Exploring the Therapeutic Potential of Ethyl 3-Hydroxybutyrate in Alleviating Skeletal Muscle Wasting in Cancer Cachexia

Abstract

Cachexia (CAC) is a debilitating metabolic syndrome. Although dietary interventions are attractive, long-term adherence to specific diets is difficult to maintain and can lead to systemic side effects. Ethyl 3-hydroxybutyrate (EHB) is a commonly used food additive found in wine and Tribolium castaneum. In this study, we investigated the effects of EHB administration in cachectic mice. After a single intraperitoneal injection of EHB into mice, 3-hydroxybutyrate (3-HB) levels were significantly increased in the serum and gastrocnemius of mice. The administration of EHB alleviated cachexia-related symptoms, ameliorated skeletal muscle atrophy, and improved survival in cachectic mice. In addition, the supplementation of cachectic mice with 3-HB by EHB administration significantly reduced tumor weights, indicating the anti-tumor effects of 3-HB. Remarkably, the addition of 3-HB to the culture medium significantly attenuated the C2C12 myotube atrophy induced by the culture supernatant of CT26 cell lines, highlighting its potential to counteract the destructive effects of tumor-derived elements on muscle tissue. NMR-based metabolomics analysis provided insights into the underlying mechanisms and revealed that the anti-cachexia effects of 3-HB treatment can be attributed to three key mechanisms: the promotion of the TCA cycle and the attenuation of proteolysis, the promotion of protein synthesis and the improvement of metabolic homeostasis, and a reduction in inflammation and an enhancement of the antioxidant capacity. This study provided compelling evidence for the protective effects of 3-HB treatment on the cachectic gastrocnemius and highlighted the efficacy of EHB administration as a ketone supplementation approach to achieve nutritional ketosis without the need for dietary restriction.

Keywords: 3-hydroxybutyrate; Ethyl 3-hydroxybutyrate; cancer cachexia; ketogenic diet; metabonomics.

Figure 1

Relative levels of 3-HB and EHB in serum and gastrocnemius over time in normal mice following a single intraperitoneal injection of EHB. (A) Serum; (B) gastrocnemius. Integrals of 3-HB and EHB were measured from 1D ¹H-NMR spectra recorded at 25 °C on a Bruker Avance III 850 MHz spectrometer. The integrals of 3-HB and EHB were normalized by those measured 0 min post-EHB administration to represent the relative 3-HB and EHB levels at different time points (n = 5 for each time point).

Figure 2

EHB administration alleviated cachectic symptoms in the mouse model of colon cancer cachexia. (A) Food intake curve; (B) ratio of change in tumor-free body weight (%); (C) epididymal fat mass; (D) gastrocnemius weight; (E) tumor weight; (F) survival curve. NOR, normal mice (n = 13); CAC, cachexia mice (n = 11); CAC-K, 3-HB-treated cachexia mice (n = 19). Experimental data are presented as mean ± SD. Statistical significances: p < 0.01, **; p < 0.001, ***; p < 0.0001, ****.

Figure 3

EHB administration decreased serum levels of inflammation factors and total antioxidant capacity in colon cancer cachexia mice. Pairwise comparisons between NOR and CAC and between CAC-K and CAC for serum levels of (A–F) inflammation factors including IFN-γ (A), TGF-β (B), IL-1 (C), IL-6 (D), TNF-α (E), TLR-4 (F), and (G) total antioxidant capacity (TAC) in gastrocnemius. Experimental data are reported as mean ± SD. Statistical significances: p > 0.05, ns; p < 0.05, *; p < 0.01, **; p < 0.001, ***.

Figure 4

EHB-administration ameliorated skeletal muscle atrophy in the mouse model of colon cancer cachexia. (A) Hematoxylin–eosin staining in gastrocnemius muscle and quantitative comparisons of cross-sectional areas of skeletal muscle fibers for NOR vs. CAC and CAC-K vs. CAC (n = 6). (B) Expressions of p-AKT, AKT, MuRF1 in gastrocnemius, and pairwise comparisons of AKT phosphorylation and MuRF1 expression between NOR and CAC and between CAC-K and CAC (n = 3). Experimental data are reported as mean ± SD. Statistical significances: p > 0.05, ns; p < 0.05, *; p < 0.01, **; p < 0.001, ***; p < 0.0001, ****.

Figure 5

Multivariate statistical analysis for identifying significant metabolites from the comparisons of CAC vs. NOR and CAC-K vs. CAC. (A,B) Score plots of the PLS-DA models for CAC vs. NOR (A) and CAC-K vs. CAC (B). (C,D) VIP score-ranking plots of the significant metabolites identified by using the criterion of VIP > 1 calculated from the PLS-DA models of CAC vs. NOR (C) and CAC-K vs. CAC (D).

Figure 6

Venn diagram of the characteristic metabolites identified from pairwise comparisons of CAC vs. NOR and CAC-K vs. CAC. Characteristic metabolites were identified by using two criteria: VIP > 1 calculated from the PLS-DA model and p < 0.05 obtained from univariate analysis. Characterized metabolites highlighted in red were shared by pairwise comparisons. Note: ↑↑↑/↓↓↓, ↑↑/↓↓, and ↑/↓ represent that A was increased compared to B with a statistical significance of p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 7

Quantitative comparisons of relative levels of the characterized metabolites shared by pairwise comparisons of CAC vs. NOR and CAC-K vs. CAC. (A) valine; (B) leucine; (C) phenylalanine; (D) fumarate; (E) glutathione; (F) 2-methylglutarate. Experimental data are represented as mean ± SD. Statistical significances: p > 0.05, ns; p <0.05, *; p < 0.01, **; p < 0.001, ***.