Skeletal muscle mitochondria demonstrate similar respiration per cristae surface area independent of training status and sex in healthy humans.

The impact of training status and sex on intrinsic skeletal muscle mitochondrial respiratory capacity remains unclear. We examined this by analysing human skeletal muscle mitochondrial respiration relative to mitochondrial volume and cristae density across training statuses and sexes. Mitochondrial cristae density was estimated in skeletal muscle biopsies originating from previous independent studies. Participants included females (n = 12) and males (n = 41) across training statuses ranging from untrained (UT, n = 8), recreationally active (RA, n = 9), active-to-elite runners (RUN, n = 27) and cross-country skiers (XC, n = 9). The XC and RUN groups demonstrated higher mitochondrial volume density than the RA and UT groups while all active groups (RA, RUN and XC) displayed higher mass-specific capacity of oxidative phosphorylation (OXPHOS) and mitochondrial cristae density than UT. Differences in OXPHOS diminished between active groups and UT when normalising to mitochondrial volume density and were lost when normalising to muscle cristae surface area density. Moreover, active females (n = 6-9) and males (n = 15-18) did not differ in mitochondrial volume and cristae density, OXPHOS, or when normalising OXPHOS to mitochondrial volume density and muscle cristae surface area density. These findings demonstrate: (1) differences in OXPHOS between active and untrained individuals may be explained by both higher mitochondrial volume and cristae density in active individuals, with no difference in intrinsic mitochondrial respiratory capacity (OXPHOS per muscle cristae surface area density); and (2) no sex differences in mitochondrial volume and cristae density or mass-specific and normalised OXPHOS. This highlights the importance of normalising OXPHOS to muscle cristae surface area density when studying skeletal muscle mitochondrial biology. KEY POINTS: Oxidative phosphorylation is the mitochondrial process by which ATP is produced, governed by the electrochemical gradient across the inner mitochondrial membrane with infoldings named cristae. In human skeletal muscle, the mass-specific capacity of oxidative phosphorylation (OXPHOS) can change independently of shifts in mitochondrial volume density, which may be attributed to variations in cristae density. We demonstrate that differences in skeletal muscle OXPHOS between healthy females and males, ranging from untrained to elite endurance athletes, are matched by differences in cristae density. This suggests that higher OXPHOS in skeletal muscles of active individuals is attributable to an increase in the density of cristae. These findings broaden our understanding of the variability in human skeletal muscle OXPHOS and highlight the significance of cristae, specific to mitochondrial respiration.

Skeletal muscle adaptations to exercise are not influenced by metformin treatment in humans: secondary analyses of 2 randomized, clinical trials.

Metformin and exercise both improve glycemic control, but in vitro studies have indicated that an interaction between metformin and exercise occurs in skeletal muscle, suggesting a blunting effect of metformin on exercise training adaptations. Two studies (a double-blind, parallel-group, randomized clinical trial conducted in 29 glucose-intolerant individuals and a double-blind, cross-over trial conducted in 15 healthy lean males) were included in this paper. In both studies, the effect of acute exercise ± metformin treatment on different skeletal muscle variables, previously suggested to be involved in a pharmaco-physiological interaction between metformin and exercise, was assessed. Furthermore, in the parallel-group trial, the effect of 12 weeks of exercise training was assessed. Skeletal muscle biopsies were obtained before and after acute exercise and 12 weeks of exercise training, and mitochondrial respiration, oxidative stress and AMPK activation was determined. Metformin did not significantly affect the effects of acute exercise or exercise training on mitochondrial respiration, oxidative stress or AMPK activation, indicating that the response to acute exercise and exercise training adaptations in skeletal muscle is not affected by metformin treatment. Further studies are needed to investigate whether an interaction between metformin and exercise is present in other tissues, e.g., the gut. Trial registration: ClinicalTrials.gov (NCT03316690 and NCT02951260). Novelty: Metformin does not affect exercise-induced alterations in mitochondrial respiratory capacity in human skeletal muscle. Metformin does not affect exercise-induced alterations in systemic levels of oxidative stress nor emission of reactive oxygen species from human skeletal muscle. Metformin does not affect exercise-induced AMPK activation in human skeletal muscle.

Induction of erythroferrone in healthy humans by micro-dose recombinant erythropoietin or high-altitude exposure.

The erythropoietin (Epo)-erythroferrone (ERFE)-hepcidin axis coordinates erythropoiesis and iron homeostasis. While mouse studies have established that Epo-induced ERFE production represses hepcidin synthesis by inhibiting hepatic BMP/SMAD signaling, evidence for the role of ERFE in humans is limited. To investigate the role of ERFE as a physiological erythroid regulator in humans, we conducted two studies: first, 24 males received six injections of saline (placebo), recombinant Epo (rhEpo) 20 UI kg-1 (micro-dose) or 50 UI kg-1 (low-dose). Second, we quantified ERFE in 22 subjects exposed to high altitude (3800 m) for 15 hours. In the first study, total hemoglobin mass (Hbmass) increased after low- but not after micro-dose injections, when compared to placebo. Serum ERFE levels were enhanced by rhEpo, remaining higher than after placebo for 48 (micro-dose) or 72 hours (low-dose) post-injections. Conversely, hepcidin levels decreased when Epo and ERFE arose, before any changes in serum iron parameters occurred. In the second study, serum Epo and ERFE increased at high altitude. The present results demonstrate that in healthy humans ERFE responds to slightly increased Epo levels not associated with Hbmass expansion and down-regulates hepcidin in an apparently iron-independent way. Notably, ERFE flags micro-dose Epo, thus holding promise as novel anti-doping biomarker.

Low doses of recombinant human erythropoietin does not affect C-terminal FGF23 in healthy men.

Recombinant human erythropoietin (rhEpo) can improve human performance, but misuse remains difficult to detect. C-terminal fibroblast growth factor 23 (cFGF23) was recently demonstrated to increase following injection of a single high dose rhEpo, but the effect of more frequent low doses is unknown. Using a randomized double-blind placebo-controlled design, we investigated whether 2 weeks of subcutaneous injections three times a week of 50 IU/kg Eprex (low-dose) or 20 IU/kg Eprex (micro-dose) increase cFGF23 levels compared with saline (placebo) injections in 24 healthy males. Venous blood was sampled at day -3, 0, 1, 3, 11, 14, 18, and 25 of the treatment and analyzed for cFGF23 and erythropoietin concentration ([EPO]). The level of cFGF23 was similar at days -3, 0, 1, 3, 11, 14, 18, and 25 with the low-dose (23 ± 4, 26 ± 5, 23 ± 7, 27 ± 6, 25 ± 8, 24 ± 10, 22 ± 5, and 24 ± 7 RU/mL, respectively), micro-dose (23 ± 6, 25 ± 5, 23 ± 8, 28 ± 9, 27 ± 7, 25 ± 9, 25 ± 5, and 23 ± 6 RU/mL, respectively) and placebo (23 ± 6, 24 ± 6, 26 ± 7, 26 ± 6, 31 ± 6, 31 ± 7, 24 ± 4, and 27 ± 8 RU/mL, respectively) treatment. The correlation coefficient between plasma [EPO] and plasma cFGF23 levels was R2 = 0.01 and insignificant. The results demonstrate that cFGF23 is not sensitive to low doses of subcutaneous rhEpo injections in healthy males.

Hematological Adaptations to Prolonged Heat Acclimation in Endurance-Trained Males.

Heat acclimation is associated with plasma volume (PV) expansion that occurs within the first week of exposure. However, prolonged effects on hemoglobin mass (Hbmass) are unclear as intervention periods in previous studies have not allowed sufficient time for erythropoiesis to manifest. Therefore, Hbmass, intravascular volumes, and blood volume (BV)-regulating hormones were assessed with 5½ weeks of exercise-heat acclimation (HEAT) or matched training in cold conditions (CON) in 21 male cyclists [(mean ± SD) age: 38 ± 9 years, body weight: 80.4 ± 7.9 kg, VO2peak: 59.1 ± 5.2 ml/min/kg]. HEAT (n = 12) consisted of 1 h cycling at 60% VO2peak in 40°C for 5 days/week in addition to regular training, whereas CON (n = 9) trained exclusively in cold conditions (<15°C). Before and after the intervention, Hbmass and intravascular volumes were assessed by carbon monoxide rebreathing, while reticulocyte count and BV-regulating hormones were measured before, after 2 weeks and post intervention. Total training volume during the intervention was similar (p = 0.282) between HEAT (509 ± 173 min/week) and CON (576 ± 143 min/week). PV increased (p = 0.004) in both groups, by 303 ± 345 ml in HEAT and 188 ± 286 ml in CON. There was also a main effect of time (p = 0.038) for Hbmass with +34 ± 36 g in HEAT and +2 ± 33 g in CON and a tendency toward a higher increase in Hbmass in HEAT compared to CON (time × group interaction: p = 0.061). The Hbmass changes were weakly correlated to alterations in PV (r = 0.493, p = 0.023). Reticulocyte count and BV-regulating hormones remained unchanged for both groups. In conclusion, Hbmass was slightly increased following prolonged training in the heat and although the mechanistic link remains to be revealed, the increase could represent a compensatory response in erythropoiesis secondary to PV expansion.

Red blood cell volume is not decreased in ESA-naive anemic chronic kidney disease patients.

Anemia is defined according to decreased blood hemoglobin concentration ([Hb]), which is considered a marker of low total red blood cell volume (RBCV). Alterations of plasma volume (PV) may also modify [Hb] without concomitant changes in RBCV. Since anemia and fluid retention are frequent complications of chronic kidney disease (CKD), we hypothesized that anemia during CKD may in part be related to expanded PV without a simultaneous decrease in RBCV. We quantified hemoglobin mass, RBCV, PV, and total blood volume (BV) using an automated carbon monoxide device in 40 consecutive stage 3-5 CKD patients not on dialysis and in seven healthy male controls of the same age range. These were compared within and to predicted volumes according to Nadler's formula. Arterial stiffness and NT-proBNP were measured. RBCV was similar to predicted values range in anemic CKD patients 2073 (1818-2704) versus, 2061 (1725-2473) mL, P > 0.05. In contrast, PV was largely increased in anemic CKD patients (3881 (3212-4352) vs. 2916 (2851-3201)), P = 0.01. Of 26 anemic patients, only six had a >20% decrease in RBCV as the cause for their anemia, whereas 14 had a >20% increase of PV as a cause for their anemia. NT-pro BNP correlated with eGFR but neither with PV nor BV, whereas arterial stiffness was not correlated to blood volumes. Anemia in CKD as diagnosed by low [Hb] is not necessarily associated to low RBCV but may reflect increased PV. This finding has implications for the treatment of CKD patients and may refrain from normalizing [Hb] levels in all CKD patients.

Changes in metabolism but not myocellular signaling by training with CHO-restriction in endurance athletes.

Carbohydrate (CHO) restricted training has been shown to increase the acute training response, whereas less is known about the acute effects after repeated CHO restricted training. On two occasions, the acute responses to CHO restriction were examined in endurance athletes. Study 1 examined cellular signaling and metabolic responses after seven training-days including CHO manipulation (n = 16). The protocol consisted of 1 h high-intensity cycling, followed by 7 h recovery, and 2 h of moderate-intensity exercise (120SS). Athletes were randomly assigned to low (LCHO: 80 g) or high (HCHO: 415 g) CHO during recovery and the 120SS. Study 2 examined unaccustomed exposure to the same training protocol (n = 12). In Study 1, muscle biopsies were obtained at rest and 1 h after 120SS, and blood samples drawn during the 120SS. In Study 2, substrate oxidation and plasma glucagon were determined. In Study 1, plasma insulin and proinsulin C-peptide were higher during the 120SS in HCHO compared to LCHO (insulin: 0 min: +37%; 60 min: +135%; 120 min: +357%, P = 0.05; proinsulin C-peptide: 0 min: +32%; 60 min: +52%; 120 min: +79%, P = 0.02), whereas plasma cholesterol was higher in LCHO (+15-17%, P = 0.03). Myocellular signaling did not differ between groups. p-AMPK and p-ACC were increased after 120SS (+35%, P = 0.03; +59%, P = 0.0004, respectively), with no alterations in p-p38, p-53, or p-CREB. In Study 2, glucagon and fat oxidation were higher in LCHO compared to HCHO during the 120SS (+26-40%, P = 0.03; +44-76%, P = 0.01 respectively). In conclusion, the clear respiratory and hematological effects of CHO restricted training were not translated into superior myocellular signaling after accustomization to CHO restriction.

Four days of bed rest increases intrinsic mitochondrial respiratory capacity in young healthy males.

Bed rest leads to impaired glucose tolerance. Whether this is linked to maladaptation's in skeletal muscle mitochondrial function and in particular to the level of reactive oxygen species (ROS) is at present unknown. The aim of this longitudinal study was to quantify skeletal muscle mitochondrial function (respiratory capacity and ROS production) together with glucose tolerance after 4 days of strict bed rest in healthy young male subjects (n = 14). Mitochondrial function was determined in permeabilized muscle fibers using high-resolution respirometry and fluorometry, mitochondrial content (citrate synthase [CS] activity) and antioxidant protein expression levels were assessed in parallel to this. Glucose tolerance was determined by means of oral glucose tolerance tests. Intrinsic mitochondrial respiratory capacity was augmented after the bed rest period (CI + IIP : 0.43 ± 0.12 vs. 0.55 ± 0.14 [pmol/sec/mg]/CS activity), due to a decreased CS activity (158 ± 39 vs. 129 ± 25 mU/mg dw.). No differences were observed in ROS production (per mg of tissue or when normalized to CS activity). Furthermore, the protein content for catalase was increased while superoxide dismutase and glutathione peroxidase remained unaffected. These findings were accompanied by an impaired glucose tolerance after the bed rest period (Matsuda index: 12 ± 6 vs. 9 ± 5). The change in intrinsic mitochondrial respiratory capacity could be an early indication in the development of impaired glucose tolerance. The increased catalase protein content might explain that no change was seen in ROS production after 4 days of bed rest. Whether these findings can be extrapolated to lifestyle-dependent decrements in physical activity and the development of type-2-diabetes remains unknown.

Determinants of maximal whole-body fat oxidation in elite cross-country skiers: Role of skeletal muscle mitochondria.

Elite endurance athletes possess a high capacity for whole-body maximal fat oxidation (MFO). The aim was to investigate the determinants of a high MFO in endurance athletes. The hypotheses were that augmented MFO in endurance athletes is related to concomitantly increments of skeletal muscle mitochondrial volume density (MitoVD ) and mitochondrial fatty acid oxidation (FAOp ), that is, quantitative mitochondrial adaptations as well as intrinsic FAOp per mitochondria, that is, qualitative adaptations. Eight competitive male cross-country skiers and eight untrained controls were compared in the study. A graded exercise test was performed to determine MFO, the intensity where MFO occurs (FatMax ), and V ˙ O 2 Max . Skeletal muscle biopsies were obtained to determine MitoVD (electron microscopy), FAOp , and OXPHOSp (high-resolution respirometry). The following were higher (P < 0.05) in endurance athletes compared to controls: MFO (mean [95% confidence intervals]) (0.60 g/min [0.50-0.70] vs 0.32 [0.24-0.39]), FatMax (46% V ˙ O 2 Max [44-47] vs 35 [34-37]), V ˙ O 2 Max (71 mL/min/kg [69-72] vs 48 [47-49]), MitoVD (7.8% [7.2-8.5] vs 6.0 [5.3-6.8]), FAOp (34 pmol/s/mg muscle ww [27-40] vs 21 [17-25]), and OXPHOSp (108 pmol/s/mg muscle ww [104-112] vs 69 [68-71]). Intrinsic FAOp (4.0 pmol/s/mg muscle w.w/MitoVD [2.7-5.3] vs 3.3 [2.7-3.9]) and OXPHOSp (14 pmol/s/mg muscle ww/MitoVD [13-15] vs 11 [10-13]) were, however, similar in the endurance athletes and untrained controls. MFO and MitoVD correlated (r2  = 0.504, P < 0.05) in the endurance athletes. A strong correlation between MitoVD and MFO suggests that expansion of MitoVD might be rate-limiting for MFO in the endurance athletes. In contrast, intrinsic mitochondrial changes were not associated with augmented MFO.

Increased capillary density in skeletal muscle is not associated with impaired insulin sensitivity induced by bed rest in healthy young men.

Physical inactivity alters glucose homeostasis in skeletal muscle, potentially developing into overt metabolic disease. The present study sought to investigate the role of skeletal muscle capillarization in glucose tolerance and insulin sensitivity (IS) using a classic human model of physical inactivity. Thirteen healthy males (age = 23 ± 2 years) underwent 4 days of full-time supervised and diet-controlled bed rest. Oral glucose tolerance test, indices of IS (quantitative insulin sensitivity check index (QUICKI), Matsuda index), as well as skeletal muscle biopsies with measurement of fiber type distribution, fiber cross-sectional area (FCSA), capillary-to-fiber ratio (C/F ratio), and capillary density (CD) were assessed prior to and after bed rest. Body weight and composition were unaltered by bed rest. Fasting glucose/insulin ratio (G0/I0 ratio) (-25%, P = 0.016), QUICKI (-7%, P = 0.023), and Matsuda index (-24%, P = 0.003) diminished with bed rest. Skeletal muscle FCSA decreased (-737.4 ± 763.2 µm-2 (-12%), P = 0.005) while C/F ratio was preserved, resulting in augmented CD (+93.9 ± 91.5 capillaries·mm-2 (+37%), P = 0.003) with bed rest. No association was detected between changes in skeletal muscle variables and metabolic outcomes. Independently of bed rest-induced effects, a positive linear relationship was detected between C/F ratio and G0/I0 ratio (ß = 17.09, P = 0.021). In conclusion, impaired glucose homeostasis with bed rest is not prevented nor associated with enhanced skeletal muscle capillarization in healthy individuals.

Sexual dimorphism of substrate utilization: Differences in skeletal muscle mitochondrial volume density and function.

NEW FINDINGS: What is the central question of this study? Females rely to a greater extent than males on fat oxidation during exercise. Whether any difference in skeletal muscle mitochondrial phenotype and oxidative capacity contributes to this sexual dimorphism remains incompletely explored. What is the main finding and its importance? Female prioritization of fat during exercise occurs in parallel to augmented mitochondrial volume density and intrinsic fatty acid and lactate oxidation in skeletal muscle fibres compared with males, independently of aerobic exercise capacity. The enlarged metabolic machinery in skeletal muscle of females is associated with lower body size and leg mass. ABSTRACT: Fat oxidation during exercise is greater in females than in males. We sought to determine whether sex differences in substrate metabolism are paralleled by distinct skeletal muscle mitochondrial volume density and oxidative capacity. Whole-body substrate (fat and carbohydrate) utilization during submaximal treadmill running was assessed, and skeletal muscle biopsies were taken to determine mitochondrial volume density and function in healthy young females (n = 12) and males (n = 12) matched by aerobic exercise capacity and exercise performance. Females presented a lower respiratory exchange ratio (0.87 ± 0.04 versus 0.91 ± 0.04, P = 0.023) and whole-body carbohydrate oxidation (27.8 ± 8.3 versus 35.8 ± 6.5 mg kg-1  min-1 , P = 0.027), whereas fat oxidation was higher (8.7 ± 2.8 versus 5.9 ± 2.6 mg kg-1  min-1 , P = 0.034) during submaximal exercise compared with males. In skeletal muscle biopsies, females demonstrated augmented mitochondrial volume density (7.51 ± 1.77 versus 5.90 ± 1.72%, P = 0.035) and oxidative capacity for fatty acid [36.6 ± 12.8 versus 24.5 ± 7.3 pmol O2  s-1  (mg wet weight)-1 , P = 0.009] and lactate [71.1 ± 24.4 versus 53.2 ± 14.6 pmol O2  s-1  (mg wet weight)-1 , P = 0.040]. No sex differences in respiratory exchange ratio, whole-body fat oxidation and skeletal muscle variables were detected when adjusted for anthropometric variables including body mass or leg mass, which were lower in females. In conclusion, female prioritization of fat over carbohydrate oxidation during exercise is underpinned by augmented body size-related mitochondrial volume density, fatty acid and lactate oxidative capacity in skeletal muscle fibres.

Reliability and validity of non-invasive determined haemoglobin mass and blood volumes.

INTRODUCTION: The carbon monoxide (CO) rebreathing method used for the determination of haemoglobin mass (Hbmass ) is associated with blood sample analysis (in this study: Radiometer ABL800). As an alternative hereto the aim of the present study was to evaluate the use of a portable and non-invasive CO pulse oximeter (Rad-57). METHOD: With simultaneous determination of CO in the circulation by ABL800 (%HbCO) and Rad-57 (SpCO), Hbmass and blood volume (BV) were determined in duplicates in 24 volunteers. Percentage of typical errors (%TE) within methods and linear correlations between the two procedures were computed. RESULTS: Hbmass (Rad-57 = 798 ± 230 g; ABL800 = 781 ± 192 g) and BV (Rad-57 = 5700 ± 1373 ml; ABL800 = 5581 ± 1096 ml) were similar between methods. However, the %TE for Hbmass was higher (P<0·001) for Rad-57 (5·84 ± 5·29%) than for ABL800 (1·35 ± 1·13%). Similarly, the %TE for BV was higher (P<0·001) for Rad-57 (6·06 ± 5·76%) than for ABL800 (1·48 ± 1·25%). Lower (P<0·05) correlation coefficients between the methods were found when Hbmass  > 905 g and BV > 6193 ml. CONCLUSION: Assessment of SpCO by Rad-57 resulted in considerably less precise determinations of Hbmass and BV, especially for high values. Thus, non-invasive assessment of Hbmass and BV cannot be recommended for scientific purposes, but may nonetheless be useful in clinical settings.

Combined effects of physical inactivity and acute hyperglycemia on arterial distensibility.

Arterial distensibility, an independent predictor of cardiovascular events, is transiently increased with acute hyperglycemia (AHG) in healthy individuals. Whether this response interacts with physical inactivity remains unknown. We examined the effects of short-term bed rest (BR) on the response of carotid artery distensibility (CD) to AHG, and the influence of underlying changes in insulin resistance and blood volume. CD was assessed with ultrasonography before as well as 30 and 120 minutes following ingestion of 75 g of glucose prior to and after 3 days of BR in 15 healthy male volunteers. Plasma insulin/glucose concentrations and blood volumes were concomitantly determined. On day 4 of BR, blood volume was re-established to pre-BR levels by albumin infusion and CD and insulin/glucose concentrations were determined as in the previous experimental days. Basal CD was not affected by BR. AHG increased CD before and after BR but reached a higher peak increment after BR (12% vs 60% at 30 min OGTT, p=0.028). BR also increased the plasma insulin concentration during AHG ( p=0.007). In regression analyses, plasma insulin and glucose concentrations were positively correlated to CD, particularly after BR ( r=0.31, p<0.05). Restoration of the BR-induced loss (5%) in blood volume did not affect the response of CD to AHG. In conclusion, short-term physical inactivity strongly accentuates the initial increase in CD in response to AHG in healthy individuals. This effect is associated with concomitant increases in circulating insulin concentration attributable to early insulin resistance.

Erythropoiesis with endurance training: dynamics and mechanisms.

The purpose of the present study was to characterize the progression of red blood cell volume (RBCV) expansion and potential volumetric and endocrine regulators of erythropoiesis during endurance training (ET). Nine healthy, untrained volunteers (age = 27 ± 4 yr) underwent supervised ET consisting of 3-4 × 60 min cycle ergometry sessions per week for 8 wk. Plasma volume (PV), RBCV, and overnight fasting hematological markers were determined before and at weeks 2, 4, and 8 of ET. In addition, plasma erythropoietin (EPO), cortisol, copeptin, and proatrial natriuretic peptide concentrations were measured during a 3-h morning period at baseline and postexercise at weeks 1 and 8 PV increased from baseline (2,405 ± 335 ml) at weeks 2, 4, and 8 (+374 ± 194, +505 ± 156, and +341 ± 160 ml, respectively, P < 0.001). Increases in RBCV from baseline (1,737 ± 442 ml) were manifested at week 4 (+109 ± 114 ml, P = 0.030) and week 8 (+205 ± 109 ml, P = 0.001). Overnight fasting plasma EPO concentration increased from baseline (11.3 ± 4.8 mIU/ml) at week 2 (+2.5 ± 2.8 mIU·ml-1, P = 0.027) and returned to baseline concentration at weeks 4 and 8 Higher 3-h-postexercise EPO concentration was observed at week 1 (11.6 mIU/ml) compared with week 8 (8.4 ± 3.9 mIU/ml, P = 0.009) and baseline (9.0 ± 4.2 mIU/ml, P = 0.019). Linear relationships between EPO concentration and hematocrit (ß = -56.2, P < 0.001) and cortisol (ß = 0.037, P < 0.001) were detected throughout the ET intervention. In conclusion, ET leads to mild, transient increases in circulating EPO concentration, concurring with early PV expansion and lowered hematocrit, preceding gradual RBCV enhancement.

Detection of blood volumes and haemoglobin mass by means of CO re-breathing and indocyanine green and sodium fluorescein injections.

The main aim of the present study was to quantify the magnitude of differences introduced when estimating a given blood volume compartment (e.g. plasma volume) through the direct determination of another compartment (e.g. red cell volume) by multiplication of venous haematocrit and/or haemoglobin concentration. However, since whole body haematocrit is higher than venous haematocrit such an approach might comprise certain errors. To test this experimentally, four different methods for detecting blood volumes and haemoglobin mass (Hbmass) were compared, namely the carbon monoxide (CO) re-breathing (for Hbmass), the indocyanine green (ICG; for plasma volume [PV]) and the sodium fluorescein (SoF; for red blood cell volume [RBCV]) methods. No difference between ICG and CO re-breathing derived PV could be established when a whole body/venous haematocrit correction factor of 0.91 was applied (p = 0.11, r = 0.43, mean difference -340 ± 612 mL). In contrast, when comparing RBCV derived by the CO re-breathing and the SoF method, the SoF method revealed lower RBCV values as compared to the CO re-breathing method (p < 0.05, r = 0.95, mean difference -728 ± 184 mL). However, compared to the ICG and the SoF methods, the typical error (%TE) and hence reliability of the CO re-breathing method was lower for all measured parameters. Therefore, estimating blood volume compartments by the direct assessment of another compartment can be considered a suitable approach. The CO re-breathing method proved accurate in determining the induced phlebotomy and is at the same time judged easier to perform than any of the other methods.

Increased Synthesis of Liver Erythropoietin with CKD.

Anemia of CKD seems to be related to impaired production of renal erythropoietin (Epo). The glycosylation pattern of Epo depends on the synthesizing cell and thus, can indicate its origin. We hypothesized that synthesis of Epo from nonkidney cells increases to compensate for insufficient renal Epo production during CKD. We determined plasma Epo levels and Epo glycosylation patterns in 33 patients with CKD before undergoing dialysis and nine patients with CKD undergoing dialysis. We compared these values with values obtained in healthy volunteers and other controls. Although patients with CKD before undergoing dialysis had median (interquartile range) Epo levels higher than those of healthy controls (13.8 IU/L; interquartile range, 10.0-20.7 IU/L versus 8.4 IU/L; interquartile range, 7.6-9.0 IU/L; P<0.01), these patients were moderately anemic (mean±SD; hemoglobin =118±17 g/L). Detected as the percentage of migrated isoforms (PMI), Epo glycosylation in patients with CKD before undergoing dialysis (PMI=36.1±11.7%) differed from that in healthy controls (PMI=9.2±3.8%; P<0.01) but not from that in umbilical cord plasma (PMI=53.9±10.6%; P>0.05), which contains mainly liver-derived Epo. Furthermore, glycosylation modification correlated with eGFR loss. These results suggest that patients with CKD maintain persistent Epo synthesis despite declining renal function, and this maintenance may result in part from increased liver Epo synthesis.

Formation of Renal Cysts and Tumors in Vhl/Trp53-Deficient Mice Requires HIF1α and HIF2α.

The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in the majority of clear cell renal cell carcinomas (ccRCC), but genetic ablation of Vhl alone in mouse models is insufficient to recapitulate human tumorigenesis. One function of pVHL is to regulate the stability of the hypoxia-inducible factors (HIF), which become constitutively activated in the absence of pVHL. In established ccRCC, HIF1α has been implicated as a renal tumor suppressor, whereas HIF2α is considered an oncoprotein. In this study, we investigated the contributions of HIF1α and HIF2α to ccRCC initiation in the context of Vhl deficiency. We found that deleting Vhl plus Hif1a or Hif2a specifically in the renal epithelium did not induce tumor formation. However, HIF1α and HIF2α differentially regulated cell proliferation, mitochondrial abundance and oxidative capacity, glycogen accumulation, and acquisition of a clear cell phenotype in Vhl-deficient renal epithelial cells. HIF1α, but not HIF2α, induced Warburg-like metabolism characterized by increased glycolysis, decreased oxygen consumption, and decreased ATP production in mouse embryonic fibroblasts, providing insights into the cellular changes potentially occurring in Vhl mutant renal cells before ccRCC formation. Importantly, deletion of either Hif1a or Hif2a completely prevented the formation of renal cysts and tumors in Vhl/Trp53 mutant mice. These findings argue that both HIF1α and HIF2α exert protumorigenic functions during the earliest stages of cyst and tumor formation in the kidney. Cancer Res; 76(7); 2025-36. ©2016 AACR.

Twenty-eight days of exposure to 3454 m increases mitochondrial volume density in human skeletal muscle.

The role of hypoxia on skeletal muscle mitochondria is controversial. Studies superimposing exercise training on hypoxic exposure demonstrate an increase in skeletal muscle mitochondrial volume density (Mito(VD)) over equivalent normoxic training. In contrast, reductions in both skeletal muscle mass and Mito(VD) have been reported following mountaineering expeditions. These observations may, however, be confounded by negative energy balance, which may obscure the results. Accordingly we sought to examine the effects of high altitude hypoxic exposure on mitochondrial characteristics, with emphasis on Mito(VD), while minimizing changes in energy balance. For this purpose, skeletal muscle biopsies were obtained from nine lowlanders at sea level (Pre) and following 7 and 28 days of exposure to 3454 m. Maximal ergometer power output, whole body weight and composition, leg lean mass and skeletal muscle fibre area all remained unchanged following the altitude exposure. Transmission electron microscopy determined that intermyofibrillar (IMF) Mito(VD) was augmented (P = 0.028) by 11.5 ± 9.2% from Pre (5.05 ± 0.9%) to 28 Days (5.61 ± 0.04%). In contrast, there was no change in subsarcolemmal (SS) Mito(VD). As a result, total Mito(VD) (IMF + SS) was increased (P = 0.031) from 6.20 ± 1.5% at Pre to 6.62 ± 1.4% at 28 Days (7.8 ± 9.3%). At the same time no changes in mass-specific respiratory capacities, mitochondrial protein or antioxidant content were found. This study demonstrates that skeletal muscle Mito(VD) may increase with 28 days acclimation to 3454 m.