Food restriction promotes damage reduction in rat models of type 2 diabetes mellitus

Abstract

There are several animal models of type 2 diabetes mellitus induction but the comparison between models is scarce. Food restriction generates benefits, such as reducing oxidative stress, but there are few studies on its effects on diabetes. The objective of this study is to evaluate the differences in physiological and biochemical parameters between diabetes models and their responses to food restriction. For this, 30 male Wistar rats were distributed in 3 groups (n = 10/group): control (C); diabetes with streptozotocin and cafeteria-style diet (DE); and diabetes with streptozotocin and nicotinamide (DN), all treated for two months (pre-food restriction period). Then, the 3 groups were subdivided into 6, generating the groups CC (control), CCR (control+food restriction), DEC (diabetic+standard diet), DER (diabetic+food restriction), DNC (diabetic+standard diet) and DNR (diabetic+food restriction), treated for an additional two months (food restriction period). The food restriction (FR) used was 50% of the average daily dietary intake of group C. Throughout the treatment, physiological and biochemical parameters were evaluated. At the end of the treatment, serum biochemical parameters, oxidative stress and insulin were evaluated. Both diabetic models produced hyperglycemia, polyphagia, polydipsia, insulin resistance, high fructosamine, hepatic damage and reduced insulin, although only DE presented human diabetes-like alterations, such as dyslipidemia and neuropathy symptoms. Both DEC and DNC diabetic groups presented higher levels of protein carbonyl groups associated to lower antioxidant capacity in the plasma. FR promoted improvement of glycemia in DNR, lipid profile in DER, and insulin resistance and hepatic damage in both diabetes models. FR also reduced the protein carbonyl groups of both DER and DNR diabetic groups, but the antioxidant capacity was improved only in the plasma of DER group. It is concluded that FR is beneficial for diabetes but should be used in conjunction with other therapies.

Fig 1. Pre-food restriction glycemia.

The glycemia of the rats was evaluated in the fasting (A) and postprandial (B) states after the initial 2 months of treatment for the control groups (C), diabetic+streptozotocin+cafeteria-style diet (DE) and diabetic+streptozotocin+nicotinamide (DN). Results expressed as mean±SE (n = 7-10/group). a p<0.05 vs C; b p<0.05 vs DE. One-way ANOVA and Tukey’s post-hoc test.

Fig 2. Glucose tolerance and insulin resistance pre-food restriction of rats (after 2 months of initial treatment).

(A) Glucose tolerance test (GTT); (B) Rate of increase of glycemia per minute during GTT; (C) insulin-tolerance test (ITT); (D) Rate of blood glucose reduction per minute during ITT. Groups: control (C), diabetic+streptozotocin+cafeteria style diet (DE) and diabetic+streptozotocin+nicotinamide (DN). Results expressed as mean±SE (n = 5/group). a p<0.05 vs group C; b p<0.05 vs DE; one-way ANOVA and Tukey’s post-hoc test.

Fig 3. Food restriction glycemia.

The glycemia of rats was evaluated in the fasting (A) and postprandial (B) states after 4 months of treatment. Groups: control (CC), control with food restriction (CCR), diabetic+streptozotocin+cafeteria-style diet (DEC); diabetic+streptozotocin+cafeteria-style diet with food restriction (DER), diabetic+streptozotocin+nicotinamide (DNC) and diabetic+streptozotocin+nicotinamide with food restriction (DNR). Results expressed as mean±SE (n = 5/group). *a p<0.05 vs CC; b p<0.05 vs DEC; c p<0.05 vs DNC. One-way ANOVA and Tukey’s post-hoc test analysis.

Fig 4. Tolerance to glucose and insulin resistance of rats after food restriction (after 4 months of treatment).

(A) Glucose tolerance test (GTT); (B) Rate of increase of glycemia per minute during GTT; (C) Insulin tolerance test (ITT); (D) Rate of blood glucose reduction per minute during ITT. Groups: control (CC), control with food restriction (CCR), diabetic+streptozotocin+cafeteria-style diet (DEC); diabetic+streptozotocin+cafeteria-style diet with food restriction (DER), diabetic+streptozotocin+nicotinamide (DNC) and diabetic+streptozotocin+nicotinamide with food restriction (DNR). Results expressed as mean±SE (n = 5/group). *a p<0.05 vs CC; b p<0.05 vs DEC; c p<0.05 vs DNC. One-way ANOVA and Tukey’s post-hoc test analysis.

Fig 5. Abdominal fat sum.

Added weight of retroperitoneal, mesenteric, periepididimal and subcutaneous fats of rats at the end of the 4 months of treatment for abdominal fat assessment. Groups: control (CC), control with food restriction (CCR), diabetic+streptozotocin+cafeteria-style diet (DEC); diabetic+streptozotocin+cafeteria-style diet with food restriction (DER), diabetic+streptozotocin+nicotinamide (DNC) and diabetic+streptozotocin+nicotinamide with food restriction (DNR). Results expressed as mean±SE (n = 5/group). *a p<0.05 vs CC. One-way ANOVA and Tukey’s post-hoc test analysis.

Fig 6. Immunostaining for pancreatic insulin-producing cells of rats after 4 months of treatment.

(A) Insulin-producing cells index; (B) Total number of insulin-producing cells; (C) Detail of a pancreatic islet, typical of a non-diabetic animal, immunostained for insulin (200x magnification); (D) Detail of a pancreatic islet, typical of a diabetic animal, immunolabelled for insulin (200x magnification). Groups: control (CC), control with food restriction (CCR), diabetic+streptozotocin+cafeteria-style diet (DEC); diabetic+streptozotocin+cafeteria-style diet with food restriction (DER), diabetic+streptozotocin+nicotinamide (DNC) and diabetic+streptozotocin+nicotinamide with food restriction (DNR). Results expressed as mean±SE (n = 5/group). *a p<0.05 vs CC; b p<0.05 vs DEC; c p<0.05 vs DNC. One-way ANOVA and Tukey’s post-hoc test analysis.

Fig 7. Radioimmunoassay for blood insulin.

(A) Amount of immunolabelled insulin in blood of rats after 2 months of treatment for groups control (C), diabetic+streptozotocin+cafeteria-style diet (DE) and diabetic+streptozotocin+nicotinamide (DN). (B) Amount of immunolabelled insulin in blood of rats after 4 months of treatment for groups control (CC), control with food restriction (CCR), diabetic+streptozotocin+cafeteria-style diet (DEC); diabetic+streptozotocin+cafeteria-style diet with food restriction (DER), diabetic+streptozotocin+nicotinamide (DNC) and diabetic+streptozotocin+nicotinamide with food restriction (DNR). Results expressed as mean±SE (n = 3-5/group). *a p<0.05 vs CC; b p<0.05 vs DEC; c p<0.05 vs DNC. One-way ANOVA and Tukey’s post-hoc test analysis.

Fig 8. Oxidative stress.

Effects of food restriction on the plasmatic oxidative status of type 2 diabetic rats. (A) Protein carbonyl groups, (B) Thiol groups and (C) Total antioxidant capacity (TAC). Oxidative status was assessed at the end of the food restriction period as described in Methods. CC, controls; CCR, controls under food restriction; DEC, diabetic (cafeteria-style diet) rats; DER, diabetic (cafeteria-style diet) rats under food restriction; DNC, diabetic (nicotinamide) rats; DNR, diabetic (nicotinamide) rats under food restriction. Results expressed as mean±SE (n = 4-5/group). *a p<0.05 vs CC; b p<0.05 vs DEC; c p<0.05 vs DNC. One-way ANOVA and Tukey’s post-hoc test analysis.