Hypoglycemia is a frequent complication of diabetes, limiting therapy and increasing morbidity and mortality. With recurrent hypoglycemia, the counterregulatory response (CRR) to decreased blood glucose is blunted, resulting in hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to these blunted effects are only poorly understood. Here, we report, with ISH, IHC, and the tissue-clearing capability of iDISCO+, that growth hormone releasing hormone (GHRH) neurons represent a unique population of arcuate nucleus neurons activated by glucose deprivation in vivo. Repeated glucose deprivation reduces GHRH neuron activation and remodels excitatory and inhibitory inputs to GHRH neurons. We show that low glucose sensing is coupled to GHRH neuron depolarization, decreased ATP production, and mitochondrial fusion. Repeated hypoglycemia attenuates these responses during low glucose. By maintaining mitochondrial length with the small molecule mitochondrial division inhibitor-1, we preserved hypoglycemia sensitivity in vitro and in vivo. Our findings present possible mechanisms for the blunting of the CRR, significantly broaden our understanding of the structure of GHRH neurons, and reveal that mitochondrial dynamics play an important role in HAAF. We conclude that interventions targeting mitochondrial fission in GHRH neurons may offer a new pathway to prevent HAAF in patients with diabetes.
Mitchell Bayne, Alexandra Alvarsson, Kavya Devarakonda, Rosemary Li, Maria Jimenez-Gonzalez, Darline Garibay, Kaetlyn Conner, Merina Varghese, Madhavika N. Serasinghe, Jerry E. Chipuk, Patrick R. Hof, Sarah A. Stanley
While autoantibodies are used in the diagnosis of rheumatoid arthritis (RA), the function of B cells in the inflamed joint remains elusive. Extensive flow cytometric characterization and SPICE algorithm analyses of single-cell synovial tissue from patients with RA revealed the accumulation of switched and double-negative memory programmed death-1 receptor–expressing (PD-1–expressing) B cells at the site of inflammation. Accumulation of memory B cells was mediated by CXCR3, evident by the observed increase in CXCR3-expressing synovial B cells compared with the periphery, differential regulation by key synovial cytokines, and restricted B cell invasion demonstrated in response to CXCR3 blockade. Notably, under 3% O2 hypoxic conditions that mimic the joint microenvironment, RA B cells maintained marked expression of MMP-9, TNF, and IL-6, with PD-1+ B cells demonstrating higher expression of CXCR3, CD80, CD86, IL-1β, and GM-CSF than their PD-1– counterparts. Finally, following functional analysis and flow cell sorting of RA PD-1+ versus PD-1– B cells, we demonstrate, using RNA-Seq and emerging fluorescence lifetime imaging microscopy of cellular NAD, a significant shift in metabolism of RA PD-1+ B cells toward glycolysis, associated with an increased transcriptional signature of key cytokines and chemokines that are strongly implicated in RA pathogenesis. Our data support the targeting of pathogenic PD-1+ B cells in RA as a focused, novel therapeutic option.
Achilleas Floudas, Nuno Neto, Viviana Marzaioli, Kieran Murray, Barry Moran, Michael G. Monaghan, Candice Low, Ronan H. Mullan, Navin Rao, Vinod Krishna, Sunil Nagpal, Douglas J. Veale, Ursula Fearon
The habenula (Hb) is a bilateral, evolutionarily conserved epithalamic structure connecting forebrain and midbrain structures that has gained attention for its roles in depression,(1) addiction,(2-5) rewards processing,(6) and motivation (7,8). Of its two major subdivisions, the medial (MHb) and lateral Hb (LHb), MHb circuitry and function is poorly understood relative to LHb (9). Prkar2a codes for cAMP-dependent protein kinase (PKA) regulatory subunit IIα (RIIα), a component of the PKA holoenzyme at the center of one of the major cell-signaling pathways conserved across systems and species. Type 2 regulatory subunits (RIIα, RIIβ) determine the subcellular localization of PKA, and unlike other PKA subunits, Prkar2a has minimal brain expression except in the MHb (10). We previously showed that RIIα knockout (RIIαKO) mice resist diet-induced obesity (DIO) (11). In the present study, we report that RIIαKO mice have decreased consumption of palatable, “rewarding” foods and increased motivation for voluntary exercise. Prkar2a deficiency led to decreased habenular PKA enzymatic activity and impaired dendritic localization of PKA catalytic subunits in MHb neurons. Re-expression of Prkar2a in the Hb rescued this phenotype confirming differential roles for Prkar2a in regulating the drives for palatable foods and voluntary exercise. Our findings show that in the MHb decreased PKA signaling and dendritic PKA activity decrease motivation for food rewards while enhancing the motivation for exercise, a desirable combination of behaviors.
Edra London, Jason C. Wester, Michelle S. Bloyd, Shelby Bettencourt, Chris J. McBain, Constantine A. Stratakis
Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects cholesterol homeostasis by targeting hepatic low-density lipoprotein receptor (LDLR) for lysosomal degradation. Clinically, PCSK9 inhibitors effectively reduce LDL cholesterol (LDL-C) level and the incidence of cardiovascular events. Because microRNAs (miRs) are integral regulators of cholesterol homeostasis, we investigated the involvement of miR-483 in regulating LDL-C metabolism. Using in silico analysis, we predicted that miR-483-5p targets the 3’UTR of PCSK9 mRNA. In HepG2 cells, miR-483-5p targeted the PCSK9 3’UTR, leading to decreased PCSK9 protein and mRNA expression, increased LDLR expression and enhanced LDL-C uptake. In hyperlipidemic mice and humans, serum levels of total cholesterol and LDL-C were inversely correlated with miR-483-5p level. In mice, hepatic miR-483 overexpression increased LDLR level by targeting Pcsk9, with a significant reduction in plasma total cholesterol and LDL-C levels. Mechanistically, the cholesterol-lowering effect of miR-483-5p was significant in mice receiving AAV8 PCSK9-3’UTR but not Ldlr-knockout mice or mice receiving AAV8 PCSK9-3’UTR (deltaBS) with the miR-483-5p targeting site deleted. Thus, exogenously administered miR-483 or similarly optimized compounds have potential to ameliorate hypercholesterolemia.
Jianjie Dong, Ming He, Jie Li, Ariane R. Pessentheiner, Chen Wang, Jin Zhang, Yameng Sun, Wei-Ting Wang, Yuqing Zhang, Junhui Liu, Shen-Chih Wang, Po-Hsun Huang, Philip L.S.M. Gordts, Zu-Yi Yuan, Sotirios Tsimikas, John Y-J Shyy
Phenylalanine hydroxylase–deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6–7. The affected pig exhibited hyperphenylalaninemia (2000–5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.
Erik A. Koppes, Bethany K. Redel, Marie A. Johnson, Kristen J. Skvorak, Lina Ghaloul-Gonzalez, Megan E. Yates, Dale W. Lewis, Susanne M. Gollin, Yijen L. Wu, Shawn E. Christ, Martine Yerle, Angela Leshinski, Lee D. Spate, Joshua A. Benne, Stephanie L. Murphy, Melissa S. Samuel, Eric M. Walters, Sarah A. Hansen, Kevin D. Wells, Uta Lichter-Konecki, Robert A. Wagner, Joseph T. Newsome, Steven F. Dobrowolski, Jerry Vockley, Randall S. Prather, Robert D. Nicholls
Symbiotic microbial colonization through the establishment of the intestinal microbiome is critical to many intestinal functions including nutrient metabolism, intestinal barrier integrity and immune regulation. Recent studies suggest that education of the intestinal immunity maybe ongoing in utero. However, the drivers of this process are unknown. The microbiome and its byproducts are one potential source. Whether a fetal intestinal microbiome exists is controversial and if microbially derived metabolites are present in utero is unknown. Here, we aimed to determine whether bacterial DNA and microbially-derived metabolites can be detected in second trimester human intestinal samples. Although, we were unable to amplify bacterial DNA from fetal intestines, we report a unique fetal metabolomic intestinal profile with an abundance of bacterially derived and host derived metabolites commonly produced in response to microbiota. Though we did not directly assess their source and function, we hypothesize that these microbial associated metabolites come either from the maternal microbiome and are vertically transmitted to the fetus to prime the fetal immune system and prepare the gastrointestinal tract for postnatal microbial encounters or are produced locally by bacteria that was below our detection threshold.
Yujia Li, Jessica M. Toothaker, Shira Ben-Simon, Lital Ozeri, Ron Schweitzer, Blake T. McCourt, Collin C. McCourt, Lael Werner, Scott B. Snapper, Dror S. Shouval, Soliman Khatib, Omry Koren, Sameer Agnihorti, George Tseng, Liza Konnikova
Cardiac energetic dysfunction has been reported in patients with type 2 diabetes (T2D) and is an independent predictor of mortality. Identification of the mechanisms driving mitochondrial dysfunction, and therapeutic strategies to rescue these modifications, will improve myocardial energetics in T2D. We demonstrate using 31P-magnetic resonance spectroscopy (31P-MRS) that decreased cardiac ATP and phosphocreatine (PCr) concentrations occurred before contractile dysfunction or a reduction in PCr/ATP ratio in T2D. Real-time mitochondrial ATP synthesis rates and state 3 respiration rates were similarly depressed in T2D, implicating dysfunctional mitochondrial energy production. Driving this energetic dysfunction in T2D was an increase in mitochondrial protein acetylation, and increased ex vivo acetylation was shown to proportionally decrease mitochondrial respiration rates. Treating T2D rats in vivo with the mitochondrial deacetylase SIRT3 activator honokiol reversed the hyperacetylation of mitochondrial proteins and restored mitochondrial respiration rates to control levels. Using 13C-hyperpolarized MRS, respiration with different substrates, and enzyme assays, we localized this improvement to increased glutamate dehydrogenase activity. Finally, honokiol treatment increased ATP and PCr concentrations and increased total ATP synthesis flux in the T2D heart. In conclusion, hyperacetylation drives energetic dysfunction in T2D, and reversing acetylation with the SIRT3 activator honokiol rescued myocardial and mitochondrial energetics in T2D.
Matthew Kerr, Jack J. Miller, Dharendra Thapa, Sophie Stiewe, Kerstin N. Timm, Claudia N. Montes Aparicio, Iain Scott, Damian J. Tyler, Lisa C. Heather
The angiopoietin-like protein ANGPTL8 (A8) is one of three ANGPTLs (A8, A3, A4) that coordinate changes in triglyceride (TG) delivery to tissues by inhibiting lipoprotein lipase (LPL), an enzyme that hydrolyzes TG. Previously we showed that A8, which is expressed in liver and adipose tissue, is required to redirect dietary TG from oxidative to storage tissues following food intake. Here we show that A8 from liver and adipose tissue have different roles in this process. Mice lacking hepatic A8 have no circulating A8, high intravascular LPL activity, low plasma TG levels, and evidence of decreased delivery of dietary lipids to adipose tissue. In contrast, mice lacking A8 in adipose tissue have higher postprandial TG levels and no alteration in fatty acid composition in adipocytes. Expression of A8, together with A4, in cultured cells reduced A4 secretion and A4-mediated LPL inhibition. Thus, hepatic A8 (with A3) acts in an endocrine fashion to inhibit intravascular LPL in oxidative tissues, whereas A8 in adipose tissue enhances LPL activity by autocrine/paracrine inhibition of A4. These combined actions of A8 ensure that TG stores are rapidly replenished and sufficient energy is available until the next meal.
Federico Oldoni, Haili Cheng, Serena Banfi, Viktoria Gusarova, Jonathan C. Cohen, Helen H. Hobbs
Orphan nuclear receptor estrogen-related receptor (ERR)γ stimulates bile acid production, however, the role and the regulatory mechanism of ERRγ in cholestatic liver disease are largely unknown. This study identifies that Sirt6 is a deacetylase of ERRγ and suggests a novel mechanism by which Sirt6 activation alleviates cholestatic liver damage and fibrosis through regulating ERRγ. We observed that hepatic expression of Sirt6 is repressed while that of ERRγ is upregulated in murine cholestasis models. Hepatocyte-specific Sirt6 knockout mice were more severely injured following a bile duct ligation (BDL) compared to wild-type mice and adenoviral re-expression of Sirt6 reversed liver damage and fibrosis as demonstrated by biochemical and histological analyses. Mechanistically, Sirt6 deacetylated ERRγ, thereby destabilized ERRγ and inhibited its transcriptional activity. Elimination of hepatic ERRγ using Ad-shERRγ abolished the deleterious effects of Sirt6 deficiency, while ERRγ overexpression aggravated cholestatic liver injury. Administration of a Sirt6 deacetylase activator prevented BDL-induced liver damage and fibrosis. In patients with cholestasis, Sirt6 expression was decreased while total- and acetylated-ERRγ levels were increased, confirming negative regulation of ERRγ by Sirt6. Thus, Sirt6 activation represents a new therapeutic strategy for treating cholestatic liver injury.
Lihua Hao, In Hyuk Bang, Jie Wang, Yuancheng Mao, Jae Do Yang, Soon-Young Na, Jeong Kon Seo, Hueng-Sik Choi, Eun Ju Bae, Byung-Hyun Park
BACKGROUND. Metabolically healthy obesity (MHO) and metabolically healthy overweight (MH-OW) have been suggested to be an important and emerging phenotype with an increased risk of cardiovascular disease (CVD). However, whether MHO and MH-OW are associated with all-cause mortality remains inconsistent. METHODS. The association of MHO and MH-OW and all-cause mortality was determined in China community-based prospective cohort study (Kailuan Study) including 93,272 adults at baseline. Data were analyzed from 2006 to 2017. Participants were categorized into six mutually exclusive groups according to the body mass index (BMI) and metabolic syndrome (MetS) status. The primary outcome is all-cause death, whereas accidental deaths were excluded. RESULTS. During a median follow-up of 11.04 years (interquartile range: 10.74-11.22 years), 8,977 deaths occurred. Compared to healthy participants with normal BMI (MH-NW), MH-OW had lowest risk of all-cause mortality (multivariate-adjusted hazard ratio [aHR]: 0.926; 95% confidence interval [CI]: 0.861 to 0.997), whereas there was no increased or decreased risk for MHO (aHR: 1.009; 95% CI: 0.886 to 1.148). Stratified analyses and sensitivity analyses further validated that nonsignificant association between MHO and all-cause mortality. CONCLUSIONS. Overweight and obesity do not predicate increased risk of all-cause mortality in metabolic healthy Chinese individuals.
Qiuyue Tian, Anxin Wang, Yingting Zuo, Shuohua Chen, Haifeng Hou, Wei Wang, Shouling Wu, Youxin Wang
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