Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatal-expressed alternatively spliced isoform of NaV1.6 (NaV1.6N), and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small but significant differences in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (WT) channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrated the value of considering molecular and cellular context when investigating SCN8A variants.
Carlos G. Vanoye, Tatiana V. Abramova, Jean-Marc Dekeyser, Nora F. Ghabra, Madeleine J. Oudin, Christopher B. Burge, Ingo Helbig, Christopher H. Thompson, Alfred L. George Jr.
Hypotrichosis is a genetic disorder which characterized by a diffuse and progressive loss of scalp and/or body hair. Nonetheless, the causative genes for several affected individuals remain elusive, and the underlying mechanisms have yet to be fully elucidated. Here, we discovered a dominant variant in ADAM17 gene caused hypotrichosis with woolly hair. Adam17 (p.D647N) knock-in mice model mimicked the hair abnormality in patients. ADAM17 (p.D647N) mutation led to hair follicle stem cells (HFSCs) exhaustion and caused abnormal hair follicles, ultimately resulting in alopecia. Mechanistic studies revealed that ADAM17 binds directly to E3 ubiquitin ligase TRIM47. ADAM17 (p.D647N) variant enhanced the association between ADAM17 and TRIM47, leading to an increase in ubiquitination and subsequent degradation of ADAM17 protein. Furthermore, reduced ADAM17 protein expression affected Notch signaling pathway, impairing the activation, proliferation, and differentiation of HFSCs during hair follicle regeneration. Overexpression of NICD rescued the reduced proliferation ability caused by Adam17 variant in primary fibroblast cells.
Xiaoxiao Wang, Chaolan Pan, Luyao Zheng, Jianbo Wang, Quan Zou, Peiyi Sun, Kaili Zhou, Anqi Zhao, Qiaoyu Cao, Wei He, Yumeng Wang, Ruhong Cheng, Zhirong Yao, Si Zhang, Hui Zhang, Ming Li
Alloreactive memory, unlike naïve, CD8+ T cells resist transplantation tolerance protocols and are a critical barrier to long-term graft acceptance in the clinic. We here show that semi-allogeneic pregnancy successfully reprogrammed memory fetus/graft-specific CD8+ T cells (TFGS) towards hypofunction. Female C57BL/6 mice harboring memory CD8+ T cells generated by the rejection of BALB/c skin grafts and then mated with BALB/c males achieved rates of pregnancy comparable to naive controls. Post-partum fetus/graft-specific CD8+ T cells (TFGS) from skin-sensitized dams upregulated expression of T cell exhaustion (TEX) markers (Tox, Eomes, PD-1, TIGIT, and Lag3). Transcriptional analysis corroborated an enrichment of canonical T exhaustion (TEX) genes in post-partum memory TFGS and additionally, revealed a downregulation of a subset of memory-associated transcripts. Strikingly, pregnancy induced extensive epigenetic modifications of exhaustion- and memory-associated genes in memory TFGS, whereas minimal epigenetic modifications were observed in naive TFGS cells. Finally, post-partum memory TFGS durably expressed the exhaustion-enriched phenotype, and their susceptibility to transplantation tolerance was significantly restored compared to memory TFGS. These findings advance the concept of pregnancy as an epigenetic modulator inducing hypofunction in memory CD8+ T cells that has relevance not only for pregnancy and transplantation tolerance, but also for tumor immunity and chronic infections.
Jared M. Pollard, Grace Hynes, Dengping Yin, Malay Mandal, Fotini Gounari, Maria-Luisa Alegre, Anita S. Chong
Glycogen storage disease type III (GSDIII) is a rare metabolic disorder due to glycogen debranching enzyme (GDE) deficiency. Reduced GDE activity leads to pathological glycogen accumulation responsible for impaired hepatic metabolism and muscle weakness. To date, there is no curative treatment for GSDIII. We previously reported that two distinct dual AAV vectors encoding for GDE were needed to correct liver and muscle in a GSDIII mouse model. Here, we evaluated the efficacy of rapamycin in combination with AAV gene therapy. Simultaneous treatment with rapamycin and a novel dual AAV vector expressing GDE in the liver and muscle resulted in a synergic effect demonstrated at biochemical and functional levels. Transcriptomic analysis confirmed synergy and suggested a putative mechanism based on the correction of lysosomal impairment. In GSDIII mice liver, dual AAV gene therapy combined with rapamycin reduced the impact of the immune response to AAV observed in this disease model. These data provide proof of concept of an approach exploiting the combination of gene therapy and rapamycin to improve efficacy and safety and support clinical translation.
Louisa Jauze, Mallaury Vie, Quentin Miagoux, Lucille Rossiaud, Patrice Vidal, Valle Montalvo-Romeral, Hanadi Saliba, Margot Jarrige, Helene Polveche, Justine Nozi, Pierre-Romain Le Brun, Luca Bocchialini, Amandine Francois, Jeremie Cosette, Jérémy Rouillon, Fanny Collaud, Fanny Bordier, Emilie Bertil-Froidevaux, Christophe Georger, Laetitia Van Wittenberghe, Adeline Miranda, Nathalie Daniele, David Gross, Lucile Hoch, Xavier Nissan, Giuseppe Ronzitti
Diabetes increases the risk of both cardiovascular disease and kidney disease. Notably, most of the excess cardiovascular risk in people with diabetes is in those with kidney disease. Apolipoprotein C3 (APOC3) is a key regulator of plasma triglycerides, and it has recently been suggested to play a role in both type 1 diabetes-accelerated atherosclerosis and kidney disease progression. To investigate if APOC3 plays a role in kidney disease in people with type 2 diabetes, we analyzed plasma levels of APOC3 from the Veterans Affairs Diabetes Trial (VADT). Elevated baseline APOC3 levels predicted a greater loss of renal function. To mechanistically test if APOC3 plays a role in diabetic kidney disease and associated atherosclerosis, we treated BTBR wildtype (WT) and leptin-deficient (OB; diabetic) mice, a model of type 2 diabetes, with an antisense oligonucleotide (ASO) to APOC3 or a control ASO (cASO), all in the setting of human-like dyslipidemia. Silencing APOC3 prevented diabetes-augmented albuminuria, renal glomerular hypertrophy, monocyte recruitment, and macrophage accumulation, partly driven by reduced ICAM1 expression. Furthermore, reduced levels of APOC3 suppressed atherosclerosis associated with diabetes. This suggests that targeting APOC3 might benefit both diabetes-accelerated atherosclerosis and kidney disease.
Jocelyn Cervantes, Juraj Koska, Farah Kramer, Shreeram Akilesh, Charles E. Alpers, Adam E. Mullick, Peter Reaven, Jenny E. Kanter
Juvenile Dermatomyositis (JDM) is one of several childhood-onset autoimmune disorders characterized by a type I interferon response and autoantibodies. Treatment options are limited due to incomplete understanding of how the disease emerges from dysregulated cell states across the immune system. We therefore investigated the blood of JDM patients at different stages of disease activity using single-cell transcriptomics paired with surface protein expression. By immunophenotyping peripheral blood mononuclear cells, we observed skewing of the B cell compartment towards an immature naive state as a hallmark of JDM at diagnosis. Furthermore, we find that these changes in B cells are paralleled by T cell signatures suggestive of Th2-mediated inflammation that persist despite disease quiescence. We applied network analysis to reveal that hyperactivation of the type I interferon response in all immune populations is coordinated with previously masked cell states including dysfunctional protein processing in CD4+ T cells and regulation of cell death programming in NK, CD8+ T cells and gdT cells. Together, these findings unveil the coordinated immune dysregulation underpinning JDM and provide insight into strategies for restoring balance in immune function.
Gabrielle Rabadam, Camilla Wibrand, Emily Flynn, George C. Hartoularos, Yang Sun, Chioma Madubata, Gabriela K. Fragiadakis, Jimmie Ye, Susan Kim, Zev J. Gartner, Marina Sirota, Jessica Neely
Applying advanced molecular profiling together with highly specific targeted therapies offers the possibility to better dissect the mechanisms underlying immune mediated inflammatory diseases such as systemic lupus erythematosus (SLE) in humans. Here we apply a combination of single cell RNA sequencing and T/B cell repertoire analysis to perform an in-depth characterization of molecular changes in the immune-signature upon CD19 CAR T cell-mediated depletion of B cells in SLE patients. The resulting datasets do not only confirm a selective CAR T cell-mediated reset of the B cell response, but simultaneously reveal consequent changes in the transcriptional signature of monocyte and T cell subsets that respond with a profound reduction in type 1 interferon signaling. Our current data thus provide evidence for a causal relationship between the B cell response and the increased interferon signature observed in SLE and additionally demonstrate the usefulness of combining targeted therapies and novel analytic approaches to decipher molecular mechanisms of immune-mediated inflammatory diseases in humans.
Artur Wilhelm, David Chambers, Fabian Müller, Aline Bozec, Ricardo Grieshaber-Bouyer, Thomas Winkler, Dimitrios Mougiakakos, Andreas Mackensen, Georg Schett, Gerhard Krönke
Thermogenesis in beige/brown adipose tissues can be leveraged to combat metabolic disorders such as type 2 diabetes and obesity. The complement system plays pleiotropic roles in metabolic homeostasis and organismal energy balance with canonical effects on immune cells and non-canonical effects on non-immune cells. The adipsin/C3a/C3aR1 pathway stimulates insulin secretion and sustains pancreatic beta cell mass. However, its role in adipose thermogenesis has not been defined. Here, we show that male Adipsin/Cfd knockout mice exhibit increased energy expenditure and white adipose tissue (WAT) browning. In addition, male adipocyte-specific C3aR1 knockout mice exhibit enhanced WAT thermogenesis and increased respiration. In stark contrast, female adipocyte-specific C3aR1 knockout mice display decreased brown fat thermogenesis and are cold intolerant. Female mice express lower levels of Adipsin in thermogenic adipocytes and adipose tissues than males. C3aR1 is also lower in female subcutaneous adipose tissue than males. Collectively, these results reveal sexual dimorphism in the adipsin/C3a/C3aR1 axis in regulating adipose thermogenesis and defense against cold stress. Our findings establish a newly discovered role of the alternative complement pathway in adaptive thermogenesis and highlight sex-specific considerations in potential therapeutic targets for metabolic diseases.
Lunkun Ma, Ankit Gilani, Alfonso Rubio-Navarro, Eric Cortada, Ang Li, Shannon M. Reilly, Liling Tang, James C. Lo
Inhibition of Bruton's tyrosine kinase (BTK) through covalent modifications of its active site (e.g., ibrutinib [IBT]) is a preferred treatment for multiple B cell malignancies. However, IBT-treated patients are more susceptible to invasive fungal infections, although the mechanism is poorly understood. Neutrophils are the primary line of defense against these infections; therefore, we examined the impact of IBT on primary human neutrophil effector activity against Aspergillus fumigatus. IBT significantly impaired the ability of neutrophils to kill A. fumigatus and potently inhibited reactive oxygen species (ROS) production, chemotaxis, and phagocytosis. Importantly, exogenous TNFα fully compensated for defects imposed by IBT and newer-generation BTK inhibitors and restored the ability of neutrophils to contain A. fumigatus hyphal growth. Blocking TNFα did not impact ROS production in healthy neutrophils but prevented exogenous TNFα from rescuing the phenotype of IBT-treated neutrophils. The restorative capacity of TNFα was independent of transcription. Moreover, the addition of TNFα immediately rescued ROS production in IBT-treated neutrophils indicating that TNFα worked through a BTK-independent signaling pathway. Finally, TNFα restored effector activity of primary neutrophils from patients on IBT therapy. Altogether, our data indicate that TNFα rescues the antifungal immunity block imposed by inhibition of BTK in primary human neutrophils.
Diego A. Vargas-Blanco, Olivia W. Hepworth, Kyle J. Basham, Patricia Simaku, Arianne J. Crossen, Kyle D. Timmer, Alex Hopke, Hannah Brown Harding, Steven R. Vandal, Kirstine N. Jensen, Daniel J. Floyd, Jennifer L. Reedy, Christopher Reardon, Michael K. Mansour, Rebecca A. Ward, Daniel Irimia, Jeremy S. Abramson, Jatin M. Vyas
Astrocyte activation is a common feature of neurodegenerative diseases. However, the ways in which dying neurons influence the activity of astrocytes is poorly understood. Receptor interacting protein kinase-3 (RIPK3) signaling has recently been described as a key regulator of neuroinflammation, but whether this kinase mediates astrocytic responsiveness to neuronal death has not yet been studied. Here, we used the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease to show that activation of astrocytic RIPK3 drives dopaminergic cell death and axon damage. Transcriptomic profiling revealed that astrocytic RIPK3 promoted gene expression associated with neuroinflammation and movement disorders, and this coincided with significant engagement of damage associated molecular pattern (DAMP) signaling. In mechanistic experiments, we show that factors released from dying neurons signal through receptor for advanced glycation endproducts (RAGE) to induce astrocytic RIPK3 signaling, which conferred inflammatory and neurotoxic functional activity. These findings highlight a mechanism of neuron-glia crosstalk in which neuronal death perpetuates further neurodegeneration by engaging inflammatory astrocyte activation via RIPK3.
Nydia P. Chang, Evan M. DaPrano, Marissa Lindman, Irving Estevez, Tsui-Wen Chou, Wesley R. Evans, Marialaina Nissenbaum, Micheal McCourt, Diego Alzate, Colm Atkins, Alexander W. Kusnecov, Rafiq Huda, Brian P. Daniels
Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned hIL-6 transgenic NSG mice (NSG+hIL6) reliably support the engraftment of malignant and pre-malignant human plasma cells including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and post-relapse myeloma, plasma cell leukemia, and AL amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells, developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single cell RNA sequencing showed non-malignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma engrafted mice given CAR T-cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results established NSG+hIL6 mice as an effective patient derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.
Zainul S. Hasanali, Alfred L. Garfall, Lisa Burzenski, Leonard D. Shultz, Yan Tang, Siddhant Kadu, Neil C. Sheppard, Wei Liu, Derek Dopkin, Dan T. Vogl, Adam D. Cohen, Adam J. Waxman, Sandra P. Susanibar-Adaniya, Martin Carroll, Edward A. Stadtmauer, David Allman
While sclerostin-neutralizing antibodies (Scl-Ab) transiently stimulate bone formation by activating Wnt signaling in osteoblast lineage cells, they exert sustained inhibition of bone resorption, suggesting an alternate signaling pathway by which Scl-Ab control osteoclast activity. Since sclerostin can activate platelet-derived growth factor receptors (PDGFRs) in osteoblast lineage cells in vitro and PDGFR signaling in these cells induces bone resorption through M-CSF secretion, we hypothesized that the prolonged anti-catabolic effect of Scl-Ab could result from PDGFR inhibition. We show here that inhibition of PDGFR signaling in osteoblast lineage cells is sufficient and necessary to mediate prolonged Scl-Ab effect on M-CSF secretion and osteoclast activity in mice. Indeed, sclerostin co-activates PDGFRs independently of Wnt/β-catenin signaling inhibition, by forming a ternary complex with LRP6 and PDGFRs in pre-osteoblasts. In turn, Scl-Ab prevents sclerostin-mediated co-activation of PDGFR signaling and consequent M-CSF up-regulation in pre-osteoblast cultures, thereby inhibiting osteoclast activity in pre-osteoblast/osteoclast co-culture assays. These results provide a new potential mechanism explaining the dissociation between anabolic and anti-resorptive effects of long-term Scl-Ab.
Cyril Thouverey, Pierre Apostolides, Julia Brun, Joseph Caverzasio, Serge Ferrari
The homeostasis of immunoglobulin G (IgG) is maintained by the neonatal Fc receptor, FcRn. Consequently, antagonism of FcRn to reduce endogenous IgG levels is an emerging strategy for treating antibody-mediated autoimmune disorders using either FcRn-specific antibodies or an engineered Fc fragment. For certain FcRn-specific antibodies, this approach has resulted in reductions in the levels of serum albumin, the other major ligand transported by FcRn. Cellular and molecular analyses of a panel of FcRn antagonists have been carried out to elucidate the mechanisms leading to their differential effects on albumin homeostasis. These analyses have identified two processes underlying decreases in albumin levels during FcRn blockade: increased degradation of FcRn and competition between antagonist and albumin for FcRn binding. These findings have potential implications for the design of drugs to modulate FcRn function.
Guanglong Ma, Andrew R. Crowley, Liesbeth Heyndrickx, Ilse Rogiers, Eef Parthoens, Jolien Van Santbergen, Raimund J. Ober, Vladimir Bobkov, Hans de Haard, Peter Ulrichts, Erik Hofman, Els Louagie, Bianca Balbino, E. Sally Ward
Activation of brown adipose tissue (BAT) thermogenesis increases energy expenditure and alleviates obesity. Here we discover that histone methyltransferase suppressor of variegation 4-20 homolog 2 (Suv420h2) expression parallels that of Ucp1 in brown and beige adipocytes and that Suv420h2 knockdown significantly reduces, whereas Suv420h2 overexpression significantly increases Ucp1 levels in brown adipocytes. Suv420h2 knockout (H2KO) mice exhibit impaired cold-induced thermogenesis and are prone to diet-induced obesity. In contrast, mice with specific overexpression of Suv420h2 in adipocytes display enhanced cold-induced thermogenesis and are resistant to diet-induced obesity. Further study shows that Suv420h2 catalyzes H4K20 trimethylation at eukaryotic translation initiation factor 4E-binding protein 1 (4e-bp1) promoter, leading to down-regulated expression of 4e-bp1, a negative regulator of the translation initiation complex. This in turn up-regulates PGC1α protein levels, which is associated with increased expression of thermogenic program. We conclude that Suv420h2 is a key regulator of brown/beige adipocyte development and thermogenesis.
Xin Cui, Qiang Cao, Fenfen Li, Jia Jing, Zhixue liu, Xiaosong Yang, Gary J. Schwartz, Liqing Yu, Huidong Shi, Hang shi, Bingzhong Xue
Clinical trials delivering high doses of adeno-associated viruses (AAVs) expressing truncated dystrophin molecules (micro-dystrophins) are underway for individuals with Duchenne muscular dystrophy (DMD). We examined the efficiency and efficacy of this strategy with four micro-dystrophin constructs (three in clinical trials and a variant of the largest clinical construct), in a severe mouse model of DMD, using doses of AAV comparable to those used in the clinical trials. We achieved high levels of micro-dystrophin expression in striated muscle with cardiac expression ~10 fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of the skeletal muscle disease was observed. Surprisingly, a lethal acceleration of cardiac disease progression occurred with two of the micro-dystrophins. The detrimental impact on the heart appears to be caused by the high levels of micro-dystrophin resulting in variable competition (dependent on the design of the micro-dystrophin) between micro-dystrophin and utrophin at the cardiomyocyte membrane. There may also be a contribution from an overloading of protein degradation. The significance of these observations for patients currently being treated with AAV-micro-dystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown. However, it suggests that micro-dystrophin treatments need to avoid excessively high levels of expression in the heart and cardiac function should be carefully monitored in these patients.
Cora C. Hart, Young il Lee, Jun Xie, Guangping Gao, Brian L. Lin, David W. Hammers, H. Lee Sweeney
Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, the cellular source and expression regulation of COL4 in liver diseases is unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA-sequencing showed increased COL4 expression, which was further confirmed via immunofluorescence staining. scRNA-sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a tumor necrosis factor α–nuclear factor–κB dependent manner through an epigenetic mechanism in liver sinusoidal endothelial cells in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB-mediated epigenome editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.
Can Gan, Usman Yaqoob, Jianwen Lu, Man Xie, Abid A. Anwar, Nidhi Jalan-Sakrikar, Sofia Jerez, Tejasav Sehrawat, Amaia Navarro-Corcuera, Enis Kostallari, Nawras W. Habash, Sheng Cao, Vijay H. Shah
A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole sporozoite PfSPZ Vaccine in African infants. Innate immune activation and myeloid signatures at pre-vaccination baseline correlated with protection from Pf parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ Vaccine dose. Machine learning identified spliceosome, proteosome, and resting dendritic cell signatures as pre-vaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline CSP-specific IgG predicted non-protection. Pre-vaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8+ T-cell responses post-vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naïve mice while diminishing the CD8+ T-cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity of whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8+ T cell responses suggest that PfSPZ Vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.
Leetah Senkpeil, Jyoti Bhardwaj, Morgan R. Little, Prasida Holla, Aditi Upadhye, Elizabeth M. Fusco, Phillip A. Swanson II, Ryan E. Wiegand, Michael D. Macklin, Kevin Bi, Barbara J. Flynn, Ayako Yamamoto, Erik L. Gaskin, D. Noah Sather, Adrian L. Oblak, Edward Simpson, Hongyu Gao, W. Nicholas Haining, Kathleen B. Yates, Xiaowen Liu, Tooba Murshedkar, Thomas L. Richie, B. Kim Lee Sim, Kephas Otieno, Simon Kariuki, Xiaoling Xuei, Yunlong Liu, Rafael B. Polidoro, Stephen L. Hoffman, Martina Oneko, Laura C. Steinhardt, Nathan W. Schmidt, Robert A. Seder, Tuan M. Tran
Fetuses with growth restriction (FGR) have an early activation of hepatic glucose production (HGP), a hallmark of type 2 diabetes (T2D). Here we used fetal hepatic catheterization to directly measure HGP and substrate flux in an FGR sheep model. We hypothesized that FGR fetuses would have increased hepatic lactate and amino acid uptake to support increased HGP. Indeed, FGR compared to normal (CON) fetuses had increased HGP and activation of gluconeogenic genes. Unexpectedly, hepatic pyruvate output was increased while hepatic lactate and gluconeogenic amino acid uptake rates were decreased in FGR fetal liver. Hepatic oxygen consumption and total substrate uptake rates were lower. In FGR liver tissue, metabolite abundance, 13C-metabolite labeling, enzyme activity, and gene expression support decreased pyruvate oxidation and increased lactate production. Isolated hepatocytes from FGR fetuses had greater intrinsic capacity for lactate-fueled glucose production. FGR livers also had lower energy (ATP) and redox state (NADH:NAD+). Thus, reduced hepatic oxidative metabolism may make carbons available for increased HGP but also produces nutrient and energetic stress in FGR fetal liver. Intrinsic programming of these pathways regulating HGP in the FGR fetus may underlie increased HGP and T2D risk postnatally.
Laura D. Brown, Paul J. Rozance, Dong Wang, Evren C. Eroglu, Randall B. Wilkening, Ashley Solmonson, Stephanie R. Wesolowski
Manganese is an essential yet potentially toxic metal. Initially reported in 2012, mutations in SLC30A10 are the first known inherited cause of manganese excess. SLC30A10 is an apical membrane protein that exports manganese from hepatocytes into bile and from enterocytes into the lumen of the gastrointestinal tract. SLC30A10 deficiency results in impaired gastrointestinal manganese excretion, leading to manganese excess, neurologic deficits, liver cirrhosis, polycythemia, and erythropoietin excess. Neurologic and liver disease are attributed to manganese toxicity. Polycythemia is attributed to erythropoietin excess. The goal of this study was to determine the basis of erythropoietin excess in SLC30A10 deficiency. Here we demonstrate that transcription factors hypoxia-inducible factor 1a (Hif1a) and 2a (Hif2a), key mediators of the cellular response to hypoxia, are both upregulated in livers of Slc30a10-deficient mice. Hepatic Hif2a deficiency corrected erythropoietin expression and polycythemia and attenuated aberrant hepatic gene expression in Slc30a10-deficient mice, while hepatic Hif1a deficiency had no discernible impact. Hepatic Hif2a deficiency also attenuated manganese excess, although the underlying cause of this is not clear at this time. Overall, our results indicate that hepatic HIF2 is a key determinant of pathophysiology in SLC30A10 deficiency and expand our understanding of the contribution of HIFs to human disease.
Milankumar Prajapati, Jared Z. Zhang, Lauren Chiu, Grace S. Chong, Courtney J. Mercadante, Heather L. Kowalski, Bradley S. Delaney, Jessica A Anderson, Shuling Guo, Mariam Aghajan, Thomas B. Bartnikas
Chronic kidney disease (CKD) causes an accumulation of uremic metabolites that negatively impact skeletal muscle function. Tryptophan-derived uremic metabolites are agonists of the aryl hydrocarbon receptor (AHR) which has been shown to be activated in the blood of CKD patients. This study investigated the role of the AHR in skeletal muscle pathology of CKD. Compared to control participants with normal kidney function, AHR-dependent gene expression (CYP1A1 and CYP1B1) was significantly upregulated in skeletal muscle of patients with CKD (P=0.032) and the magnitude of AHR activation was inversely correlated with mitochondrial respiration (P<0.001). In mice with CKD, muscle mitochondrial oxidative phosphorylation (OXPHOS) was significantly impaired and strongly correlated with both the serum level of tryptophan-derived uremic metabolites and AHR activation. Muscle-specific deletion of the AHR significantly improved mitochondrial OXPHOS in male mice with the greatest uremic toxicity (CKD+probenecid) and abolished the relationship between uremic metabolites and OXPHOS. The uremic metabolite-AHR-mitochondrial axis in skeletal muscle was further confirmed using muscle-specific AHR knockdown in C57BL6J that harbour a high-affinity AHR allele, as well as ectopic viral expression of constitutively active mutant AHR in mice with normal renal function. Notably, OXPHOS changes in AHRmKO mice were only present when mitochondria were fueled by carbohydrates. Further analyses revealed that AHR activation in mice led to significant increases in Pdk4 expression (P<0.05) and phosphorylation of pyruvate dehydrogenase enzyme (P<0.05). These findings establish a uremic metabolite-AHR-Pdk4 axis in skeletal muscle that governs mitochondrial deficits in carbohydrate oxidation during CKD.
Trace Thome, Nicholas A. Vugman, Lauren E. Stone, Keon Wimberly, Salvatore T. Scali, Terence E. Ryan