Septic shock is a life threatening condition that can develop subsequent to infection. It is a subset of sepsis with underlying circulatory and cellular/metabolic abnormalities associated with higher mortality rates (Angus et al., 2001; Singer et al., 2016). Septic shock causes progressive failure of vital homeostatic mechanisms culminating in immunosuppression, coagulopathy and microvascular dysfunction, which can lead to refractory hypotension, organ failure and death.

Early supportive therapy with fluid resuscitation and vasopressors to restore hemodynamics and reduce tissue hypoperfusion is decisive for the patient’s outcome and has figured in treatment guidelines for decades (Dellinger et al., 2013). However, mortality rate for septic shock may reach 40% even in the era of early recognition and treatment with today’s poor prognosis, mainly related to multi-organ dysfunction syndrome (MODS) (Rhee et al., 2017). The multiple organ involvement, which can lead to death within a few days, suggests that this concomitant process is systemic in nature and initiated in parallel with inflammatory response.

Given the pathophysiological complexity of sepsis spectrum and progression in clinical settings, animal models become essential tools to improve patient care, and to understand key mechanisms that may remain masked from the heterogeneity of clinical practice.

Several swine models of sepsis and septic shock have been developed to mimic the human disease progression and clinical settings by utilizing standard clinical treatments such as fluid resuscitation, antibiotics, and mechanical ventilation (Kubiak et al., 2011; Li et al., 2013; Revie et al., 2013; Corrêa et al., 2014; Chalkias et al., 2015; Vassal et al., 2015; Zhao et al., 2015; Duburcq et al., 2017). However, critiques on the relevance of experimental data regarding the translation to clinical sepsis/septic shock have been raised in the scientific community (Piper et al., 1996; Deitch, 1998; Esmon, 2004; Dyson and Singer, 2009; Opal and Patrozou, 2009). Aware of such criticism, our main goal in this study was to investigate whether our previous metabolic features characterizing septic shock patients (Ferrario et al., 2016; Cambiaghi et al., 2017) would have been observed also in a swine model of septic shock.

Previously, we found that lipids species alteration played an important role in individual patient responses to infection, where reduction in circulating lysophosphatidylcholines species, less change in plasmalogens concentration in combination with larger increment of alanine were associated with non-responsiveness to therapy (Cambiaghi et al., 2017). We speculated that alanine modulation indicated a possible alteration in the glucose-alanine cycle in the liver, providing a different picture of early liver functionality disturbances in the non-responder patients. Therefore, we aim to verify whether such metabolic constellations occurring in septic shock patients appear also in the septic shock swine model as systemic markers of early disturbances in energy metabolism and hepatic homeostasis, where liver acts not only as inflammatory barrier, but as organ actively involved in energy pathways.

In order to allow a robust comparison of the metabolomics data, we applied the same mass spectrometry-based target metabolomics strategy used for our previous clinical settings. Metabolomics analyses and biochemical/physiological measurements were conducted at three time points in septic shock and sham animals, i.e., at baseline, after shock development and after fully resuscitation, in order to capture changes in metabolic and physiologic trajectories.

We performed a controlled experimental study on a large animal model of septic shock induced by a polymicrobial peritonitis on adult swine in our Experimental Laboratory of Intensive Care (LA1230336), in the Université Libre de Bruxelles.

The local animal ethics committee (Comité Ethique du Bien-Être Animal) approved the present study (protocol 641 N) and we followed the EU Directive 2010/63/EU for animal experiments and the ARRIVE guidelines for animal research.

Since the etiology and pathophysiology of septic shock are complex, it is difficult to develop a clinically accurate animal model of septic shock with relevance as translational model of sepsis-induced shock. Nevertheless, our swine model replicates the hemodynamic dysfunctions and the systemic inflammation that are typically found in clinical setting, although it does not reproduce the comorbidities that may complicate the clinical course of human septic shock.

Importantly, our model displays an altered metabolic landscape pointing toward an early hepatic impairment similarly to what we previously reported for septic shock patients (Cambiaghi et al., 2017). We confirmed here that the time course of metabolome changes in septic shock swines are mainly characterized by reduced plasma phospholipids species trajectories, altered alanine-glucose cycle and deranged inter-organ amino acid metabolism, all metabolic traits reported in septic shock patients (Bruins et al., 2003; Murch et al., 2006; Kao et al., 2009; Ferrario et al., 2016; Ahn et al., 2017; Cambiaghi et al., 2017).

To our knowledge, this is the first study in which an experimental model of septic shock recapitulates the main metabolic derangements reported in a clinical setting of septic shock.

Shock swines are characterized by reduced levels of PC, SM and of a cluster of LPC species composed by C16:0-, C16:1-, C18:0-, C18:1-, and C18:2-LPC. Further, this metabolites pool is lower in shock animals with respect to sham animals in the corresponding period of shock and after resuscitation.

LPCs have a very complex metabolism and play key roles in inflammatory responses (Lin et al., 2005; Schmitz and Ruebsaamen, 2010). Interestingly LPC species enhance the suppressive function of CD4+ CD25+ regulatory T cells (T_regs) involved in anti-inflammatory signaling (Hasegawa et al., 2011; Osborn and Olefsky, 2012). Defects in T_reg function are considered to play a role in hepatic inflammation (Speletas et al., 2011; Peiseler et al., 2012) and low levels of LPC16 and 18 species were reported in inflammatory liver disease (Tanaka et al., 2012; Lehmann et al., 2013; Maricic et al., 2014). These observations suggest that decreased circulating LPC might hamper their anti-inflammatory effects and conceivably, may promote an excessive immune response.

Because the liver is known to be a major source of circulating PC and LPC (Sekas et al., 1985; Baisted et al., 1988), it is plausible their plasma levels might be influenced by the functionality of hepatic lipid-modifying enzymes, whose expression can be regulated by pro-inflammatory cytokines such as TNF-α (Tanaka et al., 2012). It is likely that the production of pro-inflammatory cytokines devoted to regulate the hepatic response in sepsis may further injure hepatocytes.

Importantly, we found the same imbalance of LPC/PCs cycling observed in non-responsive septic shock patients (Cambiaghi et al., 2017), therefore strongly supporting that hepatic homeostasis is very early compromised. Such lipid levels might act as metabolic messengers in signaling early liver suffering (e.g., energy status) with consequent implication in the host response (Liu et al., 2014).

To note that both albumin and the observed LPC cluster decreased after shock in our model. Hypoalbuminemia is frequently observed during acute disease state, as albumin is a negative acute-phase protein. Hypoalbuminemia may be caused by the combination of increased transcapillary escape ratio, accelerated catabolism and impaired hepatic synthesis (Nicholson et al., 2000; Gatta et al., 2012). Albumin acts as fatty acid transporter (van der Vusse, 2009) and its reduction may play a critical role in the lipid economy of the body (Novak et al., 2017).

Therefore, our findings might suggest that the concomitant drop in albumin and the reduction in LPC species may represent a vicious cycle with detrimental effect on the intensity of inflammatory responses in the liver.

The plausible impairment in the early functionality of the liver in septic shock swine is also supported by the derangement of many interconnected energy-related metabolic pathways. The enhanced circulating alanine indicates an alteration in the glucose-alanine cycle as we previously observed in non-responsive septic shock patients (Cambiaghi et al., 2017). The higher plasma alanine in shock animals may be a sign of lower hepatic capacity for conversion of alanine to glucose, whose enhanced elaboration by the liver (hepatic gluconeogenesis) is a prominent feature of the solid organ response to injury and provides fuel to the cellular elements of the inflammatory response. The glucose/lactate ratio decreased after shock, indicating an imbalance between glycolysis and gluconeogenesis. This trend paralleled the increased alanine/glutamate ratio, particularly after the resuscitation. The alanine/glutamate ratio represents the alanine bioavailability from the impaired hepatic glucose-alanine cycle as part of the gluconeogenesis pathway. In fact, ALAT catalyzes the transfer of an amino group from alanine to α-ketoglutarate, to produce glutamate and pyruvate, which enters the gluconeogenesis and is used for glucose synthesis. Interestingly, we found a downward trend of ALAT in shocked and resuscitated animals, although not statistically significant.

As regards the transaminases trends, we may refer to subclinical hepatic alterations and not an acute liver dysfunction. The observational window after the insult is too short to have an hypoxic hepatitis, which is related to hypoperfusion in the presence of hypovolemia as well as inadequate cardiac output. Moreover, the increased levels of both ALAT and ASAT are observed in animal model of sepsis for longer time windows such as 24 h after the insult (Idrovo et al., 2015). However, in McGill (2016) the author reviews the limitation of using ALAT and ASAT for monitoring liver function. It is generally thought that aminotransferase elevations are due to cell damage with plasma membrane disruption. Apoptotic cell death can be thought of as cell implosion; a controlled process with minimal release of proteins into the extracellular space. However, oncotic necrosis, which can be thought of as cell explosion, can occur in some cells secondarily after induction of apoptosis. The hallmarks of apoptosis (e.g., cell shrinkage, chromatin condensation, apoptotic body formation) are clearly observable in the liver in hepatocyte apoptosis models during the early phase of injury when there is no significant elevation in serum ALAT (McGill, 2016).

Disturbance in metabolism is one of the most critical feature in sepsis/septic shock as response of the body to mobilize nutritional resources for organs and immune cells (Bruins et al., 2000, 2003; Whelan et al., 2014). In this complex and dynamic scenario, the time-course increase of plasma alanine and glutamine, major sources of nitrogen and carbon in inter-organ amino acid metabolism, may reflect a reduced rate in their utilization by organs in septic shock swine. The rise of circulating glutamine may support dysregulation in glutamine transport and extraction system, either suggesting reduced utilization by the gut and/or impaired hepatic transformation into urea cycle. Indeed, intermediates of the urea cycle were also disturbed in shock animals, where the global arginine bioavailability ratio (GABR: arginine/ornithine+citrulline) was significantly reduced compared to sham animals. GABR reduction may reduce NO, vasodilator essential to vascular homeostasis, and may lead to endothelial dysfunction (Kao et al., 2009).

The liver receives compounds absorbed or released by the gut through the portal vein before they gain access to the systemic circulation. The raised plasma level of glutamate, citrulline, arginine, seen in our septic shock swine model, are likely due to dysregulation of the gut-liver axis conversion of glutamine to citrulline and arginine. The gut accounts for the major part of this conversion, although splanchnic citrulline release to the systemic circulation may be limited by hepatic citrulline extraction (Van De Poll et al., 2007; Marini, 2016). To note that both arginine and citrulline plays an essential role in the immune response to sepsis and a disturbed arginine-nitric oxide (NO) metabolism is associated with deteriorating organ functions (Wijnands et al., 2012, 2015). Taken together our data are consistent with recent studies revealing liver dysfunction as an early event in sepsis (Nesseler et al., 2012; Recknagel et al., 2012; Wang et al., 2014).

The animal experimental data presented herein provide evidence that reduced plasma LPC/PC species trajectories, altered alanine-glucose cycle and deranged inter-organ amino acid exchange metabolism are early events in septic shock swine. These events occur within hours from infection and point to alterations in energy substrate utilization and hepatic biochemical perturbations. They may act as early metabolic features to assist in evaluating subclinical hepatic alterations. Being consistent with the metabolomics patterns differentiating non-responsive from responsive septic shock patients (Cambiaghi et al., 2017), such metabolic features may even represent therapeutic targets and therefore pave the way to improve the management of septic shock.

Metabolomics data are in the Supplementary Material; other experimental data are available upon request.

MF conceived and designed the study, did the statistical analysis, contributed to data interpretation, drafted, revised, and approved the manuscript. LB did the metabolomics analysis, contributed to data interpretation, revised, and approved the manuscript. FS and AH designed and developed the animal model, clinical animal data acquisition and interpretation, revised, and approved the manuscript. RP conceived the study, coordinated metabolomics analysis and data interpretation, drafted, and approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.