Development of muscle weakness in a mouse model of critical illness: does fibroblast growth factor 21 play a role?

BACKGROUND: Critical illness is hallmarked by severe stress and organ damage. Fibroblast growth factor 21 (FGF21) has been shown to rise during critical illness. FGF21 is a pleiotropic hormone that mediates adaptive responses to tissue injury and repair in various chronic pathological conditions. Animal studies have suggested that the critical illness-induced rise in FGF21 may to a certain extent protect against acute lung, liver, kidney and brain injury. However, FGF21 has also been shown to mediate fasting-induced loss of muscle mass and force. Such loss of muscle mass and force is a frequent problem of critically ill patients, associated with adverse outcome. In the present study, we therefore investigated whether the critical illness-induced acute rise in FGF21 is muscle-protective or rather contributes to the pathophysiology of critical illness-induced muscle weakness. METHODS: In a catheterised mouse model of critical illness induced by surgery and sepsis, we first assessed the effects of genetic FGF21 inactivation, and hence the inability to acutely increase FGF21, on survival, body weight, muscle wasting and weakness, and markers of muscle cellular stress and dysfunction in acute (30 h) and prolonged (5 days) critical illness. Secondly, we assessed whether any effects were mirrored by supplementing an FGF21 analogue (LY2405319) in prolonged critical illness. RESULTS: FGF21 was not required for survival of sepsis. Genetic FGF21 inactivation aggravated the critical illness-induced body weight loss (p = 0.0003), loss of muscle force (p = 0.03) and shift to smaller myofibers. This was accompanied by a more pronounced rise in markers of endoplasmic reticulum stress in muscle, without effects on impairments in mitochondrial respiratory chain enzyme activities or autophagy activation. Supplementing critically ill mice with LY2405319 did not affect survival, muscle force or weight, or markers of muscle cellular stress/dysfunction. CONCLUSIONS: Endogenous FGF21 is not required for sepsis survival, but may partially protect muscle force and may reduce cellular stress in muscle. Exogenous FGF21 supplementation failed to improve muscle force or cellular stress, not supporting the clinical applicability of FGF21 supplementation to protect against muscle weakness during critical illness.

Skeletal Muscle Myokine Expression in Critical Illness, Association With Outcome and Impact of Therapeutic Interventions.

Context: Muscle expresses and secretes several myokines that bring about benefits in distant organs. Objective: We investigated the impact of critical illness on muscular expression of irisin, kynurenine aminotransferases, and amylase; association with clinical outcome; and impact of interventions that attenuate muscle wasting/weakness. Methods: We studied critically ill patients who participated in 2 randomized controlled trials (EPaNIC/NESCI) and documented time profiles in critically ill mice. Included in the study were 174 intensive care unit (ICU) patients (day 8 ± 1) vs 19 matched controls, and 60 mice subjected to surgery/sepsis vs 60 pair-fed healthy mice. Interventions studied included 7-day neuromuscular electrical stimulation (NMES), and withholding parenteral nutrition (PN) in the first ICU week (late PN) vs early PN. The main outcome measures were FNDC5 (irisin- precursor), KYAT1, KYAT3, and amylase mRNA expression in skeletal muscle. Results: Critically ill patients showed 34% to 80% lower mRNA expression of FNDC5, KYAT1, and amylases than controls (P < .0001). Critically ill mice showed time-dependent reductions in all mRNAs compared with healthy mice (P ≤ .04). The lower FNDC5 expression in patients was independently associated with a higher ICU mortality (P = .015) and ICU-acquired weakness (P = .012), whereas the lower amylase expression in ICU survivors was independently associated with a longer ICU stay (P = .0060). Lower amylase expression was independently associated with a lower risk of death (P = .048), and lower KYAT1 expression with a lower risk of weakness (P = .022). NMES increased FNDC5 expression compared with unstimulated muscle (P = .016), and late PN patients had a higher KYAT1 expression than early PN patients (P = .022). Conclusion: Expression of the studied myokines was affected by critical illness and associated with clinical outcomes, with limited effects of interventions that attenuate muscle wasting or weakness.

Efficacy and safety of ketone ester infusion to prevent muscle weakness in a mouse model of sepsis-induced critical illness.

In septic mice, 3-hydroxybutyrate-sodium-salt has shown to partially prevent sepsis-induced muscle weakness. Although effective, the excessive sodium load was toxic. We here investigated whether ketone ester 3-hydroxybutyl-3-hydroxybutanoate (3HHB) was a safer alternative. In a mouse model of abdominal sepsis, the effects of increasing bolus doses of 3HHB enantiomers on mortality, morbidity and muscle force were investigated (n = 376). Next, plasma 3HB- clearance after bolus D-3HHB was investigated (n = 27). Subsequently, in septic mice, the effect on mortality and muscle force of a continuous D,L-3HHB infusion was investigated (n = 72). In septic mice, as compared with placebo, muscle force was increased at 20 mmol/kg/day L-3HHB and at 40 mmol/kg/day D- and D,L-3HHB. However, severity of illness and mortality was increased by doubling the effective bolus doses. Bolus 3HHB caused a higher 3HB- plasma peak and slower clearance with sepsis. Unlike bolus injections, continuous infusion of D,L-3HHB did not increase severity of illness or mortality, while remaining effective in improving muscle force. Treatment of septic mice with the ketone ester 3HHB partly prevented muscle weakness. Toxicity of 3HHB administered as bolus was completely avoided by continuous infusion of the same dose. Whether continuous infusion of ketone esters represents a promising intervention to also prevent ICU-acquired weakness in human patients should be investigated.

Obesity attenuates inflammation, protein catabolism, dyslipidaemia, and muscle weakness during sepsis, independent of leptin.

BACKGROUND: Muscle weakness is a frequently occurring complication of sepsis, associated with increased morbidity and mortality. Interestingly, obesity attenuates sepsis-induced muscle wasting and weakness. As the adipokine leptin is strongly elevated in obesity and has been shown to affect muscle homeostasis in non-septic conditions, we aimed to investigate whether leptin mediates the protective effect of obesity on sepsis-induced muscle weakness. METHODS: In a mouse model of sepsis, we investigated the effects of genetic leptin inactivation in obese mice (leptin-deficient obese mice vs. diet-induced obese mice) and of leptin supplementation in lean mice (n = 110). We assessed impact on survival, body weight and composition, markers of muscle wasting and weakness, inflammation, and lipid metabolism. In human lean and overweight/obese intensive care unit (ICU) patients, we assessed markers of protein catabolism (n = 1388) and serum leptin (n = 150). RESULTS: Sepsis mortality was highest in leptin-deficient obese mice (53% vs. 23% in diet-induced obese mice and 37% in lean mice, P = 0.03). Irrespective of leptin, after 5 days of sepsis, lean mice lost double the amount of lean body mass than obese mice (P < 0.0005). Also, irrespective of leptin, obese mice maintained specific muscle force up to healthy levels (P = 0.3) whereas lean mice suffered from reduced specific muscle force (72% of healthy controls, P < 0.0002). As compared with lean septic mice, both obese septic groups had less muscle atrophy, liver amino acid catabolism, and inflammation with a 50% lower plasma TNFα increase (P < 0.005). Conversely, again mainly irrespective of leptin, obese mice lost double amount of fat mass than lean mice after 5 days of sepsis (P < 0.0001), showed signs of increased lipolysis and ketogenesis, and had higher plasma HDL and LDL lipoprotein concentrations (P ≤ 0.01 for all). Muscle fibre type composition was not altered during sepsis, but a higher atrophy sensitivity of type IIb fibres compared with IIa and IIx fibres was observed, independent of obesity or leptin. After 5 days of critical illness, serum leptin was higher (P < 0.0001) and the net waste of nitrogen (P = 0.006) and plasma urea-to-creatinine ratio (P < 0.0001) was lower in overweight/obese compared with lean ICU human patients. CONCLUSIONS: Leptin did not mediate the protective effect of obesity against sepsis-induced muscle wasting and weakness in mice. Instead, obesity-independent of leptin-attenuated inflammation, protein catabolism, and dyslipidaemia, pathways that may play a role in the observed muscle protection.

Impact of Hydrocortisone and of CRH Infusion on the Hypothalamus-Pituitary-Adrenocortical Axis of Septic Male Mice.

PURPOSE: Sepsis is hallmarked by high plasma cortisol/corticosterone (CORT), low adrenocorticotropic hormone (ACTH), and high pro-opiomelanocortin (POMC). While corticotropin-releasing hormone-(CRH) and arginine-vasopressin (AVP)-driven pituitary POMC expression remains active, POMC processing into ACTH becomes impaired. Low ACTH is accompanied by loss of adrenocortical structure, although steroidogenic enzymes remain expressed. We hypothesized that treatment of sepsis with hydrocortisone (HC) aggravates this phenotype whereas CRH infusion safeguards ACTH-driven adrenocortical structure. METHODS: In a fluid-resuscitated, antibiotics-treated mouse model of prolonged sepsis, we compared the effects of HC and CRH infusion with placebo on plasma ACTH, POMC, and CORT; on markers of hypothalamic CRH and AVP signaling and pituitary POMC processing; and on the adrenocortical structure and markers of steroidogenesis. In adrenal explants, we studied the steroidogenic capacity of POMC. RESULTS: During sepsis, HC further suppressed plasma ACTH, but not POMC, predominantly by suppressing sepsis-activated CRH/AVP-signaling pathways. In contrast, in CRH-treated sepsis, plasma ACTH was normalized following restoration of pituitary POMC processing. The sepsis-induced rise in markers of adrenocortical steroidogenesis was unaltered by CRH and suppressed partially by HC, which also increased adrenal markers of inflammation. Ex vivo stimulation of adrenal explants with POMC increased CORT as effectively as an equimolar dose of ACTH. CONCLUSIONS: Treatment of sepsis with HC impaired integrity and function of the hypothalamic-pituitary-adrenal axis at the level of the pituitary and the adrenal cortex while CRH restored pituitary POMC processing without affecting the adrenal cortex. Sepsis-induced high-circulating POMC may be responsible for ongoing adrenocortical steroidogenesis despite low ACTH.

Identification of the toxic threshold of 3-hydroxybutyrate-sodium supplementation in septic mice.

BACKGROUND: In septic mice, supplementing parenteral nutrition with 150 mg/day 3-hydroxybutyrate-sodium-salt (3HB-Na) has previously shown to prevent muscle weakness without obvious toxicity. The main objective of this study was to identify the toxic threshold of 3HB-Na supplementation in septic mice, prior to translation of this promising intervention to human use. METHODS: In a centrally-catheterized, antibiotic-treated, fluid-resuscitated, parenterally fed mouse model of prolonged sepsis, we compared with placebo the effects of stepwise escalating doses starting from 150 mg/day 3HB-Na on illness severity and mortality (n = 103). For 5-day survivors, also the impact on ex-vivo-measured muscle force, blood electrolytes, and markers of vital organ inflammation/damage was documented. RESULTS: By doubling the reference dose of 150 mg/day to 300 mg/day 3HB-Na, illness severity scores doubled (p = 0.004) and mortality increased from 30.4 to 87.5 % (p = 0.002). De-escalating this dose to 225 mg still increased mortality (p ≤ 0.03) and reducing the dose to 180 mg/day still increased illness severity (p ≤ 0.04). Doses of 180 mg/day and higher caused more pronounced metabolic alkalosis and hypernatremia (p ≤ 0.04) and increased markers of kidney damage (p ≤ 0.05). Doses of 225 mg/day 3HB-Na and higher caused dehydration of brain and lungs (p ≤ 0.05) and increased markers of hippocampal neuronal damage and inflammation (p ≤ 0.02). Among survivors, 150 mg/day and 180 mg/day increased muscle force compared with placebo (p ≤ 0.05) up to healthy control levels (p ≥ 0.3). CONCLUSIONS: This study indicates that 150 mg/day 3HB-Na supplementation prevented sepsis-induced muscle weakness in mice. However, this dose appeared maximally effective though close to the toxic threshold, possibly in part explained by excessive Na+ intake with 3HB-Na. Although lower doses were not tested and thus might still hold therapeutic potential, the current results point towards a low toxic threshold for the clinical use of ketone salts in human critically ill patients. Whether 3HB-esters are equally effective and less toxic should be investigated.

Altered cholesterol homeostasis in critical illness-induced muscle weakness: effect of exogenous 3-hydroxybutyrate.

BACKGROUND: Muscle weakness is a complication of critical illness which hampers recovery. In critically ill mice, supplementation with the ketone body 3-hydroxybutyrate has been shown to improve muscle force and to normalize illness-induced hypocholesterolemia. We hypothesized that altered cholesterol homeostasis is involved in development of critical illness-induced muscle weakness and that this pathway can be affected by 3-hydroxybutyrate. METHODS: In both human critically ill patients and septic mice, the association between circulating cholesterol concentrations and muscle weakness was assessed. In septic mice, the impact of 3-hydroxybutyrate supplementation on cholesterol homeostasis was evaluated with use of tracer technology and through analysis of markers of cholesterol metabolism and downstream pathways. RESULTS: Serum cholesterol concentrations were lower in weak than in non-weak critically ill patients, and in multivariable analysis adjusting for baseline risk factors, serum cholesterol was inversely correlated with weakness. In septic mice, plasma cholesterol correlated positively with muscle force. In septic mice, exogenous 3-hydroxybutyrate increased plasma cholesterol and altered cholesterol homeostasis, by normalization of plasma mevalonate and elevation of muscular, but not hepatic, expression of cholesterol synthesis genes. In septic mice, tracer technology revealed that 3-hydroxybutyrate was preferentially taken up by muscle and metabolized into cholesterol precursor mevalonate, rather than TCA metabolites. The 3-hydroxybutyrate protection against weakness was not related to ubiquinone or downstream myofiber mitochondrial function, whereas cholesterol content in myofibers was increased. CONCLUSIONS: These findings point to a role for low cholesterol in critical illness-induced muscle weakness and to a protective mechanism-of-action for 3-hydroxybutyrate supplementation.

The role of pro-opiomelanocortin in the ACTH-cortisol dissociation of sepsis.

BACKGROUND: Sepsis is typically hallmarked by high plasma (free) cortisol and suppressed cortisol breakdown, while plasma adrenocorticotropic hormone (ACTH) is not increased, referred to as 'ACTH-cortisol dissociation.' We hypothesized that sepsis acutely activates the hypothalamus to generate, via corticotropin-releasing hormone (CRH) and vasopressin (AVP), ACTH-induced hypercortisolemia. Thereafter, via increased availability of free cortisol, of which breakdown is reduced, feedback inhibition at the pituitary level interferes with normal processing of pro-opiomelanocortin (POMC) into ACTH, explaining the ACTH-cortisol dissociation. We further hypothesized that, in this constellation, POMC leaches into the circulation and can contribute to adrenocortical steroidogenesis. METHODS: In two human studies of acute (ICU admission to day 7, N = 71) and prolonged (from ICU day 7 until recovery; N = 65) sepsis-induced critical illness, POMC plasma concentrations were quantified in relation to plasma ACTH and cortisol. In a mouse study of acute (1 day), subacute (3 and 5 days) and prolonged (7 days) fluid-resuscitated, antibiotic-treated sepsis (N = 123), we further documented alterations in hypothalamic CRH and AVP, plasma and pituitary POMC and its glucocorticoid-receptor-regulated processing into ACTH, as well as adrenal cortex integrity and steroidogenesis markers. RESULTS: The two human studies revealed several-fold elevated plasma concentrations of the ACTH precursor POMC from the acute to the prolonged phase of sepsis and upon recovery (all p < 0.0001), coinciding with the known ACTH-cortisol dissociation. Elevated plasma POMC and ACTH-corticosterone dissociation were confirmed in the mouse model. In mice, sepsis acutely increased hypothalamic mRNA of CRH (p = 0.04) and AVP (p = 0.03) which subsequently normalized. From 3 days onward, pituitary expression of CRH receptor and AVP receptor was increased. From acute throughout prolonged sepsis, pituitary POMC mRNA was always elevated (all p < 0.05). In contrast, markers of POMC processing into ACTH and of ACTH secretion, negatively regulated by glucocorticoid receptor ligand binding, were suppressed at all time points (all p ≤ 0.05). Distorted adrenocortical structure (p < 0.05) and lipid depletion (p < 0.05) were present, while most markers of adrenocortical steroidogenic activity were increased at all time points (all p < 0.05). CONCLUSION: Together, these findings suggest that increased circulating POMC, through CRH/AVP-driven POMC expression and impaired processing into ACTH, could represent a new piece in the puzzling ACTH-cortisol dissociation.

Impact of prolonged sepsis on neural and muscular components of muscle contractions in a mouse model.

BACKGROUND: Prolonged critically ill patients frequently develop debilitating muscle weakness that can affect both peripheral nerves and skeletal muscle. In-depth knowledge on the temporal contribution of neural and muscular components to muscle weakness is currently incomplete. METHODS: We used a fluid-resuscitated, antibiotic-treated, parenterally fed murine model of prolonged (5 days) sepsis-induced muscle weakness (caecal ligation and puncture; n = 148). Electromyography (EMG) measurements were performed in two nerve-muscle complexes, combined with histological analysis of neuromuscular junction denervation, axonal degeneration, and demyelination. In situ muscle force measurements distinguished neural from muscular contribution to reduced muscle force generation. In myofibres, imaging and biomechanics were combined to evaluate myofibrillar contractile calcium sensitivity, sarcomere organization, and fibre structural properties. Myosin and actin protein content and titin gene expression were measured on the whole muscle. RESULTS: Five days of sepsis resulted in increased EMG latency (P = 0.006) and decreased EMG amplitude (P < 0.0001) in the dorsal caudal tail nerve-tail complex, whereas only EMG amplitude was affected in the sciatic nerve-gastrocnemius muscle complex (P < 0.0001). Myelin sheath abnormalities (P = 0.2), axonal degeneration (number of axons; P = 0.4), and neuromuscular junction denervation (P = 0.09) were largely absent in response to sepsis, but signs of axonal swelling [higher axon area (P < 0.0001) and g-ratio (P = 0.03)] were observed. A reduction in maximal muscle force was present after indirect nerve stimulation (P = 0.007) and after direct muscle stimulation (P = 0.03). The degree of force reduction was similar with both stimulations (P = 0.2), identifying skeletal muscle, but not peripheral nerves, as the main contributor to muscle weakness. Myofibrillar calcium sensitivity of the contractile apparatus was unaffected by sepsis (P ≥ 0.6), whereas septic myofibres displayed disorganized sarcomeres (P < 0.0001) and altered myofibre axial elasticity (P < 0.0001). Septic myofibres suffered from increased rupturing in a passive stretching protocol (25% more than control myofibres; P = 0.04), which was associated with impaired myofibre active force generation (P = 0.04), linking altered myofibre integrity to function. Sepsis also caused a reduction in muscle titin gene expression (P = 0.04) and myosin and actin protein content (P = 0.05), but not the myosin-to-actin ratio (P = 0.7). CONCLUSIONS: Prolonged sepsis-induced muscle weakness may predominantly be related to a disruption in myofibrillar cytoarchitectural structure, rather than to neural abnormalities.

Early neuromuscular electrical stimulation reduces the loss of muscle mass in critically ill patients - A within subject randomized controlled trial.

PURPOSE: To investigate the effect of Neuromuscular Electrical Stimulation (NMES) on muscle thickness, strength and morphological and molecular markers of the quadriceps. MATERIALS AND METHODS: Adult critically ill patients with an expected prolonged stay received unilateral quadriceps NMES sessions for 7 consecutive days. Before and after the intervention period, quadriceps thickness was measured with ultrasound. After the intervention period, strength was assessed in cooperative patients and muscle biopsies were taken. Multivariable regression was performed to identify factors affecting muscle thickness loss. RESULTS: Muscle thickness decreased less in the stimulated leg (-6 ± 16% versus -12 ± 15%, p = 0.014, n = 47). Strength was comparable. Opioid administration, minimal muscle contraction and more muscle thickness loss in the non-stimulated muscle were independently associated with better muscle thickness preservation. Stimulated muscles showed a shift towards larger myofibers and higher MyHC-I gene expression. NMES did not affect gene expression of other myofibrillary proteins, MuRF-1 or atrogin-1. Signs of myofiber necrosis and inflammation were comparable for both muscles. CONCLUSIONS: NMES attenuated the loss of muscle mass, but not of strength, in critically ill patients. Preservation of muscle mass was more likely in patients receiving opioids, patients with a minimal muscle contraction during NMES and patients more prone to lose muscle mass. TRIAL REGISTRATION: clinicaltrials.govNCT02133300.

Adipose tissue protects against sepsis-induced muscle weakness in mice: from lipolysis to ketones.

BACKGROUND: ICU-acquired weakness is a debilitating consequence of prolonged critical illness that is associated with poor outcome. Recently, premorbid obesity has been shown to protect against such illness-induced muscle wasting and weakness. Here, we hypothesized that this protection was due to increased lipid and ketone availability. METHODS: In a centrally catheterized, fluid-resuscitated, antibiotic-treated mouse model of prolonged sepsis, we compared markers of lipolysis and fatty acid oxidation in lean and obese septic mice (n = 117). Next, we compared markers of muscle wasting and weakness in septic obese wild-type and adipose tissue-specific ATGL knockout (AAKO) mice (n = 73), in lean septic mice receiving either intravenous infusion of lipids or standard parenteral nutrition (PN) (n = 70), and in lean septic mice receiving standard PN supplemented with either the ketone body 3-hydroxybutyrate or isocaloric glucose (n = 49). RESULTS: Obese septic mice had more pronounced lipolysis (p ≤ 0.05), peripheral fatty acid oxidation (p ≤ 0.05), and ketogenesis (p ≤ 0.05) than lean mice. Blocking lipolysis in obese septic mice caused severely reduced muscle mass (32% loss vs. 15% in wild-type, p < 0.001) and specific maximal muscle force (59% loss vs. 0% in wild-type; p < 0.001). In contrast, intravenous infusion of lipids in lean septic mice maintained specific maximal muscle force up to healthy control levels (p = 0.6), whereas this was reduced with 28% in septic mice receiving standard PN (p = 0.006). Muscle mass was evenly reduced with 29% in both lean septic groups (p < 0.001). Lipid administration enhanced fatty acid oxidation (p ≤ 0.05) and ketogenesis (p < 0.001), but caused unfavorable liver steatosis (p = 0.01) and a deranged lipid profile (p ≤ 0.01). Supplementation of standard PN with 3-hydroxybutyrate also attenuated specific maximal muscle force up to healthy control levels (p = 0.1), but loss of muscle mass could not be prevented (25% loss in both septic groups; p < 0.001). Importantly, this intervention improved muscle regeneration markers (p ≤ 0.05) without the unfavorable side effects seen with lipid infusion. CONCLUSIONS: Obesity-induced muscle protection during sepsis is partly mediated by elevated mobilization and metabolism of endogenous fatty acids. Furthermore, increased availability of ketone bodies, either through ketogenesis or through parenteral infusion, appears to protect against sepsis-induced muscle weakness also in the lean.

The Hepatic Glucocorticoid Receptor Is Crucial for Cortisol Homeostasis and Sepsis Survival in Humans and Male Mice.

Sepsis is hallmarked by hypercortisolemia, a stress response essential for survival. This elevation in plasma cortisol is partially brought about by suppressed hepatic cortisol breakdown. We demonstrate that a controlled downregulation of the hepatic glucocorticoid receptor (hepatic GR) is crucial. In a mouse model of fluid-resuscitated, antibiotic-treated abdominal sepsis and in human intensive care unit patients, sepsis reduced hepatic GR expression and signaling but increased (free) plasma cortisol/corticosterone, explained by suppressed cortisol/corticosterone-binding proteins and A-ring reductases. However, further experimental inhibition of hepatic GR with short hairpin RNA (shRNA) in septic mice increased mortality fivefold. Acutely, this further hepatic GR suppression prevented the rise in total corticosterone but further reduced binding proteins, resulting in elevated free corticosterone. After 3 days of shRNA-GR inhibition in sepsis, both total and free corticosterone levels were elevated, now explained by an additional reduction in A-ring reductase expression. Hepatic GR inhibition blunted the hyperglycemic stress response without causing hypoglycemia but also markedly increased circulating and hepatic inflammation markers and caused liver destruction, the severity of which explained increased mortality. In human sepsis, glucocorticoid treatment further suppressed hepatic GR expression, which could directly predispose to worse outcomes. In conclusion, sepsis partially suppressed hepatic GR expression, which appeared crucial to upregulate free cortisol/corticosterone availability. However, further sustained hepatic GR suppression evoked lethal excessive liver and systemic inflammation, independent of systemic cortisol/corticosterone availability.

On the Role of Illness Duration and Nutrient Restriction in Cholestatic Alterations that Occur During Critical Illness.

BACKGROUND AND AIMS: Elevated markers of cholestasis are common in response to critical illness, and associated with adverse outcome. The role of illness duration and of nutrient restriction on underlying molecular pathways of such cholestatic responses have not been thoroughly investigated. METHODS: In a mouse model of surgery- and sepsis-induced critical illness, molecular pathways of cholestasis were investigated up to 7 days. To assess which changes are explained by illness-induced lack of feeding, nutrient-restricted healthy mice were studied and compared with ad libitum fed healthy mice. Furthermore, serum bile acid (BA) concentrations were quantified in 1,114 human patients with either short or long intensive care unit (ICU) stay, matched for type and severity of illness, up to ICU-day-7. RESULTS: In critically ill mice, either evoked by surgery or sepsis, circulating and hepatic BA-levels progressively increased with time from day-3 onward, preceded by unsuppressed or upregulated CYP7A1 and CYP27A1 protein expression. From 30 h onward, nuclear farnesoid-X-receptor-retinoid-X-receptor staining was significantly suppressed in both critically ill groups, followed from day-3 onward by decreased gene expression of the apical exporter BA-specific export pump and increased expression of basolateral exporters multidrug resistance-associated protein 3 (MRP3) and MRP4. Nutrient restriction in healthy mice only partly mirrored illness-induced alterations in circulating BA and BA-transporters, without changing nuclear receptors or synthesis markers expression. Also in human critically ill patients, serum BA increased with time in long-stay patients only, similarly for patients with or without sepsis. CONCLUSIONS: Circulating BA concentrations rose days after onset of sepsis- and surgery-induced, critical illness, only partially explained by lack of feeding, preceded by suppressed nuclear feedback-sensors and ongoing BA synthesis. Expression of transporters suggested ongoing reversed BA-flow toward the blood.

The Role of Autophagy in Critical Illness-induced Liver Damage.

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which activates the unfolded protein response (UPR), mediate critical illness-induced organ failure, often affecting the liver. Autophagy is known to alleviate both and suppressed or insufficiently activated autophagy in prolonged illness has shown to associate with organ failure. Whether insufficient autophagy contributes to organ failure during critical illness by affecting these underlying mechanisms is incompletely understood. In this study, we investigated whether the inability to acutely activate hepatic autophagy during critical illness aggravates liver damage by increasing hepatic mitochondrial dysfunction and affecting the UPR. In a mouse model of critical illness, induced by surgery and sepsis, we investigated the impact of inactivating hepatic autophagy on markers of hepatic mitochondrial function, the UPR and liver damage in acute (1 day) and prolonged (3 days) critical illness. Hepatic autophagy inactivation during critical illness acutely worsened mitochondrial dysfunction and time-dependently modulated the hepatic UPR. Furthermore, autophagy inactivation aggravated markers of liver damage on both time points. In conclusion, the inability to acutely activate autophagy in liver during critical illness worsened hepatic mitochondrial damage and dysfunction, partially prohibited acute UPR activation and aggravated liver damage, indicating that autophagy is crucial in alleviating critical illness-induced organ failure.

Role of Glucagon in Catabolism and Muscle Wasting of Critical Illness and Modulation by Nutrition.

RATIONALE: Critical illness is hallmarked by muscle wasting and disturbances in glucose, lipid, and amino acid homeostasis. Circulating concentrations of glucagon, a catabolic hormone that affects these metabolic pathways, are elevated during critical illness. Insight in the nutritional regulation of glucagon and its metabolic role during critical illness is lacking. OBJECTIVES: To evaluate whether macronutrient infusion can suppress plasma glucagon during critical illness and study the role of illness-induced glucagon abundance in the disturbed glucose, lipid, and amino acid homeostasis and in muscle wasting during critical illness. METHODS: In human and mouse studies, we infused macronutrients and manipulated glucagon availability up and down to investigate its acute and chronic metabolic role during critical illness. MEASUREMENTS AND MAIN RESULTS: In critically ill patients, infusing glucose with insulin did not lower glucagon, whereas parenteral nutrition containing amino acids increased glucagon. In critically ill mice, infusion of amino acids increased glucagon and up-regulated markers of hepatic amino acid catabolism without affecting muscle wasting. Immunoneutralizing glucagon in critically ill mice only transiently affected glucose and lipid metabolism, did not affect muscle wasting, but drastically suppressed markers of hepatic amino acid catabolism and reversed the illness-induced hypoaminoacidemia. CONCLUSIONS: These data suggest that elevated glucagon availability during critical illness increases hepatic amino acid catabolism, explaining the illness-induced hypoaminoacidemia, without affecting muscle wasting and without a sustained impact on blood glucose. Furthermore, amino acid infusion likely results in a further breakdown of amino acids in the liver, mediated by increased glucagon, without preventing muscle wasting. Clinical trial registered with www.clinicaltrials.gov (NCT 00512122).

Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness.

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting. Critically ill patients require intensive medical support in order to survive. While the majority of patients will recover within a few days, about a quarter of the patients need prolonged intensive care and are at high risk of dying from non-resolving multiple organ failure. Furthermore, the prolonged phase of critical illness is hallmarked by profound muscle weakness, and endocrine and metabolic changes, of which the pathogenesis is currently incompletely understood. The most widely used animal model in critical care research is the cecal ligation and puncture model to induce sepsis. This is a very reproducible model, with acute inflammatory and hemodynamic changes similar to human sepsis, which is designed to study the acute phase of critical illness. However, this model is hallmarked by a high lethality, which is different from the clinical human situation, and is not developed to study the prolonged phase of critical illness. Therefore, we adapted the technique by placing a central venous catheter in the jugular vein allowing us to administer clinically relevant supportive care, to better mimic the human clinical situation of critical illness. This mouse model requires an extensive surgical procedure and daily intensive care of the animals, but it results in a relevant model of the acute and prolonged phase of critical illness.

Premorbid obesity, but not nutrition, prevents critical illness-induced muscle wasting and weakness.

BACKGROUND: The 'obesity paradox' of critical illness refers to better survival with a higher body mass index. We hypothesized that fat mobilized from excess adipose tissue during critical illness provides energy more efficiently than exogenous macronutrients and could prevent lean tissue wasting. METHODS: In lean and premorbidly obese mice, the effect of 5 days of sepsis-induced critical illness on body weight and composition, muscle wasting, and weakness was assessed, each with fasting and parenteral feeding. Also, in lean and overweight/obese prolonged critically ill patients, markers of muscle wasting and weakness were compared. RESULTS: In mice, sepsis reduced body weight similarly in the lean and obese, but in the obese with more fat loss and less loss of muscle mass, better preservation of myofibre size and muscle force, and less loss of ectopic lipids, irrespective of administered feeding. These differences between lean and obese septic mice coincided with signs of more effective hepatic fatty acid and glycerol metabolism, and ketogenesis in the obese. Also in humans, better preservation of myofibre size and muscle strength was observed in overweight/obese compared with lean prolonged critically ill patients. CONCLUSIONS: During critical illness premorbid obesity, but not nutrition, optimized utilization of stored lipids and attenuated muscle wasting and weakness.

Neuropathological Correlates of Hyperglycemia During Prolonged Polymicrobial Sepsis in Mice.

Glucose toxicity may play a crucial role in evoking neurologic complications of critical illness. We studied whether the neuropathological alterations in fatal human critical illness observed under hyperglycemia are present and can be attenuated by maintaining normoglycemia in a mouse model of prolonged sepsis induced by cecal ligation and puncture. Mice were randomized to moderate hyperglycemia (>8.3 mmol/L, n = 8) or normoglycemia (4.4-6.7 mmol/L, n = 8). After 5 days, hippocampus and frontal cortex from septic mice were compared with those from healthy controls (n = 8). Blood glucose was 7.8 ± 1.3 mmol/L in hyperglycemic and 6.1 ± 0.7 mmol/L in normoglycemic critically ill mice (P = 0.007). The percentage of damaged neurons was twofold higher in frontal cortex (P = 0.01) and hippocampus (P = 0.06) of hyperglycemic ill mice than that of healthy mice. In frontal cortex, neuronal damage was attenuated under normoglycemia (P = 0.04). Critical illness reduced astrocyte density and activation status fourfold in hippocampus (P ≤ 0.02), but not in frontal cortex, irrespective of glycemic control. Microglia were twofold to fourfold more abundant in both brain areas of hyperglycemic critically ill mice (P ≤ 0.002), but only in frontal cortex were they reduced in number with normoglycemia (P = 0.0008). The density of apoptotic cells and abundance of carbonylated proteins were significantly higher than normal in frontal cortex of hyperglycemic ill mice only (P = 0.05). In a mouse model of prolonged polymicrobial sepsis, remarkable neuropathological changes develop with neuronal damage, impaired astrocyte activation, increased microglia, apoptosis, and accumulation of carbonylated proteins. These changes were partially prevented or attenuated when hyperglycemia was prevented with insulin. Frontal cortex appeared more vulnerable to hyperglycemic insults than hippocampus.

Impact of parenteral nutrition versus fasting on hepatic bile acid production and transport in a rabbit model of prolonged critical illness.

BACKGROUND: Cholestatic liver dysfunction frequently occurs during critical illness. Administration of parenteral nutrition (PN) is thought to aggravate this. Underlying mechanisms are not clear. METHODS: In a burn model of prolonged critical illness, rabbits were randomized to a nutritional strategy either accepting caloric deficits (fasted, n = 11) or covering caloric needs by PN (fed, n = 10). At baseline and after 7 days of critical illness, markers of hepatotoxicity, circulating bile acids, and the hepatobiliary transport system were studied. RESULTS: Fasted animals had lower circulating alanine aminotransferase/aspartate aminotransferase levels than did the fed animals at day 7. Compared with baseline values, fed animals displayed lower serum unconjugated cholic acid (CA) and deoxycholic acid (DCA) levels. Unconjugated DCA remained unaltered in fasted animals. Unconjugated lithocholic acid was increased comparably in all animals, whereas hyodeoxycholic acid was not altered. In contrast, fasting induced a shift from unconjugated CA and DCA to glyco-CA and glyco-DCA. Total bile acids did not correlate with the bile acid-producing enzyme CYP7A1, but with the basolateral efflux transporter MRP3. Fasting increased protein expression of the basolateral (MRP3) and the canalicular (BSEP) transporter, whereas the canalicular efflux pump MRP2 was suppressed. Gene expression levels of the nuclear receptor farnesoid X receptor were lower with fasting and correlated inversely with MRP3. The heterodimer partner of farnesoid X receptor, retinoid X receptor α, was increased with fasting and correlated positively with MRP3. CONCLUSIONS: During prolonged critical illness, withholding PN improved markers for hepatocyte injury and accentuated bile acid transport toward the blood. This suggests that the latter is an adaptive rather than a dysfunctional feedback to illness.

Critical illness induces nutrient-independent adipogenesis and accumulation of alternatively activated tissue macrophages.

INTRODUCTION: We previously reported that in artificially-fed critically ill patients, adipose tissue reveals an increase in small adipocytes and accumulation of M2-macrophages. We hypothesized that nutrient-independent factors of critical illness explain these findings, and that the M2-macrophage accumulation may not be limited to adipose tissue. METHODS: In a long-term cecal ligation and puncture (CLP) mouse model of sepsis, we compared the effect of parenteral nutrition (CLP-fed, n = 13) with nutrient restriction (CLP-restricted, n = 11) on body composition, adipocyte size and macrophage accumulation in adipose tissue, liver and lungs. Fed healthy mice (n = 11) were studied as controls. In a human study, in vivo adipose tissue biopsies were studied from ICU patients (n = 40) enrolled in a randomized control trial which compared early initiation of parenteral nutrition (PN) versus tolerating nutrient restriction during the first week of ICU stay. Adipose tissue morphology was compared with healthy human controls (n = 13). RESULTS: Irrespective of nutritional intake, critically ill mice lost weight, fat and fat-free mass. Adipocyte number, proliferation marker Proliferating Cell Nuclear Antigen (PCNA) and adipogenic markers PPARγ and CCAAT/enhancer binding protein-ß (C/EBPß) increased with illness, irrespective of nutritional intake. M2-macrophage accumulation was observed in adipose tissue, liver and lungs of critically ill mice. Macrophage M2-markers correlated with CCL2 expression. In adipose tissue biopsies of critically ill patients, increased adipogenic markers and M2 macrophage accumulation were present irrespective of nutritional intake. CONCLUSIONS: Adipogenesis and accumulation of tissue M2-macrophages are hallmarks of prolonged critical illness, irrespective of nutritional management. During critical illness, M2-macrophages accumulate not only in adipose tissue, but also in the liver and lungs.