Publisher Summary This chapter discusses preparation of isolated rat liver cells The early mechanical and chemical methods for liver-cell preparation were relatively successful in converting liver tissue to a suspension of isolated cells The successful preparation of intact liver cells by perfusion with collagenase is technically quite difficult The major method for preparation of nonparenchymal liver cells is based on the selective sensitivity of parenchymal cells toward proteases By incubation of rat liver minces with pronase, most of the liver parenchyma is digested, while nonparenchymal cells remain intact and can be recovered from the incubate Similar results have been reported with trypsin digestion of collagenase-dispersed liver minces, but pronase appears to be more effective The most common procedure is to perfuse the liver briefly with pronase before it is minced and incubated with the enzyme Such direct pronase methods have been used by several investigators with yields of nonparenchymal liver cells reported to be in the range 2–15 × 10 6 cells/gm liver

Preparation of isolated rat liver cells.

While virtually absent in our diet a few hundred years ago, fructose has now become a major constituent of our modern diet. Our main sources of fructose are sucrose from beet or cane, high fructose corn syrup, fruits, and honey. Fructose has the same chemical formula as glucose (C(6)H(12)O(6)), but its metabolism differs markedly from that of glucose due to its almost complete hepatic extraction and rapid hepatic conversion into glucose, glycogen, lactate, and fat. Fructose was initially thought to be advisable for patients with diabetes due to its low glycemic index. However, chronically high consumption of fructose in rodents leads to hepatic and extrahepatic insulin resistance, obesity, type 2 diabetes mellitus, and high blood pressure. The evidence is less compelling in humans, but high fructose intake has indeed been shown to cause dyslipidemia and to impair hepatic insulin sensitivity. Hepatic de novo lipogenesis and lipotoxicity, oxidative stress, and hyperuricemia have all been proposed as mechanisms responsible for these adverse metabolic effects of fructose. Although there is compelling evidence that very high fructose intake can have deleterious metabolic effects in humans as in rodents, the role of fructose in the development of the current epidemic of metabolic disorders remains controversial. Epidemiological studies show growing evidence that consumption of sweetened beverages (containing either sucrose or a mixture of glucose and fructose) is associated with a high energy intake, increased body weight, and the occurrence of metabolic and cardiovascular disorders. There is, however, no unequivocal evidence that fructose intake at moderate doses is directly related with adverse metabolic effects. There has also been much concern that consumption of free fructose, as provided in high fructose corn syrup, may cause more adverse effects than consumption of fructose consumed with sucrose. There is, however, no direct evidence for more serious metabolic consequences of high fructose corn syrup versus sucrose consumption.

http://www.perishablepundit.com/docs/fructose-and-obesity.pdf

Metabolic Effects of Fructose and the Worldwide Increase in Obesity

Adaptations of Glucose and Long-Chain Fatty Acid Metabolism in Liver of Dairy Cows during the Periparturient Period

Publisher Summary
This chapter discusses the study of the regulation of metabolism by mathematical models. There has grown a sizable literature on the theoretical analysis of biological regulation, mostly by means of mathematical models. Modeling is an advanced technique for the study of the regulation. It involves the quantitative consideration of the multitude of data and interactions characteristic for biological systems. Modeling is useful for the deduction of the essential relations in metabolism that constitute a characteristic dynamic structure. The most important precondition for modeling is the knowledge of the main metabolic routes. It requires the identification of the stoichiometric relations in the various enzymatic reactions, branching and merging points, bypasses, and most of the intermediates. The chapter discusses different types of models and the methods of modeling.

Metabolic regulation and mathematical models

Healthy subjects ingested 2H2O and after 14, 22, and 42 h of fasting the enrichments of deuterium in the hydrogens bound to carbons 2, 5, and 6 of blood glucose and in body water were determined. The hydrogens bound to the carbons were isolated in formaldehyde which was converted to hexamethylenetetramine for assay. Enrichment of the deuterium bound to carbon 5 of glucose to that in water or to carbon 2 directly equals the fraction of glucose formed by gluconeogenesis. The contribution of gluconeogenesis to glucose production was 47 +/- 49% after 14 h, 67 +/- 41% after 22 h, and 93 +/- 2% after 42 h of fasting. Glycerol's conversion to glucose is included in estimates using the enrichment at carbon 5, but not carbon 6. Equilibrations with water of the hydrogens bound to carbon 3 of pyruvate that become those bound to carbon 6 of glucose and of the hydrogen at carbon 2 of glucose produced via glycogenolysis are estimated from the enrichments to be approximately 80% complete. Thus, rates of gluconeogenesis can be determined without corrections required in other tracer methodologies. After an overnight fast gluconeogenesis accounts for approximately 50% and after 42 h of fasting for almost all of glucose production in healthy subjects.

https://dm5migu4zj3pb.cloudfront.net/manuscripts/118000/118803/JCI96118803.pdf

Contributions of gluconeogenesis to glucose production in the fasted state.

Lipogenesis in Rat Hepatocytes

Effects of aminooxyacetate on the metabolism of isolated liver cells.

The rates of O2 consumption during fatty acid oxidation to acetyl-CoA and to CO2 were determined for isolated liver cells from starved rats on the basis of measured rates of respiration and ketogenesis. About 60% of the endogenous O2 uptake was associated with acetyl-CoA formation. The remainder was assumed to represent total combustion of fatty acid to CO2 and H2O through the Krebs cycle. In the absence of added fatty acid, 1.90 μmol · min-1· g-1 of acetyl-CoA was generated, of which 1.40 μmol · min-1· g−1 gave rise to ketone bodies. O2 consumption was stimulated about 30% by the addition of 2 mM palmitate or 4 mM hexanoate. This increase was entirely due to stimulation of O2 consumption related to oxidation of fatty acid to acetyl-CoA. The extra acetyl-CoA produced was channelled into ketone body formation.



The gluconeogenic substrate lactate, which increases hepatic demand for ATP, stimulated O2 consumption almost 40%, but in this case the increase was due to an enhancement of Krebs cycle activity, and acetyl-CoA generation was depressed 25%. In the absence of added fatty acid, 83% of the total acetyl-CoA produced by cells incubated with lactate was oxidized in the Krebs cycle. Addition of fatty acid caused a further stimulation of O2 uptake which reflected a promotion of acetyl-CoA production with an associated increase in ketone body formation.



The uncoupling agent, 2,4-dinitrophenol, stimulated total O2 consumption of cells incubated without exogenous substrate or with added fatty acid. In each circumstance this stimulation of O2 uptake was related entirely to enhancement of Krebs cycle activity and depression of ketogeneis. The large increment in O2 consumption induced by the combination of lactate and 2,4-dinitrophenol in the presence of added fatty acid was likewise attributable solely to a marked stimulation of Krebs cycle flux. Added ethanol caused only a small inhibition of total acetyl-CoA production and had little effect on respiration, but considerably depressed Krebs cycle activity. This depression was relieved by 2,4-dinitrophenol.



Oligomycin and antimycin had markedly different effects on fatty acid catabolism although both inhibitors depressed O2 consumption to the same extent. Oligomycin caused only a small diminution of rates of acetyl-CoA formation but induced a strong depression of cellular ATP concentrations and of acetyl-CoA oxidation to CO2. In contrast antimycin had no effect on acetyl-CoA oxidation and brought about only a small decline in cellular ATP levels, but considerably depressed formation of acetyl-CoA.



These results are interpreted as indicating that addition of fatty acid to liver cells induces various energy-dependent processes, including ATP synthesis, reversed electron transfer and metabolite translocation, all of which contribute to the observed stimulation of O2 uptake. It is inferred that oxidation of acetyl-CoA to CO2 is preferentially coupled to ATP synthesis whereas oxidation of fatty acid to acetyl-CoA is obligatorily associated with reversed electron transfer. Thus, ketogenesis is not constrained by the hepatic phosphorylation potential and provides a mechanism for the rapid disposal of fatty acids, independent of the ATP demands of the liver.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07251.x

The Calorigenic Nature of Hepatic Ketogenesis: An Explanation for the Stimulation of Respiration Induced by Fatty Acid Substrates

Isotopic evidence for futile cycles in liver cells.

Isolated rat or hamster liver parenchymal cells or kidney cortex segments were incubated in a medium containing 3HHO and the incorporation and partial distribution of tritium in the glucose formed was determined. Gluconeogenesis from lactate or pyruvate caused about 80 to 90% of the possible amount (7 μatoms per glucose molecule) of fixation of tritium from 3HHO in glucose, with a high percentage labelling on C-6. Glucose formation from glycogen breakdown under anaerobiosis caused less than 20% of the possible fixation of tritium, with most of the labelling on C-2. This technique may be of eventual use in determining rates and sources of glucose formation.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1974.tb03703.x

Dallas G. Clark

Papers

Lipogenesis in Rat Hepatocytes

Effects of aminooxyacetate on the metabolism of isolated liver cells.

The Calorigenic Nature of Hepatic Ketogenesis: An Explanation for the Stimulation of Respiration Induced by Fatty Acid Substrates

Isotopic evidence for futile cycles in liver cells.

Glucose Synthesis in Tritiated Water