**5. TG delivery: Lipolysis**

332 Lipid Metabolism

PPAR and (muraglitazar) [72].

glucose uptake; PPARactivate only glucose uptake [69,74].

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

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

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

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

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 through phosphorylation of perilipin by LTR4 and ERK1/2.

The main inhibitor of lipolysis is insulin through the insulin-like receptor substrates 1and 2(IRS-1and 2) that activate the phosphadityl inositol 3-kinase(PI3K) complex and phosphorylate and activate the phosphodiesterase 3(PDE-3) [80]. Insulin acts also via an Akt-Independent PI3K-dependent signalling pathway which modifies PKA phosphorylation of perilipin [91]. The reduction in insulin activity is responsible for increased lipolysis in diabetes and obesity together with the reduction in the insulinsensitizing activity of adiponectin [92] probably linked to the adipose tissue dysfunction described in sepsis [17,18]. Moreover adiponectin may reduce fat deposition in visceral adipose tissue increasing the deposit in the subcutaneous compartment through PPARγ upregulation [18]. The lipolytic activity is differently regulated in subcutaneous and visceral fat: the former, more important for the basal activity, is more sensitive to the antilipolytic action of insulin, the latter is mainly activated during hormonal stimulation and provides FFA directly to the liver through the portal circulation in physiological and pathophysiological situations. In sepsis seems to be present a failure of adipocytes differentiation leading to a decreased storage ability of the subcutaneous tissue allowing FFA to be accumulated in visceral fat (with an increase in the metabolic and cardiovascular risk) and in organs giving rise to lipotoxicity for the concomitant reduction of FAO [18]. Lipolysis is increased in sepsis [14] due to a raised activity of the lipases of the adipose tissue, mainly the HSL, because of the increased plasma level of catecholamines, ILs and natriuretic peptides and the reduced activity of insulin and adiponectin (fig 3).

The Role of Altered Lipid Metabolism in Septic Myocardial Dysfunction 335

Sepsis and inflammation reproduce an atherogenic picture of serum lipoproteins [93] with an early reduction -preceding even the change in leukocytes- of total HDL- and LDLcholesterol, an increase in VLDL and TG justified by augmented liver synthesis and lipolysis, a reduction in HDL phospholipids compensated by an increase in VLDL

Until recently HDL were considered as devoted only to reverse cholesterol transport (RCT), a process through which the cholesterol is transferred from the cells to the acceptor apolipoproteinA-1 by the specific transporter A binding cassette 1(ABC1) and delivered to the liver where it is finally secreted and eliminated in the bile, the only way of its removal from the body. The recent application in this field of the Gel Filtration Chromatography based proteomics revealed that HDL contain not only apo-A1, apo-A2 and some enzymatic and transfer protein involved in cholesterol transport but more than 50 other associated proteins [94]. Many of the proteins isolated carry out functions completely different from RCT belonging to the complement proteins (C3,C1 inhibitor, factor H) and to serine protease inhibitors (SERPIN) [95-97]. The recent attainments point to an important role of HDL in immunity and in the modulation of the inflammatory response but they are also involved in a lot of disparate functions ranging from glucose metabolism to endothelial protection displaying also an antithrombotic action (for review see [98,99]). Of main importance in sepsis is the property of HDL to bind and neutralize LPS, mediated through its content in

HDLs may carry out anti-inflammatory actions in many ways: they downregulate the proinflammatory transcription factor NF-kB through a not yet completely understood mechanism -probably by the reduced degradation of Inhibitor kappa-B-a (IkB-a) [100]-, moreover they reduce the expression of the adhesion molecules CD11b and MCP-1 on human monocytes [97] and may curtail TNF-alpha production and upregulate IL-10 production by lymphocytes. The normal anti-inflammatory function of HDLs turns into a proinflammatory one during the acute phase reaction (APR) when they become dysfunctional [101]. The reasons for this change are manifold: there is a reduction in ApoA-1 levels, partially substituted by serum amyloid A apolipoprotein (Apo-SAA), their size is smaller because of a reduced activity of lecytin cholesterol acyl transferase (LCAT), enzyme deputed to esterification of cholesterol that allows the enlargement of HDL, HDLs become

The interference with the function of the inflammatory cells may be direct or mediated by the effect of the HDL on the reduction of cellular cholesterol and of cholesterol-enriched microdomains or rafts of their plasma membrane; this is also showed by the increased lethality and inflammatory response observed during sepsis in transgenic null mice for

poorer in the antioxidant enzymes paraoxanase [102,103].

critical receptors involved in cholesterol efflux as SR-BI and ACBA1 [104].

**5.1. Lipoprotein metabolism in sepsis** 

phospholipids.

phospholipids.

**5.2. HDL in sepsis** 

**Figure 3.** Lipolysis in sepsis. See text for explanation.

#### **5.1. Lipoprotein metabolism in sepsis**

Sepsis and inflammation reproduce an atherogenic picture of serum lipoproteins [93] with an early reduction -preceding even the change in leukocytes- of total HDL- and LDLcholesterol, an increase in VLDL and TG justified by augmented liver synthesis and lipolysis, a reduction in HDL phospholipids compensated by an increase in VLDL phospholipids.

#### **5.2. HDL in sepsis**

334 Lipid Metabolism

adiponectin (fig 3).

**PPAR**

**HIF-1**

**cGMP**

**Predominance of FFA uptake on oxidation**

**Figure 3.** Lipolysis in sepsis. See text for explanation.

**Adiponectin**

**Natriuretic peptides**

The main inhibitor of lipolysis is insulin through the insulin-like receptor substrates 1and 2(IRS-1and 2) that activate the phosphadityl inositol 3-kinase(PI3K) complex and phosphorylate and activate the phosphodiesterase 3(PDE-3) [80]. Insulin acts also via an Akt-Independent PI3K-dependent signalling pathway which modifies PKA phosphorylation of perilipin [91]. The reduction in insulin activity is responsible for increased lipolysis in diabetes and obesity together with the reduction in the insulinsensitizing activity of adiponectin [92] probably linked to the adipose tissue dysfunction described in sepsis [17,18]. Moreover adiponectin may reduce fat deposition in visceral adipose tissue increasing the deposit in the subcutaneous compartment through PPARγ upregulation [18]. The lipolytic activity is differently regulated in subcutaneous and visceral fat: the former, more important for the basal activity, is more sensitive to the antilipolytic action of insulin, the latter is mainly activated during hormonal stimulation and provides FFA directly to the liver through the portal circulation in physiological and pathophysiological situations. In sepsis seems to be present a failure of adipocytes differentiation leading to a decreased storage ability of the subcutaneous tissue allowing FFA to be accumulated in visceral fat (with an increase in the metabolic and cardiovascular risk) and in organs giving rise to lipotoxicity for the concomitant reduction of FAO [18]. Lipolysis is increased in sepsis [14] due to a raised activity of the lipases of the adipose tissue, mainly the HSL, because of the increased plasma level of catecholamines, ILs and natriuretic peptides and the reduced activity of insulin and

**Catecholamines**

**Mitochondrial biogenesis and FAO**

**Decreased ability of subcutaneous adipose tissue to store fat**

**Interleukins**

**Septic dysfunction of the adipose tissue**

**cAMP**

**cAMP**

**Sepsis LPS** 

**lipolysis**

**Organ damage**

**lipotoxicity**

**HSL activation**

Until recently HDL were considered as devoted only to reverse cholesterol transport (RCT), a process through which the cholesterol is transferred from the cells to the acceptor apolipoproteinA-1 by the specific transporter A binding cassette 1(ABC1) and delivered to the liver where it is finally secreted and eliminated in the bile, the only way of its removal from the body. The recent application in this field of the Gel Filtration Chromatography based proteomics revealed that HDL contain not only apo-A1, apo-A2 and some enzymatic and transfer protein involved in cholesterol transport but more than 50 other associated proteins [94]. Many of the proteins isolated carry out functions completely different from RCT belonging to the complement proteins (C3,C1 inhibitor, factor H) and to serine protease inhibitors (SERPIN) [95-97]. The recent attainments point to an important role of HDL in immunity and in the modulation of the inflammatory response but they are also involved in a lot of disparate functions ranging from glucose metabolism to endothelial protection displaying also an antithrombotic action (for review see [98,99]). Of main importance in sepsis is the property of HDL to bind and neutralize LPS, mediated through its content in phospholipids.

HDLs may carry out anti-inflammatory actions in many ways: they downregulate the proinflammatory transcription factor NF-kB through a not yet completely understood mechanism -probably by the reduced degradation of Inhibitor kappa-B-a (IkB-a) [100]-, moreover they reduce the expression of the adhesion molecules CD11b and MCP-1 on human monocytes [97] and may curtail TNF-alpha production and upregulate IL-10 production by lymphocytes. The normal anti-inflammatory function of HDLs turns into a proinflammatory one during the acute phase reaction (APR) when they become dysfunctional [101]. The reasons for this change are manifold: there is a reduction in ApoA-1 levels, partially substituted by serum amyloid A apolipoprotein (Apo-SAA), their size is smaller because of a reduced activity of lecytin cholesterol acyl transferase (LCAT), enzyme deputed to esterification of cholesterol that allows the enlargement of HDL, HDLs become poorer in the antioxidant enzymes paraoxanase [102,103].

The interference with the function of the inflammatory cells may be direct or mediated by the effect of the HDL on the reduction of cellular cholesterol and of cholesterol-enriched microdomains or rafts of their plasma membrane; this is also showed by the increased lethality and inflammatory response observed during sepsis in transgenic null mice for critical receptors involved in cholesterol efflux as SR-BI and ACBA1 [104].

The Role of Altered Lipid Metabolism in Septic Myocardial Dysfunction 337

/- mice were protected against alterations in lipid metabolism

due to the upregulation of the hepatic synthesis of TG, FFA and apoB, to the increased lipolysis, decreased lipid oxidation and inhibition of LPL [103]. The increased lipolysis in sepsis is due to the activation of the hormone-sensitive lipase (HSL) by catecholamines acting through PKA stimulation and cAMP increase. To the increased lipolysis contribute also the increased level of ILs, of natriuretic peptides and the reduction in insulin and

Feingold, in mice, showed that LPS reduced in the heart, diaphragm and kidneys [115-117] the levels of many enzymes involved in the fatty acid metabolism (FATP1,MCAD, CD36, CPT1 etc) through the reduced expression of many transcription factors: PPARand LXR, FXRpregnane X receptors and their coactivators PPAR coactivator 1and (PGC-1andsteroid coactivator receptor 1 and 2 (SRC1-2) and ERR. The downregulation of the nuclear receptors can be reproduced by IL-1 and TNF administration. Maitra and colleagues described an important role for IRAK1, a signalling component downstream of TLR4, in the downregulation of these transcription factors in

[118]. Because all these factors are deacetylated and activated by SIRT1 their downregulation in sepsis may play an important causative role [68] (see the paragraphs on

The increase in VLDL during sepsis, traditionally considered as a reaction mobilizing lipid stores to delivery an increased amount to the immune cells, may also have a protective meaning: the increased level of serum VLDL and of their content in phospholipids can

Mitochondrial dysfunction -present in sepsis but also in many other pathological states such as chronic heart failure, diabetes mellitus, metabolic syndrome and several neurodegenerative diseases- is a syndrome characterized by a picture of reduced oxygen consumption, ineffective ATP production, increased accumulation of ROS and calcium in the organelle leading to the opening in the internal mitochondrial membrane of a high conduction pore allowing the entry of solutes until a molecular weight of 1.500, with mitochondrial swelling, collapse of the electron gradient, release of proapoptotic proteins and death of the organelle (mitoptosis) followed by mitophagy: this phenomenon called mitochondrial permeability transition pore (MPTP) has an important role in sepsis and its

inhibition may experimentally improve septic myocardial dysfunction [28,121-123].

Mitochondrial functionality is regulated by the equilibrium between de novo mitochondrial formation (mitochondrial biogenesis) and mitochondrial autophagy (mitophagy), frequently preceding whole cell autophagy. This latter process, taking place only in deenergized organelles, is very important for cell survival because it allows the elimination of dysfunctional mitochondria, producing too much radical oxygen species(ROS) and assures

adiponectin activity (see paragraph on lipolysis).

metabolic switch and mitochondrial dysfunction).

substitute for the HDL in binding and neutralizing LPS [119,120].

**6. Mitochondrial dysfunction play an important role in septic** 

sepsis showing that IRAK1 -

**cardiomyopathy** 

**Figure 4.** Relationships among mitochondrial dysfunction, metabolism, and contractility. See text for explanation.

HDL may bind and inactivate LPS mainly through their phospholipid component [105,106], in sepsis and inflammation because of the HDL reduction their LPS inhibiting function is assumed by the VLDL phospholipids.

Also the endothelial function is modified through an increased production of nitric oxide (NO) by the stimulation of endothelial nitric oxide synthase (eNOS) mediated by the activation of the protective PI3K\Akt pathway leading to vasodilation, antiadhesion and anti-inflammatory effects [107]. A similar cardioprotective and antiapoptotic action of HDL have been shown through the activation of Stat3 mediated by sphingosine 1 phosphate (S1P), one of their common component [108]. The recombinant HDL(rHDL) or HDL mimetics, currently under clinical trial, are provided with the same actions attributed to the HDL [109,110]. The level of total, HDL- and LDL-cholesterol is decreased in septic and critically ill patients, as well as in experimental animals infused with LPS and proinflammatory cytokines and the decrease is negatively related to IL6 and TNFa concentrations [111]; HDL reduction is also related to mortality and severity of the disease [105,106].

The reduced level of serum HDL and cholesterol in sepsis is explained by a reduced RCT [112] mediated by the LPS-induced downregulation of the scavenger receptors SR-B1 and ABCA1 [113]. The sequestration of the cholesterol into the cell may be useful in the short term, due to the increased delivery to the immune cells, but harmful in the long run because of the decreased delivery to the steroidogenetic and liver cells and to the atherogenic picture it induces.

#### **5.3. VLDL**

VLDL are a circulant reservoir of TG and FFA able to activate PPAR and [ In clinical sepsis and experimental endotoxemia the serum level of VLDL, FFA and TG is increased due to the upregulation of the hepatic synthesis of TG, FFA and apoB, to the increased lipolysis, decreased lipid oxidation and inhibition of LPL [103]. The increased lipolysis in sepsis is due to the activation of the hormone-sensitive lipase (HSL) by catecholamines acting through PKA stimulation and cAMP increase. To the increased lipolysis contribute also the increased level of ILs, of natriuretic peptides and the reduction in insulin and adiponectin activity (see paragraph on lipolysis).

336 Lipid Metabolism

explanation.

it induces.

**5.3. VLDL** 

assumed by the VLDL phospholipids.

mortality and severity of the disease [105,106].

**Mitochondrial dysfunction**

**Figure 4.** Relationships among mitochondrial dysfunction, metabolism, and contractility. See text for

HDL may bind and inactivate LPS mainly through their phospholipid component [105,106], in sepsis and inflammation because of the HDL reduction their LPS inhibiting function is

Also the endothelial function is modified through an increased production of nitric oxide (NO) by the stimulation of endothelial nitric oxide synthase (eNOS) mediated by the activation of the protective PI3K\Akt pathway leading to vasodilation, antiadhesion and anti-inflammatory effects [107]. A similar cardioprotective and antiapoptotic action of HDL have been shown through the activation of Stat3 mediated by sphingosine 1 phosphate (S1P), one of their common component [108]. The recombinant HDL(rHDL) or HDL mimetics, currently under clinical trial, are provided with the same actions attributed to the HDL [109,110]. The level of total, HDL- and LDL-cholesterol is decreased in septic and critically ill patients, as well as in experimental animals infused with LPS and proinflammatory cytokines and the decrease is negatively related to IL6 and TNFa concentrations [111]; HDL reduction is also related to

The reduced level of serum HDL and cholesterol in sepsis is explained by a reduced RCT [112] mediated by the LPS-induced downregulation of the scavenger receptors SR-B1 and ABCA1 [113]. The sequestration of the cholesterol into the cell may be useful in the short term, due to the increased delivery to the immune cells, but harmful in the long run because of the decreased delivery to the steroidogenetic and liver cells and to the atherogenic picture

VLDL are a circulant reservoir of TG and FFA able to activate PPAR and [ In clinical sepsis and experimental endotoxemia the serum level of VLDL, FFA and TG is increased Feingold, in mice, showed that LPS reduced in the heart, diaphragm and kidneys [115-117] the levels of many enzymes involved in the fatty acid metabolism (FATP1,MCAD, CD36, CPT1 etc) through the reduced expression of many transcription factors: PPARand LXR, FXRpregnane X receptors and their coactivators PPAR coactivator 1and (PGC-1andsteroid coactivator receptor 1 and 2 (SRC1-2) and ERR. The downregulation of the nuclear receptors can be reproduced by IL-1 and TNF administration. Maitra and colleagues described an important role for IRAK1, a signalling component downstream of TLR4, in the downregulation of these transcription factors in sepsis showing that IRAK1 - /- mice were protected against alterations in lipid metabolism [118]. Because all these factors are deacetylated and activated by SIRT1 their downregulation in sepsis may play an important causative role [68] (see the paragraphs on metabolic switch and mitochondrial dysfunction).

The increase in VLDL during sepsis, traditionally considered as a reaction mobilizing lipid stores to delivery an increased amount to the immune cells, may also have a protective meaning: the increased level of serum VLDL and of their content in phospholipids can substitute for the HDL in binding and neutralizing LPS [119,120].
