BACKGROUND Diagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG.METHODS Using multistep mass spectrometry–based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag–based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion.RESULTS Of the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and increase in truncated, mannose-rich, and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man5GlcNAc2, which was not detected in controls, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2.CONCLUSION Complement C4–derived Man5GlcNAc2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs.FUNDING U54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance)
Kishore Garapati, Rohit Budhraja, Mayank Saraswat, Jinyong Kim, Neha Joshi, Gunveen S. Sachdeva, Anu Jain, Anna N. Ligezka, Silvia Radenkovic, Madan Gopal Ramarajan, Savita Udainiya, Kimiyo Raymond, Miao He, Christina Lam, Austin Larson, Andrew C. Edmondson, Kyriakie Sarafoglou, Nicholas B. Larson, Hudson H. Freeze, Matthew J. Schultz, Tamas Kozicz, Eva Morava, Akhilesh Pandey
Fibroblast growth factor 15/19 (FGF15/19, mouse/human ortholog) is expressed in the ileal enterocytes of the small intestine and released postprandially in response to bile acid absorption. Previous reports of FGF15–/– mice have limited our understanding of gut-specific FGF15’s role in metabolism. Therefore, we studied the role of endogenous gut-derived FGF15 in bile acid, cholesterol, glucose, and energy balance. We found that circulating levels of FGF19 were reduced in individuals with obesity and comorbidities, such as type 2 diabetes and metabolic dysfunction–associated fatty liver disease. Gene expression analysis of ileal FGF15-positive cells revealed differential expression during the obesogenic state. We fed standard chow or a high-fat metabolic dysfunction-associated steatohepatitis–inducing diet to control and intestine-derived FGF15-knockout (FGF15INT-KO) mice. Control and FGF15INT-KO mice gained similar body weight and adiposity and did not show genotype-specific differences in glucose, mixed meal, pyruvate, and glycerol tolerance. FGF15INT-KO mice had increased systemic bile acid levels but decreased cholesterol levels, pointing to a primary role for gut-derived FGF15 in regulating bile acid and cholesterol metabolism when exposed to obesogenic diet. These studies show that intestinal FGF15 plays a specific role in bile acid and cholesterol metabolism regulation but is not essential for energy and glucose balance.
Nadejda Bozadjieva-Kramer, Jae Hoon Shin, Ziru Li, Alan C. Rupp, Nicole Miller, Stace Kernodle, Nicolas Lanthier, Paulina Henry, Nikhil Seshadri, Andriy Myronovych, Ormond A. MacDougald, Robert W. O’Rourke, Rohit Kohli, Charles F. Burant, Amy E. Rothberg, Randy J. Seeley
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease associated with cardiomyopathy. DMD-cardiomyopathy is characterized by abnormal intracellular Ca2+ homeostasis and mitochondrial dysfunction. We used dystrophin and utrophin null (mdx:utrn–/–) mice in sarcolipin (SLN) heterozygous knockout (sln+/–) background to examine the effect of SLN reduction on mitochondrial function in the dystrophic myocardium. Germline reduction of SLN expression in mdx:utrn–/– mice improved cardiac sarco/endoplasmic reticulum (SR) Ca2+ cycling, reduced cardiac fibrosis, and improved cardiac function. At the cellular level, reducing SLN expression prevented mitochondrial Ca2+ overload, reduced mitochondrial membrane potential loss, and improved mitochondrial function. Transmission electron microscopy of myocardial tissues and proteomic analysis of mitochondria-associated membranes show that reducing SLN expression improved mitochondrial structure and SR-mitochondria interactions in dystrophic cardiac myocytes. These findings indicate that SLN upregulation plays a significant role in the pathogenesis of cardiomyopathy and that reducing SLN expression has clinical implications in the treatment of DMD-cardiomyopathy.
Satvik Mareedu, Nadezhda Fefelova, Cristi L. Galindo, Goutham Prakash, Risa Mukai, Junichi Sadoshima, Lai-Hua Xie, Gopal J. Babu
Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal β-oxidation when mitochondrial FAO is impaired.
Safaa Hammoud, Alla Ivanova, Yosuke Osaki, Steven Funk, Haichun Yang, Olga Viquez, Rachel Delgado, Dongliang Lu, Melanie Phillips Mignemi, Jane Tonello, Selene Colon, Louise Lantier, David Wasserman, Benjamin D. Humphreys, Jeffrey Koenitzer, Justin Kern, Mark de Caestecker, Toren Finkel, Agnes Fogo, Nidia Messias, Irfan J. Lodhi, Leslie Gewin
Excessive lipolysis in white adipose tissues (WAT) leads to insulin resistance (IR) and ectopic fat accumulation in insulin-sensitive tissues. However, the impact of Gi-coupled receptors in restraining adipocyte lipolysis through inhibition of cAMP production remained poorly elucidated. Given that the Gi-coupled P2Y13 receptor (P2Y13-R) is a purinergic receptor expressed in WAT, we investigated its role in adipocyte lipolysis and its effect on IR and metabolic dysfunction-associated steatotic liver disease (MASLD). In human, mRNA expression of P2Y13-R in WAT was negatively correlated to adipocytes lipolysis. In mice, adipocytes lacking P2Y13-R displayed higher intracellular cAMP levels, indicating impaired Gi signaling. Consistently, the absence of P2Y13-R was linked to increased lipolysis in adipocytes and WAT explants via hormone-sensitive lipase activation. Metabolic studies indicate that mice lacking P2Y13-R show a greater susceptibility to diet-induced IR, systemic inflammation, and MASLD compared to their wild-type counterparts. Assays conducted on precision-cut liver slices exposed to WAT conditioned medium and on liver-specific P2Y13-R knockdown mice suggested that P2Y13-R activity in WAT protects from hepatic steatosis, independently of liver P2Y13-R expression. In conclusion, our findings support the idea that targeting adipose P2Y13-R activity may represent a pharmacological strategy to prevent obesity-associated disorders, including type 2 diabetes and MASLD.
Thibaut Duparc, Emilia Gore, Guillaume Combes, Diane Beuzelin, Julie Pires Da Silva, Vanessa Bouguetoch, Marie-Adeline Marquès, Ana Velazquez, Nathalie Viguerie, Geneviève Tavernier, Peter Arner, Mikael Rydén, Dominique Langin, Nabil Sioufi, Mohamad Nasser, Cendrine Cabou, Souad Najib, Laurent O. Martinez
Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2 - a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1 knockout cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global knockout of Spns1 caused embryonic lethality between E12.5-E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1 knockout (Spns1-KO) mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1-KO mice affected liver functions and altered the PI3K-AKT signaling pathway. Furthermore, we identified three human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and exhibiting cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.
Hoa T.T. Ha, SiYi Liu, Xuan T.A. Nguyen, Linh K. Vo, Nancy C.P. Leong, Dat T. Nguyen, Shivaranjani Balamurugan, Pei Yen Lim, YaJun Wu, Eunju Seong, Toan Q. Nguyen, Jeongah Oh, Markus R. Wenk, Amaury Cazenave-Gassiot, Zuhal Yapici, Wei-Yi Ong, Margit Burmeister, Long N. Nguyen
Connexin43 (Cx43) is the most abundant gap junction protein present in the mesenchymal lineage. In mature adipocytes, Cx43 mediates white adipose tissue (WAT) “beiging” in response to cold exposure and maintains the mitochondrial integrity of brown adipose tissue (BAT). We found that genetic deletion of Gja1 (Cx43 gene) in cells that give rise to chondro-osteogenic and adipogenic precursors driven by the Dermo1/Twist2 promoter led to lower body adiposity and partial protection against the weight gain and metabolic syndrome induced by a high fat diet (HFD) in both sexes. These protective effects from obesogenic diet were related to increased locomotion, fuel utilization, energy expenditure, non-shivering thermogenesis, and better glucose tolerance in conditionally Gja1 ablated mice. Accordingly, Gja1 mutant mice exhibited reduced adipocyte hypertrophy, partially preserved insulin sensitivity, increased BAT lipolysis and decreased whitening under HFD. This metabolic phenotype was not reproduced with more restricted Gja1 ablation in differentiated adipocytes, suggesting that Cx43 in adipocyte progenitors or other targeted cells restrains energy expenditures and promotes fat accumulation. These results disclose an hitherto unknown action of Cx43 in adiposity, and offer a promising new pharmacologic target for improving metabolic balance in diabetes and obesity.
Seung-Yon Lee, Francesca Fontana, Toshifumi Sugatani, Ignacio Portales Castillo, Giulia Leanza, Ariella Coler-Reilly, Roberto Civitelli
Glucose homeostasis is achieved via complex interactions between the endocrine pancreas and other peripheral tissues and glucoregulatory neurocircuits in the brain that remain incompletely defined. Within the brain, neurons in the hypothalamus appear to play a particularly important role. Consistent with this notion, we report evidence that (pro)renin receptor (PRR) signaling within a subset of tyrosine hydroxylase (TH) neurons located in the hypothalamic paraventricular nucleus (PVNTH neurons) is a physiological determinant of the defended blood glucose level. Specifically, we demonstrate that PRR deletion from PVNTH neurons restores normal glucose homeostasis in mice with diet-induced obesity (DIO). Conversely, chemogenetic inhibition of PVNTH neurons mimics the deleterious effect of DIO on glucose. Combined with our finding that PRR activation inhibits PVNTH neurons, these findings suggest that in mice, (a) PVNTH neurons play a physiological role in glucose homeostasis, (b) PRR activation impairs glucose homeostasis by inhibiting these neurons, and (c) this mechanism plays a causal role in obesity-associated metabolic impairment.
Shiyue Pan, Lucas A.C. Souza, Caleb J. Worker, Miriam E. Reyes Mendez, Ariana Julia B. Gayban, Silvana G. Cooper, Alfredo Sanchez Solano, Richard N. Bergman, Darko Stefanovski, Gregory J. Morton, Michael W. Schwartz, Yumei Feng Earley
AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of alpha, beta, and gamma subunits. The independent functions of the two isoforms of the catalytic alpha subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons such as photoreceptors. Here we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, show altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2 deficient mice display visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor knockout mice. These findings highlight a novel, therapeutically targetable PRKAA2 isoform-specific function of AMPK in regulating photoreceptor metabolism and function through a previously uncharacterized mechanism affecting IMPDH activity.
Tae Jun Lee, Yo Sasaki, Philip A. Ruzycki, Norimitsu Ban, Joseph B. Lin, Hung-Ting Wu, Andrea Santeford, Rajendra S. Apte
BACKGROUND. Obesity is a multi-factorial disease with adverse health implications including insulin resistance (IR). In patients with obesity, the presence of high circulating levels of leptin, deemed hyperleptinemia, is associated with IR. Recent data in mice with diet-induced-obesity (DIO) shows a partial reduction in leptin levels improves IR. Additional animal studies demonstrate IL-4 decreases leptin levels. In rodents, resident adipose tissue (AT) eosinophils (EOS) are the main source of IL-4 and are instrumental in maintaining metabolic homeostasis. A marked reduction in AT-EOS content is observed in animal models of DIO. These observations have not been explored in humans. METHODS. We analyzed AT from individuals with obesity and age-matched lean counterparts for AT-EOS content, IL-4, circulating leptin levels and measures of IR. RESULTS. Our results showed that individuals with obesity (n=15) had a significant reduction in AT-EOS content (P<0.01), decreased AT-IL-4 gene expression (P=0.02), and decreased IL-4 plasma levels (P<0.05) in addition to expected IR (P<0.001) and hyperleptinemia (P<0.01) compared to lean subjects (n=15). AT-EOS content inversely correlated with BMI (P=0.002) and IR (P=0.005). Ex vivo AT explants and in vitro cell culture of primary, human mature adipocytes exposed to either IL-4 or EOS conditioned media produced less leptin (P<0.05). CONCLUSIONS. Our results suggested for IL-4 to act as a link between EOS, AT, and leptin production. Future studies exploring this interaction may identify a new avenue for the treatment of obesity and its complications through amelioration of hyperleptinemia. TRIAL REGISTRATION. Clinicaltrials.gov NCT02378077 & NCT04234295. FUNDING. Dr. Eleanna De Filippis received support by Arizona Department of Health Services, Arizona Biomedical Research Commission (ABRC) (ADHS14-00003606), the Katryn H. and Roger Penske Career Development Award in Endocrinology in Honor of Dr. Ian Hay, and Mayo Foundation, KL2 TR002379-02-01 CTSA UL1 TR002377 NCATS/NIH. Dr. Elizabeth A. Jacobsen received support from NIAID AI132840 and Mayo Foundation
James D. Hernandez, Ting Li, Hamza Ghannam, Cassandra M. Rau, Mia Y. Masuda, James A. Madura, Elizabeth A. Jacobsen, Eleanna De Filippis
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