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)

Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis.

Pyrophosphate : fructose-6-phosphate phosphotransferase (PPi-PFK) has been purified 150-fold from potato tubers and the kinetic properties of the purified enzyme have been investigated both in the forward and the reverse direction. Saturation curves for fructose 6-phosphate and also for fructose 1,6-bisphosphate were sigmoidal whereas those for PPi and Pi were hyperbolic. In the presence of fructose 2,6-bisphosphate, the affinity for fructose 6-phosphate and for fructose 1,6-bisphosphate were greatly increased and the kinetics became Michaelian. The effect of fructose 2,6-bisphosphate was increased by the presence of fructose 6-phosphate and decreased by the presence of Pi. Consequently, the Ka for fructose 2,6-bisphosphate was as low as 5 nM for the forward reaction and reached 150 nM for the reverse reaction. On the basis of these properties, a procedure allowing one to measure fructose 2,6-bisphosphate in amounts lower than a picomole, is described.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1982.tb07039.x

A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate.

The effects of polyamines on the oligomeric forms of protein phosphatase-1 (1G), protein phosphatase-2A (2A0, 2A1 and 2A2) and their free catalytic subunits (1C and 2AC) has been studied using homogeneous enzymes isolated from rabbit skeletal muscle. Spermine increased the activity of protein phosphatase-2A towards eight of nine substrates tested. Half-maximal activation was observed at 0.2 mM with optimal effects at 1–2 mM. Above 2 mM, spermine became inhibitory. The most impressive activation of protein phosphatase-2A was obtained with glycogen synthase, especially when phosphorylated at sites-3 (8–15-fold with protein phosphatase-2A1) and phenylalanine hydroxylase (6–7-fold with protein phosphatase-2A1) as substrates. Activation of protein phosphatases 2A0, 2A1 and 2A2 was greater than that observed with 2AC. Spermine was a more potent activator than spermidine, while putrescine had only a small effect. Qualitatively similar results were obtained with five other substrates, although maximal activation was much less (1.3–3-fold with protein phosphatase-2A1). The rate of dephosphorylation of glycogen phosphorylase was decreased by spermine, inhibition being more pronounced with protein phosphatase-2AC than with 2A0, 2A1 and 2A2. Spermine (I50= 0.1 mM with protein phosphatase-2AC) was a more potent inhibitor than spermidine (I50= 0.9 mM) or putrescine (I50= 8 mM). Partially purified preparations of protein phosphatases-2A0, 2A1 and 2A2 from from rat liver were affected by spermine in a similar manner to the homogeneous enzymes from rabbit skeletal muscle.



Spermine did not activate protein phosphatase-1 to the same extent as protein phosphatase-2A. Greatest stimulation (2.5-fold) was again observed with glycogen synthase labelled in sites-3, with half-maximal activation at 0.2 mM and optimal effects at 1–2 mM spermine. Spermine was a much more effective stimulator than spermidine, while putrescine was ineffective. Very similar results were obtained with protein phosphatases 1G and 1C. With four other substrates maximal activation by spermine was < 1.5-fold, while the dephosphorylation of glycogen synthase (labelled in site-2), phosphorylase kinase, pyruvate kinase and glycogen phosphorylase were inhibited. Spermine (I50= 0.04 mM) was a more potent inhibitor of the dephosphorylation of glycogen phosphorylase than spermidine (I50= 0.9 mM) or putrescine (I50= 9 mM).



There was some resemblance between the effects of Mn2+ and spermine on the activity of protein phosphatase-2A, although with the substrates inhibitor-1 and phenylalanine hydroxylase, activation by Mn2+ was greater than that observed with spermine, while with glycogen synthase the converse was true. Mn2+ could not mimic the activating effect of spermine on dephosphorylation of glycogen synthase (labelled in sites-3) by protein phosphatase 1. By contrast, spermine hardly affected the dephosphorylation of inhibitor-1 by phosphatase-1, a reaction that was almost completely dependent on Mn2+. Physiological concentrations of Mg2+ (1.0 mM) neither mimicked, nor influenced, the response to spermine with either protein phosphatase-1 or 2A.



The effects of spermine appear to result from interaction of the polyamine with both the protein phosphatases and their substrates. The possibility that activation of protein phosphatase-1 and/or 2A by spermine underlies the relatively specific dephosphorylation of sites-3 of glycogen synthase by insulin is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1985.tb08832.x

The protein phosphatases involved in cellular regulation

A mechanism is proposed for a molecular switch that can store information indefinitely, despite the complete turnover of the molecules that make up the switch. The design of the switch is based on known types of biochemical reactions. Central to the mechanism is a kinase that is activated by phosphorylation and capable of intermolecular autophosphorylation. It is shown that such a kinase and an associated phosphatase form a bistable chemical switch that can be turned on by an external stimulus and that is not reset by protein turnover.

https://www.pnas.org/content/pnas/82/9/3055.full.pdf

A mechanism for memory storage insensitive to molecular turnover: a bistable autophosphorylating kinase.

Fru-2,6-P2 (fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure-function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2. The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.

E. Fox

Papers

Regulation of rat liver fructose 2,6-bisphosphatase.

Fructose 2,6-bisphosphate: a mediator of hormone action at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.

The action of insulin on hepatic fructose 2,6-bisphosphate metabolism

Cyclic AMP-dependent phosphorylation of rat liver 6-phosphofructo 2-kinase, fructose 2,6-bisphosphatase.

Hormonal Modulation of Key Hepatic Regulatory Enzymes in the Gluconeogenic/Glycolytic Pathway