**Possible Roles of Nuclear Lipids in Liver Regeneration**

M. Viola-Magni1 and P.B. Gahan2

*1Perugia University, Enrico Puccinelli Foundation 2King's College London 1Italy 2UK* 

#### **1. Introduction**

62 Liver Regeneration

[131] Schmelzer E, Triolo F, Turner ME, Thompson RL, Zeilinger K, Reid LM, et al. Three-

Liver Cells. Tissue Eng Part A. 2010;16(6):2007-16.

Dimensional Perfusion Bioreactor Culture Supports Differentiation of Human Fetal

Although no lipids were considered to be present inside the nuclear membrane (Berg, 1951), their presence in chromatin was first demonstrated cytochemically by Chayen et al (1957) in *Vicia faba* root apices and liver nuclei. Sphingomyelin was further demonstrated biochemically to represent some 7% of isolated calf thymus nucleohistone preparations (Chayen and Gahan, 1958), the presence of sphingomyelin being confirmed by X-ray diffraction studies (Wilkins M. H. F, personal communication). Nevertheless, the lipids and carbohydrate present in the nuclei were considered to be minor components, most of them being due to contamination during chromatin separation (Tata et al.1972). In contrast, some biochemical measurements showed the presence of neutral lipids (Song and Rebel 1987) and phospholipids in nuclei and chromosomes from a large variety of tissues (Chayen et al 1959, a, b, Gahan 1965a). The criticism linked to possible contamination cannot be applied to the cytochemical evidence that showed the presence of chromatin-associated phospholipid material in a broad range of tissues (Idelman 1957, 1958a,b, Chayen et al. 1959a,b, La Cour et al. 1958, Gahan 1965a,b, Cave and Gahan 1971, Gahan et al. 1974, Gahan et al. 1987, Viola-Magni et al. 1985a). In a combined autoradiographic and biochemical analysis, it was shown that H3 -ethalomine incorporated into *Vicia faba* root nuclei was localised at the level of chromatin and nucleoli rather than at the level of the nuclear membrane. Hepatocyte nuclei treated with Triton and hypotonic solutions liberate chromatin that contains 10% of the total nuclear lipids. The composition of fatty acids demonstrated an enrichment of palmitic acid and a reduction in arachidonic acid (Albi et al. 1994) thus supporting the idea that these lipids cannot be derived from the nuclear membrane. In addition, the chromatographic separation of phospholipids has demonstrated an enrichment of both sphingomyelin and phosphatidylserine with respect to the nuclear membrane composition (Albi et al. 1994). The data were also confirmed by studying the turnover of phospholipids at the level of the microsomes, nuclear membrane and chromatin from hepatocytes (Viola-Magni et al. 1986). In rats injected with radioactive phosphorus, the peak of incorporation was observed after 6 h in microsomes and nuclear membranes, but only after 9h in the chromatin. This confirmed a lack of possible chromatin contamination.

A clear demonstration was obtained by labelling the fatty acids of the nuclear membrane by radio-iodination. Hepatocyte nuclei were separated and then radio-iodinated; the chromatin

Possible Roles of Nuclear Lipids in Liver Regeneration 65

The single phospholipids behave differently during hepatocyte regeneration after partial

Chromatin phospholipids represent about 10% of the total nuclear lipids (Viola-Magni et al, 1985a) of which sphingomeylin represents some 35% of all nuclear phospholipids (Albi et al, 1994). During liver regeneration, a decrease is observed in the amount of SM at the beginning of S-phase (Albi and Viola-Magni 1997a) followed by an increase at the end of Sphase. The approximate remaining amounts of phospholipids in the nucleus are phosphatidylethanolamine (PE) 10%, PI 19%, phosphatidylserine (PS) 22% and PC 14%

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

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

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).

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

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

hepatectomy.

(Albi et al. 1994).

**3.1 SM behaviour** 

**3.2 PS behaviour** 

**3.3 PC behaviour** 

**3.4 PE behaviour** 

**3.5 PI behaviour** 

observed.

**3. Roles of individual phospholipids** 

authors (Stillman 1996, Alessenko and Chatterjee 1995).

polymerase as has been shown *in vitro* by Manzoli et al. (1981).

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

The presence of nuclear phospholipids was also demonstrated in a large variety of tissues including tumour cells (Splanger et al. 1975, Upreti et al. 1983).

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

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, differentiation and apoptosis.

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 hepatocytes, the cell cycle and regenerating liver.

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

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.

The single phospholipids behave differently during hepatocyte regeneration after partial hepatectomy.

Chromatin phospholipids represent about 10% of the total nuclear lipids (Viola-Magni et al, 1985a) of which sphingomeylin represents some 35% of all nuclear phospholipids (Albi et al, 1994). During liver regeneration, a decrease is observed in the amount of SM at the beginning of S-phase (Albi and Viola-Magni 1997a) followed by an increase at the end of Sphase. The approximate remaining amounts of phospholipids in the nucleus are phosphatidylethanolamine (PE) 10%, PI 19%, phosphatidylserine (PS) 22% and PC 14% (Albi et al. 1994).
