Key pointsr At the end of an incremental exercise to exhaustion a large functional reserve remains in the muscles to generate power, even at levels far above the power output at which task failure occurs, regardless of the inspiratory O 2 pressure during the incremental exercise.r Exhaustion (task failure) is not due to lactate accumulation and the associated muscle acidification; neither the aerobic energy pathways nor the glycolysis are blocked at exhaustion.r Muscle lactate accumulation may actually facilitate early recovery after exhaustive exercise even under ischaemic conditions. r Although the maximal rate of ATP provision is markedly reduced at task failure, the resynthesis capacity remaining exceeds the rate of ATP consumption, indicating that task failure during an incremental exercise to exhaustion depends more on central than peripheral mechanisms.Abstract To determine the mechanisms causing task failure during incremental exercise to exhaustion (IE), sprint performance (10 s all-out isokinetic) and muscle metabolites were measured before (control) and immediately after IE in normoxia (P IO 2 : 143 mmHg) and hypoxia (P IO 2 : 73 mmHg) in 22 men (22 ± 3 years). After IE, subjects recovered for either 10 or 60 s, with open circulation or bilateral leg occlusion (300 mmHg) in random order. This was followed by a 10 s sprint with open circulation. Post-IE peak power output (W peak ) was higher than the power output reached at exhaustion during IE (P < 0.05). After 10 and 60 s recovery in normoxia, W peak was reduced by 38 ± 9 and 22 ± 10% without occlusion, and 61 ± 8 and 47 ± 10% with occlusion (P < 0.05). Following 10 s occlusion, W peak was 20% higher in hypoxia than normoxia (P < 0.05), despite similar muscle lactate accumulation ([La]) and phosphocreatine and ATP reduction. Sprint performance and anaerobic ATP resynthesis were greater after 60 s compared with 10 s occlusions, despite the higher [La] and [H + ] after 60 s compared with 10 s occlusion recovery (P < 0.05). The mean rate of ATP turnover during the 60 s occlusion was 0.180 ± 0.133 mmol (kg wet wt) −1 s −1 , i.e. equivalent to 32% of leg peak O 2 uptake (the energy expended by the ion pumps). A greater degree of recovery is achieved, however, without occlusion. In conclusion, during incremental exercise task failure is not due to metabolite accumulation or lack of energy resources. Anaerobic metabolism, despite the accumulation of lactate and H + , facilitates early Abbreviations Cr, creatine; d.w., dry weight; F IO2 , inspired oxygen fraction; HR, heart rate; HR peak , peak heart rate; Hyp, hypoxia; IE, incremental exercise to exhaustion; La, lactate; Mb, myoglobin; Nx, normoxia; PCr, phosphocreatine; P ETCO2 , end-tidal CO 2 pressure; P ETO2 , end-tidal O 2 pressure; P IO2 , partial pressure of inspired O 2 ; RER, respiratory exchange ratio; S pO2 , haemoglobin oxygen saturation measured by pulse-oximetry; TOI, tissue oxygenation index;V CO2 , CO 2 production;V CO2peak , peak CO 2 production;V E , minute ventilation;V O2 , O 2 consumpt...

Mutations in the alanine-glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt ؊/؊ mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria.gene therapy ͉ knockout mouse ͉ oxalate ͉ urolithiasis ͉ nephrocalcinosis

Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44). Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 3 Thr (I244T) mutation in the AGXT gene, with 14 of 16 patients homozygous for this mutation. Four polymorphisms within AGXT and regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect. The consequences of these amino acid changes were investigated. Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 3 Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts. Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers. The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation. This aggregation was temperature-sensitive. AGXT*LTM expressed in Escherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity. Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates. In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation.

-AMPactivated protein kinase (AMPK) is a major mediator of the exercise response and a molecular target to improve insulin sensitivity. To determine if the anaerobic component of the exercise response, which is exaggerated when sprint is performed in severe acute hypoxia, influences sprint exercise-elicited Thr 172 -AMPK␣ phosphorylation, 10 volunteers performed a single 30-s sprint (Wingate test) in normoxia and in severe acute hypoxia (inspired PO2: 75 mmHg). Vastus lateralis muscle biopsies were obtained before and immediately after 30 and 120 min postsprint. Mean power output and O2 consumption were 6% and 37%, respectively, lower in hypoxia than in normoxia. O2 deficit and muscle lactate accumulation were greater in hypoxia than in normoxia. Carbonylated skeletal muscle and plasma proteins were increased after the sprint in hypoxia. Thr 172 -AMPK␣ phosphorylation was increased by 3.1-fold 30 min after the sprint in normoxia. This effect was prevented by hypoxia. The NAD ϩ -to-NADH.H ϩ ratio was reduced (by 24-fold) after the sprints, with a greater reduction in hypoxia than in normoxia (P Ͻ 0.05), concomitant with 53% lower sirtuin 1 (SIRT1) protein levels after the sprint in hypoxia (P Ͻ 0.05). This could have led to lower liver kinase B1 (LKB1) activation by SIRT1 and, hence, blunted Thr 172 -AMPK␣ phosphorylation. Ser 485 -AMPK␣1/Ser 491 -AMPK␣2 phosphorylation, a known negative regulating mechanism of Thr 172 -AMPK␣ phosphorylation, was increased by 60% immediately after the sprint in hypoxia, coincident with increased Thr 308 -Akt phosphorylation. Collectively, our results indicate that the signaling response to sprint exercise in human skeletal muscle is altered in severe acute hypoxia, which abrogated Thr 172 -AMPK␣ phosphorylation, likely due to lower LKB1 activation by SIRT1.sprint; AMP-activated protein kinase; signaling; muscle; metabolism AMP-ACTIVATED PROTEIN KINASE (AMPK) is a metabolic energy sensor activated by Thr 172 phosphorylation of the ␣-subunit, mainly in response to an increase of the AMP-to-ATP ratio (25). AMPK is involved in the regulation of feeding and body weight (42), lipid metabolism (26), glucose homeostasis (62), and mitochondrial biogenesis (69) and is a key player in the adaptation to exercise training (48). AMPK␣ phosphorylation of Thr 172 increases markedly in response to sprint exercise (22), most likely due to the elevation of the AMP-to-ATP ratio (11). Whether free radicals may also play a role in contractionmediated Thr 172 -AMPK␣ phosphorylation in skeletal muscle remains controversial (41,52). In cell cultures, hypoxia and anoxia increase Thr 172 -AMPK␣ phosphorylation more through the release of free radicals than through an increase in the AMP-to-ATP ratio (15). In contrast, chronic hypoxia (5 and 12 days of exposure to 5,500 m above sea level) did not increase skeletal muscle Thr 172 -AMPK␣ phosphorylation in rats (10). The influence of the inspired O 2 fraction (FI O 2 ) on exerciseinduced Thr 172 -AMPK␣ phosphorylation has been scarcely studied in humans (63)....