Scope: Higher egg intake was previously associated with a lower risk of developing type 2 diabete... more Scope: Higher egg intake was previously associated with a lower risk of developing type 2 diabetes (T2D) in the prospective, population-based Kuopio Ischaemic Heart Disease Risk Factor Study (KIHD) in eastern Finland. We explored potential compounds that could explain this association using non-targeted LC-MS-based metabolic profiling. Methods and results: We analyzed 239 baseline serum samples from the KIHD in 4 groups: subjects with higher (mean intake 1 egg/d) or lower (mean intake 2 eggs/wk) egg intake who developed T2D (cases) or remained heatlhy (controls) during the mean follow-up of 19.3 years. We observed different serum profiles of subjects who had either higher or lower egg intakes, and of those who developed type 2 diabetes or remained healthy. The higher baseline tyrosine level predicted higher odds of T2D (OR 1.94; 95% CI 1.45, 2.60; P<0.001; FDR 0.023) along with an unknown hexose-containing compound (OR 2.13; 95% CI 1.57, 2.88; P<0.001; FDR 0.005). Certain predominant metabolites in T2D cases were correlated positively with ones in lower-egg-intake group and negatively with ones in higher-egg-intake group. Conclusion: Our current findings may underline some potential metabolites that could explain how egg intake was associated with a lower risk of T2D.
ABSTRACT The aim of this study was to characterize pediatric pharmacokinetics and central nervous... more ABSTRACT The aim of this study was to characterize pediatric pharmacokinetics and central nervous system exposure of naproxen after oral administration. The pharmacokinetics of naproxen was studied in 53 healthy children aged 3 months to 12 years undergoing surgery with spinal anesthesia. Children received preoperatively a single dose of 10 mg/kg oral naproxen suspension. A single cerebrospinal fluid (CSF) sample (n = 52) was collected at the induction of anesthesia, and plasma samples (n = 270) were collected before, during, and after the operation (up to 51 hours after administration). A population pharmacokinetic model was built using the NONMEM software. Naproxen concentrations in plasma were well described by a 2-compartment model. The estimated oral clearance (CL/F) was 0.62 L/h when linearly scaled by weight to 70 kg. The apparent volume of distribution at steady state (Vss/F) was 12.5 L /70 kg. The findings are consistent with previously reported pharmacokinetic parameters for children older than 5 years. Naproxen permeated into the CSF and reached CSF concentrations that were 4 times higher than unbound plasma concentrations. Based on these data, weight can be used as a basis for naproxen dosing in children older than 3 months of age.
Scope: Urinary hippuric acid has been proposed as a biomarker for fruit, vegetable and polyphenol... more Scope: Urinary hippuric acid has been proposed as a biomarker for fruit, vegetable and polyphenol consumption. We assessed how serum hippuric acid changes after a bilberryenriched diet (BB; high anthocyanin intake) and another berry diet including strawberries, raspberries and cloudberries (SRC; lower anthocyanin intake) and how these changes associate with insulin and glucose metabolism. Methods and results: Hippuric acid was measured with LC-QTOF-MS metabolite profiling analysis from fasting serum samples at baseline and after an 8-week intervention in 47
medRxiv (Cold Spring Harbor Laboratory), Apr 9, 2021
Objective To characterize serum biomarkers in mitochondrial CHCHD10-linked spinal muscular atroph... more Objective To characterize serum biomarkers in mitochondrial CHCHD10-linked spinal muscular atrophy Jokela type (SMAJ) for disease monitoring and for understanding of pathogenic mechanisms. Methods We collected serum samples from a cohort of 49 SMAJ patients, all carriers of the heterozygous c.197G>T p.G66V variant in CHCHD10. As controls, we used age-and sex-matched serum samples obtained from Helsinki Biobank. Neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) were measured with Single molecule array (Simoa), and fibroblast growth factor 21 (FGF-21) and growth differentiation factor 15 (GDF-15) with enzyme-linked immunosorbent assay. For nontargeted serum metabolite profiling, samples were analyzed with liquid chromatography-high resolution mass spectrometry. Disease severity was evaluated retrospectively by calculating a symptom-based score. Results Axon degeneration marker NfL was unexpectedly not altered in the serum of SMAJ patients, whereas astrocytic activation marker GFAP was slightly decreased. Creatine kinase was elevated in most patients, in particular males. We identified six metabolites that were significantly altered in SMAJ patients' serum compared to controls: increased creatine and pyruvate, and decreased creatinine, taurine, N-acetyl-carnosine and succinate. Creatine correlated with disease severity. Altered pyruvate and succinate indicated a metabolic response to mitochondrial dysfunction, however, lactate or mitochondrial myopathy markers FGF-21 or GDF-15 were not changed. Conclusions Biomarkers of muscle mass and damage are altered in SMAJ serum, indicating a role for skeletal muscle in disease pathogenesis in addition to neurogenic damage. Despite the minimal All rights reserved. No reuse allowed without permission.
Since the discovery that introduction of four specific genes encoding transcription factors conve... more Since the discovery that introduction of four specific genes encoding transcription factors convert mature human somatic cells into induced pluripotent stem cells (iPSCs), there has been an enormous increase in the applications of iPSC technologies to medical sciences, especially in modeling human diseases. In this review, we summarize recent advances in applying human iPSC reprogramming to generate patient-specific neural subtypes in order to reveal molecular pathways affected in various neurodegenerative diseases. Metabolites provide a functional readout of various cellular states enabling identification of biomarker candidates for early diagnosis, segregation of patient cohorts, and to follow-up disease progression or disease responses to novel therapies. With emerging technologies, that is, mass spectrometry and nuclear magnetic resonance applications to metabolomics and various software solutions of bioinformatics, it has become possible to measure thousands of metabolites simultaneously. These fascinating new techniques provide a powerful tool for setting scientific hypotheses and linking cellular pathways to biological mechanism. This review focuses on mass spectrometry-based metabolomics as a tool for iPSC research.
Because the high prevalence of nonalcoholic fatty liver disease sets diagnostic challenges to hea... more Because the high prevalence of nonalcoholic fatty liver disease sets diagnostic challenges to health care, identification of new biomarkers of the disease that in the future could have potential utility as diagnostic biomarkers of high liver fat content is important. Our results show that increased amino acid degradation products in the feces may be such biomarkers.
Journal of Pharmaceutical and Biomedical Analysis, Jun 1, 2022
Hair steroid analysis offers the possibility to evaluate long-term steroid metabolism. It is espe... more Hair steroid analysis offers the possibility to evaluate long-term steroid metabolism. It is especially beneficial when studying the steroid milieu in newborns and children, for example it can provide new insights into steroid metabolism over months and is unaffected by diurnal fluctuations in steroid concentrations. This study describes a quantitative and highly selective high-resolution mass spectrometry method for the simultaneous analysis of multiple steroids from human scalp hair. The developed method utilizes parallel reaction monitoring in the analysis of hydroxylamine derivatized steroids from hair samples first washed with isopropanol, extracted with methanol, and further purified with solid-phase extraction and liquid-liquid extraction. The method was proven suitable for the quantitative analysis of endogenous glucocorticoids (cortisol [1.5-364 pg/mg]; cortisone [3.6-355 pg/mg]; corticosterone [0.7-347 pg/mg]; 11-deoxycortisol [0.1-343 pg/mg]), androgens (testosterone [0.1-288 pg/mg]; dehydroepiandrosterone [0.3-288 pg/mg]; androstenedione [0.1-285 pg/mg]; 11β-hydroxyandrostenedione [0.3-304 pg/mg]), progestogens (pregnenolone [0.1-313 pg/mg]; progesterone [0.1-313 pg/mg]; 17α-hydroxypregnenolone [0.3-315 pg/mg]; 17α-hydroxyprogesterone [0.7-330 pg/mg]; 21-hydroxyprogesterone [0.6-320 pg/mg]), and estrogens (estrone [0.1-267 pg/mg]; estradiol [0.5-274 pg/mg]). In addition, 11-ketoandrostenedione [0.6-60 pg/mg]; dihydrotestosterone [0.3-290 pg/mg]; 11-ketotestosterone [0.1-12 pg/mg]; and 5α-androstanedione [0.6-281 pg/mg] could be analyzed semi-quantitatively. Aldosterone [3.5-346 pg/mg]; 11β-hydroxytestosterone [0.3-300 pg/mg]; and 11-ketodihydrotestosterone [0.3-300 pg/mg] were also included in the method, but their concentrations were below the lower limit of quantification in all tested hair samples. The method was applied for the analysis of authentic hair samples from different age groups ranging from newborns to adults, including mothers within 48 h after delivery. The newborn hair samples displayed the widest variety and had also the highest amounts of steroids in comparison to the samples of the other groups.
Accumulating evidence continues to link certain aspects of the endogenous cannabinoid (EC) system... more Accumulating evidence continues to link certain aspects of the endogenous cannabinoid (EC) system with alcohol dependence, negative reinforcement learning and the modulation of stress response. Specific alterations in brain regions that are related to stress and negative reinforcement learning have been reported to exist in Cloninger type 1 and 2 alcoholics. To study possible differences in profiles of ECs between Cloninger type 1 (n=9), type 2 (n=8) alcoholics and non-alcoholic control subjects (n=10), we analyzed post-mortem amygdala and hippocampus brain samples for several ECs by quantitative liquid chromatography with triple quadrupole mass spectrometric detection. A significant difference was found between these three groups in terms of EC profiles in the amygdala (p = 0.037). In particular, this difference was prominent for variations in docosahexaenoylethanolamine levels, which were significantly higher in type 1 alcoholics (p = 0.022) when compared to controls. There was also a large negative correlation between anandamide concentration and mGlu1/5 receptor density in the hippocampus of Cloninger type 1 alcoholics (R=-0.88, p=0.002), which was not seen in type 2 alcoholics or in controls. Although preliminary, and from relatively small diagnostic groups, these results suggest that the EC profile may be altered in the hippocampus and amygdala of type 1 alcoholics.
European Journal of Pharmaceutics and Biopharmaceutics, May 1, 2012
Sublingually administered midazolam is commonly used for premedication of pediatric patients. How... more Sublingually administered midazolam is commonly used for premedication of pediatric patients. However, the irritating taste and low aqueous solubility of midazolam complicate its sublingual use. Cyclodextrin complexation can be used to improve both the taste and aqueous solubility of drugs, but based on earlier studies, the complexation efficiency of midazolam is relatively low. In the present study, the complexation of midazolam with hydroxypropyl-beta-cyclodextrin (HP-b-CD) was investigated in the presence of various excipients. The aqueous solubility of midazolam improved significantly when HP-b-CD was used together with sucrose. Sucrose alone did not increase the solubility of midazolam. In addition, sucrose increased the apparent stability constant of the midazolam/HP-b-CD complex. The pharmacokinetics of midazolam in different dosage forms was investigated in rabbits (dose 1 mg/rabbit) after intravenous administration of midazolam solution and after sublingual administration of midazolam solution, midazolam/HP-b-CD/sucrose solution or midazolam/HP-b-CD/sucrose powder. Midazolam displayed rapid sublingual absorption (mean t max 6 30 min from the liquid formulations and 60 min from the solid formulation) with high absolute bioavailability (>68%) from all evaluated formulations. Based on the results, HP-b-CD and sucrose can be utilized together to prepare more concentrated and palatable midazolam formulations for sublingual administration in pediatric patients.
Endocannabinoids are the endogenous ligands of the G protein-coupled cannabinoid receptors. The p... more Endocannabinoids are the endogenous ligands of the G protein-coupled cannabinoid receptors. The principal brain endocannabinoid, 2-arachidonoylglycerol (2-AG), is enzymatically produced by postsynaptic neurons and then activates presynaptic CB 1 receptors in a retrograde manner. The primary pathway for 2-AG generation is believed to be conversion from the diacylglycerols (DAGs) by two sn-1-specific lipases, DAGLa and DAGLb. Previous studies with DAGL-deficient mice indicated that DAGLa is the major enzyme needed for retrograde synaptic 2-AG signalling. The current study investigated whether the CB 1 receptormediated G i/o protein activity is altered in brain cryosections of DAGL-deficient mice when compared to wild-type mice and whether the sn-1-specific DAGLs are able to generate 2-AG in brain cryosections. Functional autoradiography indicated that brain regional CB 1 receptor-G i/o-activity largely remained unaltered in DAGLa-knockout and DAGLb-knockout mice when compared to wild-type littermates. Following comprehensive pharmacological blockade of 2-AG hydrolysis, brain sections generated sufficient amounts of 2-AG to activate CB 1 receptors throughout the regions endowed with these receptors. As demonstrated by LC/MS/MS, this pool of 2-AG was generated via tetrahydrolipstatin-sensitive enzymatic pathways distinct from DAGLa or DAGLb. We conclude that in addition to the sn-1-specific DAGLs, additional 2-AG generating enzymatic pathways are active in brain sections.
Membrane transporters have long been utilized to improve the oral, hepatic, and renal (re)absorpt... more Membrane transporters have long been utilized to improve the oral, hepatic, and renal (re)absorption. In the brain, however, the transporter-mediated drug delivery has not yet been fully achieved due to the complexity of the blood−brain barrier (BBB). Because L-type amino acid transporter 1 (LAT1) is a good candidate to improve the brain delivery, we developed here four novel LAT1-utilizing prodrugs of four nonsteroidal anti-inflammatory drugs. As a result, all the prodrugs were able to cross the BBB and localize into the brain cells. The brain uptake of salicylic acid (SA) was improved five times, not only across the mouse BBB but also into the cultured mouse and human brain cells. The naproxen prodrug was also transported efficiently into the mouse brain achieving less peripheral exposure, but the brain release of naproxen from the prodrug was not improved. Contrarily, the high plasma protein binding of the flurbiprofen prodrug and the premature bioconversion of the ibuprofen prodrug in the mouse blood hindered the efficient brain delivery. Thus, the structure of the parent drug affects the successful brain delivery of the LAT1-utilizing prodrugs, and the small-sized LAT1-utilizing prodrug of SA constituted a successful model to specifically deliver its parent drug across the mouse BBB and into the cultured mouse and human brain cells.
The objective of this study was to evaluate the cerebrospinal fluid (CSF) permeation of indometha... more The objective of this study was to evaluate the cerebrospinal fluid (CSF) permeation of indomethacin in healthy children. The participants (n = 31, aged 4–144 months) received indomethacin (0.35 mg/kg) as a 10‐minute intravenous infusion prior to surgery under spinal anaesthesia. A single CSF and plasma sample from each individual was collected 14 to 225 minutes after the infusion. Indomethacin concentrations were determined from the CSF, plasma, and protein‐free plasma. Total plasma, protein‐free plasma, and CSF concentrations of indomethacin ranged between 90 and 2200 ng/mL (median, 780 ng/mL), 0.3 and 0.8 ng/mL (median, 0.5 ng/mL), and 0.2 and 5.0 ng/mL (median, 1.4 ng/mL), respectively. The CSF to plasma concentration ratio remained less than 0.01. There was no correlation between the administration time and CSF concentrations. Eleven children developed 12 nonserious adverse effects, from which 5 were central nervous system (CNS) effects (agitation). In conclusion, indomethacin permeated into the CSF of children, which enables both desired and adverse CNS effects of indomethacin.
The purpose of the study was to see if nematodes (Caenorhabditis elegans, Caenorhabditis briggsae... more The purpose of the study was to see if nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, and Pelodera strongyloides) produce endocannabinoids; i.e., anandamide (AEA) and 2-arachidonoylglycerol (2-AG). In this study, AEA and 2-AG were identified as endogenous products from nematodes by using electrospray-ionization ion-trap MS/MS (ESI-IT-MS) experiments operated in the positive-ionization mode. Endocannabinoids were identified by product ion scan and concentrations were measured by triple quadrupole mass spectrometry in the multiple reaction monitoring mode (MRM). Both AEA and 2-AG were identified in all of the nematode samples, even though these species lack known cannabinoid receptors. Neither AEA nor 2-AG were detected in the fat-3 mutant of C. elegans, which lacks the necessary enzyme to produce arachidonic acid, the fatty acid precursor of these endocannabinoids.
Physical activity is essential in weight management, improves overall health, and mitigates obesi... more Physical activity is essential in weight management, improves overall health, and mitigates obesityrelated risk markers. Besides inducing changes in systemic metabolism, habitual exercise may improve gut's microbial diversity and increase the abundance of beneficial taxa in a correlated fashion. Since there is a lack of integrative omics studies on exercise and overweight populations, we studied the metabolomes and gut microbiota associated with programmed exercise in obese individuals. We measured the serum and fecal metabolites of 17 adult women with overweight during a 6-week endurance exercise program. Further, we integrated the exercise-responsive metabolites with variations in the gut microbiome and cardiorespiratory parameters. We found clear correlation with several serum and fecal metabolites, and metabolic pathways, during the exercise period in comparison to the control period, indicating increased lipid oxidation and oxidative stress. Especially, exercise caused co-occurring increase in levels of serum lyso-phosphatidylcholine moieties and fecal glycerophosphocholine. This signature was associated with several microbial metagenome pathways and the abundance of Akkermansia. The study demonstrates that, in the absence of body composition changes, aerobic exercise can induce metabolic shifts that provide substrates for beneficial gut microbiota in overweight individuals. Physical activity in its various forms is essential in weight management. Habitual exercise can improve overall health and mitigate obesity-related risk markers such as insulin resistance, inflammation, and dyslipidemia 1. Even in the absence of corresponding weight loss, physical activity can lower disease risk and improve overall fitness 2. The alterations in energy balance and systemic metabolism that occur in response to acute exercise are well characterized and documented 3,4. However, within the scope of public health and sports medicine, long-term physical activity and active lifestyle are often of great interest, and their effects on wellbeing and risk factors need more clarification 1,5. Well-conducted experimental settings can elucidate the physiological mechanisms behind exercise-induced health benefits, but more studies are needed 6. An acute bout of exercise does not only affect systemic metabolism but can also induce transient changes in the composition and metabolism of the gut microbiome 7. More importantly, increase in habitual physical activity can translate to enhanced microbial diversity and leverage health-beneficial taxa. Consequently, better cardiorespiratory fitness often associates with higher microbial diversity and the abundance of certain exercise-responsive microbial taxa as well 8-10. The gut microbiome contributes to health and disease by producing bioactive compounds such as short-chain fatty acids, trimethylamine oxide, and amino acid derivatives 11. These microbes also utilize a lot of endogenous compounds such as bile acids, amino acids, and lactate 11. Recent studies in mice also indicated specific pathways through which the microbiome-derived metabolites affect motivation to exercise 12. Untargeted metabolomics, sometimes also referred to as global metabolomics 13 or metabolic fingerprinting 14 , aims to characterize large proportions of low-molecular-weight compounds, or metabolites, in a sample matrix in a hypothesis-free manner. As evidenced by the increasing number of studies and new scientific initiatives 13,15,16 ,
Zenodo (CERN European Organization for Nuclear Research), Apr 8, 2020
Metabolomiikka mittaa ja analysoi aineenvaihduntaa Metabolomiikka mittaa ja analysoi sairauden, r... more Metabolomiikka mittaa ja analysoi aineenvaihduntaa Metabolomiikka mittaa ja analysoi sairauden, ruokavalion tai lääkityksen aiheuttamia muutoksia aineenvaihdunnassa. Aineenvaihdunnan eli metabolian aikana syntyy ja hajoaa molekyylejä, joilla osalla on vaikutus myös terveyteen. Niiden pitoisuuksia mitataan verestä, virtsasta ja kudosnäytteistä.
Alcoholism: Clinical and Experimental Research, Sep 30, 2021
Background:Ghrelin may influence several alcohol-related behaviors in animals and humans by modul... more Background:Ghrelin may influence several alcohol-related behaviors in animals and humans by modulating central and/or peripheral biological pathways. The aim of this exploratory analysis was to investigate associations between ghrelin administration and the human circulating metabolome during alcohol exposure in non-treatment seeking, heavy drinking individuals with alcohol use disorder (AUD).Methods:We used serum samples from a randomized, crossover, double-blind, placebo-controlled human laboratory study with intravenous (IV) ghrelin or placebo infusion in two experiments. A loading dose (3 μg/kg) was followed by continuous infusion (16.9 ng/kg/min) of acyl-ghrelin or placebo during each session. The first experiment included an IV alcohol self-administration (IV-ASA) session, and the second experiment included an IV alcohol clamp (IV-AC) session, both under the counterbalanced infusion of ghrelin or placebo. Serum metabolite profiles were analyzed from repeated blood samples collected during each session.Results:In both experiments, ghrelin infusion was associated with altered serum metabolite profile, specifically with significantly increased levels of cortisol (IV-ASA q-value = 0.0003, and IV-AC q < 0.0001), corticosterone (IV-ASA q = 0.0202, and IV-AC q <0.0001), and glycochenodeoxycholic acid (IV-ASA q = 0.0375, and IV-AC q = 0.0013). Furthermore, in the IV-ASA experiment, increased levels of cortisone (q = 0.0352) and fatty acids 18:1 (q = 0.0406) and 18:3 (q = 0.0320) were associated with ghrelin infusion. Moreover, in the IV-AC experiment, levels of glycocholic acid (q < 0.0001) and phenylalanine (q = 0.0458) were significantly increased in relation to ghrelin infusion.Conclusion:IV ghrelin infusion, combined with IV alcohol administration, was associated with changes in the circulating metabolite levels of, among others, corticosteroids and glycine-conjugated bile acids. More studies are required to understand the role that metabolome changes may play in the complex interaction between ghrelin and alcohol.
A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method w... more A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to compare endogenous cannabinoid levels in nematodes and in brains of rats and humans, with and without prior exposure to ethanol. After liquid-liquid extraction of the lipid fraction from homogenized samples, a reversed-phase sub 2 μm column was used for separating analytes with an isocratic mobile phase. Deuterated internal standards were used in the analysis, and detection was made by triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). Ionization was performed with positive electrospray ionization (ESI). The nematode Caenorhabditis elegans fat-3 mutant, that lacks the necessary enzyme to produce arachidonic acid, the biologic precursor to 2-arachidonoyl glycerol and anandamide, was used as an analyte-free surrogate material for selectivity and calibration studies. The matrix effect was further investigated by in-source multiple reaction monitoring (IS-MRM) and standard addition studies. Selectivity studies demonstrated that the method was free from matrix effects. Good accuracy and precision were obtained for concentrations within the calibration range of 0.4-70 nM and 40-11,000 nM for monitored N-acylethanolamides (NAEs) and acyl glycerols, respectively.
Aims/hypothesis Cannabinoid type 1 receptor (CB1R) antagonists such as rimonabant (Rim) represent... more Aims/hypothesis Cannabinoid type 1 receptor (CB1R) antagonists such as rimonabant (Rim) represent a novel approach to treat obesity and related metabolic disorders. Recent data suggest that endocannabinoids are also produced by human adipocytes. Here we studied the potential involvement of endocannabinoids in the negative crosstalk between fat and muscle. Methods The protein level of CB1R in human skeletal muscle cells (SkM) during differentiation was analysed using western blotting. SkM were treated with adipocyteconditioned medium (CM) or anandamide (AEA) in combination with the CB1R antagonists Rim or AM251, and insulin-stimulated Akt phosphorylation and glucose uptake were determined. Furthermore, signalling pathways of CB1R were investigated. Results We revealed an increase of CB1R protein in SkM during differentiation. Twenty-four hour incubation of SkM with CM or AEA impaired insulin-stimulated Akt(Ser473) phosphorylation by 60% and up to 40%, respectively. Pretreatment of cells with Rim or AM251 reduced the effect of CM by about one-half, while the effect of AEA could be prevented completely. The reduction of insulinstimulated glucose uptake by CM was completely prevented by Rim. Short-time incubation with AEA activated extracellular regulated kinase 1/2 and p38 mitogen-activated protein kinase, and impaired insulin-stimulated Akt (Ser473) phosphorylation, but had no effect on Akt (Thr308) and glycogen synthase kinase 3α/β phosphorylation. In addition, enhanced IRS-1 (Ser307) phosphorylation was observed. Conclusions/interpretation Our results show that the CB1R system may play a role in the development of insulin resistance in human SkM. The results obtained with CM support the notion that adipocytes may secrete factors which are able to activate the CB1R. Furthermore, we identified two stress kinases in the signalling pathway of AEA and enhanced IRS-1(Ser307) phosphorylation, potentially underlying the development of insulin resistance. Keywords Adipocyte-conditioned medium. Anandamide. Cannabinoid receptor type 1. Endocannabinoids. Human skeletal muscle cells. Insulin resistance. Rimonabant Abbreviations AEA anandamide 2-AG 2-arachidonoylglycerol AMPKα1 AMP-activated protein kinase alpha 1 CB1R cannabinoid type 1 receptor CM adipocyte-conditioned medium DHEA docosahexaenoyl ethanolamide 2-DOG 2-deoxy-D-[ 14 C]glucose ECL enhanced chemiluminescence ERK1/2 extracellular regulated kinase 1/2
medRxiv (Cold Spring Harbor Laboratory), Apr 28, 2022
doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by pee... more doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 2 Summary NAD + precursor nicotinamide riboside (NR) has emerged as a promising compound to improve obesity-associated mitochondrial dysfunction and metabolic syndrome in mice. However, most short-term clinical trials conducted so far have failed to report positive outcomes. Therefore, we aimed to determine whether long-term NR supplementation boosts mitochondrial biogenesis and metabolic health in humans. Twins from 22 BMI-discordant monozygotic pairs were supplemented with an escalating dose of NR (250-1000 mg/day) for 5 months (clinicaltrials.gov entry NCT03951285). NR improved blood and tissue NAD + metabolism, muscle mitochondrial number, myoblast differentiation and gut microbiota composition independent of BMI. NR also showed a capacity to modulate epigenetic control of gene expression in muscle and adipose tissue. However, NR did not ameliorate adiposity or metabolic health. Overall, our results suggest that NR acts as a potent modifier of NAD + metabolism, muscle mitochondrial biogenesis and stem cell function, gut microbiota, and DNA methylation in humans irrespective of BMI.
Scope: Higher egg intake was previously associated with a lower risk of developing type 2 diabete... more Scope: Higher egg intake was previously associated with a lower risk of developing type 2 diabetes (T2D) in the prospective, population-based Kuopio Ischaemic Heart Disease Risk Factor Study (KIHD) in eastern Finland. We explored potential compounds that could explain this association using non-targeted LC-MS-based metabolic profiling. Methods and results: We analyzed 239 baseline serum samples from the KIHD in 4 groups: subjects with higher (mean intake 1 egg/d) or lower (mean intake 2 eggs/wk) egg intake who developed T2D (cases) or remained heatlhy (controls) during the mean follow-up of 19.3 years. We observed different serum profiles of subjects who had either higher or lower egg intakes, and of those who developed type 2 diabetes or remained healthy. The higher baseline tyrosine level predicted higher odds of T2D (OR 1.94; 95% CI 1.45, 2.60; P<0.001; FDR 0.023) along with an unknown hexose-containing compound (OR 2.13; 95% CI 1.57, 2.88; P<0.001; FDR 0.005). Certain predominant metabolites in T2D cases were correlated positively with ones in lower-egg-intake group and negatively with ones in higher-egg-intake group. Conclusion: Our current findings may underline some potential metabolites that could explain how egg intake was associated with a lower risk of T2D.
ABSTRACT The aim of this study was to characterize pediatric pharmacokinetics and central nervous... more ABSTRACT The aim of this study was to characterize pediatric pharmacokinetics and central nervous system exposure of naproxen after oral administration. The pharmacokinetics of naproxen was studied in 53 healthy children aged 3 months to 12 years undergoing surgery with spinal anesthesia. Children received preoperatively a single dose of 10 mg/kg oral naproxen suspension. A single cerebrospinal fluid (CSF) sample (n = 52) was collected at the induction of anesthesia, and plasma samples (n = 270) were collected before, during, and after the operation (up to 51 hours after administration). A population pharmacokinetic model was built using the NONMEM software. Naproxen concentrations in plasma were well described by a 2-compartment model. The estimated oral clearance (CL/F) was 0.62 L/h when linearly scaled by weight to 70 kg. The apparent volume of distribution at steady state (Vss/F) was 12.5 L /70 kg. The findings are consistent with previously reported pharmacokinetic parameters for children older than 5 years. Naproxen permeated into the CSF and reached CSF concentrations that were 4 times higher than unbound plasma concentrations. Based on these data, weight can be used as a basis for naproxen dosing in children older than 3 months of age.
Scope: Urinary hippuric acid has been proposed as a biomarker for fruit, vegetable and polyphenol... more Scope: Urinary hippuric acid has been proposed as a biomarker for fruit, vegetable and polyphenol consumption. We assessed how serum hippuric acid changes after a bilberryenriched diet (BB; high anthocyanin intake) and another berry diet including strawberries, raspberries and cloudberries (SRC; lower anthocyanin intake) and how these changes associate with insulin and glucose metabolism. Methods and results: Hippuric acid was measured with LC-QTOF-MS metabolite profiling analysis from fasting serum samples at baseline and after an 8-week intervention in 47
medRxiv (Cold Spring Harbor Laboratory), Apr 9, 2021
Objective To characterize serum biomarkers in mitochondrial CHCHD10-linked spinal muscular atroph... more Objective To characterize serum biomarkers in mitochondrial CHCHD10-linked spinal muscular atrophy Jokela type (SMAJ) for disease monitoring and for understanding of pathogenic mechanisms. Methods We collected serum samples from a cohort of 49 SMAJ patients, all carriers of the heterozygous c.197G>T p.G66V variant in CHCHD10. As controls, we used age-and sex-matched serum samples obtained from Helsinki Biobank. Neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) were measured with Single molecule array (Simoa), and fibroblast growth factor 21 (FGF-21) and growth differentiation factor 15 (GDF-15) with enzyme-linked immunosorbent assay. For nontargeted serum metabolite profiling, samples were analyzed with liquid chromatography-high resolution mass spectrometry. Disease severity was evaluated retrospectively by calculating a symptom-based score. Results Axon degeneration marker NfL was unexpectedly not altered in the serum of SMAJ patients, whereas astrocytic activation marker GFAP was slightly decreased. Creatine kinase was elevated in most patients, in particular males. We identified six metabolites that were significantly altered in SMAJ patients' serum compared to controls: increased creatine and pyruvate, and decreased creatinine, taurine, N-acetyl-carnosine and succinate. Creatine correlated with disease severity. Altered pyruvate and succinate indicated a metabolic response to mitochondrial dysfunction, however, lactate or mitochondrial myopathy markers FGF-21 or GDF-15 were not changed. Conclusions Biomarkers of muscle mass and damage are altered in SMAJ serum, indicating a role for skeletal muscle in disease pathogenesis in addition to neurogenic damage. Despite the minimal All rights reserved. No reuse allowed without permission.
Since the discovery that introduction of four specific genes encoding transcription factors conve... more Since the discovery that introduction of four specific genes encoding transcription factors convert mature human somatic cells into induced pluripotent stem cells (iPSCs), there has been an enormous increase in the applications of iPSC technologies to medical sciences, especially in modeling human diseases. In this review, we summarize recent advances in applying human iPSC reprogramming to generate patient-specific neural subtypes in order to reveal molecular pathways affected in various neurodegenerative diseases. Metabolites provide a functional readout of various cellular states enabling identification of biomarker candidates for early diagnosis, segregation of patient cohorts, and to follow-up disease progression or disease responses to novel therapies. With emerging technologies, that is, mass spectrometry and nuclear magnetic resonance applications to metabolomics and various software solutions of bioinformatics, it has become possible to measure thousands of metabolites simultaneously. These fascinating new techniques provide a powerful tool for setting scientific hypotheses and linking cellular pathways to biological mechanism. This review focuses on mass spectrometry-based metabolomics as a tool for iPSC research.
Because the high prevalence of nonalcoholic fatty liver disease sets diagnostic challenges to hea... more Because the high prevalence of nonalcoholic fatty liver disease sets diagnostic challenges to health care, identification of new biomarkers of the disease that in the future could have potential utility as diagnostic biomarkers of high liver fat content is important. Our results show that increased amino acid degradation products in the feces may be such biomarkers.
Journal of Pharmaceutical and Biomedical Analysis, Jun 1, 2022
Hair steroid analysis offers the possibility to evaluate long-term steroid metabolism. It is espe... more Hair steroid analysis offers the possibility to evaluate long-term steroid metabolism. It is especially beneficial when studying the steroid milieu in newborns and children, for example it can provide new insights into steroid metabolism over months and is unaffected by diurnal fluctuations in steroid concentrations. This study describes a quantitative and highly selective high-resolution mass spectrometry method for the simultaneous analysis of multiple steroids from human scalp hair. The developed method utilizes parallel reaction monitoring in the analysis of hydroxylamine derivatized steroids from hair samples first washed with isopropanol, extracted with methanol, and further purified with solid-phase extraction and liquid-liquid extraction. The method was proven suitable for the quantitative analysis of endogenous glucocorticoids (cortisol [1.5-364 pg/mg]; cortisone [3.6-355 pg/mg]; corticosterone [0.7-347 pg/mg]; 11-deoxycortisol [0.1-343 pg/mg]), androgens (testosterone [0.1-288 pg/mg]; dehydroepiandrosterone [0.3-288 pg/mg]; androstenedione [0.1-285 pg/mg]; 11β-hydroxyandrostenedione [0.3-304 pg/mg]), progestogens (pregnenolone [0.1-313 pg/mg]; progesterone [0.1-313 pg/mg]; 17α-hydroxypregnenolone [0.3-315 pg/mg]; 17α-hydroxyprogesterone [0.7-330 pg/mg]; 21-hydroxyprogesterone [0.6-320 pg/mg]), and estrogens (estrone [0.1-267 pg/mg]; estradiol [0.5-274 pg/mg]). In addition, 11-ketoandrostenedione [0.6-60 pg/mg]; dihydrotestosterone [0.3-290 pg/mg]; 11-ketotestosterone [0.1-12 pg/mg]; and 5α-androstanedione [0.6-281 pg/mg] could be analyzed semi-quantitatively. Aldosterone [3.5-346 pg/mg]; 11β-hydroxytestosterone [0.3-300 pg/mg]; and 11-ketodihydrotestosterone [0.3-300 pg/mg] were also included in the method, but their concentrations were below the lower limit of quantification in all tested hair samples. The method was applied for the analysis of authentic hair samples from different age groups ranging from newborns to adults, including mothers within 48 h after delivery. The newborn hair samples displayed the widest variety and had also the highest amounts of steroids in comparison to the samples of the other groups.
Accumulating evidence continues to link certain aspects of the endogenous cannabinoid (EC) system... more Accumulating evidence continues to link certain aspects of the endogenous cannabinoid (EC) system with alcohol dependence, negative reinforcement learning and the modulation of stress response. Specific alterations in brain regions that are related to stress and negative reinforcement learning have been reported to exist in Cloninger type 1 and 2 alcoholics. To study possible differences in profiles of ECs between Cloninger type 1 (n=9), type 2 (n=8) alcoholics and non-alcoholic control subjects (n=10), we analyzed post-mortem amygdala and hippocampus brain samples for several ECs by quantitative liquid chromatography with triple quadrupole mass spectrometric detection. A significant difference was found between these three groups in terms of EC profiles in the amygdala (p = 0.037). In particular, this difference was prominent for variations in docosahexaenoylethanolamine levels, which were significantly higher in type 1 alcoholics (p = 0.022) when compared to controls. There was also a large negative correlation between anandamide concentration and mGlu1/5 receptor density in the hippocampus of Cloninger type 1 alcoholics (R=-0.88, p=0.002), which was not seen in type 2 alcoholics or in controls. Although preliminary, and from relatively small diagnostic groups, these results suggest that the EC profile may be altered in the hippocampus and amygdala of type 1 alcoholics.
European Journal of Pharmaceutics and Biopharmaceutics, May 1, 2012
Sublingually administered midazolam is commonly used for premedication of pediatric patients. How... more Sublingually administered midazolam is commonly used for premedication of pediatric patients. However, the irritating taste and low aqueous solubility of midazolam complicate its sublingual use. Cyclodextrin complexation can be used to improve both the taste and aqueous solubility of drugs, but based on earlier studies, the complexation efficiency of midazolam is relatively low. In the present study, the complexation of midazolam with hydroxypropyl-beta-cyclodextrin (HP-b-CD) was investigated in the presence of various excipients. The aqueous solubility of midazolam improved significantly when HP-b-CD was used together with sucrose. Sucrose alone did not increase the solubility of midazolam. In addition, sucrose increased the apparent stability constant of the midazolam/HP-b-CD complex. The pharmacokinetics of midazolam in different dosage forms was investigated in rabbits (dose 1 mg/rabbit) after intravenous administration of midazolam solution and after sublingual administration of midazolam solution, midazolam/HP-b-CD/sucrose solution or midazolam/HP-b-CD/sucrose powder. Midazolam displayed rapid sublingual absorption (mean t max 6 30 min from the liquid formulations and 60 min from the solid formulation) with high absolute bioavailability (>68%) from all evaluated formulations. Based on the results, HP-b-CD and sucrose can be utilized together to prepare more concentrated and palatable midazolam formulations for sublingual administration in pediatric patients.
Endocannabinoids are the endogenous ligands of the G protein-coupled cannabinoid receptors. The p... more Endocannabinoids are the endogenous ligands of the G protein-coupled cannabinoid receptors. The principal brain endocannabinoid, 2-arachidonoylglycerol (2-AG), is enzymatically produced by postsynaptic neurons and then activates presynaptic CB 1 receptors in a retrograde manner. The primary pathway for 2-AG generation is believed to be conversion from the diacylglycerols (DAGs) by two sn-1-specific lipases, DAGLa and DAGLb. Previous studies with DAGL-deficient mice indicated that DAGLa is the major enzyme needed for retrograde synaptic 2-AG signalling. The current study investigated whether the CB 1 receptormediated G i/o protein activity is altered in brain cryosections of DAGL-deficient mice when compared to wild-type mice and whether the sn-1-specific DAGLs are able to generate 2-AG in brain cryosections. Functional autoradiography indicated that brain regional CB 1 receptor-G i/o-activity largely remained unaltered in DAGLa-knockout and DAGLb-knockout mice when compared to wild-type littermates. Following comprehensive pharmacological blockade of 2-AG hydrolysis, brain sections generated sufficient amounts of 2-AG to activate CB 1 receptors throughout the regions endowed with these receptors. As demonstrated by LC/MS/MS, this pool of 2-AG was generated via tetrahydrolipstatin-sensitive enzymatic pathways distinct from DAGLa or DAGLb. We conclude that in addition to the sn-1-specific DAGLs, additional 2-AG generating enzymatic pathways are active in brain sections.
Membrane transporters have long been utilized to improve the oral, hepatic, and renal (re)absorpt... more Membrane transporters have long been utilized to improve the oral, hepatic, and renal (re)absorption. In the brain, however, the transporter-mediated drug delivery has not yet been fully achieved due to the complexity of the blood−brain barrier (BBB). Because L-type amino acid transporter 1 (LAT1) is a good candidate to improve the brain delivery, we developed here four novel LAT1-utilizing prodrugs of four nonsteroidal anti-inflammatory drugs. As a result, all the prodrugs were able to cross the BBB and localize into the brain cells. The brain uptake of salicylic acid (SA) was improved five times, not only across the mouse BBB but also into the cultured mouse and human brain cells. The naproxen prodrug was also transported efficiently into the mouse brain achieving less peripheral exposure, but the brain release of naproxen from the prodrug was not improved. Contrarily, the high plasma protein binding of the flurbiprofen prodrug and the premature bioconversion of the ibuprofen prodrug in the mouse blood hindered the efficient brain delivery. Thus, the structure of the parent drug affects the successful brain delivery of the LAT1-utilizing prodrugs, and the small-sized LAT1-utilizing prodrug of SA constituted a successful model to specifically deliver its parent drug across the mouse BBB and into the cultured mouse and human brain cells.
The objective of this study was to evaluate the cerebrospinal fluid (CSF) permeation of indometha... more The objective of this study was to evaluate the cerebrospinal fluid (CSF) permeation of indomethacin in healthy children. The participants (n = 31, aged 4–144 months) received indomethacin (0.35 mg/kg) as a 10‐minute intravenous infusion prior to surgery under spinal anaesthesia. A single CSF and plasma sample from each individual was collected 14 to 225 minutes after the infusion. Indomethacin concentrations were determined from the CSF, plasma, and protein‐free plasma. Total plasma, protein‐free plasma, and CSF concentrations of indomethacin ranged between 90 and 2200 ng/mL (median, 780 ng/mL), 0.3 and 0.8 ng/mL (median, 0.5 ng/mL), and 0.2 and 5.0 ng/mL (median, 1.4 ng/mL), respectively. The CSF to plasma concentration ratio remained less than 0.01. There was no correlation between the administration time and CSF concentrations. Eleven children developed 12 nonserious adverse effects, from which 5 were central nervous system (CNS) effects (agitation). In conclusion, indomethacin permeated into the CSF of children, which enables both desired and adverse CNS effects of indomethacin.
The purpose of the study was to see if nematodes (Caenorhabditis elegans, Caenorhabditis briggsae... more The purpose of the study was to see if nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, and Pelodera strongyloides) produce endocannabinoids; i.e., anandamide (AEA) and 2-arachidonoylglycerol (2-AG). In this study, AEA and 2-AG were identified as endogenous products from nematodes by using electrospray-ionization ion-trap MS/MS (ESI-IT-MS) experiments operated in the positive-ionization mode. Endocannabinoids were identified by product ion scan and concentrations were measured by triple quadrupole mass spectrometry in the multiple reaction monitoring mode (MRM). Both AEA and 2-AG were identified in all of the nematode samples, even though these species lack known cannabinoid receptors. Neither AEA nor 2-AG were detected in the fat-3 mutant of C. elegans, which lacks the necessary enzyme to produce arachidonic acid, the fatty acid precursor of these endocannabinoids.
Physical activity is essential in weight management, improves overall health, and mitigates obesi... more Physical activity is essential in weight management, improves overall health, and mitigates obesityrelated risk markers. Besides inducing changes in systemic metabolism, habitual exercise may improve gut's microbial diversity and increase the abundance of beneficial taxa in a correlated fashion. Since there is a lack of integrative omics studies on exercise and overweight populations, we studied the metabolomes and gut microbiota associated with programmed exercise in obese individuals. We measured the serum and fecal metabolites of 17 adult women with overweight during a 6-week endurance exercise program. Further, we integrated the exercise-responsive metabolites with variations in the gut microbiome and cardiorespiratory parameters. We found clear correlation with several serum and fecal metabolites, and metabolic pathways, during the exercise period in comparison to the control period, indicating increased lipid oxidation and oxidative stress. Especially, exercise caused co-occurring increase in levels of serum lyso-phosphatidylcholine moieties and fecal glycerophosphocholine. This signature was associated with several microbial metagenome pathways and the abundance of Akkermansia. The study demonstrates that, in the absence of body composition changes, aerobic exercise can induce metabolic shifts that provide substrates for beneficial gut microbiota in overweight individuals. Physical activity in its various forms is essential in weight management. Habitual exercise can improve overall health and mitigate obesity-related risk markers such as insulin resistance, inflammation, and dyslipidemia 1. Even in the absence of corresponding weight loss, physical activity can lower disease risk and improve overall fitness 2. The alterations in energy balance and systemic metabolism that occur in response to acute exercise are well characterized and documented 3,4. However, within the scope of public health and sports medicine, long-term physical activity and active lifestyle are often of great interest, and their effects on wellbeing and risk factors need more clarification 1,5. Well-conducted experimental settings can elucidate the physiological mechanisms behind exercise-induced health benefits, but more studies are needed 6. An acute bout of exercise does not only affect systemic metabolism but can also induce transient changes in the composition and metabolism of the gut microbiome 7. More importantly, increase in habitual physical activity can translate to enhanced microbial diversity and leverage health-beneficial taxa. Consequently, better cardiorespiratory fitness often associates with higher microbial diversity and the abundance of certain exercise-responsive microbial taxa as well 8-10. The gut microbiome contributes to health and disease by producing bioactive compounds such as short-chain fatty acids, trimethylamine oxide, and amino acid derivatives 11. These microbes also utilize a lot of endogenous compounds such as bile acids, amino acids, and lactate 11. Recent studies in mice also indicated specific pathways through which the microbiome-derived metabolites affect motivation to exercise 12. Untargeted metabolomics, sometimes also referred to as global metabolomics 13 or metabolic fingerprinting 14 , aims to characterize large proportions of low-molecular-weight compounds, or metabolites, in a sample matrix in a hypothesis-free manner. As evidenced by the increasing number of studies and new scientific initiatives 13,15,16 ,
Zenodo (CERN European Organization for Nuclear Research), Apr 8, 2020
Metabolomiikka mittaa ja analysoi aineenvaihduntaa Metabolomiikka mittaa ja analysoi sairauden, r... more Metabolomiikka mittaa ja analysoi aineenvaihduntaa Metabolomiikka mittaa ja analysoi sairauden, ruokavalion tai lääkityksen aiheuttamia muutoksia aineenvaihdunnassa. Aineenvaihdunnan eli metabolian aikana syntyy ja hajoaa molekyylejä, joilla osalla on vaikutus myös terveyteen. Niiden pitoisuuksia mitataan verestä, virtsasta ja kudosnäytteistä.
Alcoholism: Clinical and Experimental Research, Sep 30, 2021
Background:Ghrelin may influence several alcohol-related behaviors in animals and humans by modul... more Background:Ghrelin may influence several alcohol-related behaviors in animals and humans by modulating central and/or peripheral biological pathways. The aim of this exploratory analysis was to investigate associations between ghrelin administration and the human circulating metabolome during alcohol exposure in non-treatment seeking, heavy drinking individuals with alcohol use disorder (AUD).Methods:We used serum samples from a randomized, crossover, double-blind, placebo-controlled human laboratory study with intravenous (IV) ghrelin or placebo infusion in two experiments. A loading dose (3 μg/kg) was followed by continuous infusion (16.9 ng/kg/min) of acyl-ghrelin or placebo during each session. The first experiment included an IV alcohol self-administration (IV-ASA) session, and the second experiment included an IV alcohol clamp (IV-AC) session, both under the counterbalanced infusion of ghrelin or placebo. Serum metabolite profiles were analyzed from repeated blood samples collected during each session.Results:In both experiments, ghrelin infusion was associated with altered serum metabolite profile, specifically with significantly increased levels of cortisol (IV-ASA q-value = 0.0003, and IV-AC q < 0.0001), corticosterone (IV-ASA q = 0.0202, and IV-AC q <0.0001), and glycochenodeoxycholic acid (IV-ASA q = 0.0375, and IV-AC q = 0.0013). Furthermore, in the IV-ASA experiment, increased levels of cortisone (q = 0.0352) and fatty acids 18:1 (q = 0.0406) and 18:3 (q = 0.0320) were associated with ghrelin infusion. Moreover, in the IV-AC experiment, levels of glycocholic acid (q < 0.0001) and phenylalanine (q = 0.0458) were significantly increased in relation to ghrelin infusion.Conclusion:IV ghrelin infusion, combined with IV alcohol administration, was associated with changes in the circulating metabolite levels of, among others, corticosteroids and glycine-conjugated bile acids. More studies are required to understand the role that metabolome changes may play in the complex interaction between ghrelin and alcohol.
A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method w... more A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to compare endogenous cannabinoid levels in nematodes and in brains of rats and humans, with and without prior exposure to ethanol. After liquid-liquid extraction of the lipid fraction from homogenized samples, a reversed-phase sub 2 μm column was used for separating analytes with an isocratic mobile phase. Deuterated internal standards were used in the analysis, and detection was made by triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). Ionization was performed with positive electrospray ionization (ESI). The nematode Caenorhabditis elegans fat-3 mutant, that lacks the necessary enzyme to produce arachidonic acid, the biologic precursor to 2-arachidonoyl glycerol and anandamide, was used as an analyte-free surrogate material for selectivity and calibration studies. The matrix effect was further investigated by in-source multiple reaction monitoring (IS-MRM) and standard addition studies. Selectivity studies demonstrated that the method was free from matrix effects. Good accuracy and precision were obtained for concentrations within the calibration range of 0.4-70 nM and 40-11,000 nM for monitored N-acylethanolamides (NAEs) and acyl glycerols, respectively.
Aims/hypothesis Cannabinoid type 1 receptor (CB1R) antagonists such as rimonabant (Rim) represent... more Aims/hypothesis Cannabinoid type 1 receptor (CB1R) antagonists such as rimonabant (Rim) represent a novel approach to treat obesity and related metabolic disorders. Recent data suggest that endocannabinoids are also produced by human adipocytes. Here we studied the potential involvement of endocannabinoids in the negative crosstalk between fat and muscle. Methods The protein level of CB1R in human skeletal muscle cells (SkM) during differentiation was analysed using western blotting. SkM were treated with adipocyteconditioned medium (CM) or anandamide (AEA) in combination with the CB1R antagonists Rim or AM251, and insulin-stimulated Akt phosphorylation and glucose uptake were determined. Furthermore, signalling pathways of CB1R were investigated. Results We revealed an increase of CB1R protein in SkM during differentiation. Twenty-four hour incubation of SkM with CM or AEA impaired insulin-stimulated Akt(Ser473) phosphorylation by 60% and up to 40%, respectively. Pretreatment of cells with Rim or AM251 reduced the effect of CM by about one-half, while the effect of AEA could be prevented completely. The reduction of insulinstimulated glucose uptake by CM was completely prevented by Rim. Short-time incubation with AEA activated extracellular regulated kinase 1/2 and p38 mitogen-activated protein kinase, and impaired insulin-stimulated Akt (Ser473) phosphorylation, but had no effect on Akt (Thr308) and glycogen synthase kinase 3α/β phosphorylation. In addition, enhanced IRS-1 (Ser307) phosphorylation was observed. Conclusions/interpretation Our results show that the CB1R system may play a role in the development of insulin resistance in human SkM. The results obtained with CM support the notion that adipocytes may secrete factors which are able to activate the CB1R. Furthermore, we identified two stress kinases in the signalling pathway of AEA and enhanced IRS-1(Ser307) phosphorylation, potentially underlying the development of insulin resistance. Keywords Adipocyte-conditioned medium. Anandamide. Cannabinoid receptor type 1. Endocannabinoids. Human skeletal muscle cells. Insulin resistance. Rimonabant Abbreviations AEA anandamide 2-AG 2-arachidonoylglycerol AMPKα1 AMP-activated protein kinase alpha 1 CB1R cannabinoid type 1 receptor CM adipocyte-conditioned medium DHEA docosahexaenoyl ethanolamide 2-DOG 2-deoxy-D-[ 14 C]glucose ECL enhanced chemiluminescence ERK1/2 extracellular regulated kinase 1/2
medRxiv (Cold Spring Harbor Laboratory), Apr 28, 2022
doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by pee... more doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 2 Summary NAD + precursor nicotinamide riboside (NR) has emerged as a promising compound to improve obesity-associated mitochondrial dysfunction and metabolic syndrome in mice. However, most short-term clinical trials conducted so far have failed to report positive outcomes. Therefore, we aimed to determine whether long-term NR supplementation boosts mitochondrial biogenesis and metabolic health in humans. Twins from 22 BMI-discordant monozygotic pairs were supplemented with an escalating dose of NR (250-1000 mg/day) for 5 months (clinicaltrials.gov entry NCT03951285). NR improved blood and tissue NAD + metabolism, muscle mitochondrial number, myoblast differentiation and gut microbiota composition independent of BMI. NR also showed a capacity to modulate epigenetic control of gene expression in muscle and adipose tissue. However, NR did not ameliorate adiposity or metabolic health. Overall, our results suggest that NR acts as a potent modifier of NAD + metabolism, muscle mitochondrial biogenesis and stem cell function, gut microbiota, and DNA methylation in humans irrespective of BMI.
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Papers by Marko Lehtonen