1,3-Butanediol Administration Increases β-Hydroxybutyrate Plasma Levels and Affects Redox Homeostasis, Endoplasmic Reticulum Stress, and Adipokine Production in Rat Gonadal Adipose Tissue

Abstract

Ketone bodies (KBs) are an alternative energy source under starvation and play multiple roles as signaling molecules regulating energy and metabolic homeostasis. The mechanism by which KBs influence visceral white adipose tissue physiology is only partially known, and our study aimed to shed light on the effects they exert on such tissue. To this aim, we administered 1,3-butanediol (BD) to rats since it rapidly enhances β-hydroxybutyrate serum levels, and we evaluated the effect it induces within 3 h or after 14 days of treatment. After 14 days of treatment, rats showed a decrease in body weight gain, energy intake, gonadal-WAT (gWAT) weight, and adipocyte size compared to the control. BD exerted a pronounced antioxidant effect and directed redox homeostasis toward reductive stress, already evident within 3 h after its administration. BD lowered tissue ROS levels and oxidative damage to lipids and proteins and enhanced tissue soluble and enzymatic antioxidant capacity as well as nuclear erythroid factor-2 protein levels. BD also reduced specific mitochondrial maximal oxidative capacity and induced endoplasmic reticulum stress as well as interrelated processes, leading to changes in the level of adipokines/cytokines involved in inflammation, macrophage infiltration into gWAT, adipocyte differentiation, and lipolysis.

Keywords: Nrf2; ROS; adipokines; antioxidant enzymes; endoplasmic reticulum stress; ketone bodies; mitochondrial respiratory complexes.

Figure 1

Effect of BD treatment on (D) β-OHB serum levels, detected after 3 h from BD i.p administration (BD 3 h) and after 14 days of oral administration (BD 14 d). Values represent the mean ± SE of 10 different animals. * p < 0.0001.

Figure 2

Effect of 14 days BD administration on gWAT morphology and lipolysis. (A) shows a representative histological analysis of gWAT from control (C) and BD-treated rats (BD 14 d). Below is the enlargement (100×) of the framed areas. Note the presence of capillaries (arrow) and infiltrating (inflammatory) cells/macrophages (arrowhead) around the adipocytes in BD 14 d-compared to C-gWAT. Hematoxylin and Eosin staining. (B) represents Basal and isoprotenerol-stimulated lipolysis. Values represent the mean ± ES of 8 different animals * p < 0.05, ** p < 0.01.

Figure 3

Effect of BD on gWAT redox homeostasis. Total ROS (A), lipid hydroperoxides (B), protein oxidative damage (C), and tissue soluble antioxidant capacity (D). Values are means ± SEM of 8 different rats. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Enzymatic antioxidant capacity (E). Representative Western Blot of Superoxide dismutase-2 (SOD-2), catalase (CAT), peroxiredoxin-3 (PRDX-3), and Nuclear factor erythroid-related factor 2 (Nrf2) (E). GAPDH was used as loading control (25 μg of protein/rat/lane). Histograms represent the quantification of data. Data were normalized to the value obtained for control animals, set as 100. Values represent the mean ± SEM of 6 different rats or 3 rats in the case of PRDX-3.

Figure 4

Effect of BD on gWAT levels of proteins linked to ER and cellular stress involved in ER stress and UPR^ER response detected. Representative Western blots of GRP78/BiP, Calnexin and TnF-α (A), and total and phosphorylated (ser 78) levels of eIF2α (B) were performed in gWAT lysate. GAPDH was used as loading control (25 μg of protein/rat/lane). Histograms represent the quantification of data. Data were normalized to the value obtained for control animals, set as 100. Values represent the mean ± SEM of 6 different rats. * p < 0.05, ** p < 0.01, *** p < 0.0001.

Figure 5

Effect of BD on gWAT mitochondrial respiratory complexes abundance and cytochrome oxidase activity. (A) shows representative Western Blots of specific subunits of the five mitochondrial respiratory complexes, namely CI-NDUF88, CII-SDHB, CIII-UQCRC2, CIV-MTCO1, and CV-ATP VA. GAPDH was used as loading control (25 μg of protein/rat/lane). Histograms represent the quantification of data. Data were normalized to the value obtained for control animals, set as 100. Values represent the mean ± SEM of 6 different rats. (B,C) show Cytochrome oxidase/complex IV activity reported as nmoles O/min mg tissue proteins (B) or normalized for Complex IV levels and expressed in arbitrary units (C). * p < 0.05, ** p < 0.01, *** p < 0,001, **** p < 0.0001. Values represent the mean ± SEM of 8 different rats.

Figure 6

Effect of administration of BD for 14 days on gWAT adipokines profiles. Representative adipokine profile detected in gWAT from C and BD 14 d rats, by an adipokine protein array (A). The arrowheads indicate signals with significant changes. Magnifications of the spot of proteins whose intensity resulted statistically different are reported in (B). Histograms represent the quantification of relative levels of adipokines with observable changes (C). The values represent the mean ± SEM of 3 different samples, each one obtained by a pool of two different animals. Data were normalized to the value obtained for control animals, set as 100. * p < 0.05, ** p < 0.01.