Bidirectional modulation of TCA cycle metabolites and anaplerosis by metformin and its combination with SGLT2i.

BACKGROUND: Metformin and sodium-glucose-cotransporter-2 inhibitors (SGLT2i) are cornerstone therapies for managing hyperglycemia in diabetes. However, their detailed impacts on metabolic processes, particularly within the citric acid (TCA) cycle and its anaplerotic pathways, remain unclear. This study investigates the tissue-specific metabolic effects of metformin, both as a monotherapy and in combination with SGLT2i, on the TCA cycle and associated anaplerotic reactions in both mice and humans. METHODS: Metformin-specific metabolic changes were initially identified by comparing metformin-treated diabetic mice (MET) with vehicle-treated db/db mice (VG). These findings were then assessed in two human cohorts (KORA and QBB) and a longitudinal KORA study of metformin-naïve patients with Type 2 Diabetes (T2D). We also compared MET with db/db mice on combination therapy (SGLT2i + MET). Metabolic profiling analyzed 716 metabolites from plasma, liver, and kidney tissues post-treatment, using linear regression and Bonferroni correction for statistical analysis, complemented by pathway analyses to explore the pathophysiological implications. RESULTS: Metformin monotherapy significantly upregulated TCA cycle intermediates such as malate, fumarate, and α-ketoglutarate (α-KG) in plasma, and anaplerotic substrates including hepatic glutamate and renal 2-hydroxyglutarate (2-HG) in diabetic mice. Downregulated hepatic taurine was also observed. The addition of SGLT2i, however, reversed these effects, such as downregulating circulating malate and α-KG, and hepatic glutamate and renal 2-HG, but upregulated hepatic taurine. In human T2D patients on metformin therapy, significant systemic alterations in metabolites were observed, including increased malate but decreased citrulline. The bidirectional modulation of TCA cycle intermediates in mice influenced key anaplerotic pathways linked to glutaminolysis, tumorigenesis, immune regulation, and antioxidative responses. CONCLUSION: This study elucidates the specific metabolic consequences of metformin and SGLT2i on the TCA cycle, reflecting potential impacts on the immune system. Metformin shows promise for its anti-inflammatory properties, while the addition of SGLT2i may provide liver protection in conditions like metabolic dysfunction-associated steatotic liver disease (MASLD). These observations underscore the importance of personalized treatment strategies.

SoxC transcription factors are required for neuronal differentiation in adult hippocampal neurogenesis.

Neural stem cells (NSCs) generate new hippocampal dentate granule neurons throughout adulthood. The genetic programs controlling neuronal differentiation of adult NSCs are only poorly understood. Here we show that, in the adult mouse hippocampus, expression of the SoxC transcription factors Sox4 and Sox11 is initiated around the time of neuronal commitment of adult NSCs and is maintained in immature neurons. Overexpression of Sox4 and Sox11 strongly promotes in vitro neurogenesis from adult NSCs, whereas ablation of Sox4/Sox11 prevents in vitro and in vivo neurogenesis from adult NSCs. Moreover, we demonstrate that SoxC transcription factors target the promoters of genes that are induced on neuronal differentiation of adult NSCs. Finally, we show that reprogramming of astroglia into neurons is dependent on the presence of SoxC factors. These data identify SoxC proteins as essential contributors to the genetic network controlling neuronal differentiation in adult neurogenesis and neuronal reprogramming of somatic cells.

RBPJkappa-dependent signaling is essential for long-term maintenance of neural stem cells in the adult hippocampus.

The generation of new neurons from neural stem cells in the adult hippocampal dentate gyrus contributes to learning and mood regulation. To sustain hippocampal neurogenesis throughout life, maintenance of the neural stem cell pool has to be tightly controlled. We found that the Notch/RBPJκ-signaling pathway is highly active in neural stem cells of the adult mouse hippocampus. Conditional inactivation of RBPJκ in neural stem cells in vivo resulted in increased neuronal differentiation of neural stem cells in the adult hippocampus at an early time point and depletion of the Sox2-positive neural stem cell pool and suppression of hippocampal neurogenesis at a later time point. Moreover, RBPJκ-deficient neural stem cells displayed impaired self-renewal in vitro and loss of expression of the transcription factor Sox2. Interestingly, we found that Notch signaling increases Sox2 promoter activity and Sox2 expression in adult neural stem cells. In addition, activated Notch and RBPJκ were highly enriched on the Sox2 promoter in adult hippocampal neural stem cells, thus identifying Sox2 as a direct target of Notch/RBPJκ signaling. Finally, we found that overexpression of Sox2 can rescue the self-renewal defect in RBPJκ-deficient neural stem cells. These results identify RBPJκ-dependent pathways as essential regulators of adult neural stem cell maintenance and suggest that the actions of RBPJκ are, at least in part, mediated by control of Sox2 expression.

Validation of Candidate Phospholipid Biomarkers of Chronic Kidney Disease in Hyperglycemic Individuals and Their Organ-Specific Exploration in Leptin Receptor-Deficient db/db Mouse.

Biological exploration of early biomarkers for chronic kidney disease (CKD) in (pre)diabetic individuals is crucial for personalized management of diabetes. Here, we evaluated two candidate biomarkers of incident CKD (sphingomyelin (SM) C18:1 and phosphatidylcholine diacyl (PC aa) C38:0) concerning kidney function in hyperglycemic participants of the Cooperative Health Research in the Region of Augsburg (KORA) cohort, and in two biofluids and six organs of leptin receptor-deficient (db/db) mice and wild type controls. Higher serum concentrations of SM C18:1 and PC aa C38:0 in hyperglycemic individuals were found to be associated with lower estimated glomerular filtration rate (eGFR) and higher odds of CKD. In db/db mice, both metabolites had a significantly lower concentration in urine and adipose tissue, but higher in the lungs. Additionally, db/db mice had significantly higher SM C18:1 levels in plasma and liver, and PC aa C38:0 in adrenal glands. This cross-sectional human study confirms that SM C18:1 and PC aa C38:0 associate with kidney dysfunction in pre(diabetic) individuals, and the animal study suggests a potential implication of liver, lungs, adrenal glands, and visceral fat in their systemic regulation. Our results support further validation of the two phospholipids as early biomarkers of renal disease in patients with (pre)diabetes.

Machine Learning Approaches Reveal Metabolic Signatures of Incident Chronic Kidney Disease in Individuals With Prediabetes and Type 2 Diabetes.

Early and precise identification of individuals with prediabetes and type 2 diabetes (T2D) at risk for progressing to chronic kidney disease (CKD) is essential to prevent complications of diabetes. Here, we identify and evaluate prospective metabolite biomarkers and the best set of predictors of CKD in the longitudinal, population-based Cooperative Health Research in the Region of Augsburg (KORA) cohort by targeted metabolomics and machine learning approaches. Out of 125 targeted metabolites, sphingomyelin C18:1 and phosphatidylcholine diacyl C38:0 were identified as candidate metabolite biomarkers of incident CKD specifically in hyperglycemic individuals followed during 6.5 years. Sets of predictors for incident CKD developed from 125 metabolites and 14 clinical variables showed highly stable performances in all three machine learning approaches and outperformed the currently established clinical algorithm for CKD. The two metabolites in combination with five clinical variables were identified as the best set of predictors, and their predictive performance yielded a mean area value under the receiver operating characteristic curve of 0.857. The inclusion of metabolite variables in the clinical prediction of future CKD may thus improve the risk prediction in people with prediabetes and T2D. The metabolite link with hyperglycemia-related early kidney dysfunction warrants further investigation.

Expression of Sox11 in adult neurogenic niches suggests a stage-specific role in adult neurogenesis.

In the mammalian brain, neural stem and progenitor cells in the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus generate new neurons throughout adulthood. The generation of new functional neurons is a complex process that is tightly controlled by extrinsic signals and that is characterized by stage-specific gene expression programs and cell biological processes. The transcription factors regulating such stage-specific developmental steps in adult neurogenesis are largely unknown. Here we report that Sox11, a member of the group C Sox transcription factor family, is prominently expressed in the neurogenic areas of the adult brain. Further analysis revealed that Sox11 expression is strictly confined to doublecortin-expressing neuronally committed precursors and immature neurons but that Sox11 is not expressed in non-committed Sox2-expressing precursor cells and mature neurons of the adult neurogenic lineage. Finally, overexpression of Sox11 promotes the generation of doublecortin-positive immature neurons from adult neural stem cells in vitro. These data indicate that Sox11 is involved in the transcriptional regulation of specific gene expression programs in adult neurogenesis at the stage of the immature neuron.

Ageing Investigation Using Two-Time-Point Metabolomics Data from KORA and CARLA Studies.

Ageing, one of the largest risk factors for many complex diseases, is highly interconnected to metabolic processes. Investigating the changes in metabolite concentration during ageing among healthy individuals offers us unique insights to healthy ageing. We aim to identify ageing-associated metabolites that are independent from chronological age to deepen our understanding of the long-term changes in metabolites upon ageing. Sex-stratified longitudinal analyses were performed using fasting serum samples of 590 healthy KORA individuals (317 women and 273 men) who participated in both baseline (KORA S4) and seven-year follow-up (KORA F4) studies. Replication was conducted using serum samples of 386 healthy CARLA participants (195 women and 191 men) in both baseline (CARLA-0) and four-year follow-up (CARLA-1) studies. Generalized estimation equation models were performed on each metabolite to identify ageing-associated metabolites after adjusting for baseline chronological age, body mass index, physical activity, smoking status, alcohol intake and systolic blood pressure. Literature researches were conducted to understand their biochemical relevance. Out of 122 metabolites analysed, we identified and replicated five (C18, arginine, ornithine, serine and tyrosine) and four (arginine, ornithine, PC aa C36:3 and PC ae C40:5) significant metabolites in women and men respectively. Arginine decreased, while ornithine increased in both sexes. These metabolites are involved in several ageing processes: apoptosis, mitochondrial dysfunction, inflammation, lipid metabolism, autophagy and oxidative stress resistance. The study reveals several significant ageing-associated metabolite changes with two-time-point measurements on healthy individuals. Larger studies are required to confirm our findings.

Methylation of DNA polymerase beta by protein arginine methyltransferase 1 regulates its binding to proliferating cell nuclear antigen.

DNA polymerase beta (pol beta) is a key player in DNA base excision repair (BER). Here, we describe the complex formation of pol beta with the protein arginine methyltransferase 1 (PRMT1). PRMT1 specifically methylated pol beta in vitro and in vivo. Arginine 137 was identified in pol beta as an important target for methylation by PRMT1. Neither the polymerase nor the dRP-lyase activities of pol beta were affected by PRMT1 methylation. However, this modification abolished the interaction of pol beta with proliferating cell nuclear antigen (PCNA). Together, our results provide evidence that PRMT1 methylation of pol beta might play a regulatory role in BER by preventing the involvement of pol beta in PCNA-dependent DNA metabolic events.

Night Shift Work Affects Urine Metabolite Profiles of Nurses with Early Chronotype.

Night shift work can have a serious impact on health. Here, we assess whether and how night shift work influences the metabolite profiles, specifically with respect to different chronotype classes. We have recruited 100 women including 68 nurses working both, day shift and night shifts for up to 5 consecutive days and collected 3640 spontaneous urine samples. About 424 waking-up urine samples were measured using a targeted metabolomics approach. To account for urine dilution, we applied three methods to normalize the metabolite values: creatinine-, osmolality- and regression-based normalization. Based on linear mixed effect models, we found 31 metabolites significantly (false discovery rate <0.05) affected in nurses working in night shifts. One metabolite, acylcarnitine C10:2, was consistently identified with all three normalization methods. We further observed 11 and 4 metabolites significantly associated with night shift in early and late chronotype classes, respectively. Increased levels of medium- and long chain acylcarnitines indicate a strong impairment of the fatty acid oxidation. Our results show that night shift work influences acylcarnitines and BCAAs, particularly in nurses in the early chronotype class. Women with intermediate and late chronotypes appear to be less affected by night shift work.

DNA-Methylation and Body Composition in Preschool Children: Epigenome-Wide-Analysis in the European Childhood Obesity Project (CHOP)-Study.

Adiposity and obesity result from the interaction of genetic variation and environmental factors from very early in life, possibly mediated by epigenetic processes. Few Epigenome-Wide-Association-Studies have identified DNA-methylation (DNAm) signatures associated with BMI and body composition in children. Body composition by Bio-Impedance-Analysis and genome-wide DNAm in whole blood were assessed in 374 pre-school children from four European countries. Associations were tested by linear regression adjusted for sex, age, centre, education, 6 WBC-proportions according to Houseman and 30 principal components derived from control probes. Specific DNAm variants were identified to be associated with BMI (212), fat-mass (230), fat-free-mass (120), fat-mass-index (24) and fat-free-mass-index (15). Probes in genes SNED1(IRE-BP1), KLHL6, WDR51A(POC1A), CYTH4-ELFN2, CFLAR, PRDM14, SOS1, ZNF643(ZFP69B), ST6GAL1, C3orf70, CILP2, MLLT4 and ncRNA LOC101929268 remained significantly associated after Bonferroni-correction of P-values. We provide novel evidence linking DNAm with (i) altered lipid and glucose metabolism, (ii) diabetes and (iii) body size and composition in children. Both common and specific epigenetic signatures among measures were also revealed. The causal direction with phenotypic measures and stability of DNAm variants throughout the life course remains unclear and longitudinal analysis in other populations is required. These findings give support for potential epigenetic programming of body composition and obesity.

Maternal Smoking during Pregnancy and DNA-Methylation in Children at Age 5.5 Years: Epigenome-Wide-Analysis in the European Childhood Obesity Project (CHOP)-Study.

Mounting evidence links prenatal exposure to maternal tobacco smoking with disruption of DNA methylation (DNAm) profile in the blood of infants. However, data on the postnatal stability of such DNAm signatures in childhood, as assessed by Epigenome Wide Association Studies (EWAS), are scarce. Objectives of this study were to investigate DNAm signatures associated with in utero tobacco smoke exposure beyond the 12th week of gestation in whole blood of children at age 5.5 years, to replicate previous findings in young European and American children and to assess their biological role by exploring databases and enrichment analysis. DNA methylation was measured in blood of 366 children of the multicentre European Childhood Obesity Project Study using the Illumina Infinium HM450 Beadchip (HM450K). An EWAS was conducted using linear regression of methylation values at each CpG site against in utero smoke exposure, adjusted for study characteristics, biological and technical effects. Methylation levels at five HM450K probes in MYO1G (cg12803068, cg22132788, cg19089201), CNTNAP2 (cg25949550), and FRMD4A (cg11813497) showed differential methylation that reached epigenome-wide significance according to the false-discovery-rate (FDR) criteria (q-value<0.05). Whereas cg25949550 showed decreased methylation (-2% DNAm ß-value), increased methylation was observed for the other probes (9%: cg12803068; 5%: cg22132788; 4%: cg19089201 and 4%: cg11813497) in exposed relative to non-exposed subjects. This study thus replicates previous findings in children ages 3 to 5, 7 and 17 and confirms the postnatal stability of MYO1G, CNTNAP2 and FRMD4A differential methylation. The role of this differential methylation in mediating childhood phenotypes, previously associated with maternal smoking, requires further investigation.

Connection between histone H2A variants and chromatin remodeling complexes.

The organization of the eukaryotic genome into chromatin makes it inaccessible to the factors required for gene transcription and DNA replication, recombination, and repair. In addition to histone-modifying enzymes and ATP-dependent chromatin remodeling complexes, which play key roles in regulating many nuclear processes by altering the chromatin structure, cells have developed a mechanism of modulating chromatin structure by incorporating histone variants. These variants are incorporated into specific regions of the genome throughout the cell cycle. H2A.Z, which is an evolutionarily conserved H2A variant, performs several seemingly unrelated and even contrary functions. Another H2A variant, H2A.X, plays a very important role in the cellular response to DNA damage. This review summarizes the recent developments in our understanding of the role of H2A.Z and H2A.X in the regulation of chromatin structure and function, focusing on their functional links with chromatin modifying and remodeling complexes.

Protein arginine methyltransferase 1 coactivates NF-kappaB-dependent gene expression synergistically with CARM1 and PARP1.

Nuclear factor kappa B (NF-kappaB) plays an important role in the transcriptional regulation of genes involved in inflammation and cell survival. Transcriptional coactivators that methylate histones become increasingly important. Recently, we provided evidence that coactivator-associated arginine methyltransferase 1 (CARM1) is a transcriptional coactivator of NF-kappaB and functions as a promoter-specific regulator of NF-kappaB recruitment to chromatin. Here, we show that protein arginine methyltransferase 1 (PRMT1) synergistically coactivates NF-kappaB-dependent gene expression at the macrophage inflammatory protein 2 and human immunodeficiency virus 1 long terminal repeat promoters in concert with the transcriptional coactivators p300/CREB binding protein, CARM1, and poly(ADP-ribose) polymerase 1. PRMT1 formed a complex with poly(ADP-ribose) polymerase 1 and NF-kappaB in vivo and interacted directly with the NF-kappaB subunit p65 in vitro. The methyltransferase activity of PRMT1 appeared essential for its coactivator function in context with CARM1 and p300/CREB binding protein. These results suggest that the cooperative action between PRMT1 and CARM1 is required for NF-kappaB-dependent gene expression.

Arginine methylation regulates DNA polymerase beta.

Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excision repair (BER) corrects damaged bases, apurinic sites, and single-strand DNA breaks. Here, a regulatory mechanism for DNA polymerase beta (Pol beta) is described. Pol beta was found to form a complex with the protein arginine methyltransferase 6 (PRMT6) and was specifically methylated in vitro and in vivo. Methylation of Pol beta by PRMT6 strongly stimulated DNA polymerase activity by enhancing DNA binding and processivity, while single nucleotide insertion and dRP-lyase activity were not affected. Two residues, R83 and R152, were identified in Pol beta as the sites of methylation by PRMT6. Genetic complementation of Pol beta knockout cells with R83/152K mutant revealed the importance of these residues for the cellular resistance to DNA alkylating agent. Based on our findings, we propose that PRMT6 plays a role as a regulator of BER.

Arginine methyltransferase CARM1 is a promoter-specific regulator of NF-kappaB-dependent gene expression.

Nuclear factor kappaB (NF-kappaB) plays an important role in the transcriptional regulation of genes involved in inflammation and cell survival. Here, we show that coactivator-associated arginine methyltransferase CARM1/PRMT4 is a novel transcriptional coactivator of NF-kappaB and functions as a promoter-specific regulator of NF-kappaB recruitment to chromatin. Carm1 knockout cells showed impaired expression of a subset of NF-kappaB-dependent genes upon TNFalpha or LPS stimulation. CARM1 forms a complex with p300 and NF-kappaB in vivo and interacts directly with the NF-kappaB subunit p65 in vitro. CARM1 seems to act in a gene-specific manner mainly by enhancing NF-kappaB recruitment to cognate sites. Moreover, CARM1 synergistically coactivates NF-kappaB-mediated transactivation, in concert with the transcriptional coactivators p300/CREB-binding protein and the p160 family of steroid receptor coactivators. For at least a subset of CARM1-dependent NF-kappaB target genes, the enzymatic activities of both CARM1 and p300 are necessary for the observed synergy between CARM1 and p300. Our results suggest that the cooperative action between protein arginine methyltransferases and protein lysine acetyltransferases regulates NF-kappaB-dependent gene activation in vivo.