Diets High in Carbohydrates, Fats, and their Combination Modify Zoometric Parameters

After 90 days of feeding the animals with HCD, HFD, and HCD + HFD diets, the zoometric parameters were altered with respect to CD (Table 2). Rats fed with HCD and HCD + HFD showed a significant increase in body weight (10.7% and 23%, p < 0.01; F = 3.169), BMI (12.5% and 23%, p < 0.01; F = 5.785), and as we expected, all experimental groups showed a significant increase in the Lee index: HCD (30%), HFD (28%), and HCD + HFD (35%) (p < 0.01; F = 7.913). These results indicate increased adiposity, which is associated with MS.

Table 2 Diets high in carbohydrates, fats, and their mixture modify the zoometric parameters in male rats. The results shown are the average of zoometric parameters (n = 12/group) ± SD. (**) p < 0.01 indicates significant difference compared to control group by One-way ANOVA test followed Bonferroni post hoc test. BMI: Body Mass IndexDiets High in Carbohydrates, Fats, and their Mixture Disrupt Carbohydrate and Lipid Homeostasis

The biochemical results of MS hallmarks were compared between HCD, HFD, the mixture group, and the control group (CD) (Fig. 1). Fasting glucose levels (Fig. 1A) in the HCD and HCD + HFD groups increased by 84% and 90% (p < 0.001; F = 129.3). Fasting insulin levels (Fig. 1B) increased significantly in the experimental groups: 223% in HCD, 151% in HFD, and 197% in HCD + HFD (p < 0.001; F = 123.7). Fructosamine levels (Fig. 1C) were significantly elevated in all groups: 105% in HCD, 56% in HFD, and 85% in HCD + HFD (p < 0.001; F = 98.86). Besides, the HOMA-IR index also increased significantly: 513% in HCD, 146% in HFD, and 486% in HCD + HFD (p < 0.001; F = 204.4) (Fig. 1D). Clearly indicating a loss of homeostasis in the metabolism of carbohydrates at the serum level due to the consumption of these diets.

Fig. 1

Effect of high- diet consumption on serum carbohydrate and lipid metabolism in male rats. After treatment, the animals in each experimental group (n = 12) had their blood levels determined: Glucose (A), Insulin (B), Fructosamine (C), HOMA-IR, Triglycerides, Total Cholesterol, HDL, LDL and VLDL (D). The mean of data ± SD is plotted. Data were analyzed with One-way ANOVA followed Bonferroni post hoc test *p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the CD group)

The lipid profile showed metabolic dyslipidemia (Fig. 1E). Hypertriglyceridemia was observed in all groups by 76% in HCD, 79% in HFD, and 88% in HCD + HFD (p < 0.0001; F = 36.44). Total cholesterol levels did not differ significantly between groups (p = 0.1095; F = 2.289). However, the cholesterol fractions exhibited quantitative changes. VLDL cholesterol increased by 76% in HCD, 83% in HFD, and 56% in HCD + HFD (p < 0.0001; F = 20.86). LDL cholesterol was significantly elevated only in the HCD group (45%; p < 0.05; F = 13.22). HDL cholesterol levels decreased significantly by 33% in HCD, 21% in HFD, and 28% in HCD + HFD (p < 0.01; F = 11.39). These results show evidence that chronic consumption of any of these diets causes dyslipidemia.

Oral glucose tolerance tests and corresponding insulin responses were performed for all groups (Fig. 2). At time 0, glucose levels were significantly higher in the HCD and HCD + HFD (p < 0.001; F = 129.3) groups compared to the CD group. At 30 min post-glucose loading, no significant differences were observed between the experimental groups (p = 0.0137; F = 4.620). However, at 60 and 90 min, glucose levels were significantly higher in all nutrient-rich diet groups: HCD (126% and 64%), HFD (69% and 114%), and HCD + HFD (98% and 138%) (p < 0.0001; F = 193.6 and p < 0.0001; F = 87.96).

Fig. 2

Diets rich in carbohydrates, fats, and their mixture cause peripheral insulin resistance in male rats. The glucose tolerance test and insulin response were performed in conscious animals (n = 12/group). A glucose load (1.75 g/kg) was administered orally, and blood glucose and insulin levels were measured at 0, 30, 60, and 90 min after the load. The mean of data ± SD is plotted. Data were analyzed with One-way ANOVA followed Bonferroni post hoc test; aaap < 0.001 to compare the HCD group and CD; bbbp < 0.001 to compare the HFD group and CD and cccp < 0.001 to compare the HFD group and CD

The insulin response to the glucose load also showed a significant increase at fasting (0 min) in the HCD (217%) and HCD + HFD (197%) (p < 0.001; F = 123.7) groups relative to the control. At 30, 60, and 90 min, insulin levels significantly increased in the HCD group (132%, 137%, and 184%), in the HFD group (49%, 107%, and 99%), and in the HCD + HFD group (96%, 124%, and 163%) (p < 0.0001; F = 51.15, p < 0.0001; F = 31.22 and p < 0.0001; F = 290). Collectively, our results indicate that exposure to diets high in sugars, fats, and their mixture dysregulates the regulation of carbohydrate and lipid metabolism.

Diets High in Carbohydrates, Fats, and their Mixture Increase Corticosterone and ACTH in the Blood

The study evaluated peripheral hormonal stress factors by measuring corticosterone, and ACTH levels in serum (Fig. 3A-C). The corticosterone and ACTH levels were elevated in the HCD group (198%, and 78%), in HFD (419%, and 87%), and in the HCD + HFD (461%, and 144%) (p < 0.0001; F = 74.70 and p < 0.0001; F = 123.1). These findings clearly demonstrate that the HCD, HFD, and HCD + HFD diets are an important factor in raising stress hormones, and the effect is most pronounced when animals receive the combined HCD + HFD diet.

Fig. 3

Diets high in carbohydrates, fats, and their mixture increase hypothalamic-pituitary-adrenal (HPA) axis activity in the blood of male rats. After treatment, the concentration of corticosterone (A) and ACTH (B) in the blood of each experimental group chronically exposed to diets high in carbohydrates, fats and their mixture was quantified in the animals of each experimental group (n = 6). The mean of data ± SD is plotted. Data were analyzed with One-way ANOVA followed Bonferroni post hoc test (**p < 0.01 and ***p < 0.001, compared with the CD group)

Chronic Consumption of Diets Rich in Carbohydrates, Fats, and their Mixture Induces Anxiety

Anxiety levels were evaluated under two different conditions: the ECM test to assess anxiety under non-stressful conditions, and the forced swim test to assess anxiety under stressful conditions (Fig. 4).

Fig. 4

Chronic consumption of diets high in carbohydrates, fats, and their mixture increases anxiety-like behavior in male rats. To assess anxiety-like behaviors, experimental animals were exposed to the elevated cross maze. (n = 6) and performed the forced swim test (n = 6). In the elevated cross-maze task (A-D), the number of visits and the time spent in seconds in both the open and closed arms were quantified, respectively. In the forced swim test (E), the number of limb movements per minute was quantified for each of the animals treated with diets high in carbohydrates, fats and a mixture of them. The mean of data ± SD is plotted. Data were analyzed with One-way ANOVA followed Bonferroni post hoc test (∗p < 0. 05; ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the CD group)

In the ECM test, initially, the distance traveled (Fig. 4A) and velocity (Fig. 4B) of each group of animals were quantified. The results indicate that diets high in carbohydrates, fats, and their combination, compared to the group fed with CD, did not modify the distance traveled (CD: 200.4 ± 16.67 cm; HCD: 256.9 ± 27.25 cm; HFD: 251.7 ± 19.28 cm; HCD + HFD: 257.2 ± 37.05 cm) (p = 0.3826; F = 1.074) or velocity (CD: 52 ± 8.22 cm/min; HCD: 58.5 ± 9.46 cm/min; HFD: 57.50 ± 7.60 cm/min; HCD + HFD: 59.67 ± 6.53 cm/min) (p = 0.3826; F = 1.074) during their stay in the ECM. This suggests that feeding with the different diets studied did not alter motor activity.

Furthermore, animals fed diets rich in carbohydrates, fats, and their combination showed a reduced number of visits with open arms (Fig. 4C) compared to the control group (CD: 7.8 ± 1.16; HCD: 2.6 ± 1.03; HFD: 4.8 ± 0.75; HCD + HFD: 3.3 ± 0.81) (p < 0.0001; F = 34.55). In contrast, the CD group made fewer visits into the closed arms (CD: 3.83 ± 0.75) than the experimental groups (HCD: 7.6 ± 1.2; HFD: 6.1 ± 0.75; HCD + HFD: 6.5 ± 1.04) (p < 0.0001; F = 16.74) (Fig. 4D).

The time spent in the open arms (Fig. 4E) was significantly lower in the groups with diets rich in carbohydrates, fats, and their mixture (HCD: 39.4 ± 9.5 s; HFD: 90.4 ± 9.4 s; HCD + HFD: 55.6 ± 9.2 s) than in the control group (CD: 119.6 ± 10.7 s) (p < 0.05; F = 81.38). While the time spent in the closed arm (Fig. 4F) was only significant in animals fed with HCD (199.4 ± 20.5s) and HCD + HFD (182.6 ± 33.4 s) compared to the control group (CD:120.6 ± 12.7 s) (p < 0.05; F = 11.55). Statistical analysis of the data suggests that animals fed CD tend to develop non-anxious behavior compared to animals treated with high-nutrient diets under non-stressful conditions.

In the forced swim test (Fig. 4G), during the habituation phase, animals fed HCD, HFD, and HCD + HFD showed a significant increase of 28%, 27%, and 29% in limb movements per minute compared to the group fed CD (p < 0.05; F = 6.582). This behavior was interestingly replicated 24 h after the habituation phase. The data reveal that the number of upper and lower limb movements increased by 48% in the HCD group, 48% in the HFD group, and 85% in the HCD + HFD group (p < 0.001; F = 265.4) (Fig. 4H), respectively. The results indicate that animals under stressful conditions and with chronic consumption of high-nutrient diets exhibit a greater number of limb movements in both the habituation and final evaluation phases. Consequently, it is associated that a stressor makes anxious behavior evident in the animals. The results using both models suggest that animals consuming nutrient-rich foods are more likely to develop anxiety-like behaviors, so a diet high in carbohydrates, fats, and their combination could be considered one of the multiple factors that cause anxiety.

Chronic Consumption of Diets High in Carbohydrates, Fats, and Mixed Diets Induces Neuronal Hypotrophy

To evaluate the impact of MS induced by diets rich in carbohydrates, fats, and the mixture on neuronal structure, we performed Golgi-Cox staining and analysis of total dendritic length (TDL) and branching order of pyramidal neurons in the PFC (layers III and V), the CA1 and CA3 regions of the hippocampus, granule cells in the dentate gyrus (DG) and basolateral amygdala (BLA), as well as the medium spiny neurons in the nucleus accumbens (NAcc).

Figure 5 presents representative photomicrographs of neurons from the brain regions of interest after 3 months of diet consumption. Sholl analysis to determine the branching order of pyramidal neurons of layer III pyramidal neurons in the PFC (Fig. 6A) revealed a high interaction between HCD, HFD, HCD-HFD consumption, and reduction in dendritic arborization from orders 2 to 4 in the HCD and HFD groups (F3,28 = 77.83, p < 0.001). In contrast, orders 3 and 4 in the HCD + HFD group (F3,28 = 4.88, p = 0.0077) showed a lower dependency on high-nutrient intake, despite diminished dendritic arborization. In layer V (Fig. 6B), the HCD group showed a reduction in orders 2 to 4 (F3,28 = 104.88, p = 0.0001), the HCD + HFD group from orders 2 to 4 (F3,28 = 71.23, p = 0.0001), and the HFD group from orders 2 to 4 (F3,28 = 78.81, p < 0.0001). Layer V showed a high susceptibility to metabolic conditions induced by a diet high in carbohydrates, fats, or their combination. The TDL in pyramidal neurons of layers III and V of the prefrontal cortex (PFC) of the experimental groups is shown in Fig. 7A and B. In the graphs, the small black circles represent the average TDL of each animal per group. Data analysis indicates that animals exhibit lower TDL in the HCD (36% and 31%), HFD (31% and 20%), and HCD + HFD (34% and 26%) groups (p < 0.001; F = 20.26, in layer III and p < 0.001; F = 25.16, in layer V) compared to the CD group.

Fig. 5

Diagram of the dendritic tree of limbic system neurons in male rats fed diets high in carbohydrates, fats and their mixture for 90 days. The panel shows representative neurons of the neurons of interest and a schematic of the dendritic arbors of neurons in the prefrontal cortex (PFC) layers 3 and 5; hippocampus CA1 and CA3, as well as the dentate gyrus (DG), basolateral amygdala (BLA) and nucleus accumbens (NAcc)

Fig. 6

Effects of diets high in carbohydrates, fats, and their mixture on dendritic length according to branching order in limbic brain regions of male rats. Abbreviations: (PFC) layers 3(A) and 5(B); hippocampus CA1(C) and CA3(D), as well as the dentate gyrus (DG)(E), basolateral amygdala (BLA) (F) and nucleus accumbens (NAcc)(G). The Average Length (µm) ± SD (n = 8/ group) is plotted. Data were analyzed with Two-way ANOVA; ap < 0.05, aap < 0.01 and aaap < 0.001 to compare the HCD group and CD; bp < 0.05, bbp < 0.01 and bbbp < 0.001 to compare the HFD group and CD and cp < 0.05, cccp < 0.01 and cccp < 0.001 to compare the HCD + HFD group and CD

Fig. 7

Evaluation of total dendritic length (TDL) in limbic regions of male rats fed diets high in carbohydrates, fats, and their mixture for 90 days. Abbreviations: (PFC) layers III (A) and layer V (B); hippocampus CA1(C) and CA3(D), as well as the dentate gyrus (DG)(E), basolateral amygdala (BLA) (F) and nucleus accumbens (NAcc)(G). The dots represent the average LDT of neurons per rat ± SD (n = 8/group). Data were analyzed with One-way ANOVA followed Bonferroni post hoc test ∗∗p < 0 01 and ∗∗∗p < 0 001 compared with the CD group

In the hippocampus, pyramidal neurons in the CA1 region (Fig. 6C) showed reduced dendritic complexity in the HCD group (orders 1–4; F3,28 = 140.5, p < 0.001) and in the HCD + HFD group (orders 1–3 and 5; F3,28 = 180.2, p < 0.001), while the HFD group reduced at orders 2 and 3 (F3,8 = 2.25, p < 0.001). In the CA3 region (Fig. 6D), dendritic arborization was significantly reduced at from orders 1 to 6 in the HCD and HCD + HFD groups (F3,28 = 66.24, p < 0.001), and at orders 2 to 6 in the HFD group (F3,28 = 150.1, p < 0.001). Similarly, in DG granule neurons (Fig. 6E), all groups that followed a diet high in carbohydrates, fats, and their mixture showed significant reductions in dendritic branching at orders 3 to 6 compared to controls (F3,28 = 53.02, p < 0.0001; F3,28 = 27.91, p = 0.0006; F3,28 = 50.81, p < 0.0001).

Similarly, when evaluating the average LDT of the animals in each experimental group, Fig. 7C and D show that, on average, the hippocampal neurons in the CA1, CA3, and DG regions of the animals fed high-nutrient diets decreased significantly (HCD: 32.7, 41.0, and 33.1%), (HFD: 22.1, 46.3, and 42.3%), and (HCD + HFD: 22.7, 33.8, and 33.2%)(CA1; p < 0.001; F = 18.85),(CA3; p < 0.001; F = 36.46), (DG; p < 0.001; F = 77.08). This demonstrates that chronic consumption of foods high in carbohydrates, fats, and their combination causes dendritic hypotrophy in the CA1, CA3 and DG of the animals, clearly contrasting with the neurons of the animals that consumed a standard diet (CD).

In the pyramidal neurons, the basolateral amygdala (BLA) showed decreased arborization complexity (Fig. 6F). The HCD, HFD, and HCD + HFD groups revealed significant reductions at dendritic orders 2 to 5 (F3,28 = 138.32, p < 0.0001). When analyzing the average TDL of the rats that make up each experimental group, the data confirm that there is a significant reduction in TDL of the animals in the HCD group (41.2%), HFD (36.5%) and HCD + HFD (44.2%) compared to the CD group (Fig. 7F) (p < 0.001; F = 123.6).

Finally, in the nucleus accumbens (NAcc), medium spiny neurons exhibited decreased arborization complexity (Fig. 6G). The HCD group showed significant reductions at dendritic orders 1 to 3 (F3,28 = 83.74, p < 0.0001), the HFD group at orders 2 and 3 (F3,28 = 31.23, p < 0.0001), and the HCD + HFD group at orders 2 and 3 (F3,28 = 51.74, p < 0.0001). While the average dendritic length (DL) in neurons of animals fed HCD, HFD, and HCD + HFD was significantly reduced (40.7%, 20.6%, and 18.1%) compared to the CD group (p < 0.001; F = 19.40) (Fig. 7G). Morphological analysis suggests that chronic consumption of high-nutrient diets is a critical factor in causing a loss of arborization and dendritic length in limbic system neurons in male rats.

Diets High in Carbohydrates, Fats, and their Mixture Modify Caspase-3 Immunoreactivity and Cell Viability

Caspase-3 (Casp-3) immunoreactivity was analyzed in brain regions of interest. Qualitative analysis (Fig. 8A) revealed increased immunoreactivity in all areas examined in the three experimental groups, with the most pronounced changes observed in animals exposed to the HCD. Semi-quantitative analysis (Fig. 8B) further supported these findings. By showing that in the PFC (p < 0.001; F = 50.10), CA1 (p < 0.001; F = 73.58), CA3 (p < 0.001; F = 82.28), DG (p < 0.001; F = 38.75), BLA (p < 0.001; F = 100.1)and NAcc (p < 0.001; F = 44.0) there was greater immunoreactivity to Cap-3 in the groups with HCD (44.2, 83.6, 72.9, 100, 93.2 and 118.6%,), HFD (12.1, 66.1, 86.4, 84.2, 94.3 and 123.7%) and HCD + HFD (75.8, 124.4, 202.7, 116.6, 102.1 and 100%).

Fig. 8

Diets rich in carbohydrates, fats, and their mixtures increase caspase-3 (Casp-3) immunoreactivity and decrease cell viability in the limbic system of male rats. In (A) and (C) Representative photomicrographs are shown that demonstrate immunoreactivity for casp-3 (red color) and acridine orange (green color: viable cells and red color: apoptotic cell) in the prefrontal cortex (PFC); hippocampus CA1 and CA3, as well as in the dentate gyrus (DG), basolateral amygdala (BLA) and nucleus accumbens (NAcc). (B) and (D) show the number of Casp-3 immunoreactive cells/ 500 µm2 and % cell viability/500 µm2 in the study regions of interest. The mean of data ± SD (n = 4/ group) is plotted. Data were analyzed with Kruskal-Wallis test (∗p < 0 05 and ∗∗p < 0 01, compared with the CD group)

Regarding cell viability (green color) in brain regions of the limbic system, such as PFC, CA1, CA3, DG, ABL, and NAcc in animals fed with HCD, HFD, and their combination, as well as with CD, the results are shown in Fig. 8C. Qualitative analysis showed that in the three groups of foods high in carbohydrates, fats, and their combination, the red color was more intense in all brain regions studied, while the group fed with CD showed greater intensity of green and fewer red spots in the brain regions of the limbic system. The semi-quantitative analysis of Fig. 8D shows that the % of cell viability (green color) decreased in the PFC (p < 0.001; F = 124.6), CA1 (p < 0.001; F = 119.2), CA3 (p < 0.001; F = 97.38), DG (p < 0.001; F = 138.8), BLA (p < 0.001; F = 85.02) and NAcc (p < 0.001; F = 23.31) in the group with HCD (22.9; 20.6; 22.9; 25.8; 7.7 and 27.6%), HFD (16.9; 12.3; 20.2; 22.9; 5; 19.7%) and HCD + HFD (32.9; 22.1; 24.3; 31.2; 15.7; 31.8%,). In an integrated analysis, we can suggest that the consumption of diets high in carbohydrates, fats, or their combination, could represent a factor in increasing cell death by apoptosis.