Anti-inflammatory action of β-hydroxybutyrate via modulation of PGC-1α and FoxO1, mimicking calorie restriction

Abstract

β-Hydroxybutyrate (HB) is a ketone body used as an energy source that has shown anti-inflammatory effects similar to calorie restriction (CR); Here, PGC-1α, an abundantly expressed co-factor in the kidney, was reported to interact with both FoxO1 and NF-κB although the definitive interactive mechanism has not yet been reported. In this study, we investigated whether renal aging-related inflammation is modulated by HB. We compared aged rats administered with HB to calorie restricted rats and examined the modulation of FoxO1 and the NF-κB pathway through interactions with PGC-1α. We found that in aged rats treated with HB, pro-inflammatory signaling changes were reversed and showed effects comparable to CR. As FoxO1 and its target genes catalase/MnSOD were upregulated by HB treatment and PGC-1α selectively interacted with FoxO1, not with NF-κB, and ameliorated the renal inflammatory response. These findings were further confirmed using FoxO1 overexpression and siRNA transfection in vitro. Our findings suggest that HB suppressed aging-related inflammation as a CR mimetic by enabling the co-activation and selective interaction between FoxO1 and PGC-1α. This study demonstrates the potential therapeutic role of HB as a CR mimetic, which ameliorates inflammation by a novel mechanism where FoxO1 outcompetes NF-κB by interacting with PGC-1α in aging kidneys.

Keywords: FoxO1; aging kidney; calorie restriction; inflammation; β-hydroxybutyrate.

Figure 1

Ameliorated serum in β-hydroxybutyrate (HB)-treated aged rats. HB was administered to aged rats (n = 4 each). (A) Serum insulin, (B) glucose levels, (C) Ketone body, and (D) Leptin were measured after 30 days of HB treatment. ^##p < 0.01, ^###p < 0.001 vs. Young; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Old.

Figure 2

β-hydroxybutyrate (HB) decreases the expression of inflammatory proteins and activates FoxO1 expression in the kidney of aged rats. (A) Western blotting was performed to examine the protein levels of catalase, MnSOD, COX-2, and iNOS in the kidney of aged rats treated with HB and a caloric restriction (CR). Three independent experiments were performed, and similar results were obtained. (B) Expressions of genes encoding cytokines such as IL-1β, IL-6, and TNFα were analyzed using qRT–PCR (n = 4 each). Results were normalized to the GAPDH levels. ^##p < 0.01, ^###p < 0.001 vs. Young; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Old. (C) Levels of reactive oxygen species and (D) ONOO^- assessed in the kidney of aged rats treated with HB and CR. Three independent experiments were performed, and similar results were obtained. The data are expressed as the mean ± SEM (n = 4). ^###p < 0.001 vs. Young; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Old.

Figure 3

Caloric restriction (CR) and β-hydroxybutyrate (HB) increases FoxO1 activation during aging. Western blot analyses for renal nuclear (A) FoxO1, FoxO3, and FoxO6 as well as (B) PGC-1α and p65 were performed on nuclear proteins from rats treated with Young, Old, HB, and CR. Western blot results from 3 independent experiments were quantified by densitometry. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Young; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Old. (C) Nuclear extracts were prepared from kidneys from young and aged rat. Immunoprecipitation assay showed PGC-1α was physically associated with p-serine, p-FoxO1, FoxO1, p-p65, and p65.

Figure 4

β-hydroxybutyrate (HB) decreases the expression of insulin signaling in aged rats. (A) Western blotting was performed to examine the protein levels of p-IRS-1 (Ser307), p-IRS-1 (Tyr632), IRS, p-Akt, and Akt as well as (B) p-IKK, IKK p-JNK, and JNK in the kidney of aged rats treated with HB. Three independent experiments were performed, and similar results were obtained. Western blot results from 3 independent experiments were quantified by densitometry. ^###p < 0.001 vs. Young; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Old.

Figure 5

β-hydroxybutyrate (HB) regulates insulin-induced expression of FoxO1. (A) Levels of p-FoxO1, total FoxO1, and PGC-1α were noticeably diminished after treatment with 0.1–2 mM HB for 3 h, followed by incubation with or without 100 nM of insulin for 2 h. (B) Expressions of genes encoding cytokines such as IL-1β, TNFα, and IL-6 were analyzed using qRT–PCR (n = 3 each). Results were normalized to GAPDH mRNA levels. ^#p < 0.05, ^##p < 0.01 vs. Normal; ^*p < 0.05, vs. insulin treated group. (C) HEK293T cells were transiently transfected with a catalase and catalase-containing plasmid linked to the luciferase gene, pre-incubated with HB (0.25–1 mM) for 4 h, and then treated with insulin for 24 h. Results are presented in relative luminescence units (RLU). Results were obtained using one-factor ANOVA: ^$$$p<0.001 vs. pcDNA transduced cells; ^##p<0.01 vs. catalase-luciferase transduced cells; ^*p<0.05, ^**p<0.01 vs. insulin with catalase-luciferase transduced cells. (D) HEK293T cells were pretreated with or without 0.5 mM of HB for 3 h and then treated with insulin (100 nM) for 10 min. Cells were immunostained using rabbit anti-FoxO1 antibody followed by IgG conjugated with fluorescein isothiocyanate (green). Bar = 50 µm.

Figure 6

β-hydroxybutyrate (HB) regulates insulin-induced inflammation. (A) Levels of p-p65 and total p65 noticeably diminished after treatment with 0.1–2 mM HB for 3 h, followed by incubation with or without 100 nM insulin for 2 h. (B) HEK293T cells were transiently transfected with a NF-κΒ and NF-κΒ-containing plasmid linked to the luciferase gene, pre-incubated with HB (0.25–1 mM) for 4 h, and then treated with insulin for 24 h. Results are presented in relative luminescence units (RLU). Results were obtained using one-factor ANOVA: ^$$$p<0.001 vs. pcDNA transduced cells; ^##p<0.01 vs. NF-κB-luciferase transduced cells; ^*p<0.05 vs. insulin with NF-κB-luciferase transduced cells. (C) Confocal laser microscopy analysis of intracellular ROS levels using 2’,7’-dichlorodihydrofluorescein diacetate. Cells were incubated with 100 nM insulin for 2 h after pretreatment with HB for 3 h. Panel 1, normal; panel 2, 100 nM insulin; panel 3, 100 nM insulin + 0.5 mM HB.

Figure 7

β-hydroxybutyrate (HB) regulates the activation of FoxO1 phosphorylation through the PI3K/Akt pathway. HEK293T cells were grown to 80% confluency in 100 mm dishes in DMEM medium, pre-treated (1 day) with or without CA-Akt (100 MOI), and then treated with 0.25–1 mM HB. (A) Cells were pre-transduced in the absence or presence of CA-Akt vector (100 MOI), and analyzed by western blotting. (B) Immunoprecipitation showed that FoxO1 was physically associated with phosphorylated FoxO1, FoxO1, p65, and PGC-1α after stimulation with HB (0.25–1 mM) in the absence or presence of CA-Akt (100 MOI).

Figure 8

Effect of β-hydroxybutyrate (HB) on FoxO1-dependent gene expression after FoxO1 knockdown. Western blot analysis was used to assess protein levels in FoxO1 siRNA-treated HEK293T cells. (A) FoxO1, PGC-1α, and NF-κB protein levels in cells pretreated for 3 h with HB in the absence or presence of FoxO1 siRNA-transfected cells (200 MOI) for 1 day. (B) HEK293T cells were transiently transfected with a catalase-containing plasmid linked to the luciferase gene, pre-incubated with FoxO1-siRNA (100 MOI) for 24 h, and then treated with HB for 4 h. Results are presented in relative luminescence units (RLU). Results were obtained using one-factor ANOVA: ^$$$p<0.001 vs. pcDNA transduced cells; ^###p<0.001 vs. catalase-luciferase transduced cells; ^**p<0.01, ^***p<0.001 vs. FoxO1-siRNA with catalase-luciferase transduced cells. (C) HEK293T cells were pretreated with or without 0.5 mM HB for 3 h and then treated with FoxO1-siRNA (200 MOI) for 24 h. Cells were immunostained using rabbit anti-PGC-1α antibody followed by IgG conjugated with fluorescein isothiocyanate (green). Bar = 50 µm. (D) A possible mechanism underlying the effect of HB regulate NF-κB through interaction of FoxO1 and PGC-1α in aging.