Although signal transduction mechanisms originating from receptors on the plasma membrane and targeted to metabolic and other enzymes/proteins localized in the cytoplasm or the nucleus have been extensively studied in animal cells, few such studies have focused on the mitochondrial energy producing machinery, i.e. the electron transport chain and ATP synthase complex (F0F1). Significantly, it was shown in an earlier collaborative study that platelet-derived growth factor (PDGF), which is linked in signal transduction pathways to tyrosine kinase-dependent phosphorylations, regulates the phosphorylation of the mitochondrial ATP synthase delta subunit in cortical neurons (Zhang et. al., 1995. J. Neurochem. 65, 2812-2815). This is a particularly intriguing finding in light of more recent reports demonstrating that ATP synthases are nanomotors with a central rotor, one component of which is the delta subunit. In this report, evidence is provided that the PDGF-dependent phosphorylation of the ATP synthase delta subunit is not confined to neuronal cells but can be demonstrated also in studies with PDGF-treated NIH3T3 and kidney cells. Evidence is provided also that phosphorylation of the ATP synthase delta subunit may involve its single tyrosine residue, and that this phosphorylation is modulated when the cell based assay includes lysophosphatidic acid (LPA), a phospholipid signaling molecules. Finally, results are presented of an analysis which revealed a number of potential tyrosine phosphorylation sites on three other subunits (alpha, beta, and gamma) of the F1 (catalytic) moiety of the mitochondrial ATP synthase, thus making this important complex a most attractive target for future signal transduction studies.

Mouse, chicken and Xenopus laevis homologues to rig (rat insulinoma gene) cDNA were isolated and their nucleotide sequences were determined. Each homologue encoded a 145-amino acid protein; the amino acid sequence remained invariant in the murine and avian genes, and there were only 6 amino acid substitutions in the salientian gene. The evolutionary rate calculated for rig mRNA was sufficiently low to be viewed as evidence that rig is vital to vertebrate species. Southern blot analysis indicated that haploid sets of the mammalian genomes contain several copies of rig or rig-related sequences, whereas there appeared to be only one copy in the amphibian and bird genomes. The possibility that rig belongs to the class of housekeeping genes is discussed.

As discussed in Chapter 10, insulinomas can be induced in experimental animals by diabetogenic agents such as streptozotocin and alloxan (Lazarow, 1952; Rakieten et al., 1971; Yamagami et al., 1985) and by viruses (Uchida et al., 1979). A unifying concept was proposed for the diabetogenic and oncogenic effects of the B-cytotoxins, in which the principal action of streptozotocin and alloxan was considered to be the induction of lesions in the DNA of B-cells of islets of Langerhans (see Fig. 10.5). To elucidate the molecular mechanisms of B-cell oncogenesis, we have looked for genes whose expression is altered in insulinomas. We identified a novel gene named rig (rat insulinoma gene), which is activated in chemically induced rat insulinomas (Takasawa et al., 1986). The gene was also activated in virus-induced hamster insulinomas and in spontaneously occurring human insulinomas (Inoue et al., 1987).

When hepatocytes isolated from adult rats were cultured in the presence of 10 mM nicotinamide, insulin- and epidermal growth factor-induced DNA synthesis and cell proliferation were found to be greatly stimulated, and the cells were able to be kept alive for more than one month. In the nicotinamide-treated hepatocytes, albumin and tryptophan 2,3-dioxygenase mRNAs were present at much higher levels than in the untreated control, and the inducibility of tryptophan oxygenase gene expression by dexamethasone and glucagon was also preserved. Without nicotinamide, primary cultured hepatocytes were viable for only 5-7 days and the hepatocyte-specific phenotypes were rapidly lost. The intracellular NAD level was maintained in the nicotinamide-treated hepatocytes at or above the level in intact liver but depleted in hepatocytes without nicotinamide. These results suggest that the maintenance of the intracellular NAD level is essential for the growth and functioning of hepatocytes and that nicotinamide can preserve the NAD level by blocking NAD degradation as well as by acting as a precursor for NAD synthesis.

We have isolated a novel gene, rig (rat insulinoma gene) from rat insulinomas. In the present study, rig was found to be expressed in rat regenerating liver and in primary cultured rat hepatocytes. The level of rig mRNA was increased at the proliferative phase of liver regeneration. In synchronously cultured hepatocytes, the rig mRNA level was elevated at the G1 phase of the cell cycle and the rig-protein was accumulated in the nuclei during the S phase. These results indicated that rig could be involved in a more general way in growth or cell replication.