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Epidermal growth factor (EGF) previously isolated from the submandibular gland of mice was injected ip at different circadian phases into separate subgroups of adult male CD2F1 mice. Subsequent to each of the five time points of injection (0900, 1500, 1800, 2100, and 0300 h for animals standardized to 12 h of light alternating with 12 h of darkness), five animals were killed, 4, 8, and 12 h after the injection of EGF; comparable control groups were injected only with the carrier substance. Thirty minutes before sacrifice, each mouse was injected ip with 24 muCi [3H]thymidine. Incorporation of [3H]-thymidine into the DNA of the tongue, esophagus, and stomach was determined. The results demonstrate for the first time that EGF has a strong in vivo stimulatory effect on DNA synthesis in the tongue, esophagus, and stomach (studies on other areas of the gut have not yet been completed). Under the conditions of the study, stimulatory effects occurred as soon as 4 h subsequent to injection; however, maximal stimulation occurred for all three tissues 8 h after injection. Twelve hours after injection, the levels of DNA synthesis in all tissues were generally returning to normal levels found in the control animals. The results suggest circadian variation in susceptibility to EGF in the different tissues.

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Dsk5 mice have a gain of function in the epidermal growth factor receptor (EGFR), caused by a point mutation in the kinase domain. We analyzed the effect of this mutation on liver size, histology, and composition. We found that the livers of 12-wk-old male Dsk5 heterozygotes (+/Dsk5) were 62% heavier compared with those of wild-type controls (+/+). The livers of the +/Dsk5 mice compared with +/+ mice had larger hepatocytes with prominent, polyploid nuclei and showed modestly increased cell proliferation indices in both hepatocytes and nonparenchymal cells. An analysis of total protein, DNA, and RNA (expressed relative to liver weight) revealed no differences between the mutant and wild-type mice. However, the livers of the +/Dsk5 mice had more cholesterol but less phospholipid and fatty acid. Circulating cholesterol levels were twice as high in adult male +/Dsk5 mice but not in postweaned young male or female mice. The elevated total plasma cholesterol resulted mainly from an increase in low-density lipoprotein (LDL). The +/Dsk5 adult mouse liver expressed markedly reduced protein levels of LDL receptor, no change in proprotein convertase subtilisin/kexin type 9, and a markedly increased fatty acid synthase and 3-hydroxy-3-methyl-glutaryl-CoA reductase. Increased expression of transcription factors associated with enhanced cholesterol synthesis was also observed. Together, these findings suggest that the EGFR may play a regulatory role in hepatocyte proliferation and lipid metabolism in adult male mice, explaining why elevated levels of EGF or EGF-like peptides have been positively correlated to increased cholesterol levels in human studies.

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The role(s) of the epidermal growth factor receptor (EGFR) in hepatocytes is unknown. We generated a murine hepatocyte specific-EGFR knockout (KO) model to evaluate how loss of hepatocellular EGFR expression affects processes such as EGF clearance, circulating EGF concentrations, and liver regeneration following 70% resection or CCl4-induced centrilobular injury. We were able to disrupt EGFR expression effectively in hepatocytes and showed that the ability of EGF and heregulin (HRG) to phosphorylate EGFR and ERBB3, respectively, required EGFR. Loss of hepatocellular EGFR impaired clearance of exogenous EGF from the portal circulation but paradoxically resulted in reduced circulating levels of endogenous EGF. This was associated with decreased submandibular salivary gland production of EGF. EGFR disruption did not result in increased expression of other ERBB proteins or Met, except in neonatal mice. Liver regeneration following 70% hepatectomy revealed a mild phenotype, with no change in cyclin D1 expression and slight differences in cyclin A expression compared with controls. Peak 5-bromo-2'-deoxyuridine labeling was shifted from 36 to 48 h. Centrilobular damage and regenerative response induced by carbon tetrachloride (CCl4) were identical in the KO and wild-type mice. In contrast, loss of Met increased CCl4-induced necrosis and delayed regeneration. Although loss of hepatocellular EGFR alone did not have an effect in this model, EGFR-Met double KOs displayed enhanced necrosis and delayed liver regeneration compared with Met KOs alone. This suggests that EGFR and Met may partially compensate for the loss of the other, although other compensatory mechanisms can be envisioned.

Epidermal growth factor (EGF) previously isolated from the submandibular salivary glands of mice was injected ip at five different circadian phases into separate sub-groups of adult male CD2F1 mice that had been standardized to 12 h of light alternating with 12 h of darkness. Comparable control groups were injected only with the carrier substance. Four, 8, and 12 h after each of the five injection times, subgroups of five mice were killed. Thirty minutes before sacrifice, each mouse was injected ip with 24 mu Ci tritiated thymidine ([3H]-TdR). Incorporation of [3H]TdR into the DNA of the duodenum, jejunum, ileum, cecum, colon, and rectum was determined. The results demonstrate for the first time that EGF has a stimulatory effect on DNA synthesis in these tissues, particularly in the colon and rectum. Under the conditions of this study, the stimulatory effects of EGF on DNA synthesis in the cecum, colon, and rectum were more dramatic than those in the three regions of the small intestine; in fact, DNA synthesis in the latter was occasionally statistically significantly decreased, particularly for mice killed during the dark phase. Stimulatory effects of EGF on DNA synthesis in the cecum, colon, and rectum were noticed as early as 4 h after injection; however, maximal stimulation occurred 8 and 12 h post injection.