**2. Liver metabolism in health and disease**

of which limit life span. Caloric restriction without malnutrition can extend life span while caloric excess has the opposite effect [20]. Thus, the choice of oral intake has a profound

progression results in decompensated ESLD and the development of malignancy.

72 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

**Figure 2.** (A) *Morphological changes observed in the mice model of high fat diet (HFD) plus fructose (Western Diet) in the microbiota, fat content tissue and liver.* Liver cells accumulate FA in form of TG from the spill-over of lipid excess in the fat compartment and after saturated the normal processes of liver lipid metabolism. (B) *Local liver inflammatory response from lipid excess.* Lipotoxicity an addition to increase LPS activates SEC, HSC and Kupffer cells inducing more parenchymal cells into senescence and apoptosis which changes the local mielue into an inflammatory microenvironment. Continuous HFD decreased further mitochondrial function with lower ATP production and increase collagen deposition leading to progressive liver fibrosis, liver dysfunction. The state of progressive liver fibrosis due to a local and systemic inflammatory state results in an increasing insulin resistance status with the full metabolic syndrome phenotype. Its

The free radical intermediates hypothesis of cell aging still remains the most reasonable in the induction and maintenance of the senescent status [17, 21]. ROS, reactive nitrogen sp., and lipid peroxide are important regulators of cell signaling that provides reliable maintenance of

impact on life span.

The reduced tri-peptide glutathione (GSH) is the major antioxidant in the body responsible for maintaining the intracellular redox balance. 90% of the GSH in plasma derives from the liver [24] and aging is associated with a progressive decline in the levels of GSH in humans and rodents [25]. Senescent liver cells in culture showed elevated ROS leading to a state of chronic oxidative stress. In addition, age associated decline in GSH has been linked to an activation of neural sphingolipid hydrolase enzyme (NSMase) and the accumulation of bioactive ceramide, a precursor of inflammation [25]. The availability of L-cysteine is the rate-limiting factor of GSH synthesis and oral supplementation of cysteine alleviates GSH deficiencies in humans and rodents [25]. GSH deficiency can be alleviated by the oral intake of cysteine and its restoration rates appears to be age and sex dependent. Older animal models are associated with increased cellular stress and an enhanced subcellular injury after heat stress associated with an increased iron intracellular deposition [26]. These cause damages to mitochondria and lysosomes. Although a more precise mechanism of organelle damage was not enunciated, iron deposition mediated a decrease in Transferrin-receptor-1 which upregulates the iron storage protein ferritin after heat stress. Nevertheless, the synthesis of the iron exporter protein ferroportin was delayed [26]. Effect that may explain at least in part, organelle damage in the aging cell that occurs after natural oxidants depletion (**Figure 2B**).

**2.1. In Health**

and FADH<sup>2</sup>

*2.1.1. Metabolism and inflammation*

The metabolism of FA in the mitochondrial matrix is sequentially catalyzed through a ß-oxidation process by four enzyme families: acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. While acyl-carnitine is converted back to acyl-CoA to enter the TCA cycle, dehydrogenases activity shows different affinity for short, medium, long and very long FA's [13]. Every cycle of ß-oxidation renders acyl-CoA and shortens the FA chain by 2 carbons providing the equivalents of electron donors NADH

way is an effective way of ATP production, an overload of FA may play the role of un-coupler that exercises an inhibitory effect on the respiratory chain through a proton-phoric effect on the inner mitochondrial membrane. This effect results from the implicit effect of FA on mitochondrial membrane porosity by opening of the permeability transition pores, which results in the subsequent loss of electrical gradient and arrest of the respiratory chain [13]. The above concept favors the hypotheses of mitochondrial dysfunction from FA overload as the primary step of the insulin resistance state in obese patients. Other authors have found no mitochondrial respiratory changes in the steatotic liver but in mitochondria from skeletal muscle from a rodent model of high fat diet induced obesity [27]. Authors have proposed mitochondrial changes are due to an adaptation of the mitochondria to the high lipid charge rather than a defect per se in its function. No explanation was provided by the authors regarding the increase in ROS and inflammatory changes observed. Others suggested a protected effect from caloric lipid surplus against the development of metabolic dysfunction, as long as cells maintained functional adipocyte storage with low levels of tissue inflammation [28]. Once adipose accumulation saturates cell capacity, fatty excess spills over into other tissues leading to LD accumulation with subsequent lipid oxidation and an inflammatory response which precedes the metabolic syndrome manifestations [28]. The Delete in Breast Cancer-1 (DBC-1) protein is an important regulator of fat accumulation and storage in fat tissue which exercises its action by inhibition of SIRT1. In the DBC-/KO mice exposed to HFD, it was observed high plasma levels of FA with no liver steatosis, lower expression of senescence cells and increased storage of FA in the adipocytes with no development of insulin resistance [28]. Thus, DBC-1 protected liver and adipocytes from senescence by preservation of the fat compartment function with liver sparing and insulin sensitivity. Interestingly, a comparison has been made between this rodent model and the so called "healthy obese subjects" where there is fat accumulation but no signs of metabolic syndrome or systemic inflammation.

Obesity depresses the anti-inflammatory effects of the heat shock proteins (HSP70) pathway, an inhibition that may contribute to the progression from NAFLD to NASH [29]. Excess of lipids and fuels trigger a low grade inflammatory response in both fat and liver tissues that correlates with the impaired insulin responsiveness. TBARS, a simple but fair estimate of lipoperoxidation/malondialdehyde (systemic oxidative stress) produced throughout the body was shown to be elevated in plasma from NASH patients when compared to normal subjects and its levels were correlated with insulin-resistance status [29]. The former response involved activation of the c-Jun NH2-terminal kinases (JNK's), endoplasmic reticulum (ER)

which is the driving force for the synthesis of ATP. Although the ß-oxidation path-

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75

A diet enriched in calories and lipids increases free fatty acids (FA) in plasma obligating cells to protect themselves from lipotoxicity or death by either oxidizing FA's or sequestering them as triacylglycerol (TAG) within lipid droplets (LD) [13]. PGC-1, an exercise-induced transcriptional coactivator may play an important role in coordinating intra-muscular LD-signaling with mitochondrial remodeling. TAG within lipid droplets are the major form of energy storage in the body (muscle, liver, fat tissue) and a reservoir of membrane lipid component. TAG synthesis is initiated by glycerol-3-phosphatase acetyltransferases (GPAT) at the mitochondrial and sarcoplasmic reticulum membranes and it is completed at the sarcoplasmic reticulum by the sn-1-acyl-glycerol-3-phosphatase acyltransferase (AGPAT), phosphatidic acid phosphatase (PAP) and sn-1,2-diacylglycerol acyltransferase (DGAT) [13]. Synthetized LD-TAG are localized preferentially in proximity to mitochondrial membranes named "contact zones". Once TAG's are released, they are mainly used in the mitochondria for ATP synthesis via oxidative phosphorylation from the ß-oxidation path. The "athlete paradox" states that the accumulation of TAG in the trained and insulin sensitive cells is in greater proportion than the TAG accumulation in cells from diabetic subjects with insulin resistance. This observation supports the hypothesis of mitochondrial dysfunction as a factor of TAG accumulation from a sustained lipid charge.

The protein family of perilipins (Plin) is associated with LD's and their scaffolding may affect the interaction between TAG and the mitochondria [13]. The Plin family consists of Plin1 to 5; the most common PAT (perilipin/ADRP/TIP47) interacts with LD in different proportions. In the liver, down-regulation of Plin2 promotes a reduction of hepatic steatosis and increases insulin sensitivity, albeit a reduction in both Plin2 and Plin3 is associated with insulin resistance [13]. In the heart, a Plin5 deficiency causes increased lipid oxidation, increased ROS production and decreased cardiac function. In heart and skeletal muscle TAG and FA are the main metabolic source of energy through the ß-oxidation pathway, suggesting a very tightly regulated process from cell storage to mitochondrial metabolic use. While TAG may come from LD, FA's are mainly transported in plasma as albumin-bound or as part of the very low density lipid-protein (VLDL) complex. Different transmembrane transporter systems are involved in their translocation to the inner cell compartment where the long chain fatty acid (LCFA) forms thioesters with coenzyme A (CoA). LCFA-CoA can form TAG for storage as LD, or can enter the outer mitochondrial membrane where CPT1 catalyzes the reaction of LCFA-CoA to LC-acylcarnitine. The former compound can actively cross the inner mitochondrial membrane with the exchange of carnitine for acylcarnitine. CACT is highly expressed in tissues with predominant ß-oxidation metabolism.

#### **2.1. In Health**

factor of GSH synthesis and oral supplementation of cysteine alleviates GSH deficiencies in humans and rodents [25]. GSH deficiency can be alleviated by the oral intake of cysteine and its restoration rates appears to be age and sex dependent. Older animal models are associated with increased cellular stress and an enhanced subcellular injury after heat stress associated with an increased iron intracellular deposition [26]. These cause damages to mitochondria and lysosomes. Although a more precise mechanism of organelle damage was not enunciated, iron deposition mediated a decrease in Transferrin-receptor-1 which upregulates the iron storage protein ferritin after heat stress. Nevertheless, the synthesis of the iron exporter protein ferroportin was delayed [26]. Effect that may explain at least in part, organelle dam-

A diet enriched in calories and lipids increases free fatty acids (FA) in plasma obligating cells to protect themselves from lipotoxicity or death by either oxidizing FA's or sequestering them as triacylglycerol (TAG) within lipid droplets (LD) [13]. PGC-1, an exercise-induced transcriptional coactivator may play an important role in coordinating intra-muscular LD-signaling with mitochondrial remodeling. TAG within lipid droplets are the major form of energy storage in the body (muscle, liver, fat tissue) and a reservoir of membrane lipid component. TAG synthesis is initiated by glycerol-3-phosphatase acetyltransferases (GPAT) at the mitochondrial and sarcoplasmic reticulum membranes and it is completed at the sarcoplasmic reticulum by the sn-1-acyl-glycerol-3-phosphatase acyltransferase (AGPAT), phosphatidic acid phosphatase (PAP) and sn-1,2-diacylglycerol acyltransferase (DGAT) [13]. Synthetized LD-TAG are localized preferentially in proximity to mitochondrial membranes named "contact zones". Once TAG's are released, they are mainly used in the mitochondria for ATP synthesis via oxidative phosphorylation from the ß-oxidation path. The "athlete paradox" states that the accumulation of TAG in the trained and insulin sensitive cells is in greater proportion than the TAG accumulation in cells from diabetic subjects with insulin resistance. This observation supports the hypothesis of mitochondrial dysfunction as a factor of TAG accumulation from a sustained lipid charge.

The protein family of perilipins (Plin) is associated with LD's and their scaffolding may affect the interaction between TAG and the mitochondria [13]. The Plin family consists of Plin1 to 5; the most common PAT (perilipin/ADRP/TIP47) interacts with LD in different proportions. In the liver, down-regulation of Plin2 promotes a reduction of hepatic steatosis and increases insulin sensitivity, albeit a reduction in both Plin2 and Plin3 is associated with insulin resistance [13]. In the heart, a Plin5 deficiency causes increased lipid oxidation, increased ROS production and decreased cardiac function. In heart and skeletal muscle TAG and FA are the main metabolic source of energy through the ß-oxidation pathway, suggesting a very tightly regulated process from cell storage to mitochondrial metabolic use. While TAG may come from LD, FA's are mainly transported in plasma as albumin-bound or as part of the very low density lipid-protein (VLDL) complex. Different transmembrane transporter systems are involved in their translocation to the inner cell compartment where the long chain fatty acid (LCFA) forms thioesters with coenzyme A (CoA). LCFA-CoA can form TAG for storage as LD, or can enter the outer mitochondrial membrane where CPT1 catalyzes the reaction of LCFA-CoA to LC-acylcarnitine. The former compound can actively cross the inner mitochondrial membrane with the exchange of carnitine for acylcarnitine. CACT is

highly expressed in tissues with predominant ß-oxidation metabolism.

age in the aging cell that occurs after natural oxidants depletion (**Figure 2B**).

74 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

The metabolism of FA in the mitochondrial matrix is sequentially catalyzed through a ß-oxidation process by four enzyme families: acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. While acyl-carnitine is converted back to acyl-CoA to enter the TCA cycle, dehydrogenases activity shows different affinity for short, medium, long and very long FA's [13]. Every cycle of ß-oxidation renders acyl-CoA and shortens the FA chain by 2 carbons providing the equivalents of electron donors NADH and FADH<sup>2</sup> which is the driving force for the synthesis of ATP. Although the ß-oxidation pathway is an effective way of ATP production, an overload of FA may play the role of un-coupler that exercises an inhibitory effect on the respiratory chain through a proton-phoric effect on the inner mitochondrial membrane. This effect results from the implicit effect of FA on mitochondrial membrane porosity by opening of the permeability transition pores, which results in the subsequent loss of electrical gradient and arrest of the respiratory chain [13]. The above concept favors the hypotheses of mitochondrial dysfunction from FA overload as the primary step of the insulin resistance state in obese patients. Other authors have found no mitochondrial respiratory changes in the steatotic liver but in mitochondria from skeletal muscle from a rodent model of high fat diet induced obesity [27]. Authors have proposed mitochondrial changes are due to an adaptation of the mitochondria to the high lipid charge rather than a defect per se in its function. No explanation was provided by the authors regarding the increase in ROS and inflammatory changes observed. Others suggested a protected effect from caloric lipid surplus against the development of metabolic dysfunction, as long as cells maintained functional adipocyte storage with low levels of tissue inflammation [28]. Once adipose accumulation saturates cell capacity, fatty excess spills over into other tissues leading to LD accumulation with subsequent lipid oxidation and an inflammatory response which precedes the metabolic syndrome manifestations [28]. The Delete in Breast Cancer-1 (DBC-1) protein is an important regulator of fat accumulation and storage in fat tissue which exercises its action by inhibition of SIRT1. In the DBC-/KO mice exposed to HFD, it was observed high plasma levels of FA with no liver steatosis, lower expression of senescence cells and increased storage of FA in the adipocytes with no development of insulin resistance [28]. Thus, DBC-1 protected liver and adipocytes from senescence by preservation of the fat compartment function with liver sparing and insulin sensitivity. Interestingly, a comparison has been made between this rodent model and the so called "healthy obese subjects" where there is fat accumulation but no signs of metabolic syndrome or systemic inflammation.

#### *2.1.1. Metabolism and inflammation*

Obesity depresses the anti-inflammatory effects of the heat shock proteins (HSP70) pathway, an inhibition that may contribute to the progression from NAFLD to NASH [29]. Excess of lipids and fuels trigger a low grade inflammatory response in both fat and liver tissues that correlates with the impaired insulin responsiveness. TBARS, a simple but fair estimate of lipoperoxidation/malondialdehyde (systemic oxidative stress) produced throughout the body was shown to be elevated in plasma from NASH patients when compared to normal subjects and its levels were correlated with insulin-resistance status [29]. The former response involved activation of the c-Jun NH2-terminal kinases (JNK's), endoplasmic reticulum (ER) stress, unfolded proteins response (UPR), and the ceramide pathway by blocking nuclear factor κB (NF-κB) expression at different levels [29]. In liver tissue, HSP70 downregulates TNF-α and inducible nitric oxide synthase (NOS2), genes that increase the inflammatory response in rodents. In addition, HSP70 in humans induces apoptosis and increases the concentration of cyclopentenone prostaglandins, a potent local inhibitor of inflammation. In human liver and fat tissues, the suppression of HSP70 was strongly correlated with the upregulation of JNK1 and JNK2 [29]. The authors hypothesized that the senescence-like state in fat cells have evolved in obese individuals as an adaptation to the metabolic overutilization of fat cells, supporting the observation that hepatocyte senescence predicts NAFLD progression to NASH and to cirrhosis. Patients with different grades of ESLD from NASH or HCV had significantly decreased levels of glutathione reduced and increased levels of glutathione oxidized in plasma when compared to healthy controls [30]. Therefore, a continuous increase in the cell oxidative stress consumes the antioxidant protective mechanisms and increases the spillage of oxidative molecules. Increased oxidative compounds accumulation may induce a progressive larger number of liver cells into senescence which in turn will enlarge the SASP component worsening the inflammatory environment with further increase of stressors into the liver mieleu by triggering an activation of local and systemic immune-regulators (**Figure 3**).

In old mice, hyperglycemia increased chromatin remodeling and polyploidy levels; changes observed as well in non-obese diabetic mice [31]. Genes involved in glycemic control and metabolism are also involved in inflammation such as Ppargc1a (PGC-1α). It acts on the histone deacetylase SIRT1 as a metabolic sensor in hepatocytes and increases gene activation involved in the gluconeogenesis pathway [31]. Furthermore, PGC-1α also plays a role in lipid metabolism [31]. Through the thyroid receptor pathway, it induces the expression of Srb1 (Scavenger receptor B member 1), enhances the uptake of cholesterol esters from high density lipoproteins (HDL) in the liver and inhibits the expression of Srebp-1 (sterol regulatory element-binding transcription factor-1) down-regulating fatty acid synthesis [31]. As SASP builds up due to an increasing number of cells entering senescence, an increasing insulin resistance state starts to develop with its manifestation, hyperglycemia which further favors replicative cell arrest.

> physiology. Adiponectin acts through three major physiologically different and distinctly expressed receptors: AdipoR1, AdipoR2 and T-cadherin. Adipo receptors mediate pleiotropic adiponectin actions through signaling mechanisms involving AMPK, ERK1/2, AKT and P38. In addition, Polymerase I and transcription release factor (PTRF) regulates adipocyte differentiation, perhaps fat cell senescence and thus may determine fat compartment expandability, condition that under continuous HFD exposure increase the spill-over of FFA to the liver in

> **Figure 3.** *Local and systemic responses that occur in the progression of NAFLD to NASH and ESLD associated HCC and their metabolomic print.* The liver local progression from NAFLD to NASH is associated with a local inflammatory response that eventually involves several other organs evolving into a systemic reaction. The systemic inflammatory response is associated with the development of an insulin resistance status and the metabolic syndrome phenotype: HTN, central obesity, DMType II and NASH. The continuous liver lipotoxicity decreases mitochondria function and decreases ATP production as well as enhances the secretion of lipid intermediates which are toxic to CD4 T lymphocytes. All together enhances a regenerative stimulus of senescence cells with mitochondrial dysfunction generating at some point a metabolic swap of ATP production to the cytosol, which may be associated with a mitochondrial generated apoptotic switch and in an environment of progressive fibrosis and therefore low oxygen and nutrients delivery favoring the survival of the already highly mutated cells which in turn have escaped physiological cell cycle control and immunorecognition assuring cell clone growth. Metabolic disturbances precede variations in cell cycle and genetic expression

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Rapamycin has an effect in cell life span with significant changes in the liver transcriptome, effects that are more pronounced when animals are exposed to caloric restriction [36, 37]. In a rodent model, rapamycin prevented senescent changes with significant differences by gender but with some common genetic pathways, mainly in the preservation of mitochondrial function. Those pathways included protein ubiquitination, NRF2-mediated oxidative stress response and glucocorticoid and OGF-1 signaling [36, 37]. Cell culture studies, indicated that treatment with rapamycin decreased mitochondrial membrane potential, decreased O<sup>2</sup>

combination to a pro-inflammatory adipokine repertoire [35].

*2.1.2. Signaling of energy expenditure and metabolism*

creating metabolic signatures of liver status in health and disease.

The fat compartment has emerged not only as an energy reservoir but as an endocrine organ capable of modulating metabolic states where the adiponectin/leptin ratio determines an anti or pro-inflammatory response. Leptin is a 167-aminoacid hormone expressed predominantly in adipocytes. Its signaling is an important determinant of food intake, adiposity and energy expenditure [32]. In the *ob/ob* mouse, a homozygous mutation in the gene that encodes leptin is associated with increased appetite, obesity and an insulin-resistant state. When leptin was provided to the ob/ob animal, there was a dramatic improvement in glucose homeostasis and energy metabolism. Although leptin related glucose homeostasis is largely conserved in rodents and humans, most subjects with insulin-resistant diabetes have a hyperleptinemic state with a central resistance to leptin [32]. Adiponectin is a protein hormone of 244 amino acids synthesized as a monomer of 28–30 kDa and assembled in various molecular weights: low, medium and high molecular weight (LMW, MMW and HMW) oligomers [33, 34]. HMW oligomers are the major relevant forms in terms of physiological activities of adiponectin while, low amounts of HMW oligomers represent an independent risk factor for several metabolic pathologies such as obesity-related diseases. Adiponectin plays a pivotal role in energy metabolism being an insulin-sensitizing hormone and it is involved in a wide variety of physiological cellular processes including inflammation, immunity and vascular Cellular Senescence and Their Role in Liver Metabolism in Health and Disease... http://dx.doi.org/10.5772/intechopen.71659 77

**Figure 3.** *Local and systemic responses that occur in the progression of NAFLD to NASH and ESLD associated HCC and their metabolomic print.* The liver local progression from NAFLD to NASH is associated with a local inflammatory response that eventually involves several other organs evolving into a systemic reaction. The systemic inflammatory response is associated with the development of an insulin resistance status and the metabolic syndrome phenotype: HTN, central obesity, DMType II and NASH. The continuous liver lipotoxicity decreases mitochondria function and decreases ATP production as well as enhances the secretion of lipid intermediates which are toxic to CD4 T lymphocytes. All together enhances a regenerative stimulus of senescence cells with mitochondrial dysfunction generating at some point a metabolic swap of ATP production to the cytosol, which may be associated with a mitochondrial generated apoptotic switch and in an environment of progressive fibrosis and therefore low oxygen and nutrients delivery favoring the survival of the already highly mutated cells which in turn have escaped physiological cell cycle control and immunorecognition assuring cell clone growth. Metabolic disturbances precede variations in cell cycle and genetic expression creating metabolic signatures of liver status in health and disease.

physiology. Adiponectin acts through three major physiologically different and distinctly expressed receptors: AdipoR1, AdipoR2 and T-cadherin. Adipo receptors mediate pleiotropic adiponectin actions through signaling mechanisms involving AMPK, ERK1/2, AKT and P38. In addition, Polymerase I and transcription release factor (PTRF) regulates adipocyte differentiation, perhaps fat cell senescence and thus may determine fat compartment expandability, condition that under continuous HFD exposure increase the spill-over of FFA to the liver in combination to a pro-inflammatory adipokine repertoire [35].

#### *2.1.2. Signaling of energy expenditure and metabolism*

stress, unfolded proteins response (UPR), and the ceramide pathway by blocking nuclear factor κB (NF-κB) expression at different levels [29]. In liver tissue, HSP70 downregulates TNF-α and inducible nitric oxide synthase (NOS2), genes that increase the inflammatory response in rodents. In addition, HSP70 in humans induces apoptosis and increases the concentration of cyclopentenone prostaglandins, a potent local inhibitor of inflammation. In human liver and fat tissues, the suppression of HSP70 was strongly correlated with the upregulation of JNK1 and JNK2 [29]. The authors hypothesized that the senescence-like state in fat cells have evolved in obese individuals as an adaptation to the metabolic overutilization of fat cells, supporting the observation that hepatocyte senescence predicts NAFLD progression to NASH and to cirrhosis. Patients with different grades of ESLD from NASH or HCV had significantly decreased levels of glutathione reduced and increased levels of glutathione oxidized in plasma when compared to healthy controls [30]. Therefore, a continuous increase in the cell oxidative stress consumes the antioxidant protective mechanisms and increases the spillage of oxidative molecules. Increased oxidative compounds accumulation may induce a progressive larger number of liver cells into senescence which in turn will enlarge the SASP component worsening the inflammatory environment with further increase of stressors into the liver mieleu by triggering an activation of local and systemic immune-regulators (**Figure 3**).

76 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

In old mice, hyperglycemia increased chromatin remodeling and polyploidy levels; changes observed as well in non-obese diabetic mice [31]. Genes involved in glycemic control and metabolism are also involved in inflammation such as Ppargc1a (PGC-1α). It acts on the histone deacetylase SIRT1 as a metabolic sensor in hepatocytes and increases gene activation involved in the gluconeogenesis pathway [31]. Furthermore, PGC-1α also plays a role in lipid metabolism [31]. Through the thyroid receptor pathway, it induces the expression of Srb1 (Scavenger receptor B member 1), enhances the uptake of cholesterol esters from high density lipoproteins (HDL) in the liver and inhibits the expression of Srebp-1 (sterol regulatory element-binding transcription factor-1) down-regulating fatty acid synthesis [31]. As SASP builds up due to an increasing number of cells entering senescence, an increasing insulin resistance state starts to develop with its manifestation, hyperglycemia which further favors replicative cell arrest.

The fat compartment has emerged not only as an energy reservoir but as an endocrine organ capable of modulating metabolic states where the adiponectin/leptin ratio determines an anti or pro-inflammatory response. Leptin is a 167-aminoacid hormone expressed predominantly in adipocytes. Its signaling is an important determinant of food intake, adiposity and energy expenditure [32]. In the *ob/ob* mouse, a homozygous mutation in the gene that encodes leptin is associated with increased appetite, obesity and an insulin-resistant state. When leptin was provided to the ob/ob animal, there was a dramatic improvement in glucose homeostasis and energy metabolism. Although leptin related glucose homeostasis is largely conserved in rodents and humans, most subjects with insulin-resistant diabetes have a hyperleptinemic state with a central resistance to leptin [32]. Adiponectin is a protein hormone of 244 amino acids synthesized as a monomer of 28–30 kDa and assembled in various molecular weights: low, medium and high molecular weight (LMW, MMW and HMW) oligomers [33, 34]. HMW oligomers are the major relevant forms in terms of physiological activities of adiponectin while, low amounts of HMW oligomers represent an independent risk factor for several metabolic pathologies such as obesity-related diseases. Adiponectin plays a pivotal role in energy metabolism being an insulin-sensitizing hormone and it is involved in a wide variety of physiological cellular processes including inflammation, immunity and vascular

Rapamycin has an effect in cell life span with significant changes in the liver transcriptome, effects that are more pronounced when animals are exposed to caloric restriction [36, 37]. In a rodent model, rapamycin prevented senescent changes with significant differences by gender but with some common genetic pathways, mainly in the preservation of mitochondrial function. Those pathways included protein ubiquitination, NRF2-mediated oxidative stress response and glucocorticoid and OGF-1 signaling [36, 37]. Cell culture studies, indicated that treatment with rapamycin decreased mitochondrial membrane potential, decreased O<sup>2</sup>

consumption, and increased ATP production [36, 37]. Other effects from transcriptome pathways include a decrease in proteasome activity in parallel with an increase in cell autophagy, suggesting protein quality improvement processes and increased resistance to oxidative cell stress effects associated with reduced cell aging.

acids (HETE's), while the metabolism of linoleic acids preferentially results in hydroxyloctadecadienoic acids (HODE's), metabolites known to modulate inflammation and carcinogenesis [41]. An excess of poly-unsaturated fatty acids could enhance a higher production of HETE's and/or HODE's with an override of pathways that enhances cancer development. Hepatic COX-2 overexpression induces spontaneous HCC formation in vitro and in mice through Akt, SKT33 and mTOR signaling cascades [42]. In the healthy liver, the inhibitor of the prostaglandin degrading enzyme 15-PGDH potentiates liver regeneration after partial hepatectomy when compared to control and sham animals [43]. Thus, prostaglandin active derivatives have the potential not only to modulate local inflammatory responses but to promote cell regeneration in the healthy cell and potentially reversal of cell arrest in the senes-

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Evidence seems to indicate a peculiar aging pattern for liver grafts after transplantation. Biological age of the graft does not correspond to its behavior when transplanted to a different environment of a younger recipient [44]. One of the most important intracellular protease systems is represented by the proteasome, the central catalytic unit of the ubiquitin-proteasome system (UPS). No difference in the accumulation of oxidized proteins and polyubiquitin conjugates with maintenance of their proteolytic activity was found in liver grafts after transplantation from younger donors to older recipient when compared to liver grafts from older donors placed into younger recipients. Furthermore, there was an increase of the ß5i/α4 ratio, suggesting a shift towards proteasomes containing immune-subunits [44]. Thus, it appears older liver grafts transplanted in younger recipients switched their biological metabolism to resemble the recipient's metabolic age. However, the pattern of liver cell senescence may differ. Liver biopsies, as judged by the senescent markers telomerase and SMP-30 from older transplanted livers showed histological damage in asymptomatic patients with up to 43% and 64% at 5 and

In the warm ischemic/reperfusion liver model, glycogen synthase kinase 3 (GSK-3) inhibition ameliorated liver injury upon reperfusion through an energy-dependent mitochondrial mechanism [46]. GSK-3 is a serine/threonine kinase regulated by inactivation through serine phosphorylation. GSK-3 inhibition down regulates the opening of mitochondrial permeability transition pore (MPTP) site, preventing leakage of mitochondrial respiratory chain proteins; a key step in the activation of caspase dependent apoptosis and therefore mitochondrial-dependent cell termination. This effect was present in young animals but abrogated in old animals, and a partial response was re-established in the older group by glucose infusion with hepatic glycogen build up storage [46]. Authors speculated that during reperfusion glycogen degradation provides mitochondrial fuel in forms of glutamate and α-ketoglutarate maintaining enough energy levels that preserve mitochondrial membrane integrity or mitohormesis lowering ROS production, factors needed to decrease MPTP susceptibility. Former approach in the human was entertained, where liver graft glycogen replenishment was performed during the donor phase and evaluated upon reperfusion [47, 48]. Metabolic benefit with improved organ graft function was observed only in borderline grafts and the ones with high fat content. Nevertheless, the concept of metabolic replenishment with further graft function improvement may be refined by

strategies of ex-vivo euthermic graft perfusion prior implantation [49–56].

cent cell.

**2.2. Metabolism in the liver graft**

10 years, respectively [45].

A highly conserved signaling pathway in all eukaryotic cells is the Target of Rapamycin (TOR), which plays a central role as regulator of cell growth and metabolism. The mammalian TOR complex (mTOR) encompasses two structurally and distinct proteins. While the mTORC1 is associated with anabolic processes such as protein synthesis, lipid synthesis, nutrient uptake and inhibition of catabolic processes including autophagy, mTORC2 is an insensitive to rapamycin regulation protein. mTOR2 becomes activated by a family of kinases like the serum/glucocorticoid kinase (SGK) and protein kinase C (PKC). mTORC1 is upregulated by growth factors, cellular energy status and is inhibited by the macrolide rapamycin [36–38]. Protein synthesis is one of the most energy demanding cell functions; a favorable redox status activates mTORC1 which in turn exercises its actions down-stream through the ribosomal protein 6 kinase (S6K) and the eukaryotic translation initiation factor 4E binding protein (4R-BP) [36–38]. Due to the high demand of ATP, mitochondrial function regulation is of paramount importance on mTOR signaling pathways. Moreover, mitochondrial dysregulation and continuous mTOR activation may play a metabolic central role in the transformation and survival of cancer cells. Mutated cells with malignant potential may shift their bioenergetic state from ATP mitochondrial production to cytosol ATP production through the tricarboxylic acid (TCA) cycle. A connection between mTORC2 and mitochondrial function and cancer appears to be dependent through the HK2 pathway [38]. Nevertheless, recently mTORC2 has been linked to cytoskeleton regulation through the actin remodeling pathway, which has been suggested to have an effect on insulin sensitivity/resistance balance [36, 37]. Whole energy expenditure was affected by the mTORC1 signaling pathway, as demonstrated in the tissue specific knockout mice where a down-regulation of signaling pathways on adipose tissue also impacts on thermogenesis and systemic sensitivity to insulin [38]. In addition, mTORC2 signaling pathway, in the same animal model was a crucial regulator of liver and pancreas metabolism affecting animal growth and insulin homeostasis. mTORC 1 and 2 signaling in the liver affects systemic glucose and insulin homeostasis mainly due to their effects on Akt and hepatic glucose uptake. Interestingly, liver tumors in the tissue specific raptor knockout mice, showed a shift from glucose to glutamine as the main fuel source, making tumor cells glutamine addictive with high expression of mTORC1 and FGF-21. Rapamycin treatment may be beneficial as it may inhibit growth on glutamine addictive tumors. Some liver transplant programs switch their immunosuppression protocol from tacrolimus to rapamycin in patients with high risk for HCC recurrence after transplantation. However and on retrospective studies, its effect on long term overall survival on patients after liver transplantation for HCC, have had conflicting results [39, 40].

The functional relationship between poly-unsaturated lipid metabolism, inflammation and cancer development has been discussed in multiple avenues. Cyclooxygenases (COX's) and lipoxygenases (LOX's) are enzymatic families that metabolize poly-unsaturated fatty acids. COX is present in two isoforms (COX-1 and COX-2) that produce prostaglandins (PG's) and thromboxanes, respectively [41]. LOXs constitute a family of dioxygenases that insert O<sup>2</sup> into poly-unsaturated fatty acids with regional specificity [41]. These metabolites are biologically active hydroperoxyeicosatetraenoic acids that upon reduction forms hydro-eicosatetranoic acids (HETE's), while the metabolism of linoleic acids preferentially results in hydroxyloctadecadienoic acids (HODE's), metabolites known to modulate inflammation and carcinogenesis [41]. An excess of poly-unsaturated fatty acids could enhance a higher production of HETE's and/or HODE's with an override of pathways that enhances cancer development. Hepatic COX-2 overexpression induces spontaneous HCC formation in vitro and in mice through Akt, SKT33 and mTOR signaling cascades [42]. In the healthy liver, the inhibitor of the prostaglandin degrading enzyme 15-PGDH potentiates liver regeneration after partial hepatectomy when compared to control and sham animals [43]. Thus, prostaglandin active derivatives have the potential not only to modulate local inflammatory responses but to promote cell regeneration in the healthy cell and potentially reversal of cell arrest in the senescent cell.

#### **2.2. Metabolism in the liver graft**

consumption, and increased ATP production [36, 37]. Other effects from transcriptome pathways include a decrease in proteasome activity in parallel with an increase in cell autophagy, suggesting protein quality improvement processes and increased resistance to oxidative cell

A highly conserved signaling pathway in all eukaryotic cells is the Target of Rapamycin (TOR), which plays a central role as regulator of cell growth and metabolism. The mammalian TOR complex (mTOR) encompasses two structurally and distinct proteins. While the mTORC1 is associated with anabolic processes such as protein synthesis, lipid synthesis, nutrient uptake and inhibition of catabolic processes including autophagy, mTORC2 is an insensitive to rapamycin regulation protein. mTOR2 becomes activated by a family of kinases like the serum/glucocorticoid kinase (SGK) and protein kinase C (PKC). mTORC1 is upregulated by growth factors, cellular energy status and is inhibited by the macrolide rapamycin [36–38]. Protein synthesis is one of the most energy demanding cell functions; a favorable redox status activates mTORC1 which in turn exercises its actions down-stream through the ribosomal protein 6 kinase (S6K) and the eukaryotic translation initiation factor 4E binding protein (4R-BP) [36–38]. Due to the high demand of ATP, mitochondrial function regulation is of paramount importance on mTOR signaling pathways. Moreover, mitochondrial dysregulation and continuous mTOR activation may play a metabolic central role in the transformation and survival of cancer cells. Mutated cells with malignant potential may shift their bioenergetic state from ATP mitochondrial production to cytosol ATP production through the tricarboxylic acid (TCA) cycle. A connection between mTORC2 and mitochondrial function and cancer appears to be dependent through the HK2 pathway [38]. Nevertheless, recently mTORC2 has been linked to cytoskeleton regulation through the actin remodeling pathway, which has been suggested to have an effect on insulin sensitivity/resistance balance [36, 37]. Whole energy expenditure was affected by the mTORC1 signaling pathway, as demonstrated in the tissue specific knockout mice where a down-regulation of signaling pathways on adipose tissue also impacts on thermogenesis and systemic sensitivity to insulin [38]. In addition, mTORC2 signaling pathway, in the same animal model was a crucial regulator of liver and pancreas metabolism affecting animal growth and insulin homeostasis. mTORC 1 and 2 signaling in the liver affects systemic glucose and insulin homeostasis mainly due to their effects on Akt and hepatic glucose uptake. Interestingly, liver tumors in the tissue specific raptor knockout mice, showed a shift from glucose to glutamine as the main fuel source, making tumor cells glutamine addictive with high expression of mTORC1 and FGF-21. Rapamycin treatment may be beneficial as it may inhibit growth on glutamine addictive tumors. Some liver transplant programs switch their immunosuppression protocol from tacrolimus to rapamycin in patients with high risk for HCC recurrence after transplantation. However and on retrospective studies, its effect on long term overall survival on patients

after liver transplantation for HCC, have had conflicting results [39, 40].

The functional relationship between poly-unsaturated lipid metabolism, inflammation and cancer development has been discussed in multiple avenues. Cyclooxygenases (COX's) and lipoxygenases (LOX's) are enzymatic families that metabolize poly-unsaturated fatty acids. COX is present in two isoforms (COX-1 and COX-2) that produce prostaglandins (PG's) and thromboxanes, respectively [41]. LOXs constitute a family of dioxygenases that insert O<sup>2</sup>

poly-unsaturated fatty acids with regional specificity [41]. These metabolites are biologically active hydroperoxyeicosatetraenoic acids that upon reduction forms hydro-eicosatetranoic

into

stress effects associated with reduced cell aging.

78 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

Evidence seems to indicate a peculiar aging pattern for liver grafts after transplantation. Biological age of the graft does not correspond to its behavior when transplanted to a different environment of a younger recipient [44]. One of the most important intracellular protease systems is represented by the proteasome, the central catalytic unit of the ubiquitin-proteasome system (UPS). No difference in the accumulation of oxidized proteins and polyubiquitin conjugates with maintenance of their proteolytic activity was found in liver grafts after transplantation from younger donors to older recipient when compared to liver grafts from older donors placed into younger recipients. Furthermore, there was an increase of the ß5i/α4 ratio, suggesting a shift towards proteasomes containing immune-subunits [44]. Thus, it appears older liver grafts transplanted in younger recipients switched their biological metabolism to resemble the recipient's metabolic age. However, the pattern of liver cell senescence may differ. Liver biopsies, as judged by the senescent markers telomerase and SMP-30 from older transplanted livers showed histological damage in asymptomatic patients with up to 43% and 64% at 5 and 10 years, respectively [45].

In the warm ischemic/reperfusion liver model, glycogen synthase kinase 3 (GSK-3) inhibition ameliorated liver injury upon reperfusion through an energy-dependent mitochondrial mechanism [46]. GSK-3 is a serine/threonine kinase regulated by inactivation through serine phosphorylation. GSK-3 inhibition down regulates the opening of mitochondrial permeability transition pore (MPTP) site, preventing leakage of mitochondrial respiratory chain proteins; a key step in the activation of caspase dependent apoptosis and therefore mitochondrial-dependent cell termination. This effect was present in young animals but abrogated in old animals, and a partial response was re-established in the older group by glucose infusion with hepatic glycogen build up storage [46]. Authors speculated that during reperfusion glycogen degradation provides mitochondrial fuel in forms of glutamate and α-ketoglutarate maintaining enough energy levels that preserve mitochondrial membrane integrity or mitohormesis lowering ROS production, factors needed to decrease MPTP susceptibility. Former approach in the human was entertained, where liver graft glycogen replenishment was performed during the donor phase and evaluated upon reperfusion [47, 48]. Metabolic benefit with improved organ graft function was observed only in borderline grafts and the ones with high fat content. Nevertheless, the concept of metabolic replenishment with further graft function improvement may be refined by strategies of ex-vivo euthermic graft perfusion prior implantation [49–56].

#### **2.3. The chronically diseased liver**

Hepatocyte senescence expression has been shown to be present in up to 80% of the cells in advanced liver disease [57]. The effects of insulin in the liver cell are mediated through two main cellular pathways: the phosphatidylinositol 3-kinase (PI3K)-Akt and the Ras-MAP kinase (MAPK) pathways. While both pathways are active in the regulation of cellular growth, proliferation and differentiation the PI3K-Akt mediates the metabolic actions of insulin. Those actions include activation of mTOR1 and its S6 kinase and the inactivation of glycogen synthase kinase-3 (GSK3) as well as its AS160 with nuclear exclusion of the Forkhead box protein (Fox01) [57]. In culture, HepG2 cell lines showed a signaling defect downstream of the Akt pathway with an impact upon insulin mediated Fox01 cytosol sequestration and AS160 phosphorylation; a cascade that translated into insulin resistance of older cells when compared to younger cells. Nevertheless, maintenance of the senescent state requires an active role in the transcriptional activity of Fox01 as cell cycle inhibitor, even in the presence of growth factors. Thus, it appears gluconeogenesis and insulin resistance are unwanted but unavoidable effects of Fox01 gene, which is involved in cell cycle arrest, detoxification of oxygen species, DNA repair and gluconeogenesis [57].

cells, optimal excitation-contraction coupling requires an optimal energy and O<sup>2</sup>

of election during food abundance against metabolic disease deserves further studies.

receptors), the SR Ca2+ pumps and the sarcolemmal Na+

in turn affects the Ca2+ handling at the sarcoplasm reticulum (SR) release channels (ryanodine

in average 100,000 times per day catalyzing about 6 kg of ATP to ADP. The mitochondrion provides the ATP needed for contraction (≈66%) and the ATP needed for ion transporting (≈33%) essential for the cardiac electrical activity. Thus, the link among lipid supply and mitochondrial function, insulin sensitivity/resistance and ion pump exchange is established for optimal cardiac function or dysfunction in the obese individual [13]. Perhaps, there is no argument lipid oxidation confers a metabolic advantage during starvation and exercise, but its role as the fuel

In liver, cellular senescence is associated with a pro-fibrogenic environment and the relation between advanced liver fibrosis and shortening of the cell telomere appears to be consistent [11, 60]. Telomeres are repetitive DNA sequences (TTAGGG) associated with the specialized protein shelterin. They are located at the chromosomal end acting as a cap that stabilizes and protect the chromosome from erosion and miss-identification as DNA breaks. During normal cell division, telomeres shorten due to the "end replicating problem": the inability of DNA polymerases to fully replicate the 3′ end of chromosomes [61]. Germline cells overcome this problem by expressing telomerase, a reverse transcriptase that maintains telomere length by synthetizing new DNA sequences at the end of the chromosome [60]. The telomerase complex includes a reverse transcriptase (TERT) and the RNA component (TERC) [61]. In other somatic cells, continuous cell division results in telomere shortening which in turn start signaling cell arrest mechanisms, i.e. senescence or apoptosis. Failure of cell arrest signaling, as in a silence p53 state sparks further cell proliferation with chromosomal end-to-end fusions and instability. In addition, exhaustion of liver regenerative paths and invested mechanisms of telomere repair could be overcome under continuous and chronic cell injury with subsequent acceleration of cell senescence and aging. Some studies had shown that telomere biology is involved in HCC initiation and its progression [60]. Therefore, telomere shortening is a physiological marker of cell aging signaling and/or cell arrest preventing further cell division; failure of cell arrest may end in chromosomal instability and subsequent mutations favoring tumor development [11, 61]. In fact, the strength of the DNA damage response (DDR) in the normal cell depends ultimately to the degree of p53 gene regulation: a higher p53 response is associated with apoptosis, a lower response is associated with cell senescence and a silence p53 response may favor tumor development and growth [62]. In addition, a sirtuin (SIRT7) showed an in vivo hyperacetylation of p53 and the SIRT7 knockout mice suffered among other maladies steatotic liver disease. Sirtuins were initially identified in yeast as the Silent Information Regulator (SIR). In mammals, SIRT protein family comprises seven distinct members involved in cellular survival, senescence and tumorigenesis [62]. The SIRT7 knockout mice showed a 2.5 fold increase in the liver triglyceride content and an increased accumulation of hepatocyte inflammatory markers [62]. Findings that were associated with liver cells mitochondrial dysfunction through a deacetylate GABPß1 mitochondrial protein pathway and with the development of HCC through maintaining a deacetylated state of H3K18 at promoters sites of many tumor suppressor genes [62].

Lipodystrophic syndromes are rare and heterogeneous diseases, genetic or acquired, where partial atrophy is associated with a phenotype consistent with insulin-resistant diabetes, dyslipidemia and NAFLD. Although the genetic cause of these syndromes are largely

supply which

81

/Ca++ exchanger. The heart at rest beats

http://dx.doi.org/10.5772/intechopen.71659

Cellular Senescence and Their Role in Liver Metabolism in Health and Disease...

FA overload can damage the respiratory chain in the mitochondrion through a dual role: as an un-coupler and as an inhibitor [13]. Impairment of the key respiratory state 4 → 3 can occur via inhibition of ATP-synthase thereby producing an increase production of ROS irrespective of ADP concentration. The concept of redox-optimized ROS balance (R-ORB) postulates that ROS efflux from the mitochondrion will attain a minimum at intermediate values of oxidation, when VO<sup>2</sup> reaches a maximum following ADP stimulation. Under state 3 respiration, GSH and thioredoxin systems are essential for minimizing ROS release from the mitochondria [13]. Moreover, mitochondria from cells with chronic liver disease under oxidant challenge displayed a two-fold increase in H2 O2 emission when compared to controls along with a 50% decrease in GSH [13]. Since 90% of GSH in plasma is excreted by the liver, glutathione sp. could serve as a surrogate of cell/mitochondrial stress and their ratio in plasma may reflect overall liver redox balance [24]. In animal models of liver malignancy, with or without cirrhosis glutathione sp. (glutathione reduced-GSH, glutathione oxidized-GSSG and ophthalmate) predicted the growth of malignant cells on normal livers as early as 14 days after malignant cells implantation and differentiated animals with cirrhosis by tumor status (HCC+ vs. HCC−) [58, 59]. Furthermore, glutathione sp. in plasma were part of the metabolic signature that discriminated healthy controls and subjects after liver transplantation with normal graft function from subjects with chronic liver disease (**Figure 3**). In addition, metabolic prints graded patient's degree of end stage liver disease which correlated with the MELD score, and they were able to separate patients with cirrhosis by tumor status, i.e. HCC+ vs. HCC− [30].

Others argued mitochondrial dysfunction by FA's respiratory chain uncoupling is incompatible with thermo-regulatory principles that governs mitochondrial respiratory chain through energy demand: intracellular lipids will accumulate whenever FA's supply exceeds the energy needs of the cell [13]. While TAG-LD in cells from a trained individual increases as the source of energy, in the diabetic obese subject TAG-LD are the result of accumulation with the subsequent potential overproduction of lipid derived toxins in the form of LCFA-CoA, diacylglycerides (DAG) and ceramide, metabolites responsible, at least in part for the development of insulin cell resistance [13]. The former theory is attractive in the heart and skeletal muscle. In contractile cells, optimal excitation-contraction coupling requires an optimal energy and O<sup>2</sup> supply which in turn affects the Ca2+ handling at the sarcoplasm reticulum (SR) release channels (ryanodine receptors), the SR Ca2+ pumps and the sarcolemmal Na+ /Ca++ exchanger. The heart at rest beats in average 100,000 times per day catalyzing about 6 kg of ATP to ADP. The mitochondrion provides the ATP needed for contraction (≈66%) and the ATP needed for ion transporting (≈33%) essential for the cardiac electrical activity. Thus, the link among lipid supply and mitochondrial function, insulin sensitivity/resistance and ion pump exchange is established for optimal cardiac function or dysfunction in the obese individual [13]. Perhaps, there is no argument lipid oxidation confers a metabolic advantage during starvation and exercise, but its role as the fuel of election during food abundance against metabolic disease deserves further studies.

**2.3. The chronically diseased liver**

80 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

tion, when VO<sup>2</sup>

displayed a two-fold increase in H2

Hepatocyte senescence expression has been shown to be present in up to 80% of the cells in advanced liver disease [57]. The effects of insulin in the liver cell are mediated through two main cellular pathways: the phosphatidylinositol 3-kinase (PI3K)-Akt and the Ras-MAP kinase (MAPK) pathways. While both pathways are active in the regulation of cellular growth, proliferation and differentiation the PI3K-Akt mediates the metabolic actions of insulin. Those actions include activation of mTOR1 and its S6 kinase and the inactivation of glycogen synthase kinase-3 (GSK3) as well as its AS160 with nuclear exclusion of the Forkhead box protein (Fox01) [57]. In culture, HepG2 cell lines showed a signaling defect downstream of the Akt pathway with an impact upon insulin mediated Fox01 cytosol sequestration and AS160 phosphorylation; a cascade that translated into insulin resistance of older cells when compared to younger cells. Nevertheless, maintenance of the senescent state requires an active role in the transcriptional activity of Fox01 as cell cycle inhibitor, even in the presence of growth factors. Thus, it appears gluconeogenesis and insulin resistance are unwanted but unavoidable effects of Fox01 gene, which is involved in

cell cycle arrest, detoxification of oxygen species, DNA repair and gluconeogenesis [57].

FA overload can damage the respiratory chain in the mitochondrion through a dual role: as an un-coupler and as an inhibitor [13]. Impairment of the key respiratory state 4 → 3 can occur via inhibition of ATP-synthase thereby producing an increase production of ROS irrespective of ADP concentration. The concept of redox-optimized ROS balance (R-ORB) postulates that ROS efflux from the mitochondrion will attain a minimum at intermediate values of oxida-

GSH and thioredoxin systems are essential for minimizing ROS release from the mitochondria [13]. Moreover, mitochondria from cells with chronic liver disease under oxidant challenge

decrease in GSH [13]. Since 90% of GSH in plasma is excreted by the liver, glutathione sp. could serve as a surrogate of cell/mitochondrial stress and their ratio in plasma may reflect overall liver redox balance [24]. In animal models of liver malignancy, with or without cirrhosis glutathione sp. (glutathione reduced-GSH, glutathione oxidized-GSSG and ophthalmate) predicted the growth of malignant cells on normal livers as early as 14 days after malignant cells implantation and differentiated animals with cirrhosis by tumor status (HCC+ vs. HCC−) [58, 59]. Furthermore, glutathione sp. in plasma were part of the metabolic signature that discriminated healthy controls and subjects after liver transplantation with normal graft function from subjects with chronic liver disease (**Figure 3**). In addition, metabolic prints graded patient's degree of end stage liver disease which correlated with the MELD score, and they were able to

Others argued mitochondrial dysfunction by FA's respiratory chain uncoupling is incompatible with thermo-regulatory principles that governs mitochondrial respiratory chain through energy demand: intracellular lipids will accumulate whenever FA's supply exceeds the energy needs of the cell [13]. While TAG-LD in cells from a trained individual increases as the source of energy, in the diabetic obese subject TAG-LD are the result of accumulation with the subsequent potential overproduction of lipid derived toxins in the form of LCFA-CoA, diacylglycerides (DAG) and ceramide, metabolites responsible, at least in part for the development of insulin cell resistance [13]. The former theory is attractive in the heart and skeletal muscle. In contractile

O2

separate patients with cirrhosis by tumor status, i.e. HCC+ vs. HCC− [30].

reaches a maximum following ADP stimulation. Under state 3 respiration,

emission when compared to controls along with a 50%

In liver, cellular senescence is associated with a pro-fibrogenic environment and the relation between advanced liver fibrosis and shortening of the cell telomere appears to be consistent [11, 60]. Telomeres are repetitive DNA sequences (TTAGGG) associated with the specialized protein shelterin. They are located at the chromosomal end acting as a cap that stabilizes and protect the chromosome from erosion and miss-identification as DNA breaks. During normal cell division, telomeres shorten due to the "end replicating problem": the inability of DNA polymerases to fully replicate the 3′ end of chromosomes [61]. Germline cells overcome this problem by expressing telomerase, a reverse transcriptase that maintains telomere length by synthetizing new DNA sequences at the end of the chromosome [60]. The telomerase complex includes a reverse transcriptase (TERT) and the RNA component (TERC) [61]. In other somatic cells, continuous cell division results in telomere shortening which in turn start signaling cell arrest mechanisms, i.e. senescence or apoptosis. Failure of cell arrest signaling, as in a silence p53 state sparks further cell proliferation with chromosomal end-to-end fusions and instability. In addition, exhaustion of liver regenerative paths and invested mechanisms of telomere repair could be overcome under continuous and chronic cell injury with subsequent acceleration of cell senescence and aging. Some studies had shown that telomere biology is involved in HCC initiation and its progression [60]. Therefore, telomere shortening is a physiological marker of cell aging signaling and/or cell arrest preventing further cell division; failure of cell arrest may end in chromosomal instability and subsequent mutations favoring tumor development [11, 61]. In fact, the strength of the DNA damage response (DDR) in the normal cell depends ultimately to the degree of p53 gene regulation: a higher p53 response is associated with apoptosis, a lower response is associated with cell senescence and a silence p53 response may favor tumor development and growth [62]. In addition, a sirtuin (SIRT7) showed an in vivo hyperacetylation of p53 and the SIRT7 knockout mice suffered among other maladies steatotic liver disease. Sirtuins were initially identified in yeast as the Silent Information Regulator (SIR). In mammals, SIRT protein family comprises seven distinct members involved in cellular survival, senescence and tumorigenesis [62]. The SIRT7 knockout mice showed a 2.5 fold increase in the liver triglyceride content and an increased accumulation of hepatocyte inflammatory markers [62]. Findings that were associated with liver cells mitochondrial dysfunction through a deacetylate GABPß1 mitochondrial protein pathway and with the development of HCC through maintaining a deacetylated state of H3K18 at promoters sites of many tumor suppressor genes [62].

Lipodystrophic syndromes are rare and heterogeneous diseases, genetic or acquired, where partial atrophy is associated with a phenotype consistent with insulin-resistant diabetes, dyslipidemia and NAFLD. Although the genetic cause of these syndromes are largely unknown, most of the monogenic diseases have in common primary alterations in the fat tissue consistent with disturbances of the adipogenesis process or defects in the formation, maintenance and/or regulation of the lipid droplet [63]. Acquired syndromes are seen mainly after HIV therapy with anti-retroviral agents as zidovudine and stavudine (tNRTl's). Agents known to render mitochondrial toxicity with metabolic disturbances similar to the metabolic syndrome seen in obesity. This metabolic adverse effects include premature aging associated with impaired prelamin-A maturation [63]. Lamin-A alterations could produce fragile nuclear envelopes, alter chromatin organization, increase oxidative stress and promote premature senescence at the cellular level. The metabolic disturbances observed in genetic or acquired lipodystrophic syndromes support the hypothesis of a primary fat compartment dysfunction as the source of metabolic disturbances, similar to the ones detected in obesity.

cell. Other fatty acid downregulated proteins included SORL1 (involved in the uptake of LDL), ACSM2B (a medium-chain fatty-acid-CoA ligase) and PHGDH indirectly involved in amino-acid synthesis [18]. In addition, senescent cells secreted a variety of bioactive molecules including proinflammatory cytokines and chemokines that may influence extracellular matrix and the microenvironment but as well modulate the immune response with the promotion of macrophage migration leading to further increase in the inflammatory mielue [65]. Monocyte chemotactic protein (MCP-1) could provide a signal for monocyte recruitment into the liver followed by activation of Kupffer cells with the upregulation of death ligands. The expression of Fas ligand, TNFα, and TNF-related apoptosis inducing ligand (TRAIL) further aggravates lipo-apoptosis [66]. In addition the FFA palmitate increases the expression of TRAIL and abrogation of the TRAIL

Cellular Senescence and Their Role in Liver Metabolism in Health and Disease...

http://dx.doi.org/10.5772/intechopen.71659

83

receptor expression suppresses the inflammation induced by nutrient excess in mice [66].

in the presence of oxygen [70].

Prior assumptions on cellular senescence determined that cell cycle arrest was a mechanism to protect the cell towards tumorigenesis. Nevertheless, it has been shown that the cell in cycle arrest can produce pro-inflammatory mediators, the senescence-associated secretory phenotype that promotes tumor growth [67]. During chronic liver disease, senescent machinery becomes "hijacked" perhaps triggering proliferation and transformation of hepatocytes, thus, promoting metabolic adaptation which may enhance tumor grafting and growth [68, 69]. The above metabolic paths could at least in part, be mediated by the over expression of the phosphatase and tensin homolog (PTEN) described in T-leukemia but later shown in liver tumors to inhibit the pentose phosphatase pathway (PPP) by binding to glucose-6-phophodiesterase (G6PD). With no active G6DP dimer, cells favor glycolysis with the production of lactate even

Aging and senescent liver cells have different genetic paths that may converge to similar metabolic traits. Aging liver cells have a proliferative response after injury associated with the repression of C/EBPα, Farnesoid X Receptor (FXR), telomere reverse transcriptase (TERT), and a decrease in the Wnt signaling pathway [71, 72]. A physiological Wnt signaling pathway involves a soluble ligand that binds to the Frizzled receptor (Fzd) and the LRP5/6 co-receptor on the plasma membrane; this interaction activates the cytoplasmic Disheveled protein which inhibits the ß-catenin (Ctnnb1) destruction complex (APC, GSK3ß, and Axin) by preventing Ctnnb1 phosphorylation and its subsequent destruction. Stable ß-catenin (intact Wnt signaling) translocates to the nucleus to form a complex with Lef and Tcf transcription factors that target genes as c-Myc and Cyclin D1. In cell culture and a mice model of HCC, tumor growth was ablated by the suppression of N-Myc downregulated gene 1 (NDRG1) expression; it promoted HCC cells to go into cell arrest [73]. The induction of senescence on malignant cells was accomplished by upregulation of the tumor suppressor genes p53, p21 and p16 in addition to decreased phosphorylated Rb. Senescent liver cells response to injury included transcription of Nf-kB, Myb, Nkx2-1, Nr5a2 and Ep300 factors; proteins known to be involved in inflammation, cell differentiation, lipid metabolism and chromatin remodeling. In addition, the chronic inflammatory phenotype of senescent cells induces telomere dysfunction and accelerates liver cell aging [74]. Thus, decreased physiological cell signaling that occurs with aging plus stress induced cell senescence may add to the lipid toxic microenvironment by promoting a vicious circle that overrules redundant mechanisms that prevent uncontrolled cell division. Mechanisms that imply an apoptosis "switch" from a pro-apoptotic to an anti-apoptotic status. Nonetheless, it is no clear the role of mitochondrial Bcl-2 proteins family and their expression may determine cellular fate [75].

Chronic liver disease is associated with an increased translocation of intestinal bacteria contributory to the liver inflammatory response and may promote the development of HCC [12]. Liposaccharide (LPS) produced by Gram (−) bacteria hosted in intestines from obese humans and rodents was associated with the transition of NAFLD to NASH and consequently to its progression to cirrhosis and HCC. LPS is recognized by the Toll like receptor 4 (TLR4) which is expressed upon cell activation on migrating and local macrophages (Kupffer cells). TLR4 is central for the secretion of TNF-ß and IL-6, cytokines present in the chronic inflammatory environment that precedes the detection of malignancy [12]. Further support to the role of LPS was found by interventions such as gut sterilization, removal of LPS or inactivation of TLR4; maneuvers that diminished tumor growth in chronically injured livers [12]. In experimental models, dietary or genetic obesity alterations on the gut microbiota increased levels of metabolites like deoxycholic acid (DCA) that in turn damages DNA. The enterohepatic circulation may further enhance the concentration of such metabolites by both encouraging the senescent-associated secretory phenotype response and favoring a tumor-promoting environment.
