**1. Introduction**

322 Lipid Metabolism

86:7473-7483.

[206] Miller ME, Adhikary S, Kolokoltsov AA, Davey RA (2012) Ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection. J Virol.

[207] Ciesek S, Steinmann E, Manns MP, Wedemeyer H, Pietschmann T (2008) The suppressive effect that myriocin has on hepatitis C virus RNA replication is independent of inhibition of serine palmitoyl transferase. J Infect Dis 198: 1091-1093.

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Despite the novel pathogenetic and therapeutic acquisitions sepsis remains the main cause of death in the critically ill patient (p.). The incidence of sepsis is increasing, with more than 750 000 cases occurring in the United States every year; sepsis is complicated by organ dysfunction giving rise to severe sepsis which causes more than 200 000 deaths each year [1]. The cardiovascular syndrome is the cause of the major part of the fatalities leading to the picture of the septic shock with 90,000 deaths annually. The myocardial dysfunction in sepsis has been widely studied and many causes of heart dysfunction have been described. The presence of a metabolic compromise is well known in chronic heart failure and has been considered as a basis for contractile failure, interestingly similar alterations have been recently described also in sepsis. The purpose of this study is to examine the current available data on this topic.

Sepsis has been defined as the systemic host reaction to an infection [2]. The typical immune response in sepsis presents a first stage, characterized by an increased production of proinflammatory interleukins (ILs) by monocytes, macrophages. lymphocytes and endothelial cells in response to a few molecules, common to all virulent pathogens, called pathogenassociated molecular patterns (PAMPs) [3]. They are recognized by receptors on the surface of the immune cells, named Pattern Recognition Receptors (PRP) the most important being the Toll-like Receptors (TLRs), existing in 13 different types each recognizing a different microbial constituent. TLR are an important component of immune activity programmed to respond quickly to the infectious challenges by recognizing PAMP. Lipopolysaccharide (LPS) in particular is recognized by the complex TLR4-CD14 in the plasma membrane and the inflammatory signal is transduced by the recruitment of the adaptor molecules that associate with the intracellular Toll/interleukin-1 receptor (TIR) domain of the TLR to initiate signal transduction. Of these adaptor molecules myeloid differentiation primary- response protein 88 (MyD88) is associated with all TLRs except TLR3 while the TIR-domain containing adaptor

© 2013 Siracusano and Girasole, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

inducing interferon (IFN)-β (TRIF) is only associated to TLR3 [3]. Both of them lead to nuclear factor-κB (NF-kB) activation and induction of over 150 inflammatory and procoagulant genes with increased expression of the proinflammatory ILs tumor necrosis factor TNF), IL1, IL6, interferon (IFN), chemokines and cell adhesion molecules. This phase, named Systemic Inflammatory Reaction Syndrome (SIRS) is followed by a second phase, the compensatory anti- inflammatory response syndrome (CARS), that modulates SIRS through death by apoptosis of immune cells, gene silencing of the pro-inflammatory genes and increased production of the contra-inflammatory ILs IL10, IL4 and TGF[4-6] Through this biphasic reaction in the largest part of the cases the homeostasis is re-established. It is possible however that during the proinflammatory phase because of the "cytokine storm"-induced organ damage, further increased through the release by the injured tissue of the endogenous damage-associated molecular pattern (DAMPs or alarmins such as hig mobility group protein B1(HMBG1), hyaluronan, HSP, proteins S100 etc), as more frequently occurs in young and reactive people, or during the contra-inflammatory phase, due to immunosuppression and diffuse infection, death may ensue [4]. The normal biphasic reaction may be altered when sepsis is present in very old and diseased persons that frequently present a mild phase of SIRS or do not present it at all; in the later stages it seems that immunosuppression is almost always present [7]. Indeed from what has been described it seems that in both the cases sepsis is characterized by an "altered immune response", unbalanced [8] with prevalence of an excessive proinflammatory reaction in the early phase and a prevalent immunosuppression in the late phase [8.] The definition of sepsis based on the recent achievement on this topic could be reconsidered focusing more on the immune status, we propose the sequent definition: " sepsis is the altered immune response to an infectious challenge, either increased or decreased, and followed by a compromised organ function". The passage from the SIRS to the CARS is marked by the phenomenon of LPS tolerance [9]: the prolonged stimulation of TLR4, the specific receptor for endotoxin, by LPS leads to the silencing of proinflammatory genes and to the stimulation of the contra-inflammatory ones through a process of gene reprogramming [10] with increased expression of IL-10, TGF-β, and type I IFNs. Neutrophil function is seriously impaired in sepsis with the development of a decreased ability to migrate into the infection site, related to the severity of the disease [for review see 11]. During this stage macrophages exhibit an increased phagocytic ability but an altered capacity of antigen presentation due to reduced expression of several major histocompatibility complex (MHC) Class II molecules (e.g. HLA-DRs) with impaired T-cell proliferation as well as reduced production of IFN. This phase corresponds also to a stage of ischemic tolerance caused by the reduced production of NF-kB responsible for the amplification of the damage induced by ischemia [12]. TLRs are indeed involved in the tissue damage caused by non-infectious challenges such as ischemia-reperfusion injury (IRI), burns, surgery, trauma etc. because they mediate the inflammatory reaction to the alarmins released by these noxae: the deletion of TLR2 and TLR4 may reduce the cellular damage in these settings [13-14].The mitochondrial DNA released after tissue damage may be recognized by TLRs, behaving as a DAMP for its far origin by bacteria according to endosymbiotic theory; its experimental infusion may cause a picture similar to the ARDS [15]. TLRs are also important to explain the link between inflammation and metabolic alterations [16] and the state of low grade inflammation present for so far unknown reasons in obesity, metabolic syndrome, diabetes and in chronic The Role of Altered Lipid Metabolism in Septic Myocardial Dysfunction 325

myocardial ischemia; moreover they may also be involved in the adipose tissue dysfunction described during sepsis [17,18]. One of the main problems in the study of sepsis has been its experimental reproduction: the LPS infusion method, used also at low doses in human volunteers, is not reliable because it reproduces only the proinflammatory phase and led in the past to misunderstand the complex regulation of immunity in sepsis, moreover in this model is lacking an infectious focus. The availability of successive different models such as the infusion of living bacteria and the cecal ligation and puncture (CLP), at present considered a good reproduction of human septic peritonitis, allowed further progress supplying models

The first phase of sepsis, coincident with SIRS and also called flow phase, is characterized by a state of hypercatabolism with negative nitrogen balance induced by the increase in proinflammatory ILs and stress hormones, while the anabolic ones are reduced. This phase is also marked by hyperglycemia, hyperlactatemia and increased energy expenditure varying with the nature of the injurious stress (15-20% after surgery, 20-40% in sepsis and up to 120% in burns [20]). Injury in general and sepsis in particular are associated with an increased protein breakdown (mainly derived by the muscle representing the main store of proteins in the body, up to 40% of total body proteins), protein catabolism in sepsis may reach 260 gr\day [21] corresponding to a loss of 1 Kg muscle, with negative nitrogen balance, an increased blood pool of amino acids (a.a.), used in the liver for neoglucogenesis and the production of acute phase proteins, and reduced uptake of a.a. by the muscle. The protein utilization as a fuel is opposite to the normal and rapidly depletes body lean mass. The increased production of IL-1, IL-6 and TNFcauses protein catabolism both directly and indirectly through the stimulation of the HypothalamusPituitary-Adrenal (HPA) axis. The increased protein

Insulin resistance may be defined as the inability of insulin to adequately stimulate glucose uptake in the muscle or to inhibit gluconeogenesis in the liver [23]. The pathogenesis of the insulin resistance is no more interpreted on the basis of the glucocentric but of the lipocentric theory [24] stating that the excess of circulating free fatty acids (FFA), the main competitive inhibitors of glucose utilization in the cell, when present in excess in plasma because of increased lipolysis, decreased fat storing capacity of adipose tissue or reduced fatty acid oxydation (FAO) may accumulate in the organs (liver, myocardium, pancreas etc) giving origin to reduced sensitivity to insulin, increased hepatic glucose output and to the phenomenon of lipotoxicity, syndrome characterized by accumulation of FFA in the cell, increased production of reactive oxygen species (ROS), activation of apoptosis (lipoapoptosis) and organ dysfunction [25]. The etiology of insulin resistance in sepsis is related to the increase of the contra-insular substances (glucagon, TNF, growth hormone, cortisol) and to the reduced expression of the insulin-sensitizing adiponectin while the endogenous insulin secretion in the critically ill p. is increased as occurs in the early phase of type II diabetes mellitus, on the contrary of what was suggested in the past [26]. The consequences of the

similar to human sepsis [19].

**2. Metabolic response in sepsis and injury** 

catabolism may be reproduced by counter-regulatory hormones infusion.

**2.1. Glucose metabolism and insulin sensitivity in sepsis** 

myocardial ischemia; moreover they may also be involved in the adipose tissue dysfunction described during sepsis [17,18]. One of the main problems in the study of sepsis has been its experimental reproduction: the LPS infusion method, used also at low doses in human volunteers, is not reliable because it reproduces only the proinflammatory phase and led in the past to misunderstand the complex regulation of immunity in sepsis, moreover in this model is lacking an infectious focus. The availability of successive different models such as the infusion of living bacteria and the cecal ligation and puncture (CLP), at present considered a good reproduction of human septic peritonitis, allowed further progress supplying models similar to human sepsis [19].

#### **2. Metabolic response in sepsis and injury**

324 Lipid Metabolism

inducing interferon (IFN)-β (TRIF) is only associated to TLR3 [3]. Both of them lead to nuclear factor-κB (NF-kB) activation and induction of over 150 inflammatory and procoagulant genes with increased expression of the proinflammatory ILs tumor necrosis factor TNF), IL1, IL6, interferon (IFN), chemokines and cell adhesion molecules. This phase, named Systemic Inflammatory Reaction Syndrome (SIRS) is followed by a second phase, the compensatory anti- inflammatory response syndrome (CARS), that modulates SIRS through death by apoptosis of immune cells, gene silencing of the pro-inflammatory genes and increased production of the contra-inflammatory ILs IL10, IL4 and TGF[4-6] Through this biphasic reaction in the largest part of the cases the homeostasis is re-established. It is possible however that during the proinflammatory phase because of the "cytokine storm"-induced organ damage, further increased through the release by the injured tissue of the endogenous damage-associated molecular pattern (DAMPs or alarmins such as hig mobility group protein B1(HMBG1), hyaluronan, HSP, proteins S100 etc), as more frequently occurs in young and reactive people, or during the contra-inflammatory phase, due to immunosuppression and diffuse infection, death may ensue [4]. The normal biphasic reaction may be altered when sepsis is present in very old and diseased persons that frequently present a mild phase of SIRS or do not present it at all; in the later stages it seems that immunosuppression is almost always present [7]. Indeed from what has been described it seems that in both the cases sepsis is characterized by an "altered immune response", unbalanced [8] with prevalence of an excessive proinflammatory reaction in the early phase and a prevalent immunosuppression in the late phase [8.] The definition of sepsis based on the recent achievement on this topic could be reconsidered focusing more on the immune status, we propose the sequent definition: " sepsis is the altered immune response to an infectious challenge, either increased or decreased, and followed by a compromised organ function". The passage from the SIRS to the CARS is marked by the phenomenon of LPS tolerance [9]: the prolonged stimulation of TLR4, the specific receptor for endotoxin, by LPS leads to the silencing of proinflammatory genes and to the stimulation of the contra-inflammatory ones through a process of gene reprogramming [10] with increased expression of IL-10, TGF-β, and type I IFNs. Neutrophil function is seriously impaired in sepsis with the development of a decreased ability to migrate into the infection site, related to the severity of the disease [for review see 11]. During this stage macrophages exhibit an increased phagocytic ability but an altered capacity of antigen presentation due to reduced expression of several major histocompatibility complex (MHC) Class II molecules (e.g. HLA-DRs) with impaired T-cell proliferation as well as reduced production of IFN. This phase corresponds also to a stage of ischemic tolerance caused by the reduced production of NF-kB responsible for the amplification of the damage induced by ischemia [12]. TLRs are indeed involved in the tissue damage caused by non-infectious challenges such as ischemia-reperfusion injury (IRI), burns, surgery, trauma etc. because they mediate the inflammatory reaction to the alarmins released by these noxae: the deletion of TLR2 and TLR4 may reduce the cellular damage in these settings [13-14].The mitochondrial DNA released after tissue damage may be recognized by TLRs, behaving as a DAMP for its far origin by bacteria according to endosymbiotic theory; its experimental infusion may cause a picture similar to the ARDS [15]. TLRs are also important to explain the link between inflammation and metabolic alterations [16] and the state of low grade inflammation present for so far unknown reasons in obesity, metabolic syndrome, diabetes and in chronic

The first phase of sepsis, coincident with SIRS and also called flow phase, is characterized by a state of hypercatabolism with negative nitrogen balance induced by the increase in proinflammatory ILs and stress hormones, while the anabolic ones are reduced. This phase is also marked by hyperglycemia, hyperlactatemia and increased energy expenditure varying with the nature of the injurious stress (15-20% after surgery, 20-40% in sepsis and up to 120% in burns [20]). Injury in general and sepsis in particular are associated with an increased protein breakdown (mainly derived by the muscle representing the main store of proteins in the body, up to 40% of total body proteins), protein catabolism in sepsis may reach 260 gr\day [21] corresponding to a loss of 1 Kg muscle, with negative nitrogen balance, an increased blood pool of amino acids (a.a.), used in the liver for neoglucogenesis and the production of acute phase proteins, and reduced uptake of a.a. by the muscle. The protein utilization as a fuel is opposite to the normal and rapidly depletes body lean mass. The increased production of IL-1, IL-6 and TNFcauses protein catabolism both directly and indirectly through the stimulation of the HypothalamusPituitary-Adrenal (HPA) axis. The increased protein catabolism may be reproduced by counter-regulatory hormones infusion.

#### **2.1. Glucose metabolism and insulin sensitivity in sepsis**

Insulin resistance may be defined as the inability of insulin to adequately stimulate glucose uptake in the muscle or to inhibit gluconeogenesis in the liver [23]. The pathogenesis of the insulin resistance is no more interpreted on the basis of the glucocentric but of the lipocentric theory [24] stating that the excess of circulating free fatty acids (FFA), the main competitive inhibitors of glucose utilization in the cell, when present in excess in plasma because of increased lipolysis, decreased fat storing capacity of adipose tissue or reduced fatty acid oxydation (FAO) may accumulate in the organs (liver, myocardium, pancreas etc) giving origin to reduced sensitivity to insulin, increased hepatic glucose output and to the phenomenon of lipotoxicity, syndrome characterized by accumulation of FFA in the cell, increased production of reactive oxygen species (ROS), activation of apoptosis (lipoapoptosis) and organ dysfunction [25]. The etiology of insulin resistance in sepsis is related to the increase of the contra-insular substances (glucagon, TNF, growth hormone, cortisol) and to the reduced expression of the insulin-sensitizing adiponectin while the endogenous insulin secretion in the critically ill p. is increased as occurs in the early phase of type II diabetes mellitus, on the contrary of what was suggested in the past [26]. The consequences of the metabolic storm in sepsis are an increased hepatic production of glucose, a reduced glucose uptake by insulin-dependent tissues, increased levels of FFA- consequence of the increased lipolysis- reduced FAO and reduced insulin sensitivity at the muscular and hepatic level [27]. The high levels of blood glucose allow a high uptake by the non-insulin-dependent cells because of the increased expression of the stress- and Ils-dependent glucose transporter GLUT1 [27]. In this way a high rate of glucose oxidation through mitochondria is made possible leading to an enhanced production of ROS: the increased oxidative and nitrosative stress, chiefly in presence of a reduction in ROS scavengers, -mainly glutathione[28]- typical of the critical states, may damage mainly the Complexes I and III of the electron transport chain (ETC) initiating the mitochondrial dysfunction. Insulin is provided with an anti-inflammatory action that is missing if a reduced sensitivity to its action is present, such as in critical illness, further increasing inflammation [27]. Proinflammatory ILs interfere with insulin activity through multiple mechanisms ; 1) TNF and other cytokines activate Jun-N-terminal kinase (JNK) responsible for serine phosphorylation of the insulin receptor substrate 1 (IRS-1) at Ser 307, so disrupting insulin signalling [29] 2) furthermore JNK activation has been showed, in LPS-induced experimental sepsis, to depress PPAR expression reducing FFA oxidation [30]; JNK inhibition prevented cardiac dysfunction, despite continued induction of inflammatory markers, demonstrating that there is a dysmetabolic basis for LPS-induced cardiac dysfunction in this experimental setting [30]. An important clinical application of the role played by the altered glucose metabolism in the critically ill p. derived from the Leuven Study [31] that showed a striking reduction from 8% to 4,6% in the mortality of critically ill surgical p. receiving mechanical ventilation by maintaining glycemia between 80 and 110 mg% with intensive insulin therapy. However in 2009 the NICE SUGAR Study showed that a blood glucose target of 180 mg% or less per deciliter resulted in lower mortality than did a target of 81 to 108 mg range, probably by reducing the incidence of hypoglycaemia [32]. A diffuse coverage of this matter is beyond the scope of this report (for review see reference [33]).

The Role of Altered Lipid Metabolism in Septic Myocardial Dysfunction 327

low-density lipoprotein, VLDL) through the endothelial lipoprotein lipase (LPL), enzyme present in the capillary endothelium and bound to it through a proteoglycan, the Glycosylphosphatidyl-inositol high density lipoprotein-binding protein 1 (GPIHBP1), which anchors LPL to endothelium and serves as a bridge allowing LP uptake[40]. The myocardial cells may take up FFA also from the albumin-bound pool (10 time less abundant). FFA obtained from hydrolysis of TG by LPL or transported by albumin are then taken up by the myocardiocyte, through transporters, mainly FAT\CD36 (50-60%) but also by fatty acid binding protein (FABP) and fatty acid transport plasma membrane (FATPpm), only a very small part is taken up by diffusion [41]. Once transported into the cytosol, FFAs are esterified to long chain acyl CoA by fatty acyl CoA synthase (FACS) and transported for FFA oxidation(FAO) across mitochondrial membrane. This process is carried out in three steps: at first acyl-CoA is metabolized into acyl-carnitine by the enzyme carnitine palmitoyl transferase-1 (CPT-1), located on the outer mitochondrial membrane. Acyl-carnitine is hence transported across the inner mitochondrial membrane by carnitine translocase (CAT) and again transformed into long chain Acyl-CoA and carnitine by CPT- 2 (carnitine shuttle) [42]. The step catalyzed by CPT-1 is the rate-limiting reaction of FAO and the activity of this enzyme is strictly regulated by malonyl-CoA (the product of carboxylation of acetyl-CoA), whose levels are controlled by a balance between the activity of the enzymes involved in its metabolism, the acetyl CoA carboxylase (ACC) and malonyl CoA decarboxylase. AMPactivated protein kinase (AMPK), a fundamental energy sensor in the cell, controls this reaction phosphorylating and inhibiting ACC whenever it senses a decrease in the ATP\AMP ratio [42]. In the normal heart, 75% of the fatty acids taken up are immediately oxidized. Acetyl-CoA has a central role in the regulation of the utilization of the substrates: it is the metabolic product of both glucose (through pyruvate) and fatty acid utilization. It may be utilized for oxidation in the Krebs cycle or, if the cellular level of ATP is high, used for FFA synthesis. Acetyl-CoA may also be transformed into pyruvate and lactate by LDH in the presence of hypoxia but also when it is produced in huge quantities. More than 40 years ago Randle described the reciprocal inhibition exercised by glucose and FFA utilization [43]. He displayed in the isolated heart and in a skeletal muscle preparation that the oxidation of one substrate inhibited directly, without hormonal intermediation, the use of the other (Randle cycle). So an increase in FAO causes a proportional decrease in glucose oxidation and uptake leading to hyperglycemia. The actual interpretation of Randle cycle is that an increase in serum concentration and uptake of FFA activates FAO and causes accumulation of its byproducts as ROS, ceramides, diacylglycerol, acyl-carnitine and fatty acyl-CoAs inside the skeletal muscle so depressing glucose uptake, whereas in the liver they inhibit insulin-mediated suppression of glycogenolysis and gluconeogenesis [44-45]. Also the increased lipolysis present in many conditions (fasting, exercise, inflammatory conditions, diabetes and obesity) leading to an increased FAO may downregulate glucose utilization through increased serum FFA and reduced insulin sensitivity. All these signals, particularly diacylglycerol, interfere with insulin signalling activating PKC novel isoforms () causing a reduced tyrosine phosphorylation of the IRS-1 which is normally responsible for the activation through phosphatidyl inositol 3-kinase (PI3K) and Akt of the membrane translocation of the insulin-dependent glucose transporter 4 (GLUT4)[46]. So an

Hyperlactatemia in sepsis has been considered as a consequence of the establishment of an hypoxic environment, this concept is however no longer tenable, except for the p. in septic shock, because in many organs an increased oxygen saturation has been showed both in clinical sepsis and after LPS infusion [34]. Currently the cause of hyperlactatemia is considered to be the increased expression of Hypoxia-inducible factor1 (HIF-1)- and of cytokines activating PDHK4, an enzyme inhibiting PDH (pyruvate dehydrogenase) through phosphorylation so preventing the transformation of pyruvate into acetyl-CoA and its oxidation through the TCA (Tricarboxilic acid Cycle); the excess of pyruvate is transformed into lactate by Lactate dehydrogenase (LDH) through a process called accelerated aerobic glycolysis [35-38]. The plasma levels of lactate seems to be related to the outcome [35].

#### **2.2. TG and FFA utilization in the normal heart**

In a physiological environment rich in oxygen but also in pathophysiological conditions (like diet rich in lipids, starvation or diabetes) the heart can utilize all metabolites but it prefers FFA oxidation, from which it gets 60-90% of the necessary energy, obtaining the remaining part from the oxidation of glucose and ketone bodies [39]. The myocardiocyte gets FFA mainly taking up tryglicerides (TG)-rich lipoproteins (LP) (chylomicrons and very low-density lipoprotein, VLDL) through the endothelial lipoprotein lipase (LPL), enzyme present in the capillary endothelium and bound to it through a proteoglycan, the Glycosylphosphatidyl-inositol high density lipoprotein-binding protein 1 (GPIHBP1), which anchors LPL to endothelium and serves as a bridge allowing LP uptake[40]. The myocardial cells may take up FFA also from the albumin-bound pool (10 time less abundant). FFA obtained from hydrolysis of TG by LPL or transported by albumin are then taken up by the myocardiocyte, through transporters, mainly FAT\CD36 (50-60%) but also by fatty acid binding protein (FABP) and fatty acid transport plasma membrane (FATPpm), only a very small part is taken up by diffusion [41]. Once transported into the cytosol, FFAs are esterified to long chain acyl CoA by fatty acyl CoA synthase (FACS) and transported for FFA oxidation(FAO) across mitochondrial membrane. This process is carried out in three steps: at first acyl-CoA is metabolized into acyl-carnitine by the enzyme carnitine palmitoyl transferase-1 (CPT-1), located on the outer mitochondrial membrane. Acyl-carnitine is hence transported across the inner mitochondrial membrane by carnitine translocase (CAT) and again transformed into long chain Acyl-CoA and carnitine by CPT- 2 (carnitine shuttle) [42]. The step catalyzed by CPT-1 is the rate-limiting reaction of FAO and the activity of this enzyme is strictly regulated by malonyl-CoA (the product of carboxylation of acetyl-CoA), whose levels are controlled by a balance between the activity of the enzymes involved in its metabolism, the acetyl CoA carboxylase (ACC) and malonyl CoA decarboxylase. AMPactivated protein kinase (AMPK), a fundamental energy sensor in the cell, controls this reaction phosphorylating and inhibiting ACC whenever it senses a decrease in the ATP\AMP ratio [42]. In the normal heart, 75% of the fatty acids taken up are immediately oxidized. Acetyl-CoA has a central role in the regulation of the utilization of the substrates: it is the metabolic product of both glucose (through pyruvate) and fatty acid utilization. It may be utilized for oxidation in the Krebs cycle or, if the cellular level of ATP is high, used for FFA synthesis. Acetyl-CoA may also be transformed into pyruvate and lactate by LDH in the presence of hypoxia but also when it is produced in huge quantities. More than 40 years ago Randle described the reciprocal inhibition exercised by glucose and FFA utilization [43]. He displayed in the isolated heart and in a skeletal muscle preparation that the oxidation of one substrate inhibited directly, without hormonal intermediation, the use of the other (Randle cycle). So an increase in FAO causes a proportional decrease in glucose oxidation and uptake leading to hyperglycemia. The actual interpretation of Randle cycle is that an increase in serum concentration and uptake of FFA activates FAO and causes accumulation of its byproducts as ROS, ceramides, diacylglycerol, acyl-carnitine and fatty acyl-CoAs inside the skeletal muscle so depressing glucose uptake, whereas in the liver they inhibit insulin-mediated suppression of glycogenolysis and gluconeogenesis [44-45]. Also the increased lipolysis present in many conditions (fasting, exercise, inflammatory conditions, diabetes and obesity) leading to an increased FAO may downregulate glucose utilization through increased serum FFA and reduced insulin sensitivity. All these signals, particularly diacylglycerol, interfere with insulin signalling activating PKC novel isoforms () causing a reduced tyrosine phosphorylation of the IRS-1 which is normally responsible for the activation through phosphatidyl inositol 3-kinase (PI3K) and Akt of the membrane translocation of the insulin-dependent glucose transporter 4 (GLUT4)[46]. So an

326 Lipid Metabolism

metabolic storm in sepsis are an increased hepatic production of glucose, a reduced glucose uptake by insulin-dependent tissues, increased levels of FFA- consequence of the increased lipolysis- reduced FAO and reduced insulin sensitivity at the muscular and hepatic level [27]. The high levels of blood glucose allow a high uptake by the non-insulin-dependent cells because of the increased expression of the stress- and Ils-dependent glucose transporter GLUT1 [27]. In this way a high rate of glucose oxidation through mitochondria is made possible leading to an enhanced production of ROS: the increased oxidative and nitrosative stress, chiefly in presence of a reduction in ROS scavengers, -mainly glutathione[28]- typical of the critical states, may damage mainly the Complexes I and III of the electron transport chain (ETC) initiating the mitochondrial dysfunction. Insulin is provided with an anti-inflammatory action that is missing if a reduced sensitivity to its action is present, such as in critical illness, further increasing inflammation [27]. Proinflammatory ILs interfere with insulin activity through multiple mechanisms ; 1) TNF and other cytokines activate Jun-N-terminal kinase (JNK) responsible for serine phosphorylation of the insulin receptor substrate 1 (IRS-1) at Ser 307, so disrupting insulin signalling [29] 2) furthermore JNK activation has been showed, in LPS-induced experimental sepsis, to depress PPAR expression reducing FFA oxidation [30]; JNK inhibition prevented cardiac dysfunction, despite continued induction of inflammatory markers, demonstrating that there is a dysmetabolic basis for LPS-induced cardiac dysfunction in this experimental setting [30]. An important clinical application of the role played by the altered glucose metabolism in the critically ill p. derived from the Leuven Study [31] that showed a striking reduction from 8% to 4,6% in the mortality of critically ill surgical p. receiving mechanical ventilation by maintaining glycemia between 80 and 110 mg% with intensive insulin therapy. However in 2009 the NICE SUGAR Study showed that a blood glucose target of 180 mg% or less per deciliter resulted in lower mortality than did a target of 81 to 108 mg range, probably by reducing the incidence of hypoglycaemia [32]. A diffuse

coverage of this matter is beyond the scope of this report (for review see reference [33]).

**2.2. TG and FFA utilization in the normal heart** 

Hyperlactatemia in sepsis has been considered as a consequence of the establishment of an hypoxic environment, this concept is however no longer tenable, except for the p. in septic shock, because in many organs an increased oxygen saturation has been showed both in clinical sepsis and after LPS infusion [34]. Currently the cause of hyperlactatemia is considered to be the increased expression of Hypoxia-inducible factor1 (HIF-1)- and of cytokines activating PDHK4, an enzyme inhibiting PDH (pyruvate dehydrogenase) through phosphorylation so preventing the transformation of pyruvate into acetyl-CoA and its oxidation through the TCA (Tricarboxilic acid Cycle); the excess of pyruvate is transformed into lactate by Lactate dehydrogenase (LDH) through a process called accelerated aerobic glycolysis [35-38]. The plasma levels of lactate seems to be related to the outcome [35].

In a physiological environment rich in oxygen but also in pathophysiological conditions (like diet rich in lipids, starvation or diabetes) the heart can utilize all metabolites but it prefers FFA oxidation, from which it gets 60-90% of the necessary energy, obtaining the remaining part from the oxidation of glucose and ketone bodies [39]. The myocardiocyte gets FFA mainly taking up tryglicerides (TG)-rich lipoproteins (LP) (chylomicrons and very increase in FAO causes a proportional decrease in glucose uptake and oxidation with hyperglycemia. This mechanism has been considered important in the pathophysiology of diabetes mellitus type2 (T2DM). Conversely glucose uptake and oxidation to acetyl CoA and pyruvate involves the conversion to malonylCoA by ACC leading to inhibition of CPT-1 and consequently of FAO; moreover insulin decreases lipolysis and the level of FFA. During starvation increased lipolysis raises FFA uptake and oxidation inhibiting glucose oxidation and favouring accumulation of pyruvate and lactate redirected to glycogen synthesis allowing also glucose sparing for the brain. Conversely after a glucose rich meal the increased uptake and utilization of glucose inhibit the utilization of FFA through the accumulation of malonylCoA. The normal heart responds to an increase in workload by switching from FFA to preferential glucose utilization. The Randle cycle explains how the heart regulates the fuel selection in the short term to face physiologic challenges however the long-term adaptation to physiologic and pathophysiologic conditions is mediated by translational and post-translational modifications rather than by the metabolic ones. A fitting example is the metabolic switch occurring after birth. During the fetal life the heart, probably because of the relatively hypoxic medium, relies more on carbohydrate metabolism, whereas in the adult myocardiocyte glycogen occupies less than 2% of the cell volume, in the fetal cells it represents more than 30% of the cell volume [47]. After birth the heart, due probably to the increased availability of oxygen, prefers FFA oxidation because of the discontinuous availability of metabolites and the elevated fat content of mother's milk; the metabolic shift is accompanied by increased expression of the enzymes involved in FAO. The utilization of both lipid and carbohydrates has advantages and drawbacks: the complete oxidation of a molecule of palmitic acid (molecular mass 256,43) yields more ATP (105 molecules vs 31) but consumes much more oxygen (46 atoms vs 12) than the oxidation of a molecule of glucose. Moreover the oxidation of lipid produces more oxidative stress, a drawback partially corrected by the increased production of the uncoupling protein 3(UCP3) and differently from anaerobic glycolysis it is not available in hypoxic or ischemic settings. Such long term substrate modifications are transcriptionally regulated and the switch to carbohydrate utilization is due to a downregulation of the nuclear receptors PPAR involved in the transcription of the enzymes of FFA transport and utilization.

The Role of Altered Lipid Metabolism in Septic Myocardial Dysfunction 329

which can also continue in an environment poor in oxygen. Even though glycolysis allows a much lesser production of energy (2 molecules of ATP instead of 36 per molecule of glucose oxidized) through the transformation of pyruvate into lactate by LDH it presents the advantage to go on also in a hypoxic setting; however this process, which in the short-term is adaptive, in the long-term becomes maladaptive because of the reduced energy production [49]. The better the heart defends itself in the presence of stressful events, the more easily it is able to switch to the type of metabolism more efficient in that situation and the more it is provided with metabolic flexibility. Thus the survival of the myocardium depends, in these conditions, on an increased supply of glucose that is assured by the augmentation of the main transmembrane glucose transporters, GLUT1 and GLUT4. GLUT1 is present ubiquitously, augments in presence of reduced glucose levels and during inflammatory, metabolic and osmotic stress [50]; GLUT4 is present in the adipose tissue, heart, liver and in the striated muscle and is the typical insulin-dependent transporter. The increase in glucose transporters, mainly in GLUT1, has an adaptive meaning in the stressed heart; its overexpression protects against experimental ventricular hypertrophy, at least until cardiac failure appears, causing its downregulation [51]. This metabolic remodelling seems to be controlled by the fetal gene program, so called because it is present in the fetal environment, relatively hypoxic, and is reactivated whenever a stressful situation is present [47,52]. It modifies the expression of contractile proteins switching the expression of heavy myosin chain (HMC) from the more efficient but more energy expensive alpha to the slower but less energy consuming beta isoform, and increases the production of some humoral factors (brain natriuretic peptide, BNP ) and of several protective proteins (heat shock proteins, HSP). The cardioprotective pathway of the PI3K and Akt, activated during pre- and post-conditioning, is also activated by the fetal gene program supplying an antiapoptotic protection and leading to an overlapping between cardioprotection in chronic ischemia and ischemic preconditioning [52]. The metabolic component is mainly regulated by the enzyme pyruvate deydrogenase kinase (PDHK4) phosphorylating and inactivating pyruvate deydrogenase (PDH) and inhibiting the transformation into Acetyl-CoA that could enter the TCA; it is instead transformed into lactate by LDH allowing a continued production of ATP also in low oxygen settings. This activation of glycolysis, such as the dependent increase in GLUT1,4, in LDH and glycolytic enzymes is mainly controlled by the transcription factor HIF-1 that is upregulated in the settings of ischemia\hypoxia but also in non-hypoxic conditions such as during inflammatory states and tumours because its expression is increased through a cross-talk with NF-kB [53]. On the other hand hypoxia and inflammation are not completely separated conditions as showed by the induction of TLR4 and TLR6 by HIF-1 during hypoxia [54]. HIF-1 is composed by a constitutively expressed HIF-1 subunit and an oxygen-sensitive HIF-1 subunit, target under normoxic conditions of three prolyl hydroxilases 1,2,3(PDH1,2,3), the hydroxylation allows the binding of the von Hippel-Lindau protein (VHL) and the consequent ubiquitination and proteasome degradation of HIF-1. On the contrary during hypoxia PDH2 activity is reduced with accumulation of HIF-1, its

dimerization with HIF-1 and the expression of many target genes [55].

The increased expression of HIF-1 causes at the same time a downregulation of mitochondrial lipid oxidation depressing the expression of the transcription factor PPAR

The key enzyme in the regulation of pyruvate metabolism is the PDH. The activity of cardiac PDH is mainly modulated by the PDH kinase (PDHK4), an isoform of the kinase phosphorylating and transforming PDH from the active to the inactive form. Short-term metabolic inhibition of PDHK4 is affected by pyruvate while its activation is affected by acetyl CoA and NADH. The long-term regulation is determined by many conditions characterized by insulin deficiency (diabetes) or resistance (starvation, high-fat diet, and hyperthyroidism) that increase PDHK expression, while insulin suppresses its production [35,36,38].

#### **3. The metabolic myocardial switch**

In pathological conditions a long term modification in substrate preference may be present: the diabetic heart relies almost exclusively on FAO [48], on the contrary the myocardium submitted to mechanical, ischemic\hypoxic or inflammatory stress switches to the anaerobic glycolysis, which can also continue in an environment poor in oxygen. Even though glycolysis allows a much lesser production of energy (2 molecules of ATP instead of 36 per molecule of glucose oxidized) through the transformation of pyruvate into lactate by LDH it presents the advantage to go on also in a hypoxic setting; however this process, which in the short-term is adaptive, in the long-term becomes maladaptive because of the reduced energy production [49]. The better the heart defends itself in the presence of stressful events, the more easily it is able to switch to the type of metabolism more efficient in that situation and the more it is provided with metabolic flexibility. Thus the survival of the myocardium depends, in these conditions, on an increased supply of glucose that is assured by the augmentation of the main transmembrane glucose transporters, GLUT1 and GLUT4. GLUT1 is present ubiquitously, augments in presence of reduced glucose levels and during inflammatory, metabolic and osmotic stress [50]; GLUT4 is present in the adipose tissue, heart, liver and in the striated muscle and is the typical insulin-dependent transporter. The increase in glucose transporters, mainly in GLUT1, has an adaptive meaning in the stressed heart; its overexpression protects against experimental ventricular hypertrophy, at least until cardiac failure appears, causing its downregulation [51].

328 Lipid Metabolism

increase in FAO causes a proportional decrease in glucose uptake and oxidation with hyperglycemia. This mechanism has been considered important in the pathophysiology of diabetes mellitus type2 (T2DM). Conversely glucose uptake and oxidation to acetyl CoA and pyruvate involves the conversion to malonylCoA by ACC leading to inhibition of CPT-1 and consequently of FAO; moreover insulin decreases lipolysis and the level of FFA. During starvation increased lipolysis raises FFA uptake and oxidation inhibiting glucose oxidation and favouring accumulation of pyruvate and lactate redirected to glycogen synthesis allowing also glucose sparing for the brain. Conversely after a glucose rich meal the increased uptake and utilization of glucose inhibit the utilization of FFA through the accumulation of malonylCoA. The normal heart responds to an increase in workload by switching from FFA to preferential glucose utilization. The Randle cycle explains how the heart regulates the fuel selection in the short term to face physiologic challenges however the long-term adaptation to physiologic and pathophysiologic conditions is mediated by translational and post-translational modifications rather than by the metabolic ones. A fitting example is the metabolic switch occurring after birth. During the fetal life the heart, probably because of the relatively hypoxic medium, relies more on carbohydrate metabolism, whereas in the adult myocardiocyte glycogen occupies less than 2% of the cell volume, in the fetal cells it represents more than 30% of the cell volume [47]. After birth the heart, due probably to the increased availability of oxygen, prefers FFA oxidation because of the discontinuous availability of metabolites and the elevated fat content of mother's milk; the metabolic shift is accompanied by increased expression of the enzymes involved in FAO. The utilization of both lipid and carbohydrates has advantages and drawbacks: the complete oxidation of a molecule of palmitic acid (molecular mass 256,43) yields more ATP (105 molecules vs 31) but consumes much more oxygen (46 atoms vs 12) than the oxidation of a molecule of glucose. Moreover the oxidation of lipid produces more oxidative stress, a drawback partially corrected by the increased production of the uncoupling protein 3(UCP3) and differently from anaerobic glycolysis it is not available in hypoxic or ischemic settings. Such long term substrate modifications are transcriptionally regulated and the switch to carbohydrate utilization is due to a downregulation of the nuclear receptors

PPAR involved in the transcription of the enzymes of FFA transport and utilization.

that increase PDHK expression, while insulin suppresses its production [35,36,38].

**3. The metabolic myocardial switch** 

The key enzyme in the regulation of pyruvate metabolism is the PDH. The activity of cardiac PDH is mainly modulated by the PDH kinase (PDHK4), an isoform of the kinase phosphorylating and transforming PDH from the active to the inactive form. Short-term metabolic inhibition of PDHK4 is affected by pyruvate while its activation is affected by acetyl CoA and NADH. The long-term regulation is determined by many conditions characterized by insulin deficiency (diabetes) or resistance (starvation, high-fat diet, and hyperthyroidism)

In pathological conditions a long term modification in substrate preference may be present: the diabetic heart relies almost exclusively on FAO [48], on the contrary the myocardium submitted to mechanical, ischemic\hypoxic or inflammatory stress switches to the anaerobic glycolysis, This metabolic remodelling seems to be controlled by the fetal gene program, so called because it is present in the fetal environment, relatively hypoxic, and is reactivated whenever a stressful situation is present [47,52]. It modifies the expression of contractile proteins switching the expression of heavy myosin chain (HMC) from the more efficient but more energy expensive alpha to the slower but less energy consuming beta isoform, and increases the production of some humoral factors (brain natriuretic peptide, BNP ) and of several protective proteins (heat shock proteins, HSP). The cardioprotective pathway of the PI3K and Akt, activated during pre- and post-conditioning, is also activated by the fetal gene program supplying an antiapoptotic protection and leading to an overlapping between cardioprotection in chronic ischemia and ischemic preconditioning [52]. The metabolic component is mainly regulated by the enzyme pyruvate deydrogenase kinase (PDHK4) phosphorylating and inactivating pyruvate deydrogenase (PDH) and inhibiting the transformation into Acetyl-CoA that could enter the TCA; it is instead transformed into lactate by LDH allowing a continued production of ATP also in low oxygen settings. This activation of glycolysis, such as the dependent increase in GLUT1,4, in LDH and glycolytic enzymes is mainly controlled by the transcription factor HIF-1 that is upregulated in the settings of ischemia\hypoxia but also in non-hypoxic conditions such as during inflammatory states and tumours because its expression is increased through a cross-talk with NF-kB [53]. On the other hand hypoxia and inflammation are not completely separated conditions as showed by the induction of TLR4 and TLR6 by HIF-1 during hypoxia [54]. HIF-1 is composed by a constitutively expressed HIF-1 subunit and an oxygen-sensitive HIF-1 subunit, target under normoxic conditions of three prolyl hydroxilases 1,2,3(PDH1,2,3), the hydroxylation allows the binding of the von Hippel-Lindau protein (VHL) and the consequent ubiquitination and proteasome degradation of HIF-1. On the contrary during hypoxia PDH2 activity is reduced with accumulation of HIF-1, its dimerization with HIF-1 and the expression of many target genes [55].

The increased expression of HIF-1 causes at the same time a downregulation of mitochondrial lipid oxidation depressing the expression of the transcription factor PPAR

(possessing a DNA consensus motif for HIF-1)[56] and an upregulation of PPAR activating fatty acid uptake and glycerolipid biosynthesis so leading to lipotoxicity [57]. HIF-1 stimulates also mitochondrial autophagy to rebalance the reduced oxidation with mitochondrial mass[ 58]. The stimulation of glycolysis by HIF-1 has an important role in sepsis because the phagocytes use huge quantities of glucose through glycolysis in the hypoxic environment of the inflamed tissues either for cell migration either for phagocytosis and bacterial killing [59]. The activity of HIF-1 is regulated by acetylation performed by CBP\p300 and inhibited by deacetylation by SIRT1. The inverse shift to mitochondrial lipid oxidation is controlled by SIRT1 which increases the expression of PGC-1 with simultaneous activation of both mitochondrial biogenesis and lipid oxidation through PPAR upregulation. SIRT1 and HIF-1 are reciprocally regulated so that in the hypoxic\inflammatory setting HIF- is activated while Sirt1 is decreased due to NAD+ scarcity so inhibiting HIF-deacetylation and inactivation while in normoxic environment HIF-is inactivated by oxygen-dependent prolyl hydroxylases PHD2 activated by SIRT1.

The Role of Altered Lipid Metabolism in Septic Myocardial Dysfunction 331

healthy aging. Sirtuins exist in seven forms with different subcellular localization: SIRT1,6,7 present in nucleus, SIRT2 in cytoplasm, SIRT3,4,5 in the mitochondria. Because they are mainly regulated by the levels of NAD+, the AMPK that increases its concentration upregulates their expression while the DNA-repairing enzyme poly(ADP-ribose) polymerase 1 (PARP), activated whenever there is an oxidative stress and consuming for its activity high quantities of NAD+, downregulates sirtuin expression . SIRT1 is able to deacetylate and activate a series of transcription factors involved in many steps of metabolism (AMPK, FOXO, HIF-1, PGC-1, p53NF-κBPPAR and , LXR,FXR,ERR). Mitochondrial sirtuins are in a critical location to regulate metabolism and biogenesis of the organelles controlling oxidative phosphorylation (OXPHOS), intracellular Calcium and the production of ROS [65]. SIRT1 is also regulated via microRNA: microRNA34a downregulates its expression metabolically and microRNA199a in the hypoxic setting [66,67]. Statins have been showed to upregulate SIRT1 expression in endothelial cells

**downregulation upregulation**

**SIRT1**

**NAD+**

**Increased oxidative and nitrosative stress**

**Damage of nuclear and mitochondrial DNA**

**Activation of the nuclear DNA-repairing enzyme PARP**

**p53 FOXO**

**4. Transcriptional regulation of FFA transport, uptake and oxidation** 

Transport, uptake and oxidation of FFA in the cell is a process tightly regulated by several transcription factors belonging to the nuclear hormone receptor superfamily. The nuclear receptors involved include the peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), farnesoid X receptor (FXR) and ERR (estrogen related receptor), which

**PPAR PPAR**

**Deacetylation**

**Nutrient deprivation**

**eNOS**

**LXR FXR ERR**

**MIrna34a Starvation Exercise**

contributing possibly to improve clinically endothelial function [68].

**Ventricular hypertrophy**

**Figure 2.** Regulation of SIRT1 expression. See text for explanation.

**Mirna 199a Inflammation Hypoxia**

**HIF-1 PGC-1 NF-kB AMPK**

**Figure 1.** Metabolic switch. See text for explanation.

Sirtuins are class III NAD+-dependant histone deacetylases (HDAC) targeting histones, transcription factors and enzymes regulating in this way gene expression and metabolic function in relation to the energy status of the cell [60-64]. They have been related to the regulation of metabolism, apoptosis and inflammation, to extending life span and to the healthy aging. Sirtuins exist in seven forms with different subcellular localization: SIRT1,6,7 present in nucleus, SIRT2 in cytoplasm, SIRT3,4,5 in the mitochondria. Because they are mainly regulated by the levels of NAD+, the AMPK that increases its concentration upregulates their expression while the DNA-repairing enzyme poly(ADP-ribose) polymerase 1 (PARP), activated whenever there is an oxidative stress and consuming for its activity high quantities of NAD+, downregulates sirtuin expression . SIRT1 is able to deacetylate and activate a series of transcription factors involved in many steps of metabolism (AMPK, FOXO, HIF-1, PGC-1, p53NF-κBPPAR and , LXR,FXR,ERR). Mitochondrial sirtuins are in a critical location to regulate metabolism and biogenesis of the organelles controlling oxidative phosphorylation (OXPHOS), intracellular Calcium and the production of ROS [65]. SIRT1 is also regulated via microRNA: microRNA34a downregulates its expression metabolically and microRNA199a in the hypoxic setting [66,67]. Statins have been showed to upregulate SIRT1 expression in endothelial cells contributing possibly to improve clinically endothelial function [68].

330 Lipid Metabolism

**Environment rich in oxygen, diet rich in lipids, starvation, diabetes**

**Increase**

**Figure 1.** Metabolic switch. See text for explanation.

**Metabolic switch**

**PGC-1 SIRT1 AMPK**

**Upregulation of FAO enzymes and mitochondrial biogenesis**

**PPAR PGC-1 S AMPK**

**Downregulation of FAO (fatty acid oxidation) enzymes and mitochondrial biogenesis**

> **Preferential utilization of lipid**

> > **mitochondrial transcription factor A**

**PPAR nuclear respiratory factors**

**NRF1, NRF2 mtTFA**

Sirtuins are class III NAD+-dependant histone deacetylases (HDAC) targeting histones, transcription factors and enzymes regulating in this way gene expression and metabolic function in relation to the energy status of the cell [60-64]. They have been related to the regulation of metabolism, apoptosis and inflammation, to extending life span and to the

**Ischemia-hypoxia Hypertrophy Inflammation Mechanical overload**

**HIF-1**

(possessing a DNA consensus motif for HIF-1)[56] and an upregulation of PPAR activating fatty acid uptake and glycerolipid biosynthesis so leading to lipotoxicity [57]. HIF-1 stimulates also mitochondrial autophagy to rebalance the reduced oxidation with mitochondrial mass[ 58]. The stimulation of glycolysis by HIF-1 has an important role in sepsis because the phagocytes use huge quantities of glucose through glycolysis in the hypoxic environment of the inflamed tissues either for cell migration either for phagocytosis and bacterial killing [59]. The activity of HIF-1 is regulated by acetylation performed by CBP\p300 and inhibited by deacetylation by SIRT1. The inverse shift to mitochondrial lipid oxidation is controlled by SIRT1 which increases the expression of PGC-1 with simultaneous activation of both mitochondrial biogenesis and lipid oxidation through PPAR upregulation. SIRT1 and HIF-1 are reciprocally regulated so that in the hypoxic\inflammatory setting HIF- is activated while Sirt1 is decreased due to NAD+ scarcity so inhibiting HIF-deacetylation and inactivation while in normoxic environment HIF-is inactivated by oxygen-dependent prolyl hydroxylases PHD2 activated by SIRT1.

**Transcriptional and genetic regulation of substrate preference in the heart**

Fetal gene program

Reduce

**Glucose utilization via glycolysis**

**Figure 2.** Regulation of SIRT1 expression. See text for explanation.

#### **4. Transcriptional regulation of FFA transport, uptake and oxidation**

Transport, uptake and oxidation of FFA in the cell is a process tightly regulated by several transcription factors belonging to the nuclear hormone receptor superfamily. The nuclear receptors involved include the peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), farnesoid X receptor (FXR) and ERR (estrogen related receptor), which

#### 332 Lipid Metabolism

bind and are activated by lipids, acting as sensors of endocrine signals and of dietary components and planners of the physiological response to nutrients. PPARs, the most important of them, are present in three types (alpha, beta and gamma), besides their action as lipids sensors, they are involved in the regulation of lipid and glucose metabolism, insulin sensitivity and modulation of inflammation (for review see [69-71]); their actions are partially overlapping with a certain degree of redundancy but they are not coincident. In elucidating the function of PPARs an important role has been played by the studies in genetically modified mice with overexpression or deletion of these nuclear receptors. PPARs are activated by endogenous ligands, FFA or their derivatives (arachidonic acid, eicosanoids, leukotrienes and oxidized lipoproteins) but there are also known exogenous ligands, used as drugs, for PPAR (fibrates) and PPAR (thiazolidinediones) or for both PPAR and (muraglitazar) [72].

The Role of Altered Lipid Metabolism in Septic Myocardial Dysfunction 333

macrophages, involved in the low grade inflammation and important in dysmetabolic conditions such as reduced insulin sensitivity, metabolic syndrome and atherosclerosis on the other side it may be of critical interest in regulating inflammatory reaction in sepsis [78]. LXRs and FXR heterodimerize also with RXR and are activated respectively by oxysterol and bile acids: both exist in the isoforms and , contribute to lipid and lipoprotein regulation and modulate inflammation. In the regulation of FAO a fundamental role is also played by the orphan receptor (transcription factors without an identified endogenous ligand) ERR (estrogen related receptor) involved also in the regulation of mitochondrial biogenesis, gluconeogenesis and oxidative phosphorylation

The amount of lipid in the myocardial cell is regulated by the balance between TG delivery to the heart through the VLDL- regulated by lipolysis- and the oxidation of TG in the myocardiocyte. Considering their importance in cellular signalling, lipid synthesis and as energy substrates, the level of circulating TG and FFA is tightly regulated [80]. Adipocytes are the only cells able to hydrolize TG giving rise to FFA and glycerol utilized by other tissues. This process is initiated by the adipose triglyceride lipase (ATGL) catalysing the hydrolysis of triglycerides to diglycerides followed by the hydrolysis of diglycerides to monoglycerides by the hormone-sensitive lipase (HSL) able also to catalyse the first phase and of monoglycerides to TG and glycerol by the monoglyceride lipase (MGL)[81-83]. ATGL deficiency is associated with fat deposition in all tissues indicating that ATGL is rate limiting in the catabolism of cellular fat depots [84]. Lipolysis is also regulated by perilipin , a protein coating the lipid

Many substances may activate physiologically and in diseases HSL, the more important enzyme for its sensitivity to the hormonal regulation but also for its action modulating adipogenesis and adipose metabolism [86]. Catecholamines acting through β1, β2 and also <sup>3</sup> receptors coupled to a G stimulating (Gs) protein are the main stimulators of HSL; the alpha2 receptors inhibit instead lipolysis through a G inhibitory (Gi) protein, this effect may be antagonized by administration of beta-blockers. Also natriuretic peptides are able to enhance lipolysis by activating guanylyl cyclase and increasing cGMP [83]. TNF IL1 and IL6 increase HSL activity through the stimulation of several kinases of the MAP kinase cascade (janus kinases (JNK), p44\42 and extracellular signal-regulated kinase-1/2 ( ERK1/2) and the consequent increase in cAMP [87,88]. These kinases phosphorylate perilipin allowing HSL and

LPS may activate lipolysis by increasing ILs level, the demonstration however that the inhibition of TNF-alpha, IL1 and cathecolamines could not prevent lipolysis in endotoxemic rats, led to show a direct lipolytic action carried out by low-dose endotoxin [90].This action is not mediated by an increase in cAMP, activation of PKA or PKC or inhibition of NFkB but

droplets, that exposes TG of a lipid droplet to the action of the lipases [85].

ATGL to access and hydrolyse TG; this effect was prevented by metformin [89].

through phosphorylation of perilipin by LTR4 and ERK1/2.

by interacting with PPAR and and PGC-1 [79].

**5. TG delivery: Lipolysis** 

The PPARs form heterodimers with retinoid X receptors (RXR) and bind to consensus DNA sequences in the promoter of the target genes, the PPAR response binding element (PPRE), formed by two hexameric nucleotides sequences separated by one base pair. In the absence of ligands the heterodimer binds with the co-repressors, blocking gene transcription; when activated by ligands, the complex, because of a change of conformation, releases the corepressors and recruits the co-activators and the RNA polymerase giving rise to transcription. PPAR are expressed mainly in tissues with high oxidative metabolism such as heart, muscle and liver. PPAR are expressed at high levels only in adipose tissue where they regulate the maturation and fat deposition in adipocytes so indirectly regulating, through the plasma level of FFA, the insulin sensitivity; they are however expressed at low level in many organs among which the heart [73]. Whereas PPAR and share the ability to activate the transcription of proteins involved in FFA uptake, storage and oxidation, PPAR only activate the proteins involved in FFA oxidation. Also different are the effects on glucose metabolism: PPAR depress glucose oxidation whereas PPAR activate glycolysis (through PDHK4 activation) and glucose uptake; PPARactivate only glucose uptake [69,74].

PPAR\ are expressed in various tissues where they regulate lipid but even glucose metabolism. PPAR also regulate mitochondrial biogenesis through interaction with the PPAR coactivator 1 (PGC-1) network (see below)[74]. Because FAO takes mainly place in mitochondria (only the breakdown of very long chain fatty acids, subsequently converted to medium chain fatty acids and shuttled to mitochondria, takes place in the peroxisomes) there is a common regulatory mechanism for FAO and mitochondrial biogenesis controlled by PPARs and by the network of PGC-1. The PPAR mainly but also the PPAR have been showed, in several model systems, to modulate inflammation by transrepressing target genes of the transcription factors NF-κB, nuclear factor of activated T cells (NFAT), activator protein 1 (AP1) and signal transducers and activators of transcription (STATs) through a process called transrepression [75-77] so modulating the action of lymphocytes, macrophages and dendritic cells. This process has twofold importance, on the one side it may regulate the inflammation induced by tissue macrophages, involved in the low grade inflammation and important in dysmetabolic conditions such as reduced insulin sensitivity, metabolic syndrome and atherosclerosis on the other side it may be of critical interest in regulating inflammatory reaction in sepsis [78]. LXRs and FXR heterodimerize also with RXR and are activated respectively by oxysterol and bile acids: both exist in the isoforms and , contribute to lipid and lipoprotein regulation and modulate inflammation. In the regulation of FAO a fundamental role is also played by the orphan receptor (transcription factors without an identified endogenous ligand) ERR (estrogen related receptor) involved also in the regulation of mitochondrial biogenesis, gluconeogenesis and oxidative phosphorylation by interacting with PPAR and and PGC-1 [79].
