BACKGROUND:Sepsis HDL- and LDL-hypocholesterolemia associate with adverse outcomes, but whether this necessitates supplementation or merely reflects disease severity remains unclear. We hypothesize that sustained hypocholesterolemia can contribute to ICU-acquired weakness and adrenal dysfunction, and that cholesterol supplementation can improve tissue cholesterol availability, muscle and adrenal integrity. METHODS:In a catheterized mouse model of cecal-ligation-and-puncture-induced sepsis (5-days), septic mice received continuous infusion with an LDL- (mouse study 1; n = 51) (3.5 mg/d) or HDL-cholesterol (mouse study 2; n = 47) (5 mg/d) enriched cholesterol mixture, compared to placebo, and healthy reference mice. Plasma HDL-, LDL-cholesterol, CORT, TNF-α and total bile acids were measured, in addition to muscle force, myofiber cholesterol, ex-vivo adrenal ACTH response, adrenal cholesterol and structure. Gene expression markers of cholesterol synthesis were measured in liver, adrenal and muscle tissue. RESULTS:LDL-cholesterol infusion in septic mice increased plasma LDL-, but not HDL-cholesterol, whereas HDL-cholesterol infusion increased plasma HDL- and LDL-cholesterol (P < 0.0001 versus placebo). Cholesterol supplementation attenuated sepsis-induced adrenal cholesterol depletion (P < 0.05), without improving adrenocortical structure or the adrenal ACTH response. Cholesterol supplementation did not affect muscle mass loss, force or myofiber cholesterol, but increased plasma bile acids and reduced markers of cholesterol synthesis in liver, adrenal and muscle versus placebo (P ≤ 0.05). No additional effect on elevated plasma CORT and TNF-α was observed. CONCLUSION:Cholesterol supplementation reversed sepsis-induced hypocholesterolemia, without reversing the sepsis-induced adrenal or muscle phenotype. Together with suppressed markers of cholesterol uptake, synthesis and increased bile acid formation, these findings argue against the need to treat hypocholesterolemia during prolonged sepsis.

Critically ill patients requiring treatment in the intensive care unit (ICU) suffer from muscle weakness that persists for years. As compared with healthy subjects, skeletal muscle of patients biopsied five years post-ICU revealed an abnormal transcriptome partially associated with poor muscle strength. We now hypothesized that skeletal muscle of long-term ICU survivors is "epigenetically aged", as determined by a muscle-specific epigenetic clock, and that such accelerated epigenetic aging contributes to their long-term muscle weakness. Muscle DNA-methylation data from former ICU patients at 5-year follow-up (N = 118) and healthy controls (N = 160), aged 18-89 years, were analyzed by the MEATv2 epigenetic clock. First, epigenetic age (DNAmAge), epigenetic minus chronological age (AADiff) and epigenetic age acceleration (AAResid) were compared between 97 former patients and 97 controls, propensity score-matched for age and sex. Next, the impact of any muscle-specific epigenetic aging of ICU survivors was investigated, via multivariable models, as a potential contributor to the altered transcriptome and reduced muscle strength. Former ICU patients showed a significantly higher muscle DNAmAge, AADiff, and AAResid than matched controls. In adjusted models, higher muscle DNAmAge, AADiff, or AAResid did not substantially contribute to differentially expressed muscle RNAs in former patients as compared with controls and was not associated with the poor long-term muscle strength. In conclusion, five years after ICU discharge, former patients showed accelerated epigenetic aging in skeletal muscle. However, the muscle-specific epigenetic clock did not capture molecular changes that are associated with long-term muscle weakness, which highlights the need for other muscle-specific biological predictors of age-related physical impairment. Trail Registration: ClinicalTrials.gov: NCT00512122.

Administration of ketone bodies attenuated the severity of sepsis-induced muscle weakness in preclinical studies. Whether lipid-rich emulsions may likewise mitigate such muscle weakness by stimulating the endogenous ketogenic capacity remains uncertain, especially in relation to glucose, a critical suppressor of ketogenesis. This study investigated the ketogenic potential of parenteral nutrition rich in long- and/or medium-chain triglycerides with differing glucose content on sepsis-induced muscle weakness. We used a parenterally fed murine model of prolonged sepsis-induced muscle weakness to investigate specific lipid mixtures in two consecutive studies. Septic mice receiving standard total parenteral nutrition (TPN) and healthy control (HC) animals were included as references in both studies. In a first study, septic mice received pure long-chain triglycerides (LCT) or long-chain triglycerides supplemented with glucose (gLCT). The second study compared a gLCT mixture to a mixed medium- and long-chain triglyceride emulsion supplemented with glucose (gMCT). After 5 days of sepsis, markers of ketone body metabolism, muscle function, and muscle and liver metabolomics were measured. In study one, ketosis was undetectable with TPN-treatment, but substantially increased with pure LCT (median 1.39 mmol/L, p < 0.001). Supplemental glucose suppressed ketosis sixfold (median 0.24 mmol/L, p < 0.001). The sepsis-induced muscle weakness was exacerbated in LCT mice, while muscle force was comparable between TPN-treated and gLCT mice (TPN 60.9

Hypocholesterolemia hallmarks sepsis, though its pathophysiology and tissue-specific consequences are unclear. As low circulating cholesterol may reflect impaired endogenous cholesterol synthesis, we hypothesized that infusion of the cholesterol precursor mevalonate can reverse sepsis-induced hypocholesterolemia, whereby beneficially affecting adrenal and muscle integrity. In a catheterized mouse model of cecal ligation and puncture-induced sepsis (male 24-wk-old C57BL/6J mice), septic mice received either 5-day mevalonate infusion (78 mg/day) or placebo versus healthy controls (n = 50). Plasma high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, corticosterone, total bile acids, adrenocortical lipids, myofiber cholesterol, and muscle force were quantified. Expression markers of cholesterol homeostasis and structural integrity were investigated in adrenal, muscle and liver tissue. Liver mevalonate metabolites were quantified with liquid chromatography-mass spectrometry (LC-MS). Next, a secondary analysis on a prospective observational human study on the time course of adrenal function in the intensive care unit was performed to assess the association between plasma cholesterol and cortisol (n = 47). Also, plasma mevalonate was quantified with LC-MS. In septic mice, 5-day mevalonate infusion worsened HDL and LDL hypocholesterolemia versus placebo (P < 0.05). Decreased hepatic cholesterol synthesis expression markers, apolipoproteins, and hepatic cholesterol concentrations were observed in mevalonate-infused septic mice versus placebo (P < 0.05). No additional effect on plasma corticosterone, bile acids, myofiber cholesterol, and loss of muscle force and adrenocortical lipid depletion was observed. In prolonged sepsis patients, plasma mevalonate was increased, whereas plasma HDL- and LDL-cholesterol were low (P < 0.05) but did not correlate with plasma cortisol. To conclude, mevalonate infusion worsened sepsis-induced hypocholesterolemia, possibly due to increased feedback on hepatic cholesterol synthesis, without aggravating the adrenal or muscle sepsis phenotype.NEW & NOTEWORTHY Prolonged infusion with the cholesterol precursor, mevalonate, worsened hypocholesterolemia in a mouse model of sepsis-induced critical illness, likely due to increased feedback on hepatic cholesterol synthesis. Loss of muscle force and mass was not further affected, nor did mevalonate affect adrenal gland steroidogenic markers and the loss of adrenocortical lipids. In prolonged sepsis patients, sustained HDL and LDL hypocholesterolemia was observed in the face of high plasma mevalonate concentrations, but did not correlate with plasma cortisol.

BACKGROUND:Critically ill patients requiring intensive care unit (ICU) admission suffer from muscle weakness that persists for years. Recently, altered RNA expression was documented in muscle of former ICU patients 5 years after critical illness that suggested disrupted mitochondrial function, disturbed lipid metabolism and fibrosis, of which many associated with the former patients' long-term loss of muscle strength. We hypothesized that abnormal DNA methylation detectable years after critical illness associates with these abnormal RNA expression patterns, as a potential biological basis for the persistent loss of muscle strength. METHODS:Genome-wide DNA methylation was assessed (Infiniumv2-HumanMethylationEPIC-BeadChips) in skeletal muscle biopsies from 118 former ICU patients harvested 5 years after critical illness (79.6% male, median 58 years, median BMI 27.3 kg/m2) and 30 controls who never required ICU admission (76.7% male, median 61 years, median BMI 26.4 kg/m2). Differentially methylated positions (DMPs) in former patients versus controls were identified, adjusting for age, sex, and BMI (minfi-package in R, Benjamini-Hochberg false-discovery-rate < 0.05), followed by pathway over-representation of affected genes. Spearman correlations between DMP methylation and RNA expression were compared among groups of RNA with Z-test and Kolmogorov-Smirnov test. Risk factors for abnormal DNA methylation were identified with multivariable linear regression. RESULTS:As compared with controls, former ICU patients showed 7379 DMPs (average difference 2.6% ranging up to 24.9%). They were associated with 1334 unique genes, enriched for muscle contraction, vascular development, cell differentiation and signal transduction. DMPs correlated more strongly with differentially expressed RNAs (DERNAs) than with non-differentially expressed RNAs (18.1% vs. 1.7% correlations with |rho| > 0.3, p < 2.2 × 10-16). Such correlations were more abundant among DERNAs associated with reduced muscle strength vs. those not associated (24.4% vs. 12.5%), also within the previously identified disrupted pathways (mitochondrial function 23.3% vs. 10.9%, lipid metabolism 15.9% vs. 7.2%, fibrosis 44.3% vs. 5.8%, all p < 2.2 × 10-16). Older age, female sex, in-ICU treatment with glucocorticoids, benzodiazepines, early parenteral nutrition and opioids and insulin and antipsychotic medication at follow-up were most notably associated with more abnormal DNA methylation. CONCLUSIONS:Abnormal DNA methylation in muscle biopsied 5 years after critical illness associated with long-term altered RNA expression that has been linked to lower muscle strength. These data suggest a possible epigenetic basis for this long-term sequel after critical illness. Abnormal DNA methylation was also found to associate with (possibly) avoidable risk factors during and after ICU stay. These findings may open perspectives for prevention and possibly treatment of long-term muscle weakness after critical illness.

ABSTRACT Background Suppression of the peroxisome proliferator‐activated receptor alpha (PPARα) has been related to poor outcomes in sepsis and may compromise ketogenesis during critical illness. Infusion of 3‐hydroxybutyrate (3HB) was shown to attenuate muscle weakness in septic mice. We hypothesise that endogenous ketogenesis induced by pharmacological PPARα activation, either alone or combined with ketogenic nutrition, is safe and can also mitigate muscle weakness in septic mice. Methods In a fluid‐resuscitated, antibiotic‐treated mouse model of prolonged sepsis, we first (Study 1) assessed the safety and effectiveness (impact on ketosis and muscle weakness) of the PPARα agonist pemafibrate (1 mg/kg/d, n = 16), versus placebo (n = 15) combined with standard balanced parenteral nutrition (PN), composed of glucose, amino acids and long‐chain triglycerides (LCT) (balanced‐TPN). We subsequently (Study 2) evaluated the impact of pemafibrate combined with four types of PN on ketosis and muscle weakness: balanced‐TPN (n = 18), TPN with extra LCTs (TPN + LCT, n = 18), low‐dose pure LCT emulsion (Low‐LCT, n = 16) and high‐dose pure LCT emulsion (High‐LCT n = 18). Carbohydrates and amino acids were omitted in the pure LCT groups. Healthy control mice (HC, n = 19) served as controls. Ex vivo muscle force was measured as the primary outcome. Metabolic, inflammatory and microstructural parameters were assessed on plasma and in muscle and liver tissue by targeted metabolomics, gene expression analysis, biochemical and metabolite assays and histological assessment. Results Pemafibrate treatment with balanced‐TPN upregulated hepatic gene expression of PPARα (Ppara) and its downstream genes (Cd36, Cpt1a, Atgl, Acadl, Hadha, Acox1, and Hmgcs2) (p < 0.0001) and was well tolerated. However, pemafibrate treatment with the use of balanced‐TPN administration did not induce detectable ketosis or improve muscle weakness. In combination with pemafibrate, TPN + LCT also did not induce ketosis, nor did it affect muscle weakness. In contrast, 3‐hydroxybutyrate plasma concentrations increased with High‐LCT (95‐fold) and Low‐LCT (10‐fold) (p < 0.0001) in combination with pemafibrate, yet muscle force declined further (High‐LCT 25.0%, Low‐LCT 10.7% of HC, p < 0.0001). Blood glucose was lowered with pure High‐LCT and Low‐LCT (High‐LCT 86.9%, Low‐LCT 55.1% of TPN, p < 0.05), while plasma lipids and LC‐carnitines were increased (p < 0.0001). Markers of hepatic protein catabolism were upregulated with High‐LCT and Low‐LCT (p < 0.007), while muscle glycolytic intermediates (p < 0.0001) and ATP levels (p < 0.0001) were depleted. Conclusions In septic mice, pemafibrate combined with balanced‐TPN or lipid‐rich TPN induced PPARα activation but did not result in ketosis nor affect muscle weakness. Pemafibrate combined with pure LCTs induced ketosis in sepsis but worsened muscle weakness, possibly explained by muscular bioenergetic failure.

Disclosure: C. Lauwers: None. J. Gunst: None. S. Derde: None. I. Derese: None. S. Vander Perre: None. L. Pauwels: None. S. El Dawy: None. G.H. Van Den Berghe: None. M.P. Casaer: None. L. Langouche: None. Introduction: Ketogenic diets may mitigate age-related declines in muscle function. It remains unclear whether such diets may also attenuate critical illness-induced muscle weakness, and whether medium-chain triglycerides (MCTs) are more effective than long-chain triglycerides (LCTs) at inducing ketogenesis. We studied the impact of MCT- and LCT-rich nutrition as compared to standard feeding preparations on ketosis and muscle weakness in septic mice. Methods: Sepsis was induced in C57Bl/6j mice by caecal ligation and puncture. After 24h fluid resuscitation, mice were randomized to receive 3 types of parenteral nutrition (PN) with different ketogenic potential. The first group received total PN (TPN, composing 49% glucose, 35% LCT and 16% amino acids, n=16), the second an LCT-rich emulsion (LCT, composing 90% LCT and 10% glucose, n=17) and the third group a mixed MCT/LCT emulsion (MCT/LCT, composing 45% MCT, 45% LCT and 10% glucose, n=16). Healthy control mice (HC) were included to provide healthy reference values (n=17). After 5 days of sepsis, ex-vivo muscle force (Aurora Scientific®) was assessed as the primary outcome, and plasma 3-hydroxybutyrate (3HB), plasma lipids (TG, LDL- and HDL-cholesterol) were measured. Hepatic gene expression of ketogenic markers including the peroxisome-proliferator receptor α (PPARα, the transcriptional regulator of ketogenesis) and 3-hydroxymethylglutaryl-CoA synthase-2 (Hmgcs2, the rate-limiting enzyme of ketogenesis) was assessed, and immunoblotting was conducted for Hmgcs2 on liver tissue. Results: After 5 days of sepsis, survival was similar in the three groups (TPN: 84.2%; MCT/LCT: 66.2%; LCT: 85.0%; p=0.6). Muscle force was equally reduced in septic mice as compared with HC (TPN: 68.1%; MCT/LCT: 60.5%; LCT: 74.0% of 144.5 mN/mm²; p>0.05). Plasma 3HB levels were below the detection limit (0.005 mmol/L) in the TPN group. In LCT mice, 3HB levels increased to median 0.11 mmol/L (p<0.001 compared with TPN), while they were lower in MCT/LCT mice (median 0.02 mmol/L; p<0.001 compared with TPN). Plasma TG, glycerol, and HDL- and LDL-cholesterol were suppressed in TPN mice (p<0.03) but equally increased in both lipid-rich groups. Despite differences in plasma 3HB, hepatic gene expression of ketogenic markers was similarly upregulated in the lipid-rich groups compared to the TPN group (p<0.001). Yet, Hmgcs2 protein expression was suppressed in the TPN group as compared to the HC (p=0.009), but increased stepwise in the MCT/LCT and LCT mice (p=0.04 and p<0.001, resp., compared to TPN). Further gene expression and metabolomics analyses in liver and muscle tissue are ongoing. Conclusion: The tested ketogenic diets did not attenuate sepsis-induced muscle weakness. An MCT-rich emulsion was less effective at inducing ketosis than a pure LCT emulsion. Presentation: Monday, July 14, 2025

Abstract Background Sepsis-induced cardiomyopathy (SICM) often occurs in the acute phase of sepsis and is associated with increased mortality due to cardiac dysfunction. The pathogenesis remains poorly understood, and no specific treatments are available. Although SICM is considered reversible, emerging evidence suggests potential long-term sequelae. We hypothesized that metabolic and inflammatory cardiac changes, previously observed in acute sepsis as potential drivers of SICM, partially persist in prolonged sepsis. Methods In 24-week-old C57BL/6J mice, sepsis was induced by cecal ligation and puncture, followed by intravenous fluid resuscitation, subcutaneous analgesics and antibiotics, and, in the prolonged phase, by parenteral nutrition. Mice were killed after 5 days of sepsis (prolonged sepsis, n = 15). For comparison, we included acutely septic mice killed at 30 h (acute sepsis, n = 15) and healthy controls animals (HC, n = 15). Cardiac tissue was collected for assessment of inflammatory and metabolic markers through gene expression, metabolomic analysis and histological assessment. Results In prolonged sepsis, cardiac expression of IL-1β and IL-6 and macrophage infiltration remained upregulated (p ≤ 0.05). In contrast, tissue levels of Krebs cycle intermediates and adenosine phosphates were normal, whereas NADPH levels were low in prolonged sepsis (p ≤ 0.05). Gene expression of fatty acid transporters and of the glucose transporter Slc2a1 was upregulated in prolonged sepsis (p ≤ 0.01). Lipid staining and glycogen content were elevated in prolonged sepsis together with increased gene expression of enzymes responsible for lipogenesis and glycogen synthesis (p ≤ 0.05). Intermediate glycolytic metabolites (hexose-phosphates, GADP, DHAP) were elevated (p ≤ 0.05), but gene expression of several enzymes for glycolysis and mitochondrial oxidation of pyruvate, fatty-acyl-CoA and ketone bodies to acetyl-CoA were suppressed in prolonged sepsis (p ≤ 0.05). Key metabolic transcription factors PPARα and PGC-1α were downregulated in acute, but upregulated in prolonged, sepsis (p ≤ 0.05 for both). Ketone body concentrations were normal but ketolytic enzymes remained suppressed (p ≤ 0.05). Amino acid metabolism showed mild, mixed changes. Conclusions Our results suggest myocardial lipid and glycogen accumulation and suppressed mitochondrial oxidation, with a functionally intact Krebs cycle, in the prolonged phase of sepsis, together with ongoing myocardial inflammation. Whether these alterations have functional consequences and predispose to long-term sequelae of SICM needs further research.

Critically ill patients requiring intensive care unit (ICU) admission suffer from muscle weakness that persists for years, compromising quality-of-life. The pathophysiology of this long-term weakness remains unclear. We hypothesized that former ICU-patients show a long-term abnormal RNA-expression profile, which may contribute to lower long-term strength and for which modifiable risk factors can be identified. This pre-planned secondary analysis of the EPaNIC-trial compared muscle transcriptomes of 115 former ICU-patients 5 years after critical illness and 30 matched controls with RNA-sequencing, followed by pathway over-representation and differential co-expression analyses of the differentially expressed RNAs. We used multivariable linear regression analyses to identify which of the abnormal RNA-expressions associated with the long-term muscle strength of the patients and to identify potential risk factors for the abnormal RNA-expressions. In former patients, 234 down-regulated and 116 up-regulated RNAs were identified after adjustment for age, sex, and BMI. Pathway over-representation and further molecular and histological analyses indicated impaired mitochondrial energy metabolism, disturbed lipid metabolism, and increased collagen formation/fibrosis in former patients. Abnormal muscle RNA-expression in former patients correlated with lower long-term muscle strength. Several treatments given in-ICU and at 5-year follow-up associated with abnormal RNA-expression, most notably in-ICU early parenteral nutrition (early PN) and glucocorticoid use. Abnormal RNA-expression profiles 5 years after critical illness suggest disrupted mitochondrial function, disturbed lipid metabolism, and fibrosis, associated with lower long-term muscle strength and partly attributable to possibly avoidable risk factors. These findings open perspectives for prevention and possibly treatment of long-term muscle weakness after critical illness. ClinicalTrials.gov-NCT00512122, July 31, 2007.

INTRODUCTION:Intensive-care-unit-acquired-muscle-weakness is a debilitating complication of sepsis, characterized by loss of muscle mass and functionality. Immobilization is an important trigger, but the role of disturbed mechanical signaling is incompletely understood. In health, the integrin-receptor-complex with key components Kindlin2 (KIND2/Fermt2) and integrin-linked-kinase (ILK/Ilk1) converses mechanical forces into biochemical signals to regulate muscle mass. We hypothesize that this complex, through key elements KIND2 and ILK, plays a role in sepsis-induced-muscle-weakness. METHODS:AAV2/9-vectors expressing shRNA-sequences against Ilk1, Fermt2 or noncoding-control-gene were injected in tibialis anterior (TA) muscles of 24w-old male C57BL/6J mice. Two-weeks-post-injection, after knockdown validation, mice were made septic by cecal ligation and puncture. Five-days-post-sepsis muscle force, mass and fiber size were quantified and expression of mechanosensitive elements and downstream pathways of the integrin-receptor-complex was assessed. RESULTS:Two-weeks-post-injection the respective sh-targets were strongly suppressed (mRNA Ilk1-44%, Fermt2-76%, protein ILK -34%, KIND2-70%). In rAAV-sh-controls, sepsis induced upregulation across TA and EDL muscle of Ilk1 and Fermt2 and integrin-receptor-complex-related genes Itga7, ItgB1, Tln1, Lims1, Lims2, Parva (P < 0.001), whereas in SOL muscle Lims1, Lims2 and Fermt2 were not and Vcl1 (P < 0.001) was upregulated. In TA and EDL, but not in SOL, rAAV-shIlk1 and rAAV-shFermt2 attenuated upregulation of respective targets down to healthy controls, but without affecting expression of other integrin-receptor-complex-related genes. TA muscle force or weight were not affected by rAAV-shIlk1 or rAAV-shFermt2 (P > 0.05), whereas muscle fiber size reduction (-20.7% in Sepsis shControl) was attenuated up to -13.4% (Sepsis shIlk1, P < 0.001) and -12.3% (Sepsis shFermt2, P < 0.001). Sepsis or sh-treatment did not shift TA fiber types. Expression of markers of atrophy, inflammation, autophagy, protein synthesis and regeneration were affected by sepsis, but not by sh-treatment. Only markers of metabolism Slc2a4 (P < 0.05) and Rac1 (P < 0.01) were further affected by sh-treatment. CONCLUSIONS:Sepsis induced upregulation of integrin-receptor-complex-related genes but attenuating the upregulation of Ilk1 or Fermt2 did not affect the development of muscle weakness, although muscle fiber size was better preserved, arguing against a key role for Ilk1 or Fermt2.

Background Critically ill children suffer from impaired physical/neurocognitive development 2 years later. Glucocorticoid treatment alters DNA methylation within the hypothalamus–pituitary–adrenal (HPA) axis which may impair normal brain development, cognition and behaviour. We tested the hypothesis that paediatric-intensive-care-unit (PICU) patients, sex- and age-dependently, show long-term abnormal DNA methylation within the HPA-axis layers, possibly aggravated by glucocorticoid treatment in the PICU, which may contribute to the long-term developmental impairments. Results In a pre-planned secondary analysis of the multicentre PEPaNIC-RCT and its 2-year follow-up, we identified differentially methylated positions and differentially methylated regions within HPA-axis genes in buccal mucosa DNA from 818 former PICU patients 2 years after PICU admission ( n = 608 no glucocorticoid treatment; n = 210 glucocorticoid treatment) versus 392 healthy children and assessed interaction with sex and age, role of glucocorticoid treatment in the PICU and associations with long-term developmental impairments. Adjusting for technical variation and baseline risk factors and correcting for multiple testing (false discovery rate < 0.05), former PICU patients showed abnormal DNA methylation of 26 CpG sites (within CRHR1, POMC, MC2R, NR3C1, FKBP5, HSD11B1, SRD5A1, AKR1D1, DUSP1, TSC22D3 and TNF ) and three DNA regions (within AVP, TSC22D3 and TNF ) that were mostly hypomethylated. These abnormalities were sex-independent and only partially age-dependent. Abnormal methylation of three CpG sites within FKBP5 and one CpG site within SRD5A1 and AKR1D1 was partly attributable to glucocorticoid treatment during PICU stay. Finally, abnormal methylation within FKBP5 and AKR1D1 was most robustly associated with long-term impaired development. Conclusions Two years after critical illness in children, abnormal methylation within HPA-axis genes was present, predominantly within FKBP5 and AKR1D1 , partly attributable to glucocorticoid treatment in the PICU, and explaining part of the long-term developmental impairments. These data call for caution regarding liberal glucocorticoid use in the PICU.

Hypocholesterolemia hallmarks critical illness though the underlying pathophysiology is incompletely understood. As low circulating cholesterol levels could partly be due to an increased conversion to cortisol/corticosterone, we hypothesized that glucocorticoid treatment, via reduced de novo adrenal cortisol/corticosterone synthesis, might improve cholesterol availability and as such affect adrenal gland and skeletal muscle function. In a matched set of prolonged critically ill patients (n = 324) included in the EPaNIC RCT, a secondary analysis was performed to assess the association between glucocorticoid treatment and plasma cholesterol from ICU admission to day five. Next, in a mouse model of cecal ligation and puncture-induced sepsis, septic mice were randomized to receive either hydrocortisone (1.2 mg/day) (n = 17) or placebo (n = 15) for 5 days, as compared with healthy mice (n = 18). Plasma corticosterone, cholesterol, and adrenocortical and myofiber cholesterol were quantified. Adrenal structure and steroidogenic capacity were evaluated. Muscle force and markers of atrophy, fibrosis and regeneration were quantified. In a consecutive mouse study with identical design (n = 24), whole body composition was assessed by EchoMRI to investigate impact on lean mass, fat mass, total and free water. In human patients, glucocorticoid treatment was associated with higher plasma HDL- and LDL-cholesterol from respectively ICU day two and day three, up to day five (P < 0.05). Plasma corticosterone was no longer elevated in hydrocortisone-treated septic mice compared to placebo, whereas the sepsis-induced reduction in plasma HDL- and LDL-cholesterol and in adrenocortical cholesterol was attenuated (P < 0.05), but without improving the adrenocortical ACTH-induced CORT response and with increased adrenocortical inflammation and apoptosis (P < 0.05). Total body mass was further decreased in hydrocortisone-treated septic mice (P < 0.01) compared to placebo, with no additional effect on muscle mass, force or myofiber size. The sepsis-induced rise in markers of muscle atrophy and fibrosis was unaffected by hydrocortisone treatment, whereas markers of muscle regeneration were suppressed compared to placebo (P < 0.05). An increased loss of lean body mass and total and free water was observed in hydrocortisone-treated septic mice compared to placebo (P < 0.05). Glucocorticoid treatment partially attenuated critical illness-induced hypocholesterolemia, but at a cost of impaired adrenal function, suppressed muscle regeneration and exacerbated loss of body mass.

In critically ill adults, high plasma cortisol in the face of low ACTH coincides with high pro-opiomelanocortin (POMC) levels. Glucocorticoids further lower ACTH without affecting POMC. We hypothesized that in pediatric cardiac surgery-induced critical illness, plasma POMC is elevated, plasma ACTH transiently rises intraoperatively but becomes suppressed post-operatively, and glucocorticoid administration amplifies this phenotype. From 53 patients (0–36 months), plasma was obtained pre-operatively, intraoperatively, and on post-operative days 1 and 2. Plasma was also collected from 24 healthy children. In patients, POMC was supra-normal pre-operatively (P < 0.0001) but no longer thereafter (P > 0.05). ACTH was never high in patients. While in glucocorticoid-naive patients ACTH became suppressed by post-operative day 1 (P < 0.0001), glucocorticoid-treated patients had already suppressed ACTH intraoperatively (P ≤ 0.0001). Pre-operatively high POMC, not accompanied by increased plasma ACTH, suggests a centrally activated HPA axis with reduced pituitary processing of POMC into ACTH. Increasing systemic glucocorticoid availability with glucocorticoid treatment accelerated the suppression of plasma ACTH. Significance statement Glucocorticoids are often administered during pediatric cardiac surgery. In critically ill children, endogenous systemic glucocorticoid availability is elevated already upon ICU admission while ACTH levels are normal. This hormonal constellation suggests the presence of active feedback inhibition of ACTH. In this study, we have documented that intraoperative administration of glucocorticoids accelerates the suppression of ACTH, resulting in low plasma ACTH already upon ICU admission. Pre-operative plasma POMC, the ACTH precursor, but not ACTH, was increased. This is compatible with a centrally activated HPA axis prior to surgery in young children but reduced processing of POMC into ACTH within the pituitary. These findings suggest that glucocorticoid treatment in the context of pediatric cardiac surgery may amplify pre-existing impaired pituitary processing of the prohormone POMC.

Abstract Disclosure: L. De Bruyn: None. S. Derde: None. F. Van Beek: None. S. Vander Perre: None. I. Derese: None. L. Pauwels: None. G. Van den Berghe: None. L. Langouche: None. Introduction: Sepsis is hallmarked by an immediate and sustained reduction in total-, HDL- and LDL-cholesterol (-C), associated with illness severity and a potential contributor to the development of adrenal failure and muscle weakness. The underlying mechanisms that might be involved in sepsis-induced hypocholesterolemia are multifactorial and partly understood. We hypothesized that treatment with the cholesterol precursor mevalonate might improve cholesterol availability, thereby improving adrenal gland and skeletal muscle function. Our hypothesis was tested in a validated and clinically relevant mouse model of prolonged sepsis. Methods: In a catheterized mouse model of cecal ligation and puncture-induced, fluid resuscitated and antibiotics treated polymicrobial abdominal sepsis (5 d), septic mice received continuous infusion with either mevalonate (78 mg/d) or placebo solution, whereas healthy mice served as controls (N=50). Plasma total-C (HDL- + LDL-C), CORT and total bile acids were measured, in addition to ex vivo muscle force and adrenocortical cholesterol ester content. Gene expression markers of inflammation (Tnf-α, Il-6) were measured in the adrenal gland, as well as hepatic mRNA expression of cholesterol synthesis enzymes (Hmgcs1, Hmgcr, Fdft1). Metabolites of the mevalonate pathway (mevalonic acid, squalene, lanosterol, desmosterol and (7-dehydro) cholesterol) were quantified in the liver with LC-MS. Results: The sepsis-induced reduction in plasma total-C was further decreased in mevalonate-treated septic mice (26±3 mg/dL) as compared with placebo (53±3 mg/dL) and healthy controls (108±3 mg/dL) (p<0.0001). Metabolites of the mevalonate pathway were all increased in the liver after mevalonate treatment (p<0.01), whereas hepatic cholesterol content was decreased as compared with placebo (p<0.05). Also, gene expression markers of cholesterol synthesis enzymes were further decreased in the liver of mevalonate-treated septic mice as compared with placebo (p<0.05). In the context of the unexpected further reduction in plasma total-C, no additional effect on plasma CORT, total bile acids, and sepsis-induced loss of muscle force and depletion in adrenocortical cholesterol ester content was observed. In addition, the sepsis-induced rise in markers of adrenal inflammation (Tnf-α, Il-6) was not further affected by mevalonate treatment. Conclusion: Mevalonate treatment further reduced the sepsis-induced hypocholesterolemia in prolonged septic mice, potentially due to increased feedback inhibition on hepatic cholesterol biosynthesis, but without further affecting adrenal and muscle function. Other therapies such as substitution with bovine serum HDL-C are currently investigated to improve altered cholesterol availability during sepsis. Presentation: 6/2/2024

Abstract Disclosure: C. Lauwers: None. M.P. Casaer: None. W. Vankrunkelsven: None. S. Derde: None. I. Derese: None. S. Vander Perre: None. P. Vermeersch: None. G.H. Van Den Berghe: None. L. Langouche: None. Introduction: In septic mice, infusion of ketone bodies (beta-hydroxybutyrate (BHB)) attenuated muscle weakness, a debilitating complication of critical illness. Interestingly, in critically ill children, fasting-induced ketosis also improved outcomes, but in adults ketosis upon fasting was impaired, likely due to suppression of PPARα, a key transcriptional regulator of ketogenesis. In septic mice, pharmacological induction of PPARα by pemafibrate (PF) alone, however, was ineffective in promoting ketosis. Therefore, we here hypothesized that PPARα activation by PF combined with ketogenic nutrition can more effectively induce ketosis and hereby reduce muscle weakness in sepsis. Methods: In a fluid-resuscitated and antibiotic-treated mouse model of prolonged (5 days) sepsis (male C57BL/6J), the impact of PF (1mg/kg/d) combined with 4 types of parenteral nutrition (PN) was studied. Mice were either allocated to PF + total PN (composed of glucose, long-chain triglycerides (LCTs) and amino acids) (TPN, n = 18), PF + TPN with an extra LCT emulsion (TPN + LCTs, n = 18), PF + a pure low dose LCT emulsion (Low LCT, n = 16) or PF + a pure high dose LCT emulsion (High LCT n = 18). Healthy control mice on standard chow served as a reference (HC, n=19). After 5 days of sepsis, ex vivo muscle force (aurora scientific®), plasma BHB and lipids (TG, FFA, LDL- and HDL-cholesterol; commercial assays), and liver gene expression levels were measured. Metabolomics of muscle tissue was assessed by LC-MS and plasma acylcarnitines by LC-MS-MS. Results: Liver PPARα gene expression was higher in all PF-treated mice than in HCs. Compared to HCs, specific muscle force was reduced in all septic mice, but mostly so in both PF + LCT groups (PF + Low LCT: 10.7%, PF + High LCT: 25.0%, PF+TPN + LCTs: 44.6%, PF+TPN: 58.4% of HC: 124.7 mN/mm²; p = 0.0001). PF + TPN + LCTs did not induce ketosis, whereas BHB plasma levels increased 95-fold in the PF + High LCT and 10-fold in the PF + Low LCT group (p < 0.0001 vs. other groups). Glycemia was lower in both PF + LCT groups relative to the other septic mice (PF + Low LCT: 52 mg/dl; PF + High LCT: 80 mg/dl; PF + TPN + LCTs: 108 mg/dl; PF + TPN: 102 mg/dl; p < 0.0001). Compared to the PF + TPN group, plasma lipids and long-chain acylcarnitines were increased in all other septic mice with the highest levels in the PF + High LCT group. Muscular glycolytic intermediates and ATP levels were depleted in both PF + LCT groups in comparison with all other septic mice and HCs. Conclusion: In septic mice, PF-induced PPARα activation combined with TPN, irrespective of the amount of LCTs, was unable to induce ketosis and did not attenuate muscle weakness. By contrast, PF-induced PPARα activation in combination with pure LCTs resulted in ketosis, but aggravated rather than improved muscle weakness, irrespective of the dose. In these groups, metabolic analyses suggested a bioenergetic failure of muscle fibers with an accumulation of plasma lipids. Presentation: 6/1/2024

Background Altered DNA-methylation affects biological ageing in adults and developmental processes in children. DNA-methylation is altered by environmental factors, trauma and illnesses. We hypothesised that paediatric critical illness, and the nutritional management in the paediatric intensive care unit (PICU), affects DNA-methylation changes that underly the developmental processes of childhood ageing. Results We studied the impact of critical illness, and of the early use of parenteral nutrition (early-PN) versus late-PN, on “epigenetic age-deviation” in buccal mucosa of 818 former PICU-patients (406 early-PN, 412 late-PN) who participated in the 2-year follow-up of the multicentre PEPaNIC-RCT (ClinicalTrials.gov-NCT01536275), as compared with 392 matched healthy children, and assessed whether this relates to their impaired growth. The epigenetic age-deviation (difference between PedBE clock-estimated epigenetic age and chronological age) was calculated. Using bootstrapped multivariable linear regression models, we assessed the impact hereon of critical illness, and of early-PN versus late-PN. As compared with healthy children, epigenetic age of patients assessed 2 years after PICU-admission deviated negatively from chronological age ( p < 0.05 in 51% of bootstrapped replicates), similarly in early-PN and late-PN groups. Next, we identified vulnerable subgroups for epigenetic age-deviation using interaction analysis. We revealed that DNA-methylation age-deceleration in former PICU-patients was dependent on age at time of illness ( p < 0.05 for 83% of bootstrapped replicates), with vulnerability starting from 6 years onwards. Finally, we assessed whether vulnerability to epigenetic age-deviation could be related to impaired growth from PICU-admission to follow-up at 2 and 4 years. Multivariable repeated measures ANOVA showed that former PICU-patients, as compared with healthy children, grew less in height ( p = 0.0002) and transiently gained weight ( p = 0.0003) over the 4-year time course. Growth in height was more stunted in former PICU-patients aged ≥ 6-years at time of critical illness ( p = 0.002) than in the younger patients. Conclusions As compared with healthy children, former PICU-patients, in particular those aged ≥ 6-years at time of illness, revealed epigenetic age-deceleration, with a physical correlate revealing stunted growth in height. Whether this vulnerability around the age of 6 years for epigenetic age-deceleration and stunted growth years later relates to altered endocrine pathways activated at the time of adrenarche requires further investigation.

Background Former critically ill children show an epigenetic age deceleration 2 years after paediatric intensive care unit (PICU) admission as compared with normally developing healthy children, with stunted growth in height 2 years further in time as physical correlate. This was particularly pronounced in children who were 6 years or older at the time of critical illness. As this age roughly corresponds to the onset of adrenarche and further pubertal development, a relation with altered activation of endocrine pathways is plausible. We hypothesised that children who have been admitted to the PICU, sex- and age-dependently show long-term abnormal DNA methylation within genes involved in steroid hormone synthesis or steroid sulphation/desulphation, possibly aggravated by in-PICU glucocorticoid treatment, which may contribute to stunted growth in height further in time after critical illness. Results In this preplanned secondary analysis of the multicentre PEPaNIC-RCT and its follow-up, we compared the methylation status of genes involved in the biosynthesis of steroid hormones (aldosterone, cortisol and sex hormones) and steroid sulphation/desulphation in buccal mucosa DNA (Infinium HumanMethylation EPIC BeadChip) from former PICU patients at 2-year follow-up ( n = 818) and healthy children with comparable sex and age ( n = 392). Adjusting for technical variation and baseline risk factors and corrected for multiple testing (false discovery rate < 0.05), former PICU patients showed abnormal DNA methylation of 23 CpG sites (within CYP11A1 , POR , CYB5A , HSD17B1 , HSD17B2 , HSD17B3 , HSD17B6 , HSD17B10 , HSD17B12 , CYP19A1 , CYP21A2 , and CYP11B2 ) and 4 DNA regions (within HSD17B2 , HSD17B8 , and HSD17B10 ) that were mostly hypomethylated. These abnormalities were partially sex- (1 CpG site) or age-dependent (7 CpG sites) and affected by glucocorticoid treatment (3 CpG sites). Finally, multivariable linear models identified robust associations of abnormal methylation of steroidogenic genes with shorter height further in time, at 4-year follow-up. Conclusions Children who have been critically ill show abnormal methylation within steroidogenic genes 2 years after PICU admission, which explained part of the stunted growth in height at 4-year follow-up. The abnormalities in DNA methylation may point to a long-term disturbance in the balance between active sex steroids and mineralocorticoids/glucocorticoids after paediatric critical illness, which requires further investigation.

Critically ill children requiring intensive care suffer from impaired physical/neurocognitive development 2 y later, partially preventable by omitting early use of parenteral nutrition (early-PN) in the paediatric intensive-care-unit (PICU). Altered methylation of DNA from peripheral blood during PICU-stay provided a molecular basis hereof. Whether DNA-methylation of former PICU patients, assessed 2 y after critical illness, is different from that of healthy children remained unknown. In a pre-planned secondary analysis of the PEPaNIC-RCT (clinicaltrials.gov-NCT01536275) 2-year follow-up, we assessed buccal-mucosal DNA-methylation (Infinium-HumanMethylation-EPIC-BeadChip) of former PICU-patients (N = 406 early-PN; N = 414 late-PN) and matched healthy children (N = 392). CpG-sites differentially methylated between groups were identified with multivariable linear regression and differentially methylated DNA-regions via clustering of differentially methylated CpG-sites using kernel-estimates. Analyses were adjusted for technical variation and baseline risk factors, and corrected for multiple testing (false-discovery-rate <0.05). Differentially methylated genes were functionally annotated (KEGG-pathway database), and allocated to three classes depending on involvement in physical/neurocognitive development, critical illness and intensive medical care, or pre-PICU-admission disorders. As compared with matched healthy children, former PICU-patients showed significantly different DNA-methylation at 4047 CpG-sites (2186 genes) and 494 DNA-regions (468 genes), with most CpG-sites being hypomethylated (90.3%) and with an average absolute 2% effect-size, irrespective of timing of PN initiation. Of the differentially methylated KEGG-pathways, 41.2% were related to physical/neurocognitive development, 32.8% to critical illness and intensive medical care and 26.0% to pre-PICU-admission disorders. Two years after critical illness in children, buccal-mucosal DNA showed abnormal methylation of CpG-sites and DNA-regions located in pathways known to be important for physical/neurocognitive development.

Background Withholding parenteral nutrition (PN) until one week after PICU admission facilitated recovery from critical illness and protected against emotional and behavioral problems 4 years later. However, the intervention increased the risk of hypoglycemia, which may have counteracted part of the benefit. Previously, hypoglycemia occurring under tight glucose control in critically ill children receiving early PN did not associate with long-term harm. We investigated whether hypoglycemia in PICU differentially associates with outcome in the context of withholding early PN, and whether any potential association with outcome may depend on the applied glucose control protocol. Methods In this secondary analysis of the multicenter PEPaNIC RCT, we studied whether hypoglycemia in PICU associated with mortality ( N = 1440) and 4-years neurodevelopmental outcome ( N = 674) through univariable comparison and multivariable regression analyses adjusting for potential confounders. In patients with available blood samples ( N = 556), multivariable models were additionally adjusted for baseline serum NSE and S100B concentrations as biomarkers of neuronal, respectively, astrocytic damage. To study whether an association of hypoglycemia with outcome may be affected by the nutritional strategy or center-specific glucose control protocol, we further adjusted the models for the interaction between hypoglycemia and the randomized nutritional strategy, respectively, treatment center. In sensitivity analyses, we studied whether any association with outcome was different in patients with iatrogenic or spontaneous/recurrent hypoglycemia. Results Hypoglycemia univariably associated with higher mortality in PICU, at 90 days and 4 years after randomization, but not when adjusted for risk factors. After 4 years, critically ill children with hypoglycemia scored significantly worse for certain parent/caregiver-reported executive functions (working memory, planning and organization, metacognition) than patients without hypoglycemia, also when adjusted for risk factors including baseline NSE and S100B. Further adjustment for the interaction of hypoglycemia with the randomized intervention or treatment center revealed a potential interaction, whereby tight glucose control and withholding early PN may be protective. Impaired executive functions were most pronounced in patients with spontaneous or recurrent hypoglycemia. Conclusion Critically ill children exposed to hypoglycemia in PICU were at higher risk of impaired executive functions after 4 years, especially in cases of spontaneous/recurrent hypoglycemia.