Presurgery health influences outcomes following vertical sleeve gastrectomy in adolescents.

OBJECTIVE: Weight loss following vertical sleeve gastrectomy (VSG) in youth can range from 10% to 50%. We examined whether there are differences in demographic or metabolic parameters before VSG in youth who achieve above-average weight loss (AAWL) versus below-average weight loss (BAWL) at 1 year post VSG and if youth with BAWL still achieve metabolic health improvements at 1 year post VSG. METHODS: Demographic, anthropometric, and clinical lab data were collected before VSG and at 1, 3, 6, and 12 months after VSG. RESULTS: Forty-three youth with a mean age of 16.9 (SD 1.7) years before VSG were studied; 70% were female, 19% non-Hispanic Black, 58% non-Hispanic White, and 23% mixed/other race. Mean baseline BMI was 51.1 (SD 10.5) kg/m2. Average weight loss was 25.8%. The AAWL group lost 18.6 kg/m2 (35.3%) versus the BAWL group, who lost 8.8 kg/m2 (17.5%). BMI, age, race, sex, and socioeconomic status at baseline were similar between AAWL and BAWL groups; however, the BAWL group had a higher frequency of pre-VSG dysglycemia, steatotic liver disease, and dyslipidemia. At 1 year post VSG, fewer youth in the BAWL group achieved ideal health parameters, and they had less resolution of comorbidities. CONCLUSIONS: The presence of comorbidities before VSG is associated with less weight loss and reduced resolution of metabolic conditions at 1 year post VSG.

A mitochondrial NADPH-cholesterol axis regulates extracellular vesicle biogenesis to support hematopoietic stem cell fate.

Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal; however, the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here, we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination.

Extranodal NK/T-cell lymphoma with localized relapse in bone marrow of lower leg detected using PET-CT.

Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) is a rare subtype of non-Hodgkin lymphoma (NHL) with poor prognosis, particularly in relapsed or refractory patients. Thus, timely detection of relapse and appropriate disease management are crucial. We present two patients with ENKTL, wherein positron emission tomography-computed tomography (PET-CT) with total-body coverage after induction therapy, detected newly relapsed regions in the bone marrow of the lower leg prior to progression. Case 1: A 47-year-old woman with nasal obstruction, showing 18F-fluoro-deoxyglucose (FDG) uptake in the nasal cavity (Lugano stage IE). After induction therapy (RT-2/3 DeVIC), PET-CT revealed abnormal uptake only in the right fibula. Case 2: A 68-year-old man with a skin nodule/ulcer and an enlarged right inguinal lymph node was diagnosed with advanced ENKTL. A PET-CT scan revealed abnormal uptake in the subcutaneous mass of the right medial thigh, lymph nodes, and descending colon (Lugano stage IV). After induction therapy, PET-CT revealed new abnormal uptake only in the left tibia. In both patients, CT-guided biopsy confirmed ENKTL recurrence. Moreover, PET-CT with whole-body coverage was useful for the timely assessment of relapse and detection of asymptomatic bone involvement. This approach allowed for modifications to treatment strategies in certain patients.

Management of Acquired Factor X Deficiency Caused by In Vitro Non-neutralizing Anti-factor X Autoantibodies with Pre-emptive Corticosteroid Therapy.

Coagulation factor X (FX) deficiency causes severe hemorrhagic symptoms. We herein report a 90-year-old man with hemorrhagic symptoms and prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT). Cross-mixing tests showed a factor deficiency pattern, but administration of plasma products was not effective. Acquired coagulation factor deficiency was suspected, and immunosuppressive therapy was started. After the intervention, his hemorrhagic symptoms improved. A decrease in FX activity was later confirmed, and anti-FX autoantibody was retrospectively detected by an enzyme-linked immunosorbent assay. Immediate intervention is important for patients suspected of having acquired coagulation factor deficiency.

Mechanistic insights into the peroxisome proliferator-activated receptor alpha as a transcriptional suppressor.

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent hepatic disorders that 20-30% of the world population suffers from. The feature of NAFLD is excess lipid accumulation in the liver, exacerbating multiple metabolic syndromes such as hyperlipidemia, hypercholesterolemia, hypertension, and type 2 diabetes. Approximately 20-30% of NAFLD cases progress to more severe chronic hepatitis, known as non-alcoholic steatohepatitis (NASH), showing deterioration of hepatic functions and liver fibrosis followed by cirrhosis and cancer. Previous studies uncovered that several metabolic regulators had roles in disease progression as key factors. Peroxisome proliferator-activated receptor alpha (PPARα) has been identified as one of the main players in hepatic lipid homeostasis. PPARα is abundantly expressed in hepatocytes, and is a ligand-dependent nuclear receptor belonging to the NR1C nuclear receptor subfamily, orchestrating lipid/glucose metabolism, inflammation, cell proliferation, and carcinogenesis. PPARα agonists are expected to be novel prescription drugs for NASH treatment, and some of them (e.g., Lanifibranor) are currently under clinical trials. These potential novel drugs are developed based on the knowledge of PPARα-activating target genes related to NAFLD and NASH. Intriguingly, PPARα is known to suppress the expression of subsets of target genes under agonist treatment; however, the mechanisms of PPARα-mediated gene suppression and functions of these genes are not well understood. In this review, we summarize and discuss the mechanisms of target gene repression by PPARα and the roles of repressed target genes on hepatic lipid metabolism, fibrosis and carcinogenesis related to NALFD and NASH, and provide future perspectives for PPARα pharmaceutical potentials.

[Adult T-cell leukemia/lymphoma diagnosed by RNA in situ hybridization for HTLV-1 bZIP factor].

A 62-year-old man visited the Department of Otorhinolaryngology at our hospital with a chief complaint of a pharyngeal mass. He was admitted to our department with a diagnosis of T-cell lymphoma based on a biopsy of a mesopharyngeal tumor. Although clonality analysis was not performed due to the lack of an appropriate sample, we considered the possibility of lymphoma-type (Lugano classification stage II) adult T-cell leukemia-lymphoma (ATL), as the anti-HTLV-1 antibody was positive. During the course of the disease, the peripheral blood smear revealed atypical lymphocytes with cleaved nuclei, and inverse PCR was performed with DNA extracted from those cells; however, the result showed that the pattern of HTLV-1 proviral DNA integration sites was polyclonal. Further, we performed RNA in situ hybridization targeting HTLV-1 bZIP factor (HBZ-ISH) using the formalin-fixed paraffin-embedded (FFPE) tissue samples of the mesopharyngeal tumor, and a high expression of HBZ was found in the tumor cells, leading to the diagnosis of ATL. These findings suggest the effectiveness of the novel diagnostic method using FFPE tissue samples for ATL.

A Label-Free Electrical Impedance Spectroscopy for Detection of Clusters of Extracellular Vesicles Based on Their Unique Dielectric Properties.

Extracellular vesicles (EVs) have gained considerable attention as vital circulating biomarkers since their structure and composition resemble the originating cells. The investigation of EVs' biochemical and biophysical properties is of great importance to map them to their parental cells and to better understand their functionalities. In this study, a novel frequency-dependent impedance measurement system has been developed to characterize EVs based on their unique dielectric properties. The system is composed of an insulator-based dielectrophoretic (iDEP) device to entrap and immobilize a cluster of vesicles followed by utilizing electrical impedance spectroscopy (EIS) to measure their impedance at a wide frequency spectrum, aiming to analyze both their membrane and cytosolic charge-dependent contents. The EIS was initially utilized to detect nano-size vesicles with different biochemical compositions, including liposomes synthesized with different lipid compositions, as well as EVs and lipoproteins with similar biophysical properties but dissimilar biochemical properties. Moreover, EVs derived from the same parental cells but treated with different culture conditions were characterized to investigate the correlation of impedance changes with biochemical properties and functionality in terms of pro-inflammatory responses. The system also showed the ability to discriminate between EVs derived from different cellular origins as well as among size-sorted EVs harbored from the same cellular origin. This proof-of-concept approach is the first step towards utilizing EIS as a label-free, non-invasive, and rapid sensor for detection and characterization of pathogenic EVs and other nanovesicles in the future.

Hepatic Ago2 Regulates PPARα for Oxidative Metabolism Linked to Glycemic Control in Obesity and Post Bariatric Surgery.

Argonaute 2 (Ago2) is the main component of the RNA-induced silencing complex. We recently showed that liver-specific Ago2-deficiency in mice (L-Ago2 knockout [KO] mice) enhances mitochondrial oxidation and alleviates obesity-associated pathophysiology. However, the precise mechanisms behind the role of hepatic Ago2 in regulating the mitochondrial oxidation associated with glucose metabolism are still unclear. Here, we show that hepatic Ago2 regulates the function of peroxisome proliferator-activated receptor α (PPARα) for oxidative metabolism. In both genetically and diet-induced severe obese conditions, L-Ago2 KO mice developed obesity and hepatic steatosis but exhibited improved glucose metabolism accompanied by lowered expression levels of pathologic microRNAs (miRNAs), including miR-802, miR-103/107, and miR-152, and enhanced expression of PPARα and its target genes regulating oxidative metabolism in the liver. We then investigated the role of hepatic Ago2 in the outcomes of vertical sleeve gastrectomy (VSG) in which PPARα plays a crucial role in a drastic transcription reprogram associated with improved glycemia post VSG. Whereas VSG reduced body weight and improved fatty liver in wild-type mice, these effects were not observed in hepatic Ago2-deficient mice. Conversely, glucose metabolism was improved in a hepatic Ago2-dependent manner post VSG. Treating Ago2-deficient primary hepatocytes with WY-14643, a PPARα agonist, showed that Ago2-deficiency enhances sensitivity to WY-14643 and increases expression of PPARα target genes and mitochondrial oxidation. Our findings suggest that hepatic Ago2 function is intrinsically associated with PPARα that links Ago2-mediated RNA silencing with mitochondrial functions for oxidation and obesity-associated pathophysiology.

Modeling Human Bile Acid Transport and Synthesis in Stem Cell-Derived Hepatocytes with a Patient-Specific Mutation.

The bile salt export pump (BSEP) is responsible for the export of bile acid from hepatocytes. Impaired transcellular transport of bile acids in hepatocytes with mutations in BSEP causes cholestasis. Compensatory mechanisms to regulate the intracellular bile acid concentration in human hepatocytes with BSEP deficiency remain unclear. To define pathways that prevent cytotoxic accumulation of bile acid in hepatocytes, we developed a human induced pluripotent stem cell-based model of isogenic BSEP-deficient hepatocytes in a Transwell culture system. Induced hepatocytes (i-Heps) exhibited defects in the apical export of bile acids but maintained a low intracellular bile acid concentration by inducing basolateral export. Modeling the autoregulation of bile acids on hepatocytes, we found that BSEP-deficient i-Heps suppressed de novo bile acid synthesis using the FXR pathway via basolateral uptake and export without apical export. These observations inform the development of therapeutic targets to reduce the overall bile acid pool in patients with BSEP deficiency.

Adaptive Thermogenesis in Mice Is Enhanced by Opsin 3-Dependent Adipocyte Light Sensing.

Almost all life forms can detect and decode light information for adaptive advantage. Examples include the visual system, in which photoreceptor signals are processed into virtual images, and the circadian system, in which light entrains a physiological clock. Here we describe a light response pathway in mice that employs encephalopsin (OPN3, a 480 nm, blue-light-responsive opsin) to regulate the function of adipocytes. Germline null and adipocyte-specific conditional null mice show a light- and Opn3-dependent deficit in thermogenesis and become hypothermic upon cold exposure. We show that stimulating mouse adipocytes with blue light enhances the lipolysis response and, in particular, phosphorylation of hormone-sensitive lipase. This response is Opn3 dependent. These data establish a key mechanism in which light-dependent, local regulation of the lipolysis response in white adipocytes regulates energy metabolism.

Rapid and label-free isolation of small extracellular vesicles from biofluids utilizing a novel insulator based dielectrophoretic device.

Exosomes are nano-scale membrane-encapsulated vesicles produced by the majority of cells and have emerged as a rich source of biomarkers for a wide variety of diseases. Although many approaches have been developed for exosome isolation from biofluids, most of them have substantial shortcomings including long processing time, inefficiency, high cost, lack of specificity and/or surface marker-dependency. To address these issues, here we report a novel insulator-based dielectrophoretic (iDEP) device predicated on an array of borosilicate micropipettes to rapidly isolate exosomes from conditioned cell culture media and biofluids, such as plasma, serum, and saliva. The device is capable of exosome isolation from small sample volumes of 200 µL within 20 minutes under a relatively low (10 V cm-1) direct current (DC). This device is easy to fabricate thus, no cleanroom facility and expensive equipment are needed. Therefore, the iDEP device offers a rapid and cost-effective strategy for exosome isolation from biofluids in timely manner while maintaining the yield and purity.

RNAs and RNA-Binding Proteins in Immuno-Metabolic Homeostasis and Diseases.

The increasing prevalence of worldwide obesity has emerged as a major risk factor for type 2 diabetes (T2D), hepatosteatosis, and cardiovascular disease. Accumulating evidence indicates that obesity has strong inflammatory underpinnings tightly linked to the development of metabolic diseases. However, the molecular mechanisms by which obesity induces aberrant inflammation associated with metabolic diseases are not yet clearly defined. Recently, RNAs have emerged as important regulators of stress responses and metabolism. RNAs are subject to changes in modification status, higher-order structure, and cellular localization; all of which could affect the affinity for RNA-binding proteins (RBPs) and thereby modify the RNA-RBP networks. Proper regulation and management of RNA characteristics are fundamental to cellular and organismal homeostasis, as well as paramount to health. Identification of multiple single nucleotide polymorphisms (SNPs) within loci of fat mass- and obesity-associated protein (FTO) gene, an RNA demethylase, through genome-wide association studies (GWAS) of T2D, and functional assessments of FTO in mice, support the concept that disruption in RNA modifications leads to the development of human diseases including obesity and metabolic disorder. In obesity, dynamic alterations in modification and localization of RNAs appear to modulate the RNA-RBP networks and activate proinflammatory RBPs, such as double-stranded RNA (dsRNA)-dependent protein kinase (PKR), Toll-like receptor (TLR) 3 and TLR7, and RNA silencing machinery. These changes induce aberrant inflammation and the development of metabolic diseases. This review will describe the current understanding of the underlying causes of these common and altered characteristics of RNA-RBP networks which will pave the way for developing novel approaches to tackle the pandemic issue of obesity.

Extracellular Vesicles: A Potential Novel Regulator of Obesity and Its Associated Complications.

Childhood obesity continues to be a major public health concern. Obesity causes various metabolic complications, including insulin resistance, type 2 diabetes mellitus (T2DM), non-alcoholic fatty liver disease (NAFLD), dyslipidemia, and cardiovascular disease. However, currently, we have a limited understanding of the pathophysiology in the development of these processes. Extracellular vesicles (EVs) are nano-sized vesicles secreted by different cell types that travel to various organ systems carrying molecular and genetic information. These vesicles have been proposed as a novel intercellular communication mode in systemic metabolic regulation and in several pathophysiologic processes. In particular, recent studies indicate that EVs play a critical role in the pathogenesis of obesity and its metabolic complications. In this study, we reviewed the current literature that supports the role of EVs in the regulation of metabolic homeostasis and pathogenesis of obesity and its associated metabolic complications, with a short discussion about future directions in the EV research field.

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Hepatic Ago2-mediated RNA silencing controls energy metabolism linked to AMPK activation and obesity-associated pathophysiology.

RNA silencing inhibits mRNA translation. While mRNA translation accounts for the majority of cellular energy expenditure, it is unclear if RNA silencing regulates energy homeostasis. Here, we report that hepatic Argonaute 2 (Ago2)-mediated RNA silencing regulates both intrinsic energy production and consumption and disturbs energy metabolism in the pathogenesis of obesity. Ago2 regulates expression of specific miRNAs including miR-802, miR-103/107, and miR-148a/152, causing metabolic disruption, while simultaneously suppressing the expression of genes regulating glucose and lipid metabolism, including Hnf1ß, Cav1, and Ampka1. Liver-specific Ago2-deletion enhances mitochondrial oxidation and ATP consumption associated with mRNA translation, which results in AMPK activation, and improves obesity-associated pathophysiology. Notably, hepatic Ago2-deficiency improves glucose metabolism in conditions of insulin receptor antagonist treatment, high-fat diet challenge, and hepatic AMPKα1-deletion. The regulation of energy metabolism by Ago2 provides a novel paradigm in which RNA silencing plays an integral role in determining basal metabolic activity in obesity-associated sequelae.

An Hsp20-FBXO4 Axis Regulates Adipocyte Function through Modulating PPARγ Ubiquitination.

Exposure to cold temperature is well known to upregulate heat shock protein (Hsp) expression and recruit and/or activate brown adipose tissue and beige adipocytes in humans and animals. However, whether and how Hsps regulate adipocyte function for energy homeostatic responses is poorly understood. Here, we demonstrate a critical role of Hsp20 as a negative regulator of adipocyte function. Deletion of Hsp20 enhances non-shivering thermogenesis and suppresses inflammatory responses, leading to improvement of glucose and lipid metabolism under both chow diet and high-fat diet conditions. Mechanistically, Hsp20 controls adipocyte function by interacting with the subunit of the ubiquitin ligase complex, F-box only protein 4 (FBXO4), and regulating the ubiquitin-dependent degradation of peroxisome proliferation activated receptor gamma (PPARγ). Indeed, Hsp20 deficiency mimics and enhances the pharmacological effects of the PPARγ agonist rosiglitazone. Together, our findings suggest a role of Hsp20 in mediating adipocyte function by linking ß-adrenergic signaling to PPARγ activity.

Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models.

Obesity and type 2 diabetes are frequently associated with peripheral neuropathy. Though there are multiple methods for diagnosis and analysis of morphological changes of peripheral nerves and blood vessels, three-dimensional high-resolution imaging is necessary to appreciate the pathogenesis with an anatomically recognizable branching morphogenesis and patterning. Here we established a novel technique for whole-mount imaging of adult mouse ear skin to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels. Whole-mount immunostaining of adult mouse ear skin showed that peripheral sensory and sympathetic nerves align with large-diameter blood vessels. Diet-induced obesity (DIO) mice exhibit defective vascular smooth muscle cells (VSMCs) coverage, while there is no significant change in the amount of peripheral nerves. The leptin receptor-deficient db/db mice, a severe obese and type 2 diabetic mouse model, exhibit defective VSMC coverage and a large increase in the amount of smaller-diameter nerve bundles with myelin sheath and unmyelinated nerve fibers. Interestingly, an increase in the amount of myeloid immune cells was observed in the DIO but not db/db mouse skin. These data suggest that our whole-mount imaging method enables us to investigate the neuro-vascular and neuro-immune phenotypes in the animal models of obesity and diabetes.

Cellular Approaches in Investigating Argonaute2-Dependent RNA Silencing.

In mammals, there are four Argonaute (Ago) family proteins that play crucial roles in RNA silencing, a process wherein microRNA (miRNA) mediates inhibition of target mRNA translation. Among the Ago proteins, Argonaute2 (Ago2) uniquely possesses an endoribonuclease (slicer) activity that is critical for the biogenesis of specific miRNAs and mRNA cleavage. This Ago2 slicer activity is required for postnatal development. Despite its important roles, there are still gaps in our understanding of the mechanistic basis of Ago2's unique functions in vivo due to a limited availability of experimental tools. In order to investigate Ago2's functions, we generated a new cellular model of Ago2-deficiency in 3T3 mouse embryonic fibroblasts (MEFs). This cell line can be used for investigating general Ago2 functions, but also for further understanding of Ago2's unique characteristics including the slicer activity, specific amino acid residues, and domains in Ago2 by reconstitution of Ago2 mutants. Here, we describe the methods for establishing Ago2-deficient MEFs and for reconstituting the MEFs with an Ago2 mutant lacking its slicer activity by means of a retrovirus-mediated gene transfer.

A critical role for PKR complexes with TRBP in Immunometabolic regulation and eIF2α phosphorylation in obesity.

Aberrant stress and inflammatory responses are key factors in the pathogenesis of obesity and metabolic dysfunction, and the double-stranded RNA-dependent kinase (PKR) has been proposed to play an important role in integrating these pathways. Here, we report the formation of a complex between PKR and TAR RNA-binding protein (TRBP) during metabolic and obesity-induced stress, which is critical for the regulation of eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation and c-Jun N-terminal kinase (JNK) activation. We show that TRBP phosphorylation is induced in the setting of metabolic stress, leading to PKR activation. Suppression of hepatic TRBP reduced inflammation, JNK activity, and eIF2α phosphorylation and improved systemic insulin resistance and glucose metabolism, while TRBP overexpression exacerbated the impairment in glucose homeostasis in obese mice. These data indicate that the association between PKR and TRBP integrates metabolism with translational control and inflammatory signaling and plays important roles in metabolic homeostasis and disease.