**3. Roles of individual phospholipids**

#### **3.1 SM behaviour**

64 Liver Regeneration

extracted from them was unlabelled, whereas all the label present in the nuclei was recovered from the nuclear membrane fraction. Radio-iodination of isolated chromatin

The presence of nuclear phospholipids was also demonstrated in a large variety of tissues

Cocco et al. (1988) demonstrated the presence of phosphoinositides which may act as nuclear signals through the generation of DAG (diacylglycerol) due to specific phospholipase activity (D' Santos et al. 1998, Cocco et al. 2001, Martelli et al. 2001, Irvine 2003). The presence of neutral lipids was demonstrated by Song and Rebel (1987) and of cholesterol (CHO) by Albi and Viola-Magni (2002). The presence in chromatin of the enzymes sphingomyelinase (SMase), sphingomyelin synthase (SMsynthase), phospholipases of phosphatidylcholine (PC) and phosphatidylinositol (PI) and sphingomyelin reverse synthase associated demonstrated the existence of a metabolic cycle for such phospholipids.

There is evidence for the presence of a phospholipid-calcium-dependent protein kinase C (PKC) in nuclei together with the enzymes involved with phospholipid turnover (Alessenko and Burlakova (2002). Protein kinase C interacts with the nuclear phosphoinositol and sphingomyelin cycle products. This fact implies the possibility that signal transduction events could also occur at the nuclear level during the induction of cell proliferation,

In this review, it is intended to consider the composition of the lipids present in chromatin, the enzymes associated with the metabolism of these lipids, their possible roles in normal

After 70% hepatectomy, the liver first regenerates the hepatocytes prior to regenerating the other cell types. The first peak of new hepatocytes is observed after 24 h in 30 day-old rats with a second peak occurring after 36 h. The other cell types, including Kupffer cells and endothelial cells, blood vessels and others, proliferate on the third day (Bresnick 1971).

The synthesis of phospholipids was studied after partial hepatectomy (Viola-Magni et al. 1985 b) in the both hepatocyte nuclei and chromatin. The whole nuclei showed an increase in phospholipid synthesis after six hours reaching a peak at 12 h, after which, a constant level was maintained until 48 h. The synthesis of phospholipids in the chromatin increased at 12h to reach a peak at 18h, which level remained until 24 h. This was followed by a peak at 30 h, a timing that marks the end of the first proliferation peak and the start of the second wave of mitosis (Viola-Magni et al, 1985b). It is to be noted that DNA synthesis starts at 12h after partial hepatectomy to reach a maximum at about 24h (Viola-Magni 1985b). This shows that the initiation of both DNA and phospholipid synthesis are occurring at about the same time. Since the second peak of DNA synthesis starts at 30 h, the end of the DNA synthesis of the first peak and the initiation of the second happens at the same time as the consequencial

peak of lipids observed is the algebraic summation of the two events.

**2. Composition of the chromatin associated phospholipids in normal v** 

showed the presence of label thus confirming the presence of lipids. (Albi et al.1994).

including tumour cells (Splanger et al. 1975, Upreti et al. 1983).

(Albi and Viola-Magni 2004, Albi 2011).

hepatocytes, the cell cycle and regenerating liver.

differentiation and apoptosis.

**regenerating liver** 

It has been hypothesized that SM may have a role in stabilising the DNA molecule. The decrease of SM at the start of the S phase may be associated with the unwinding of the DNA helix and the increase of SM at the end of S-phase may be linked to the rewinding of the DNA helix. A similar behaviour of SM was also observed in other models by different authors (Stillman 1996, Alessenko and Chatterjee 1995).

### **3.2 PS behaviour**

PS is also one the PLs present in a higher amount in chromatin with respect to the level seen in the nuclear membrane. PS increases when DNA synthesis starts during liver regeneration. A possible role for PS in the chromatin may be the stimulation of DNA polymerase as has been shown *in vitro* by Manzoli et al. (1981).

#### **3.3 PC behaviour**

This PL is mostly present in the nuclear membrane with only a small amount in the chromatin. The chromatin PC has a different composition to that of the microsomal fraction in that it contains many unsaturated forms of the monoenic fraction with respect to the microsomal PC that was enriched with tetraene and exaene fractions (Albi et al. 1994).

The chromatin PC does not present a particular modification during liver regeneration except that DAG, a product of PC, increases at 12 h hand 30 h in parallel with the initiation of the two waves of proliferating activity when DNA synthesis starts (Viola-Magni et al.1985b).

#### **3.4 PE behaviour**

Although PE represents 22% of the total PL present in chromatin, its behaviour is similar to that of SM. However, no precise indication as to its role(s) in liver regeneration has been observed.

#### **3.5 PI behaviour**

PI represents 19% of the PLs present in chromatin (Viola-Magni et al. 1985a, Albi et al. 1994). No specific variations in the amounts of PI have been described during liver regeneration

Possible Roles of Nuclear Lipids in Liver Regeneration 67

1990) from which it can be transported to the plasma membrane by vesicular flow (Koval

The evaluation of SM synthase activity in the nuclear membrane and chromatin fractions showed this enzyme to have different characteristics for each fraction. In the nuclear

The Km was 1.68 x 10-4 and 3.59 x10-5 for the nuclear membrane and chromatin fractions, respectively. SM synthase activity was 770 pmol/mg protein/min (Vmax 1.1nmol/mg protein/min) in the nuclear membrane and 288 pmol/mg protein/min (Vmax 297 pmol/mg protein/min) in the chromatin. These characteristics exclude a possible contamination by the cytoplasmic structures since the specific activity is higher both in nuclear membrane and in the chromatin with respect to that found in the whole homogenate (Albi and Viola-Magni

The presence of this enzyme, together with SMase, can help to explain the possible

This enzyme utilises SM as a source of phosphorylcholine and is one of the mechanisms involved in PC synthesis. Other mechanisms for the biosynthesis of PC are the Kennedy pathway (Kent 1990), phosphatidylethanolamine methylation (Stetten 1941), lyso-PCacylation (Marinetti et al. 1958) and base- exchange from phophatidylserine (Bijerve 1971). It is difficult to suppose that PC will be synthesized in the cytoplasm and transferred to the nuclei since the PC modifications observed occur in a very short time. The base- exchange component was demonstrated in the nucleus (Albi and Viola-Magni 1997b). The presence of reverse SM-synthase may favour a more rapid exchange of PC by using DAG and

The presence of this enzyme was demonstrated both in the nuclear membrane and in the isolated chromatin (Albi et al. 2003a). The activity found in the whole homogenate was 0.93 pmol/mg protein/min, in the cytosol 2.61 pmol/mg protein/min and in the nuclear membrane 0.87 pmol/mg protein/min. A higher level of activity was observed in the chromatin at 37.09 pmol/mg protein/min. The optimum pH was 8.4 as for the other chromatin enzymes probably because the maximum solubilisation of chromatin observed at

The reaction was linear with respect to both time and protein concentration. The activity was 9.5 pmol/mg protein/min when DAG was added and increased to 50 pmol/mg protein/min in the presence of SM. Equally, the Km values were 3.56 x 10-5 M for exogenous SM and 1.12 x 10-4 for exogenous DAG so obeying the Michaelis-Menten kinetics (Table 1). It is not clear at the moment if the SM-synthase and reverse -SM-synthase are the same enzyme or are two different enzymes. The ratio DAG/ceramide depends upon their activities and, therefore, it is necessary to take into account eventual differences (Table 1).

The activity of SM-synthase was measured in the various sub-cellular fractions i.e. whole homogenate, cytosol, nuclear membrane and chromatin fractions. The ratio between SMsynthase /reverse SM-synthase was also determined for these fractions. The higher ratio

membrane the optimum pH was 7.6 whereas in the chromatin it was pH 8.4.

variations in chromatin SM content as observed during liver regeneration.

and Pagano 1991;.van Meer and Burger 1992).

**4.3 Reverse sphingomyelin-synthase** 

phosphorylcholine derived from SM.

this pH may favour enzyme activity expression.

1999a, Table 1).

although it may have a role through its degradation enzymes by producing DAG (Albi et al. 2003a).

## **4. Phospholipid-associated enzymes**

#### **4.1 Sphingomyelinase**

Sphingolmyelinase was first demonstrated in chromatin by Albi and Viola Magni (1997a). This enzyme is well known as a lysosomal enzyme in the acid form and as a cytoplasmic enzyme in the neutral form (Slife et al. 1989). It is present in many tissues e.g. hepatocytes, the nervous system and various cell cultures.

The hydrolysis of SM by SMAse results in the production of ceramide that has many physiological functions. This reaction is stimulated by many factors including interferon, (Kim et al. 1991), interleukin1 (Ballou et al. 1992), 1-25 OH vitamin D (Okasaki et al. 1989, 1990) and TNF (Dressler et al. 1992, Jayadev et al. 1994). Ceramide can be further hydrolysed to sphingosine that inhibits the protein kinase C present in the hepatocyte nuclei.

The enzyme was evaluated in both the nuclear membrane and chromatin fractions isolated from the hepatocytes. The enzyme activity reached a maximum at pH 7.6 in the nuclear membrane fraction and at pH 8.4 in the chromatin fraction. The reactions versus protein content show linear reactions for each enzyme up to 400 mg protein. In contrast, the reactions versus time showed that the nuclear membrane enzyme rose linearly from zero time whilst the chromatin enzyme remained low until 90 minutes when it rose sharply to reach its maximum value. The Km of the nuclear membrane enzyme is 3.9 x 10-4 M and that of the chromatin enzyme is 2.4 x 10-5 M implying that the nuclear membrane enzyme is more similar to that of the plasma membrane. In contrast, the chromatin enzyme appears to be similar to that present in the microsomal fraction.. The specific activity is 9.12 nmoles/10 minutes for the nuclear membrane SMase and 1.39 nmoles /90 minutes for the chromatin SMase (Albi and Viola-Magni 1997a, Table 1).

Generally, the production of ceramides results in a block at G0/G1 in the cell cycle (Riboni et al. 1992; Gomez-Munoz et al. 1995). However, the increased ceramide levels at 12 h after partial hepatectomy coincide with the start of DNA synthesis. Given the differences between the SMases present in the nuclear membrane and chromatin fractions, it is possible to hypothesis that the two enzymes are different and that the enzyme present in the chromatin may play a different role to that of the nuclear membrane and so may not necessarily result in a G0/G1 block.

#### **4.2 Sphingomyelin synthase**

The synthesis of sphingomyelin may be obtained through two pathways:

The first involves the reaction between CDP- choline and N-acylsphingosine (Scribney and Kennedy 1958) whilst the second consists of phosphocholine transfer from lecithin to ceramide. This reaction is catalysed by the enzyme phosphatidylcholine:ceramide phosphocholine transferase or sphingomyelin synthase (Diringer et al.1972). SM synthase was found, initially in the microsomes of kidney, lung, liver, spleen and heart (Ullman et al. 1974). Its subcellular localisation is in the Golgi apparatus (Jeckel et al. 1990; Futeman et al.

although it may have a role through its degradation enzymes by producing DAG (Albi et al.

Sphingolmyelinase was first demonstrated in chromatin by Albi and Viola Magni (1997a). This enzyme is well known as a lysosomal enzyme in the acid form and as a cytoplasmic enzyme in the neutral form (Slife et al. 1989). It is present in many tissues e.g. hepatocytes,

The hydrolysis of SM by SMAse results in the production of ceramide that has many physiological functions. This reaction is stimulated by many factors including interferon, (Kim et al. 1991), interleukin1 (Ballou et al. 1992), 1-25 OH vitamin D (Okasaki et al. 1989, 1990) and TNF (Dressler et al. 1992, Jayadev et al. 1994). Ceramide can be further hydrolysed

The enzyme was evaluated in both the nuclear membrane and chromatin fractions isolated from the hepatocytes. The enzyme activity reached a maximum at pH 7.6 in the nuclear membrane fraction and at pH 8.4 in the chromatin fraction. The reactions versus protein content show linear reactions for each enzyme up to 400 mg protein. In contrast, the reactions versus time showed that the nuclear membrane enzyme rose linearly from zero time whilst the chromatin enzyme remained low until 90 minutes when it rose sharply to reach its maximum value. The Km of the nuclear membrane enzyme is 3.9 x 10-4 M and that of the chromatin enzyme is 2.4 x 10-5 M implying that the nuclear membrane enzyme is more similar to that of the plasma membrane. In contrast, the chromatin enzyme appears to be similar to that present in the microsomal fraction.. The specific activity is 9.12 nmoles/10 minutes for the nuclear membrane SMase and 1.39 nmoles /90 minutes for the chromatin

Generally, the production of ceramides results in a block at G0/G1 in the cell cycle (Riboni et al. 1992; Gomez-Munoz et al. 1995). However, the increased ceramide levels at 12 h after partial hepatectomy coincide with the start of DNA synthesis. Given the differences between the SMases present in the nuclear membrane and chromatin fractions, it is possible to hypothesis that the two enzymes are different and that the enzyme present in the chromatin may play a different role to that of the nuclear membrane and so may not

The first involves the reaction between CDP- choline and N-acylsphingosine (Scribney and Kennedy 1958) whilst the second consists of phosphocholine transfer from lecithin to ceramide. This reaction is catalysed by the enzyme phosphatidylcholine:ceramide phosphocholine transferase or sphingomyelin synthase (Diringer et al.1972). SM synthase was found, initially in the microsomes of kidney, lung, liver, spleen and heart (Ullman et al. 1974). Its subcellular localisation is in the Golgi apparatus (Jeckel et al. 1990; Futeman et al.

The synthesis of sphingomyelin may be obtained through two pathways:

to sphingosine that inhibits the protein kinase C present in the hepatocyte nuclei.

2003a).

**4.1 Sphingomyelinase** 

**4. Phospholipid-associated enzymes** 

the nervous system and various cell cultures.

SMase (Albi and Viola-Magni 1997a, Table 1).

necessarily result in a G0/G1 block.

**4.2 Sphingomyelin synthase** 

1990) from which it can be transported to the plasma membrane by vesicular flow (Koval and Pagano 1991;.van Meer and Burger 1992).

The evaluation of SM synthase activity in the nuclear membrane and chromatin fractions showed this enzyme to have different characteristics for each fraction. In the nuclear membrane the optimum pH was 7.6 whereas in the chromatin it was pH 8.4.

The Km was 1.68 x 10-4 and 3.59 x10-5 for the nuclear membrane and chromatin fractions, respectively. SM synthase activity was 770 pmol/mg protein/min (Vmax 1.1nmol/mg protein/min) in the nuclear membrane and 288 pmol/mg protein/min (Vmax 297 pmol/mg protein/min) in the chromatin. These characteristics exclude a possible contamination by the cytoplasmic structures since the specific activity is higher both in nuclear membrane and in the chromatin with respect to that found in the whole homogenate (Albi and Viola-Magni 1999a, Table 1).

The presence of this enzyme, together with SMase, can help to explain the possible variations in chromatin SM content as observed during liver regeneration.

#### **4.3 Reverse sphingomyelin-synthase**

This enzyme utilises SM as a source of phosphorylcholine and is one of the mechanisms involved in PC synthesis. Other mechanisms for the biosynthesis of PC are the Kennedy pathway (Kent 1990), phosphatidylethanolamine methylation (Stetten 1941), lyso-PCacylation (Marinetti et al. 1958) and base- exchange from phophatidylserine (Bijerve 1971).

It is difficult to suppose that PC will be synthesized in the cytoplasm and transferred to the nuclei since the PC modifications observed occur in a very short time. The base- exchange component was demonstrated in the nucleus (Albi and Viola-Magni 1997b). The presence of reverse SM-synthase may favour a more rapid exchange of PC by using DAG and phosphorylcholine derived from SM.

The presence of this enzyme was demonstrated both in the nuclear membrane and in the isolated chromatin (Albi et al. 2003a). The activity found in the whole homogenate was 0.93 pmol/mg protein/min, in the cytosol 2.61 pmol/mg protein/min and in the nuclear membrane 0.87 pmol/mg protein/min. A higher level of activity was observed in the chromatin at 37.09 pmol/mg protein/min. The optimum pH was 8.4 as for the other chromatin enzymes probably because the maximum solubilisation of chromatin observed at this pH may favour enzyme activity expression.

The reaction was linear with respect to both time and protein concentration. The activity was 9.5 pmol/mg protein/min when DAG was added and increased to 50 pmol/mg protein/min in the presence of SM. Equally, the Km values were 3.56 x 10-5 M for exogenous SM and 1.12 x 10-4 for exogenous DAG so obeying the Michaelis-Menten kinetics (Table 1). It is not clear at the moment if the SM-synthase and reverse -SM-synthase are the same enzyme or are two different enzymes. The ratio DAG/ceramide depends upon their activities and, therefore, it is necessary to take into account eventual differences (Table 1).

The activity of SM-synthase was measured in the various sub-cellular fractions i.e. whole homogenate, cytosol, nuclear membrane and chromatin fractions. The ratio between SMsynthase /reverse SM-synthase was also determined for these fractions. The higher ratio

Possible Roles of Nuclear Lipids in Liver Regeneration 69

This behaviour indicates the presence of at least two different isoforms that are quantitatively different between the two fractions. The presence of two isoforms, beta1 in the chromatin and gamma1 in both the nuclear membrane and chromatin fractions, were demonstrated using specific antibodies coupled with electron microscopy (Neri et al. 1997). However, the delta1 isoform that is present in the cytoplasm was absent from the nuclei.

The PI content in the nuclear membrane fraction was 15.2 µg/mg protein and 1.05 µg/mg protein in the chromatin fraction i.e. fifteen times less. The enzymatic activity evaluated under optimal conditions was 121.43 pmol/mg protein/min in the nuclear membrane fraction and 369.05 pmol/mg protein/min in the chromatin fraction i.e. more than three times higher in the chromatin with respect to nuclear membrane. The Km was 5.77x10-5M for the chromatin associated PI-PLC and 3.89.x10-3M for this enzyme associated with the nuclear membrane fraction having a Vmax of 3.3 nmol/mg protein/min and 0.034 nmol/mg protein/min, respectively (Table 1). These results indicate a greater substrate affinity of the chromatin-associated enzyme. It has been demonstrated that this enzyme has

Enzymes pH Km Sp. activity pH Km Sp. activity

protein/min

protein/min

protein/min

protein/min

Table 1. Characteristic differences of nuclear membrane and chromatin PLs enzymes

**5. The roles of phospholipid-associated enzymes in normal hepatocytes** 

The role of the phospholipid-associated enzymes present in the chromatin seems to be related to the control of a number of cell events through the balance between the levels of ceramide and DAG in the nucleus (Albi and Viola-Magni 2003c, Albi et al. 2008 ). When the ceramide increases, the SM synthase is stimulated to produce DAG. When there is an increase in DAG, reverse-SM-synthase is activated together with SMase to yield an increased production of ceramide in order to reach an equilibrium. It is possible that the increase in ceramide may favour the production of sphingosine that can act as a pro-

8.4 2.4x10-5M 1.39 nmoles/90 m

protein/min

protein/min

protein/min

8.4 3.59x10-5M 288 pmol/mg

8.4 \* 3.56 x10-5M 37.09 pmol/mg

8.4 7.83 x10-5M 21 pmol/mg

7.6 – 8.6 5.77 x10-5M 369.05 pmol/mg

protein/min \*\*1.12 x10-4M

a role in cell proliferation (Sun et al. 1997).

SM synthase

Reverse SM synthase

\*: V/SM substrate conc. \*\*: V/DAG substrate conc.

SMase 7.6 3.9x10-4M 9.12 moles/10

PC-PLC 7.6 2.46x10-4M 1.76nmol/mg

PI-PLI 7.6,8.4-8.8 3.89x10-3M 121.43 pmol/mg

apoptotic stimulus. (Tsugane et al.1999).

Nuclear membranes Chromatin

7.6 1.68x10-4M 770 pmol/mg

7.6 ------------- 0.87 pmol/mg

min

value was observed in the nuclear membrane fraction of 885.05 indicating that the synthesis of PC may be due to an alternative enzymatic reaction. The SM-synthase activity in the chromatin fraction was only 7.49 higher with respect to the reverse SM-synthase with a consequently lower value for the DAG/ceramide ratio.

#### **4.4 Phosphorylcholine-dependent phospholipase C**

This enzyme hydrolyses PC to produce phosphorylcholine that may be used for SM synthesis and DAG that may control many cellular functions (Exton 1990).

Phosphorylcholine-dependent phospholipase C has been determined in hepatocytes and especially in the nuclear membrane and chromatin fractions in which two different isoforms were demonstrated (Baldassarre et al. 1997). In fact the PC present in these two fractions differs in content and turnover (Viola-Magni et al. 1985b, 1986). Since other enzymes such as SMase and SM-synthase were demonstrated, the presence of additional enzymes may help to understand the nuclear DAG/ceramide ratio and how it may be involved in regulating different cellular functions such cell duplication, differentiation and apoptosis. Therefore, the hepatocyte nuclei were separated and the chromatin and nuclear membrane fractions extracted for the determination of the presence and activity of phosphorylcholinedependent phospholipase C. The enzyme activity in the nuclear membrane was 1.76 nmol/mgprotein/min (Vmax 3.01 nmol/mg protein/min) whilst that in the chromatin fraction was 8.4 times lower (Vmax 0.22 nmol/mg protein/min). The phosphorylcholinedependent phospholipase C had a pH optimum of 7.6 in the nuclear membrane and 8.4 in the chromatin; its activity was linear during the first 45 min of incubation in the range from 100 to 400mg protein. The enzyme activity followed regular Michaelis-Menten kinetics in both preparations the Km values being 2.46 x 10-4 M for the nuclear membrane fraction and 7.8 3 x10-5 M for the chromatin fraction (Albi and Viola-Magni. 1999b, Table 1).

This enzyme is Ca++ independent and, therefore, may stimulate protein kinase C present in the nuclei since there is no variation in the Ca++ concentration that may interfere with its activity (Buchner 1995). The existence of nuclear PKC forms has been shown in the liver (Rogue et al. 1990) and their function may be in maintaining DNA structure or favouring DNA synthesis and repair through the action on laminin B which is localised at the sites of DNA replication (Moir et al. 1994).

#### **4.5 Phosphatidylinositol-dependent phospholipase C**

The amount and turnover of phosphatidylinositol in the chromatin fraction were different with respect to those of the nuclear membrane fraction (Viola-Magni et al. 1986). This could be due to a different enzyme activity such as that of phosphatidylinositol-dependent phospholipase C since various enzyme isoforms exist that may be activated by different stimuli (Martelli et al. 2000, 2001, Santi et al. 2001).

The activity of phosphatidylinositol-dependent-phospholipase C was determined in both the nuclear membrane and the chromatin fractions. Two peaks of pH were present in the nuclear membrane fraction, a first peak appearing at pH 7.6 followed by a second peak at pH 8.4-8 (Albi et al. 2003b).

In contrast, the chromatin fraction showed only a small peak at pH 7.6 with a sharper peak at pH 8.6.

value was observed in the nuclear membrane fraction of 885.05 indicating that the synthesis of PC may be due to an alternative enzymatic reaction. The SM-synthase activity in the chromatin fraction was only 7.49 higher with respect to the reverse SM-synthase with a

This enzyme hydrolyses PC to produce phosphorylcholine that may be used for SM

Phosphorylcholine-dependent phospholipase C has been determined in hepatocytes and especially in the nuclear membrane and chromatin fractions in which two different isoforms were demonstrated (Baldassarre et al. 1997). In fact the PC present in these two fractions differs in content and turnover (Viola-Magni et al. 1985b, 1986). Since other enzymes such as SMase and SM-synthase were demonstrated, the presence of additional enzymes may help to understand the nuclear DAG/ceramide ratio and how it may be involved in regulating different cellular functions such cell duplication, differentiation and apoptosis. Therefore, the hepatocyte nuclei were separated and the chromatin and nuclear membrane fractions extracted for the determination of the presence and activity of phosphorylcholinedependent phospholipase C. The enzyme activity in the nuclear membrane was 1.76 nmol/mgprotein/min (Vmax 3.01 nmol/mg protein/min) whilst that in the chromatin fraction was 8.4 times lower (Vmax 0.22 nmol/mg protein/min). The phosphorylcholinedependent phospholipase C had a pH optimum of 7.6 in the nuclear membrane and 8.4 in the chromatin; its activity was linear during the first 45 min of incubation in the range from 100 to 400mg protein. The enzyme activity followed regular Michaelis-Menten kinetics in both preparations the Km values being 2.46 x 10-4 M for the nuclear membrane fraction and

synthesis and DAG that may control many cellular functions (Exton 1990).

7.8 3 x10-5 M for the chromatin fraction (Albi and Viola-Magni. 1999b, Table 1).

This enzyme is Ca++ independent and, therefore, may stimulate protein kinase C present in the nuclei since there is no variation in the Ca++ concentration that may interfere with its activity (Buchner 1995). The existence of nuclear PKC forms has been shown in the liver (Rogue et al. 1990) and their function may be in maintaining DNA structure or favouring DNA synthesis and repair through the action on laminin B which is localised at the sites of

The amount and turnover of phosphatidylinositol in the chromatin fraction were different with respect to those of the nuclear membrane fraction (Viola-Magni et al. 1986). This could be due to a different enzyme activity such as that of phosphatidylinositol-dependent phospholipase C since various enzyme isoforms exist that may be activated by different

The activity of phosphatidylinositol-dependent-phospholipase C was determined in both the nuclear membrane and the chromatin fractions. Two peaks of pH were present in the nuclear membrane fraction, a first peak appearing at pH 7.6 followed by a second peak at

In contrast, the chromatin fraction showed only a small peak at pH 7.6 with a sharper peak

consequently lower value for the DAG/ceramide ratio.

**4.4 Phosphorylcholine-dependent phospholipase C** 

DNA replication (Moir et al. 1994).

pH 8.4-8 (Albi et al. 2003b).

at pH 8.6.

**4.5 Phosphatidylinositol-dependent phospholipase C** 

stimuli (Martelli et al. 2000, 2001, Santi et al. 2001).

This behaviour indicates the presence of at least two different isoforms that are quantitatively different between the two fractions. The presence of two isoforms, beta1 in the chromatin and gamma1 in both the nuclear membrane and chromatin fractions, were demonstrated using specific antibodies coupled with electron microscopy (Neri et al. 1997). However, the delta1 isoform that is present in the cytoplasm was absent from the nuclei.

The PI content in the nuclear membrane fraction was 15.2 µg/mg protein and 1.05 µg/mg protein in the chromatin fraction i.e. fifteen times less. The enzymatic activity evaluated under optimal conditions was 121.43 pmol/mg protein/min in the nuclear membrane fraction and 369.05 pmol/mg protein/min in the chromatin fraction i.e. more than three times higher in the chromatin with respect to nuclear membrane. The Km was 5.77x10-5M for the chromatin associated PI-PLC and 3.89.x10-3M for this enzyme associated with the nuclear membrane fraction having a Vmax of 3.3 nmol/mg protein/min and 0.034 nmol/mg protein/min, respectively (Table 1). These results indicate a greater substrate affinity of the chromatin-associated enzyme. It has been demonstrated that this enzyme has a role in cell proliferation (Sun et al. 1997).


\*: V/SM substrate conc.

\*\*: V/DAG substrate conc.

Table 1. Characteristic differences of nuclear membrane and chromatin PLs enzymes

### **5. The roles of phospholipid-associated enzymes in normal hepatocytes**

The role of the phospholipid-associated enzymes present in the chromatin seems to be related to the control of a number of cell events through the balance between the levels of ceramide and DAG in the nucleus (Albi and Viola-Magni 2003c, Albi et al. 2008 ). When the ceramide increases, the SM synthase is stimulated to produce DAG. When there is an increase in DAG, reverse-SM-synthase is activated together with SMase to yield an increased production of ceramide in order to reach an equilibrium. It is possible that the increase in ceramide may favour the production of sphingosine that can act as a proapoptotic stimulus. (Tsugane et al.1999).

Possible Roles of Nuclear Lipids in Liver Regeneration 71

fold rise in the cyclin mRNA level, its level remaining high between 24 and 48 h and

During S-phase, cyclin A is associated with p32cdk2 kinase, whereas during the transition

The mRNA levels of cyclin B1 and p34cdc2 behave in a similar manner remaining low for the S-phase period and increasing only after 20 h to reach a peak at 26 h. This is followed by a decline at 34 h with a new peak forming between 38 h and 44 h. The two peaks correspond

The great majority of p34cdc2 is linked to cyclin B1 whereas only 25% is linked to cyclin A (Loyer et al. 1994). The presence of the phosphorylated form of cyclin A may represent an

The increase in chromatin-bound CHO during the first six hours activates the kinases with PS favouring DNA polymerase alpha synthesis and PIP. At 12 h post-hepatectomy a transient increase of DAG is due to the hydrolysis of PI followed the activation of the

The increase of DAG favours the translocation of PKC into the nucleus (Divecha et al. 1991). The synthesis of cyclin A increases as it complexes with p32cdk2 kinase. When the S-phase starts, a more consistent DAG peak is evident due to the hydrolysis of PC by the enzyme PC-PLC. At the same time, there is a decrease in SM due to the activation of SMase and

12 increase of DAG due to PI PLI, and then to PC-PLI nuclear translocation of protein

18 DNA synthesis starts, cyclin A complex activity increases, increase of SM due to

Decrease of SM due to SMase, increase of free CHO which stimulates Cyclin A

to the G2-M phases of the first wave and second waves of the hepatocyte cell cycle.

**7.2 Cell cycle regulation by chromatin-associated phospholipids (Table 2)** 

6 increase of bound CHO, activation of PS, synthesis of DNA polymerase

the activity of SM synthase, decrease of free CHO fraction

22 decrease of cyclin A activity, increase of cyclin B complex

26 cyclin B peak, cell transition from G2 to M

38-44 second peak of cyclin B and end of second S-phase

Table 2. Molecular events in relation to the time after hepatectomy

returning to the normal value only after 72 h.

G2-M it forms a complex with p34cdc2 (Pagano et al. 1992).

inactivation of this cyclin during the G2-M transition.

consequently an increase in the free CHO present in the nucleus.

chromatin enzyme PI-PLC.

kinase C

complex

24 DNA synthesis peak

34 decrease of cyclin B

36 second peak of DNA synthesis

hours Events

This enzyme system may also be involved with gene expression by controlling the transfer of RNA to the cytoplasm. After enzymatic digestion with DNase and RNase, it was possible to isolate a complex containing a small amount of DNA, RNA, proteins and PLs. The RNA is RNase insensitive and behaves as double-stranded RNA. There are only two PLs invloved, namely, SM and PC (Albi et al. 1996; Micheli et al.1998).

The enzymes SMase, SM synthase and reverse SM-synthase are present. If the complex is treated with SMase, the undigested RNA becomes RNase sensitive. Therefore, the presence of SM appears to protect the RNA from digestion. SMase aids the digestion by causing a decrease of SM that returns to the normal value through the activation of SM-synthase that exploits the PC derived from phosphorylcholine. The amount of PC may be restored through the activation of the enzyme reverse SM-synthase (Micheli et al. 1998).

The products of PL metabolism may act also as internal signals by activating other nuclear proteins such as PKC or favouring the synthesis of polymerases through the presence of PS (Albi et al. 1991).
