Gabriel Baverel

Intrinsic Gluconeogenesis Is Enhanced in Renal Proximal Tubules of Zucker Diabetic Fatty Rats

Recent studies indicate that renal gluconeogenesis is substantially stimulated in patients with type 2 diabetes, but the mechanism that is responsible for such stimulation remains unknown. Therefore, this study tested the hypothesis that renal gluconeogenesis is intrinsically elevated in the Zucker diabetic fatty rat, which is considered to be an excellent model of type 2 diabetes. For this, isolated renal proximal tubules from diabetic rats and from their lean nondiabetic littermates were incubated in the presence of physiologic gluconeogenic precursors. Although there was no increase in substrate removal and despite a reduced cellular ATP level, a marked stimulation of gluconeogenesis was observed in diabetic relative to nondiabetic rats, with near-physiologic concentrations of lactate (38%), glutamine (51%) and glycerol (66%). This stimulation was caused by a change in the fate of the substrate carbon skeletons resulting from an increase in the activities and mRNA levels of the key gluconeogenic enzymes that are common to lactate, glutamine, and glycerol metabolism, i.e., mainly of phosphoenolpyruvate carboxykinase and, to a lesser extent, of glucose-6-phosphatase and fructose-1,6-bisphosphatase. Experimental evidence suggests that glucocorticoids and cAMP were two factors that were responsible for the long-term stimulation of renal gluconeogenesis observed in the diabetic rats. These data provide the first demonstration in an animal model that renal gluconeogenesis is upregulated by a long-term mechanism during type 2 diabetes. Together with the increased renal mass (38%) observed, they lend support to the view so far based only on in vivo studies performed in humans that renal gluconeogenesis may be stimulated by and crucially contribute to the hyperglycemia of type 2 diabetes.

Inhibition of Glutamine Synthetase in the Mouse Kidney A NOVEL MECHANISM OF ADAPTATION TO METABOLIC ACIDOSIS

As part of a study on the regulation of renal ammoniagenesis in the mouse kidney, we investigated the effect of chronic metabolic acidosis on glutamine synthesis by isolated mouse renal proximal tubules. The results obtained reveal that, in tubules from control mice, glutamine synthesis occurred at high rates from glutamate and proline and, to a lesser extent, from ornithine, alanine, and aspartate. A 48 h, metabolic acidosis caused a marked inhibition of glutamine synthesis from near-physiological concentrations of both alanine and proline that were avidly metabolized by the tubules; metabolic acidosis also greatly stimulated glutamine utilization and metabolism. These effects were accompanied by a large increase (i) in alanine, proline, and glutamine gluconeogenesis and (ii) in ammonia accumulation from proline and glutamine. In the renal cortex of acidotic mice, the activity of phosphoenolpyruvate carboxykinase increased 4-fold, but that of glutamate dehydrogenase did not change; in contrast with what is known in the rat renal cortex, metabolic acidosis markedly diminished the glutamine synthetase activity and protein level, but not the glutamine synthetase mRNA level in the mouse renal cortex. These results strongly suggest that, in the mouse kidney, glutamine synthetase is an important regulatory component of the availability of the ammonium ions to be excreted for defending systemic acid-base balance. Furthermore, they show that, in rodents, the regulation of renal glutamine synthetase is species-specific.

Transport and utilization of α-ketoglutarate by the rat kidney in vivo

In order to establish the characteristics of net renal transport and utilization of α-ketoglutarate (α-KG) in the rat, we have precisely quantified the renal blood flow, the urinary flow and the rates of α-KG delivery, filtration, reabsorption or secretion, excretion, uptake or production by an in vivo rat kidney preparation. In normal rats, α-KG uptake was higher than α-KG reabsorption at both endogenous and elevated plasma α-KG concentrations; thus, a net peritubular transport, which was the main supplier of α-KG to the renal cells, took place. Saturation of reabsorption and peritubular transport of α-KG occurred at blood α-KG concentrations about 30 and 150 times above normal, respectively. Acute metabolic acidosis was found to have no effect on renal handling of α-KG. At endogenous plasma α-KG concentrations, alkalosis converted net renal uptake into net renal production of α-KG resulting in addition of α-KG by the renal cells both to blood and to the luminal fluid. Elevation of blood α-KG concentration restored the renal uptake of α-KG. This uptake, which was entirely accounted for by the peritubular transport of α-KG, reached a maximum which was lower than that observed in normal and acidotic rats.

Effect of starvation on glutamine ammoniagenesis and gluconeogenesis in isolated mouse kidney tubules.

It has been shown recently that glutamine is taken up by the mouse kidney in vivo. However, knowledge about the fate of this amino acid and the regulation of its metabolism in the mouse kidney remains poor. Given the physiological and pathophysiological importance of renal glutamine metabolism and the increasing use of genetically modified mice in biological research, we have conducted a study to characterize glutamine metabolism in the mouse kidney. Proximal tubules isolated from fed and 48 h-starved mice and then incubated with a physiological concentration of glutamine, removed this amino acid and produced ammonium ions at similar rates. In agreement with this observation, activities of the ammoniagenic enzymes, glutaminase and glutamate dehydrogenase, were not different in the renal cortex of fed and starved mice, but the glutamate dehydrogenase mRNA level was elevated 4.5-fold in the renal cortex from starved mice. In contrast, glucose production from glutamine was greatly stimulated whereas the glutamine carbon removed, that was presumably completely oxidized in tubules from fed mice, was virtually suppressed in tubules from starved animals. In accordance with the starvation-induced stimulation of glutamine gluconeogenesis, the activities and mRNA levels of glucose-6-phosphatase, and especially of phosphoenolpyruvate carboxykinase, but not of fructose-1,6-bisphosphatase, were increased in the renal cortex of starved mice. On the basis of our in vitro results, the elevated urinary excretion of ammonium ions observed in starved mice probably reflected an increased transport of these ions into the urine at the expense of those released into the renal veins rather than a stimulation of renal ammoniagenesis.

Toxicity of chloroacetaldehyde is similar in adult and pediatric kidney tubules

Abstract The nephrotoxicity of chloroacetaldehyde (CAA), one of the main products of hepatic ifosfamide metabolism, was compared in isolated human pediatric and adult renal tubules. Tubules metabolizing lactate were incubated in the presence of various concentrations of CAA (0.1–0.5 mM). Both at low, clinically relevant (0.2 mM), and at higher concentrations (0.3, 0.4 and 0.5 mM), CAA induced a cellular depletion of thiol compounds, i.e. glutathione, coenzyme A and acetyl-coenzyme A that are involved in CAA detoxication and cellular energy metabolism, respectively. The toxicity to renal cells was clearly observed in the presence of 0.4 and 0.5 mM CAA, which led to a fall of the cellular ATP level, to the accumulation of pyruvate and the inhibition of glucose synthesis from lactate. Inhibition of lactate uptake and an increase in the release of lactate dehydrogenase were observed only in the presence of 0.5 mM CAA. The sensitivity of pediatric tubules to the toxic effects of CAA and the rate of their CAA uptake were not statistically different from those found in adult tubules. It is concluded that an increased susceptibility of pediatric tubules to CAA toxicity cannot be put forward to explain the increased risk for ifosfamide-induced nephrotoxicity in children relative to adults.

Characteristics of glutamine metabolism in human precision-cut kidney slices: a 13C-NMR study

The metabolism of glutamine, a physiological substrate of the human kidney, plays a major role in systemic acid-base homoeostasis. Not only because of the limited availability of human renal tissue but also in part due to the lack of adequate cellular models, the mechanisms regulating the renal metabolism of this amino acid in humans have been poorly characterized. Therefore given the renewed interest in their use, human precision-cut renal cortical slices were incubated in Krebs-Henseleit medium (118 mM NaCl, 4.7 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4*7H2O, 24.9 mM NaHCO3 and 2.5 mM CaCl2*2H2O) with 2 mM unlabelled or 13C-labelled glutamine residues. After incubation, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. Glutamate accumulation tended to plateau but glutamine removal and ammonia, alanine and lactate production as well as flux through GLDH (glutamate dehydrogenase) increased to various extents with time for up to 4 h of incubation indicating the metabolic viability of the slices. Valproate, a stimulator of renal glutamine metabolism, markedly and in a dose-dependent fashion increased ammonia production. With [3-13C]glutamine as a substrate, and in the absence and presence of valproate, [13C]glutamate, [13C]alanine and [13C]lactate accounted for 81 and 96%, 34 and 63%, 30 and 46% of the glutamate, alanine and lactate accumulations measured enzymatically respectively. The slices also metabolized glutamine and retained their reactivity to valproate during incubations lasting for up to 48 h. These results demonstrate that, although endogenous metabolism substantially operates in the presence of glutamine, human precision-cut renal cortical slices are metabolically viable and strongly respond to the ammoniagenic effect of valproate. Thus, this experimental model is suitable for metabolic and pharmaco-toxicological studies.

Effect of the antiepileptic drug sodium valproate on glutamine and glutamate metabolism in isolated human kidney tubules

We studied the effects of sodium valproate, a widely used antiepileptic drug and a hyperammonemic agent, on L-[1-14C]glutamine and L-[1-14C]glutamate metabolism in isolated human kidney-cortex tubules. Valproate markedly stimulated glutamine removal as well as the formation of ammonia, 14CO2, pyruvate, lactate and alanine, but it inhibited glucose synthesis; the increase in ammonia formation was explained by a stimulation by valproate mainly of flux through glutaminase (EC 3.5.1.2) and to a much lesser extent of flux through glutamate dehydrogenase (EC 1.4.1.3). By contrast, valproate did not stimulate glutamate removal or ammonia formation, suggesting that the increase in flux through glutamate dehydrogenase observed with glutamine as substrate was secondary to the increase in flux through glutaminase. Accumulation of pyruvate, alanine and lactate in the presence of valproate was less from glutamate than from glutamine. Inhibition by aminooxyacetate of accumulation of alanine from glutamine caused by valproate did not prevent the acceleration of glutamine utilization and the subsequent stimulation of ammonia formation. It is concluded from these data, which are the first concerning the in vitro metabolism of glutamine and glutamate in human kidney-cortex tubules, that the stimulatory effect of valproate is primarily exerted at the level of glutaminase in human renal cortex.

Glutamine gluconeogenesis in the small intestine of 72 h-fasted adult rats is undetectable

Recent reports have indicated that 48-72 h of fasting, Type 1 diabetes and high-protein feeding induce gluconeogenesis in the small intestine of adult rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic and (ii) have major consequences in the metabolism, nutrition and diabetes fields, we have thoroughly re-examined this question in the situation reported to induce the highest rate of gluconeogenesis. For this, metabolically viable small intestinal segments from 72 h-fasted adult rats were incubated with [3-13C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR spectroscopic methods. Although the segments utilized [13C]glutamine at high rates and accumulated 13C-labelled products linearly for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re-utilization of [13C]glucose initially synthesized from [13C]glutamine. Arteriovenous metabolite concentration difference measurements across the portal vein-drained viscera of 72 h-fasted Wistar and Sprague-Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore we challenge the view that the small intestine of the adult rat is a gluconeogenic organ.

The Rabbit Kidney Tubule Simultaneously Degrades and Synthesizes Glutamate A 13C NMR STUDY

The rabbit kidney does not readily metabolize but synthesizes glutamine at high rates by pathways that remain poorly defined. Therefore, the metabolism of variously labeled [13C]- and [14C]glutamates has been studied in isolated rabbit kidney tubules with and without acetate. CO2, glutamine, and alanine were the main carbon and nitrogenous end products of glutamate metabolism but no ammonia accumulated. Absolute fluxes through enzymes involved in glutamate metabolism, including enzymes of four different cycles operating simultaneously, were assessed by combining mainly the 13C NMR data with a new model of glutamate metabolism. In contrast to a previous conclusion of Klahr et al. (Klahr, S., Schoolwerth, A. C., and Bourgoignie, J. J. (1972) Am. J. Physiol. 222, 813-820), glutamate metabolism was found to be initiated by glutamate dehydrogenase at high rates. Glutamate dehydrogenase also operated at high rates in the reverse direction; this, together with the operation of the glutamine synthetase reaction, masked the release of ammonia. Addition of acetate stimulated the operation of the “glutamate → α-ketoglutarate → glutamate” cycle and the accumulation of glucose but reduced both the net oxidative deamination of glutamate and glutamine synthesis. Acetate considerably increased flux through α-ketoglutarate dehydrogenase and citrate synthase at the expense of flux through phosphoenolpyruvate carboxykinase; acetate also caused a large decrease in flux through alanine aminotransferase, pyruvate dehydrogenase, and the “substrate cycle” involving oxaloacetate, phosphoenolpyruvate, and pyruvate.

Lactate and pyruvate metabolism in isolated human kidney tubules

To our knowledge, uptake of lactate and pyruvate by the human kidney in vivo in normal subjects has not as yet been studied. These substrates are not taken up from the blood by the kidneys of patients afflicted with diabetes mellitus [I]. However, they are removed by the human kidney in vivo in certain other pathological states. This was first demonstrated in patients with various renal diseases [2] and, later observed [3] in obese patients following prolonged starvation. Since most of our knowledge concerning the metabolism of lactate and pyruvate has been derived from arterio-venous measurements, the metabolic fate of these substrates in the human kidney remains uncertain. Complete oxidation of lactate was calculated [2] to account for 35% of renal oxygen consumption. However, it was concluded [3] that all the lactate and pyruvate carbon skeletons removed by the kidney were converted to glucose. In an attempt to clarify this subject, we studied the metabolism of lactate and pyruvate in isolated human kidney tubules. The data indicate that lactate and pyruvate may serve as the carbon sources of glucose and alanine in human kidneys, and that, at physiological concentration (1 mM), complete oxidation of lactate is very limited.