Studies on gluconeogenesis and ketogenesis in isolated hepatocytes from alloxan diabetic rats.

Gluconeogenesis and ketogenesis were studied in isolated hepatocytes obtained from normal and alloxan diabetic rats. Insulin treatment maintained near-normal blood glucose levels and caused an increase in glycogen deposition. The third day after insulin withdrawal the rats displayed a diabetic syndrome marked by progressive hyperglycemia and glycogen depletion. Net glucose production in liver cells isolated from alloxan diabetic rats progressively increased with time up to 72 hr after the last in vivo insulin injection. Maximal glucose production was observed at 72 hr with 10 mM alanine, lactate, pyruvate, or fructose. Glucose production decreased at 96 hr. The same pattern was observed with the incorporation of labeled bicarbonate into glucose. Ketogenesis in liver cells and hepatic lipid content also peaked at 72 hr.     

The role of leucine in ketogenesis in starved rats.

The quantitative significance of the conversion in vivo of L-[U-14C]leucine to ketone bodies was determined in rats starved for 3 or 48 h. In animals starved for 3 h, 4.4% of ketone-body carbon is derived from the metabolism of leucine, and in rats starved for 48 h the corresponding value is 2.3%. This conversion occurs rapidly, and the specific radioactivity of ketone bodies in blood is maximal at 2 min after the intravenous injection of labelled leucine for both periods of starvation. The flux of leucine in the blood is 1.01 and 1.04 mumol/min per 100 g body wt. respectively for animals starved for 3 and 48 h. The specific radioactivity of blood ketone bodies was compared at 2 min after the injection of labelled leucine, lysine and phenylalanine. The specific radioactivity was 4-5 fold higher with leucine than with lysine or phenylalanine.     

Differential inhibition of ketogenesis by malonyl-CoA in mitochondria from fed and starved rats.

Rates of ketogenesis in mitochondria from fed or starved rats were identical at optimal substrate concentrations, but responded differently to inhibition by malonyl-CoA. Kinetic data suggest that the K1 for malonyl-CoA is greater in the starved animal. These results indicate that, for the regulation of ketogenesis in the starved state, the lower sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA may be more important than the concentration of malonyl-CoA.     

The effects of inhibition of gluconeogenesis in suckling newborn rats.

Inhibition of gluconeogenesis with 3-mercaptopicolinate in suckling newborn rats caused a fall in blood [glucose], but no change in their high plasma [free fatty acid] and blood [ketone bodies]. Active gluconeogenesis seems to be more important than sparing of glucose by high concentrations of fat-derived substrates for the maintenance of normal blood [glucose] in suckling newborn rats.     

Distinctive effects of glucagon on gluconeogenesis and ketogenesis in hepatocytes isolated from normal and biotin-deficient rats.

In hepatocytes from 48 h-starved rats identical glucagon dose-response curves were obtained for the stimulation of gluconeogenesis from lactate, for ketogenesis and for the decreasing of the C5-dicarboxylate pool. Glucagon (20 nM) caused a 5-fold increase in 3-hydroxybutyrate formation, but decreased acetoacetate production 50% of that of the control. In hepatocytes from biotin-deficient rats glucagon no longer stimulated gluconeogenesis from lactate, but still produced its effects on the mitochondrial redox state and the C5-dicarboxylate pool. The results suggest that the primary site of the hormone action on gluconeogenesis is located within the mitochondria rather than in the cytosol.     

Relationship between ketogenesis and gluconeogenesis in isolated hepatocytes from newborn rats.

In hepatocytes from 1-day-old rats, active gluconeogenesis occurs in parallel with active ketogenesis, although the carbon atoms of non-esterified fatty acids do not participate in glucose synthesis. Once a significant ketogenesis is established, a further increase does not enhance gluconeogenesis. Indeed, octanoate is more ketogenic than oleate, but stimulates gluconeogenesis to a similar extent.     

Effects of adrenaline on ketogenesis from long- and medium-chain fatty acids in starved rats.

1. Injection of adrenaline into 24 h-starved rats caused a 69% decrease in blood [ketone-body] (3-hydroxybutyrate plus acetoacetate), accompanied by a decreased [3-hydroxybutyrate]/[acetoacetate] ratio. Blood [glucose] and [lactate] increased, but [alanine] was unchanged. 2. Adrenaline also decreased [ketone-body] after intragastric feeding of both long- and medium-chain triacylglycerol. The latter decrease was observed after suppression of lipolysis with 5-methylpyrazole-3-carboxylic acid, indicating that the antiketogenic action of adrenaline was not dependent on the chain length of the precursor fatty acid. 3. The actions of adrenaline to decrease blood [ketone-body] and to increase blood [glucose] were not observed after administration of 3-mercaptopicolinate, an inhibitor of gluconeogenesis. This suggests that these effects of the hormone are related. 4. The possible clinical significance of the results is discussed with reference to the restricted ketosis often observed after surgical or orthopaedic injury.     

Stimulation by glucose of gluconeogenesis in hepatocytes isolated from starved rats.

Control properties of the gluconeogenic pathway in hepatocytes isolated from starved rats were studied in the presence of glucose. The following observations were made. (1) Glucose stimulated the rate of glucose production from 20 mM-glycerol, from a mixture of 20 mM-lactate and 2 mM-pyruvate, or from pyruvate alone; no stimulation was observed with 20 mM-alanine or 20 mM-dihydroxyacetone. Maximal stimulation was obtained between 2 and 5 mM-glucose, depending on the conditions. At concentrations above 6 mM, gluconeogenesis declined again, so that at 10 mM-glucose the glucose production rate became equal to that in its absence. (2) With glycerol, stimulation of gluconeogenesis by glucose was accompanied by oxidation of cytosolic NADH and reduction of mitochondrial NAD+ and was insensitive to the transaminase inhibitor amino-oxyacetate; this indicated that glucose accelerated the rate of transport of cytosolic reducing equivalents to the mitochondria via the glycerol 1-phosphate shuttle. (3) With lactate plus pyruvate (10:1) as substrates, stimulation of gluconeogenesis by glucose was almost additive to that obtained with glucagon. From an analysis of the effect of glucose on the curves relating gluconeogenic flux and the steady-state intracellular concentrations of gluconeogenic intermediates under various conditions, in the absence and presence of glucagon, it was concluded that addition of glucose stimulated both phosphoenolpyruvate carboxykinase and pyruvate carboxylase activity.     

Liver and kidney cortex gluconeogenesis from L-alanine in fed and starved rats

Circulating [14C]glucose 2, 5 and 10 min after intravenous injection of [U-14C]-L-alanine was greater in 24 hr starved than in fed rats. In vitro uptake of [14C]alanine by liver and kidney cortex slices from 24 hr starved and fed rats rose in parallel with increased medium substrate concentration. Formation of [14C]glucose from 1mM [14C]alanine was similar in liver and kidney cortex slices and increased in tissues from 24 hr starved compared with fed rats. With 5 mM [14C]alanine more [14C]glucose was produced by liver than by kidney cortex slices from 24 hr starved rats. Liver slices always produced more [14C]lactate and less [14C]-CO2 from [14C]alanine than kidney cortex slices. It is proposed that under physiological conditions, the kidneys cortex actively participates in glucose production from alanine.     

Comparative acute effects of leptin and insulin on gluconeogenesis and ketogenesis in perfused rat liver

The acute effects of physiological levels of leptin (10 ng ml(-1)) and insulin (20 microU ml(-1)) on hepatic gluconeogenesis and ketogenesis were compared. Leptin or insulin alone decreased (p<0.05) the activation of hepatic glucose, L-lactate and urea production from L-alanine. However, the hepatic glucose production was not modified if leptin was combined with insulin. These results indicated that both, i.e. leptin and insulin, could promote a non-additive reduction in the rate of catabolism of L-alanine. However, in contrast with insulin (p<0.05), leptin did not inhibit the activation of hepatic glucose production from pyruvate or glycerol. On the other hand, activation of hepatic production of acetoacetate and beta-hydroxybutyrate from octanoate was not affected by leptin or insulin. Thus, our data demonstrate that the acute effect of leptin on hepatic metabolism was partially similar to insulin (activation of glucose production from L-alanine and activation of acetoacetate or beta-hydroxybutyrate production from octanoate) and partially different from insulin (activation of glucose production from pyruvate or glycerol).     

Effect of lactation on gluconeogenesis and ketogenesis in ovine hepatocytes

1. Rates of glucose synthesis from radioactive precursors and ketogenesis were determined in hepatocytes from control and lactating sheep. 2. Gluconeogenesis from propionate was the same in both groups. Gluconeogenesis from lactate + pyruvate was three-fold higher in hepatocytes from lactating sheep. Palmitate stimulated gluconeogenesis from lactate + pyruvate in both groups. 3. Rates of ketogenesis from palmitate but not butyrate were slightly higher in hepatocytes from lactating sheep. No other differences in the metabolism of palmitate or butyrate were seen in the two groups. Exogenous carnitine stimulated ketogenesis from palmitate. Propionate inhibited ketogenesis from palmitate and butyrate. Lactate + pyruvate also inhibited ketogenesis slightly but stimulated oxidation and esterification. 4. It is concluded that the major changes in glucose and ketone production seen in the lactating ruminant are not the result of long-term changes within the hepatocyte but occur because of the changes in substrate supply to the liver and changes in intracellular concentrations of metabolites.     

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.     

Effect of L-alanine infusion on gluconeogenesis and ketogenesis in the rat in vivo.

1. In 48 h-starved 6-week-old rats the 14C incorporation in vivo into blood glucose from a constant-specific-radioactivity pool of circulating [14c]actateconfirmed that lactate is the preferred gluconeogenic substrate. 2. Increasing the blood [alanine] to that occurrring in the fed state increased 14C incorporation into blood glucose 2.3-fold from [14c]alanine and 1.7-fold from [14c]lactate. 3. When the blood [alanine] was increased to that in the fed state, the 14C incorporation into liver glycogen from circulating [14c]alanine or [14c]lactate increased 13.5- and 1.7-fold respectively. 4. The incorporation of 14C into blood acetoacetate and 3-hydroxybutyrate from a constant-specific-radioactivity pool of circulating [14c]oleate was virtually abolished by increasing the blood [alanine] to that existing in the fed state. However, the [acetoacetate] remained unchanged, whereas [3-hydroxybutyrate] decreased, although less rapidly than did its radiochemical concentration. 5. It is concluded that during starvation in 6-week-old rats, the blood [alanine] appears to influence ketogenesis for circulating unesterfied fatty acids and inversely affects gluconeogenesis from either lactate or alanine. A different pattern of gluconeogenesis may exist for alanine and lactate as evidenced by comparative 14C incorporation into liver glycogen and blood glucose.