No abstract

Understanding the regulation of hepatic glucose metabolism had its foundation in the elucidation of several pathways, but recent advances have come from the application of molecular genetics. Five years ago little was known about the primary structure of the key regulatory enzymes. Since then, the primary sequence of liver GK, 6-PF-1-K, Fru-1,6-P2ase, PK, PEPCK, and 6-PF-2-K/Fru-2,6-P2ase have been derived from cDNA sequences and/or determined by direct protein sequencing. This has provided new insights into the molecular mechanisms of catalysis and the regulation of these enzymes by covalent modification. Isolation of the cDNAs for these enzymes also has allowed for the quantitation of specific mRNAs and permitted analysis of hormonal control of specific gene expression. The genes for these enzymes have been isolated and sequenced, and their promoter regions are being identified and characterized. Hormone response elements have been delineated in several of the promoters. The promoter regions for 6-PF-2-K/Fru-2,6-P2ase and Fru-1,6-P2ase have also been identified, and future research will focus on the elucidation of the mechanisms whereby hormones regulate the expression of these genes. A number of generalizations can be made about the regulation of gene expression of glycolytic/gluconeogenic enzymes. First, there is coordinate hormonal regulation of gene expression and these effects are consonant with their physiologic actions. Insulin induces the mRNAs that encode glycolytic enzymes and represses the mRNAs that encode gluconeogenic enzymes; cAMP has opposite effects. Both can increase or decrease transcription. Whereas insulin and cAMP affect all of these mRNAs, glucocorticoids appear to have a more restricted action. Second, transcriptional and posttranscriptional regulatory mechanisms are involved. The synthesis of all of the mRNAs discussed is regulated by hormones. Relatively little is known about how mRNA stability is regulated in general, but it is clear that PEPCK mRNA is stabilized by agents that increase the rate of transcription of the gene. Under appropriate metabolic signals this dual control of mRNA synthesis and stability provides for a long-term increase in PEPCK mRNA and protein. Studies with PK mRNA are less direct, but suggest a similar dual mechanism. It will be interesting to see whether multilevel regulation is restricted to these two mRNAs, both of which are involved in the same substrate cycle, or whether the stability of other mRNAs involved in hepatic glucose metabolism is also affected. Third, glucose appears to be important in the regulation of these hepatic genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Maturity-onset diabetes of the young (MODY) is a form of non-insulin-dependent diabetes mellitus (NIDDM) characterized by early onset, usually before 25 years of age and often in adolescence, and by autosomal dominant inheritance [1]. Although commonly thought to be a relatively rare form of NIDDM, recent studies suggest that it may not be that uncommon and 2-5 % of patients with NIDDM may in fact have MODY [2]. Mutations in genes on chromosomes 20, 7 and 12, designated MODY1, MODY2/glucokinase (GCK) and MODY3, respectively, can cause this form of diabetes [3][4][5]. Moreover, there are likely to be additional MODY genes since there are families in which MODY does not cosegregate with markers tightly linked to the three known MODY loci [5]. Diabetologia (1997) Summary Mutations in glucokinase are associated with defects in insulin secretion and hepatic glycogen synthesis resulting in mild chronic hyperglycaemia, impaired glucose tolerance or diabetes mellitus. We screened members of 35 families with features of maturity-onset diabetes of the young for mutations in the glucokinase gene and found 16 different mutations. They included 14 new mutations in the glucokinase gene: 9 missense mutations (A53S, G80A, H137R, T168P, M210T, C213R, V226M, S336L and V367M); 2 nonsense mutations (E248X and S360X); a deletion of one nucleotide resulting in a frameshift (V401del1); a substitution of a conserved nucleotide at a splice acceptor site (L122-1G → T); and a 10 base pair deletion that removed the GT of the splice donor site and the following eight nucleotides (K161 + 2del10). In addition, we found two previously identified mutations: R186X and G261R. Study of 260 subjects with glucokinase-deficient hyperglycaemia from 42 families with 36 different GCK mutations made it possible to define the clinical profile of this subtype of non-insulin-dependent diabetes mellitus (NIDDM). Hyperglycaemia due to glucokinase deficiency is often mild (fewer than 50 % of subjects have overt diabetes) and is evident during the early years of life. Despite the long duration of hyperglycaemia, glucokinase-deficient subjects have a low prevalence of micro-and macro-vascular complications of diabetes. Obesity, arterial hypertension and dyslipidaemia are also uncommon in this form of NIDDM. [Diabetologia (1997) 40: 217-224]

Adult rat hepatocytes have been previously isolated and maintained in monolayer culture, but attempts to stimulate DNA synthesis have been unsuccessful. Hormonal conditions are now described which induce DNA synthesis in cultured hepatocytes from partially hepatectomized rats. DNA synthesis was determined autoradiographically by the incorporation of [3H~thymidine into nuclei of morphologically distinct hepatocytes. Insulin (4-4000 nM) or epidermal growth factor (10 ng/ml) alone caused significant increases in the labeling index. The two hormones together acted synergistically to produce labeling indices of 35-50% on the third day of culture, compared with 2-7% in control cultures. The addition of glucagon (400 nM) further increased the labeling index. Dexamethasone (80 ng/ml) inhibited DNA synthesis but, under certain conditions, enhanced cell attachment. Growth hormone and triiodothyronine had no significant effect on DNA synthesis. The mixture of epidermal growth factor, insulin, and glucagon also stimulated incorporation of [3H~thymidine into phenol-extracted DNA. Although DNA synthesis was stimulated, cell division occurred infrequently. These data suggest a prominent role for epidermal growth factor in promoting hepatic DNA synthesis by acting in concert with insulin and glucagon.Liver regeneration after partial hepatectomy has been employed widely as an experimental model of mammalian cell division (1, 2). However, the regulatory mechanisms that stimulate quiescent hepatocytes to proliferate are poorly understood. Cross circulation studies between partially hepatectomized rats and intact animals suggest the involvement of humoral stimuli (3). Although several hormones have been implicated (4-9), definitive studies have been difficult to carry out because of complex hormonal interactions in vivo. The development of cell cultures of normal adult hepatocytes would provide a system to characterize those factors necessary for DNA synthesis and cell division. However, while fetal liver (10) and hepatoma cells (11) can be grown in culture, there have been, as yet, no successful attempts to induce DNA synthesis or cell division in adult liver cells in culture. Studies with hepatocytes from partially hepatectomized rats have shown that DNA synthesis declines progressively with the time in culture (12), and that only 50-60% of the isolated hepatocytes adhere to the substratum.We report conditions that increase to 90% the number of hepatocytes that adhere to flasks as well as conditions which stimulate DNA synthesis. Epidermal growth factor (EGF), in combination with insulin and glucagon, has been found to greatly enhance DNA synthesis. MATERIALS AND METHODSIsolation and Plating of Liver Cells. Male rats (150-300 g, Sprague-Dawley, Madison, Wisc.) were subjected to partial hepatectomy (13) and then starved for 18-24 hr. The remaining liver fragments were perfused with collagenase and hyaluronidase as previously described (14), except that after catheterization of the portal and superior vena cava veins the fr...