Hepatic glucose cycling does not contribute to the development of hyperglycemia in Zucker diabetic fatty rats.

Hepatic glucose cycling, whereby glucose is taken up by the liver, partially metabolized, then recycled to glucose, makes a substantial contribution to the development of hyperglycemia in IDDM. This stimulation of glucose cycling appears to be associated with elevated rates of fatty acid oxidation. Whether hepatic glucose cycling also contributes to the development of hyperglycemia in NIDDM is unclear. Using a model of NIDDM, the Zucker diabetic fatty (ZDF) rat, we determined whether glucose cycling was enhanced. Hepatocytes from ZDF rats exhibited higher rates of glucose phosphorylation and glycolysis, but there was no increase in the rate of cycling between glucose and glucose-6-phosphate or between glycolytically derived pyruvate and glucose. Despite the increased rates of glycolysis, the production of CO2 in liver cells from ZDF rats was no different from rates measured in cells from control animals. Instead, there was a large increase in the accumulation of lactate and pyruvate in the ZDF liver cells. The addition of 2-bromopalmitate, an inhibitor of fatty acid oxidation that inhibited glucose cycling in hepatocytes from IDDM rats, had no effect on glucose cycling in cells from ZDF rats. We therefore conclude that, unlike in IDDM, hepatic glucose cycling does not contribute to the development of hyperglycemia in the NIDDM Zucker rat.

Suppression of glycolysis is associated with an increase in glucose cycling in hepatocytes from diabetic rats.

Rates of cycling between glucose and glucose 6-phosphate and between glucose and pyruvate, and the effects of these cycles on glucose metabolism, were compared in hepatocytes isolated from fasted normal or streptozotocin-induced diabetic rats. In diabetic hepatocytes the rate of glucose phosphorylation was 30% lower than that in normal hepatocytes, and there was a doubling of the rate of glucose/glucose 6-phosphate cycling. In addition, the rate of glycolysis was 60% lower in diabetic hepatocytes. This inhibition of glycolysis and stimulation of glucose/glucose 6-phosphate cycling appeared to be a consequence of the elevated rates of endogenous fatty acid oxidation observed in diabetic hepatocytes. The proportion of glycolytically derived pyruvate that was recycled to glucose was more than doubled in hepatocytes from diabetic rats compared with normal animals. This increase also appeared to be linked to the high rates of endogenous fatty acid oxidation in diabetic cells. As a consequence of the increased rates of both these cycles, 85% of all glucose molecules taken up by diabetic hepatocytes were recycled to glucose, compared with only 50% in normal hepatocytes. Glucose cycling is therefore likely to make a substantial contribution to the hyperglycemia of diabetes.

Effects of thyroid status on glucose cycling by isolated rat hepatocytes.

The effects of alterations in thyroid status on glucose metabolism have been investigated in rat hepatocytes. Addition of 10 or 40 mmol/L glucose induced increases in respiration rate that were significantly larger in cells from hyperthyroid rats than from hypothyroid animals. The responses of hepatocytes from euthyroid rats were intermediate. In cells from hyperthyroid rats, most of the increase occurred upon addition of 10 mmol/L glucose, with only a further small stimulation resulting when glucose concentration was increased to 40 mmol/L. For a given glucose concentration, glycolytic rates, determined by measuring release of tritium from [6-3H]glucose, were comparable in all thyroid states. Studies with 10 mmol/L [2-3H]glucose showed that cycling between glucose-6-phosphate and glucose was almost twofold higher in euthyroid and hyperthyroid states as compared with the hypothyroid state, although the magnitude of the increase in cycling rate was only approximately 0.2 mumol glucose.min-1.g-1. When 40 mmol/L [2-3H]glucose was added, over 44% of the glucose that was phosphorylated to glucose-6-phosphate was cycled back to glucose, but this cycling was independent of thyroid status. Cycling between fructose-1,6-bisphosphate and fructose-6-phosphate was negligible in all thyroid states. Rates of glycogen synthesis were comparable in hypothyroid and hyperthyroid states and slightly less than in the euthyroid state. Glycolytically formed pyruvate was cycled back to glucose in hepatocytes from hypothyroid, euthyroid, and hyperthyroid rats. During a 60-minute incubation period, cycling to glucose in the presence of 10 mmol/L or 40 mmol/L glucose was up to twofold higher in cells from euthyroid and hyperthyroid rats than in hepatocytes from hypothyroid animals. The measured increases in cycling rates induced by thyroid hormone were small and in theory could have been satisfied by a much smaller increase in respiration rate than was observed.

Stimulation of gluconeogenesis leads to an increased rate of beta-oxidation in hepatocytes from fasted diabetic but not from fasted normal rats.

We have investigated the effects of imposing an ATP demand, generated by the addition of lactate, on hepatocytes isolated from fasted normal and streptozocin-induced diabetic rats. The stimulation of O2 consumption upon lactate addition was much greater in hepatocytes from diabetic rats, as a result of a lactate-induced stimulation of beta-oxidation that was not observed in control cells. This lactate-induced increment in beta-oxidation was extremely sensitive to inhibition by low levels of a number of inhibitors of energy transduction, implying that the increment was tightly coupled to ATP synthesis. Such sensitivity of the beta-oxidative pathway to the addition of similar low concentrations of these inhibitors was not seen in control cells. Inhibitors of the gluconeogenic pathway were also more effective in decreasing beta-oxidation in cells from diabetic animals than in cells from normal rats. The increment in beta-oxidation was not accompanied by increased rates of glucose synthesis, fatty acid esterification or ureogenesis. We propose that it may be associated with higher rates of glucose cycling in cells from diabetic rats.

The contribution of glucose cycling to the maintenance of steady-state levels of lactate by hepatocytes during glycolysis and gluconeogenesis.

When hepatocytes from fasted rats were incubated with 10 mM glucose, there was a linear accumulation of lactate and pyruvate for about 80 min after which steady-state concentrations of these metabolites became established. The rate of glycolysis, determined with [6-3H]glucose, was constant over the entire incubation period and was 50% greater than that calculated from carbon balance studies. This suggests that one-third of the glycolytic products formed were recycled to glucose. To enable study of the factors associated with the generation and maintenance of the lactate steady state and to measure accurately the carbon balance, incubations were performed using supraphysiological concentrations of glucose (20-80 mM). Under these conditions the initial rate of lactate accumulation and its concentration at steady state were shown to be dependent on the concentration of extracellular glucose. Rates of glycolysis were also measured using 40 mM [6-3H]glucose and [U-14C]glucose added alone, or in combination with a steady-state lactate concentration (3 mM). There was no effect on the rate of glycolysis determine with [6-3H]glucose, even when lactate was present in the medium. The difference in rates between measurements with the two isotopes reflect the apparent degree of glucose recycling which in the absence and presence of added lactate increased from 0.26 to 0.54 mumol C3 equivalents min-1.g-1 respectively. Identical studies employing [U-14C]lactate showed that glucose and CO2 were the major products of lactate metabolism under steady-state conditions and that the formation of lactate from [U-14C]glucose exactly balanced the rate of lactate removal as a result of oxidation and gluconeogenesis. These studies provide evidence for the concomitant operation of glycolysis and gluconeogenesis, even in the presence of high glucose concentrations. They also demonstrate that lactate steady states are achieved not by the cessation of glycolysis but rather by the removal of lactate and pyruvate at a rate equal to that of their production.

Effects of fatty acid oxidation on glucose utilization by isolated hepatocytes.

We have studied the inhibitory action of long- and short-chain fatty acids on hepatic glucose utilization in hepatocytes isolated from fasted rats. The rates of hepatic glucose phosphorylation and glycolysis were determined from the tritiated products of [2-3H] and [6-3H]glucose metabolism, respectively. The difference between these was taken as an estimate of the 'cycling' between glucose and glucose-6-phosphate. In the presence of 40 mM glucose this cycling was estimated at 0.68 mumol/min/g wet wt. Glucose phosphorylation was unaffected during palmitate and hexanoate oxidation to ketone bodies but glycolysis was inhibited. The rate of glucose cycling was increased during this phase to 1.25 mumol/min/g. Following the complete metabolism of the fatty acids, glycolysis was reinstated and cycling rates returned to control levels. Hepatic glucose cycling appears to be an important component of the glucose/fatty acid cycle.

Effect of palmitate concentration on the relative contributions of the beta-oxidation pathway and citric acid cycle to total O2 consumption of isolated rat hepatocytes.

The relative contributions of beta-oxidation and citric acid cycle activity to total O2 consumption during fatty acid oxidation were examined in isolated hepatocytes. When hepatocytes were incubated with palmitate alone, a rise in fatty acid concentration induced an increase in O2 uptake that reflected a large stimulation of beta-oxidation and an accompanying smaller inhibition of citric acid cycle oxidation. In the presence of lactate, successive increments in palmitate concentration over the range from 0 to 1.0 mM stimulated glucose synthesis and brought about a concomitant incremental stimulation of both beta-oxidation and citric acid cycle flux. However, above 1.5 mM palmitate, additional increments in fatty acid concentration depressed gluconeogenesis and citric acid cycle activity but induced a further stimulation of beta-oxidation. These findings demonstrate that, during fatty acid oxidation, the rate of citric acid cycle turnover is more closely linked to the rate of glucose synthesis than is the rate of beta-oxidation. This may be relevant to observations that the stimulation of hepatic O2 consumption, induced by fatty acid oxidation, is much greater than can be explained in terms of the ATP-demand arising from exposure of hepatocytes to fatty acid.

Relationship between the stimulation of citric acid cycle oxidation and the stimulation of fatty acid esterification and inhibition of ketogenesis by lactate in isolated rat hepatocytes.

Isolated hepatocytes from fasted rats were used to study the effects of lactate on palmitate metabolism. Lactate was found to stimulate fatty acid esterification and citric acid cycle oxidation and to inhibit ketone body synthesis. These effects of lactate were largely maintained when gluconeogenesis was inhibited with either quinolinate or perfluorosuccinate, but were overcome by alpha-cyano-4-hydroxycinnamate. However, the responses of hepatocytes to lactate could be restored in the presence of alpha-cyano-4-hydroxycinnamate by the further addition of propionate. The stimulation of triacylglycerol synthesis by lactate was not associated with an increase in the concentration of glycerol 3-phosphate. Rather, there was a correlation between flux through the citric acid cycle and the rate of triacylglycerol synthesis. In all instances reduction of ketone body formation in the presence of lactate was accompanied by a stimulation of citric acid cycle oxidation.

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: further evidence for thermodynamic control of metabolism.

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.

Evidence that stimulation of gluconeogenesis by fatty acid is mediated through thermodynamic mechanisms.

We have studied the stimulatory effects of palmitate on the rate of glucose synthesis from lactate in isolated hepatocytes. Control of the metabolic flow was achieved by modulating the activity of enolase using graded concentrations of fluoride. Unexpectedly, palmitate stimulated gluconeogenesis even when enolase was rate-limiting. This stimulation was also observed when the activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase were modulated using graded concentrations of quinolinate and aminooxyacetate, respectively. Linear force-flow relationships were found between the rate of gluconeogenesis and indicators of cellular energy status (i.e. mitochondrial membrane and redox potentials and cellular phosphorylation potential). These findings suggest that the fatty acid stimulation of glucose synthesis is in part mediated through thermodynamic mechanisms.

Linear relationships between mitochondrial forces and cytoplasmic flows argue for the organized energy-coupled nature of cellular metabolism.

We have studied rates of formation of glucose, urea and lactate by isolated hepatocytes incubated with a variety of inhibitors of energy transduction. Linear relationships have been found between these metabolic rates and mitochondrial forces (membrane, redox and phosphorylation potentials). The findings are suggestive of extensive enzyme organization within these metabolic pathways.

Preparation of cytoplasmic bodies (cytospheres) from isolated hepatocytes and their biochemical properties.

The controlled centrifugation of isolated rat hepatocytes at 260 000 g results in the formation of membrane-bounded cell fragments that we have termed 'cytospheres'. A method is described for the isolation of these cytospheres. Cytospheres are spherical, have a mean diameter of 9.2 +/- 3.2 microns (SD) and a protein content of 225 +/- 12 mg/g wet wt. About 3% of the protein from the original isolated hepatocyte suspension is recoverable. Transmission electron microscopy (TEM) shows cytospheres to possess a trilaminar membrane, and a finely granular hyaloplasm generally devoid of organelles, filaments and microtubules. Freeze-fracture studies reveal a membrane structure typical of a plasma membrane. Ouabain and wheat germ agglutinin (WGA)-binding studies indicate that the original orientation of the plasma membrane is maintained throughout the formation of the cytospheres. The cytospheres have also been characterized biochemically. Cytospheres are enriched in the enzymes normally associated with the hyaloplasm, whereas the activities of enzymes localized in organelles are greatly diminished. Lipid analysis of the cytosphere membrane indicates that it is derived from the plasma membrane of the hepatocyte. Cytospheres are sensitive to changes in the osmolarity and ionic composition of their environment. Cytospheres should therefore prove a useful preparation for the study of hyaloplasm metabolism and of plasma membrane receptor and permeability properties.