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KEY POINTS: This is the first long-term human clinical trial to report on effects of NR on skeletal muscle mitochondrial function, content and morphology •NR supplementation decreases NAMPT protein abundance in skeletal muscle •NR supplementation do not affect NAD metabolite concentrations in skeletal muscle •Respiration, distribution, and quantity of muscle mitochondria are unaffected by NR •NAMPT in skeletal muscle correlates positively with OXPHOS Complex I, SIRT3, and SDH ABSTRACT: Preclinical evidence suggest that the nicotinamide adenine dinucleotide (NAD+ ) precursor nicotinamide riboside (NR) boosts NAD+ levels and improves diseases associated with mitochondrial dysfunction. We aimed to determine if dietary NR supplementation in middle-aged, obese, insulin-resistant men affects mitochondrial respiration, content, and morphology in skeletal muscle. In a randomized, placebo-controlled clinical trial, 40 participants received 1,000 mg NR or placebo twice daily for 12 weeks. Skeletal muscle biopsies were collected before and after the intervention. Mitochondrial respiratory capacity was determined by high-resolution respirometry on single muscle fibres. Protein abundance and mRNA expression were measured by Western blot and qPCR analyses, respectively, and in a subset of the participants (placebo n = 8; NR n = 8) we quantified mitochondrial fractional area and mitochondrial morphology by laser scanning confocal microscopy. Protein levels of nicotinamide phosphoribosyltransferase (NAMPT), an essential NAD+ biosynthetic enzyme in skeletal muscle, decreased 14% with NR. However, steady-state NAD+ levels as well as gene expression and protein abundance of other NAD+ biosynthetic enzymes remained unchanged. Neither respiratory capacity of skeletal muscle mitochondria nor abundance of mitochondrial associated proteins were affected by NR. Moreover, no changes in mitochondrial fractional area or network morphology were observed. Our data do not support the hypothesi

Fasting in human subjects shifts skeletal muscle metabolism toward lipid utilization and accumulation, including intramyocellular lipid (IMCL) deposition. Growth hormone (GH) secretion amplifies during fasting and promotes lipolysis and lipid oxidation, but it is unknown to which degree lipid deposition and metabolism in skeletal muscle during fasting depends on GH action. To test this, we studied nine obese but otherwise healthy men thrice: (a) in the postabsorptive state (“CTRL”), (b) during 72-hr fasting (“FAST”), and (c) during 72-hr fasting and treatment with a GH antagonist (GHA) (“FAST + GHA”). IMCL was assessed by magnetic resonance spectroscopy (MRS) and blood samples were drawn for plasma metabolomics assessment while muscle biopsies were obtained for measurements of regulators of substrate metabolism. Prolonged fasting was associated with elevated GH levels and a pronounced GHA-independent increase in circulating medium- and long-chain fatty acids, glycerol, and ketone bodies indicating increased supply of lipid intermediates to skeletal muscle. Additionally, fasting was associated with a release of short-, medium-, and long-chain acylcarnitines to the circulation from an increased β-oxidation. This was consistent with a ≈55%–60% decrease in pyruvate dehydrogenase (PDHa) activity. Opposite, IMCL content increased ≈75% with prolonged fasting without an effect of GHA. We suggest that prolonged fasting increases lipid uptake in skeletal muscle and saturates lipid oxidation, both favoring IMCL deposition. This occurs without a detectable effect of GHA on skeletal muscle lipid metabolism.

Hyperpolarized [1-13C]pyruvate magnetic resonance (MR) spectroscopy has the unique ability to detect real-time metabolic changes in vivo owing to its high sensitivity compared with thermal MR and high specificity compared with other metabolic imaging methods. The aim of this study was to explore the potential of hyperpolarized MR spectroscopy for quantification of liver pyruvate metabolism during a hyperinsulinemic isoglycemic clamp in mice. Hyperpolarized [1-13C]pyruvate was used for in vivo MR spectroscopy of liver pyruvate metabolism in mice. Mice were divided into two groups: i) non-stimulated 5-hour fasted mice and ii) hyperinsulinemic isoglycemic clamped mice. During clamp conditions, insulin and donor blood were administered at a constant rate whereas glucose were infused to maintain isoglycemia. When steady state was reached, insulin-stimulated mice were rapidly infused with hyperpolarized [1-13C]pyruvate for real-time tracking of the dynamic distribution of metabolic derivatives from pyruvate, such as [1-13C]lactate, [1-13C]alanine, and [13C]bicarbonate. Isotopomer analysis of plasma glucose confirmed 13C-incorporation from [1-13C]pyruvate into glucose was increased in fasted mice compared with insulin-stimulated mice, demonstrating an increased gluconeogenesis in fasted mice. The AUC ratios for [1-13C]alanine/[1-13C]pyruvate (38.2%), [1-13C]lactate/[1-13C]pyruvate (41.8%), and [13C]bicarbonate/[1-13C]pyruvate (169%) all increased significantly during insulin stimulation. Hyperpolarized [1-13C]pyruvate can be used for in vivo MR spectroscopy of liver pyruvate metabolism during hyperinsulinemic isoglycemic clamp conditions. Under these conditions, insulin decreased gluconeogenesis and increased [1-13C]alanine, [1-13C]lactate, and [13C]bicarbonate after a [1-13C]pyruvate bolus. This application of in vivo spectroscopy has the potential to identify impairments in specific metabolic pathways in the liver associated with obesity, insulin resistance, and non-a

Background: Animal studies suggest a positive role for nicotinamide riboside (NR) on insulin sensitivity and hepatic steatosis in models of obesity and type 2 diabetes. NR, an NAD+ precursor, is a member of the vitamin B-3 family now available as an over-the-counter supplement. Although data from preclinical trials appear consistent, potential effects and safety need to be evaluated in human clinical trials.Objective: The aim of this study was to test the safety of dietary NR supplementation over a 12-wk period and potential to improve insulin sensitivity and other metabolic parameters in obese, insulin-resistant men.Design: In an investigator-initiated randomized, placebo-controlled, double-blinded, and parallel-group designed clinical trial, forty healthy, sedentary men with a body mass index (BMI) > 30 kg/m2, age-range 40-70 y were randomly assigned to 12 wk of NR (1000 mg twice daily) or placebo. We determined the effects of NR supplementation on insulin sensitivity by a hyperinsulinemic euglycemic clamp and substrate metabolism by indirect calorimetry and labeled substrates of tritiated glucose and palmitate. Body composition and fat mass distribution were determined by whole-body dual-energy X-ray absorptiometry (DXA) and MRI scans, and measurements of intrahepatic lipid content were obtained by MR spectroscopy.Results: Insulin sensitivity, endogenous glucose production, and glucose disposal and oxidation were not improved by NR supplementation. Similarly, NR supplementation had no effect on resting energy expenditure, lipolysis, oxidation of lipids, or body composition. No serious adverse events due to NR supplementation were observed and safety blood tests were normal.Conclusion: 12 wk of NR supplementation in doses of 2000 mg/d appears safe, but does not improve insulin sensitivity and whole-body glucose metabolism in obese, insulin-resistant men. This trial was registered at clinicaltrials.gov as NCT02303483.

Purpose: Low intake of dietary fibre is associated with the development of type 2 diabetes. Dyslipidaemia plays a key role in the pathogenesis of type 2 diabetes. Knowledge of the impact of dietary fibres on postprandial lipaemia is, however, sparse. This study aimed in subjects with metabolic syndrome to assess the impact on postprandial lipaemia and features of the metabolic syndrome of a healthy carbohydrate diet (HCD) rich in cereal fibre, arabinoxylan and resistant starch compared to a refined-carbohydrate western-style diet (WSD). Methods: Nineteen subjects completed the randomised, crossover study with HCD and WCD for 4-week. Postprandial metabolism was evaluated by a meal-challenge test and insulin sensitivity was assessed by HOMA-IR and Matsuda index. Furthermore, fasting cholesterols, serum-fructosamine, circulating inflammatory markers, ambulatory blood pressure and intrahepatic lipid content were measured. Results: We found no diet effects on postprandial lipaemia. However, there was a significant diet × statin interaction on total cholesterol (P = 0.02) and LDL cholesterol (P = 0.002). HCD decreased total cholesterol (−0.72 mmol/l, 95% CI (−1.29; −0.14) P = 0.03) and LDL cholesterol (−0.61 mmol/l, 95% CI (−0.86; −0.36) P = 0.002) compared with WSD in subjects on but not without statin treatment. We detected no other significant diet effects. Conclusions: In subjects with metabolic syndrome on statins a 4-week diet rich in arabinoxylan and resistant starch improved fasting LDL and total cholesterol compared to subjects not being on statins. However, we observed no diet related impact on postprandial lipaemia or features of the metabolic syndrome. The dietary fibre x statin interaction deserves further elucidation.

Context: Low birth weight (LBW; <2500 g) is linked to the development of insulin resistance and limbic-hypothalamic-pituitary-adrenal (LHPA) axis hyperactivity.Objective: Our first aim was to study insulin action, LHPA axis function, and limbic brain structures in young, healthy LBW men vs normal birthweight (NBW) controls (part 1). Our second aim was to investigate the effects of escitalopram vs placebo in LBW men in the LHPA axis and insulin sensitivity (part 2).Design Setting, Participants, and Intervention: The maximal (Rdmax) and submaximal (Rdsubmax) rates of insulin-stimulated glucose turnover, LHPA axis, and brain morphology were examined in 40 LBW men and 20 matched NBW men using two-stage hyperinsulinemic euglycemic clamp, 24-hour hormone plasma profiles, and magnetic resonance imaging. Subsequently, all LBW subjects underwent randomized and double-blind treatment with escitalopram 20 mg/d or placebo for 3 months followed by a complete reexamination.Main Outcome Measures (Part 2): Changes in Rdmax/Rdsubmax and plasma-free cortisol 24-hour area under the curve.Results: In LBW vs NBW, Rdsubmax and Rdmax were ∼16% (P = 0.01) and ∼12% (P = 0.01) lower, respectively, and 24-hour free cortisol levels were ∼20% higher (P = 0.02), primarily driven by a ∼99% increase at 05:00 am (P < 0.001). Furthermore, these changes were related to structural alterations within left thalamus and ventromedial prefrontal cortex. However, in LBW men, exposure to escitalopram normalized the free cortisol levels and improved the Rdsubmax by ∼24% (P = 0.04) compared with placebo.Conclusions: LBW vs NBW displayed alterations in key brain structures modulating the LHPA axis, elevated free cortisol levels, and insulin resistance. Escitalopram administration ameliorated these defects, suggesting a potential for LHPA axis modulation compounds to improve insulin action in LBW subjects.

The early progression of diabetic nephropathy is notoriously difficult to detect and quantify before the occurrence of substantial histological damage. Recently, hyperpolarized [1-(13)C]pyruvate has demonstrated increased lactate production in the kidney early after the onset of diabetes, implying increased lactate dehydrogenase activity as a consequence of increased nicotinamide adenine dinucleotide substrate availability due to upregulation of the polyol pathway, i.e., pseudohypoxia. In this study, we investigated the role of oxidative stress in mediating these metabolic alterations using state-of-the-art hyperpolarized magnetic resonance (MR) imaging. Ten-week-old female Wistar rats were randomly divided into three groups: healthy controls, untreated diabetic (streptozotocin treatment to induce insulinopenic diabetes), and diabetic, receiving chronic antioxidant treatment with TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) via the drinking water. Examinations were performed 2, 3, and 4 wk after the induction of diabetes by using a 3T Clinical MR system equipped with a dual tuned (13)C/(1)H-volume rat coil. The rats received intravenous hyperpolarized [1-(13)C]pyruvate and were imaged using a slice-selective (13)C-IDEAL spiral sequence. Untreated diabetic rats showed increased renal lactate production compared with that shown by the controls. However, chronic TEMPOL treatment significantly attenuated diabetes-induced lactate production. No significant effects of diabetes or TEMPOL were observed on [(13)C]alanine levels, indicating an intact glucose-alanine cycle, or [(13)C]bicarbonate, indicating normal flux through the Krebs cycle. In conclusion, this study demonstrates that diabetes-induced pseudohypoxia, as indicated by an increased lactate-to-pyruvate ratio, is significantly attenuated by antioxidant treatment. This demonstrates a pivotal role of oxidative stress in renal metabolic alterations occurring in early diabetes.

Objective: To evaluate the potential of magnetic resonance imaging (MRI) for evaluation of velocity fields downstream of prosthetic aortic valves. Furthermore, to provide comparative data from bileaflet aortic valve prostheses in vitro and in patients. Methods: A pulsatile flow loop was set up in a 7.0 Tesla MRI scanner to study fluid velocity data downstream of a 25 mm aortic bileaflet heart valve prosthesis. Three dimensional surface plots of velocity fields were displayed. In six NYHA class I patients blood velocity profiles were studied downstream of their St. Jude Medical aortic valves using a 1.5 Tesla MRI whole-body scanner. Blood velocity data were displayed as mentioned above. Results: Fluid velocity profiles obtained from in vitro studies 0.25 valve diameter downstream of the valve exhibited significant details about the cross sectional distribution of fluid velocities. This distribution completely reflected the valve design. Blood velocity profiles in humans were considerably smoother and in some cases skewed with the highest velocities toward the anterior-right ascending aortic wall. Conclusion: Display and interpretation of fluid and blood velocity data obtained downstream of prosthetic valves is feasible both in vitro and in vivo using the MRI technique. An in vitro model with a straight tube and the test valve oriented orthogonally to the long axis of the test tube does not entail fluid velocity profiles which are compatible to those obtained from humans, probably due to the much more complex human geometry, and variable alignment of the valve with the ascending aorta. With the steadily improving quality of MRI scanners this technique has significant potential for comparative in vitro and in vivo hemodynamic evaluation of heart valves

Purpose: Our aim was to assess a novel 13C radial fast spin echo golden ratio single shot method for interrogating early renal changes in the diabetic kidney, using hyperpolarized (HP) [13C,15N2]urea as a T2 relaxation based contrast bio-probe. Methods: A novel HP 13C MR contrast experiment was conducted in a group of streptozotocin type-1 diabetic rat model and age matched controls. Results: A significantly different relaxation time (P=0.004) was found in the diabetic kidney (0.49±0.03 s) compared with the controls (0.64±0.02 s) and secondly, a strong correlation between the blood oxygen saturation level and the relaxation times were observed in the healthy controls. Conclusion: HP [13C,15N2]urea apparent T2 mapping may be a useful for interrogating local renal pO2 status and renal tissue alterations.

This study investigated how one bout (1EX) and three bouts (3EX) of strenuous resistance exercise affected the cross-sectional area (CSA) and water content (WC) of the quadriceps muscle and patella tendon (PT), 4 h and 52 h after the last exercise bout. Ten healthy untrained male subjects performed 1EX with one leg and 3EX with the other leg. CSA and WC were measured with magnetic resonance imaging 10, 20 and 30 cm proximal to the tibia plateau (TP) for the muscle, and at the proximal, central and distal site for the PT prior to exercise, and 4 h and 52 h after the last exercise bout. Ten centimeter above the TP, muscle CSA was significantly increased at 4 h (1EX: 13 ± 5%; 3EX: 13 ± 4%) and 52 h (1EX: 16 ± 5%; 3EX: 16 ± 5%) compared with baseline. Muscle WC was significantly increased at 4 h (1EX: 7 ± 1%; 3EX: 6 ± 2%) and 52 h (1EX: 8 ± 2%; 3EX: 8 ± 3%) compared to baseline. PT central CSA was significantly reduced at 52 h (3EX: 14 ± 2%) compared with baseline and (3EX: 13 ± 1%) compared with 4 h. Present data demonstrate that strenuous resistance exercise results in an acute increase in muscle WC and underlines the importance of ensuring sufficient time between the last exercise bout and the determination of anatomical dimensions in muscles.