ASXL2 is an ETP family protein that interacts with PPARγ. We find that ASXL2−/− mice are insulin resistant, lipodystrophic, and fail to respond to a high-fat diet. Consistent with genetic variation ...at the ASXL2 locus and human bone mineral density, ASXL2−/− mice are also severely osteopetrotic because of failed osteoclast differentiation attended by normal bone formation. ASXL2 regulates the osteoclast via two distinct signaling pathways. It induces osteoclast formation in a PPARγ/c-Fos-dependent manner and is required for RANK ligand- and thiazolidinedione-induced bone resorption independent of PGC-1β. ASXL2 also promotes osteoclast mitochondrial biogenesis in a process mediated by PGC-1β but independent of c-Fos. Thus, ASXL2 is a master regulator of skeletal, lipid, and glucose homeostasis.
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•ASXL2-null mice are osteopetrotic, lipodystrophic, and insulin resistant•Attenuated osteoclastogenesis in ASXL2 KO mice reflects failure of PPARγ activation•c-Fos and PGC1β differentially regulate osteoclast formation and function•ASXL2 independently regulates skeletal and metabolic homeostasis
ASXL2 regulates glucose homeostasis, adipogenesis, and osteoclast differentiation by activating PPARγ. Izawa et al. find that ASXL2-deficient mice are insulin resistant, lipodystrophic, and osteopetrotic. ASXL2 promotes osteoclast formation in a Fos-dependent manner independent of PGC-1β. ASXL2 enhances osteoclast mitochondrial biogenesis in a process mediated by PGC-1β but independent of c-Fos.
Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell ...death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.
Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of ...protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire—pre- and post-vaccination—and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.
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•Serum vaccine response is dominated by a small number of abundant antibody clonotypes•Vaccine-boosted antibodies predominantly target conserved norovirus epitopes•Identified cross-genogroup and strain-specific epitopes•Discovered a pandemic-genotype neutralizing antibody recognizing a conserved epitope
Human norovirus (HuNoV) is a leading cause of gastroenteritis. Lindesmith et al. identify circulating serum antibodies following experimental HuNoV vaccination in humans and map them to viral epitopes. One antibody recognizes a neutralizing epitope conserved across three decades of pandemic strains and neutralizes virus in vitro, demonstrating that vaccination can elicit pandemic-strain neutralizing antibody responses in some individuals.
Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt ...signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Holstein steers (n = 40; initial BW = 84.9 ± 7.1 kg) were used to study the genesis of liver abscesses (LA) using an acidotic diet challenge with or without intraruminal bacterial inoculation. Steers ...were housed in individual pens inside a barn and randomly assigned to one of three treatments: (1) low-starch control diet comprised primarily of dry-rolled corn and wet corn gluten feed (CON); (2) high-starch acidotic diet with steam-flaked corn (AD); or (3) acidotic diet plus intraruminal inoculation with Fusobacterium necrophorum subsp. necrophorum (9.8 × 108 colony forming units CFU/mL), Trueperella pyogenes (3.91 × 109 CFU/mL), and Salmonella enterica serovar Lubbock (3.07 × 108 CFU/mL), previously isolated from LA (ADB). Steers in AD and ADB were fed the acidotic diet for 3 d followed by 2 d of the CON diet, and this cycle was repeated four times. On day 23, ADB steers were intraruminally inoculated with the bacteria. At necropsy, gross pathology of livers, lungs, rumens, and colons was noted. Continuous data were analyzed via mixed models as repeated measures over time with individual steer as the experimental unit. Mixed models were also used to determine the difference in prevalence of necropsy scores among treatments. Ruminal pH decreased in AD and ADB steers during each acidotic diet cycle (P ≤ 0.05). LA prevalence was 42.9% (6 of 14) in ADB vs. 0% in AD or CON treatments (P < 0.01). Ruminal damage was 51.1% greater in ADB than in AD (P ≤ 0.04). Culture of LA determined that 100% of the abscesses contained F. necrophorum subsp. necrophorum, 0% contained T. pyogenes, 50% contained Salmonella, and 50% contained a combination of F. necrophorum subsp. necrophorum and Salmonella. The F. necrophorum subsp. necrophorum was clonally identical to the strain used for the bacterial inoculation based on phylogenetic analysis of the whole genome. This experimental model successfully induced rumenitis and LA in Holstein steers and confirms the central dogma of LA pathogenesis that acidosis and rumenitis lead to the entry of F. necrophorum into the liver to cause abscesses. Our findings suggest that an acidotic diet, in conjunction with intraruminal bacterial inoculation, is a viable model to induce LA. Further research is needed to determine the repeatability of this model, and a major application of the model will be in evaluations of novel interventions to prevent LA.
Abstract
Holstein steers (n = 40; initial body weight BW = 96.0 ± 10.5 kg) were individually housed in a climate-controlled barn to evaluate potential models for the genesis of liver abscesses (LA). ...In this 2 × 2 factorial, steers were balanced by BW and randomly assigned to one of two treatments: 1) intravenous saline injection followed by intraruminal bacterial inoculation with Fusobacterium necrophorum subsp. necrophorum (1 × 109 colony forming unit CFU/mL) and Salmonella enterica serovar Lubbock (1 × 106 CFU/mL; CON; n = 20 steers); or 2) intravenous injection with 0.25 µg/kg BW of lipopolysaccharide (LPS; Escherichia coli O111:B4) followed by intraruminal bacterial inoculation of F. necrophorum subsp. necrophorum (1 × 109 CFU/mL) and S. enterica serovar Lubbock (1 × 106 CFU/mL; LBI; n = 20 steers) and 1 of 2 harvest dates (3 or 10 d post LPS infusion). Body weights were recorded on days −4, −1, 3, and 10, and blood was collected for hematology on days −4, 3, and 10, relative to LPS infusion on day 0. Intraruminal bacterial inoculation occurred on day 1. Steers from each treatment group were harvested at two different time points on day 3 or 10 to perform gross pathological examination of the lung, rumen, liver, LA (if present), and colon. Feed disappearance was less for LBI than CON (P < 0.01); however, BW did not differ (P = 0.33) between treatments. Neither treatment nor time differed for hematology (P ≥ 0.13), and no gross pathological differences were noted in the lung, liver, LA, or colon (P ≥ 0.25). A treatment × harvest date interaction was noted for ruminal pathology in which LBI had an increased percentage of abnormal rumen scores on day 3 (P < 0.01). These results suggest that an LPS challenge in combination with intraruminal bacterial inoculation of pathogens commonly isolated from LA was not sufficient to induce LA in steers within 3 or 10 d (P = 0.95) when compared to CON. Further evaluation is needed to produce a viable model to investigate the genesis and prevention of LA in cattle.
Lipopolysaccharide in combination with intraruminal bacterial inoculation of Fusobacterium necrophorum and Salmonella enterica is not a viable model to induce liver abscesses in steers within 10 d. Bacterial inoculation alone was insufficient at inducing liver abscesses, and further research to determine the time necessary to induce liver abscesses is needed.
Lay Summary
Liver abscesses in feedlot cattle can cause a decrease in feed intake, average daily gain, feed efficiency, and hot carcass weight. At harvest, liver abscesses result in liver condemnations, carcass trimming, and a potential decrease in quality grade, with an estimated economic cost to packers of $41.6 million annually. Our objective was to evaluate an intravenous endotoxin challenge followed by intraruminal inoculation of bacteria commonly isolated from liver abscesses over a 10-d period as a potential model to understand the genesis and etiology of liver abscesses in cattle and evaluate possible preventative interventions. Results suggest that an endotoxin challenge in combination with intraruminal bacterial inoculation is not a viable model to induce liver abscesses in steers, and bacterial inoculation alone was insufficient to induce liver abscesses. The length of time necessary to induce liver abscesses is also unknown. Based on our results, more research is needed to develop a noninvasive model to induce liver abscesses in cattle.
Abstract
Reliable predictions of metabolizable energy (ME) from digestible energy (DE) are necessary to prescribe nutrient requirements of beef cattle accurately. A previously developed database that ...included 87 treatment means from 23 respiration calorimetry studies has been updated to evaluate the efficiency of converting DE to ME by adding 47 treatment means from 11 additional studies. Diets were fed to growing-finishing cattle under individual feeding conditions. A citation-adjusted linear regression equation was developed where dietary ME concentration (Mcal/kg of dry matter DM) was the dependent variable and dietary DE concentration (Mcal/kg) was the independent variable: ME = 1.0001 × DE – 0.3926; r2 = 0.99, root mean square prediction error RMSPE = 0.04, and P < 0.01 for the intercept and slope. The slope did not differ from unity (95% CI = 0.936 to 1.065); therefore, the intercept (95% CI = −0.567 to −0.218) defines the value of ME predicted from DE. For practical use, we recommend ME = DE – 0.39. Based on the relationship between DE and ME, we calculated the citation-adjusted loss of methane, which yielded a value of 0.2433 Mcal/kg of dry matter intake (DMI; SE = 0.0134). This value was also adjusted for the effects of DMI above maintenance, yielding a citation-adjusted relationship: CH4, Mcal/kg = 0.3344 – 0.05639 × multiple of maintenance; r2 = 0.536, RMSPE = 0.0245, and P < 0.01 for the intercept and slope. Both the 0.2433 value and the result of the intake-adjusted equation can be multiplied by DMI to yield an estimate of methane production. These two approaches were evaluated using a second, independent database comprising 129 data points from 29 published studies. Four equations in the literature that used DMI or intake energy to predict methane production also were evaluated with the second database. The mean bias was substantially greater for the two new equations, but slope bias was substantially less than noted for the other DMI-based equations. Our results suggest that ME for growing and finishing cattle can be predicted from DE across a wide range of diets, cattle types, and intake levels by simply subtracting a constant from DE. Mean bias associated with our two new methane emission equations suggests that further research is needed to determine whether coefficients to predict methane from DMI could be developed for specific diet types, levels of DMI relative to body weight, or other variables that affect the emission of methane.
Signaling Elements Involved in the Metabolic Regulation of mTOR by Nutrients, Incretins, and Growth Factors in Islets
Guim Kwon 1 ,
Connie A. Marshall 1 ,
Kirk L. Pappan 1 ,
Maria S. Remedi 2 and
...Michael L. McDaniel 1
1 Departments of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri
2 Departments of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri
Address correspondence and reprint requests to Michael L. McDaniel, Washington University School of Medicine, Campus Box 8118,
660 South Euclid Ave., St. Louis, MO 63110. E-mail: mcdaniel{at}pathology.wustl.edu
Abstract
Mammalian target of rapamycin (mTOR) is a protein kinase that integrates signals from mitogens and the nutrients, glucose
and amino acids, to regulate cellular growth and proliferation. Previous findings demonstrated that glucose robustly activates
mTOR in an amino acid-dependent manner in rodent and human islets. Furthermore, activation of mTOR by glucose significantly
increases rodent islet DNA synthesis that is abolished by rapamycin. Glucagon-like peptide-1 (GLP-1) agonists, through the
production of cAMP, have been shown to enhance glucose-dependent proinsulin biosynthesis and secretion and to stimulate cellular
growth and proliferation. The objective of this study was to determine if the glucose-dependent and cAMP-mediated mechanism
by which GLP-1 agonists enhance β-cell growth and proliferation is mediated, in part, through mTOR. Our studies demonstrated
that forskolin-generated cAMP resulted in activation of mTOR at basal glucose concentrations as assessed by phosphorylation
of S6K1, a downstream effector of mTOR. Conversely, an adenylyl cyclase inhibitor partially blocked glucose-induced S6K1 phosphorylation.
Furthermore, the GLP-1 receptor agonist, Exenatide, dose-dependently enhanced phosphorylation of S6K1 at an intermediate glucose
concentration (8 mmol/l) in a rapamycin-sensitive manner. To determine the mechanism responsible for this potentiation of
mTOR, the effects of intra- and extracellular Ca 2+ were examined. Glyburide, an inhibitor of ATP-sensitive K + channels (K ATP channels), provided partial activation of mTOR at basal glucose concentrations due to the influx of extracellular Ca 2+ , and diazoxide, an activator of K ATP channels, resulted in partial inhibition of S6K1 phosphorylation by 20 mmol/l glucose. Furthermore, Exenatide or forskolin
reversed the inhibition by diazoxide, probably through mobilization of intracellular Ca 2+ stores by cAMP. BAPTA, a chelator of intracellular Ca 2+ , resulted in inhibition of glucose-stimulated S6K1 phosphorylation due to a reduction in cytosolic Ca 2+ concentrations. Selective blockade of glucose-stimulated Ca 2+ influx unmasked a protein kinase A (PKA)-sensitive component involved in the mobilization of intracellular Ca 2+ stores, as revealed with the PKA inhibitor H-89. Overall, these studies support our hypothesis that incretin-derived cAMP
participates in the metabolic activation of mTOR by mobilizing intracellular Ca 2+ stores that upregulate mitochondrial dehydrogenases and result in enhanced ATP production. ATP can then modulate K ATP channels, serve as a substrate for adenylyl cyclase, and possibly directly regulate mTOR activation.
AMPK, AMP-dependent protein kinase
CMRL, CMRL-1066 medium
cCMRL, CMRL plus 5.6 mmol/l glucose, supplemented with 10% FCS, 2 mmol/l l-glutamine, 100 units/ml penicillin, and 100 μg/ml
streptomycin
eIF-4E, eukaryotic initiation factor 4E
GLP-1, glucagon-like peptide-1
IRS, insulin receptor substrate
KATP channel, ATP-sensitive K+ channel
mTOR, mammalian target of rapamycin
PDK1, 3-phosphoinositide-dependent kinase
PI3K, phosphoinositide-3 kinase
PKA, protein kinase A
PKB, protein kinase B
Footnotes
This article is based on a presentation at a symposium. The symposium and the publication of this article were made possible
by an unrestricted educational grant from Servier.
Accepted April 27, 2004.
Received February 28, 2004.
DIABETES
Major bottlenecks in the expansion of human β-cell mass are limited proliferation, loss of β-cell phenotype, and increased apoptosis. In our previous studies, activation of Wnt and mTOR signaling ...significantly enhanced human β-cell proliferation. However, isolated human islets displayed insulin signaling pathway resistance, due in part to chronic activation of mTOR/S6K1 signaling that results in negative feedback of the insulin signaling pathway and a loss of Akt phosphorylation and insulin content. We evaluated the effects of a new generation insulin sensitizer, MSDC-0160, on restoring insulin/IGF-1 sensitivity and insulin content in human β-cells. This novel TZD has low affinity for binding and activation of PPARγ and has insulin-sensitizing effects in mouse models of diabetes and ability to lower glucose in Phase 2 clinical trials. MSDC-0160 treatment of human islets increased AMPK activity and reduced mTOR activity. This was associated with the restoration of IGF-1-induced phosphorylation of Akt, GSK-3, and increased protein expression of Pdx1. Furthermore, MSDC-0160 in combination with IGF-1 and 8 mM glucose increased β-cell specific gene expression of insulin, pdx1, nkx6.1, and nkx2.2, and maintained insulin content without altering glucose-stimulated insulin secretion. Human islets were unable to simultaneously promote DNA synthesis and maintain the β-cell phenotype. Lithium-induced GSK-3 inhibition that promotes DNA synthesis blocked the ability of MSDC-0160 to maintain the β-cell phenotype. Conversely, MSDC-0160 prevented an increase in DNA synthesis by blocking β-catenin nuclear translocation. Due to the counteracting pathways involved in these processes, we employed a sequential ex vivo strategy to first induce human islet DNA synthesis, followed by MSDC-0160 to promote the β-cell phenotype and insulin content. This new generation PPARγ sparing insulin sensitizer may provide an initial tool for relieving inherent human islet insulin signaling pathway resistance that is necessary to preserve the β-cell phenotype during β-cell expansion for the treatment of diabetes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Glycogen Synthase Kinase-3 and Mammalian Target of Rapamycin Pathways Contribute to DNA Synthesis, Cell Cycle Progression,
and Proliferation in Human Islets
Hui Liu 1 ,
Maria S. Remedi 2 ,
Kirk L. ...Pappan 1 ,
Guim Kwon 3 ,
Nidhi Rohatgi 1 ,
Connie A. Marshall 1 and
Michael L. McDaniel 1
1 Department of Pathology and Immunology, Washington University, St. Louis, Missouri
2 Department of Cell Biology and Physiology, Washington University, St. Louis, Missouri
3 Department of Pharmaceutical Sciences, School of Pharmacy, Southern Illinois University, Edwardsville, Illinois
Corresponding author: Michael L. McDaniel, mmcdaniel{at}wustl.edu
Abstract
OBJECTIVE— Our previous studies demonstrated that nutrient regulation of mammalian target of rapamycin (mTOR) signaling promotes regenerative
processes in rodent islets but rarely in human islets. Our objective was to extend these findings by using therapeutic agents
to determine whether the regulation of glycogen synthase kinase-3 (GSK-3)/β-catenin and mTOR signaling represent key components
necessary for effecting a positive impact on human β-cell mass relevant to type 1 and 2 diabetes.
RESEARCH DESIGN AND METHODS— Primary adult human and rat islets were treated with the GSK-3 inhibitors, LiCl and the highly potent 1-azakenpaullone (1-Akp),
and with nutrients. DNA synthesis, cell cycle progression, and proliferation of β-cells were assessed. Measurement of insulin
secretion and content and Western blot analysis of GSK-3 and mTOR signaling components were performed.
RESULTS— Human islets treated for 4 days with LiCl or 1-Akp exhibited significant increases in DNA synthesis, cell cycle progression,
and proliferation of β-cells that displayed varying degrees of sensitivity to rapamycin. Intermediate glucose (8 mmol/l) produced
a striking degree of synergism in combination with GSK-3 inhibition to enhance bromodeoxyuridine (BrdU) incorporation and
Ki-67 expression in human β-cells. Nuclear translocation of β-catenin responsible for cell proliferation was found to be particularly
sensitive to rapamycin.
CONCLUSIONS— A combination of GSK-3 inhibition and nutrient activation of mTOR contributes to enhanced DNA synthesis, cell cycle progression,
and proliferation of human β-cells. Identification of therapeutic agents that appropriately regulate GSK-3 and mTOR signaling
may provide a feasible and available approach to enhance human islet growth and proliferation.
Footnotes
Published ahead of print at http://diabetes.diabetesjournals.org on 10 December 2008.
H.L., M.S.R., and K.L.P. contributed equally to this work.
The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views
of the National Institutes of Health (NIH) or American Diabetes Association (ADA).
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work
is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Accepted November 30, 2008.
Received August 27, 2007.
DIABETES
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