Immune-Modulating Effects of Low-Carbohydrate Ketogenic Foods in Healthy Canines

Abstract

Background: Ketogenic foods limit digestible carbohydrates but contain high fat, and have antioxidant and anti-inflammatory effects as well as improving mitochondrial function. β-Hydroxybutyrate (BHB), 1 of the ketone bodies, reduces the proinflammatory NLR family pyrin domain containing 3 inflammasomes, as well as chemokines in cultures.

Objectives: We assessed the immune-modulating effects of 2 low-carbohydrate (LoCHO) foods varying in protein and fat and compared their effects with a food replete with high-carbohydrate (HiCHO) in healthy canines.

Methods: Dogs were fed control food [HiCHO; ketogenic ratio (KR: 0.46) followed by LoCHO_PROT (KR: 0.97), then LoCHO_FAT (KR: 1.63) or LoCHO_FAT followed by LoCHO_PROT. Each food was fed for 5 wk, with collections in the 5th wk; 15 wk feeding total. Gene expression for circulating inflammatory cytokines from 10 dogs was assessed using the Canine RT² Profiler polymerase chain reaction array, and fold changes were calculated using the ΔΔCt method.

Results: LoCHO_FAT significantly increased circulating β-hydroxybutyrate compared with both HiCHO and LoCHO_PROT. When compared with HiCHO, there was a significant decrease in several proinflammatory cytokines/chemokines in LoCHO_PROT and LoCHO_FAT groups, including chemokine (C-C motif) ligand (CCL)1, CCL8, CCL13, CCL17, CCL24, chemokine (C-X3-C motif) ligand 1, chemokine (C-X-C motif) receptor 1, Interleukin-10 receptor alpha ((IL)-10RA), IL-1 receptor antagonist, IL-5, and secreted phosphoprotein 1 (all P < 0.05). Interestingly, a subset of inflammatory proteins that decreased in LoCHO_PROT but not in LoCHO_FAT included IL-33, IL-6 receptor, IL-7, IL-8, Nicotinamide phosphoribosyltransferase, and tumor necrosis factor (TNF) receptor superfamily member 11B. In contrast, the decrease in inflammatory markers in LoCHO_FAT, but not in LoCHO_PROT, included complement component 5, granulocyte colony-stimulating factor or G-CSF, interferon-γ, IL-3, IL-10RB, IL-17C, Tumor necrosis factor superfamily (TNFSF)13, TNFSF13B, and TNFSF14. Decreased concentrations of selected cytokines indicate that both low-carbohydrate foods exert an anti-inflammatory effect and provide a strong rationale for testing their efficacy in dogs with inflammatory conditions.

Conclusions: Both LoCHO_PROT and LoCHO_FAT foods might be important as part of immune-modulating therapeutic nutritional strategies to reduce inflammation to maintain health in canines. Our study identifies several inflammatory genes that are reduced when fed ketogenic food that were not previously reported.

Keywords: PCR; canines; histopathology; immune; inflammation; renal.

FIGURE 1

(A) A pie chart depicting the breakdown of macronutrients in the foods. Dogs (n = 35) were fed HiCHO food, which had a ketogenic ratio (KR) of 0.46; low carbohydrate with higher protein (LoCHO_PROT): KR = 0.97, and low carbohydrate with higher fat (LoCHO_FAT): KR = 1.63. (B) Circulating concentrations of β-hydroxybutyrate were increased significantly with LoCHO_FAT food but not with LoCHO_PROT when compared with HiCHO. Data are shown as mean ± SEM in both LoCHO diets and HiCHO. ∗P < 0.05. HiCHO, high carbohydrate; P/F/C, protein/fat/carbohydrate; SEM, standard error of the mean.

FIGURE 2

Effect of HiCHO, LoCHO_PROT, and LoCHO_FAT foods (n = 35) on laboratory measurements on complete blood chemistry values (CBC). Data are presented as mean + SEM (∗P < 0.05). ALB/GLOB, albumin/globulin; HiCHO, high carbohydrate; LoCHO_FAT, low carbohydrate_fat; LoCHO_PROT, low carbohydrate_protein; SEM, standard error of the mean; WBC, white blood cell.

FIGURE 3

(A) Heat map showing the fold change in gene expression in LoCHO_PROT when compared with HiCHO. Gene expression studies were conducted in a subset of 10 dogs randomly selected from the 35 dogs (see text for details). Data are presented as mean fold change (ΔΔCt) normalized to β2-microglobulin (B2M), the housekeeping gene. Fold-change values >1 indicate an upregulation. Fold-change values <1 indicate a negative or downregulation, and the fold-regulation is the negative inverse of the fold-change (Qiagen). The individual squares in a heat map are scaled with a range of colors proportional to gene expression values. The figure below (Rows A–G) corresponds to the squares above, and each square indicates the mean values for that gene, with numerically positive values indicating up-regulation and negative values indicating down-regulation. Row H included the housekeeping genes ACTB, B2M, GAPDH, HPRT1, and RPLP1 in the assay. Other controls in row H included those for detection of genomic DNA contamination, reverse transcription control, and positive PCR control. Circled genes = P < 0.05. (B) It is the same as (A) but shows changes in LoCHO_FAT when compared with HiCHO. DNA, deoxyribonucleic acid; HiCHO, high carbohydrate; LoCHO_FAT, low carbohydrate_fat; LoCHO_PROT, low carbohydrate_protein; ACTB, actin beta; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; HPRT1, hypoxanthine phosphoribosyltransferase 1 and RPLP1, Ribosomal Protein Lateral Stalk Subunit P1.

FIGURE 4

Venn diagram (n = 10) representing significant down-regulation of cytokines/chemokines observed in either LoCHO_PROT or LoCHO_FAT as well as significantly decreased expression in both diets when compared with the control food (HiCHO, P < 0.5). CCL, chemokine (C-C motif) ligand; CX3CL1, chemokine (C-X3-C motif) ligand 1; CXCR1, chemokine (C-X-C motif) receptor 1; HiCHO, high carbohydrate; IL-6R, interleukin 6 receptor; LoCHO_FAT, low carbohydrate_fat; LoCHO_PROT, low carbohydrate_protein; NAMPT, nicotinamide phosphoribosyltransferase; SPP1, secreted phosphoprotein 1; TNFRSF11B, tumor necrosis factor receptor superfamily member 11B; TNFSF13, tumor necrosis factor superfamily member 13; IL-10RA, Interleukin-10 receptor alpha; IL-10RB, Interleukin-10 receptor beta.

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