Lactate Gluconeogenesis Research Articles

Short-term effects of sodium arsenite (AsIII) and sodium arsenate (AsV) on carbohydrate metabolism in the perfused rat liver

The actions of arsenite and arsenate on carbohydrate metabolism in the once-through perfused rat liver were investigated. The compound inhibited lactate gluconeogenesis with an IC50 of 25 µM. It also increased glycolysis and fructolysis at concentrations between 10 and 100 µM. This effect was paralleled by strong inhibition of pyruvate carboxylation (IC50 = 4.25 µM) and by a relatively moderate diminution in the ATP levels. The inhibitory action of arsenate on pyruvate carboxylation and lactate gluconeogenesis was 103 times less effective than that of arsenite. For realistic doses and concentrations («1 mM), impairment of metabolism by arsenate can be expected to occur solely after its reduction to arsenite. Arsenite, on the other hand, can be regarded as a strong short-term modifier of lactate gluconeogenesis and other pathways. The main cause of the former is inhibition of pyruvate carboxylation, a hitherto unknown effect of arsenic compounds.

Kinetics of the metabolic effects, distribution spaces and lipid-bilayer affinities of the organo-chlorinated herbicides 2,4-D and picloram in the liver

Tordon® is the commercial name of a mixture of two organo-chlorinated herbicides, 2,4-D and picloram. Both compounds affect energy transduction in isolated mitochondria and the present study aimed at characterizing the actions of these two compounds on liver metabolism and their cellular distribution in the isolated perfused rat liver. 2,4-D, but not picloram, increased glycolysis in the range from 10 to 400 μM. The redox potential of the cytosolic NAD+-NADH couple was also increased by 2,4-D. Both compounds inhibited lactate gluconeogenesis. Inhibitions by 2,4-D and picloram were incomplete, reaching maximally 46% and 23%, respectively. Both compounds diminished the cellular ATP levels. No synergism between the actions of 2,4-D and picloram was detected. Biotransformations of 2,4-D and picloram were slow, but their distributions occurred at high rates and were concentrative. Molecular dynamics simulations revealed that 2,4-D presented low affinity for the hydrophobic lipid bilayers, the opposite occurring with picloram. Inhibition of energy metabolism is possibly a relevant component of the toxicity of 2,4-D and of the commercial product Tordon®. Furthermore, the interactions of 2,4-D with the membrane lipid bilayer can be highly destructive and might equally be related to its cellular toxicity at high concentrations.

Cadmium chloride inhibits lactate gluconeogenesis in mouse renal proximal tubules: An in vitro metabolomic approach with (13)C NMR.

Using isolated mouse renal proximal tubules incubated with lactate as substrate, we have found that the addition of 1-50 μM cadmium chloride (CdCl2) caused a concentration-dependent decrease in lactate utilization, in glucose production and in the cellular level of ATP, coenzyme A, acetyl-coenzyme A and glutathione (reduced and oxidized forms). Combining enzymatic and (13)C NMR measurements in a cellular metabolomic approach, we have shown that, in the presence of 10 μM CdCl2, fluxes through the key-enzymes of gluconeogenesis, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were greatly depressed by cadmium. This was accompanied by a reduction in fluxes through the enzymes of the tricarboxylic acid cycle. Comparing the mouse and human renal metabolic responses to cadmium, it is interesting to observe that the mouse renal proximal tubule was much more sensitive than the human renal proximal tubule to the adverse effects of CdCl2. As far as renal gluconeogenesis is concerned, the mouse seems to be an appropriate and convenient animal model to study the mechanism of cadmium nephrotoxicity. However, the data obtained in the mouse should be extrapolated to humans with caution because the inhibition of fluxes through the enzymes of the tricarboxylic acid cycle in mouse tubules were not observed in human tubules.

Molecular action of metformin in hepatocytes: an updated insight.

Although, metformin is a drug of the first choice in the treatment of type 2 diabetes mellitus, its molecular action is not fully determined. It is widely accepted that the antihyperglycemic effect of metformin is a result of a decrease in hepatic glucose production, and several cellular targets of the drug have been proposed. The reduction of gluconeogenesis evoked by metformin may be a result of an energy deficit evoked through the inhibition of mitochondrial respiratory chain complex I and/or increased cytosolic redox state and decreased mitochondrial redox state elicited by the inhibition of mitochondrial glycerophosphate dehydrogenase (mGPD). Metformin mediated reduction of hepatic gluconeogenesis was found to be AMP-activated protein kinase (AMPK) dependent and independent, including the inhibition of gluconeogenesis gene expression and allosteric regulation of key gluconeogenesis enzymes. Recently, it was reported that inhibition of mGPD by metformin decreases the level of dihydroxyacetone phosphate and reduces the conversion of lactate to pyruvate, that in consequence diminishes the utilization of glycerol and lactate for gluconeogenesis. The purpose of this paper is to discus molecular mechanisms responsible for the metabolic action of metformin.

N-Octyl gallate as inhibitor of pyruvate carboxylation and lactate gluconeogenesis.

The alkyl gallates are found in several natural and industrial products. In the latter products, these compounds are added mainly for preventing oxidation. In the present work, the potencies of methyl gallate, n-propyl gallate, n-pentyl gallate, and n-octyl gallate as inhibitors of pyruvate carboxylation and lactate gluconeogenesis were evaluated. Experiments were done with isolated mitochondria and the isolated perfused rat liver. The potency of the gallic acid esters as inhibitors of pyruvate carboxylation in isolated mitochondria obeyed the following decreasing sequence: n-octyl gallate > n-pentyl gallate > n-propyl gallate > methyl gallate. A similar sequence of decreasing potency for lactate gluconeogenesis inhibition in the perfused liver was found in terms of the portal venous concentration. Both actions correlate with the lipophilicity of the compounds. The effects are harmful at high concentrations. At appropriate concentrations, however, octyl gallate should act therapeutically because its inhibitory action on gluconeogenesis will contribute further to its proposed antihyperglycemic effects.

Magnitude and mechanisms of glucose counterregulation following islet transplantation in patients with type 1 diabetes suffering from severe hypoglycaemic episodes

Pancreatic islet transplantation stabilises glycaemic control in type 1 diabetes mellitus patients with neuroglycopoenia, despite them not achieving insulin independence because of limited graft function. However, the extent and underlying metabolic pathways of restored glucose counterregulation are unknown. We therefore compared systemic glucose turnover, including lactate gluconeogenesis (GN) and muscle glucose uptake, in individuals with type 1 diabetes who were transplant recipients with partial graft function (T1DM/ITx(+)), matched non-transplanted individuals with type 1 diabetes (T1DM/ITx(-)) and matched healthy non-diabetic individuals. Participants (n = 12 in each group) underwent a euglycaemic and a hypoglycaemic (2.5-2.8 mmol/l) hyperinsulinaemic clamp (0.8 mU kg(-1) min(-1)) in a randomised crossover fashion. Systemic and skeletal muscle glucose and lactate kinetics were assessed using a combination of isotopic and forearm balance techniques. Whole-body glucose counterregulation, the difference in glucose infusion rates required to maintain the glycaemic goal between the hypoglycaemic and euglycaemic clamps, was improved in T1DM/ITx(+) (7.8 ± 1.3 μmol kg(-1) min(-1)) compared with T1DM/ITx(-) (0.3 ± 0.9 μmol kg(-1) min(-1)), but was ~45% lower than in controls (14.1 ± 2.1 μmol kg(-1) min(-1)). Increased endogenous glucose production (EGP) and decreased systemic glucose disposal accounted for 49% and 39% of glucose counterregulation in T1DM/ITx(+), respectively, compared with 60% and 36% in controls. Lactate GN increased in T1DM/ITx(+) (2.7 ± 0.4 μmol kg(-1) min(-1)) and controls (1.7 ± 0.5 μmol kg(-1) min(-1)), such that it accounted for 70% and 20% of the increased EGP, respectively. Skeletal muscle accounted for similar proportions of the decrease in systemic glucose disposal in controls (49%) and T1DM/ITx(+) (41%). Partial islet graft function improves hypoglycaemia counterregulation by increasing EGP, largely via lactate GN and decreasing systemic glucose disposal. This may explain the reduction in severe hypoglycaemic events in T1DM/ITx(+) individuals. ClinicalTrials.gov NCT01668485.

The metabolic effects of prolonged starvation and refeeding in sturgeon and rainbow trout

The present study examines the particular metabolic strategies of the sturgeon Acipenser naccarii in facing a period of prolonged starvation (72days) and subsequent refeeding (60days) compared to the trout Oncorhynchus mykiss response under similar conditions. Plasma metabolites, endogenous reserves, and the activity of intermediate enzymes in liver and white muscle were evaluated. This study shows the mobilization of tissue reserves during a starvation period in both species with an associated enzymatic response. The sturgeon displayed an early increase in hepatic glycolysis during starvation. The trout preferentially used lactate for gluconeogenesis in liver and white muscle. The sturgeon had higher lipid-degradation capacity and greater synthesis of hepatic ketone bodies than the trout, although this latter species also showed strong synthesis of ketone bodies during starvation. During refeeding, the metabolic activity present before starvation was recovered in both fish, with a reestablishment of tissue reserves, plasmatic parameters (glucemia and cholesterol), and enzymatic activities in the liver and muscle. A compensatory effect in enzymes regarding lipids, ketone bodies, and oxidative metabolism was displayed in the liver of both species. There are metabolic differences between sturgeon and trout that support the contention that the sturgeon has common characteristics with elasmobranchs and teleosts.

Cadmium chloride inhibits lactate gluconeogenesis in isolated human renal proximal tubules: a cellular metabolomic approach with 13C-NMR

As part of a study on cadmium nephrotoxicity, we studied the effect of cadmium chloride (CdCl2) in isolated human renal proximal tubules metabolizing the physiological substrate lactate. Dose-effect experiments showed that 10-500 μM CdCl2 reduced lactate removal, glucose production and the cellular levels of ATP, coenzyme A, acetyl-coenzyme A and of reduced glutathione in a dose-dependent manner. After incubation with 5 mM L: -[1-(13)C]-, or L: -[2-(13)C]-, or L: -[3-(13)C] lactate or 5 mM L: -lactate plus 25 mM NaH(13)CO3 as substrates, substrate utilization and product formation were measured by both enzymatic and carbon 13 NMR methods. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism previously validated showed that 100 μM CdCl2 caused an inhibition of flux through lactate dehydrogenase and alanine aminotransferase and through the entire gluconeogenic pathway; fluxes were diminished by 19% (lactate dehydrogenase), 28% (alanine aminotransferase), 28% (pyruvate carboxylase), 42% (phosphoenolpyruvate carboxykinase), and 52% (glucose-6-phosphatase). Such effects occurred without altering the oxidation of the lactate carbons or fluxes through enzymes of the tricarboxylic acid cycle despite a large fall of the cellular ATP level, a marker of the energy status and of the viability of the renal cells. These results that were observed at clinically relevant tissue concentrations of cadmium provide a biochemical basis for a better understanding of the cellular mechanism of cadmium-induced renal proximal tubulopathy in humans chronically exposed to cadmium.

The antidiabetic metformin is toxic to liver mitochondria of Zucker Diabetic Fatty (ZDF) rats: a cellular metabolomic study with 13C‐lactate and carbon 13 NMR

Metformin decreases hyperglycemia in diabetic subjects and animals. This effect results in part from the inhibition of hepatic gluconeogenesis. The objective of this study was to investigate in vitro the effect of metformin on hepatic lactate gluconeogenesis by using an innovative approach.Precision‐cut liver slices from adult male ZDF rats were incubated with variously 13C‐labeled lactates (5 mM) or with 5 mM unlabeled lactate plus 25 mM 13C‐bicarbonate. Substrate removal and product formation were measured by enzymatic and 13C‐NMR methods.In slices from fasted lean ZDF rats, metformin dose‐dependently inhibited 13C‐glucose synthesis from [2‐13C]‐lactate (IC50 2.3 mM). The labeling patterns of the glucoses synthesized indicated that lactate gluconeogenesis involved not only the passage of carbons through pyruvate carboxylase and the reversible equilibration of oxaloacetate with fumarate but also the entire tricarboxylic acid cycle.In slices from fed diabetic ZDF rats, 2 mM metformin inhibited lactate consumption, glucose production, the synthesis of all the 13C‐labeled glucose carbons, the production of 13CO2 and reduced the cellular ATP level. By contrast, metformin dramatically increased lactic acid production and ketogenesis.These results strongly suggest that a compound found to be toxic to liver mitochondria in vitro should not necessarily be withdrawn from drug development.

Uranyl nitrate inhibits lactate gluconeogenesis in isolated human and mouse renal proximal tubules: A 13C-NMR study

As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate reduced lactate removal and gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs–Henseleit medium with 5 mM l-[1- 13C]-, or l-[2- 13C]-, or l-[3- 13C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. In the presence of 3 mM uranyl nitrate, glucose production and the intracellular ATP content were significantly reduced in both human and mouse tubules. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic enzymes in the presence of 3 mM uranyl nitrate; in human and mouse tubules, fluxes were lowered by 20% and 14% (lactate dehydrogenase), 27% and 32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase), and 39% and 45% (glucose-6-phosphatase), respectively. These results indicate that natural uranium is an inhibitor of renal lactate gluconeogenesis in both humans and mice.

The action of n-propyl gallate on gluconeogenesis and oxygen uptake in the rat liver

In the present study the metabolic actions of n-propyl gallate on hepatic gluconeogenesis, oxygen uptake and related parameters were investigated. Experiments were done in the isolated perfused rat liver. n-Propyl gallate inhibited gluconeogenesis and stimulated oxygen uptake at concentrations up to 200 μM. The inhibitory effects on lactate gluconeogenesis (ED 50 86.4 μM) and alanine gluconeogenesis were considerably more pronounced than those on glycerol and fructose gluconeogenesis. n-Propyl gallate also stimulated oxygen uptake in both the mitochondrial (63%) and microsomal (37%) electron transport chains. The first one is due mainly to the oxidation of n-propanol, as a metabolite of the first step of n-propyl gallate transformation. The second one results from a direct stimulation of the microsomal electron transport chain. n-Propyl gallate inhibited pyruvate carboxylation (ED 50 142.2 μM) in consequence of an inhibition of pyruvate transport into the mitochondria an effect not found for gallic acid. This is probably the main cause for glucose output inhibition. Secondary causes are (1) deviation of intermediates for the production of NADPH to be used in microsomal electron transport; (2) deviation of glucose 6-phosphate for glucuronidation reactions; (3) gluconeogenesis inhibition by n-propanol, produced intracellularly from n-propyl gallate. Inhibition of mitochondrial energy metabolism is not significant in the range up to 200 μM, as indicated by the very small effect on the cellular ATP levels (5% decreased). n-Propyl gallate can be considered a kind of metabolic effector, whose actions on the liver metabolism are relatively mild although they can become harmful for the organ and the whole organism at high doses and concentrations.

A Potential Important Role of Skeletal Muscle in Human Counterregulation of Hypoglycemia

During hypoglycemia, systemic glucose uptake (SGU) decreases and endogenous glucose release (EGR) increases. Skeletal muscle appears to be primarily responsible for the reduced SGU and may be important for the increased EGR by providing lactate for gluconeogenesis (GN). The objective of the study was to test the hypothesis that reduced muscle glucose uptake and increased muscle lactate release both make major contributions to glucose counterregulation using systemic isotopic techniques in combination with forearm net balance measurements. The study was conducted at the University of Giessen Clinical Research Center. Nine healthy volunteers participated in the study. A 2-h hyperinsulinemic euglycemic clamp (blood glucose approximately 4.4 mm) was followed by a 90-min hypoglycemic clamp (blood glucose approximately 2.6 mm). Compared with the euglycemic clamp, SGU decreased (21.0 +/- 2.0 vs. 29.6 +/- 1.8 micromol.kg body weight(-1).min(-1); P < 0.001), whereas EGR (11.2 +/- 1.7 vs. 4.9 +/- 1.3 micromol.kg body weight(-1) .min(-1); P < 0.003), arterial lactate concentrations (1051 +/- 162 vs. 907 +/- 115 microm; P < 0.02), systemic lactate release (23.5 +/- 0.9 vs. 17.1 +/- 0.9 micromol.kg body weight(-1).min(-1); P < 0.001), and lactate GN (4.50 +/- 0.60 vs. 2.74 +/- 0.30 micromol.kg body weight(-1).min(-1); P < 0.02) increased during hypoglycemia; the proportion of lactate used for GN remained unchanged (38 +/- 4 vs. 32 +/- 3%; P = 0.27). Whole-body muscle glucose uptake decreased approximately 50% during hypoglycemia (6.4 +/- 1.9 vs. 13.6 +/- 2.9 micromol.kg body weight(-1).min(-1); P < 0.001), which accounted for approximately 85% of the reduction of SGU. Whole-body muscle lactate release increased 6.6 +/- 1.6 micromol.kg body weight(-1). min(-1) (P < 0.01), which could have accounted for all the increase in systemic lactate release and, considering the proportion of lactate used for GN, contributed 1.4 +/- 0.4 micromol.kg body weight(-1).min(-1) (approximately 25%) to the increase in EGR. Reduced muscle glucose uptake and increased muscle lactate release both make major contributions to glucose counterregulation in humans.

Type 2 diabetic patients have increased gluconeogenic efficiency to substrate availability, but intact autoregulation of endogenous glucose production

Objective. An autoregulatory mechanism involving a reciprocal relationship between gluconeogenesis and glycogenolysis regulates endogenous glucose production (EGP) in healthy individuals. In type 2 diabetes, fasting hyperglycemia may be due to increased EGP. Material and methods. To examine gluconeogenesis and autoregulation of EGP in type 2 diabetes, 9 type 2 diabetics and 8 healthy controls were studied during a 3‐h infusion of 30 µmol/kg/min Na‐lactate. The diabetics were also studied during a control infusion of Na‐bicarbonate. To standardize levels of glucoregulatory hormones, plasma insulin, growth hormone, and glucagon were clamped at identical levels during the three experiments. Glucagon levels were elevated from basal levels to ∼330 ng/l when the lactate or bicarbonate infusions were started, in order to mimic the hyperglucagonemia often seen in diabetes. Lactate gluconeogenesis and total EGP were measured by infusions of [6‐3H] glucose and [U‐14C] lactate. Results. In the bicarbonate experiments, hyperglugagonemia increased lactate gluconeogenesis in the diabetic patients from 4.3±1.8 to 6.1±2.4 µmol/kg/min (p = 0.04). EGP did not change significantly (basal EGP: 15.3±3.9, EGP at the end of the study: 14.2±3.9 µmol/kg/min, p = 0.14). During both lactate experiments, plasma lactate increased more than 4‐fold. The increase in lactate gluconeogenesis was significantly higher in diabetics than in controls (values obtained at the end of experiments minus basal values: 10.8±3.6 versus 6.4±3.6 µmol/kg/min, p = 0.03). Compared with normal subjects, the diabetic patients had higher EGP values both at basal conditions (p = 0.001) and during lactate infusion (p = 0.005). Despite augmented gluconeogenesis, EGP did not change during lactate and glucagon infusion in any of the groups (diabetics, basal EGP: 15.4±2.7 versus EGP at the end of experiments: 15.6±3.6 µmol/kg/min, p>0.30. Controls, basal EGP: 11.8±0.8 versus EGP at the end of experiments: 11.6±1.9 µmol/kg/min, p>0.30). Conclusions. Althought type 2 diabetics have increased EGP and increased lactate gluconeogenesis, the hepatic autoregulation of EGP during increased substrate‐induced gluconeogenesis seems to be intact.

Metabolic viability and pharmaco-toxicological reactivity of cryopreserved human precision-cut renal cortical slices

We have tested the suitability of cryopreserved human precision-cut renal cortical slices for metabolic and pharmaco-toxicological studies. The viability of these slices and their pharmaco-toxicological reactivity were assessed using intracellular ATP and protein contents, lactate dehydrogenase (LDH) leakage, lactate and glutamine metabolism and the ammoniagenic effect of valproate. Despite a decrease in ATP and protein contents when compared with those of fresh slices, cryopreserved slices did not show any LDH leakage and retained the capacity to metabolize glutamine and lactate. Glutamine removal and ammonia, lactate and alanine production were similar in fresh and cryopreserved slices; by contrast, cryopreserved slices accumulated more glutamate as a result of decreased flux through glutamate dehydrogenase which catalyses an oxygen-dependent reaction. Valproate markedly and similarly stimulated glutamine metabolism in fresh and cryopreserved slices. Cryopreservation did not alter lactate removal but inhibited lactate gluconeogenesis. In conclusion, these results demonstrate that, although their mitochondrial oxidative metabolism seems to be diminished, cryopreserved human precision-cut renal cortical slices remain metabolically viable and retain the capacity to respond to the ammoniagenic effect of valproate. Thus, this experimental model may be helpful to optimize the use of human renal tissue for metabolic and pharmaco-toxicological studies.

Lactate carbon does not enter the sugars of lipopolysaccharide when gonococci are grown in a medium containing glucose and lactate: implications in vivo

In media containing glucose, lactate stimulates the metabolism of gonococci at concentrations that simulate conditions in vivo. Nuclear magnetic resonance (NMR) spectroscopy of 13C-labelled lipids obtained from gonococci grown in a synthetic medium with 13C-labelled lactate and unlabelled glucose (culture A), 13C-labelled glucose alone (culture B) or 13C-labelled glucose and unlabelled lactate (culture C) showed lactate carbon was not present in glycerol/ethanolamine residues of lipids from culture A. This indicated that, in the presence of glucose, lactate gluconeogenesis is shut down. Hence, the stimulation of metabolism could result from the production of extra energy because lactate is used solely for conversion to acetyl-CoA, the precursor of fatty acid synthesis and the components of the tricarboxylic acid cycle. In this paper, additional evidence for lack of gluconeogenesis has been sought using a different approach. The carbohydrate moieties of lipopolysaccharide (LPS) have been examined for lactate carbon after gonococci were grown with lactate and glucose. Two methods were used: NMR spectroscopy of 13C-labelled lipopolysaccharide purified from the three cultures described above showed that, in the presence of glucose, lactate carbon, in contrast to glucose carbon, was not in the carbohydrate moiety. Also, 14C-labelled lactate was added to a culture containing unlabelled glucose and lactate (culture A) and [ 14C]glucose to cultures containing unlabelled glucose without unlabelled lactate (culture B) and with unlabelled lactate (culture C). When LPS samples purified from these cultures were subjected to hydrazinolysis, the ratio of the radioactivity of water-soluble products (carbohydrate moieties) to those of chloroform-soluble products (fatty acids) was much lower when [ 14C]lactate was used in culture A, than when [ 14C]glucose was used in cultures B and C. Thus, in the presence of glucose, lactate carbon, unlike glucose carbon, is incorporated predominantly into fatty acids of LPS, not into its carbohydrate moieties. There is no doubt, therefore, that gluconeogenesis is shut off when lactate is present with glucose and there is a consequent stimulation of metabolism. This probably occurs in vivo on mucous surfaces, where gonococci are surrounded by a mixture of glucose and lactate in the secretions.

Renal substrate exchange and gluconeogenesis in normal postabsorptive humans.

Release of glucose by the kidney in postabsorptive normal humans is generally regarded as being wholly due to gluconeogenesis. Although lactate is the most important systemic gluconeogenic precursor and there is appreciable net renal lactate uptake, renal lactate gluconeogenesis has not yet been investigated. The present studies were therefore undertaken to quantitate the contribution of lactate to renal gluconeogenesis and the role of the kidney in lactate metabolism. We determined systemic and renal lactate conversion to glucose as well as renal lactate net balance, fractional extraction, uptake, and release in 24 postabsorptive humans by use of a combination of isotopic and renal balance techniques. For comparative purposes, accumulated similar data for glutamine, alanine, and glycerol are also reported. Systemic lactate gluconeogenesis (1.97 +/- 0.12 micromol x kg(-1) x min(-1)) was about threefold greater than that from glycerol, glutamine, and alanine. The sum of gluconeogenesis from these precursors, uncorrected for tricarboxylic acid (TCA) cycle carbon exchange, explained 34% of systemic glucose release. Renal lactate uptake (3.33 +/- 0.28 micromol x kg(-1) x min(-1)) accounted for nearly 30% of its systemic turnover. Renal gluconeogenesis from lactate (0.78 +/- 0.10 micromol x kg(-1) x min(-1)) was 3.5, 2.5, and 9.6-fold greater than that from glycerol, glutamine, and alanine. The sum of renal gluconeogenesis from these precursors equaled approximately 40% of the sum of their systemic gluconeogenesis. When the isotopically determined rates of systemic and renal gluconeogenesis were corrected for TCA cycle carbon exchange, gluconeogenesis from these precursors accounted for 43% of systemic glucose release and 89% of renal glucose release. We conclude that 1) in postabsorptive normal humans, lactate is the dominant precursor for both renal and systemic gluconeogenesis; 2) the kidney is an important organ for lactate disposal; 3) under these conditions, renal glucose release is predominantly, if not exclusively, due to gluconeogenesis; and 4) liver and kidney are similarly important for systemic gluconeogenesis.

Acidotic pH augments glucagon secretion and gluconeogenesis in the isolated perfused rat pancreas and liver.

To study effects of acidosis on glucagon secretion and gluconeogenetic action of glucagon, rat pancreas and liver were perfused with media of pH 6.4, 6.9, 7.4 and 7.9. Glucagon secretion from the pancreas during glucopenic perfusion (1 mmol/l) was blunted at alkalotic pH, and was augmented at acidotic pH; 0.28+/-0.18 at pH 7.9 (P<0.01), 3.57+/-0.94 at pH 6.9 (P<0.01) and 1.72+/-0.36 at pH 6.4 (P<0.01), vs. 0.66+/-0.25 pmol for 15 min at pH 7.4. Incorporation rate of 14C of lactate-U-14C into glucose carbon one was decreased at pH 7.9 (1.2+/-0.2% for 15 min, P<0.05) and was increased at pH 6.9 (2.8+/-0.5%, P<0.05) compared to that at pH 7.4 (1.9+/-0.3%). Percent increasing rate of lactate gluconeogenesis by 1 nmol/l glucagon was not different within a range of pH 6.4-7.9. Thus, glucagon-stimulated gluconeogenesis from lactate was smaller at pH 7.9 (2.2+/-0.6%) and was significantly greater at pH 6.9 (4.9+/-0.9%, P<0.05) than that at pH 7.4 (3.2+/-0.6%). These results suggest that the pancreatic glucagon secretion and the glucagon-stimulated hepatic gluconeogenesis play more important roles in the maintainance of blood glucose level in the stress states associated with acidosis than without acidosis.

Increased Glucagon Action on Lactate Gluconeogenesis in Perfused Liver of Dexamethasone-Treated Rats

To clearly understand the hyperglycemic action of glucocorticoids, we studied the action of glucagon on lactate gluconeogenesis in the liver of rats 7 days after adrenalectomy and after treatment with 1 mg/kg dexamethasone for 7 days. The liver was isolated and cyclically perfused at 20 ml/min with 25 ml of perfusion medium containing 5 mM lactate, [U-14C]lactate, and 0–100 ng/ml glucagon. In the absence of glucagon, incorporation of [14C]lactate into glucose carbon 1 did not change significantly in the adrenalectomized rat liver (1.66 ± 0.12% of total radioactivity for 5 min) and increased in the dexamethasone-treated rat liver (3.61 ± 0.54%,P< 0.01) compared to the normal rat liver (1.99 ± 0.28%). The response of lactate gluconeogenesis to glucagon was extremely blunted in the adrenalectomized rat liver and was much larger in the dexamethasone-treated rat than in the normal rat liver (at a glucagon concentration of 100 ng/ml, 2.13 ± 0.33, 8.55 ± 1.06, and 4.61 ± 0.53% for 5 min, respectively). Glucagon binding to liver plasma membrane was not changed by adrenalectomy and was decreased by dexamethasone treatment. These results suggest that glucocorticoids induce hyperglycemia by increasing the response to glucagon, together with the high basal activity of hepatic gluconeogenesis. In addition, these effects do not occur through changes in glucagon binding to receptors.

Stable isotope determination of plasma lactate conversion into glucose in fasting infants.

To quantify lactate gluconeogenesis, we developed a gas chromatography-mass spectrometry method based on the infusion of [6,6-2H2]glucose and [3-13C]lactate tracers to 12 infants aged 1-25 mo fasting for 11.5 +/- 1.5 h. Both rates of appearance of plasma glucose (26.7 +/- 2.6 mumol.kg-1.min-1, 4.8 +/- 0.5 mg.kg-1.min-1) and lactate (30.8 +/- 3.1 mumol.kg-1.min-1, 2.8 +/- 0.3 mg.kg-1.min-1) were remarkably elevated compared with adult values. The interconversion of plasma lactate and glucose was determined by 1) measuring the incorporation of 13C from [3-13C]lactate into plasma glucose; 2) correcting for the metabolic exchange of carbon atoms in the tricarboxylic acid cycle. For this purpose, an additional group of six infants was infused with [3-13C]lactate, and the distribution of 13C at specific carbon positions in the glucose molecule was determined using relevant ions in the electron-impact mass spectrum of its 1,2,5,6-diisopropylidene-3-O-acetyl-alpha-furanosyl derivative; and 3) measuring the reverse conversion of glucose to lactate in five other infants infused with [1-13C]glucose. We found that 54 +/- 2% of glucose was derived from plasma lactate (14.4 +/- 1.3 mumol.kg-1.min-1, 2.6 +/- 0.2 mg.kg-1.min-1). Lactate and glucose rates of appearance were correlated (r = 0.58, P < 0.05) and decreased with fasting duration (r = 0.66, P < 0.02). The correction factor for carbon exchange in the tricarboxylic acid cycle was 1.14 +/- 0.11.(ABSTRACT TRUNCATED AT 250 WORDS)

A new family of protein kinases— The mitochondrial protein kinases

Molecular cloning has provided evidence for a new family of protein kinases in eukaryotic cells. These kinases show no sequence similarity with other eukaryotic protein kinases, but are related by sequence to the histidine protein kinases found in prokaryotes. These protein kinases, responsible for phosphorylation and inactivation of the branched-chain α-ketoacid dehydrogenase and pyruvate dehydrogenase complexes, are located exclusively in mitochondrial matrix space and have most likely evolved from genes originally present in respiration-dependent bacteria endocytosed by primitive eukaryotic cells. Long-term regulatory mechanisms involved in the control of the activities of these two kinases are of considerable interest. Dietary protein deficiency increases the activity of branched-chain α-ketoacid dehydrogenase kinase associated with the branched-chain α-ketoacid dehydrogenase complex. The amount of branched-chain α-ketoacid dehydrogenase kinase protein associated with the branched-chain α-ketoacid dehydrogenase complex and the message level for branched-chain α-ketoacid dehydrogenase kinase are both greatly increased in the liver of rats starved for protein, suggesting increased expression of the gene encoding branched-chain α-ketoacid dehydrogenase kinase. The increase in branched-chain α-ketoacid dehydrogenase kinase activity results in greater phosphorylation and lower activity of the branched-chain α-ketoacid dehydrogenase complex. The metabolic consequence is conservation of branched chain amino acids for protein synthesis during periods of dietary protein deficiency. Two isoforms of pyruvate dehydrogenase kinase have been identified and cloned. Pyruvate dehydrogenase kinase 1, the first isoform cloned, corresponds to the 48 kDa subunit of the pyruvate dehydrogenase kinase isolated from rat heart tissue. Pyruvate dehydrogenase kinase 2, the second isoform cloned, corresponds to the 45 kDa subunit of this enzyme. In addition, it also appears to correspond to a possibly free or soluble form of pyruvate dehydrogenase kinase that was originally named kinase activator protein. Assuming that differences in kinetic and/or regulatory properties of these isoforms exist, tissue specific expression of these enzymes and/or control of their association with the complex will probably prove to be important for the long term regulation of the activity of the pyruvate dehydrogenase complex. Starvation and the diabetic state are known to greatly increase activity of the pyruvate dehydrogenase kinase in the liver, heart and muscle of the rat. This contributes in these states to the phosphorylation and inactivation of the pyruvate dehydrogenase complex and conservation of pyruvate and lactate for gluconeogenesis. Whether the mechanism responsible for the increase in activity of pyruvate dehydrogenase kinase involves an induced increase in protein amount of pyruvate dehydrogenase kinase (as found for the branched-chain α-ketoacid dehydrogenase kinase in response to dietary protein deficiency), an isoenzyme shift, or activation of the pyruvate dehydrogenase kinases by unknown mechanisms remains to be established.