Caloric restriction favorably impacts metabolic and immune/inflammatory profiles in obese mice but curcumin/piperine consumption adds no further benefit

Abstract

Background: Obesity is associated with low-grade inflammation and impaired immune response. Caloric restriction (CR) has been shown to inhibit inflammatory response and enhance cell-mediated immune function. Curcumin, the bioactive phenolic component of turmeric spice, is proposed to have anti-obesity and anti-inflammation properties while piperine, another bioactive phenolic compound present in pepper spice, can enhance the bioavailability and efficacy of curcumin. This study sought to determine if curcumin could potentiate CR's beneficial effect on immune and inflammatory responses in obesity developed in mice by feeding high-fat diet (HFD).

Methods: Mice were fed a HFD for 22 wk and then randomized into 5 groups: one group remained on HFD ad libitum and the remaining 4 groups were fed a 10% CR (reduced intake of HFD by 10% but maintaining the same levels of micronutrients) in the presence or absence of curcumin and/or piperine for 5 wk, after which CR was increased to 20% for an additional 33 wk. At the end of the study, mice were sacrificed, and spleen cells were isolated. Cells were stimulated with T cell mitogens, anti-CD3/CD28 antibodies, or lipopolysaccharide to determine T cell proliferation, cytokine production, and CD4+ T cell subpopulations.

Results: Compared to HFD control group, all CR mice, regardless of the presence of curcumin and/or piperine, had lower body weight and fat mass, lower levels of blood glucose and insulin, and fewer total spleen cells but a higher percentage of CD4+ T cells. Additionally, they demonstrated lower production of pro-inflammatory cytokines IL-1β and TNF-α, a trend toward lower IL-6, and lower production of PGE2, a lipid molecule with pro-inflammatory and T cell-suppressive properties. Mice with CR alone had higher splenocyte proliferation and IL-2 production, but this effect of CR was diminished by spice supplementation. CR alone or in combination with spice supplementation had no effect on production of cytokines IL-4, IL-10, IFN-γ, and IL-17, or the proportion of different CD4+ T cell subsets.

Conclusion: CR on an HFD favorably impacts both metabolic and immune/inflammatory profiles; however, the presence of curcumin and/or piperine does not amplify CR's beneficial effects.

Figure 1

Effect of CR and spice compounds on body weight and fat mass. Mice were fed experimental diets for 60 wk as described in the “Materials and methods” section. Body weight and fat mass were recorded at the time points as indicated. Values are means ± SEM, n = 9-10 mice/group. HFD: high fat diet, CR: caloric restriction, C: curcumin, P: piperine.

Figure 2

Effect of CR and spice compounds on plasma glucose and insulin concentrations. Plasma was collected and the glucose (A) and insulin (B) levels were determined by using Glucose Colorimetric Assay Kit and Ultra Sensitive Mouse Insulin ELISA Kit, respectively. Values are means ± SEM, n = 8 mice/group. Means without a common letter differ at P < 0.05. HFD: high fat diet, CR: caloric restriction, C: curcumin, P: piperine.

Figure 3

Effect of CR and spice compounds on total number of spleen cells and proportion of CD4⁺ T cells. Isolated splenocytes were counted under microscope for total spleen cells (A) and stained with fluorescence-conjugated anti-CD4 antibody, followed by flow cytometry to determine the splenic CD4⁺ T cell levels (B). Values are means ± SEM, n = 9 mice/group. Means without a common letter differ at P < 0.05. HFD: high fat diet, CR: caloric restriction, C: curcumin, P: piperine.

Figure 4

Effect of CR and spice compounds on inflammatory cytokine production by splenocytes. Spleen cells were stimulated with LPS for 24 h and supernatant was used to determine production of IL-1β (A), TNF-α (B), and IL-6 (C) using ELISA. Values are means ± SEM (n = 9 mice/group). Means without a common letter differ at P < 0.05. HFD: high fat diet, CR: caloric restriction, C: curcumin, P: piperine.

Figure 5

Effect of CR and spice compounds on splenocyte proliferation. Splenocytes were stimulated with T cell mitogen Con A or PHA, or anti-CD3/CD28 at their optimal concentrations (1.5, 5, 5/1 μg/ml, respectively) for 72 h, and proliferation were determined by [³H]-thymidine incorporation. Values are means ± SEM (n = 9 mice/group). Means without a common letter differ at P < 0.05. HFD: high fat diet, CR: caloric restriction, C: curcumin, P: piperine.

Figure 6

Effect of CR and spice compounds on T cell cytokine production. Splenocytes were stimulated with Con A (1.5 μg/ml) and plate-coated anti-CD3 (5 μg/ml)/anti-CD28 (1 μg/ml) for 48 h and supernatant was collected to determine production of T cell cytokines IL-2 (A), IFN-γ (B), IL-4 (C), IL-10 (D) using ELISA. Values are means ± SEM (n = 9 mice/group). Means without a common letter differ at P < 0.05. HFD: high fat diet, CR: caloric restriction, C: curcumin, P: piperine.

Figure 7

Effect of CR and spice compounds on intracellular levels of T cell cytokines. Splenocytes were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of Golgi Stop for 4 h and intracellular IL-2 and IFN-γ (A), IL-4 and IL-10 (B), and IL-17 (C) were determined using flow cytometry. Values are means ± SEM (n = 9 mice/group). Means without a common letter differ at P < 0.05. HFD: high fat diet, CR: caloric restriction, C: curcumin, P: piperine.

Figure 8

Effect of CR and spice compounds on LPS-stimulated PGE₂ production. Spleen cells were stimulated with LPS for 24 h and supernatant was used to determine PGE₂ production using a MSD kit and platform. Values are means ± SEM (n = 6 mice/group). Means without a common letter differ at P < 0.05. HFD: high fat diet, CR: caloric restriction, C: curcumin, P: piperine.

Figure 9

Effect of CR and spice compounds on CD4⁺ T cell subpopulations. Splenocytes were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of Golgi Stop for 4 h. After that, appropriate surface and intracellular stainings were performed to identify CD4⁺ T cell subpopulations as defined by their cytokine production pattern: Th1 (IL-2 and IFN-γ) (A), Th2 (IL-4 and IL-10) (B), Th17 (IL-17) (C), and Treg (CD4⁺CD25⁺Foxp3⁺) (D) using flow cytometry. All analyses were performed within the gated CD4⁺ T cells. Values are means ± SEM (n = 9 mice/group). Means without a common letter differ at P < 0.05. HFD: high fat diet, CR: caloric restriction, C: curcumin, P: piperine.