Vitamin C decreases the obesogenic and hyperglycemic effect of invert sugar in prediabetic rats

1 Universidade de Santa Cruz do Sul, Departamento de Educação Física e Saúde, Programa de Pós-Graduação em Promoção da Saúde. Av. Independência, 2293, Sala 4206, Campus Universitário, 96815-900, Santa Cruz do Sul, RS, Brasil. Correspondência para/Correspondence to: SIR FRANKE. E-mail: <silviafr@unisc.br>. 2 Universidade de Santa Cruz do Sul, Departamento de Educação Física e Saúde, Laboratório de Nutrição Experimental. Santa Cruz do Sul, RS, Brasil. 3 Universidade de Santa Cruz do Sul, Departamento de Biologia e Farmácia, Curso de Ciências Biológicas. Santa Cruz do Sul, RS, Brasil. Article based on the master’s theses of P. MOLZ, entitled “Influência in vivo de nutrientes sobre a citotoxicidade e estabilidade genômica associadas à sobrecarga de açúcar”. Universidade de Santa Cruz do Sul; 2015. Suport: Conselho Nacional de Desenvolvimento Científico e Tecnológico (Process no 479554/2013-7), by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul, and by the Laboratory of Experimental Nutrition of Universidade de Santa Cruz do Sul. ORIGINAL | ORIGINAL

Vitamin C decreases the obesogenic and hyperglycemic effect of invert sugar in prediabetic rats

A B S T R A C T Objective
To evaluate whether vitamin C can help to prevent obesity and hyperglycemia in Wistar rats treated with excess invert sugar to induce prediabetes.

Methods
One hundred-day-old Male Wistar rats with a mean weight of 336.58±23.43gwere randomly assigned to the following groups: (1) control, receiving water (C); (2) invert sugar control, receiving a 32% watery solution of invert sugar; (3) vitamin C control, receiving a watery solution of vitamin C (60mg/L), and (4) vitamin C plus invert sugar, receiving a watery solution of vitamin C and invert sugar.All animals had access to chow and water ad libitum and were treated for 17 weeks.Prediabetes was assessed according to two criteria: obesity (based on body mass indexand peritoneal fat content) and impaired glucose tolerance (assessed by the intraperitoneal glucose tolerance test and expressed as area under the curve).

Results
Group invert sugar control gained significantly more weight (p=0.035) and visceral fat (p<0.001)than groups vitamin C control and vitamin C plus invert sugar.Consequently, groups vitamin C control and vitamin C plus invert sugar had gained as little body mass index as group C by the end of the experiment.Vitamin C decreased the fasting glycemia of both groups supplemented with vitamin C and normalized the glucose tolerance of group vitamin C plus invert sugar, whose area under the curve matched that of group C.

Conclusion
Vitamin C has anti-obesogenic and glycemia-lowering effects in Wistar rats, which might be promising to prediabetics.Future studies are needed to understand the anti-obesogenic and anti-hyperglycemic mechanisms of vitamin C in prediabetes.Keywords: Hyperglycemia.Obesity.Prediabetes states.Vitamin C.

I N T R O D U C T I O N
Vitamin C, also known as ascorbic acid, is one of the main dietary vitamins.It is present in a variety of foods, such as fruits and vegetables 1 .Moreover, vitamin C is a natural antioxidant whose action has been studied because of its numerous important biological effects 2 .
One of the possible beneficial effects of vitamin C is prevention and treatment of obesity 3 .Human 4 and animal 5 studies have found that vitamin C supplementation has effectively reduced body weight.Still in this context, vitamin C may also have a protective role in the stages of induction and development of type 2 Diabetes Mellitus (DM2) 6 , helping to regulate glucose https://doi.org/10.1590/1678-98652017000100003transport in the cell 7 , contributing to better glycemic control in DM2.
Prediabetes occurs before the onset of DM2.It is an intermediate hyperglycemic state where individuals present low glucose tolerance and initial insulin resistance 8 .It is during this stage that individuals are more likely to develop functional or pathological changes, before the establishment of DM2 diagnosis 9 .This stage is also closely related to an increase in overweight and obesity, a predictor for the development of DM2 and its complications 10,11 .Excess energy intake and a sedentary lifestyle are among the main risk factors for obesity and the development of DM2.In this context, excess sugar intake has an important role in the growing global prevalence of both conditions [11][12][13] .In most situations, higher dietary glycemic load leads to a positive energy balance, increasing body fat and insulin resistance 14 .
More studies are needed on factors that minimize the adverse health effects caused by excess sugar intake, and vitamin C may be a nutritional strategy to reduce weight gain, visceral fat, Body Mass Index (BMI), glycemia, and low glucose tolerance in prediabetes, given that this stage is still little explored.Therefore, the objective of this study was to assess whether vitamin C can prevent obesity and hyperglycemia in Wistar rats given excess invert sugar to induce prediabetes.

M E T H O D S Animals
This study was approved by the Animal Research Ethics Committee of Universidade de Santa Cruz do Sul (under Protocol nº 14/2013).Thirty-two male Wistar rats with approximately 100 days age and average body weight of 336.58±23.43g were placed in individual cages at the university's vivarium at a temperature of 22±3°C, 12:12-hour light-to-dark cycle, and relative humidity of 60%.

Treatment
The animals were allowed to adapt to their new environment for seven days, during which they received a chow with normal protein and energy contents (Nuvilab ® , Quimtia, Colombo, Brazil; pertinent composition presented in Table 1) and water ad libitum.After this time, the 32 animals were divided into four groups (8 animals in each group) to receive the following treatments for 17 weeks: 1) Control group (group C), given only water; 2) group Invert Sugar (IS) control, given a watery solution of invert sugar; 3) group Vitamin C (VitC) control, given a watery solution of vitamin C; and 4) group Vitamin C + Invert Sugar (VitCIS), given a watery solution of vitamin C and invert sugar.Eight animals were placed in each group based on the research group's past experience, always trying to use fewer animals than similar experiments 15,16 .
Water was used as the vehicle for vitamin C and invert sugar administration, which was provided ad libitum to the animals, along with the chow, the same one with normal protein and energy contents used during the adaptation

Test substances: Induction of prediabetes and vitamin C supplementation
In order to induce prediabetes in the animals we used an overload of invert sugar (Pantec ® , São Paulo, São Paulo, Brazil) dissolved in water at a concentration of 32% 17 .
Vitamin C supplementation (Sigma A4544) was based on the concentrations of the Tolerable Upper Intake Level for humans (Upper Level [UL]) 18 adapted for rats.UL is the maximum tolerable daily intake to avoid the chance of a nutrient causing adverse health effects.Thus, to avoid health risks to the animals caused by excess vitamin C (UL according to the Dietary Reference Intakes=2,000 mg/day 18 ), the animals were given only 0.5UL.In humans, 0.5UL is equivalent to 14mg/kg for an adult male weighing 70kg, so extrapolating to rats, which weigh on average 400g and consume approximately 100mL of fluids per day 19 , the amount provided was approximately 60mg/L.A watery solution of vitamin C remained available for one week at a controlled temperature of 22±3°C.The solution was protected from light to minimize vitamin C loss.At the end of the experiment, the mean fluid intake was measured, and the vitamin C intake of each group was calculated.
Two criteria were used for confirming prediabetes: obesity and low glucose tolerance, adapted from Hafizur et al. 15 , who presented a 'humanized' prediabetes model in rats.

Obesogenic markers
A rodent-specific BMI was used for assessing obesity.It is given by the relationship between the animal's weight and length.The rats' BMI were calculated weekly as recommended by Novelli et al. 20 .Rats with BMI>0.67g/cm 2 were considered obese.The animals were weighed by an analytical balance (Toledo ® , São Bernardo do Campo, São Paulo, Brazil) with minimum accuracy of 25.00g and maximum load of 35.00kg.An anthropometric tape measure (Carci ® , São Paulo, São Paulo, Brazil) measured the length of the animal's body from nose to anus.
At the end of the experiment, the body fat of the animals was collected as recommended by Cinti 21 , which involves collecting the visceral fat deposits, located in the thorax and abdominal cavity, to weigh and assess the amount of visceral fat.

Hyperglycemia marker
Glucose tolerance was assessed by the Intraperitoneal Glucose Tolerance Test (ipGTT) 22 at the end of the experiment (17 th week).The ipGTT curve was measured by the portable glucometer Accu-Chek ® (São Paulo, São Paulo, Brazil), using blood collected from the tail's vein after a 6-hour fast (time 0).Next, intraperitoneal glucose was administered (1mg of glucose per g of rat weight) and glycemia was measured 5, 15, 30, 60, and 120 minutes later.Blood glucose changes during the ipGTT were expressed as glucose Area Under the Curve (AUC).

Statistical analysis
The data were tabulated and treated by the software GraphPad Prism 5.01 (GraphPad Software, Inc.; San Diego, California), using oneway Analysis of Variance (one-way Anova) followed by the Bonferroni post hoc test or Student's t-test for independent samples.Pearson's correlation analysis was also used.All data were expressed as mean and standard deviation after being checked for normality and homoscedasticity.The significance level was set at 5% (p<0.05).

D I S C U S S I O N
Treatment with an overloaded watery solution (32% concentrated) of invert sugar was capable of inducing prediabetes in rats, as their tolerance to glucose decreased according to the ipGTT, as well as obesity.Invert sugar was used for inducing prediabetes because it is present in sugary beverages.It consists of a mixture of equal parts (33% of each with 33% of inversion) of glucose, fructose, and invert sucrose 23,24 .Moreover, studies have found that sugar overload can promote metabolic, hemodynamic, structural, and functional disorders in rodents, increasing plasma insulin, leptin, triglycerides, glucose, and free fatty acids, leading to low glucose tolerance 25 , typical prediabetes characteristics.
Finding nutritional alternatives that can minimize the adverse health effects caused by excess sugar can be an important strategy as it is extremely challenging to reduce the intake of high-sugar foods 26 .In this sense, vitamin C could easily be included in the composition of commercially prepared foods or added to the diet as a supplement.Vitamin C may benefit all individuals, including nondiabetics (vitamin C control group) as found by the present and other studies [3][4][5] , reducing the amount of weight and visceral fat gain, normalizing BMI, and acting as a blood glucose-lowering agent.
The groups given invert sugar (group IS and VitCIS) had higher liquid and lower chow intakes.The energy intake of these two groups were similar, but the weight gained by the rats co-treated with vitamin C (group VitCIS) was lower than that gained by the group given only IS.Studies have reported that high energy intake is the main cause of excess weight 27 .Sugary beverages increase appetite more than solid foods given the low satiety and incomplete compensation of fluid calories, being an important contributor to higher fat deposition 14,28 .Since vitamin C reduces weight and visceral fat gain 3,4 , it may have helped to reduce weight gain in the study rats.
Co-treatment with vitamin C was capable of reducing induced weight gain promoted by invert sugar intake.At the same time, vitamin C effectively reduced visceral fat accumulation.These effects may be related to the antioxidant nature of vitamin C, as demonstrated by Garcia--Diaz et al. 3 .In the present study, both lower weight gain and lower visceral fat in the group co-treated with vitamin C and invert sugar are similar to those of the control group.Both parameters (weight gain and visceral fat) effectively reduced the BMI gain of group VitCIS.BMI is an important tool to assess obesity 20 as obesity is one of the risk factors for DM2 development 10,27 .
Al-Shamsi et al. 5 found that weight loss in streptozotocin-induced diabetic Wistar rats was associated with vitamin C dose.Moderate and high doses (50mg/kg; 100mg/kg) of vitamin C for 10 days slowed down weight gain more effectively, especially after DM onset 5 .Still in this context, Leffa et al. 29 assessed the effect of vitamin C (1mg/kg) in rats given a cafeteria diet for 13 weeks and also found that animals co-treated with vitamin C gained less weight.Nonetheless, the mean energy intake of the group fed the cafeteria diet was lower than that of the control group, despite the cafeteria diet's high energy density and low nutrient density.The cafeteria diet consists of chocolate, biscuits, cookies, hot dogs, potato chips, and carbonated beverages, among others 25 .
Even though Al-Shamsi et al. 5 used relatively high doses of vitamin C (50 mg/kg; 100 mg/kg) to slow down weight gain, the treatment time (10 days) was much shorter than that of the present study.On the other hand, Leffa et al. 29 not only used a lower dose of vitamin C (1 mg/kg) than the dose used herein, but also treated their rats for a shorter period (13 weeks).In the present study, the difference in weight gained by groups VitCIS and IS only appeared in week twelve.
The mechanisms by which vitamin C slows down weight gain have not been fully elucidated, but Garcia-Diaz et al. 3 suggest that this beneficial effect may be associated with vitamin C ability to modulate adipocyte lipolysis, regulate the release of glucocorticoids by the adrenal glands, inhibit glucose metabolism and leptin release by isolated adipocytes, reduce hyperglycemia and glycosylation in obese-diabetic models, and reduce the inflammatory response.Carillon et al. 30 pointed out that obesity induced by an inadequate diet may be associated with oxidative stress, reducing the antioxidant defenses and allowing vitamin C to act as a natural antioxidant, which helps to treat obesity.Still in this context, in oxidant conditions, obesity may favor the development of insulin resistance by deregulating pro-inflammatory adipokines and cytokines 31 .Hence, dietary antioxidant supplementation, especially of vitamin C, may be beneficial.
Little is known about the effects of vitamin C in prediabetes, especially because controlled DM2-prevention studies using vitamin C are still needed 32 .In the present study, both groups given vitamin C had significantly lower fasting glycemia than the control group, demonstrating a glycemia-lowering effect of the vitamin.Likewise, groups VitC and C had similar glucose AUC.The AUC of the animals treated with invert sugar and vitamin C was lower than that of group IS.Both AUC showed that vitamin C reduced glucose intolerance.Leffa et al. 29 found that the group given vitamin C supplements or vitamin C-containing juices did not have smaller glucose AUC than the group treated with the cafeteria diet.Corroborating the present study, Al-Shamsi et al. 5 found that 10mg/kg of vitamin C reduced fasting glycemia and glucose AUC.However, higher doses of vitamin C (100mg/kg) did not have a glycemia-lowering effect 5 .
There is evidence that vitamin C can improve DM2-related endothelial dysfunction and improve insulin resistance by improving insulin signaling 3,33 .Another hypothesis for the ability of vitamin C to reduce glycemia is related to lower oxidative stress, preventing the development of DM2 3 .Nevertheless, the serum vitamin C of the https://doi.org/10.1590/1678-98652017000100003study animals was not measured, but it would provide better evidence of the vitamin C effects found by the present study.Unlike humans, rats are capable of synthesizing vitamin C, even though most humans can easily consume the recommended daily dose of vitamin C. The present study was careful not to use a high dose of vitamin C, using only 0.5UL, taking into account the rat's endogenous vitamin C production, as high doses of vitamin C (exceeding the UL) can adversely affect the animal's health, promoting pro-oxidant effects 2 .

C O N C L U S I O N
Vitamin C intake by rats with prediabetes induced by invert sugar overload was antiobesogenic and anti-hyperglycemic.This is an important finding given that epidemiological data have shown alarming rates of obesity with simultaneous increase of sugary beverages.These factors have increased the prediabetic population, and vitamin C can be a strategy to prevent the development of DM2 without the need of high doses.Additional studies are needed to understand the anti-obesogenic and antihyperglycemic mechanisms of vitamin C in prediabetes.

Figure 1 .
Figure 1.Difference between food (A), liquids (B), and energy (C) intake of groups: Control groups (C), Invert Sugar (IS) control, group Vitamin C (VitC) control, and Vitamin C+ control Invert Sugar (VitCIS), and of vitamin C intake (D) of the groups VitC and VitCIS.Laboratory of Experimental Nutrition of the Universidade de Santa Cruz do Sul (RS), Brazil, 2013.Note: * p<0.05; ** p<0.001.p: Significance level according to on-way Analysis of Variance followed by the Bonferroni post hoc test or Student's t-test.

Figure 2 .
Figure 2. Difference between body weight (A) and visceral fat (B) of the groups: Control groups (C), Invert Sugar (IS) control, group Vitamin C (VitC) control, and Vitamin C+ control Invert Sugar (VitCIS).Laboratory of Experimental Nutrition of the Universidade de Santa Cruz do Sul, Santa Cruz do Sul (RS), Brazil, 2013.Note: * p<0.05; ** p<0.01; *** p<0.001.p: Significance level according to one-way Analysis of Variance followed by Bonferroni post hoc test.

Figure 3 .
Figure 3. Evolution of body mass index of groups Control groups (C), Invert Sugar (IS) control, group Vitamin C (VitC) control, and Vitamin C+ control Invert Sugar (VitCIS).Laboratory of Experimental Nutrition of the Universidade de Santa Cruz do Sul, Rio Grande do Sul (RS), Brazil.2013.Note: r: Correlation coefficient and significance level of p according to the Pearson's correlation test.The horizontal dashed line indicates the body mass index cut-off point for the classification of obesity in rats according to Novelli et al. 20 .
A C K N O W L E D G M E N T S Colleagues at the Laboratory of Histology and Pathology, Laboratory of Biotechnology, and Laboratory of Experimental Nutrition of Universidade de Santa Cruz for help to sacrifice the animals.C O N T R I B U T O R S P MOLZ helped to conceive the study and experimental design, analyze and interpret data, and review the manuscript; AN RAEL and MQ FISCHER helped in the experimental design and reviewed the manuscript; LB LIMBERGER helped in the experimental design; D PRÁ helped to conceive the study and experimental design, and review the manuscript; SIR FRANKE helped to conceive the study and experimental design, interpret the data, and review the manuscript.

Table 1 .
Nutritional composition of the chow Presence byNuvilab ://doi.org/10.1590/1678-98652017000100003period.Food and water intake were measured weekly by subtracting the volumes not ingested from the volumes provided.