Jan Gunst

Effect of tolerating macronutrient deficit on the development of intensive-care unit acquired weakness: a subanalysis of the EPaNIC trial

BACKGROUND Patients who are critically ill can develop so-called intensive-care unit acquired weakness, which delays rehabilitation. Reduced muscle mass, quality, or both might have a role. The Early Parenteral Nutrition Completing Enteral Nutrition in Adult Critically Ill Patients (EPaNIC) trial (registered with ClinicalTrials.gov, number NCT00512122) showed that tolerating macronutrient deficit for 1 week in intensive-care units (late parenteral nutrition [PN]) accelerated recovery compared with early PN. The role of weakness was unclear. Our aim was to assess whether late PN and early PN differentially affect muscle weakness and autophagic quality control of myofibres. METHODS In this prospectively planned subanalysis of the EPaNIC trial, weakness (MRC sum score) was assessed in 600 awake, cooperative patients. Skeletal muscle biopsies, harvested from 122 patients 8 days after randomisation and from 20 matched healthy controls, were studied for autophagy and atrophy. We determined the significance of differences with Mann-Whitney U, Median, Kruskal-Wallis, or χ(2) (exact) tests, as appropriate. FINDINGS With late PN, 105 (34%) of 305 patients had weakness on first assessment (median day 9 post-randomisation) compared with 127 (43%) of 295 patients given early PN (absolute difference -9%, 95% CI -16 to -1; p=0·030). Weakness recovered faster with late PN than with early PN (p=0·021). Myofibre cross-sectional area was less and density was lower in critically ill patients than in healthy controls, similarly with early PN and late PN. The LC3 (microtubule-associated protein light chain 3) II to LC3I ratio, related to autophagosome formation, was higher in patients given late PN than early PN (p=0·026), reaching values almost double those in the healthy control group (p=0·0016), and coinciding with less ubiquitin staining (p=0·019). A higher LC3II to LC3I ratio was independently associated with less weakness (p=0·047). Expression of mRNA encoding contractile myofibrillary proteins was lower and E3-ligase expression higher in muscle biopsies from patients than in control participants (p≤0·0006), but was unaffected by nutrition. INTERPRETATION Tolerating a substantial macronutrient deficit early during critical illness did not affect muscle wasting, but allowed more efficient activation of autophagic quality control of myofibres and reduced weakness. FUNDING UZ Leuven, Research Foundation-Flanders, the Flemish Government, and the European Research Council.

Insufficient Activation of Autophagy Allows Cellular Damage to Accumulate in Critically Ill Patients

CONTEXT Responses to critical illness, such as excessive inflammation and hyperglycemia, may trigger detrimental chain reactions that damage cellular proteins and organelles. Such responses to illness contribute to the risk of (nonresolving) multiple organ dysfunction and adverse outcome. OBJECTIVE We studied autophagy as a bulk degradation pathway able to remove toxic protein aggregates and damaged organelles and how these are affected by preventing hyperglycemia with insulin during critical illness. DESIGN AND SETTING Patients participated in a randomized study, conducted at a university hospital surgical/medical intensive care unit. PATIENTS We studied adult prolonged critically ill patients vs. controls. INTERVENTIONS Tolerating excessive hyperglycemia was compared with intensive insulin therapy targeting normoglycemia. MAIN OUTCOME MEASURES We quantified (ultra)structural abnormalities and hepatic and skeletal muscle protein levels of key players in autophagy. RESULTS Morphologically, both liver and muscle revealed an autophagy-deficiency phenotype. Proteins involved in initiation and elongation steps of autophagy were induced 1.3- to 6.5-fold by critical illness (P ≤ 0.01), but mature autophagic vacuole formation was 62% impaired (P = 0.05) and proteins normally degraded by autophagy accumulated up to 97-fold (P ≤ 0.03). Mitophagy markers were unaltered or down-regulated (P = 0.05). Although insulin preserved hepatocytic mitochondrial integrity (P = 0.05), it further reduced the number of autophagic vacuoles by 80% (P = 0.05). CONCLUSIONS Insufficient autophagy in prolonged critical illness may cause inadequate removal of damaged proteins and mitochondria. Such incomplete clearance of cellular damage, inflicted by illness and aggravated by hyperglycemia, could explain lack of recovery from organ failure in prolonged critically ill patients. These data open perspectives for therapies that activate autophagy during critical illness.

Early parenteral nutrition evokes a phenotype of autophagy deficiency in liver and skeletal muscle of critically ill rabbits.

Muscular and hepatic abnormalities observed in artificially fed critically ill patients strikingly resemble the phenotype of autophagy-deficient mice. Autophagy is the only pathway to clear damaged organelles and large ubiquitinated proteins and aggregates. Fasting is its strongest physiological trigger. Severity of autophagy deficiency in critically ill patients correlated with the amount of infused amino acids. We hypothesized that impaired autophagy in critically ill patients could partly be evoked by early provision of parenteral nutrition enriched with amino acids in clinically used amounts. In a randomized laboratory investigation, we compared the effect of isocaloric moderate-dose iv feeding with fasting during illness on the previously studied markers of autophagy deficiency in skeletal muscle and liver. Critically ill rabbits were allocated to fasting or to iv nutrition (220 kcal/d, 921 kJ/d) supplemented with 50 kcal/d (209 kJ/d) of either glucose, amino acids, or lipids, while maintaining normoglycemia, and were compared with healthy controls. Fasted critically ill rabbits revealed weight loss and activation of autophagy. Feeding abolished these responses, with most impact of amino acid-enriched nutrition. Accumulation of p62 and ubiquitinated proteins in muscle and liver, indicative of insufficient autophagy, occurred with parenteral feeding enriched with amino acids and lipids. In liver, this was accompanied by fewer autophagosomes, fewer intact mitochondria, suppressed respiratory chain activity, and an increase in markers of liver damage. In muscle, early parenteral nutrition enriched with amino acids or lipids aggravated vacuolization of myofibers. In conclusion, early parenteral nutrition during critical illness evoked a phenotype of autophagy deficiency in liver and skeletal muscle.

Insufficient autophagy contributes to mitochondrial dysfunction, organ failure, and adverse outcome in an animal model of critical illness.

Objective:Increasing evidence implicates mitochondrial dysfunction as an early, important event in the pathogenesis of critical illness-induced multiple organ failure. We previously demonstrated that prevention of hyperglycemia limits damage to mitochondria in vital organs, thereby reducing morbidity and mortality. We now hypothesize that inadequate activation of mitochondrial repair processes (clearance of damaged mitochondria by autophagy, mitochondrial fusion/fission, and biogenesis) may contribute to accumulation of mitochondrial damage, persistence of organ failure, and adverse outcome of critical illness. Design:Prospective, randomized studies in a critically ill rabbit model. Setting:University laboratory. Subjects:Three-month-old male rabbits. Interventions:We studied whether vital organ mitochondrial repair pathways are differentially affected in surviving and nonsurviving hyperglycemic critically ill animals in relation to mitochondrial and organ damage. Next, we investigated the impact of preventing hyperglycemia over time and of administering rapamycin as an autophagy activator. Measurements and Main Results:In both liver and kidney of hyperglycemic critically ill rabbits, we observed signs of insufficient autophagy, including accumulation of p62 and a concomitant decrease in the microtubule-associated protein light-chain-3-II/microtubule-associated protein light-chain-3-I ratio. The phenotype of insufficient autophagy was more pronounced in nonsurviving than in surviving animals. Molecular markers of insufficient autophagy correlated with impaired mitochondrial function and more severe organ damage. In contrast, key players in mitochondrial fusion/fission or biogenesis were not significantly different regarding survival status. Therefore, we focused on autophagy to study the impact of preventing hyperglycemia. Both after 3 and 7 days of illness, autophagy was better preserved in normoglycemic than in hyperglycemic rabbits, which correlated with improved mitochondrial function and less organ damage. Stimulation of autophagy in kidney with rapamycin correlated with protection of renal function. Conclusions:Our findings put forward insufficient autophagy as a potentially important contributor to mitochondrial and organ damage in critical illness and open perspectives for therapies that activate autophagy during critical illness.

Hyperglycemic kidney damage in an animal model of prolonged critical illness

Acute kidney injury frequently complicates critical illness and increases mortality; maintaining normoglycemia with insulin has been shown to reduce the incidence of intensive care unit (ICU)-acquired kidney injury. Here we tested the mechanisms by which this intervention might achieve its goal, using a rabbit model of burn-induced prolonged critical illness in which blood glucose and insulin were independently regulated at normal or elevated levels. Hyperglycemia caused elevated plasma creatinine and severe morphological kidney damage that correlated with elevated cortical glucose levels. Renal cortical perfusion and oxygen delivery were lower in hyperglycemic/hyperinsulinemic rabbits, compared to other groups, but this did not explain the elevated creatinine. Mitochondrial respiratory chain activities were severely reduced in the hyperglycemic groups (30-40% residual activity), and were inversely correlated with plasma creatinine and cortical glucose. These activities were much less affected by normoglycemia, and hyperinsulinemia was not directly protective. Mitochondrial damage, evident at day 3, preceded the structural injury evident at 7 days. Our study found that hyperglycemia evoked cellular glucose overload in the kidneys of critically ill rabbits, and this was associated with mitochondrial dysfunction and renal injury. Normoglycemia, independent of insulinemia, protected against this damage.

Blood glucose control in the intensive care unit: benefits and risks.

Abnormal blood glucose levels are common during critical illness and are associated with outcomes that correspond to a J‐shaped curve, the lowest risk associated with normoglycemia. Three proof‐of‐concept randomized‐controlled‐trials performed in the surgical, medical, and pediatric intensive care units of the Leuven University Hospital in Belgium demonstrated that maintaining strict age‐adjusted normal fasting levels of glycemia (80–110 mg/dl in adults, 70–100 mg/dl in children, 50–80 mg/dl in infants) with intensive insulin therapy reduced morbidity and mortality as compared with tolerating stress hyperglycemia as a potentially beneficial response. Recently, concern has risen about the safety of this intervention, as a multicenter adult study reported an, as yet unexplained, increased mortality with targeting normoglycemia as compared with an intermediate blood glucose level of around 140 mg/dl. This apparent contradiction may be explained by several methodological differences among studies, comprising, among others, different glucose target ranges in the control groups, different feeding policies, and variable accuracy of tools used for glucose measurement and insulin infusion. Hence, efficacy and safety of intensive insulin therapy may be affected by patient‐related and ICU setting‐related variables. Therefore, no single optimal blood glucose target range for ICU patients can be advocated. It appears safe not to embark on targeting “age‐normal” levels in intensive care units (ICUs) that are not equipped to accurately and frequently measure blood glucose, and have not acquired extensive experience with intravenous insulin administration using a customized guideline. A simple fallback position could be to control blood glucose levels as close to normal as possible without evoking unacceptable blood glucose fluctuations, hypoglycemia, and hypokalemia.

Alterations in adipose tissue during critical illness: An adaptive and protective response?

RATIONALE Critical illness is characterized by lean tissue wasting, whereas adipose tissue is preserved. Overweight and obese critically ill patients may have a lower risk of death than lean patients, suggestive of a protective role for adipose tissue during illness. OBJECTIVES To investigate whether adipose tissue could protectively respond to critical illness by storing potentially toxic metabolites, such as excess circulating glucose and triglycerides. METHODS We studied adipose tissue morphology and metabolic activity markers in postmortem biopsies of 61 critically ill patients and 20 matched control subjects. Adipose morphology was also studied in in vivo biopsies of 27 patients and in a rabbit model of critical illness (n = 22). MEASUREMENTS AND MAIN RESULTS Adipose tissue from critically ill patients revealed a higher number and a smaller size of adipocytes and increased preadipocyte marker levels as compared with control subjects. Virtually all adipose biopsies from critically ill patients displayed positive macrophage staining. The animal model demonstrated similar changes. Glucose transporter levels and glucose content were increased. Glucokinase expression was up-regulated, whereas glycogen and glucose-6-phosphate levels were low. Acetyl CoA carboxylase protein and fatty acid synthase activity were increased. Hormone-sensitive lipase activity was not altered, whereas lipoprotein lipase activity was increased. A substantially increased AMP-activated protein kinase activity may play a crucial role. CONCLUSIONS Postmortem adipose tissue biopsies from critically ill patients displayed a larger number of small adipocytes in response to critical illness, revealing an increased ability to take up circulating glucose and triglycerides. Similar morphologic changes were present in vivo. Such changes may render adipose tissue biologically active as a functional storage depot for potentially toxic metabolites, thereby contributing to survival.

Impact of Early Parenteral Nutrition on Metabolism and Kidney Injury

A poor nutritional state and a caloric deficit associate with increased morbidity and mortality, but a recent multicenter, randomized controlled trial found that early parenteral nutrition to supplement insufficient enteral nutrition increases morbidity in the intensive care unit, including prolonging the duration of renal replacement therapy, compared with withholding parenteral nutrition for 1 week. Whether early versus late parenteral nutrition impacts the incidence and recovery of AKI is unknown. Here, we report a prespecified analysis from this trial, the Early Parenteral Nutrition Completing Enteral Nutrition in Adult Critically Ill Patients (EPaNIC) study. The timing of parenteral nutrition did not affect the incidence of AKI, but early initiation seemed to slow renal recovery in patients with stage 2 AKI. Early parenteral nutrition did not affect the time course of creatinine and creatinine clearance but did increase plasma urea, urea/creatinine ratio, and nitrogen excretion beginning on the first day of amino acid infusion. In the group that received late parenteral nutrition, infusing amino acids after the first week also increased ureagenesis. During the first 2 weeks, ureagenesis resulted in net waste of 63% of the extra nitrogen intake from early parenteral nutrition. In conclusion, early parenteral nutrition does not seem to impact AKI incidence, although it may delay recovery in patients with stage 2 AKI. Substantial catabolism of the extra amino acids, which leads to higher levels of plasma urea, might explain the prolonged duration of renal replacement therapy observed with early parenteral nutrition.

Impact of hyperglycemia on neuropathological alterations during critical illness

CONTEXT Although preventing excessive hyperglycemia during critical illness may provide clinical neuroprotection, it remains debated whether normoglycemia is without risk for the brain. OBJECTIVE To address this question, we compared the neuropathological alterations in microglia, astrocytes, and neurons, with uncontrolled hyperglycemia, moderately controlled hyperglycemia, and normoglycemia during human critical illness. We further investigated the time course in an animal model. DESIGN AND SETTING We analyzed brain specimens from patients who died in the intensive care unit and from critically ill rabbits randomized to hyper- or normoglycemia. PATIENTS/OTHER PARTICIPANTS: We compared 10 critically ill patients randomized to normoglycemia (104 ±9 mg/dl) or moderate hyperglycemia (173 ±32 mg/dl), and five patients with uncontrolled hyperglycemia (254 ±83 mg/dl) with 16 controls (out of hospital sudden deaths). Critically ill rabbits were randomized to hyperglycemia (315 ±32 mg/dl) or normoglycemia (85 ±13 mg/dl) and studied after 3 and 7 d. INTERVENTIONS Insulin was infused to control blood glucose. MAIN OUTCOME MEASURES AND RESULTS Patients with uncontrolled hyperglycemia showed 3.7-6-fold increased microglial activation, 54-95% reduced number and activation of astrocytes, more than 9-fold increased neuronal and glial apoptosis, and a 1.5-2-fold increase in damaged neurons in hippocampus and frontal cortex (all P ≤ 0.05). Most of these abnormalities were attenuated with moderate hyperglycemia and virtually absent with normoglycemia. Frontal cortex of hyperglycemic rabbits that had been critically ill for 3 d only revealed microglial activation, followed after 7 d by astrocyte and neuronal abnormalities similar to those observed in patients, all prevented by normoglycemia. CONCLUSIONS Preventing hyperglycemia with insulin during critical illness reduced neuropathological abnormalities, with microglial activation being the earliest preventable event. Whether these pathological findings associate with neurological outcome remains unknown.

Mitochondrial fusion, fission, and biogenesis in prolonged critically ill patients.

CONTEXT Critical illness induces swelling, enlargement, and dysfunction of mitochondria, which in liver, but not in muscle, is aggravated by excessive hyperglycemia. We previously demonstrated impaired autophagic clearance of damaged mitochondria in fed prolonged critically ill patients. Impaired fusion/fission-mediated repair and/or renewal through biogenesis may further accentuate mitochondrial abnormalities. OBJECTIVE We studied mitochondrial fusion/fission and biogenesis and how these are affected by preventing hyperglycemia with insulin during critical illness. DESIGN AND SETTING Patients admitted to a university hospital surgical/medical intensive-care unit participated in a randomized study. PATIENTS We studied adult prolonged critically ill patients vs. controls. INTERVENTION Tolerating hyperglycemia up to 215 mg/dl was compared with intensive insulin therapy targeting normoglycemia (80-110 mg/dl). MAIN OUTCOME MEASURES In liver and skeletal muscle, we quantified levels of several proteins involved in mitochondrial fusion/fission and biogenesis. RESULTS Key players in mitochondrial fusion/fission and biogenesis were up-regulated in postmortem liver (1.4- to 3.7-fold) and rectus abdominis (1.2- to 4.2-fold) but not in in vivo or postmortem vastus lateralis biopsies of critically ill patients. Maintaining normoglycemia with insulin attenuated the hepatic response in the mitochondrial fusion/fission process but did not affect the markers of mitochondrial biogenesis in liver or muscle. CONCLUSIONS Our observations suggest tissue-dependent attempts of compensatory activation of mitochondrial repair mechanisms during critical illness. Considering the previously observed persistent mitochondrial damage, this activation may be insufficient and contribute to mitochondrial dysfunction. Suppressed activation of fusion/fission when excessive hyperglycemia is prevented with insulin may reflect reduced need for diluting (less) damage during normoglycemia or, alternatively, a suppressive effect of insulin on repair.