This laboratory has reported previously that a cytoplasmic extract of weanling or regenerating adult rat liver (but not normal rat liver) will produce a 2.5-fold increase in the incorporation of tritiated thymidine ([3H]dThd) into liver DNA of a 34%-hepatectomized test animal. (J. Physiol. London 248: 273-284, 1975). The present study showed that hepatic stimulator substance (HSS) will stimulate DNA synthesis in normal adult rats and CF1 mice as well. The increased incorporation of [3H]dThd into DNA produced in the normal, nonhepatectomized adult rat was comparable with that induced by a 34% hepatectomy. Autoradiographic studies revealed that the [3H]dThd was incorporated into nuclear DNA and that the stimulation occurred almost exclusively in parenchymal cells. HSS was shown to be heat stable (100 degrees C for 15 min) and was precipitated but not inactivated by alcohol. Ultrafiltration and dialysis studies suggested a molecular weight slightly greater than 10,000. HSS proved to be organ specific, stimulating the liver but not the kidney, bone marrow, or spleen. HSS was found to contain no insulin, glucagon, epidermal growth factor, or peptides of the nonsuppressible insulinlike/multiplication-stimulating activity (somatomedin) group.

An extract of normal weanling or adult regenerating rat liver called hepatic stimulator substance (HSS) stimulates incorporation of tritiated thymidine ([3H]dThd) into liver DNA. In vivo, it is organ specific, species nonspecific, displays diurnal rhythm, and has a molecular weight of approximately 10,000-20,000. It has now been further characterized in vitro. HSS retained organ specificity, stimulating normal adult hepatocytes in primary culture, the HTC, PLC, and MH1C1 hepatoma cell lines, and the PRC rat liver cell line. It did not stimulate human or mouse lymphocytes, 3T3 mouse fibroblasts, Y1BS1 mouse adrenal cells, Chinese hamster ovary cells, rat hypernephroma cells, C6 rat glial cells, or L1210 mouse leukemic cells. More detailed studies of the response of HTC cells to HSS revealed a dose-dependent increase in [3H]dThd incorporation (up to 30-fold) with a response to as little as 140-ng purified extract/ml culture medium. Autoradiography confirmed that the thymidine was taken up by nuclear DNA. This represented a true stimulation of growth as shown by an almost twofold increase in cell number after 6 days of exposure to low doses of HSS.

The regenerating rat liver was used as a model to investigate the necessity for new cholesterol synthesis prior to the onset of cell division. Plasma cholesterol levels in partially hepatectomized rats were significantly decreased 24 and 48 h after surgery compared with levels in sham-operated animals. Hepatic cholesteryl ester content was also significantly increased in livers from partially hepatectomized animals, but the hepatic content of unesterified cholesterol was not affected. Hepatic triglyceride content was significantly increased within 6 h after surgery in the regenerating liver. The triglyceride levels reached a peak at 24 h, and by 72 h they had decreased back to levels that were no different from control. In the regenerating liver, microsomal 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity was increased 12 h after surgery. The activity of this enzyme remained significantly elevated throughout the 72-h period after surgery. In contrast, 12 h after partial hepatectomy the rate of hepatic cholesterol synthesis was significantly lower than that observed in livers from sham-operated rats. An increase in the rate of cholesterol synthesis was not observed until 48 h after partial hepatectomy, some 32 h after the start of DNA synthesis. Microsomal acyl-CoA:cholesterol acyltransferase activity was unchanged except for a 28% decrease at 72 h after partial hepatectomy. The data suggest that new cholesterol synthesis is not a requirement prior to the initiation of DNA synthesis in the regenerating rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic stimulator substance is a unique, 12,000- to 18,000-dalton peptide found in the liver of weanling and regenerating, but not normal, adult rats. It has also been demonstrated in dogs, rabbits, and pigs. It is organ specific, both in vivo and in vitro, but species nonspecific. It induces liver growth after a 10- to 12-hr lag period, during which time new protein and RNA synthesis are required. The initial events induced by HSS in HTC hepatoma cells in vitro include a rapid influx of Na+ via the Na+/H+ antiport and a rapid influx of extracellular Ca2+. The rise in intracellular Ca2+ appears to be dependent on the influx of Na+ and the influx of Na+ is necessary, but not in itself sufficient, to stimulate DNA synthesis.

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