European Journal of Nutrition
Anti-diabetic effect of a preparation of vitamins, minerals and trace elements in diabetic rats
--Manuscript Draft--
Manuscript Number:
Full Title: Anti-diabetic effect of a preparation of vitamins, minerals and trace elements in diabetic rats
Article Type: Original Contribution
Keywords: multivitamin, multimineral, prevention, streptozotocin-induced diabetes mellitus Corresponding Author: Peter Ferdinandy, M.D., Ph.D, MBA
Semmelweis University, Faculty of Medicine Budapest, HUNGARY
Corresponding Author Secondary Information:
Corresponding Author's Institution: Semmelweis University, Faculty of Medicine Corresponding Author's Secondary
Institution:
First Author: Marta Sarkozy, M.D.
First Author Secondary Information:
Order of Authors: Marta Sarkozy, M.D.
Veronika Fekete, M.D.
Gergo Szucs, M.D.
Szilvia Torok, M.Sc.
Csilla Szucs, Pharm.D.
Judit Barkanyi, Pharm.D Zoltan V Varga, M.D.
Csaba Csonka, M.D., Ph.D.
Csaba Konya, M.D.
Tamas Csont, M.D., Ph.D.
Peter Ferdinandy, M.D., Ph.D, MBA Order of Authors Secondary Information:
Abstract: Purpose: Although multivitamin products are widely used as dietary supplements to maintain health or as special medical food in certain diseases, the effects of these products were not investigated in diabetes. Therefore, here we investigated if a preparation of different minerals, vitamins, and trace elements (MVT) for human use affects the severity of experimental diabetes mellitus. Methods: Two days old neonatal Wistar rats were injected with 100 mg/kg of streptozotocin or its vehicle to induce diabetes. At week 4, rats were fed with an MVT preparation or placebo for 8 weeks.
Serum glucose during oral glucose tolerance test, insulin and HbA1c levels were measured at week 4, 8 and 12. Results: Oral glucose tolerance test showed an impaired glucose tolerance in streptozotocin-treated rats in both genders at week 4. In males, fasting blood glucose and HbA1c were significantly increased and glucose tolerance and serum insulin was decreased at week 12 in the diabetic placebo group as compared to the non-diabetic placebo group; however, all of these parameters were significantly improved by MVT treatment. In females, streptozotocin treatment resulted in a less severe diabetic phenotype as only glucose tolerance and HbA1c were altered by the end of the study in the diabetic placebo group as compared to the non-diabetic placebo group. MVT treatment failed to improve these parameters in female
streptozotocin-treated rats. Conclusion: This is the first demonstration that MVT significantly attenuates the progression of diabetes in male rats.
Suggested Reviewers: Arpad Tosaki, M.D., Ph.D., MBA
Head of department, Faculty of Pharmacy, University of Debrecen, Nagyer tosaki.arpad@pharm.unideb.hu
Pal Pacher, M.D., Ph.D., MBA Bethesda, MD 20892-9413 pacher@mail.nih.gov
Edit Bodolay, M.D., Ph.D., MBA
Medical and Health Science Center University of Debrecen edit.bodolay@gmail.com
Anti-diabetic effect of a preparation of vitamins, minerals and trace
1
elements in diabetic rats
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3
Márta Sárközy1, Veronika Fekete1, Gergő Szűcs1, Szilvia Török1, Csilla Szűcs3, Judit Bárkányi3, Zoltán V.
4
Varga1,4, Csaba Csonka1,2, Csaba Kónya3, Tamás Csont1,2,*, Péter Ferdinandy2,4,*
5 6
1Cardiovascular Research Group, Department of Biochemistry, Faculty of Medicine, University of Szeged
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2Pharmahungary Group, Szeged, Hungary
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3Béres Pharmaceuticals Ltd, Budapest, Hungary
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4Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Budapest
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*These authors equally contributed to the paper
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13
Address for correspondence:
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Péter Ferdinandy MD, PhD, MBA
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Pharmahungary Group
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6 Hajnóczy Street, Szeged, H-6722, Hungary
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Tel: +36 62 545096, Fax: +36 1 2422249
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E-mail: peter.ferdinandy@pharmahungary.com
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20 21 22 23 24 25
ManuscriptClick here to download Manuscript: SarkozyM et al_EJN_MS_final_2013jun19_sm.docx Click here to view linked References
Abstract
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Purpose: Although multivitamin products are widely used as dietary supplements to maintain health or as special
28
medical food in certain diseases, the effects of these products were not investigated in diabetes. Therefore, here we
29
investigated if a preparation of different minerals, vitamins, and trace elements (MVT) for human use affects the
30
severity of experimental diabetes mellitus. Methods: Two days old neonatal Wistar rats were injected with 100 mg/kg
31
of streptozotocin or its vehicle to induce diabetes. At week 4, rats were fed with an MVT preparation or placebo for 8
32
weeks. Serum glucose during oral glucose tolerance test, insulin and HbA1c levels were measured at week 4, 8 and 12.
33
Results: Oral glucose tolerance test showed an impaired glucose tolerance in streptozotocin-treated rats in both genders
34
at week 4. In males, fasting blood glucose and HbA1c were significantly increased and glucose tolerance and serum
35
insulin was decreased at week 12 in the diabetic placebo group as compared to the non-diabetic placebo group;
36
however, all of these parameters were significantly improved by MVT treatment. In females, streptozotocin treatment
37
resulted in a less severe diabetic phenotype as only glucose tolerance and HbA1c were altered by the end of the study in
38
the diabetic placebo group as compared to the non-diabetic placebo group. MVT treatment failed to improve these
39
parameters in female streptozotocin-treated rats. Conclusion: This is the first demonstration that MVT significantly
40
attenuates the progression of diabetes in male rats.
41 42
Key words: multivitamin, multimineral, prevention, streptozotocin-induced diabetes
43
44
Introduction
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The rapid increase in the prevalence of diabetes mellitus across the world gives diabetes the status of an
46
epidemic in the 21st century [1]. In the last decades, there was an explosive increase in the number of people diagnosed
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with diabetes worldwide due to aging as well as increasing prevalence of obesity and physical inactivity [1-3]. The total
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number of people with diabetes is projected to rise from 347 million in 2008 [4] to 552 million in 2030 [5].
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Not only total energy intake and macronutrients including carbohydrates, protein and fat, but also
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micronutrients including vitamins, minerals and trace elements have effects on the severity of diabetes mellitus. Clinical
51
studies have shown that some individual vitamins e.g. A [6], B1 [7], B3 [8], C [9,10], D [11] and E [12], minerals e.g.
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calcium [13], magnesium [14] and trace elements e.g. zinc [15], chrome [16] beneficially affect the complications of
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diabetes mellitus. In these clinical studies, effects of individual vitamins, minerals and trace elements or combination of
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two or three components were investigated on diabetes. Surprisingly, there is no literature data available on the effects
55
of multivitamin, minerals and trace elements containing preparations that can be used for human treatment in diabetes
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mellitus.
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Regular consumption of vitamin/mineral supplements is common in developed countries [17] to maintain
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general health. In the United States, more than half of adult population use dietary supplements [18,19] primarily in the
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form of multivitamins with or without minerals [20]. In Germany, a study in 1998 reported that 18% of men and 25% of
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women were regular users of multivitamins in a sample of population aged 18–79 years [21]. Moreover, MVT
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preparations appeared on the market as medical food for diabetics; however, no literature data supports the beneficial
62
effect of these preparations in preclinical or clinical studies.
63
Therefore, here we aimed to investigate if an MVT preparation containing 26 different minerals, vitamins and
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vitamin-like antioxidants, as well as trace elements affects the progression of diabetes in an experimental model of
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diabetes in rats.
66 67
Experimental methods
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This investigation conforms to the National Institutes of Health Guide for the Care and Use of Laboratory
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Animals (NIH Pub. No. 85-23, Revised 1996) and was approved by the Animal Research Ethics Committee of the
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University of Szeged.
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Neonatal male and female Wistar rats were used in this study. Lactating females with their litters were
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separately housed in individually ventilated cages (Sealsafe IVC system, Italy) and were maintained in a temperature-
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controlled room with a 12:12 h light: dark cycles for four weeks. After separation from the mother, littermates were
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housed in pairs under the same circumstances as mentioned above until 12 weeks of age. Standard rat chow and tap
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water were supplied ad libitum throughout the study.
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Experimental protocol
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Two day old neonatal male and female Wistar rats were injected with 100 mg/kg of streptozotocin (STZ) or its
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vehicle (ice-cold citrate buffer) to induce experimental diabetes mellitus (Figure 1). At week 4, fasting blood glucose
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measurement and an oral glucose tolerance test (OGTT) were performed in order to verify the development of impaired
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glucose tolerance or diabetes mellitus (Figure 1). Both non-diabetic and diabetic rats were then fed with a MVT
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preparation (253.3 mg/kg/day) to be registered as medical food for human use (Diacomplex film-coated tablet, Béres
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Pharmaceuticals, Budapest, Hungary; for content see Table 1) or placebo (157 mg/kg/day) for eight weeks (Figure 1).
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To conform to the human daily dose of the preparation, rat daily dose was adjusted according to the ratio of human and
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rat body surface areas. Fasting blood glucose measurement was performed in every second week and OGTT in every
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fourth week until week 12 to monitor the effect of multivitamin treatment on diabetes mellitus (Figure 1). Serum insulin
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and hemoglobinA1c level were measured at week 12 as well (Figure 1).
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89
Serum glucose level measurements and oral glucose tolerance test (OGTT)
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Rats were fasted overnight (12 h) prior to serum glucose level measurements (week 4, 6, 8, 10 and 12) and OGTTs
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(week 4, 8 and 12) in order to verify the development of diabetes mellitus and to monitor the effect of multivitamin
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treatment on diabetes. Blood samples were collected from the saphenous vein. Blood glucose levels were measured
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using Accucheck blood glucose monitoring systems (Roche Diagnostics Corporation, USA, Indianapolis) [22,23].
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OGTT was performed as follows. After measurement of baseline glucose concentrations, glucose at 1.5 g/kg body
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weight was administered via oral gavage and plasma glucose levels were measured 30, 60 and 120 minutes later and
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area under the curve was determined [22,23].
97 98
Hemoglobin A1c
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In order to monitor the chronic effect of MVT containing preparation on diabetes mellitus, hemoglobin A1c was
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measured from whole venous blood with an in vitro test (Bio-Rad in2it (I) System) according to the instructions of the
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manufacturer. The test is based on single wave length photometry (440 nm) to detect glycated fraction separated from
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the non-glycated fraction by boronate affinity chromatography.
103 104
Measurement of serum insulin levels
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To monitor the effect of MVT treatment on diabetes mellitus, serum insulin levels were measured by an enzyme
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immunoassay (Mercodia, Ultrasensitive Rat Insulin ELISA) in triplicates according to the manufacturer’s instructions
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as described [22].
108 109
Statistical analysis
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Statistical analysis was performed by using Sigmaplot 12.0 for Windows (Systat Software Inc). All values are
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presented as mean±SEM. Two way repeated measures ANOVA was used to determine the effect of diabetes or MVT
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on body weight, fasting serum glucose levels and glucose levels during oral glucose tolerance test in the entire
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population as well as in males or females, respectively. Two-Way ANOVA was used to determine the effect of diabetes
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or MVT on glucose tolerance test AUC, HbA1c and serum glucose level in the entire population as well as in males or
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females, respectively. P<0.05 was accepted as a statistically significant difference.
116 117
Results
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In order to verify the development of diabetes mellitus in the STZ-treated rats, concentrations of several
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plasma metabolites and body weight were measured at week 4 and during the treatment and follow-up period at week 6,
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8, 10 and 12 (Figure 1). In the entire population of experimental animals of mixed genders, STZ-treated rats showed
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lower body weight from week 4 to week 12, and increased blood glucose at week 6, 10, 12 confirming the development
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of diabetes mellitus (Figure 2). In STZ-treated group, serum glucose levels were significantly decreased by MVT
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treatment at week 6, 10 and 12 (Figure 2). However, male STZ-treated rats showed a significant rise in serum fasting
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glucose level and a marked decrease in body weight from week 4 to the end of the follow up period (Figure 3a and 3c)
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showing the development of diabetes mellitus. Female STZ-treated rats failed to show elevated serum fasting glucose
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level and reduced body weight during the whole follow up period (Figure 3b and 3d).
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Oral glucose tolerance test showed increased area under the curve (AUC) in mixed genders, male and female
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in STZ-treated rats at week 4, 8 and 12 showing impaired glucose tolerance (Figure 4-6). MVT treatment showed
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borderline significance (p=0.054) in case of mixed genders at week 12 (Figure 4). Separating the genders, multivitamin
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treatment decreased significantly the OGTT AUC only in male STZ-treated rats at week 8 and 12 proving anti-diabetic
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effect of multivitamin treatment (Figure 5). However, MVT treatment did not change OGTT AUC in male control rats
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(Figure 5). Interestingly, AUC remained unchanged by multivitamin treatment in female animals both in STZ-treated
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and control groups as well as at week 8 and 12 (Figure 6).
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HbA1c level was significantly higher in STZ-induced diabetes in mixed genders, as well as in males and
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females respectively as compared to controls (Figure 7) at week 12. Interestingly, multivitamin treatment has
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significantly reduced HbA1c level only in STZ-treated males without having any effect in STZ-treated females and
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control animals in both genders (Figure 7).
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Serum insulin concentration was significantly decreased in STZ-treated animals proving beta cell damage
139
(Figure 8). MVT treatment showed a statistically not significant increasing tendency in STZ-treated animals (Figure 8).
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Separate evaluation of the data of the genders revealed that multivitamin treatment improved serum insulin level in
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STZ-treated males; however, serum insulin levels remained unaffected by the multivitamin treatment in control males
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(Figure 8). Neither diabetes mellitus, nor multivitamin treatment had any effect on serum insulin levels in female
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animals (Figure 8).
144 145
Discussion
146
Here we have shown that chronic treatment with an MVT preparation improved diabetic markers such as serum
147
fasting glucose, HbA1c, glucose tolerance, serum insulin levels in male diabetic rats. This is the first demonstration,
148
that an MVT preparation attenuates the progression of experimental diabetes. Furthermore, we have shown here that
149
females developed a less severe diabetic phenotype in response to 100 mg/kg neonatal STZ-treatment as only OGTT
150
AUC and HbA1c were increased. In these animals, the MVT preparation failed to attenuate the progression of diabetes
151
significantly.
152
Regular consumption of MVT preparations as food supplements or medical food for diabetics is common in
153
developed countries. However, surprisingly preclinical or clinical evaluation of such preparations is not available in the
154
literature. Therefore, here we evaluated the effect of chronic treatment with an MVT preparation containing 15 vitamins
155
and vitamin-like substances such as lutein and rutoside, 3 minerals, and 8 micro/trace elements in experimental diabetic
156
rats. The MVT preparation showed beneficial effects on major markers of diabetes in male but not in female rats in the
157
present study. This is the first evidence in the literature that a complex MVT preparation developed for human
158
consumption significantly delayed progression of diabetes in an animal model. The reason why the MVT preparation
159
was ineffective in female rats is not known, however, it should be emphasized that female rats developed only an
160
impaired glucose tolerant state characterized by an increased OGTT AUC and HbA1c levels in the present study. It is
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well known that the progression of diabetes occurs at a later age and becomes milder in females compared to age-
162
matched males in rodent models of diabetes (see for review [24]). This could be the reason why the MVT preparation
163
used in our present study was unable to improve the biochemical markers of early diabetes significantly.
164
In different experimental diabetes models and clinical studies involving limited number of patients, several data are
165
available on the effect of individual vitamins, minerals, trace elements, or the combination of limited number of them.
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The individual components of the MVT preparation investigated in the present study were selected by the manufacturer
167
on the basis of their preclinical and clinical data in different diabetic animal models or patient populations. Daily doses
168
of all components of the preparation were set below the human upper safe level [25].
169
Effects of individual vitamins on diabetes and/or its complications have been investigated in a number of
170
preclinical and clinical trials. It was demonstrated that a 2-week administration of vitamin B1 in a high dose (0.2%
171
thiamin in drinking water) prevents diabetes-induced cardiac fibrosis without reducing the blood glucose level in male
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diabetic rats [7]. A randomized double-blind placebo-controlled clinical pilot study recruiting 40 type 2 diabetics with
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microalbuminuria demonstrated that a high-dose vitamin B1 therapy (300 mg/day) for 3 months produced a regression
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of urinary albumin excretion without any effect on plasma glucose or HbA1c levels [26]. Vitamin D deficiency is a
175
known risk factor of diabetes mellitus. A double-blind parallel group placebo-controlled randomized trial involving 87
176
type 2 diabetics reported that a single large dose (100,000 IU) of vitamin D2 improved endothelial function 8 weeks
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after the administration, however, HbA1c and HOMA-IS were unaffected by vitamin D2 therapy [27]. Additionally,
178
another randomized, placebo-controlled clinical trial with 81 participants showed that vitamin D3 supplementation
179
(4000 IU) for 6 months significantly improved insulin sensitivity and fasting insulin level in type 2 diabetic women
180
[11].
181
A number of preclinical and clinical trials have investigated the effects of individual minerals on diabetes and/or
182
its complications. A randomized controlled, single-blinded trial with 31 patients demonstrated that oral calcium
183
supplementation (1500 mg/day) for 2 months improves insulin sensitivity in patients with type 2 diabetes and
184
hypertension, however, both fasting blood glucose and HbA1c levels were unaffected by the calcium supplementation
185
[28]. Furthermore, it was shown that dietary calcium supplementation (600 mg/day) for 3 months significantly reduced
186
vascular resistance and induced partial regression of left ventricular hypertrophy in hypertensive non-insulin-dependent
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diabetic Afro-Americans [13]. A clinical randomized double-blind placebo-controlled trial recruiting 63 type 2 diabetic
188
patients with hypomagnesemia receiving glibenclamide has shown that supplementation of magnesium (2.5 g
189
MgCl2/day) for 4 months improved HbA1c, HOMA-IR, fasting glucose as well as insulin levels [29]. Another double-
190
blind placebo-controlled clinical trial enrolling 82 diabetic hypertensive adults with hypomagnesemia receiving
191
captopril demonstrated that oral magnesium supplementation (2.5 g MgCl2/day) for 4 months significantly reduced
192
fasting plasma glucose as well as HbA1c levels, systolic and diastolic blood pressure [14]. In contrast, a randomized
193
clinical study involving 97 patients showed that chronic supplementation of magnesium (300 mg/day) for 5 years
194
attenuated the evolution of polyneuropathy in type 1 diabetics with magnesium deficiency without reducing HbA1c
195
level [30].
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Limited number of clinical data on some individual trace elements has shown beneficial effects on diabetes and its
197
complications. A clinical pilot study involving 22 patients demonstrated that zinc supplementation (30 mg/day) for 3
198
months decreased lipid peroxidation in type 1 diabetes mellitus [31]. A prospective double-blind placebo-controlled
199
crossover study involving 30 participants demonstrated that supplementation of chromium for 2 months significantly
200
reduced serum triglyceride level in type 2 diabetic patients without any effect on serum glucose level [32]. In contrast,
201
another randomized clinical study involving 180 patients with type 2 diabetes showed that chromium administration
202
(1000 μg/day) for 4 months had beneficial effects on HbA1c, glucose, insulin, and cholesterol variables [16].
203
Limited data on combinations of few numbers of vitamins, minerals and trace elements have shown beneficial
204
effects on diabetes and/or its complications. A randomized, double-blind, placebo-controlled clinical trial showed that a
205
combination of 200 mg magnesium, 30 mg zinc, 200 mg vitamin C, and 100 IU vitamin E significantly improved
206
glomerular function [6], blood pressure [33] and increased HDL-c and apo A1 level [33] without beneficially affecting
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serum glucose and HbA1c levels in 69 patients with type 2 diabetes mellitus after 3 months daily treatment. A
208
randomized clinical trial enrolling 64 children with recent onset of type 1 diabetes (IMDIAB IX) demonstrated that
209
implementation of insulin therapy with vitamin B3 (25 mg/kg body weight) alone or in combination with vitamin E (15
210
mg/kg body weight) for 2 years preserved baseline C-peptide secretion without any effect on HbA1c level [34]. In
211
contrast, a placebo-controlled double-blind randomized clinical trial involving 348 participants has reported that statin
212
therapy co-supplemented with biotin (2 mg/day) and chromium (600 μg/day) for 3 months has significantly decreased
213
serum glucose, HbA1c, LDL-cholesterol, total cholesterol, and VLDL-cholesterol levels in type 2 diabetic patients [35].
214
In many of the abovementioned studies [6,11,26,27,31,33,35], some of the individual vitamins, minerals and trace
215
elements were used in a daily dose above the upper safe level [25]. A pilot clinical study in Sri Lanka involving 96
216
patients demonstrated that a 15-component MVT preparation significantly reduced serum glucose and lipid levels in
217
adult diabetic patients after 4 months of supplementation [36], however, the daily dose of 3 components of this
218
preparation was above the upper safe level and no gender difference was investigated [36].
219
The limitation of the present study is that it does not provide evidence on the mechanism of the effect of the MVT
220
preparation and the individual contribution of the 26 components to the anti-diabetic effect of the preparation. The
221
potential interactions of these components and their combined effect rather than the value of a single component could
222
be responsible for the beneficial effects of the MVT preparation on the severity of diabetes; however, effects of each
223
components and their different variation of combinations were out of the scope of the present study.
224 225
Conclusion
226
Although MVT preparations are widely used by diabetics, our present study is the first demonstration that a MVT
227
preparation attenuates the progression of experimental diabetes. Further studies are needed to optimize the composition
228
and to elucidate the efficacy, safety and the mechanism of the effect of MVT preparations in diabetics.
229 230
Acknowledgments
231
232
Source of funding: This work was supported by grants from the National Development Agency (MED_FOOD
233
TECH_08-A1-2008-0275, Baross DA-TECH-07-2008-0041, TÁMOP-4.2.1/B-09/1/KONV-2010-0005, TÁMOP-
234
4.2.2/B-10/1-2010-0012), the Hungarian Scientific Research Fund (OTKA K79167), and co-financed by the European
235
Regional Development Fund and VÁTI Hungarian Nonprofit LLC for Regional Development and Town Planning
236
(HURO/0901/137/2.2.2-HU-RO-TRANS-MED). T. Csont held a "János Bolyai Felowship" of the Hungarian Academy
237
of Sciences. We acknowledge the technical support of Judit Pipis for blood sampling and serum insulin measurements.
238
Author contributions: C.K., T.C. and P.F conception and design of research; M.S., V.F., G.S., T.S., C.S., J.B. and
239
Z.V.V. performed experiments; M.S., V.F., G.S., T.S., C.S., J.B. and C.C. analyzed data; M.S., V.F., G.S. J.B. and T.C.
240
interpreted results of experiments; M.S. prepared figures; M.S., T.C. and P.F. drafted manuscript; M.S., T.C. and P.F.
241
edited and revised manuscript; M.S., V.F., G.S., T.S., C.S., J.B., Z.V.V., C.C., C.K., T.C. and P.F. approved final
242
version of manuscript.
243 244
Conflicts of interests
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Béres Pharmaceuticals Ltd. was the leader of the consortial project funded by the National Development Agency
246
(MED_FOOD TECH_08-A1-2008-0275).
247
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27. Sugden JA, Davies JI, Witham MD et al (2008) Vitamin D improves endothelial function in patients with Type 2
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34. Crino A, Schiaffini R, Manfrini S et al (2004) A randomized trial of nicotinamide and vitamin E in children with
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35. Albarracin C, Fuqua B, Geohas J et al (2007) Combination of chromium and biotin improves coronary risk factors
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36. Gunasekara P, Hettiarachchi M, Liyanage C et al (2011) Effects of zinc and multimineral vitamin supplementation
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Figure legends
330
Fig. 1 Two day old neonatal male and female Wistar rats were injected with 100 mg/kg of streptozotocin (STZ or its
331
vehicle to induce experimental diabetes mellitus. At week 4, fasting blood glucose measurement and an oral glucose
332
tolerance test (OGTT. were performed in order to verify the development of diabetes mellitus. Both non-diabetic and
333
diabetic rats were then fed with a mixture of vitamins, minerals, and trace elements (MVT or placebo for eight weeks)
334
Fasting blood glucose measurement was performed in every second week and OGTT at week 4, 8 and 12 to monitor the
335
effect of MVT treatment on diabetes mellitus. Serum insulin and hemoglobinA1c level were measured at week 12 as
336
well
337
338
Fig. 2 Body weight (panel a, n=19-20) and fasting serum glucose level (panel b, n=19-20. in mixed genders. Values are
339
means±SEM, *p<0.05 control vs. diabetes; #p≤0.05 placebo vs. vitamin
340
341
Fig. 3 Body weight in males (panel a, n=11 in each group and females, panel b, n=8-9 in each group. and fasting serum
342
glucose level in males, panel c, n=11 in each group and females, panel d, n=8-9 in each group). Values are
343
means±SEM, *p<0.05 control vs. diabetes; # p<0.05 placebo vs. vitamin
344
345
Fig. 4 Blood glucose and OGTT AUC in both genders at week 4, 8 and 12. Values are means±SEM, *p<0.05 control
346
vs. diabetes; n=19-20 in each group (p=0.054 placebo vs. vitamin, OGTT AUC glucose levels at week 12)
347
348
Fig. 5 Blood glucose and OGTT AUC in males at week 4, 8 and 12. Values are means±SEM, *p<0.05 control vs.
349
diabetes; #p<0.05 placebo vs. vitamin, n=11 in each group
350
351
Fig. 6 Blood glucose and OGTT AUC in females at week 4, 8 and 12. Values are means±SEM, *p<0.05 control vs.
352
diabetes, n=8-9 in each group
353
354
Fig. 7 HbA1c levels in mixed genders (n=19-20 in each group, males (n=11 in each group) and females (n=8-9 in each
355
group) at week 12. Values are means±SEM, *p<0.05 control vs. diabetes; #p<0.05 placebo vs. vitamin. (p=0.097
356
placebo vs. vitamin in both genders)
357
358
Fig. 8 Serum insulin levels in mixed genders (n=19-20 in each group, males (n=11 in each group) and females (n=8-9 in
359
each group) at week 12. Values are means±SEM, *p<0.05 vs. control placebo; #p<0.05 vs. diabetes placebo
360
Table 1
Ingredients of the MVT preparation
Active ingredients Amount of ingredient/
1 g product Daily dose*
Vitamin A1 (Retinol) 329 μg/g
(1097 IU/g)
83.3 μg/kg/day (278 IU/kg/day)
Vitamin B1 (Thiamin) 2.30 mg/g 0.58 mg/kg/day
Vitamin B2 (Riboflavin) 2.63 mg/g 0.67 mg/kg/day
Vitamin B3 (Nicotinamide) 11.8 mg/g 2.99 mg/kg/day
Vitamin B5 (Pantothenic acid) 3.95 mg/g 1.00 mg/kg/day
Vitamin B6 (Pyridoxine) 3.29 mg/g 0.83 mg/kg/day
Vitamin B12 (Cyanocobalamin) 3 μg/g 0.76 μg/kg/day
Folic acid 197 μg/g 49.9 μg/kg/day
Biotin 99 μg/g 25.1 μg/kg/day
Vitamin D3 (Cholecalciferol) 3 μg/g
(120 IU/g)
0.76 μg/kg/day (30.4 IU/kg/day)
Vitamin K1 (Phyllokinone) 26 μg/g 6.59 μg/kg/day
Rutoside 3.29 mg/g 0.83 mg/kg/day
Vitamin C 65.8 mg/g 16.7 mg/kg/day
Vitamin E 32.9 mg/g 8.33 mg/kg/day
Lutein 1.97 mg/g 0.50 mg/kg/day
Chrome 39 μg/g 9.88 μg/kg/day
Zinc 9.87 mg/g 2.50 mg/kg/day
Selenium 26 μg/g 6.59 μg/kg/day
Iron 2.63 mg/g 0.67 mg/kg/day
Iodine 66 μg/g 16.7 μg/kg/day
Manganese 0.66 mg/g 0.17 mg/kg/day
Copper 921 μg/g 233 μg/kg/day
Molybdenum 49 μg/g 12.4 μg/kg/day
Magnesium 65.8 mg/g 16.7 mg/kg/day
Calcium 132 mg/g 33.4 mg/kg/day
Phosphorus 102 mg/g 25.8 mg/kg/day
i.
* To conform to the human daily dose of the preparation, rat daily dose was adjusted according to the ratio of human and rat body surface areas.
Table
Fig. 1
Experimental protocol
day 2
placebo multivitamin CONTROL
(ip. vehicle on day 2)
DIABETES
(ip. streptozotocin on day 2)
4 6 8 10 12
0 2 wk
○
◊
○
placebo multivitamin
○
◊
●
○
◊
○
ip. injection of streptozotocin or vehicle body weight, serum glucose
oral glucose tolerance test
HbA1c, serum and pancreatic insulin
○ ◊
●
Figure
Fig. 2
a) b)
0 4 8 12 16
4 6 8 10 12
Seru m gluc o se (mmol /L )
* *
wk Mixed genders
* *
*
*
*
*
#
p=0.050
# control placebo
control vitamin
diabetes placebo diabetes vitamin
wk 0
100 200 300 400 500
4 6 8 10 12
B o dy w ei gh t (g)
Figure
Fig. 3
0 4 8 12 16
Seru m gluc o se (mmol /L )
Female d)
0 4 8 12 16
Se rum gl uc o se ( mmo l/L )
Male c)
*
*
Female b)
wk
#
control placebo control vitamin
diabetes placebo diabetes vitamin
a) Male
* *
*
*
*
# #
#
0 100 200 300 400 500
4 6 8 10 12
Bo d y w eigh t (g)
wk
0 100 200 300 400 500
4 6 8 10 12
Bo d y w eigh t (g)
Figure
Fig. 4
Mixed genders
0 10 20 30
0 30 60 90 120
*
week 4
Bl oo d g lu co se (m m ol /L )
Time (min)
0 500 1000 1500 2000 2500
O GT T A UC glu co se (m in *mm ol /L )
Plac Vit Plac Vit
0 10 20 30
0 30 60 90 120
Time (min) week 8
0 500 1000 1500 2000 2500
Plac Vit Plac Vit
O GT T A UC glu co se (m in *mm ol /L )
0 10 20 30
0 30 60 90 120
week 12
Time (min)
Bl oo d g lu co se (m m ol /L ) Bl oo d g lu co se (m m ol /L )
0 500 1000 1500 2000 2500
Plac Vit Plac Vit
O GT T A UC glu co se (m in *mm ol /L )
a) b) c)
e) f)
d)
*
*
*
*
*
*
* control placebo (Plac)
control vitamin (Vit)
diabetes placebo (Plac) diabetes vitamin (Vit)
* *
*
*
*
*
*
*
*
Figure
Fig. 5
Male
0 10 20 30
0 30 60 90 120
Bl oo d g lu co se ( m m ol /L )
Time (min)
a)
week 4
b) c)
0 10 20 30
0 30 60 90 120
Bl oo d g lu co se ( m m ol /L )
Time (min)
*
*
#
#
week 8
0 10 20 30
0 30 60 90 120
Bl oo d g lu co se ( m m ol /L )
Time (min) week 12
*
*
# *
# #
*
*
control placebo (Plac) control vitamin (Vit)
diabetes placebo (Plac) diabetes vitamin (Vit)
0 500 1000 1500 2000 2500
0 500 1000 1500 2000 2500
e) f)
O GT T A UC glu co se (m in *mm ol /L )
#
O GT T A UC glu co se (m in *mm ol /L )
#
Plac Vit Plac Vit Plac Vit Plac Vit
O GT T A UC glu co se (m in *mm ol /L )
d)
Plac Vit Plac Vit
*
* *
0 500 1000 1500 2000 2500
*
* *
*
*
*
Figure
Fig. 6
0 10 20 30
0 30 60 90 120
Bl oo d g lu co se (m m ol /L )
Time (min)
Female a)
week 4
*
*
b) c)
0 10 20 30
0 30 60 90 120
Bl oo d g lu co se ( m m ol /L )
Time (min) week 8
*
*
Time (min)
control placebo (Plac) control vitamin (Vit)
diabetes placebo (Plac) diabetes vitamin (Vit)
week 4
d) e) f)
week 8 week 12
O G T T A UC gl u co se (mi n * mmol /L) O G T T A UC gl u co se (mi n * mmol /L)
Plac Vit Plac Vit Plac Vit Plac Vit
Plac Vit Plac Vit O
G T T A UC gl u co se (mi n * mmol /L)
*
*
*
*
*
*
*
* *
0 10 20 30
0 30 60 90 120
Bloo d gl uc ose (m m ol /L )
*
*
week 12
0 500 1000 1500 2000 2500
0 500 1000 1500 2000 2500
0 500 1000 1500 2000 2500
Figure
Fig. 7
Male Female
#
Control Diabetes Plac Vit Plac Vit
Control Diabetes Plac Vit Plac Vit
Mixed genders
HbA1c (%)
0 4 8 12
Control Diabetes Plac Vit Plac Vit
p=0.097
*
*
*
Figure
Fig. 8
Male Female
Control Diabetes Plac Vit Plac Vit
#
Seru m insu lin ( µg /ml)
Control Diabetes Plac Vit Plac Vit Control Diabetes
Plac Vit Plac Vit Mixed genders 0.6
0.4
0.2
0.0
*
*
Figure
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