Introduction
Limb and respiratory muscle weakness develop in the majority of critically ill patients who require a prolonged stay in the intensive care unit (ICU), referred to as ICU-acquired weakness. The pathophysiology of ICU-acquired weakness is complex and multifactorial with underlying mechanisms that negatively affect muscle function independently from loss of muscle mass [
1]. This debilitating complication is associated with greater post-ICU functional impairment, prolonged hospitalization, delayed rehabilitation and late death. Effective therapies, other than avoiding modifiable risk factors, are currently lacking [
1‐
4].
Recently, we have shown in critically ill mice that supplementation with 3-hydroxybutyrate (3HB) protects against muscle weakness [
5]. In this sepsis-induced mouse model of critical illness, 3HB supplementation did not affect the illness-induced loss of muscle mass, whereas it did attenuate illness-induced loss of muscle force. Key underlying mechanisms of muscle weakness, such as atrophy, autophagy and inflammation, were not affected by 3HB and 3HB also did not appear to serve as an alternative energy substrate [
5]. Remarkably, 3HB treatment increased plasma LDL cholesterol up to normal levels [
5]. This was a surprising observation, as critical illness in humans and in mice is typically characterized by a robust, immediate and sustained decrease in plasma cholesterol concentrations (LDL and HDL), which is proportional to the severity of illness and the risk of death [
6‐
9]. Ketone bodies can theoretically serve as precursors of cholesterol, after conversion into acetyl-CoA. Indeed, earlier studies that mainly focused on brain and liver have shown that ketone bodies, under specific circumstances such as during development and starvation, can be a preferred substrate for cholesterogenesis [
10‐
15].
There is emerging evidence also pointing to a role for cholesterol in controlling muscle function. For example, cholesterol-lowering statin therapy can evoke muscle weakness and aches [
16]. Such side effects of statins have been linked to impaired mitochondrial function caused by reduced ubiquinone levels, a derivative of the cholesterol precursor mevalonate and an essential co-factor in the mitochondrial respiratory chain [
16,
17]. In skeletal muscle, cholesterol also plays an important role in the regulation of myofiber membrane fluidity and signal transduction processes. Depletion of myofiber membrane cholesterol has been shown to hamper muscle contractions [
18‐
20], whereas hereditary muscular dystrophy is associated with increased myofiber membrane cholesterol [
21,
22]. Despite emerging evidence highlighting a role of cholesterol in controlling muscle function and the known hypocholesterolemia of critical illness, the link between low cholesterol and ICU-acquired weakness has not been investigated.
We hypothesized that altered cholesterol homeostasis plays a role in the development of ICU-acquired weakness and that the protective effect of 3HB supplementation on weakness is related to its effects on cholesterol homeostasis. These hypotheses were tested in a human study and in a clinically relevant and validated mouse model of critical illness evoked by sepsis.
Discussion
We here demonstrated that altered cholesterol homeostasis is involved in the development of critical illness-induced muscle weakness, in both human critically ill patients and septic mice. Furthermore, the previously observed muscle protection of 3HB supplementation during sepsis could be linked to effects on cholesterol homeostasis. In mice, critical illness-induced hypocholesterolemia was prevented with 3HB supplementation. Supplemented 3HB was preferentially taken up by skeletal muscle and used as substrates for cholesterogenesis. Not ubiquinone formation with downstream muscular mitochondrial function, but myofiber cholesterol content was increased and associated with muscle force.
Acute and sustained low circulating cholesterol concentrations are a hallmark of critical illness and considered a marker of poor prognosis [
6‐
9]. The acute decrease in cholesterol has been interpreted as linked to increased cortisol production and to endotoxin-scavenging functions, but the exact pathophysiology remains unclarified [
31‐
35]. We demonstrated in critically ill patients that reduced circulating cholesterol levels were associated with the subsequent development of muscle weakness, and this independently from the severity or type of critical illness and independently from whether patients received early parenteral nutrition. Also in septic mice, the low plasma cholesterol concentrations and the low myofiber cholesterol content correlated with impaired muscle force generation. In addition, 3HB supplementation of septic mice was able to normalize cholesterol levels in plasma and in skeletal muscle and its protective effects on muscle force could be linked to increased myofiber cholesterol content.
Statin-induced lowering of cholesterol has been linked to statin-induced muscle aches and muscle weakness [
16]. In this context, research has mainly focused on ubiquinone, a derivative of the cholesterol precursor mevalonate, which is an essential co-factor in the mitochondrial respiratory chain [
16,
17]. Ubiquinone deficiency and impaired mitochondrial function have been described during sepsis [
36,
37]. Plasma ubiquinone is often used as clinical proxy for functional tissue levels [
34]. Remarkably, we found increased plasma ubiquinone concentrations in all septic mice, unaffected by 3HB supplementation. Possibly, these high plasma levels are due to leaking of ubiquinone into the circulation after sepsis-induced mitochondrial and/or tissue damage. Mitochondrial function was indeed found to be impaired in skeletal muscle of the septic mice, but it was unaffected by 3HB supplementation and mitochondrial functional test results did not correlate with muscle force. In contrast, total cholesterol content in myofibers was lowered by sepsis, increased by 3HB supplementation and positively correlated with muscle force. Furthermore, increased gene expression of
Nceh1 in muscle may suggest enhanced hydrolysis of de novo synthetized cholesterol, and such hydrolyzed cholesterol is the main form of cholesterol present in cell membranes. Depletion of myofiber membrane cholesterol has indeed been shown to impair muscle contraction [
18‐
20].
In mice, 3HB supplementation not only increased cholesterol content in myofibers, also plasma cholesterol concentration was increased. However, how 3HB supplementation affected plasma cholesterol concentrations remains speculative. Hepatic cholesterol homeostasis was largely unaffected by 3HB supplementation, suggesting that the liver was not involved in altered plasma cholesterol. Possibly, increased uptake of 3HB in skeletal muscle, followed by ketone-to-cholesterol metabolism, may have reduced the need for muscular cholesterol uptake from the circulation. Whether this was sufficient to increase plasma cholesterol levels, or whether other mechanisms in other organs may be involved remains to be investigated further. In general, high-fat ketogenic diets tend to increase circulating cholesterol concentrations, possibly through higher consumption of dietary cholesterol in combination with enhanced ketone-to-cholesterol metabolism [
38,
39]. Conversely, exogenous administration of ketone esters has been shown to either not affect [
40] or reduce [
41] plasma cholesterol concentrations. However, the experiments with ketone esters were performed in healthy full-fed rodents and humans, which is quite a different context than that of critical illness which is hallmarked by low circulating cholesterol. Additionally, in healthy individuals, ketone bodies are primarily shuttled into the TCA cycle, which could theoretically reduce the availability of acetyl-CoA for cholesterogenesis [
42]. In septic mice however, it has already been observed that ketone bodies are preferential used as signaling molecules, not energy substrates, indicating that other metabolic pathways may be activated [
5]. Indeed, with state-of-the art tracer technology we here could demonstrate that conversion of 3HB to mevalonate was several-fold higher than conversion to the TCA cycle metabolites citrate and malate in septic animals, though not in healthy mice.
Whether direct cholesterol substitution therapy could benefit the critically ill patient has not been investigated. Therapies that mimic the endotoxin-scavenging role of cholesterol, such as treatment with a phospholipid emulsion or polymyxin B hemoperfusion, have been unsuccessful in improving outcome [
43,
44]. At present, a Phase I/II feasibility trial is ongoing to test whether cholesterol levels can be stabilized with a lipid emulsion in septic patients, but no clinical endpoints are investigated yet [
45]. Therefore, the novel finding presented here, an effect of 3HB supplementation on cholesterol homeostasis during sepsis, is quite promising. In our mice experiments, 3HB did not cause the hepatic adversities associated with infusion of high lipid doses [
5], and the local effect of 3HB on cholesterol homeostasis in skeletal muscle may be an advantage. In addition, 3HB supplementation in septic mice has previously been shown to stimulate markers of muscle regeneration, a process that is impaired in patients suffering from ICU-acquired weakness [
5,
46]. This dual effect of 3HB on muscle force generation and muscle regeneration may support the therapeutic potential, which should be further investigated.
This study also has limitations. First, we used a multivariate logistic regression model to assess the independent association of plasma cholesterol with weakness in human critically ill patients. Due to the inherent limitations of such a statistical analysis, we cannot exclude that the observed effect of cholesterol on muscle weakness is partly confounded by other factors. The presence of sepsis was the strongest determinant for muscle weakness and also is a strong suppressor of plasma cholesterol [
9]. However, the independent association, observed in our controlled septic animal model, strongly argues against this confounder. Second, mice typically have much lower plasma LDL cholesterol and higher plasma HDL compared to humans [
47]. We assessed total plasma cholesterol in both the human and animal study, but extrapolations on the contribution of the different lipoproteins between these species have to be done with caution. Future studies on the role of cholesterol in sepsis should consider using humanized mice models to better mimic the human setting [
48,
49].
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.