Evaluating the Impact of Different Hypercaloric Diets on Weight Gain, Insulin Resistance, Glucose Intolerance, and Its Comorbidities in Rats

Abstract

Animal experimentation has a long history in the study of metabolic syndrome-related disorders. However, no consensus exists on the best models to study these syndromes. Knowing that different diets can precipitate different metabolic disease phenotypes, herein we characterized several hypercaloric rat models of obesity and type 2 diabetes, comparing each with a genetic model, with the aim of identifying the most appropriate model of metabolic disease. The effect of hypercaloric diets (high fat (HF), high sucrose (HSu), high fat plus high sucrose (HFHSu) and high fat plus streptozotocin (HF+STZ) during different exposure times (HF 3 weeks, HF 19 weeks, HSu 4 weeks, HSu 16 weeks, HFHSu 25 weeks, HF3 weeks + STZ) were compared with the Zucker fatty rat. Each model was evaluated for weight gain, fat mass, fasting plasma glucose, insulin and C-peptide, insulin sensitivity, glucose tolerance, lipid profile and liver lipid deposition, blood pressure, and autonomic nervous system function. All animal models presented with insulin resistance and dyslipidemia except the HF+STZ and HSu 4 weeks, which argues against the use of these models as metabolic syndrome models. Of the remaining animal models, a higher weight gain was exhibited by the Zucker fatty rat and wild type rats submitted to a HF diet for 19 weeks. We conclude that the latter model presents a phenotype most consistent with that observed in humans with metabolic disease, exhibiting the majority of the phenotypic features and comorbidities associated with type 2 diabetes in humans.

Keywords: adipose tissue; animal models; glucose tolerance; hypertension; insulin sensitivity; metabolic syndrome; obesity; type 2 diabetes; weight gain.

Figure 1

Weight gain in animal models of type 2 diabetes and obesity. (A) Growth curves. (B) Weight increase in animal groups expressed as g/day. All groups of hypercaloric diet animals and their aged-matched controls as well as Zucker diabetic rat and its lean controls of 17 weeks included 6–8 animals per group; Zucker diabetic rat of 23 weeks included 3 animals per group, and its lean control only one animal. Bars represent means ± SEM. Two-way ANOVA with Bonferroni’s multiple comparisons test: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 comparing age-matched controls with disease models; ^# p < 0.05, ^### p < 0.001, and ^#### p < 0.0001 comparing HF3 weeks with HF19 weeks, HF+STZ, HSu 4 weeks, HSu 4 weeks, HFHSu 25 weeks, and Zucker 17 weeks, respectively; ^ȠȠȠȠ p < 0.0001 comparing HF19 weeks with HSu 4 weeks, HSu 16 weeks, HFHSu 25 weeks, and Zucker 17 weeks; ^ϕϕϕϕ p < 0.0001 comparing HF+STZ with HSu 4 weeks, HSu 16 weeks, and Zucker 17 weeks; ^ΔΔ p < 0.01 comparing HSu 4 weeks with HSu 16 weeks and Zucker 17 weeks; ^θθθθ p < 0.0001 comparing HFHSu 25 weeks with Zucker 17 weeks; ^ΓΓΓΓ p < 0.0001 comparing HSu 16 weeks with Zucker 17 weeks.

Figure 2

Glucose metabolism and insulin action and secretion in animal models of type 2 diabetes and obesity. (A) Fasting glycemia levels; (B) insulin sensitivity expressed as the constant of insulin tolerance test (K_ITT); (C) glucose tolerance depicted as glucose excursion curves (left panel) and as the area under the curve (AUC) obtained from the glucose excursion curves (right panel); (D) fasting insulin (left panel) and C-peptide (right panel) levels in animal models of obesity and type 2 diabetes. All groups of hypercaloric diet animals and its aged-matched controls as well as Zucker diabetic rat and its lean controls of 17 weeks included 6–8 animals per group; Zucker diabetic rat of 23 weeks included 3 animals per group, and its lean control only one animal. Bars represent means ± SEM; two-way ANOVA with Bonferroni’s multiple comparisons test: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 comparing age-matched controls with disease models; ^# p< 0.05, ^## p < 0.01, and ^#### p < 0.0001 comparing HF 3 weeks group with HF 19 weeks, HF+STZ, HSu 4 weeks, and Zucker 17 weeks; ^Ƞ p < 0.05, ^ȠȠ p < 0.01, ^ȠȠȠ p < 0.001, and ^ȠȠȠȠ p < 0.0001 comparing HF 19 weeks with HF+STZ, HSu 4 weeks, HFHSu 25 weeks, and Zucker 17 weeks; ^ϕ p < 0.05, ^ϕϕ p < 0.01, ^ϕϕϕ p < 0.001, and ^ϕϕϕϕ p < 0.0001 comparing HF+STZ with HSu 16 weeks, HFHSu 25 weeks, and Zucker 17 weeks; ^∆ p < 0.05; ^∆∆ p < 0.01, and ^∆∆∆∆ p < 0.0001 comparing HSu 4 weeks with HFHSu 25 weeks and Zucker 17 weeks; ^Γ p < 0.05, ^ΓΓ p < 0.01, and ^ΓΓΓ p < 0.001 comparing HSu 16 weeks group with Zucker 17 weeks; ^θ p < 0.05, ^θθ p < 0.01, and ^θθθθ p < 0.0001 comparing HFHSu 25 weeks with Zucker 17 weeks.

Figure 3

Alterations in the expression of proteins involved in insulin signaling in insulin-sensitive tissues in animal models of obesity and type 2 diabetes. (A) Total insulin receptor (IR) expression (B) phosphorylated IR expression; (C) total AKT expression; (D) phosphorylated AKT expression; (E) Glu4 expression. Below the graphs, representative bands from the Western blots for the correspondent proteins are shown. Bars represent means ± SEM; one- and two-way ANOVA with Bonferroni’s multiple comparisons test: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 comparing age-matched controls with disease models; ^# p < 0.05 and ^## p < 0.01 comparing HF 3 weeks with HSu 16 weeks; ^ΔΔ p < 0.01 and ^ΔΔΔ p < 0.001 comparing HSu 4 weeks with HSu 16 weeks.

Figure 4

Mean arterial pressure levels in the distinct models of obesity and type 2 diabetes. All groups of hypercaloric diet animals and its aged-matched controls as well as Zucker diabetic rat and its lean controls of 17 weeks included 6–8 animals per group; Zucker diabetic rat of 23 weeks included 3 animals per group, and its lean control only one animal. Bars represent means ± SEM; two-way ANOVA with Bonferroni’s multiple comparisons test: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 comparing age-matched controls with disease models.

Figure 5

Lipid deposition in the liver in the distinct models of obesity and type 2 diabetes. (A) Hematoxicilin and eosin images of liver slices from HF 19 weeks (A1), HFHSu 25 weeks (A2), and Zucker 23 weeks (A3) and their respective controls. (B) Percentage of lipid deposition in the liver quantified by the Folch method [26]. All groups of hypercaloric diet animals and its age-matched controls as well as Zucker diabetic rat and its lean controls of 17 weeks included 6–8 animals per group; Zucker diabetic rat of 23 weeks included 3 animals per group, and its lean control only one animal. Bars represent means ± SEM; two-way ANOVA with Bonferroni’s multiple comparisons test: ** p < 0.01, *** p < 0.001, and **** p < 0.0001 comparing age-matched controls with disease models; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 comparing HF 3 weeks with HF 19 weeks, HSu 4 weeks, and HFHSu 25 weeks; ^Ƞ p < 0.05 and ^ȠȠȠȠ p < 0.0001 comparing HF 19 weeks with HF+STZ, HSu 4 weeks, and HFHSu 25 weeks; ^ϕ p < 0.05, ^ϕϕ p < 0.01 comparing HF+STZ with HSu 4 weeks and HFHSu 25 weeks; ^∆ p < 0.05 comparing HSu 4 weeks with Zucker 17 weeks; ^Γ p < 0.05 comparing HSu 16 weeks with Zucker 17 weeks; ^θθ p < 0.01 comparing HFHSu 25 weeks with Zucker 17 weeks. Black arrowheads represent lipidosis and white arrowheads fibrosis. Scale bar, 100 μm; original magnification, 20×.

Figure 6

Effect of hypercaloric diets and genetic deletion of leptin receptors on sympathetic activity evaluated by spectral analysis of the heart rate. (A) shows representative experiments of power spectral density (PSD) calculated in control, in HF 3 weeks, and in HF+ STZ animals. (B,C) Autonomic function assessed by the ratio between the percentage of low frequencies (LF) that represents the sympathetic component of the autonomic nervous system and the percentage of high frequencies (HF) that represents the parasympathetic component of the autonomic nervous system. Frequencies are presented in normalized units. All groups of hypercaloric diet animals and their aged-matched controls as well as Zucker diabetic rat and its lean controls of 17 weeks included 6–8 animals per group; Zucker diabetic rat of 23 weeks included 3 animals per group, and its lean control only one animal. Bars represent means ± SEM; two-way ANOVA with Bonferroni’s multiple comparisons test: * p < 0.05 and ** p < 0.01 comparing age-matched controls with disease models.