Neurotransmitter glutamate has been thought to derive mainly from glutamine via the action of glutaminase type 1 (GLS1). To address the importance of this pathway in glutamatergic transmission, we knocked out GLS1 in mice. The insertion of a STOP cassette by homologous recombination produced a null allele that blocked transcription, encoded no immunoreactive protein, and abolished GLS1 enzymatic activity. Null mutants were slightly smaller, were deficient in goal-directed behavior, hypoventilated, and died in the first postnatal day. No gross or microscopic defects were detected in peripheral organs or in the CNS. In cultured neurons from the null mutants, miniature EPSC amplitude and duration were normal; however, the amplitude of evoked EPSCs decayed more rapidly with sustained 10 Hz stimulation, consistent with an observed reduction in depolarization-evoked glutamate release. Because of this activitydependent impairment in glutamatergic transmission, we surmised that respiratory networks, which require temporal summation of synaptic input, would be particularly affected. We found that the amplitude of inspirations was decreased in vivo, chemosensitivity to CO 2 was severely altered, and the frequency of pacemaker activity recorded in the respiratory generator in the pre-Bötzinger complex, a glutamatergic brainstem network that can be isolated in vitro, was increased. Our results show that although alternate pathways to GLS1 glutamate synthesis support baseline glutamatergic transmission, the GLS1 pathway is essential for maintaining the function of active synapses, and thus the mutation is associated with impaired respiratory function, abnormal goal-directed behavior, and neonatal demise.

To elucidate the sequence of myosin heavy chain (MHC) transitions in fast-to-slow transforming rabbit muscle, direct reverse transcriptase-polymerase chain reaction was applied for detecting mRNAs specific to five MHC isoforms in single fibers from control and low-frequency-stimulated tibialis anterior muscles. The detection of MHCIIb, MHCIId(x), MHCIα, and MHCIβ mRNAs was based on previously published methods. The RT-PCR assay for MHCIIa mRNA was based on the identification of a cDNA sequence in the 3′-region from which specific primers were derived. Comparisons between rat, rabbit, and human MHCIIa sequences revealed high degrees of sequence identities. MHC mRNA isoform patterns in single fibers from stimulated muscles showed hybrid fibers expressing the following combinations: MHCIId(x) + MHCIIa, MHCIId(x) + MHCIIa + MHCIα, MHCIId(x) + MHCIIa + MHCIα + MHCIβ, MHCIIa + MHCIα, MHCIIa + MHCIα + MHCIβ, and MHCIα + MHCIβ. The combination MHCIIa + MHCIβ without MHCIα was never seen. These coexpression patterns suggest that the fast-to-slow fiber transition results from sequential isoform expressions in the order MHCIId(x) → MHCIIa → MHCIα → MHCIβ. The allocation of MHCIα between MHCIIa and MHCIβ seems to be in line with graded differences in sequence identity of the 3′-regions of these mRNA isoforms.

Energy-rich phosphates, [ATP]/[ADPfree] ratios, and the myosin heavy chain (MHC) complement were determined in single fibres from normal rabbit muscles, and in fibres isolated from tibialis anterior muscle undergoing fast-to-slow conversion by chronic low-frequency stimulation (CLFS). In normal muscles, energy-rich phosphate contents and [ATP]/[ADPfree] ratios could thus be assigned to different MHC-based fibre types. Phosphocreatine (PCr) contents and [ATP]/[ADPfree] ratios differed markedly between fast- and slow-twitch fibres, as well as within the fast fibre subtypes. Both magnitudes were approximately twofold higher in the fastest (type IIB) fibres as compared to the slowest (type I) fibres. According to PCr contents and [ATP]/[ADPfree] ratios pure and hybrid fibres were aligned in an order similar to that determined by their contractile properties and myofibrillar ATPase activities. CLFS for up to 30 days induced pronounced decreases in PCr and [ATP]/[ADPfree] which attained levels twofold lower than in normal slow-twitch fibres. In both normal and stimulated muscles, PCr and [ATP]/[ADPfree] ratios were correlated, indicating their equilibrium in the different fibre types. The relationship detected between MHC isoform expression and the [ATP]/[ADPfree] ratio suggests that the drastic and persistent depression of the cellular energy state may act as an important signal initiating fast-to-slow transformation processes in muscle fibres.

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.

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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.

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.

Recent studies in vivo have suggested that, in humans in the postabsorptive state, the kidneys contribute a significant fraction of systemic gluconeogenesis, and that the stimulation of renal gluconeogenesis may fully explain the increase in systemic gluconeogenesis during adrenaline infusion. Given the potential importance of human renal gluconeogenesis in various physiological and pathophysiological situations, we have conducted a study in vitro to further characterize this metabolic process and its regulation. For this, successive segments (S1, S2 and S3) of human proximal tubules were dissected and incubated with physiological concentrations of glutamine or lactate, two potential gluconeogenic substrates that are taken up by the human kidney in vivo, and glucose production was measured. The effects of adrenaline, noradrenaline and cAMP, a well established stimulator of gluconeogenesis in animal kidney tubules, were also studied in suspensions of human renal proximal tubules. The results indicate that the three successive segments have about the same capacity to synthesize glucose from glutamine; by contrast, the S2 and S3 segments synthesize more glucose from lactate than the S1 segment. In the S2 and S3 segments, lactate appears to be a better gluconeogenic precursor than glutamine. The addition of cAMP, but not of adrenaline or noradrenaline, led to the stimulation of gluconeogenesis from lactate and glutamine by human proximal tubules. These results indicate that, in the human kidney in vivo, lactate might be the main gluconeogenic precursor, and that the stimulation of renal gluconeogenesis observed in vivo upon adrenaline infusion may result from an indirect action on the renal proximal tubule.