Unique features of ß-cell metabolism are lost in type 2 diabetes.

Pancreatic ß cells play an essential role in the control of systemic glucose homeostasis as they sense blood glucose levels and respond by secreting insulin. Upon stimulating glucose uptake in insulin-sensitive tissues post-prandially, this anabolic hormone restores blood glucose levels to pre-prandial levels. Maintaining physiological glucose levels thus relies on proper ß-cell function. To fulfill this highly specialized nutrient sensor role, ß cells have evolved a unique genetic program that shapes its distinct cellular metabolism. In this review, the unique genetic and metabolic features of ß cells will be outlined, including their alterations in type 2 diabetes (T2D). ß cells selectively express a set of genes in a cell type-specific manner; for instance, the glucose activating hexokinase IV enzyme or Glucokinase (GCK), whereas other genes are selectively "disallowed", including lactate dehydrogenase A (LDHA) and monocarboxylate transporter 1 (MCT1). This selective gene program equips ß cells with a unique metabolic apparatus to ensure that nutrient metabolism is coupled to appropriate insulin secretion, thereby avoiding hyperglycemia, as well as life-threatening hypoglycemia. Unlike most cell types, ß cells exhibit specialized bioenergetic features, including supply-driven rather than demand-driven metabolism and a high basal mitochondrial proton leak respiration. The understanding of these unique genetically programmed metabolic features and their alterations that lead to ß-cell dysfunction is crucial for a comprehensive understanding of T2D pathophysiology and the development of innovative therapeutic approaches for T2D patients.

Low sphingolipid levels predict poor survival in patients with alcohol-related liver disease.

Background & Aims: Alcohol-related hepatitis (AH) and alcohol-related cirrhosis are grave conditions with poor prognoses. Altered hepatic lipid metabolism can impact disease development and varies between different alcohol-related liver diseases. Therefore, we aimed to investigate lipidomics and metabolomics at various stages of alcohol-related liver diseases and their correlation with survival. Methods: Patients with newly diagnosed alcohol-related cirrhosis, who currently used alcohol (ALC-A), stable outpatients with decompensated alcohol-related cirrhosis with at least 8 weeks of alcohol abstinence (ALC), and patients with AH, were compared with each other and with healthy controls (HC). Circulating lipids and metabolites were analysed using HPLC and mass spectrometry. Results: Forty patients with ALC, 95 with ALC-A, 30 with AH, and 42 HC provided plasma. Lipid levels changed according to disease severity, with generally lower levels in AH and cirrhosis than in the HC group; this was most pronounced for AH, followed by ALC-A. Nine out of 10 free fatty acids differed between cirrhosis groups by relative increases of 0.12-0.66 in ALC compared with the ALC-A group (p <0.0005). For metabolomics, total bile acids increased by 19.7, 31.3, and 80.4 in the ALC, ALC-A, and AH groups, respectively, compared with HC (all p <0.0001). Low sphingolipid ([d42:1] and [d41:1]) levels could not predict 180-day mortality (AUC = 0.73, p = 0.95 and AUC = 0.73, p = 0.95) more accurately than the model for end-stage liver disease score (AUC = 0.71), but did predict 90-day mortality (AUC d42:1 = 0.922, AUC d41:1 = 0.893; pd42:1 = 0.005, pd41:1 = 0.007) more accurately than the MELD score AUCMELD = 0.70, pMELD = 0.19). Conclusions: Alcohol-related severe liver disease is characterised by low lipid levels progressing with severity of liver disease, especially low sphingomyelins, which also associate to poor prognoses. Impact and implications: Lipidomics has the potential to diagnose and risk stratify patients with liver diseases. Lipidomics differed between patients with alcohol-related hepatitis and alcohol-related cirrhosis with and without recent alcohol use. Furthermore, lipidomics could predict short-term mortality and might be suitable as a prognostic tool in the future. Clinical Trials Registration: Scientific Ethics Committee of the Capital Region of Denmark, journal no. H-21013476.

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.

Atorvastatin for patients with cirrhosis. A randomized, placebo-controlled trial.

BACKGROUND: Patients with cirrhosis and portal hypertension face a high risk of complications. Besides their anti-inflammatory and antifibrotic effects, statins may reduce portal pressure and thus the risk of complications and mortality. We aimed to investigate the effects of atorvastatin on hospital admissions, mortality, inflammation, and lipidomics in cirrhosis with portal hypertension. METHODS: We performed a double-blinded, randomized, placebo-controlled clinical trial among patients with cirrhosis and portal hypertension. Atorvastatin (10-20 mg/d) was administered for 6 months. We measured splanchnic hemodynamics, analyzed inflammatory markers, and performed lipidomics at baseline and after 6 months. RESULTS: Seventy-eight patients were randomized, with 38 patients allocated to atorvastatin and 40 patients to placebo. Fifty-nine patients completed 6 months of intervention. Comparisons between changes in each group were calculated. Liver-related complications and mortality were similar between the groups. The HVPG and Model for End-stage Liver Disease score did not change between groups (p=0.95 and 0.87, respectively). Atorvastatin decreased 3 of 42 inflammatory markers, CD62-L-selectin, matrix metalloproteinases-2, and TNF-α (p-values: 0.005, 0.011, and 0.023, respectively), while lipidomics was not significantly changed. CONCLUSIONS: In patients with cirrhosis, atorvastatin was safe to use, but did not reduce mortality, the risk of liver-related complications, or the HVPG. Atorvastatin induced minor anti-inflammatory effects and minor effects on lipids during a 6-month treatment period.

Scalable generation of functional human iPSC-derived CAR-macrophages that efficiently eradicate CD19-positive leukemia.

BACKGROUND: Macrophages have recently become attractive therapeutics in cancer immunotherapy. The potential of macrophages to infiltrate and influence solid malignancies makes them promising targets for the chimeric antigen receptor (CAR) technology to redirect their stage of polarization, thus enhancing their anticancer capacities. Given the emerging interest for CAR-macrophages, generation of such cells so far mainly depends on peripheral blood monocytes, which are isolated from the respective donor prior to genetic manipulation. This procedure is time-intensive and cost-intensive, while, in some cases, insufficient monocyte amounts can be recovered from the donor, thus hampering the broad applicability of this technology. Hence, we demonstrate the generation and effectiveness of CAR-macrophages from various stem cell sources using also modern upscaling technologies for next generation immune cell farming. METHODS: Primary human hematopoietic stem and progenitor cells and induced pluripotent stem cells were used to derive anti-CD19 CAR-macrophages. Anticancer activity of the cells was demonstrated in co-culture systems, including primary material from patients with leukemia. Generation of CAR-macrophages was facilitated by bioreactor technologies and single-cell RNA (scRNA) sequencing was used to characterize in-depth response and behavior of CAR-macrophages. RESULTS: Irrespective of the stem-cell source, CAR-macrophages exhibited enhanced and antigen-dependent phagocytosis of CD19+ target cancer cells with increased pro-inflammatory responses. Phagocytic capacity of CAR-macrophages was dependent on target cell CD19 expression levels with superior function of CAR-macrophages against CD19+ cancer cell lines and patient-derived acute lymphocytic leukemia cancer cells. scRNA sequencing revealed CAR-macrophages to be distinct from eGFP control cells after co-culture with target cells, which includes the activation of pro-inflammatory pathways and upregulation of chemokines and cytokines associated with adaptive immune cell recruitment, favoring the repolarization of CAR-macrophages to a pro-inflammatory state. Taken together, the data highlight the unique features of CAR-macrophages in combination with the successful upscaling of the production pipeline using a three-dimensional differentiation protocol and intermediate scale bioreactors. CONCLUSION: In summary, our work provides insights into the seminal use and behavior of CAR-macrophages which are derived from various sources of stem cells, while introducing a unique technology for CAR-macrophage manufacturing, all dedicated to the clinical translation of CAR-macrophages within the field of anticancer immunotherapies.

Serotonin receptor 4 agonism prevents high fat diet induced reduction in GLP-1 in mice.

Hormone-producing enteroendocrine cells (EECs) are present throughout the gastrointestinal tract and respond to various nutrient and gut microbiota produced metabolites stimuli. Two important EEC subtypes, Glucagon like peptide-1 (GLP-1) producing L-cells and serotonin (5-HT) producing enterochromaffin (EC) cells interact via paracrine signaling and exhibit bidirectional regulation of expression and secretion of produced hormones. Accordingly, in vitro studies suggest potential to modulate 5-HT secretion by GLP-1 receptor agonism, and L-cell differentiation via serotonin receptor 4 agonism. However, the importance of this cellular signaling on host metabolism is poorly understood. In this study, we found that two weeks of high fat diet (HFD) feeding reduced RNA expression of gut hormones, including proglucagon (Gcg) gene encoding GLP-1 and Tryptophan hydroxylase1 (Tph1) gene encoding rate limiting enzyme in 5-HT synthesis, specifically in the colon and reduced plasma GLP-1 levels. Levels of propionate and butyrate were also reduced following HFD. However, supplementation of sodium propionate did not improve HFD induced reduction in GLP-1. In contrast, chemical induction of serotonin receptor 4 promoted GLP-1 levels, colonic Gcg RNA expression accompanied by improvement in glucose tolerance in HFD-fed mouse. Thus, this study suggests a novel mechanism to improve glucose tolerance via serotonin receptor 4 stimulation in the HFD induced obese mouse model.

Microbial metabolite p-cresol inhibits gut hormone expression and regulates small intestinal transit in mice.

p-cresol is a metabolite produced by microbial metabolism of aromatic amino acid tyrosine. p-cresol and its conjugated forms, p-cresyl sulfate and p-cresyl glucuronide, are uremic toxins that correlate positively with chronic kidney disease and diabetes pathogenesis. However, how p-cresol affects gut hormones is unclear. Here, we expose immortalized GLUTag cells to increasing concentrations of p-cresol and found that p-cresol inhibited Gcg expression and reduced glucagon-like peptide-1 (GLP-1) secretion in vitro. In mice, administration of p-cresol in the drinking water for 2 weeks reduced the transcript levels of Gcg and other gut hormones in the colon; however, it did not affect either fasting or glucose-induced plasma GLP-1 levels. Furthermore, it did not affect glucose tolerance but promoted faster small intestinal transit in mice. Overall, our data suggest that microbial metabolite p-cresol suppresses transcript levels of gut hormones and regulates small intestinal transit in mice.

Glucose Controls Glucagon Secretion by Regulating Fatty Acid Oxidation in Pancreatic α-Cells.

Whole-body glucose homeostasis is coordinated through secretion of glucagon and insulin from pancreatic islets. When glucose is low, glucagon is released from α-cells to stimulate hepatic glucose production. However, the mechanisms that regulate glucagon secretion from pancreatic α-cells remain unclear. Here we show that in α-cells, the interaction between fatty acid oxidation and glucose metabolism controls glucagon secretion. The glucose-dependent inhibition of glucagon secretion relies on pyruvate dehydrogenase and carnitine palmitoyl transferase 1a activity and lowering of mitochondrial fatty acid oxidation by increases in glucose. This results in reduced intracellular ATP and leads to membrane repolarization and inhibition of glucagon secretion. These findings provide a new framework for the metabolic regulation of the α-cell, where regulation of fatty acid oxidation by glucose accounts for the stimulation and inhibition of glucagon secretion. ARTICLE HIGHLIGHTS: It has become clear that dysregulation of glucagon secretion and α-cell function plays an important role in the development of diabetes, but we do not know how glucagon secretion is regulated. Here we asked whether glucose inhibits fatty acid oxidation in α-cells to regulate glucagon secretion. We found that fatty acid oxidation is required for the inhibitory effects of glucose on glucagon secretion through reductions in ATP. These findings provide a new framework for the regulation of glucagon secretion by glucose.

Structural elucidation of 3-nitrophenylhydrazine derivatives of tricarboxylic acid cycle acids and optimization of their fragmentation to boost sensitivity in liquid chromatography-mass spectrometry.

Carboxylic acids participate in many metabolic pathways including tricarboxylic acid (TCA) cycle. Therefore, there have been ongoing attempts to develop sensitive liquid chromatography-mass spectrometry methods over the last decades. Derivatization of the carboxylic acids with 3-nitrophenylhydrazine presents a well-established methodology, and yet the derivatized species of polycarboxylic acids and their fragmentation in collision-induced dissociation have not been fully studied before. In our study, we elucidated how annotation of most abundant 3-nitrophenylhydrazine derivatives and optimization of their fragmentation in multiple reaction monitoring can boost the sensitivity, especially for polycarboxylic acids. Finally, the optimized liquid chromatography-tandem mass spectrometry method allowed for low detection limits ranging from 10 pM for 2-oxoglutaric acid to 800 pM for pyruvic acid. All TCA carboxylates were quantified in 20 µL of human plasma and the targeted method was validated in the same matrix. The same methodology with a modified gradient elution was also applied to untargeted screening of fatty acids by using high-resolution mass spectrometry enabling identification of 29 medium- to long-chain fatty acids in human plasma. The TCA carboxylates were also quantified in 105 of C2C12 mouse myuotube cells grown under different treatments to proof applicability of the methodology to biological studies in a wider sense. However, unfortunately all the TCA carboxylates were also found in the derivatized blanks in substantial amounts, which prevents from using the methodology for quantification of the carboxylates in less than 105 cells.

Targeted Delivery of Butyrate Improves Glucose Homeostasis, Reduces Hepatic Lipid Accumulation and Inflammation in db/db Mice.

Butyrate produced by the gut microbiota has beneficial effects on metabolism and inflammation. Butyrate-producing bacteria are supported by diets with a high fiber content, such as high-amylose maize starch (HAMS). We investigated the effects of HAMS- and butyrylated HAMS (HAMSB)-supplemented diets on glucose metabolism and inflammation in diabetic db/db mice. Mice fed HAMSB had 8-fold higher fecal butyrate concentration compared to control diet-fed mice. Weekly analysis of fasting blood glucose showed a significant reduction in HAMSB-fed mice when the area under the curve for all five weeks was analyzed. Following treatment, fasting glucose and insulin analysis showed increased homeostatic model assessment (HOMA) insulin sensitivity in the HAMSB-fed mice. Glucose-stimulated insulin release from isolated islets did not differ between the groups, while insulin content was increased by 36% in islets of the HAMSB-fed mice. Expression of insulin 2 was also significantly increased in islets of the HAMSB-fed mice, while no difference in expression of insulin 1, pancreatic and duodenal homeobox 1, MAF bZIP transcription factor A and urocortin 3 between the groups was observed. Hepatic triglycerides in the livers of the HAMSB-fed mice were significantly reduced. Finally, mRNA markers of inflammation in liver and adipose tissue were reduced in mice fed HAMSB. These findings suggest that HAMSB-supplemented diet improves glucose metabolism in the db/db mice, and reduces inflammation in insulin-sensitive tissues.

Oak stands along an elevation gradient have different molecular strategies for regulating bud phenology.

BACKGROUND: Global warming raises serious concerns about the persistence of species and populations locally adapted to their environment, simply because of the shift it produces in their adaptive landscape. For instance, the phenological cycle of tree species may be strongly affected by higher winter temperatures and late frost in spring. Given the variety of ecosystem services they provide, the question of forest tree adaptation has received increasing attention in the scientific community and catalyzed research efforts in ecology, evolutionary biology and functional genomics to study their adaptive capacity to respond to such perturbations. RESULTS: In the present study, we used an elevation gradient in the Pyrenees Mountains to explore the gene expression network underlying dormancy regulation in natural populations of sessile oak stands sampled along an elevation cline and potentially adapted to different climatic conditions mainly driven by temperature. By performing analyses of gene expression in terminal buds we identified genes displaying significant dormancy, elevation or dormancy-by-elevation interaction effects. Our Results highlighted that low- and high-altitude populations have evolved different molecular strategies for minimizing late frost damage and maximizing the growth period, thereby increasing potentially their respective fitness in these contrasting environmental conditions. More particularly, population from high elevation overexpressed genes involved in the inhibition of cell elongation and delaying flowering time while genes involved in cell division and flowering, enabling buds to flush earlier were identified in population from low elevation. CONCLUSION: Our study made it possible to identify key dormancy-by-elevation responsive genes revealing that the stands analyzed in this study have evolved distinct molecular strategies to adapt their bud phenology in response to temperature.

Full activation of thermogenesis in brown adipocytes requires Basigin action.

Exploring mechanisms responsible for brown adipose tissue's (BAT) high metabolic activity is crucial to exploit its energy-dissipating ability for therapeutic purposes. Basigin (Bsg), a multifunctional highly glycosylated transmembrane protein, was recently proposed as one of the 98 critical markers allowing to distinguish 'white' and 'brown' adipocytes, yet its function in thermogenic brown adipocytes is unknown. Here, we report that Bsg is negatively associated with obesity in mice. By contrast, Bsg expression increased in the mature adipocyte fraction of BAT upon cold acclimation. Additionally, Bsg levels were highly induced during brown adipocyte maturation in vitro and were further increased upon ß-adrenergic stimulation in a HIF-1α-dependent manner. siRNA-mediated Bsg gene silencing in cultured brown adipocytes did not impact adipogenesis nor mitochondrial function. However, a significant decrease in mitochondrial respiration, lipolysis and Ucp1 transcription was observed in adipocytes lacking Bsg, when activated by norepinephrine. Furthermore, using gas chromatography/mass spectrometry-time-of-flight analysis to assess the composition of cellular metabolites, we demonstrate that brown adipocytes lacking Bsg have lower levels of intracellular lactate and acetoacetate. Bsg was additionally required to regulate intracellular AcAc and tricarboxylic acid cycle intermediate levels in NE-stimulated adipocytes. Our study highlights the critical role of Bsg in active brown adipocytes, possibly by controlling cellular metabolism.

Xylan glucuronic acid side chains fix suberin-like aliphatic compounds to wood cell walls.

Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood. We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L. × tremuloides Michx.) and Arabidopsis (Arabidopsis thaliana (L.) Heynh), to decrease contents of the 4-O-methyl glucuronopyranose acid (mGlcA) substituent of xylan, to elucidate mGlcA's functions. The enzyme affected the content of aliphatic insoluble cell wall components having composition similar to suberin, which required mGlcA for binding to cell walls. Such suberin-like compounds have been previously identified in decayed wood, but here, we show their presence in healthy wood of both hardwood and softwood species. By contrast, γ-ester bonds between mGlcA and lignin were insensitive to cell wall-localized α-glucuronidase, supporting the intracellular formation of these bonds. These findings challenge the current view of the wood cell wall composition and reveal a novel function of mGlcA substituent of xylan in fastening of suberin-like compounds to cell wall. They also suggest an intracellular initiation of lignin-carbohydrate complex assembly.

Type 2 diabetes is associated with increased circulating levels of 3-hydroxydecanoate activating GPR84 and neutrophil migration.

Obesity and diabetes are associated with inflammation and altered plasma levels of several metabolites, which may be involved in disease progression. Some metabolites can activate G protein-coupled receptors (GPCRs) expressed on immune cells where they can modulate metabolic inflammation. Here, we find that 3-hydroxydecanoate is enriched in the circulation of obese individuals with type 2 diabetes (T2D) compared with nondiabetic controls. Administration of 3-hydroxydecanoate to mice promotes immune cell recruitment to adipose tissue, which was associated with adipose inflammation and increased fasting insulin levels. Furthermore, we demonstrate that 3-hydroxydecanoate stimulates migration of primary human and mouse neutrophils, but not monocytes, through GPR84 and Gαi signaling in vitro. Our findings indicate that 3-hydroxydecanoate is a T2D-associated metabolite that increases inflammatory responses and may contribute to the chronic inflammation observed in diabetes.

The human batokine EPDR1 regulates ß-cell metabolism and function.

OBJECTIVE: Ependymin-Related Protein 1 (EPDR1) was recently identified as a secreted human batokine regulating mitochondrial respiration linked to thermogenesis in brown fat. Despite that EPDR1 is expressed in human pancreatic ß-cells and that glucose-stimulated mitochondrial metabolism is critical for stimulus-secretion coupling in ß-cells, the role of EPDR1 in ß-cell metabolism and function has not been investigated. METHODS: EPDR1 mRNA levels in human pancreatic islets from non-diabetic (ND) and type 2 diabetes (T2D) subjects were assessed. Human islets, EndoC-ßH1 and INS1 832/13 cells were transfected with scramble (control) and EPDR1 siRNAs (EPDR1-KD) or treated with human EPDR1 protein, and glucose-stimulated insulin secretion (GSIS) assessed by ELISA. Mitochondrial metabolism was investigated by extracellular flux analyzer, confocal microscopy and mass spectrometry-based metabolomics analysis. RESULTS: EPDR1 mRNA expression was upregulated in human islets from T2D and obese donors and positively correlated to BMI of donors. In T2D donors, EPDR1 mRNA levels negatively correlated with HbA1c and positively correlated with GSIS. EPDR1 silencing in human islets and ß-cell lines reduced GSIS whereas treatment with human EPDR1 protein increased GSIS. Epdr1 silencing in INS1 832/13 cells reduced glucose- and pyruvate- but not K+-stimulated insulin secretion. Metabolomics analysis in Epdr1-KD INS1 832/13 cells suggests diversion of glucose-derived pyruvate to lactate production and decreased malate-aspartate shuttle and the tricarboxylic acid (TCA) cycle activity. The glucose-stimulated rise in mitochondrial respiration and ATP/ADP-ratio was impaired in Epdr1-deficient cells. CONCLUSION: These results suggests that to maintain glucose homeostasis in obese people, upregulation of EPDR1 may improve ß-cell function via channelling glycolysis-derived pyruvate to the mitochondrial TCA cycle.

The PI3K inhibitor pictilisib and the multikinase inhibitors pazopanib and sorafenib have an impact on Rac1 level and migration of medulloblastoma in vitro.

Metastatic disease is the leading cause of death in children suffering from medulloblastoma and a major treatment challenge. The evidence of leptomeningeal dissemination defines the most aggressive tumours and is associated with increased mortality; thus, inhibition of migration as a factor involved in the process of metastatic disease is fundamental for the treatment and prevention of metastatic dissemination. Targeting the small Rho GTPases Rac1 has been shown to effectively impair medulloblastoma cell migration in vitro. Yet clinically applicable selective Rac1 inhibitors are still lacking. In view of the pertinent oncogenic role of the PI3K signalling cascade and tyrosine kinase-mediated signalling pathways in medulloblastoma, we explored clinically available targeted therapeutics to this effect. Here, we show that Rac1 is expressed in both the cytoplasm and nucleus in the medulloblastoma cell lines Daoy and MEB-Med-8A representative of two high risk medulloblastoma entities. We demonstrate that activated Rac1 is subject to substantial downmodulation following administration of the clinically available inhibitor of the PI3K pathway Pictilisib (GDC-0941) and the multityrosine kinase inhibitors Pazopanib and Sorafenib. The application of those drugs was associated with reduced mobility of the medulloblastoma cells and alterations of the actin skeleton. Of note, PI3K inhibition reveals the strongest anti-migratory effect in Daoy cells. Thus, our in vitro observations provide new insights into different strategies of blocking Rac1 and inhibiting migration in medulloblastoma employing clinically available agents paving the way for confirmatory studies in in vivo models.

A randomized placebo-controlled trial of nicotinamide riboside and pterostilbene supplementation in experimental muscle injury in elderly individuals.

BACKGROUNDDuring aging, there is a functional decline in the pool of muscle stem cells (MuSCs) that influences the functional and regenerative capacity of skeletal muscle. Preclinical evidence has suggested that nicotinamide riboside (NR) and pterostilbene (PT) can improve muscle regeneration, e.g., by increasing MuSC function. The objective of this study was to investigate if supplementation with NR and PT (NRPT) promotes skeletal muscle regeneration after muscle injury in elderly individuals by improved recruitment of MuSCs.METHODSThirty-two elderly individuals (55-80 years of age) were randomized to daily supplementation with either NRPT (1,000 mg NR and 200 mg PT) or matched placebo. Two weeks after initiation of supplementation, skeletal muscle injury was induced by electrically induced eccentric muscle work. Skeletal muscle biopsies were obtained before, 2 hours after, and 2, 8, and 30 days after injury.RESULTSA substantial skeletal muscle injury was induced by the protocol and associated with release of myoglobin and creatine kinase, muscle soreness, tissue edema, and a decrease in muscle strength. MuSC content, proliferation, and cell size revealed a large demand for recruitment after injury, but this was not affected by NRPT. Furthermore, histological analyses of muscle fiber area, central nuclei, and embryonic myosin heavy chain showed no NRPT supplementation effect.CONCLUSIONDaily supplementation with 1,000 mg NR and 200 mg PT is safe but does not improve recruitment of the MuSC pool or other measures of muscle recovery in response to injury or subsequent regeneration in elderly individuals.TRIAL REGISTRATIONClinicalTrials.gov NCT03754842.FUNDINGNovo Nordisk Foundation (NNF17OC0027242) and Novo Nordisk Foundation CBMR.

Targeted biallelic integration of an inducible Caspase 9 suicide gene in iPSCs for safer therapies.

Drug-inducible suicide systems may help to minimize risks of human induced pluripotent stem cell (hiPSC) therapies. Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed. We have introduced a drug-inducible Caspase 9 suicide system (iCASP9) into the AAVS1 safe-harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro. After transplantation into mice, drug treatment generally led to rapid elimination of teratomas, but single animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ∼ 3 × 10-8. Besides transgene elimination, presumably via loss of heterozygosity (LoH), silencing via aberrant promoter methylation was identified as a major underlying mechanism. In contrast to monoallelic iCASP9 hiPSCs, no escapees from biallelic iCASP9 cells were observed after treatment of up to 0.8 billion hiPSCs. The highly increased safety level provided by biallelic integration of the iCASP9 system may substantially contribute to the safety level of iPSC-based therapies.