Metallothioneins (MTs) are intracellular, low molecular, low molecular weight, cysteine-rich proteins. Ubiquitous in eukaryotes, MTs have unique structural characteristics to give potent metal-binding and redox capabilities. A primary role has not been identified, and remains elusive, as further functions continue to be discovered. The most widely expressed isoforms in mammals, MT-1 and MT-2, are rapidly induced in the liver by a wide range of metals, drugs and inflammatory mediators. In teh gut and pancreas, MT responds mainly to Zn status. A brain isoform, MT-3, has a specific neuronal growth inhibitory activity, while MT-1 and MT-2 have more diverse functions related to their thiolate cluster structure. These include involvement in Zn homeostasis, protection against heavy metal (especially Cd) and oxidant damage, and metabolic regulation via Zn donation, sequestration and/or redox control. Use of mice with altered gene expression has enhance our understanding of the multifaceted role of MT, emphasised in this review.

Metallothionein: the multipurpose protein

Zinc transporters and the cellular trafficking of zinc

Endophilin I is a presynaptic protein of unknown function that binds to dynamin, a GTPase that is implicated in endocytosis and recycling of synaptic vesicles. Here we show that endophilin I is essential for the formation of synaptic-like microvesicles (SLMVs) from the plasma membrane. Endophilin I exhibits lysophosphatidic acid acyl transferase (LPAAT) activity, and endophilin-I-mediated SLMV formation requires the transfer of the unsaturated fatty acid arachidonate to lysophosphatidic acid, converting it to phosphatidic acid. A deletion mutant lacking the SH3 domain through which endophilin I interacts with dynamin still exhibits LPAAT activity but no longer mediates SLMV formation. These results indicate that endophilin I may induce negative membrane curvature by converting an inverted-cone-shaped lipid to a cone-shaped lipid in the cytoplasmic leaflet of the bilayer. We propose that, through this action, endophilin I works with dynamin to mediate synaptic vesicle invagination from the plasma membrane and fission.

/pdf/endophilin-i-mediates-synaptic-vesicle-formation-by-transfer-4d04qj4t2o.pdf

Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid

Coordination dynamics of zinc in proteins.

Schild, Detlev, and Diego Restrepo. Transduction Mechanisms in Vertebrate Olfactory Receptor Cells. Physiol. Rev. 78: 429–466, 1998. — Considerable progress has been made in the understanding of tr...

Transduction Mechanisms in Vertebrate Olfactory Receptor Cells

Because of differences in the pattern of enzyme activities involved in glycolysis and gluconeo-genesis between rat and guinea pig liver, the regulation of gluconeogenesis in guinea pig liver was studied.



The following results were obtained:



1
Starvation increases the capacity for gluconeogenesis from pyruvate in vivo.

2
Gluconeogenesis from l-lactate and from pyruvate in isolated perfused livers is greater in the guinea pig than in the rat, whereas gluconeogenesis from other precursors is similar in both species.

3
Long chain and medium chain fatty acids inhibit gluconeogenesis from l-lactate in isolated perfused rat liver. In the guinea pig liver the difference does not result from a lower rate of fatty acid oxidation but from a greater reduction in the cytoplasmic NAD+/NADH system.

During enhanced fatty acid oxidation in guinea pig liver and in rat liver, similar changes in the concentrations of both acetyl-S-CoA and CoA-SH are observed.

With high concentrations of pyruvate as the gluconeogenic precursor, both fatty acids and ethanol lead to a slight stimulation of glucose formation by supplying hydrogen equivalents to the cytosolic NAD+/NADH system.

4
Both glucagon and dibutyryl-Ado-3′:5′-P stimulate ureogenesis and ketogenesis in isolated perfused guinea pig liver. However, the stimulation of gluconeogenesis by glucagon and Ado-3′:5′-P normally observed in isolated perfused rat livers was not found in isolated perfused guinea pig livers.

5
Partially purified pyruvate carboxylases from rat and guinea pig liver do not differ with respect to the apparent Km values for pyruvate and ATP, and also the apparent Ka values for acetyl-S-CoA at different pH values. At constant concentrations of MgATP2− or MnATP2− the pH-optima for the guinea pig enzyme are significantly lower.

6
5-Methoxyindole-2-carbonic acid inhibits gluconeogenesis in guinea pig liver in the same way as in rat liver. Quinolinate, on the other hand is far less inhibitory in guinea pig liver and does not inhibit at all in isolated perfused pigeon livers.

7
Major species differences are found when the activities of the rate limiting enzymes involved in gluconeogenesis are measured under Vmax conditions. Pyruvate carboxylase activity is in the same range in rat and guinea pig liver; pyruvate kinase activity is considerably lower, phospho-enolpyruvate carboxykinase activity is considerably higher in guinea pig liver.





According to our results gluconeogenesis in guinea pig liver from lactate or pyruvate is mainly regulated by the concentration of pyruvate.



The difference in the regulation of gluconeogenesis between rat and guinea pig liver probably results from a difference in the compartmentalization of phosphoenopyruvate carboxykinase. A higher rate of futile cycling between phosphoenolpyruvate and pyruvate in rat liver, due to the higher activity of pyruvate kinase in relation to the activities of phosphoenolpyruvate carboxykinase and pyruvate carboxylase, may also contribute to the observed differences.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1970.tb01084.x

Regulation of Gluconeogenesis in the Guinea Pig Liver

Gluconeogenesis was studied in isolated perfused livers from pigeons, guinea pigs and rats in order to evaluate the role of intramitochondrial formation of phosphoenolpyruvate and the rate and importance of “futile cycling” of carbon for the regulation of glucose formation. The net formation of glucose from lactate (20 mM) by pigeon liver is about twice as high as in guinea pig liver and about 3 to 4 times higher than in rat liver, although the intracellular ATP/ADP ratio in pigeon liver is extremely low (< 1) in the presence and absence of gluconeogenesis.



In contrast to experiments with rat livers, but in accordance with those with guinea pig livers, oleate (2 mM) failed to stimulate gluconeogenesis from lactate in pigeon liver.



With pyruvate (20 mM) there is no net formation of glucose by pigeon livers in the absence or presence of hexanoate, oleate or xylitol. In the presence of ethanol the rate of glucose formation from pyruvate increased but did not exceed 30% of the rate observed with lactate as precursor.



In contrast to the results obtained in rat liver experiments the transaminase inhibitor (aminooxy)acetate did not inhibit gluconeogenesis from lactate in pigeon liver although soluble and mitochondrial glutamate oxaloacetate aminotransferase from pigeon liver were strongly inhibited by (aminooxy)acetate. On the other hand, n-butylmalonate (5 mM) inhibited strongly gluconeogenesis from lactate in isolated perfused rat and pigeon livers. This inhibition was accompanied by an inhibition of oxygen uptake in pigeon but not in rat liver. Benzene 1,2,3-tricarboxylate, an inhibitor of the tricarboxylate translocator, had no effect on gluconeogenesis from lactate in isolated perfused rat and pigeon livers. Gluconeogenesis from propionate (10 mM) decreased in the order guinea pig-rat-pigeon.



The activity of phosphoenolpyruvate carboxykinase as well as the phosphoenolpyruvate carboxykinase/pyruvate kinase ratio in pigeon liver were significantly higher than in rat and guinea pig liver. The activities of phosphoenolpyruvate carboxykinase as well as pyruvate carboxylase in pigeon liver were almost completely located intramitochondrially. Malic enzyme activity in pigeon liver was about 33 times higher than in rat liver and about 260 times higher than in guinea pig liver. However, the sum of the activities of the 4 NADPH-generating enzymes, malic enzyme + isocitrate dehydrogenase (NADP) + glucose-6-phosphate dehydrogenase + 6-phosphogluconate dehydrogenase was similar in pigeon and guinea pig liver, but somewhat lower in rat liver.



Measurement of oxygen uptake during gluconeogenesis from intraportally infused lactate+pyruvate (10/1) was performed with isolated perfused livers from pigeon, rats and guinea pigs. The ratio oxygen used/glucose formed was 2.47 in pigeon liver, 2.42 in rat liver, and 2.35 in guinea pig liver.



These results are very close to the theoretically expected value of 2.



The following conclusions have been made: In pigeon liver phosphoenolpyruvate formed intramitochondrially is used for gluconeogenesis. There is no correlation between the overall ATP/ADP ratio and the rate of glucose formation. The regulation of gluconeogenesis is strongly species-dependent as can be demonstrated for instance by the inability of pigeon liver to form glucose from pyruvate. The rate of “futile cycling” of carbon between phosphoenolpyruvate and pyruvate cannot exceed 40% of the rate of pyruvate carboxylation. The significant species difference in the maximum capacity for glucose formation from lactate cannot be explained on the basis of different degrees of “futile cycling”.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1973.tb02980.x

Relationship between Intracellular Distribution of Phosphoenolpyruvate Carboxykinase, Regulation of Gluconeogenesis, and Energy Cost of Glucose Formation

Intracellular Zinc Movement and Its Effect on the Carbohydrate Metabolism of Isolated Rat Hepatocytes

The effects of intraportal infusion of 0.18–7.20 mmole/h of sodium caproate on gluconeogenesis were studied in isolated perfused livers of fed male rats.



Even 0.18 mmole/h of sodium caproate enhanced the incorporation of carbon from the C-2 and the C-1 position of the pyruvate-lactate pool into glucose and glycogen, increased the specific activity of glucose and glycogen, and stimulated the net uptake of lactate and pyruvate as well as the net production of glucose. Net changes in liver glycogen concentration were the same with and without the infusion of caproate.



With 0.72 mmole/h of sodium caproate, the ratio of carbon incorporated into glucose to carbon incorporated into CO2 had increased about 8 times for carbon from the C-2 position and about 4 times for carbon from the C-1 position of the lactate-pyruvate pool.



An increase in the lactate/pyruvate and the β-hydroxybutyrate/acetoacetate ratio in the liver and in the perfusion medium occurred only with 0.72mmole/h or more of sodium caproate, whereas the CoASH/CoASAc ratio had already been decreased significantly with 0.18 mmole/h. A similar decrease in the CoASH/CoASAc ratio was observed in vivo in livers of fat-fed rats. Fat-feeding led to a 300% increase in the steady-state concentration of CoASH plus CoASAc in vivo, whereas the steady-state concentration of CoASH plus CoASAc in perfused livers did not change either with or without caproate. In contrast to this, the total concentration of ATP plus ADP was decreased by 40–50% after 30 min of perfusion with and without caproate. The ATP/ADP ratio was not changed by the intraportal infusion of caproate.



From this it has been concluded, that:



1
The primary mechanism by which fatty acids enhance gluconeogenesis is a stimulation of the pyruvate carboxylase reaction and an inhibition of pyruvate oxidation. This is accomplished by an increase in the steady-state concentration of CoASAc and a decrease in the CoASH/CoASAc ratio, rather than a stimulation of the triosephosphate dehydrogenase reaction by a decreased cytoplasmic NAD+/NADH ratio.

2
The chance that oxaloacetate formed by carboxylation of pyruvate will be converted to phosphoenolpyruvate is higher in liver than in kidney.

3
Changes in the ATP/ADP ratio are not directly related to the regulation of gluconeogenesis by fatty acid oxidation.

Regulation of gluconeogenesis by fatty acid oxidation in isolated perfused livers of non-starved rats.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2879%2980494-9

Subcellular compartmentation of guanine nucleotides and functional relationships between the adenine and guanine nucleotide systems in isolated hepatocytes

Jochen Kleineke

Papers

Regulation of Gluconeogenesis in the Guinea Pig Liver

Relationship between Intracellular Distribution of Phosphoenolpyruvate Carboxykinase, Regulation of Gluconeogenesis, and Energy Cost of Glucose Formation

Intracellular Zinc Movement and Its Effect on the Carbohydrate Metabolism of Isolated Rat Hepatocytes

Regulation of gluconeogenesis by fatty acid oxidation in isolated perfused livers of non-starved rats.

Subcellular compartmentation of guanine nucleotides and functional relationships between the adenine and guanine nucleotide systems in isolated hepatocytes