Sepsis increases muscle proteolysis in severely burned adults, but does not impact whole-body lipid or carbohydrate kinetics.

Abstract

Sepsis is a common and often fatal consequence of severe burn injury, but its exact effects on whole body and muscle metabolism in the burn patient is unclear. To address this, 13 septic and 11 non-septic patients (age: 36.9 ±13.0 years) with burns encompassing >30% of their total body surface area underwent muscle protein kinetic studies under post-absorptive conditions using bolus injections of ring-¹³C₆ and ¹⁵N phenylalanine isotopes. In parallel, whole-body lipid and carbohydrate kinetics were assessed using constant-infusions of [U-¹³C₆]palmitate, [6,6-²H₂]glucose and [²H₅]glycerol, and during a 2 hour hyperinsulinemic euglycemic clamp. Muscle mRNA levels of genes implicated in the development of muscle cachexia were assessed by qPCR. Fractional breakdown rates of mixed-muscle proteins were found to be 2.4-fold greater in septic versus non-septic patients (P<0.05). No discernable differences in fractional synthetic rate of mixed muscle proteins or rate of appearance of plasma free fatty acids, glycerol, or glucose could be observed between patient groups, although the latter was significantly associated with burn size (P<0.05). Hyperinsulinemia stimulated whole-body glucose uptake and suppressed endogenous glucose production and whole-body lipolytic rate to equivalent degrees in both groups. Muscle mRNA levels of genes spanning autophagy, lysosomal, and ubiquitin-proteasome mediated proteolysis were not enhanced in septic versus non-septic patients. Our results demonstrate that accelerated muscle proteolysis appears to be the principal metabolic consequence of sepsis in severe burn patients and could be a contributing factor to the accelerated loss of muscle mass in these individuals. The exact mechanistic basis for these changes remain unclear.

INTRODUCTION

Severe burn trauma results in a profound and prolonged hypermetabolic response of a magnitude unseen following any other trauma event or disease (1). While tremendous strides have been made in reducing the mortality seen following burns, increased energy expenditure and the ensuing catabolism of fat and protein stores remain a clinical challenge (2). Consequently, the loss of skeletal muscle mass is common in these patients (1) and is associated with poor wound healing, functional impairment, and delayed integration of the patient to society (3).

It has previously been argued that the strongest predictor of muscle catabolism following burn injury is the presence of sepsis, more so than burn size, time until excision of the burn post-injury, and resting energy expenditure (4). Furthermore, Hart and colleagues have shown net protein balance to be negative in the muscle of fed adult burn patients, which is further exacerbated with sepsis (4). However, from the data reported, it remains unknown if the accelerated protein catabolism seen in septic burn patients is a consequence of accelerated muscle proteolysis, an impaired capacity to synthesize new muscle proteins, or a combination of the two.

Non-septic burn patients are known to lose muscle protein content as a consequence of muscle proteins being degraded at a rate that exceeds the synthesis of new muscle proteins, despite the latter being elevated due to increased amino acid recycling within the muscle cell (5). In contrast, animal models of sepsis are characterized by a rapid and profound suppression of muscle protein synthesis rates, due in part to an impairment in cell signaling events associated with the induction of translation initiation (6). Thus, we hypothesize that sepsis-induced blunting of muscle protein synthesis in burned patients is in part responsible for the enhanced protein loss observed in the muscle of these critically-ill patients.

Sepsis and burn injury are independently associated with the induction of adipocyte lipolysis and whole-body insulin resistance (7, 8), often leading to the loss of glucose homeostasis (9). Of concern, the presence of hyperglycemia in the burn patient is connected to the rejection of skin grafts and frequency of wound infection (9, 10). Whether the presence of sepsis in burn patients further compounds the issues of lipolysis and whole-body insulin resistance is currently unclear. Thus, the aims of the current study were two-fold. Firstly, to establish the basis for the enhanced muscle protein catabolism observed with sepsis in severe burn patients and secondly, to determine the wider impact of sepsis on metabolism following burn injury.

METHODS

Patients

Twenty-four adult patients enrolled on a Phase-II clinical trial aiming to assess the safety and efficacy of propranolol following major burn injury (ClinicalTrial.gov ID: NCT0190810), were recruited from the Blocker Burn Unit at The University of Texas Medical Brach (Galveston, TX). The inclusion criteria were as follows: 18 years of age or older, burns covering a minimum of 30% of the total body surface area (TBSA), and admitted to the burn unit within 72 hours of injury. Individuals were excluded from participating if they had a diagnosis of cancer within the last 5 years, a condition prior to injury requiring glucocorticoid treatment, or a history of catabolic disease (e.g. HIV or AIDS). The study was approved by the Institutional Review Board at the University of Texas Medical Branch and informed consent was obtained from patients.

All patients received our institution’s standard of care for burn injuries, including fluid resuscitation and wound excision within two days of arrival. During their stay, nutrition in the form of Vivonex total enteral nutrition (82% carbohydrate, 15% protein and 3% fat by energy; Nestle Healthcare Nutrition Inc.) was provided via continuous nasoduodenal tube administration with patients receiving 1500 kcal.m^-2 total body surface area + 1500 kcal.m^-2 total body surface area burned per day, or at a rate of 1.4–1.6 time resting energy expenditure which was determined weekly by indirect calorimetry. A subset of patients (n = 13), also received the non-selective β-blocker propranolol throughout their hospitalization (0.5 mg.kg^-1 every 6 h, titrated to reduce heart rate by 20% of the patient’s admission value), a requirement of the parent trial from which patients were recruited. In the current trial, propranolol administration was approximately equivalent between septic and non-septic patients, with 7 non-septic patients and 6 septic patients treated with propranolol.

At the time of enrolment, patients were assessed for clinical signs of sepsis using the American Burn Association published criteria commonly employed by burn clinicians (11). The criteria required the positive identification of the source of infection and for patients to display three or more of the following symptoms: fever (> 39°C), hypothermia (< 36.5°C), progressive tachycardia (> 110 bpm), progressive tachypnea (not ventilated: > 25 breaths.min^-1 or ventilated: > 12 L.min^-1), thrombocytopenia (< 100,000 platelets.μl^-1 after ≥ 3 days following initial resuscitation), hyperglycemia (either: >200 mg.dL^-1, requiring 7 units insulin.hr^-1 or >25% increase in daily insulin requirements) or an inability to tolerate enteral feedings for more than 24 hours due to abdominal distension or uncontrollable diarrhea. Patients meeting the criteria for sepsis received aggressive antibiotic therapy to treat the underlying source of infection; confirmed or suspected gram positive infections were treated with vancomycin, gram-negative infections with imipenem-cilastatin and in the case of multi-drug resistant bacteria, colistin (a detailed list of the drugs patients were administered appears in Table 1). Of the patients recruited to the study, 13 (54%) met the stated criteria for sepsis and underwent detailed metabolic assessment within 3 days following classification. The remaining patients underwent the same battery of assessments, but failed to meet the criteria for sepsis for a minimum of 3 days following assessment and were considered free of sepsis at time of assessment.

Table 1:

Subject characteristics. No significant differences observed between groups.

	Non-septic (n = 11)	Septic (n = 13)
Age at burn (years)	37.5 ± 13.2	36.3 ± 13.3
Gender (M:F)	10:1	12:1
TBSA (%)	50 ± 18	63 ± 22
Full thickness burn (%)	37 ± 21	50 ± 26
Presence of inhalation injury	7 (64%)	9 (69%)
Length of stay (days)	47 ± 43	77 ± 44 (C)
Mortality	0 (0%)	3 (23%)
Received propranolol	Total: 7 (64%)	Total: 6 (46%)
Days post burn at time of study	14 ± 6	15 ± 7
Nutrition received in days preceding metabolic assessment (kcal.day-1)	3087 ±1228	3831 ±1119
Number of operations received in the course of treatment (A)	2.7 ±1.2	3.8 ±1.5
REE (% of predicted)	154 ±61	179 ±47
Average heart rate (bpm) (A)	98.6 ±17.3	118.7 ±16.4 *
Average body temperature (°C) (A)	38.2 ±0.6	38.9 ±0.7 *
Drugs administered prior to study (number of patients) (B)	Primaxin® (imipenem and cilastatin): 9; vancomycin: 8; insulin: 3; levofloxacin: 3; dobutamine: 2; fluconazole: 2; caspofungin: 1; ceftazidime: 1; levothyroxine: 1; metronidazole: 1; micafungin: 1; voriconazole: 1	Vancomycin: 10; insulin: 9; Primaxin ® (imipenem and cilastatin): 7; tobramycin: 7; levofloxacin: 5; colistin: 4; voriconazole: 4; epinephrine: 3; Zosyn ® (piperacillin and tazobactam): 2; acyclovir: 2; Bactrim ® (sulfamethoxazole and trimethoprim): 2; dobutamine: 2; polymyxin B: 2; digoxin: 1; filgrastim: 1; imipenem: 1; levothyroxine: 1; linezolid: 1; metformin: 1; verapamil: 1

Means ± SD

^(A): as determined at time of study

^(B): assessed over a three day period immediately preceding the study visit

^(C): does not include data from 3 patients that died during their hospitalization.

^*significantly different from non-septic patients (P < 0.05).

Metabolic assessment

Approximately 13 ±4.5 days (±SD) post burn, patients underwent metabolic assessment using previously published protocols involving isotopically-labelled tracers (12, 13) to delineate the effects of sepsis on whole-body and muscle metabolism, with slight modifications as described below (Figure 1). The protocol adopted was originally devised for the parent trial described above and from which patients were recruited, but nevertheless, represented an optimal strategy for ascertaining the impact of sepsis on muscle and whole-body substrate kinetics. In short, nasogastric feeding was stopped a minimum of four hours prior to metabolic assessment to allow initial measurements to be performed under post-absorptive conditions. On the morning of assessment, blood samples were collected from a previously implanted venous cannula to allow the determination of background enrichment of the isotopically-labelled tracers and circulating cytokine concentrations. Afterwards, primed-constant infusions of [U-¹³C₆]palmitate (NaH¹³CO₃ prime: 150 μmol.kg^-1; [U-¹³C₆]palmitate infusion: 0.08 μmol.kg^-1.min^-1), [6,6-²H₂]glucose (prime: 20 μmol.kg^-1; infusion: 0.44 μmol.kg^-1.min^-1) and [²H₅]glycerol (prime: 1.5 μmol.kg^-1; infusion: 0.12 μmol.kg^-1.min^-1) were initiated (t = 0 hrs) and continued for 4 hours. At the outset of the infusion protocol, a 50 μmol.kg^-1 bolus of [ring-¹³C₆]phenylalanine was administered intravenously, followed 30 minutes later by a 50 μmol.kg^-1 bolus of [¹⁵N]phenylalanine to allow the assessment of muscle protein turnover.

Figure 1: Schematic of infusion protocol.

Muscle protein turnover assessed in the fasted state using two separate bolus injections of alternative stable-isotopes of phenylalanine. Whole-body lipid and carbohydrate kinetics were assessed over a 4 hour period, with a hyperinsulinemic clamp performed in the final two hours. REE: Resting energy expenditure.

Two hours following the commencement of stable isotope administration, a hyperinsulinemic euglycaemic clamp was initiated to assess the impact of sepsis on peripheral-glucose uptake, hepatic glucose production, and lipolysis under both basal and insulin-stimulated conditions. To accomplish this, a primed (1.5 mU.kg^-1) constant infusion (1.5 mU.kg^-1.min^-1) of insulin was administered which results in a steady-state serum insulin concentration of ~50 mIU.ml^-1 with 20% dextrose administered intravenously as required to maintain blood glucose concentrations at pre-clamp concentrations. At t = 4 hours, insulin and tracer infusions were stopped and the patient reverted to standard care.

Throughout the four-hour period of assessment, blood samples were collected at frequent intervals, centrifuged at 3000 rpm for 20 minutes, and the resulting plasma stored at -20°C for subsequent analysis of tracer enrichments. A single blood sample collected under basal conditions, and an additional sample obtained during the insulin clamp, were collected from each participant and submitted to the University of Texas Medical Branch Pathology Department for the determination of serum insulin concentrations. Two muscle biopsy specimens were obtained under local anesthesia from the vastus lateralis muscle at 10 min and 1 hour from the start of the infusion protocol using the Bergstrom percutaneous needle approach. On collection, muscle samples were immediately washed in ice-cold saline to remove visible blood, frozen in liquid nitrogen, and stored at -80°C for subsequent processing.

Resting energy expenditure was assessed using an indirect calorimeter (Sensor-Medics 2900 metabolic cart, Yorba Linda, Ca) for a 20 minute period during the initial phase of the infusion protocol when patients were in a fasted state. All measurements were made with patients in a room temperature of ~30 °C, standard environmental conditions for our adult burn ICU. In parallel, the patients’ resting energy expenditure was calculated using the Harris-Benedict equation (14) and the measured resting energy expenditure expressed as a percentage of the calculated expenditure.

Processing of plasma samples

Plasma triglyceride concentrations and the percent contribution of palmitate to total plasma free fatty acids (FFA) were quantified by gas chromatography, using previously established methods (15). In separate aliquots, the enrichment (tracer-to-tracee ratio) of glucose, glycerol, phenylalanine and palmitate were determined by gas chromatography mass spectrometry (GC-MS), as described previously (15, 16). Prior to analysis, samples were treated to produce tert-butyldimethylsilyl, tris-trimethylsilyl, penta-acetate and methyl ester derivatives of phenylalanine, glycerol, glucose and FFA, respectively. Derivatized samples were analyzed on an Agilent 6890 GC-MS as described previously (12, 15, 16).

Processing of muscle samples

Muscle samples were processed for the determination of phenylalanine concentration and tracer incorporation as described previously (12). In short, 3 μmol/l of [ring-¹³C₆, ¹⁵N]phenylalanine was added to 30 mg of muscle tissue as an internal control before samples were homogenized with 10% perchloric acid and centrifuged at 3000 rpm for 10 min. The resultant supernatant was used for the determination of free intracellular amino acids after being processed in the same manner as the plasma samples described above. Muscle pellets were washed with saline and alcohol before being dried overnight at 50°C, followed by hydrolyzing of the dry pellet by incubating in 6N HCl overnight at 110°C. Afterwards, the hydrolyzed proteins were treated in the same manner as the supernatant samples. The derivatized blood and muscle samples were analyzed by GC-MS for the determination of isotopic enrichments and free intracellular muscle phenylalanine concentrations.

Calculations

Mixed-muscle protein fractional breakdown rate (FBR) was determined using the below formula (Equation 1), an approach which relies on repeated assessment of arterial (EA) and muscle intracellular (EM) enrichment as opposed to attaining an isotopic plateau (17). Given that the two phenylalanine tracers share the same metabolic fate, and thus decay pattern (17), the decay curve of L-[ring-¹³C₆]phenylalanine can be determined by assessing enrichment of the muscle intracellular free pool from the biopsies obtained at 10 and 60 minutes. Following administration of L-[ring-¹³C₆]phenylalanine at t=0, the first biopsy allowed the assessment of phenylalanine tracer decay over the initial 10 minute period (t₁). The second biopsy, in conjunction with the bolus administration of [¹⁵N]phenylalanine at t = 30 minutes, allowed the determination of tracer decay at 30 (t₂) and 50 (t₃) minutes by assessing enrichment of the muscle intracellular free pool for [¹⁵N]phenylalanine and L-[ring-¹³C₆]phenylalanine, respectively. Utilizing this information, the precursor was taken as the area under the decay curve for intracellular phenylalanine enrichment (12). Thus, the formula below provides FBR over the first hour of the stable-isotope infusion protocol, where QM/T is the ratio of intracellular free tracee content to protein-bound tracee in the muscle.

$$FBR= \frac{\left[EM\left(t2\right)-EM\left(t1\right)\right]{{\displaystyle \int }}_{t2}^{t3}\left[EA\left(t\right)-EM\left(t\right)\right]\Delta t-\left[EM\left(t3\right)-EM\left(t2\right)\right]{{\displaystyle \int }}_{t1}^{t2}\left[EA\left(t\right)-EM\left(t\right)\right]\Delta t}{{{\displaystyle \int }}_{t2}^{t3}EM\left(t\right)\Delta t{{\displaystyle \int }}_{t1}^{t2}EA\left(t\right)\Delta t- {{\displaystyle \int }}_{t1}^{t2}EM\left(t\right)\Delta t{{\displaystyle \int }}_{t2}^{t3}EA\left(t\right)\Delta t}.\left(\frac{QM}{T}\right)$$ (1)

Muscle protein fractional synthesis rate (FSR) was calculated over the same period by assessing the rate of incorporation of L-[ring¹³C₆]-phenylalanine from the intracellular pool into the protein-bound pool, as described previously (12). FSR was calculated utilizing the below equation (Equation 2), which determines the increase in enrichment of the protein bound pool (EB) over time (t), expressed relative to the average enrichment in the free pool (EF).

$$FSR= \frac{EB\left(t\right)-EB\left(0\right)}{{{\displaystyle \int }}_{t0}^{t1}EF\left(t\right)\Delta t}$$ (2)

Assessment of whole-body lipid and glucose kinetics was performed under steady state conditions, with the rate of appearance of glycerol calculated using the formula:

$$Ra= \frac{I}{TTR}$$ (3)

Where Ra represents the rate of appearance of the tracee in the circulation, I represents the rate of ²H₅-glycerol infusion, and TTR represents the plasma tracer-to-tracee ratio determined by GCMS as described above. The rate of appearance of FFAs into the systemic circulation was calculated using the same approach after adjusting for the percentage contribution that palmitate makes to the total plasma FFA pool. The glucose rate of appearance in the plasma was calculated using the glucose tracer infusion rate divided by plateau enrichment (TTR), and Ra of glucose calculated to determine endogenous glucose production.

Serum cytokine concentrations

Serum cytokine concentrations of patients were assessed from serum samples collected during the stable-isotope infusion trial using a human cytokine panel (Bio-Plex) and analyzed on a Bio-Plex Suspension Array System (Bio-Rad, Hercules, CA). Concentrations of the following 17 inflammatory markers were determined: interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (G-MCSF), interferon-γ (IFNγ), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1 β), and tumor necrosis factor-α (TNFα). The assay was performed in accordance with the manufacturer’s instructions.

Quantification of mRNA levels of atrophy-associated genes

Total RNA was extracted from muscle biopsies taken at t = 10 mins using the Pure Link RNA isolation kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Because of the limited size of the muscle samples available, only a subset of patients could be examined (5 per group, of which 3 out of 5 non-septic, and 2 out of 5 septic patients did receive propranolol). Quantification and confirmation of the quality of the extracted RNA was determined spectrophotometrically, after which 100 ng of total RNA was used as the template for cDNA synthesis using a high capacity RNA to cDNA synthesis kit from Thermo Scientific (Waltham, MA).

From the synthesized cDNA, the mRNA levels of 18 genes associated with protein turnover were assessed using pre-designed commercial Taqman® primer and probe sets (Applied Biosystems, USA; see Table, Supplementary Digital Content 1, which lists details of the primer/probe targets). Samples were analyzed in duplicate, with each reaction including the parallel determination of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels using probes incorporating an alternative fluorescent chemistry to that of the target gene. The cycle threshold values obtained for GAPDH were not found to differ between septic and non-septic patients, confirming its appropriateness as a housekeeping gene. Differences in mRNA levels between patient groups were calculated using the ΔΔt approach, with data normalized to the non-septic group.

Statistics

Differences in demographics and muscle gene expression data between septic and non-septic patient groups were assessed by use of an unpaired t-test, with the exception of categorical data which was analyzed using the chi-square test. Multiple regression analysis was employed to determine the impact of burn size and sepsis on serum cytokine concentrations and protein, glucose, and lipid kinetics, with non-normally distributed data log-transformed prior to analysis. Other potential confounders including number of operations, activity levels of patients, or drugs received, were not controlled for. Thus, the collective impact of sepsis was instead considered, incorporating the clinical care received by the patient which could differ between individuals. When a main effect of the insulin clamp was observed (lipid and glucose kinetics), a Mann-Whitney test was performed on the non-logged data to locate differences. Except where stated otherwise, tabular data within the report is presented as means ± standard deviation, while graphical data is presented as means ± standard error of mean. Multiple regression analysis was performed in the statistical package R (v3.3.2; www.r-project.org), all other tests were performed in Prism (v7.03; GraphPad Software, USA). Significance was accepted when P < 0.05.

RESULTS

Patient demographics

Overall, 24 subjects were recruited on to the trial, 13 of which met the study’s criteria for sepsis. Positives cultures were obtained from clinical samples collected from soft tissue (n = 7), blood (n = 7), respiratory tract (n = 1) and catheter sites (n = 5) of septic patients. Of these, 3 patients displayed positive cultures in both soft tissue and blood specimens, 1 patient with soft tissue and catheter tip positive cultures, 1 patient with soft tissue and respiratory tract positive cultures, and 1 patient with positive cultures from blood, catheter tip, and soft tissue samples. Across groups, patients were predominantly male (92%), 36.9 ± 13.0 (range: 21 to 68) years of age, and primarily injured by flame exposure (1 patient in the non-septic group had suffered an electrical flame burn). Groups were evenly matched in relation to age, presence of inhalation injury, prior number of operations, nutrition administered preceding assessment, numbers that received propranolol, and the time at which the metabolic assessment was conducted post-injury (Table 1).

Patients identified as septic tended to have larger burns (63 ± 22% TBSA) compared to their non-septic counterparts (50 ± 18% TBSA), but this was not statistically significant. Similarly, the recovery of septic patients appeared more protracted (septic: 77 ± 44 vs. non-septic: 47 ± 43 days), but this also failed to reach significance (p = 0.11). At time of metabolic assessment, the average heart rate and body temperature of septic patients was significantly greater than that of their non-septic counterparts (Table 1; P < 0.05). Following the trial, 3 septic patients (23%) succumbed to their injuries, dying 10, 36, and 285 days post-injury. In two cases, multiple organ failure was recorded as the basis for death, while pneumonia was the cause of death in the third patient. No deaths were observed amongst the non-septic patients.

Circulating cytokine concentrations

Given the known ability of cytokines to profoundly impact muscle and whole-body metabolism, a 17-panel cytokine array was used to provide insight on the impact of sepsis on circulating cytokine concentrations in major burn patients. Eight of the cytokines included in the array were below the limits of detection for most participants across both patient groups and were not considered further (IL-5, GM-CSF, IL-2, IL-1B, IL-13, IL-4, IL-7 and IL-12p70). Similarly, a patient with a 99% TBSA burn and septic at the time of study, showed a strikingly different cytokine expression profile from that of the other participants (identified via Grubbs’ test; p < 0.01) and was removed as an outlier. Of the remaining 9 cytokines studied, circulating concentrations of G-CSF, IFNγ and MCP-1 were elevated by the presence of sepsis (Table 2). In contrast, concentrations of IL-6, IL-8, IL-10, IL-17, MIP-1B and TNFα appeared unaffected by sepsis in burn patients (P > 0.05). Burn size appeared to have no impact on the circulating concentration of the cytokines studied.

Table 2:

Serum cytokine concentrations of septic and non-septic burn patients

Cytokine	Serum concentrations (pg.ml-1)
	Non-septic	Septic
G-CSF	24.9 ± 55.6	100 ± 70.8 *
IFNγ	0.93 ± 1.2	3.0 ± 1.6 *
IL-6	14.2 ± 10.7	23.0 ± 10.5
IL-8	168 ± 229	175 ± 186
IL-10	3.4 ± 2.7	4.6 ± 3.0
IL-17	2.5 ± 2.4	3.4 ± 3.8
MCP-1	54.0 ± 23.9	126 ± 39.6 **
MIP-1β	16.8 ± 13.3	9.3 ± 4.1
TNFα	26.5 ± 14.0	17.0 ± 4.2

Values represent mean ± SD.* denotes significantly different from non-septic patients

^*P < 0.05

^**P < 0.01. Degree of TBSA burned had no significant effect on the concentrations of any of the listed cytokines. N = 7 – 9 per group.

Muscle Protein turnover

Net muscle protein balance under post-absorptive conditions was significantly reduced by the presence of sepsis (septic: -0.245 ± 0.082% vs. non-septic: -0.078 ± 0.038%; P < 0.05), the result of an enhancement of mixed muscle FBR in septic patients compared to their non-septic counterparts (0.329 ± 0.081% and 0.136 ± 0.035%, respectively; Figure 2). The impact of sepsis following burns on muscle FBR remained significant (P < 0.05), and a trend towards decreased net muscle balance still seen (P = 0.058), when propranolol administration was included as a variable in the multiple regression model adopted. Thus, despite propranolol’s known effects on the hypermetabolic response to burns (18), differences in propranolol administration between groups was not responsible for the observed effects of sepsis on protein turnover. Similarly, when incorporating body temperature as a variable so as to account for differences in fever extent between patients, a trend towards increased muscle FBR (P = 0.053) and decreased net protein balance (P = 0.060) remained. Contrary to expectations, no difference in the FSR of mixed muscle proteins could be discerned between groups (septic: 0.084 ± 0.013 vs. non-septic: 0.058 ± 0.011%; non-significant). Likewise, burn size had no detectable impact on any parameter of muscle protein turnover examined.

Figure 2: Effect of sepsis during burn injury on mixed muscle protein turnover and atrophy-gene expression.

Fractional synthetic rate (FSR; A), fractional breakdown rate (FBR; B) and net protein balance (C) in burn patients with and without sepsis. In a subset of patients (n = 5 per group) muscle mRNA levels of key atrophy-associated genes were examined by qPCR (D). mRNA levels reported as the average expression in septic patients expressed relative to non-septic burn patients. All data presented as means ± SEM. * denotes significantly different from non-septic burn patients (P < 0.05). N = 10 – 11 per group.

To try and understand the molecular basis for the enhanced proteolysis seen in the muscle of septic burn patients, the expression of key genes implicated in protein degradation and associated signaling processes were examined. Due to the limited biopsy tissue available, qPCR analysis could only be performed in a subset of patients (n = 5 per group). Crucially, muscle protein breakdown and net protein balance were found to be significantly perturbed in the smaller cohort when compared to their non-septic counterparts (P < 0.05), confirming that they were a suitable group for detailed analysis and reflective of the larger population. Despite many of the included genes being widely implicated in the loss of muscle mass during catabolic disease, including sepsis (19), no robust changes indicative of enhanced proteolysis could be observed (Figure 2D). In contrast, mRNA levels of MAFbx (a.k.a. atrogin-1), a muscle-specific ubiquitin ligase whose expression appears enhanced in multiple conditions characterized by cachexia (20) and is associated with the targeting of muscle proteins for ubiquitin-proteasome mediated proteolysis, was decreased in septic patients compared to non-septic burn patients (P < 0.05).

REE and whole-body lipid and carbohydrate kinetics

Indirect calorimetry revealed the REE of patients to be significantly greater than would be predicted using established equations for healthy populations (Table 1). However, the elevated REE of patients was not impacted by the TBSA burned or the presence of sepsis (septic: 179 ± 47% vs. non-septic: 154 ± 61% of predicted REE; non-significant). In keeping with sepsis failing to impact on energy demands, the contribution of energy substrates from hepatic glucose production and whole-body lipolysis, determined by the rate of appearance (Ra) of circulating glycerol, glucose, and FFA, were equivalent between groups under basal conditions (Table 3). Notably, the Ra of glycerol and FFA were approximately 100% and 75% greater than previously reported in fasted healthy adults (21), consistent with the enhanced lipolysis seen in major burn patients (8).

Table 3:

Impact of sepsis on lipid and carbohydrate whole-body kinetics in burn patients.

	Non-septic		Septic
	Basal	Insulin clamp	Basal	Insulin clamp
FFA Ra (μmol.kg-1.min-1)	8.9 ±2.5	3.0 ±1.6 **	10.0 ±4.4	3.1 ±1.7 **
Glycerol Ra (μmol.kg-1.min-1)	7.5 ±10.3	4.2 ±4.6 ***	4.1 ±1.3	2.4 ±0.7 **
Endogenous glucose Ra (μmol.kg-1.min-1) †	18.1 ±3.9	2.0 ±6.2 **	25.4 ±10.4	7.0 ±12.7 **
Average glucose infusion rate (mg.kg-1.min-1)	-	4.5 ±1.5	-	4.2 ±3.6

Data presented as mean ± SD. † significant effect associated with TBSA burned

^†< 0.05

^*significantly different compared to basal state

^**< 0.01

^***< 0.001. No significant effect of sepsis detected for any of the parameters examined. FFA: Free fatty acids, Ra: Rate of appearance. N = 10 – 13 per group.

The intravenous administration of insulin was successful at elevating serum insulin concentrations in both patient groups to comparable degrees (non-septic: 51.9 ±18.2 mIU.ml^-1 vs. septic: 51.3 ±14.4 mIU.ml^-1). Under these conditions, the Ra of glycerol and FFA were significantly reduced irrespective of the presence of sepsis, indicative of their depressed endogenous production. Under hyperinsulinemic clamp conditions, the average rate of dextrose administration to maintain blood glucose concentrations, an index of peripheral glucose uptake, was equivalents between groups (Table 3). Similarly, endogenous glucose production was reduced by insulin administration and was similar between septic and non-septic patients, but positively associated with the TBSA burned (Table 3; P<0.001).

DISCUSSION

Sepsis remains the biggest clinical threat to patients with large burns and has been reported to account for almost 50% of patient deaths (22). Notably, previous research has identified sepsis as the single greatest contributor to negative protein balance in burn patients (4), further exacerbating the rapid and debilitating loss of muscle seen following burn injury. However, the exact basis for the accelerated muscle catabolism seen in septic burn patients and the wider consequences of sepsis on the hypermetabolic burn patient remains largely unknown. Our results demonstrate that accelerated muscle proteolysis is the principal metabolic challenge faced by the septic burn patient in addition to the original hypermetabolic response to thermal injury. The exact mechanistic basis for this response remains elusive but does not appear to involve the transcriptional upregulation of traditional atrophy-inducing genes.

As early as the 1970s it was observed that septic burn patients displayed altered protein kinetics compared to their non-septic counterparts. In one of the earliest reports on the topic, it was demonstrated that septic burn patients display greater fasting serum phenylalanine concentrations compared to non-septic burn patients, but comparable tyrosine appearance in serum following the administration of an oral phenylalanine bolus (23). From these findings, it was postulated that the increased phenylalanine serum concentrations of septic patients was likely the consequence of enhanced muscle proteolysis as opposed to reduced hepatic conversion of phenylalanine to tyrosine. More recently, authors of a report describing increased 3-methylhistidine urinary excretion in septic versus non-septic burn patients have gone further, concluding that muscle proteolysis is enhanced with sepsis, despite the known caveats of relying on urinary 3-methylhistidine as a marker of myofibrillar protein breakdown (24). Thus, our findings are the first to utilize contemporary isotope approaches to confirm that sepsis does indeed increase muscle protein breakdown rates in major burn patients during the hypermetabolic phase of their recovery.

Contrary to expectations, muscle protein synthesis was unaffected by the presence of sepsis compared to burn injury alone. In pediatric populations, it has been observed that muscle protein synthesis rates are lower with very large burns (>80% TBSA burned) compared to smaller sized burns (40–60% TBSA burned) and notably, septic complications are more commonly observed with larger burns (25). While the incidence of sepsis was not accounted for in the aforementioned study, it was postulated that the dual insult of sepsis and multiple organ failure could blunt ATP consuming processes, including protein synthesis. In adults at least, our results demonstrate that sepsis has no impact on muscle protein synthesis of burn patients in the fasted state. Whether the same is true in the fed state, a clinically pertinent question given the emphasis placed on providing nutrition to meet enhanced energy demands, remains unresolved and an important focus for future work. Likewise, given our findings are based on acute measures of muscle protein turnover, what chronic metabolic changes occur in response to sepsis remain unclear and requires further investigation.

Despite observing a robust increase in muscle proteolysis rates in septic burn patients, we were unable to identify the underlying mechanisms underpinning this response. Previous studies have described a coordinated program of transcriptional events occur in muscle in response to atrophying conditions, including sepsis (20). Indeed, it has been suggested that the ubiquitin proteasome system underpins the bulk of the proteolysis that occurs in most cachectic states (see (26)), with the transcriptional upregulation of a small number of muscle specific ubiquitin ligases a defining feature (20, 27). We failed to observe such transcriptional changes in our septic burn patients. In contrast, Chai and colleagues have described increased muscle mRNA levels of ubiquitin, the ubiquitin conjugating enzyme E2–14K, and the C2 subunit of the proteasome in response to burn sepsis in both patient (28) and experimental animal models of thermal injury (29). While the reasons for the discrepancy between findings is unclear, it is noteworthy that they observed a profound (> 6-fold) increase in plasma concentrations of the cachectic-inducing cytokine, TNFα, in their septic versus non-septic burn patients (28), a finding not observed in the current study. It is unclear if the authors controlled for burn size between groups, but given that we have previously shown burn size to be associated with serum TNFα concentrations (30), it may represent one possible explanation for the discord between findings.

While the components of the ubiquitin proteasome system studied in the current report differ from that described by Chai and colleagues (28, 29), reflecting the shift in emphasis in recent years to the role of select ubiquitin ligases in the induction of cachexia, we do not believe our focus on different ubiquitin proteasome system targets is responsible for the discrepancy in findings. We have previously shown in an animal model of endotoxemia that muscle mRNA levels of the ubiquitin ligases MAFbx and MuRF1 precede increases in the mRNA and protein abundance of proteasome subunits, and remain elevated during declines in muscle protein content (31). Furthermore, multiple investigators have reported MAFbx and MuRF1 to be transcriptionally upregulated during ubiquitin proteasome-mediated muscle atrophy (20, 27, 31), and knockout of either ligase blunts muscle loss under atrophying conditions (27), reinforcing the contribution of the ligases to muscle catabolism.

It is clear from our findings that further work is warranted to unravel the mechanistic basis for the enhanced proteolysis seen in septic burn patients. We speculate that components of the proteolytic machinery, predominately the ubiquitin proteasome system, are already enhanced because of the original burn injury, and that further enhancement of proteolytic rates are not underpinned by transcriptional events. However, given the small number of patients in which we were able to study muscle gene expression changes, the absence of any robust change with sepsis post-burn should be viewed with caution until larger studies can be performed. Nevertheless, what signals the acceleration of proteolysis represents an important question to resolve in the quest to develop effective therapeutic interventions to impede the loss of muscle and aid recovery in these patients.

Whilst sepsis had no additive impact on circulating concentrations of TNFα or IL-6, two cytokines often implicated in muscle cachexia, septic burn patients did display higher serum concentrations of G-CSF, MCP-1 and IFNγ, the latter of which could be of importance. Previous studies utilizing IFNγ knockout animals subjected to burn injury, have reported ex vivo muscle protein breakdown rates to be suppressed compared to wild type animals (32). Viewed in conjunction with the findings reported in the current study, IFNγ could be a potential mediator for the additive effect of sepsis on burn-induced muscle proteolysis and warrants further study. Moreover, given that transgenic knockout of the IFNγ receptor in mice prevents the hypermetabolic response to lipopolysacharride administration (33), it is permissible that IFNγ may feature as a regulator of both muscle proteolysis and hypermetabolism and represent an attractive therapeutic target. In contrast, G-CSF and MCP-1 have been linked with beneficial effects on muscle recovery in response to injury (34, 35) and are unlikely to be major contributors to the protein catabolism that occurs in septic burn patients.

In contrast to muscle protein kinetics, the presence of sepsis appeared to have little additional effect on whole-body lipolysis and hepatic glucose production. It is widely acknowledged that both major burns and sepsis are each individually associated with the increased hydrolysis of adipocyte-derived triglycerides, releasing FFAs and glycerol into the circulation triggered by a surge in catecholamine release (7, 8). Our results demonstrate that under basal conditions, sepsis has no additive effect on lipolytic rate or hepatic glucose production beyond that caused by burn injury alone, although the latter did appear related to the size of burn injury. Similarly, the ability of hyperinsulinemia to blunt endogenous glucose production and lipolytic rate appeared unimpaired by sepsis, which is in agreement with a previous report demonstrating that burn patients and septic patients display a similar capacity to suppress endogenous glucose production in response to insulin infusion (36). While it has been previously reported that insulin-stimulated glucose-uptake is impaired by sepsis following burn injury (36), a finding that contrasts sharply with our own observations, the limited number of patients studied in the original report (3 per group) in combination with the notable differences in weight status between recruited septic and non-septic patients, questions the reliability of this historical finding. Notably, the average glucose disposal observed in the current trial in response to a 1.5 mU.kg^-1.min^-1 insulin infusion was 4.3 ±3.0 mg.kg^-1.min^-1, a value that is in strict agreement with previous reports investigating the impact of similar insulin infusion rates on whole-body glucose disposal in burn patients (37) and substantially lower than seen in healthy non-burned volunteers (37). Thus, while burn injury appears to impair the ability of peripheral tissues to uptake glucose in response to insulin, it does not appear to be exacerbated by the presence of sepsis. It has been previously reported that poor glycaemic control resulting in hyperglycaemia is associated with increased frequency of wound infection (9), with the authors concluding that hyperglycemia has a detrimental effect on antimicrobial defense. This has led some to speculate that sepsis could exacerbate hyperglycemia in the burn patient (7), although our findings would appear to discredit this possibility.

Limitations

While providing unique insight into the metabolic consequences of sepsis in the severely burned patient, the study is not without its limitations. First and foremost, the study reports data from a small number of patients and therefore, its applicability to the wider burn population remains to be confirmed. However, despite this caveat, our ability to detect notable changes in muscle protein breakdown rates with sepsis reinforces the potential importance of this phenomenon. Restrictions on tissue availability prevented a detailed inspection of the mechanisms responsible and should be the focus of future efforts. Importantly, muscle protein kinetics were assessed in the post-absorptive state, which could be argued is not a true reflection of the hospitalized critical-ill patient. It is permissible that under fed conditions, differences in net protein balance between septic and non-septic burn patients become even more pronounced, with the anabolic response to feeding blunted in septic patients. Likewise, sepsis severity could influence the magnitude of response observed and should be a focus of future, larger trials.

Conclusions

Our results demonstrate that the additive effect of sepsis on whole-body metabolism following major burn injury appears modest, but does elicit a pronounced increase in muscle proteolysis. The latter observation explains the enhanced decline in muscle net protein balance reported in septic versus non-septic burn patients. Further studies are required to understand the exact mechanism responsible for this effect, although the role of increased concentrations of the pro-inflammatory cytokine IFN-γ would appear a judicious starting point.

List of abbreviations

FBR

Fractional breakdown rate

FFA

Free fatty acids

FSR

Fractional synthesis rate

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

G-CSF

Granulocyte colony-stimulating factor

G-MCSF

Granulocyte-macrophage colony stimulating factor

GC-MS

Gas chromatography mass spectrometry

IFNγ

Interferon-gamma

IL

Interleukin

MCP-1

Monocyte chemoattractant protein-1

MIP-1β

Macrophage inflammatory protein-1 beta

Ra

Rate of appearance

REE

Resting energy expenditure

TBSA

Total body surface area

TNFα

Tumor necrosis factor-alpha