Dual-release hydrocortisone improves body composition and the glucometabolic profile in patients with secondary adrenal insufficiency.

PURPOSE: Studies have suggested improved metabolic profiles in patients with adrenal insufficiency treated with dual-release hydrocortisone (DR-HC) compared with conventional hydrocortisone (C-HC). This study investigates the effect of DR-HC compared with C-HC treatment on five health variables: diurnal salivary cortisol/cortisone, body composition, bone health, glucose metabolism, lipids, and blood pressure. METHODS: Prospective study of 27 participants (24 men) with secondary adrenal insufficiency with measurements during stable C-HC and 16 weeks after treatment switch to DR-HC. OUTCOMES: Diurnal salivary-cortisol/cortisone, body composition assessed by Dual-Energy X-ray absorptiometry scan, bone status indices (serum type I N-terminal procollagen [PINP], collagen type I cross-linked C-telopeptide [CTX], osteocalcin, receptor activator kappa-B [RANK] ligand, osteoprotegerin, and sclerostin), lipids, haemoglobin A1c (HbA1c), and 24-hour blood pressure. RESULTS: After the switch to DR-HC, the diurnal salivary-cortisol area under the curve (AUC) decreased non-significantly (mean difference: -55.9 nmol/L/day, P = 0.06). The salivary-cortisone-AUC was unchanged. Late-evening salivary-cortisol and cortisone were lower (-1.6 and -1.7 nmol/L, P = 0.002 and 0.004). Total and abdominal fat mass (-1.5 and -0.5 kg, P = 0.003 and 0.02), HbA1c (-1.2 mmol/mol, P = 0.02), and osteocalcin decreased (-7.0 µg/L, P = 0.03) whereas sclerostin increased (+41.1 pg/mL, P = 0.0001). The remaining bone status indices, lipids, and blood pressure were unchanged. CONCLUSION: This study suggests that switching to DR-HC leads to lower late-evening cortisol/cortisone exposure and a more favourable metabolic profile and body composition. In contrast, decreased osteocalcin with increasing sclerostin might indicate a negative impact on bones. CLINICAL TRIAL REGISTRATION: EudraCT201400203932.

Ventricular CSF proteomic profiles and predictors of surgical treatment outcome in chronic hydrocephalus.

BACKGROUND: By applying an unbiased proteomic approach, we aimed to search for cerebrospinal fluid (CSF) protein biomarkers distinguishing between obstructive and communicating hydrocephalus in order to improve appropriate surgical selection for endoscopic third ventriculostomy vs. shunt implants. Our second study purpose was to look for potential CSF biomarkers distinguishing between patients with adult chronic hydrocephalus benefitting from surgery (responders) vs. those who did not (non-responders). METHODS: Ventricular CSF samples were collected from 62 patients with communicating hydrocephalus and 28 patients with obstructive hydrocephalus. CSF was collected in relation to the patients' surgical treatment. As a control group, CSF was collected from ten patients with unruptured aneurysm undergoing preventive surgery (vascular clipping). RESULTS: Mass spectrometry-based proteomic analysis of the samples identified 1251 unique proteins. No proteins differed significantly between the communicating hydrocephalus group and the obstructive hydrocephalus group. Four proteins were found to be significantly less abundant in CSF from communicating hydrocephalus patients compared to control subjects. A PCA plot revealed similar proteomic CSF profiles of obstructive and communicating hydrocephalus and control samples. For obstructive hydrocephalus, ten proteins were found to predict responders from non-responders. CONCLUSION: Here, we show that the proteomic profile of ventricular CSF from patients with hydrocephalus differs slightly from control subjects. Furthermore, we find ten predictors of response to surgical outcome (endoscopic third ventriculostomy or ventriculo-peritoneal shunt) in patients with obstructive hydrocephalus.

Effect of aerobic exercise training on the fat fraction of the liver in persons with chronic hepatitis B and hepatic steatosis: Trial protocol for a randomized controlled intervention trial- The FitLiver study.

BACKGROUND: The global prevalence of chronic hepatitis B is more than 300 million people, and in Denmark, 17,000 people are estimated to have chronic hepatitis B. Untreated, chronic hepatitis B can lead to the development of liver cirrhosis and liver cancer. There is no curable therapy. In persons with obesity and chronic hepatitis B infection, the development of hepatic steatosis imposes a double burden on the liver, leading to an increased risk of cirrhosis and liver cancer. In patients without chronic hepatitis B, exercise interventions have shown beneficial effects on hepatic steatosis through improvements in fat fraction of the liver, insulin resistance, fatty acid metabolism, and glucose metabolism, as well as activation of liver-induced regulatory protein secretion (hepatokines) after the exercise intervention. OBJECTIVE: To investigate in persons with chronic hepatitis B and hepatic steatosis: Primary: Whether exercise will decrease the fat fraction of the liver. Secondary: If exercise will affect hepatokine secretion and if it will improve lipid- and glucose metabolism, liver status, markers of inflammation, body composition, and blood pressure. METHODS: A randomized, controlled, clinical intervention trial consisting of 12 weeks of aerobic exercise training or no intervention. Thirty persons with chronic hepatitis B and hepatic steatosis will be randomized 1:1. Before and after the intervention, participants will undergo an MRI scan of the liver, blood sampling, oral glucose tolerance test, fibroscan, VO2max test, DXA scan, blood pressure measurements, and optional liver biopsy. Lastly, a hormone infusion test with somatostatin and glucagon to increase the glucagon/insulin ratio for stimulating secretion of circulating hepatokines will be performed. The training program includes three weekly training sessions of 40 min/session over 12 weeks. DISCUSSION: This trial, investigating high-intensity interval training in persons with chronic hepatitis B and hepatic steatosis, is the first exercise intervention trial performed on this group of patients. If exercise reduces hepatic steatosis and induces other beneficial effects of clinical markers in this group of patients, there might be an indication to recommend exercise as part of treatment. Furthermore, the investigation of the effect of exercise on hepatokine secretion will provide more knowledge on the effects of exercise on the liver. TRIAL REGISTRATION: Danish Capital Regions committee on health research ethics reference: H-21034236 (version 1.4 date: 19-07-2022) and ClinicalTrials.gov: NCT05265026.

Lowered muscle glycogen reduces body mass with no effect on short-term exercise performance in men.

Performance in short-duration sports is highly dependent on muscle glycogen, but the total degradation is only moderate and considering the water-binding property of glycogen, unnecessary storing of glycogen may cause an unfavorable increase in body mass. To investigate this, we determined the effect of manipulating dietary carbohydrates (CHO) on muscle glycogen content, body mass, and short-term exercise performance. In a randomized and counterbalanced cross-over design, twenty-two men completed two maximal cycle tests of either 1-min (n = 10) or 15-min (n = 12) duration with different pre-exercise muscle glycogen levels. Glycogen manipulation was initiated three days prior to the tests by exercise-induced glycogen depletion followed by ingestion of a moderate (M-CHO) or high (H-CHO) CHO-diet. Subjects were weighed before each test, and muscle glycogen content was determined in biopsies from m. vastus lateralis before and after each test. Pre-exercise muscle glycogen content was lower following M-CHO than H-CHO (367 mmol · kg-1 DW vs. 525 mmol · kg-1 DW, p < 0.00001), accompanied by a 0.7 kg lower body mass (p < 0.00001). No differences were observed in performance between diets in neither the 1-min (p = 0.33) nor the 15-min (p = 0.99) test. In conclusion, pre-exercise muscle glycogen content and body mass were lower after ingesting moderate compared with high amounts of CHO, while short-term exercise performance was unaffected. This demonstrates that adjusting pre-exercise glycogen levels to the requirements of competition may provide an attractive weight management strategy in weight-bearing sports, particularly in athletes with high resting glycogen levels.

Validation of a novel automated system, Fluispotter®, for serial sampling of dried blood spots.

Repeated blood sampling is required in certain clinical and research settings, which is currently performed by drawing blood from venous catheters requiring manual handling of each sample at the time of collection. A novel body-worn device for repeated serial samples, Fluispotter®, with automated extraction, collection, and storage of up to 20 venous dried blood spot samples over the course of 20 h may overcome problems with current methods for serial sampling. The purpose of this study was to assess the performance and safety of Fluispotter for the first time in healthy subjects. Fluispotter consists of a cartridge with tubing, a reservoir for flushing solution, pumps and filterpaper, and a multi-lumen catheter placed in the brachial vein. We recruited healthy subjects for testing in an in-hospital setting. Fluispotter was attached by an anesthesiologist to 22 healthy subjects of which 9/22 (40.9%) participants had all 20 samples taken, which was lower than the goal of complete sampling in 80% of the subjects (P = 0.02). The main reason for sample failure was clogging of blood flow which was observed in 11/22 (50%) of the participants. No serious adverse events occurred, and the participants rated the pain from the insertion and the removal of catheter as very low. A cortisol profile showed nadir values at midnight and highest values at 05:00 h. Although full sampling was not successful in all participants, the Fluispotter technology proved safe and highly acceptable to the participants producing the expected cortisol profile without the requirement of staff during sample collection.

Differential proteomic profile of lumbar and ventricular cerebrospinal fluid.

BACKGROUND: Pathological cerebral conditions may manifest in altered composition of the cerebrospinal fluid (CSF). Although diagnostic CSF analysis seeks to establish pathological disturbances in the brain proper, CSF is generally sampled from the lumbar compartment for reasons of technical ease and ethical considerations. We here aimed to compare the molecular composition of CSF obtained from the ventricular versus the lumbar CSF compartments to establish a relevance for employing lumbar CSF as a proxy for the CSF bathing the brain tissue. METHODS: CSF was collected from 46 patients with idiopathic normal pressure hydrocephalus (iNPH) patients during their diagnostic workup (lumbar samples) and in connection with their subsequent CSF diversion shunt surgery (ventricular samples). The mass-spectrometry-based proteomic profile was determined in these samples and in addition, selected biomarkers were quantified with ELISA (S100B, neurofilament light (NfL), amyloid-ß (Aß40, Aß42), and total tau (T-tau) and phosphorylated tau (P-tau) forms). The latter analysis was extended to include paired porcine samples obtained from the lumbar compartment and the cerebromedullary cistern closely related to the ventricles. RESULTS: In total 1231 proteins were detected in the human CSF. Of these, 216 distributed equally in the two CSF compartments, whereas 22 were preferentially (or solely) present in the ventricular CSF and four in the lumbar CSF. The selected biomarkers of neurodegeneration and Alzheimer's disease displayed differential distribution, some with higher (S100B, T-tau, and P-tau) and some with lower (NfL, Aß40, Aß42) levels in the ventricular compartment. In the porcine samples, all biomarkers were most abundant in the lumbar CSF. CONCLUSIONS: The overall proteomic profile differs between the ventricular and the lumbar CSF compartments, and so does the distribution of clinically employed biomarkers. However, for a range of CSF proteins and biomarkers, one can reliably employ lumbar CSF as a proxy for ventricular CSF if or a lumbar/cranial index for the particular molecule has been established. It is therefore important to verify the compartmental preference of the proteins or biomarkers of interest prior to extrapolating from lumbar CSF to that of the ventricular fluid bordering the brain.

Impact of Aging and Lifelong Exercise Training on Mitochondrial Function and Network Connectivity in Human Skeletal Muscle.

Aging is associated with metabolic decline in skeletal muscle, which can be delayed by physical activity. Moreover, both lifelong and short-term exercise training have been shown to prevent age-associated fragmentation of the mitochondrial network in mouse skeletal muscle. However, whether lifelong endurance exercise training exerts the same effects in human skeletal muscle is still not clear. Therefore, the aim of the present study was to examine the effect of volume-dependent lifelong endurance exercise training on mitochondrial function and network connectivity in older human skeletal muscle. Skeletal muscle complex I+II-linked mitochondrial respiration per tissue mass was higher, but intrinsic complex I+II-linked mitochondrial respiration was lower in highly trained older subjects than in young untrained, older untrained, and older moderately trained subjects. Mitochondrial volume and connectivity were higher in highly trained older subjects than in untrained and moderately trained older subjects. Furthermore, the protein content of the ADP/ATP exchangers ANT1 + 2 and VDAC was higher and of the mitophagic marker parkin lower in skeletal muscle from the highly trained older subjects than from untrained and moderately trained older subjects. In contrast, H2O2 emission in skeletal muscle was not affected by either age or exercise training, but SOD2 protein content was higher in highly trained older subjects than in untrained and moderately trained older subjects. This suggests that healthy aging does not induce oxidative stress or mitochondrial network fragmentation in human skeletal muscle, but high-volume exercise training increases mitochondrial volume and network connectivity, thereby increasing oxidative capacity in older human skeletal muscle.

GDF15 is an exercise-induced hepatokine regulated by glucagon and insulin in humans.

Objective: Growth differentiation factor (GDF)-15 is implicated in regulation of metabolism and circulating GDF15 increases in response to exercise. The source and regulation of the exercise-induced increase in GDF15 is, however not known. Method: Plasma GDF15 was measured by ELISA under the following conditions: 1) Arterial-to-hepatic venous differences sampled before, during, and after exercise in healthy male subjects (n=10); 2) exogenous glucagon infusion compared to saline infusion in resting healthy subjects (n=10); 3) an acute exercise bout with and without a pancreatic clamp (n=6); 4) healthy subjects for 36 hours (n=17), and 5) patients with anorexia nervosa (n=25) were compared to healthy age-matched subjects (n=25). Tissue GDF15 mRNA content was determined in mice in response to exhaustive exercise (n=16). Results: The splanchnic bed released GDF15 to the circulation during exercise and increasing the glucagon-to-insulin ratio in resting humans led to a 2.7-fold (P<0.05) increase in circulating GDF15. Conversely, inhibiting the exercise-induced increase in the glucagon-to-insulin ratio blunted the exercise-induced increase in circulating GDF15. Fasting for 36 hours did not affect circulating GDF15, whereas resting patients with anorexia nervosa displayed elevated plasma concentrations (1.4-fold, P<0.05) compared to controls. In mice, exercise increased GDF15 mRNA contents in liver, muscle, and adipose tissue. Conclusion: In humans, GDF15 is a "hepatokine" which increases during exercise and is at least in part regulated by the glucagon-to-insulin ratio. Moreover, chronic energy deprivation is associated with elevated plasma GDF15, which supports that GDF15 is implicated in metabolic signalling in humans.

DPYD genotyping and dihydropyrimidine dehydrogenase (DPD) phenotyping in clinical oncology. A clinically focused minireview.

BACKGROUND: In clinical oncology, systemic 5-fluorouracil (5-FU) and its oral pro-drugs are used to treat a broad group of solid tumours. Patients with dihydropyrimidine dehydrogenase (DPD) enzyme deficiency are at elevated risk of toxicity if treated with standard doses of 5-FU. DPYD genotyping and measurements of plasma uracil concentration (DPD phenotyping) can be applied as tests for DPD deficiency. In April 2020, the European Medicines Agency recommended pre-treatment DPD testing to reduce the risk of 5-FU-related toxicity. OBJECTIVES: The objective of this study is to present the current evidence for DPD testing in routine oncological practice. METHODS: Two systematic literature searches were performed following the PRISMA guidelines. We identified studies examining the possible benefit of DPYD genotyping or DPD phenotyping on the toxicity risk. FINDINGS: Nine and 12 studies met the criteria for using DPYD genotyping and DPD phenotyping, respectively. CONCLUSIONS: The evidence supporting either DPYD genotyping or DPD phenotyping as pre-treatment tests to reduce 5-FU toxicity is poor. Further evidence is still needed to fully understand and guide clinicians to dose by DPD activity.

Acute effects on glucose tolerance by neprilysin inhibition in patients with type 2 diabetes.

AIMS: Sacubitril/valsartan is a neprilysin-inhibitor/angiotensin II receptor blocker used for the treatment of heart failure. Recently, a post-hoc analysis of a 3-year randomized controlled trial showed improved glycaemic control with sacubitril/valsartan in patients with heart failure and type 2 diabetes. We previously reported that sacubitril/valsartan combined with a dipeptidyl peptidase-4 inhibitor increases active glucagon-like peptide-1 (GLP-1) in healthy individuals. We now hypothesized that administration of sacubitril/valsartan with or without a dipeptidyl peptidase-4 inhibitor would lower postprandial glucose concentrations (primary outcome) in patients with type 2 diabetes via increased active GLP-1. METHODS: We performed a crossover trial in 12 patients with obesity and type 2 diabetes. A mixed meal was ingested following five respective interventions: (a) a single dose of sacubitril/valsartan; (b) sitagliptin; (c) sacubitril/valsartan + sitagliptin; (d) control (no treatment); and (e) valsartan alone. Glucose, gut and pancreatic hormone responses were measured. RESULTS: Postprandial plasma glucose increased by 57% (incremental area under the curve 0-240 min) (p = .0003) and increased peak plasma glucose by 1.7 mM (95% CI: 0.6-2.9) (p = .003) after sacubitril/valsartan compared with control, whereas postprandial glucose levels did not change significantly after sacubitril/valsartan + sitagliptin. Glucagon, GLP-1 and C-peptide concentrations increased after sacubitril/valsartan, but insulin and glucose-dependent insulinotropic polypeptide did not change. CONCLUSIONS: The glucose-lowering effects of long-term sacubitril/valsartan treatment reported in patients with heart failure and type 2 diabetes may not depend on changes in entero-pancreatic hormones. Neprilysin inhibition results in hyperglucagonaemia and this may explain the worsen glucose tolerance observed in this study. CLINICALTRIALS: gov (NCT03893526).

Amino Acid Metabolism and Protein Turnover in Lean and Obese Humans During Exercise-Effect of IL-6 Receptor Blockade.

CONTEXT: Interleukin-6 (IL-6) is implicated in skeletal muscle wasting and in regulating skeletal muscle hypertrophy in the healthy state. OBJECTIVE: This work aimed to determine the role of IL-6 in regulating systemic protein and amino acid metabolism during rest, exercise, and recovery in lean and obese humans. METHODS: In a nonrandomized, single-blind design, 12 lean and 9 obese individuals were infused first with 0.9% saline (Saline), secondly with the IL-6 receptor antibody tocilizumab (Acute IL-6R ab), and 21 days later with saline while still under tocilizumab influence (Chronic IL-6R ab). Outcome measures were determined before, during, and after 90 minutes of exercise at 40% Wattmax by isotope dilution technique, using primed continuous infusion of L-[ring-D5]phenylalanine and L-[D2]tyrosine. Main outcomes measures included systemic protein turnover and plasma amino acid concentrations. RESULTS: We saw no effect of acute or chronic IL-6 receptor blockade on protein turnover. In lean individuals, chronic IL-6 receptor blockade increased plasma concentrations of total amino acids (rest Δ + 186 µmol/L; 95% CI, 40-332; recovery Δ + 201 µmol/L; 95% CI, 55-347) and essential amino acids (rest Δ + 43 µmol/L; 95% CI, 12-76; recovery Δ + 45 µmol/L; 95% CI, 13-77) independently of exercise but had no such effect in obese individuals (total amino acids rest Δ + 63 µmol/L; 95% CI, -170 to 295, recovery Δ - 23 µmol/L, 95% CI, -256 to 210; essential amino acids rest Δ + 26 µmol/L; 95% CI, -21 to 73, recovery Δ + 11 µmol/L; 95% CI, -36 to 58). CONCLUSION: IL-6 receptor blockade has no effect on protein turnover in fasting lean and obese humans during rest, exercise, and recovery. Chronic IL-6 receptor blockade increases total and essential amino acid concentrations only in lean individuals.

Circulating Concentrations of C-Type Natriuretic Peptides Increase with Sacubitril/Valsartan Treatment in Healthy Young Men.

BACKGROUND: C-type natriuretic peptide (CNP) is a cardioprotective peptide with high affinity for the ectoenzyme neutral endopeptidase (neprilysin). We aimed to determine whether angiotensin receptor-neprilysin inhibitor treatment acutely affects circulating concentrations of bioactive CNP and its molecular amino-terminal precursor (NT-proCNP). METHODS: We included 9 and 10 healthy young men in 2 randomized crossover trials with sacubitril/valsartan vs control (Trial 1) and sacubitril/valsartan and sitagliptin vs sitagliptin (Trial 2). The participants were randomized to a single dose of sacubitril/valsartan (194/206 mg) or control at the first visit 30 min prior to a standardized meal intake. We obtained blood samples at 12 time points over 5 h and measured plasma concentrations of NT-proCNP in both trials and CNP in Trial 2. RESULTS: NT-proCNP concentrations increased 3.5 h after sacubitril/valsartan treatment, and at 4.5 h concentrations were 42% and 65% higher compared with control in Trial 1 and Trial 2, respectively. The total area under the curve (tAUC)15-270 min was 22% higher (P = 0.007) in Trial 1 and 17% higher with treatment (P = 0.017) in Trial 2. Concentrations of bioactive CNP followed a similar temporal pattern with an increase of 93% at 4.5 h and a 31% higher tAUC15-270 min compared with control (P = 0.001) in Trial 2. CONCLUSIONS: Sacubitril/valsartan augments circulating concentrations of both bioactive CNP and NT-proCNP in healthy young men. The increase in bioactive CNP is most likely caused by de novo synthesis and secretion rather than diminished breakdown through neprilysin inhibition.ClinicalTrials.gov registration number NCT03717688.

Ameliorating Effects of Lifelong Physical Activity on Healthy Aging and Mitochondrial Function in Human White Adipose Tissue.

Growing old is patently among the most prominent risk factors for lifestyle-related diseases and deterioration in physical performance. Aging in particular affects mitochondrial homeostasis, and maintaining a well-functioning mitochondrial pool is imperative in order to avoid age-associated metabolic decline. White adipose tissue (WAT) is a key organ in energy balance, and impaired mitochondrial function in adipocytes has been associated with increased low-grade inflammation, altered metabolism, excessive reactive oxygen species (ROS) production, and an accelerated aging phenotype. Exercise training improves mitochondrial health but whether lifelong exercise training can sufficiently maintain WAT mitochondrial function is currently unknown. Therefore, to dissect the role and dose-dependence of lifelong exercise training on aging WAT metabolic parameters and mitochondrial function, young and older untrained, as well as moderately and highly exercise trained older male subjects were recruited and abdominal subcutaneous (s)WAT biopsies and venous blood samples were obtained to measure mitochondrial function and key metabolic factors in WAT and plasma. Mitochondrial intrinsic respiratory capacity was lower in sWAT from older than from young subjects. In spite of this, maximal mitochondrial respiration per wet weight, markers of oxidative capacity, and mitophagic capacity were higher in sWAT from the lifelong highly exercise trained group than all other groups. Furthermore, ROS emission was generally lower in sWAT from lifelong highly exercise trained subjects than older untrained subjects. Taken together, aging reduces intrinsic mitochondrial respiration in human sWAT, but lifelong high-volume exercise training increases oxidative capacity by increasing mitochondrial volume likely contributing to healthy aging.

Blocking endogenous IL-6 impairs mobilization of free fatty acids during rest and exercise in lean and obese men.

Lack of interleukin-6 (IL-6) leads to expansion of adipose tissue mass in rodents and humans. The exact underlying mechanisms have not been identified. In this placebo-controlled, non-randomized, participant-blinded crossover study, we use the IL-6 receptor antibody tocilizumab to investigate the role of endogenous IL-6 in regulating systemic energy metabolism at rest and during exercise and recovery in lean and obese men using tracer dilution methodology. Tocilizumab reduces fatty acid appearance in the circulation under all conditions in lean and obese individuals, whereas lipolysis (the rate of glycerol appearance into the circulation) is mostly unaffected. The fact that fatty acid oxidation is unaffected by IL-6 receptor blockade suggests increased re-esterification of fatty acids. Glucose kinetics are unaffected. We find that blocking endogenous IL-6 signaling with tocilizumab impairs fat mobilization, which may contribute to expansion of adipose tissue mass and, thus, affect the health of individuals undergoing anti-IL-6 therapy (Clinicaltrials.gov: NCT03967691).

Neprilysin Inhibition Increases Glucagon Levels in Humans and Mice With Potential Effects on Amino Acid Metabolism.

CONTEXT: Inhibitors of the protease neprilysin (NEP) are used for treating heart failure, but are also linked to improvements in metabolism. NEP may cleave proglucagon-derived peptides, including the glucose and amino acid (AA)-regulating hormone glucagon. Studies investigating NEP inhibition on glucagon metabolism are warranted. OBJECTIVE: This work aims to investigate whether NEP inhibition increases glucagon levels. METHODS: Plasma concentrations of glucagon and AAs were measured in eight healthy men during a mixed meal with and without a single dose of the NEP inhibitor/angiotensin II type 1 receptor antagonist, sacubitril/valsartan (194 mg/206 mg). Long-term effects of sacubitril/valsartan (8 weeks) were investigated in individuals with obesity (n = 7). Mass spectrometry was used to investigate NEP-induced glucagon degradation, and the derived glucagon fragments were tested pharmacologically in cells transfected with the glucagon receptor (GCGR). Genetic deletion or pharmacological inhibition of NEP with or without concomitant GCGR antagonism was tested in mice to evaluate effects on AA metabolism. RESULTS: In healthy men, a single dose of sacubitril/valsartan significantly increased postprandial concentrations of glucagon by 228%, concomitantly lowering concentrations of AAs including glucagonotropic AAs. Eight-week sacubitril/valsartan treatment increased fasting glucagon concentrations in individuals with obesity. NEP cleaved glucagon into 5 inactive fragments (in vitro). Pharmacological NEP inhibition protected both exogenous and endogenous glucagon in mice after an AA challenge, while NEP-deficient mice showed elevated fasting and AA-stimulated plasma concentrations of glucagon and urea compared to controls. CONCLUSION: NEP cleaves glucagon, and inhibitors of NEP result in hyperglucagonemia and may increase postprandial AA catabolism without affecting glycemia.

The Regulation of Circulating Hepatokines by Fructose Ingestion in Humans.

CONTEXT: Fibroblast growth factor 21 (FGF21), follistatin, angiopoietin-like 4 (ANGPTL4), and growth differential factor 15 (GDF15) are regulated by energy metabolism. Recent findings in humans demonstrate that fructose ingestion increases circulating FGF21, with increased response in conditions of insulin resistance. OBJECTIVE: This study examines the acute effect of fructose and somatostatin on circulating FGF21, follistatin, ANGPTL4, and GDF15 in humans. METHODS: Plasma FGF21, follistatin, ANGPTL4, and GDF15 concentrations were measured in response to oral ingestion of 75 g of fructose in 10 young healthy males with and without a 15-minute infusion of somatostatin to block insulin secretion. A control infusion of somatostatin was also performed in the same subjects. RESULTS: Following fructose ingestion, plasma FGF21 peaked at 3.7-fold higher than basal concentration (P < 0.05), and it increased 4.9-fold compared with basal concentration (P < 0.05) when somatostatin was infused. Plasma follistatin increased 1.8-fold after fructose ingestion (P < 0.05), but this increase was blunted by concomitant somatostatin infusion. For plasma ANGPTL4 and GDF15, no increases were obtained following fructose ingestion. Infusion of somatostatin alone slightly increased plasma FGF21 and follistatin. CONCLUSION: Here we show that in humans (1) the fructose-induced increase in plasma FGF21 was enhanced when somatostatin was infused, suggesting an inhibitory role of insulin on the fructose-induced FGF21 increase; (2) fructose ingestion also increased plasma follistatin, but somatostatin infusion blunted the increase; and (3) fructose ingestion had no stimulating effect on ANGPTL4 and GDF15 levels, demonstrating differences in the hepatokine response to fructose ingestion.

Glycogen supercompensation is due to increased number, not size, of glycogen particles in human skeletal muscle.

NEW FINDINGS: What is the central question of this study? Glycogen supercompensation after glycogen-depleting exercise can be achieved by consuming a carbohydrate-enriched diet, but the associated effects on the size, number and localization of intramuscular glycogen particles are unknown. What is the main finding and its importance? Using transmission electron microscopy to inspect individual glycogen particles visually, we show that glycogen supercompensation is achieved by increasing the number of particles while keeping them at submaximal sizes. This might be a strategy to ensure that glycogen particles can be used fast, because particles that are too large might impair utilization rate. ABSTRACT: Glycogen supercompensation after glycogen-depleting exercise can be achieved by consuming a carbohydrate-enriched diet, but the associated effects on the size, number and localization of intramuscular glycogen particles are unknown. We investigated how a glycogen-loading protocol affects fibre type-specific glycogen volume density, particle diameter and numerical density in three subcellular pools: between (intermyofibrillar) or within (intramyofibrillar) the myofibrils or beneath the sarcolemma (subsarcolemmal). Resting muscle biopsies from 11 physically active men were analysed using transmission electron microscopy after mixed (MIX), LOW or HIGH carbohydrate consumption separated by glycogen-lowering cycling at 75% of maximal oxygen consumption until exhaustion. After HIGH, the total volumetric glycogen content was 40% [95% confidence interval 16, 68] higher than after MIX in type I fibres (P < 0.001), with little to no difference in type II fibres (9% [95% confidence interval -9, 27]). Median particle diameter was 22.5 (interquartile range 20.8-24.7) nm across glycogen pools and fibre types, and the numerical density was 61% [25, 107] and 40% [9, 80] higher in the subsarcolemmal (P < 0.001) and intermyofibrillar (P < 0.01) pools of type I fibres, respectively, with little to no difference in the intramyofibrillar pool (3% [-20, 32]). In LOW, total glycogen was in the range of 21-23% lower, relative to MIX, in both fibre types, reflected in a 21-46% lower numerical density across pools. In comparison to MIX, particle diameter was unaffected by other diets ([-1.4, 1.3] nm). In conclusion, glycogen supercompensation after prolonged cycling is exclusive to type I fibres, predominantly in the subsarcolemmal pool, and involves an increase in the numerical density rather than the size of existing glycogen particles.

Quantification of biotin in plasma samples by column switching liquid chromatography - tandem mass spectrometry.

Biotin (or Vitamin B7) is a vitamin where deficiency can be caused by inadequate intake. Biotin deficiency is rare, as most people get enough biotin from diet, since many foods contain biotin. In addition to biotin from food, intestinal bacteria can synthesize biotin, which can then be absorbed by the body. Supplementation with biotin has been advocated, mainly due to proposed beneficial effects on skin, nail and hair growth. There is no evidence that high biotin intakes are toxic, but a high intake may interfere with diagnostic assays that use biotin-streptavidin technology. These tests are commonly used to measure plasma concentrations of a wide range of hormones. Erroneous results may lead to misdiagnosis of various endocrine disorders. Supplementation with high-dose biotin has been used experimental for the treatment of diseases (e.g. multiple sclerosis) and high doses are used to obtain effect on nail and hair growth. On this background a demand for tests to determine if there is a risk of obtaining false test results when using biotin-streptavidin based tests have appeared. In this paper we present a method based on column switching liquid chromatography tandem mass spectrometry for the quantification of biotin in plasma and serum and explore the effects of biotin on an immunoassay based on biotin strept(avidin) chemistry.

Heterogeneity in subcellular muscle glycogen utilisation during exercise impacts endurance capacity in men.

KEY POINTS: When muscle biopsies first began to be used routinely in research on exercise physiology five decades ago, it soon become clear that the muscle content of glycogen is an important determinant of exercise performance. Glycogen particles are stored in distinct pools within the muscles, but the role of each pool during exercise and how this is affected by diet is unknown. Here, the effects of diet and exercise on these pools, as well as their relation to endurance during prolonged cycling were examined. We demonstrate here that an improved endurance capacity with high carbohydrate loading is associated with a temporal shift in the utilisation of the distinct stores of glycogen pools and is closely linked to the content of the glycogen pool closest to actin and myosin (intramyofibrillar glycogen). These findings highlight the functional importance of distinguishing between different subcellular microcompartments of glycogen in individual muscle fibres. ABSTRACT: In muscle cells, glycogen is stored in three distinct subcellular pools: between or within myofibrils (inter- and intramyofibrillar glycogen, respectively) or beneath the sarcolemma (subsarcolemmal glycogen) and these pools may well have different functions. Here, we investigated the effect of diet and exercise on the content of these distinct pools and their relation to endurance capacity in type 1 and 2 muscle fibres. Following consumption of three different diets (normal, mixed diet = MIX, high in carbohydrate = HIGH, or low in carbohydrate = LOW) for 72 h, 11 men cycled at 75% of V̇O2 max until exhaustion. The volumetric content of the glycogen pools in muscle biopsies obtained before, during, and after exercise were quantified by transmission electron micrographs. The mean (SD) time to exhaustion was 150 (30), 112 (22), and 69 (18) minutes in the HIGH, MIX and LOW trials, respectively (P < 0.001). As shown by multiple regression analyses, the intramyofibrillar glycogen content in type 1 fibres, particularly after 60 min of exercise, correlated most strongly with time to exhaustion. In the HIGH trial, intramyofibrillar glycogen was spared during the initial 60 min of exercise, which was associated with levels and utilisation of subsarcolemmal glycogen above normal. In all trials, utilisation of subsarcolemmal and intramyofibrillar glycogen was more pronounced than that of intermyofibrillar glycogen in relative terms. In conclusion, the muscle pool of intramyofibrillar glycogen appears to be the most important for endurance capacity in humans. In addition, a local abundance of subsarcolemmal glycogen reduces the utilisation of intramyofibrillar glycogen during exercise.