross-sectional study and

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prospective study [10,11]. However, most intervention studies have failed to demonstrate a beneficial effect of vitamin D supplementation on body weight [12]. As compared to humans, mice have higher levels of 1,25-dihydroxy cholecalciferol (1,25-(OH)2-D), the active form of vitamin D, and mice with human VDR overexpression not only exhibit increased

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adipogenesis and expression of typical adipocyte genes, but also decreased energy expenditure due to lower expression of uncoupling proteins [13,14]. Moreover, VDR

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knockout mice lose fat mass over time owing to an increase in energy expenditure, whereas

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mice with increased VDR mediated signaling in adipose tissue become obese [13,15]. Thus, VDR signaling in mice may be negatively associated with body fat mass, at least partially, by

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decreasing energy expenditure. Vitamin D status is also associated with glucose metabolism. In a systematic review and meta-analysis, Forouhi et al. [16] found that only prospective studies showed inverse associations between circulating vitamin D levels and incidence of type 2 diabetes. In a crosssectional survey, the National Health and Nutrition Examination Survey (NHNES) III in the USA found an inverse relationship between vitamin D status and the incidence of type 2 diabetes [17]. However, the inverse association was only found in young women and old men in the Korean KNHANES study, indicating gender and age specificity [18]. However, 4

ACCEPTED MANUSCRIPT George et al. [19] demonstrated that in randomized trials of vitamin D supplementation there is no evidence to recommend vitamin D supplementation for patients with type 2 diabetes or

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impaired glucose intolerance for treating diabetes and/or improving glycemic control.

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Therefore, it remains unclear whether vitamin D supplementation is beneficial for type 2 diabetes. However, a systematic review by Mitri et al. [20] suggests that there is little

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association between vitamin D intake, at least at the levels in those studies, and type 2

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diabetes. However, low vitamin D status based, on serum 25-OH-D levels, was highly associated with the prevalence of type 2 diabetes in most studies. Therefore, much of the uncertainty may be due to a possible disconnect between vitamin D intake and vitamin D status.

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We hypothesized that vitamin D deficiency and supplementation might modify energy and glucose metabolism, possibly through modulation of PPAR-γ activation, in a type 2

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diabetic animal model. The purpose of the study was to test the hypothesis and to explore

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which physiological pathways may be involved using the Goto-Kakizaki (GK) rat, which is a

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non-obese Wistar substrain that develops type 2 diabetes mellitus early in life.

Materials and methods

2.1. Animals and Ethics Eight week old male GK rats (weighing 236±19 g) were housed individually in stainless steel cages in a controlled environment (23°C and with a 12-h light/dark cycle). All experimental procedures were performed according to the guidelines of the Animal Care and Use Review Committee of Hoseo University, Korea (2013-03).

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ACCEPTED MANUSCRIPT 2.2. Experimental design Forty-eight GK rats were randomly assigned to the following three groups according to

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vitamin D (cholecalciferol; Sigma, St. Louise, MA, USA) contents in the diet: 1) vitamin D

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deficient (VD-low; 25 IU cholecalciferol/kg diet); 2) vitamin D sufficient (VD-normal; 1,000 cholecalciferol IU/kg diet), and 3) excessive vitamin D (VD-high; 10,000 cholecalciferol

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IU/kg diet). All experimental animals were given free access to water and a moderately high-

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fat Western type diet containing the assigned cholecalciferol contents during the 8 week experimental period. The high-fat diet was a modified semi-purified AIN-93 formulation for experimental animals [21]. The diet consisted of 40 percent energy (En%) from carbohydrates, 20 En% from protein and 40 En% from fats (Table 1). The major

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carbohydrate, protein and fat sources were starch plus sugar, casein (milk protein) and lard (CJ Co., Seoul, Korea), respectively.

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Overnight-fasted serum glucose levels, food and water intakes, and body weights were

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measured every Tuesday at 10 am. Insulin resistance was determined using the homeostasis model assessment estimate of insulin resistance (HOMA-IR) [HOMA-IR = fasting serum

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insulin (µIU/ml) × fasting serum glucose (mM) / 22.5]. At the end of the study, rats were anesthetized with ketamine and xylazine (100 and 10 mg/kg body weight, respectively). Epididymal and retroperitoneal fat pads were then removed and weighed. After blood collection by abdominal cardiac puncture, human insulin (5U/kg body weight) was injected through the inferior vena cava to determine insulin signaling in the liver. Serum 25-OH-D levels were measured using an electrochemiluminescence immunoassay (ECLIA; Molecular Analytics E170 Roche Diagnostics, Germany). The inter-assay coefficients of variation were 2.6% and the antibody is specific for 25-OH-D. Serum samples were then stored at -70°C for 6

ACCEPTED MANUSCRIPT biochemical analysis.

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2.3. Energy expenditure analysis by indirect calorimetry

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After 7 weeks of the assigned treatment, energy expenditure was assessed at the beginning of the dark phase of the light/dark cycle after 6 h of fasting. The rats were placed

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into metabolic chambers (airflow = 800 ml/min) with a computer-controlled O2 and CO2

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measurement system (BIOPAC Systems, Inc., Goleta, CA) to determine their calorimetric parameters. The respiratory quotients (RQ) and resting energy expenditures (REE) were calculated using previously reported equations [22,23].

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2.4. Bone mineral density (BMD) and lean and fat mass measurements A densitometer was calibrated daily with a phantom supplied by the manufacturer to

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measure body composition. Two days later after measuring energy expenditure, rats were

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anesthetized with ketamine and xylazine (100 and 10 mg/kg body weight, respectively), and each rat was laid in a prone position with their hind legs maintained in external rotation with

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tape. Hip, knee and ankle articulations were in 90° flexion. Upon completion of scanning, BMD was determined in the designated and equivalent areas of the right femur and lumbar spine in all rats by dual-energy X-ray absorptiometry (DEXA) using an absorptiometer (pDEXA Sabre; Norland Medical Systems Inc., Fort Atkinson, WI, USA), which was equipped with the appropriate software for assessment of bone density in small animals [23]. Similarly, abdominal fat mass and lean mass were measured by DEXA.

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ACCEPTED MANUSCRIPT 2.5. Insulin resistance as assessed by euglycemic hyperinsulinemic clamp After measuring body composition by DEXA, catheters were surgically implanted into

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the right carotid artery and left jugular vein of the rats from each group. At five to six days

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after catheterization, 8 overnight-fasted free-moving rats in each group had a euglycemic hyperinsulinemic clamp and the remaining 8 rats from each group were used in a

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hyperglycemic clamp study. A euglycemic hyperinsulinemic clamp was performed to

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measure insulin resistance by continuous infusion of [3-3H] glucose (NEN Life Science, Boston, MA) during a 4-hour period at the rate of 0.05 μCi/min [24,25]. Basal hepatic glucose output was measured at 100 and 120 minutes after the initiation of the [3-3H] glucose infusion. Then, a primed continuous infusion of human regular insulin (Humulin; Eli Lilly,

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Indianapolis, IN) was initiated at a rate of 20 pmol × kg–1 × min–1 to raise plasma insulin concentrations to approximately 1100 pM at 200-240 minutes during the clamp. Glucose

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(25%) was infused at variable rates as needed to clamp glucose levels at about 6 mM. While

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serum glucose and insulin levels were steady between 200 and 240 minutes, the rate of hepatic glucose production was determined by measuring the blood levels of [3-3H] glucose

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and 3H2O every ten minutes. Clamped hepatic glucose output was calculated by subtracting glucose infusion rates from the rates of glucose appearance. Glucose infusion rate was expressed in terms of mg of glucose per kg of body weight per minute required to maintain euglycemia during hyperinsulinemia. After completing the clamp, tissues were rapidly collected and frozen in liquid nitrogen, and stored at –70oC for further analysis.

2.6. Glucose-stimulated insulin secretion during hyperglycemic clamp A hyperglycemic clamp was performed in ten free-moving and overnight-fasted rats to 8

ACCEPTED MANUSCRIPT determine insulin secretory capacity as described in previous studies [25,26]. During the clamp, glucose was infused to maintain serum glucose levels of 5.5 mM above the baseline

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and serum insulin levels were measured at designated times. After the clamp, rats were freely

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provided with food and water for 2 days and on the next day they were deprived of food for 16 hours. Cerebrospinal fluid was then obtained from each rat by cisternal puncture. Prior

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to sacrificing the rats, they were anesthetized with a mixture of ketamine and xylazine and

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5U insulin /kg body weight was injected through the inferior vena cava for insulin stimulation. Fifteen min later, they were killed by decapitation and tissues were rapidly dissected and frozen in liquid nitrogen, and stored at -70 oC for further analysis.

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2.7. Isolation of total RNA in tissues and real-time PCR

The liver, epididymal fat pads and brown adipose tissues were collected at the end of the

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treatment period, powdered with a cold steel mortar and pestle, and then mixed with a

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monophasic solution of phenol and guanidine isothiocyanate (TRIzol reagent; Gibco-BRL, Rockville, MD) for total RNA extraction, according to the manufacturer’s instructions. RNA

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was determined using a Lamda 850 spectrophotometer (Perkin Elmer, Waltham, MA, USA) and cDNA was synthesized from 1 μg RNA extracted from individual rats using a superscript III reverse transcriptase kit (Life Science Technology). Five different cDNAs were made from each group and each cDNA was used for realtime PCR. Equal amounts of cDNA and primers for specific genes were mixed with SYBR Green mix (Bio-Rad, Richmond, CA) in duplicate and amplified using a real-time PCR instrument (Bio-Rad) according to the manufacturer’s manual. To assess changes in the expressions of genes related to fatty acid synthesis and oxidation, the mRNA expressions of sterol regulatory element-binding protein9

ACCEPTED MANUSCRIPT 1c (SREBP-1c), fatty acid synthase (FAS), peroxisome proliferator-activated receptor (PPAR)-α, and carnitine palmitoyltransferase-1 (CPT-1) in the liver, PPAR-γ, CPT-1 and

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tumor necrosis factor (TNF)-α in the adipose tissues and uncoupling protein (UCP)-1 in the

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brown adipocytes were measured with corresponding primers as previously described [23,26]. Cycle of threshold (CT) for each sample was determined. The gene expression levels in

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unknown samples were quantified using the comparative CT method (ΔΔCT method) [27].

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ΔCT was calculated via formula: ΔCT = CT (target gene) – CT (endogenous reference gene, β-actin). Relative fold-changes in expression were calculated by the equation of ΔΔCt = ΔCttreatment − ΔCtcontrol. Results were presented as 2-ΔΔCT.

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2.8. Immunoblot analysis

The frozen liver tissues collected after 10 min of insulin stimulation were lysed with a 20

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mM Tris buffer (pH 7.4) containing 2 mM EDTA, 137 mM NaCl, 1% NP40, 10% glycerol,

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and 12 mM α-glycerol phosphate and protease inhibitors. The protein concentrations of the lysates were determined using a protein assay kit (Bio-Rad). Lysate samples with equivalent

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protein levels (30‒50 μg) were directly resolved by SDS-PAGE. Immunoblotting was performed using specific antibodies against protein kinase B (PKB or Akt), phosphorylated PKBSer473,

glycogen

synthase-3β

(GSK-3β),

phosphorylated

GSK-3βser9,

phosphoenolpyruvate carboxykinase (PEPCK) and β-actin (Cell Signaling Technology, Beverly, MA, USA) as described previously [25]. The intensity of protein expression was determined using the Imagequant TL software package (Amersham Biosciences, Piscataway, NJ, USA). These experiments were repeated three times for each group.

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ACCEPTED MANUSCRIPT 2.9. Immunohistochemistry and islet morphometry At the end of the 8-week experimental period, six rats from each group were injected

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with BrdU (100 µg/kg body weight). Six hours post-injection, each rat was anesthetized

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with an intraperitoneal injection of ketamine and xylazine, and the pancreas was immediately dissected. The pancreas was fixed with 4% paraformaldehyde and paraffin-embedded, as

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described in previous studies [25]. Two serial 5-μm paraffin-embedded tissue sections were

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selected out of each seventh or eighth section to avoid counting the same islet twice. Endocrine β-cells were identified by applying guinea pig anti-insulin antibodies to the sections. BrdU incorporation in β-cells was determined by staining rehydrated paraffin sections with anti-insulin and anti-BrdU antibodies [25]. Apoptosis of β-cells was determined

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by TUNEL kit (Roche Molecular Biochemicals, Indianapolis, IN) and counterstained with hematoxylin to visualize nucleus [25]. Pancreatic β-cell area was measured by examining all

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non-overlapping images in two insulin-stained sections of each rat at a magnification of 10x

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with a Zeiss Axiovert microscope (Carl Zeiss Microimaging, Thornwood, New York). The βcell quantification was expressed as the percentage of the total surveyed area containing

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insulin-positive cells, measured by IP Lab Spectrum software (Scanalytics Inc., Fairfax, VA). Pancreatic β-cell mass was calculated by multiplying the percentage of insulin-positive areas by the weight of the corresponding pancreatic portion [25]. The individual β-cell size was determined as the insulin-positive area divided by the number of nuclei counted in the corresponding insulin-positive structures in randomly immunofluoresence-stained sections.

2.10. PPAR-γ activity and triacylglycerol accumulation in 3T3-L1 adipocytes Human embryonic kidney 293 cells were transiently transfected with a PPRE-luciferase 11

ACCEPTED MANUSCRIPT construct (firefly pGL3-DR-1-luciferase; 0.12 μg DNA∙well-1), pSV-SPORT-PPAR-γ expression vector (0.12 μg DNA∙well-1) and pSV-SPORT-retinoid X receptor-α vector (0.08

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μg DNA∙well-1) with Lipofectamine PLUS reagent (Invitrogen, Carlsbad, CA) according to

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the manufacturer's protocol. PPAR-γ activity was determined as previously described [25,26]. Briefly, for an assessment of transfection efficiency, renilla phRL-TK vector (10 ng

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DNA∙well-1, Promega, Madison, WI) was also transfected. After 2 h of transfection, vehicle

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(DMSO), 1,25-(OH)2-D (Sigma), or selective PPAR-γ antagonist, a blocker of transcriptional activity of PPAR-γ (T 0070907; TOCRIS Bioscience, Bristol) were added into media for 40 h and the media was changed to serum-free DMEM containing 0.1 % BSA, which also contained the respective compounds, for 12 h [27]. Cell lysates were assayed for both firefly

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(PPRE-luciferase) and renilla luciferase activities using the Dual-Luciferase Reporter Assay System (Promega) and an Aureon PhL luminometer (Aureon Biosystems, Vienna, Austria).

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Ratios of firefly luciferase activity and renilla luciferase activity were calculated for results.

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Vehicle (DMSO), 1,25-(OH)2-D (Sigma), or selective PPAR-γ antagonist, a blocker of transcriptional activity of PPAR-γ (T 0070907; TOCRIS Bioscience, Bristol) were added into

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media with the differentiation inducers for 4 days during the differentiation of 3T3-L1 fibroblasts, and then the cells were treated with vehicle or 1,25-(OH)2-D (Sigma), or selective PPAR-γ antagonist without differentiation inducers for 6 additional days. At the end of incubation, the cells were harvested with a lysis buffer without glycerol and the triacylglycerol contents were measured using a Trinder kit (Young Dong Pharmaceutical Co., Seoul, Korea) as previously described [27]. The results were expressed as the percentage of change in triacylglycerol concentration from the baseline (vehicle treatment).

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ACCEPTED MANUSCRIPT 2.11.

Statistical analysis

Statistical analysis was performed using SAS, version 7.0 (SAS, Inc. Cary, NC USA).

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Results are expressed as means ± standard deviations. The significant differences among VD-

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low, VD-normal, and VD-high were analyzed by one-way analysis of variance (ANOVA). Multiple comparisons among the groups were identified by Tukey’s tests. P<0.05 was

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3.

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considered statistically significant.

Results

3.1. Energy metabolism

Serum 25-OH-D levels, an index of vitamin D status, were lower in the descending order

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of VD-high, VD-normal group, and VD-low. Although vitamin D intake was 400 times higher in the VD-high group than the VD-low group, there was only a 10-fold difference in

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serum concentrations. Body weight at the end of experiment was not significantly different

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among groups according to vitamin D status. In addition, visceral fat mass, the sum of epididymal and retroperitoneal fat pad weights was higher in the VD-high group than the

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VD-normal group and VD-low (Table 2). However, serum leptin levels exhibited the same tendency (Table 2). Since energy balance is the net of food intake and energy expenditure, both were measured, but did not differ among the groups (Table 2). Energy expenditure from carbohydrate was not different among the groups, but energy expenditure from fat was greater in the VD-normal and high groups than VD-low (Table 2).

3.2. Vitamin D deficiency altered body composition There was no significant difference in BMD of the femur and spine among the different 13

ACCEPTED MANUSCRIPT VD groups (Fig. 1A). Without changing body weight, body composition of lean and fat mass was altered. Vitamin D deficiency lowered lean mass in the abdomen and leg but increased

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fat mass only in the legs compared to the VD-normal group (Fig. 1B and 1C). However,

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neither lean body mass nor fat mass of the VD-high group was significantly different from

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deficiency

exacerbated

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resistance

during

euglycemic

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3.3. Vitamin

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either the VD-high or VD-low group.

hyperinsulinemic clamp

During euglycemic hyperinsulinemic clamp, serum glucose and insulin levels were maintained from 90 to 120 min at 5.6±0.7 mmol/L and 1153±254 pmol/L, respectively,

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and were not significantly different among groups (data not shown). Whole body glucose uptake during euglycemic hyperinsulinemic clamp was not significantly

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different among the groups (Fig. 2A). However, the glucose infusion rates were lower in

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the descending order of the VD-normal group, the VD-high group and the VD-low group. Hepatic glucose outputs at the basal (fasting) state tended to be higher in the VD-low

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group than the other groups, but it was not statistically significant (p=0.08); whereas hepatic glucose output in the hyperinsulinemic state was significantly greater in the VD-low group than the VD-normal and VD-high groups (Fig. 2B). The higher hepatic glucose output during the hyeprinsulinemic clamp indicates an attenuation of hepatic insulin sensitivity.

3.4. Vitamin D deficiency attenuated hepatic insulin signaling Hepatic insulin sensitivity is associated with hepatic insulin signaling that modulates PEPCK expression, which in turn regulates hepatic glucose output at the 14

ACCEPTED MANUSCRIPT hyperinsulinemic state. VD-low reduced the phosphorylation of Akt in comparison to VD-normal (Fig. 2C, p<0.05). Furthermore, the phosphorylation of GSK-3β was also

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3.5. Vitamin D deficiency impaired lipid metabolism

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lower in the VD-low group than in the VD-normal group.

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attenuated in VD-low, compared to VD-normal (Fig. 2C). PEPCK protein levels were

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Serum triglyceride levels were significantly higher in the VD-low group than the other groups (Table 3). The triglyceride contents in the liver and gastrocnemius muscles were also greater in the vitamin D deficient status than the other groups (Table 3). However, those in the quadriceps muscle were lower in the VD-high group than the

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other groups.

In comparison to the VD-low, the lower hepatic triglyceride contents in the VD-

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normal and VD-high groups were related to the higher expression of genes associated

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with fatty acid oxidation such as PPAR-α and CPT-1 and the lower the expression of genes involved in fatty acid synthesis such as FAS and SREBP-1c (Fig. 3A).

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The changes in abdominal fat mass, especially epididymal fat, were also associated with the expressions of genes involved in fatty acid synthesis and oxidation. VD-low had lower PPAR-γ expression than VD-normal and VD-high whereas the expression of genes associated with fatty acid oxidation, such as CPT-1, were not significantly different according to the vitamin D status (Fig. 3B). However, UCP1 expression, which is related to thermogenesis, increased in brown adipocytes of VD-normal, but not VD-high, in comparison to VD-low. In particular the expression of TNF-α, a pro-inflammatory cytokine, was higher in VD-low than the other groups (Fig. 3B). 15

ACCEPTED MANUSCRIPT 3.6. Vitamin D deficiency impaired insulin secretion Overnight-fasted serum glucose levels were not different among the groups whereas

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serum insulin levels at the fasting state were higher in the VD-normal group than the

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VD-low group (Table 4). HOMA-IR, an index of insulin resistance calculated from fasted serum glucose and insulin levels, were greater in the VD-low group than the

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other groups (Table 4). During hyperglycemic clamp, serum glucose levels gradually

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increased up to 5.5 mM above the fasting serum glucose levels until 50 min and the serum glucose levels were maintained at 300 mg/dL from 60 min to 120 min in all groups (data not shown). Serum insulin levels were increased right after glucose infusion and decreased at 10 min, representing the first phase insulin secretion, and the levels tended to decrease at 60 and

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90 min (Fig. 4A). Overall, insulin secretory capacity, represented by the area under the curve of serum insulin levels in the first (0 to 10 min) and the second phase (60-120 min) phases,

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was lower in the VD-low group than the other groups (Fig. 4B; Table 4). Especially, the

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increment of serum insulin levels at the first phase was very low in the VD-low group. The glucose infusion rates during the hyperglycemic clamp state were decreased in

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the descending order of the VD-normal group, the VD-high group and VD-low group. Insulin sensitivity at hyperglycemic states, calculated as the ratio of glucose infusion rates to steady-state serum insulin levels, exhibited the same trend as glucose infusion rates (Table 4). Thus, vitamin D deficiency impaired β-cell function and insulin sensitivity at the hyperglycemic state.

3.7. Vitamin D deficiency reduced β-cell mass The percentage of β-cell area in the total pancreas area of a section was smaller in the 16

ACCEPTED MANUSCRIPT VD-low groups than the VD-normal and VD-high groups (Fig. 4C, Table 5). Since pancreas weight was not significantly different according to the vitamin D status (data not shown), β-

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cell mass showed a similar pattern as the percentage of β-cell area. The percentage of β-cell

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area is determined by the net number of β-cells and individual size of β-cells. Individual βcell size was greater in the VD-low group than the VD-high group (Table 5). Vitamin D status

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affected the proliferation of β-cells but not apoptosis: vitamin D deficiency lowered β-cell

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proliferation compared to the other groups (Table 5). These results indicated that low vitamin D status modulated islet morphometry by impairing β-cell proliferation.

3.8. PPAR-γ activity and triacylglycerol accumulation in 3T3-L1 adipocytes

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Treatment with 1,25-(OH)2-D increased PPAR-γ activity in a dose-dependent manner in HEK 293 cells transfected with pSV-SPORT-PPAR-γ expression vector, pSV-SPORT-retinoid X

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receptor (RXR)-α vector and renilla phRL-TK vector (Fig. 5A), although the increase with

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1,25-(OH)2-D was not as great as with rosiglitazone treatment (a commercial PPAR-γ activator). However, the increase by 1,25-(OH)2-D treatment was abolished by adding a

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PPAR-γ antagonist (T0070907). This indicated that vitamin D acts through PPAR-γ activation. In addition, 1,25-(OH)2-D treatment elevated triglyceride accumulation at high doses, but co-treatment with PPAR-γ antagonist and vitamin D blocked the deposition (Fig. 5B). Furthermore, insulin-stimulated glucose uptake also increased with 1,25-(OH)2-D treatment in a dose-dependent manner (data not shown).

4.

Discussion Although observational human studies support a link between vitamin D status and 17

ACCEPTED MANUSCRIPT diabetes, little is known about the molecular pathways involved and few studies have been performed to investigate the effects of vitamin D on energy and glucose metabolism in type 2

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diabetic animal models. The present study was a novel exploration of the effects of vitamin D

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status on the integration of energy, glucose and lipid metabolism in GK rats, non-obese type 2 diabetic rats that have similar characteristics as Asian type 2 diabetes [29,30]. The present

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study demonstrated that varying dietary vitamin D intakes could modify serum 25-OH-

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D levels in GK rats, resulting in profound metabolic disturbances. Vitamin D deficiency impaired energy, lipid and glucose metabolism in GK rats compared to rats with normal vitamin D status. However, excessive supplementation of vitamin D tended to have less benefit for insulin resistance, and most metabolic parameter values were intermediate

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between rats with low and normal vitamin D intakes and they were not significantly different from either. Therefore, vitamin D intake needs to be in an optimal range that is

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neither too low nor too high.

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Vitamin D status is assessed by serum 25-OH-D levels that are positively correlated with dietary intakes of vitamin D [31]. In the present study, serum 25-OH-D levels in

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the rats were positively associated with dietary vitamin D, although the association was not linear. Drincic et al. [31] has also shown that serum 25-OH-D levels in response to vitamin D intake is directly related to dose and body size with about 2.5 IU/kg body weight required for every unit increment in serum levels of 25-OH-D in obese adults. However, our data indicated that a high dosage of vitamin D intake might result in lower utilization of vitamin D including absorption rates and hydroxylation in the liver than do lower intakes since the increase of serum 25-OH-D levels were only slightly higher in VD-high than VD-normal. Thus, vitamin D intakes did not linearly increase 18

ACCEPTED MANUSCRIPT serum 25-OH-D levels in the rats, and is probably also true for humans within certain ranges.

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Vitamin D deficiency is also reported to be associated with the loss of skeletal

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muscle mass and strength especially in the elderly [32,33], but no intervention trials have been performed to determine the impact of vitamin D supplementation on body

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composition in humans. The present study found that vitamin D sufficiency preserved

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lean body mass that was lower in vitamin D deficient rats and intermediate with excessive vitamin D supplementation. BMD also exhibited a similar tendency to that of lean body mass, but it was not significantly different. VDR knockout mice exhibit atrophy of type I and II muscle fibers at 8 weeks of age due to insufficient myogenesis

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in comparison to the age-matched wild type mice [34] and also have impaired overall motor performance [35]. Thus, adequate vitamin D status may be needed to maintain

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lean body mass and proper skeletal function.

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There are some inconsistent results on the association of vitamin D status and fat mass in human and animal studies [36-39]. Cross-sectional and prospective human

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studies have revealed a beneficial effect of vitamin D for decreasing visceral fat mass [9,36] but randomized clinical trials of vitamin D supplementation have shown inconsistent effects on body fat mass in obese adults [37,38]. The present study provided a clue for why there are inconsistent results in human vitamin D supplementation studies. It may be related to the dosage. The VD-high group had higher epididymal fat mass compared to the VD-normal rats, and the VD-low group tended to be higher also, but was not significantly different from either group. The fat mass increase may be the net of fat synthesis and oxidation. These results may be associated 19

ACCEPTED MANUSCRIPT with the role of vitamin D in not only fat synthesis, but also fat oxidation. VD-normal and VD-high groups had both higher fat oxidation and lower expressions of genes

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related to fatty acid synthesis as compared to VD-low. In the cell-based study, 1,25-

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(OH)2 -D increased triglyceride accumulation in 3T3-L1 adipocytes in a dose-dependent manner, and PPAR-γ antagonist blocked the increase. Thus, triglyceride deposition with

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vitamin D treatment was increased through PPAR-γ in adipocytes; however,

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physiological levels of vitamin D intake may not alter PPAR-γ activation in type 2 diabetic rats, even though its superphysiological dosage may increase it to elevate fat mass in cells.

Vitamin D deficiency is associated with the development of type 2 diabetes and with

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increased insulin resistance and impaired insulin secretion in human and animal studies [3640]. Cross-sectional and prospective studies have revealed that vitamin D deficiency is

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involved in the derangement of insulin resistance and insulin secretion which is commonly

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involved in the etiology of type 2 diabetes mellitus [9,16-18]. A systematic review and metaanalysis of prospective studies suggested that maintaining adequate levels of vitamin D may

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be a useful preventive measure for metabolic diseases including type 2 diabetes [9]. A few studies have evaluated the alteration of glucose metabolism in type 2 diabetic rats, but the mechanisms were not studied [37,41]. Vitamin D consumption (10 IU/kg body weight) for 60 days decreased fasting plasma glucose levels, HbA1c and insulin resistance index in male Wistar rats intraperitoneally injected with a single low dose of streptozotocin (35 mg/kg body weight) [41]. The present study showed that HOMA-IR, an insulin resistance index, was higher and whole body glucose infusion rates at euglycemic and hyperglycemic states were lower in VD-low than VD-normal rats. In addition, hepatic glucose outputs at 20

ACCEPTED MANUSCRIPT hyperinsulinemic states were higher in VD-low than VD-normal. This indicated that vitamin D deficiency exacerbated both whole body and hepatic insulin resistance during euglycemia

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and hyperglycemia. However, the beneficial effects of vitamin D on insulin sensitivity were

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attenuated in the VD-high group in comparison to the sufficient state of vitamin D, as shown by the increased triglyceride storage in the liver and skeletal muscles as with vitamin D

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deficiency. These effects on insulin sensitivity may be related to the increase in visceral fat

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mass in VD-high. In addition, PPAR-γ is associated with glucose metabolism and insulin resistance in some tissues especially skeletal muscles and adipose tissues [42]. The relationship between PPAR-γ and vitamin D is controversial: 1,25-(OH)2-cholecalciferol at 0.5 nmol/l increased apoptosis and inhibited lipid accumulation in 3T3-L1 adipocytes [43]

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but 1-α hydroxyvitamin D3 supplementation significantly increases the relative expressions of VDR, PGC1α, and PPAR-γ genes and decreases IL-6, and pro-cytokines which are elevated

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in the peripheral blood mononuclear cells of obese people [44].

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Many studies have found that intracellular accumulation of triglyceride in the liver and skeletal muscles are characteristics of insulin resistance due to impaired insulin signaling

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[45,46]. In the present study, PPAR-γ expression in the epidydimal fat pads was increased in the VD-normal and VD-high groups of the GK rats compared to the VD-low. Vitamin D deficiency is reported to increase non-alcoholic steatosis in humans and animals along with high fat diet and/or obesity [47-49]. In addition, vitamin D deficiency increased hepatic fat accumulation in GK rats fed a high fat diet, and suppressed expression of genes related to fatty acid oxidation and increased expression of lipogenic genes in the liver. Several studies have found that vitamin D deficiency induces hepatic steatosis by decreasing fatty acid oxidation and increasing lipogenesis, and recently it was associated with the impairment of 21

ACCEPTED MANUSCRIPT enterohepatic circulation of fat [47-49]. However, excessive vitamin D (220-270 IU/day/rat) did not reduce hepatic fat accumulation in the present study. Thus, a proper amount of

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vitamin D (20-30 IU/day in this rat model) is beneficial for improving lipid metabolism and

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preventing hepatic steatosis, but excessive vitamin D may reverse those benefits. In addition to insulin resistance vitamin D influences β-cell function and mass. Vitamin

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D is known to regulate islet physiology directly by binding to its receptors [50]. The present

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study is the first study to examine the effect if vitamin D on β-cell function and mass in GK rats, a non-obese type 2 diabetic animal model. We found that vitamin D deficiency suppressed glucose-stimulated insulin secretion during hyperglycemic clamp in GK rats, although fasting plasma glucose levels in VD-low were higher than VD-normal and this

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indicated that vitamin D may be important for glucose-stimulated insulin secretion. In addition, vitamin D deficiency decreased β-cell mass due to decreased β-cell proliferation,

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but not apoptosis. These results suggested that vitamin D may be associated with β-cell

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proliferation which is needed to maintain β-cell mass. Consistent with our results, Kampmann et al. [51] demonstrated that vitamin D supplementation improves glucose-

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stimulated insulin secretion; however it did not affect insulin resistance and HbA1c levels in type 2 diabetic patients in randomized controlled clinical trials. In addition, treatment with 1,25-(OH)2-vitamin D prevents β-cell apoptosis induced by cytokines and restores insulin secretion in human and mouse islets, which is related to anti-inflammatory activity of vitamin D. However, inconsistent with our results, Ayesha et al. [52] reported that the impairment of insulin secretion by vitamin D deficiency was not specific to glucose stimulation, although vitamin D deficiency decreases insulin secretion and vitamin D supplementation reverses it. Thus, the effect of vitamin D on type 2 diabetes and its mechanism remains uncertain. 22

ACCEPTED MANUSCRIPT The rodent model used in this study has limited utility for extrapolation to human dose responses to vitamin D. However, the metabolic pathways described in this study for vitamin

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D regulation of energy metabolism suggests a mechanism for how the dose response to

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vitamin D may be mediated. It also supports the existence of a J-shaped dose response curve,

elucidate the dose response principals involved.

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as suggested by Sanders et al. [53], for mvtabolic effects of vitamin D and therefore helps

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In conclusion, vitamin D deficiency resulted in the dysregulation of glucose metabolism in GK rats, a non-obese type 2 diabetes model, through impaired glucose-stimulated insulin secretion during hyperglycemic clamp and decreased β-cell mass by reducing β-cell proliferation. In addition, it simultaneously impaired insulin sensitivity by decreasing adipose

research

was

supported

by

grants

from

National

Research

Foundation

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This

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Acknowledgement

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PPAR-γ expression.

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(2012R1A1A3009100).

References

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Bone mineral density (BMD), lean mass and fat mass measured by DEXA

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Goto-Kakizaki rats were randomly divided into three groups according to dietary vitamin D

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(cholecalciferol) contents: vitamin D deficiency (VD-low; 25 IU cholecalciferol /kg diet), sufficient vitamin D (VD-normal; 1,000 cholecalciferol IU/kg diet), and excessive vitamin D

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(VD-high; 10,000 cholecalciferol IU/kg diet). At the end of the experimental period, BMD in

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the spine and leg regions (A) and lean mass (B) and fat mass (C) in the abdominal and leg regions were measured by DEXA. Bars and error bars are expressed as means ± SD (n=16). a-c

Significantly different among the three groups according to dietary vitamin D contents at

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P<0.05 (Tukey’s test).

Figure 2. Glucose disposal rates, glucose uptake and hepatic glucose output (HGO) at basal

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state and clamped state during euglycemic hyperinsulinemic clamp

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Goto-Kakizaki rats were randomly divided into three groups according to dietary vitamin D (cholecalciferol) contents: vitamin D deficiency (VD-low; 25 IU cholecalciferol /kg diet),

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sufficient vitamin D (VD-normal; 1,000 cholecalciferol IU/kg diet), and excessive vitamin D (VD-high; 10,000 cholecalciferol IU/kg diet). At the end of experimental periods, to determine glucose infusion rates and whole body glucose uptake (A) and HGO at basal and clamped states (B) at 1100 pM serum insulin, a euglycemic hyperinsulinemic clamp was performed in overnight fasted Goto-Kakizaki rats from vitamin D deficiency (VD-low; 25 IU cholecalciferol /kg diet), sufficient vitamin D (VD-normal; 1,000 cholecalciferol IU/kg diet), and excessive vitamin D (VD-high; 10,000 cholecalciferol IU/kg diet) (n=8). Hepatic insulin signaling was measured by immunoblotting (C) (n=6). Bars and error bars represent the mean 30

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Significantly different among the three groups according to dietary vitamin D contents at

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P<0.05 (Tukey’s test).

Figure 3. Expression of hepatic and epididymal fat genes involved in fatty acid utilization

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measured by realtime PCR

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Goto-Kakizaki rats were randomly divided into three groups according to dietary vitamin D (cholecalciferol) contents: vitamin D deficiency (VD-low; 25 IU cholecalciferol /kg diet), sufficient vitamin D (VD-normal; 1,000 cholecalciferol IU/kg diet), and excessive vitamin D (VD-high; 10,000 cholecalciferol IU/kg diet). After the experimental period, mRNA levels of

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genes involved in fatty acid synthesis and oxidation in the liver (A) and mRNA levels of PPAR-γ, CPT-1 and TNF-α in epididymal fat pads and UCP1 in brown adipose tissues (B)

Significantly different among the three groups according to dietary vitamin D contents at

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a-c

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were measured by real-time PCR. Bars and error bars represent the mean ± SD (n=5).

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P<0.05 (Tukey’s test).

Figure 4. The changes of serum insulin levels during hyperglycemic clamp and islet morphometry At the end of experimental periods of Goto-Kakizaki rats from three groups vitamin D deficiency (VD-low; 25 IU cholecalciferol /kg diet), sufficient vitamin D (VD-normal; 1,000 cholecalciferol IU/kg diet), and excessive vitamin D (VD-high; 10,000 cholecalciferol IU/kg diet), a hyperglycemic clamp to maintain blood glucose levels 100 mg/dL above fasting levels was performed in overnight fasted rats to determine insulin secretion patterns (A) and 31

ACCEPTED MANUSCRIPT insulin secretion capacity calculated by area under the curve of serum insulin levels during hyperglycemic clamp (B). In the islet morphometry the brown staining (dab) was insulin area

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and blue dots were nucleus in the magnification of 100 (C). Each dot and bar and error bars

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represent the mean ± SD (n=8).

Significantly different among the three groups according to dietary vitamin D contents at

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P<0.05 (Tukey’s test).

Figure 5. PPAR-γ activity and triglyceride accumulation during the differentiation from 3T3L1 fibroblast to adipocytes with 1,25-(OH)2-D

HEK 293 cells were transfected with PPRE-luciferase construct, pSV-SPORT-PPAR-γ

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expression vector, pSV-SPORT-retinoid X receptor (RXR)-α vector and renilla phRL-TK

D

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vector for 2 h and then they were treated with vehicle (DMSO), 0, 1, 10, 100 nM 1,25-(OH)2with and without PPAR-γ antagonist T0070907 (10 nM) in serum-free DMEM

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containing 0.1% BSA for 40 h. At the end of the incubation, the cells were assayed for both

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firefly (PPRE-luciferase) and renilla luciferase activities using the Dual-Luciferase Reporter Assay System. Ratios of firefly luciferase activity and renilla luciferase activity as PPAR-γ activity were calculated (A). Two days at post-confluence 3T3-L1 fibroblasts in 6 well plates were treated with differentiation inducers for 4 days and vehicle (DMSO), 0, 1, 10, 100 nM 1,25-(OH)2-D with and without PPAR-γ antagonist T0070907 (10 nM) and then the cells were treated with the previously assigned compounds without differentiation inducers for 6 additional days. At the end of treatments, the cells were harvested with lysis buffer without glycerol. The triglyceride contents (B) in the cells were also determined. The treatment of 2 μM rosiglitazone (RGZ) was considered as the positive control. Bars and error bars represent 32

ACCEPTED MANUSCRIPT the mean ± SD (n=8). a-e

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ACCEPTED MANUSCRIPT Table 1. Composition of experimental diets

VD-normal

VD-high

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280

286

200

200

34

13

12

50

40

38

Shortening (g)

160

150

150

Mineral mixture (g)

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31

30

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10

10

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25

1,000

10,000

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Vitamin mixture without

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ACCEPTED MANUSCRIPT Table 2. Parameters related to energy metabolism at the end of experimental period VD-normal

VD-high

(n=16)

(n=16)

(n=16)

8.1±0.12c

55.4±4.2b

335±23

333±21

339±20

89.6±5.6a

8.3±0.9b

9.2±1.1a

11.7±1.3

11.1±1.3

12.7±1.4

20.5±1.9ab

19.4±1.9b

21.9±2.0a

Serum leptin levels (ng/ml)

3.8±0.5

3.7±0.5

4.0±0.6

Food intake at 8th week

14.2±1.4

13.8±1.3

14.4±1.5

109±18

119±17

113±19

6.1±0.7

5.7±0.7

5.5±0.7

5.4±0.7b

7.0±0.8a

6.6±0.7a

Serum 25-OH-D (nmol/L) Body weight (g)

8.7±1.0ab

Retroperitoneum fat pads (g)

MA NU

Epididymal fat pads (g)

SC

RI P

T

VD-low

ED

Visceral fat mass (g)

(kcal/ kg0.75/day)

CE

Energy expenditure

PT

(g/day)

AC

Carbohydrate oxidation (mg/ kg0.75/min) Fat oxidation (mg/ kg0.75/min)

Values are mean±SD.

48

ACCEPTED MANUSCRIPT a-c

Values in the same row with different superscripts were significantly different in Tukey test

RI P

T

at P<0.05.

Table 3. Lipid metabolism

116.7±12.2b

114.6±12.8b

triglyceride

1.31±0.23a

0.95±0.17b

1.07±0.18b

1.48±0.17b

1.58±0.19ab

1.97±0.20b

2.13±0.28ab

the

muscles

ED

gastrocnemius

in

1.74±0.20a

PT

Triglyceride

CE

(mg/g tissue) in

the

2.34±0.26a

muscles

AC

quadriceps

(n=16)

127.6±12.9a

(mg/g tissue)

Triglyceride

(n=16)

triglyceride

(mg/dL) Hepatic

VD-high

MA NU

(n=16) Serum

VD-normal

SC

VD-low

(mg/g tissue)

Values are mean±SD. a,b

Values in the same row with different superscripts were significantly different in Tukey test

at P<0.05.

49

ACCEPTED MANUSCRIPT Table 4. Insulin secretion capacity during hyperglycemic clamp

(n=8)

(n=8)

(ng/mL)

4.8±0.6a

188±17

MA NU

Serum insulin at basal state

RI P

204±19

(mg/mL)

T

VD-normal

SC

Serum glucose at basal state

VD-low

VD-high (n=8) 194±17

3.9±0.4b

4.4±0.5ab

10.8±1.2a

8.4±0.9b

9.2±1.0b

Serum insulin at first phase

5.0±0.7b

5.8±0.7a

5.8±0.8a

3.7±0.5b

4.2±0.7ab

4.4±0.7a

19.7±2.8b

23.5±3.0a

25.6±3.6a

161±23b

203±36a

225±39a

6.0±0.7c

10.4±1.2a

9.1±1.0b

ED

HOMA-IR

PT

(ng/mL)

(ng/mL)

CE

Serum insulin at second phase

AC

Area under the curve of first phase insulin (AU)

Area under the curve of second phase insulin (AU) Glucose infusion rate (mg/kg bw/min)

50

ACCEPTED MANUSCRIPT

Insulin

sensitivity

(µmol

5.7±0.7c

8.7±1.0a

7.3±0.9b

T

glucose · min-1 · 100 g-1 per

Values are mean±SD.

SC

RI P

µmol insulin/L)

First phase insulin secretion was defined as the average of serum

MA NU

insulin levels at 2 and 5 mins, with second phase at 60, 90 and 120 mins.

Insulin sensitivity

at hyperglycemic state was calculated as the ratio of glucose infusion rate to steady-state plasma insulin levels. a-c

Values in the same row with different superscripts were significantly different in Tukey test

AC

CE

PT

ED

at P<0.05.

51

ACCEPTED MANUSCRIPT Table 5. The modulation of islet morphometry at the end of experiment

VD-normal

(n=6)

(n=6)

β-cell area (%)

5.7±0.8b

6.9±0.8a

Absolute β-cell mass (mg)

22.2±3.0b

β-cell

RI P

0.92±0.11

(n=6) 6.5±0.7a

25.9±2.9a

225±27b

241±28ab

0.97±0.11a

0.96±0.12a

0.87±0.10

0.89±0.10

AC

CE

Apoptosis (% apoptotic bodies of islets)

0.85±0.10b

PT

cells of islets)

ED

(μm2)

BrdU+ cells (% BrdU+

SC

252±28a

size

VD-high

27.6±3.1a

MA NU

Individual

T

VD-low

Values are mean±SD. a,b

Values in the same row with different superscripts were significantly different in Tukey test

at P<0.05.

52