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Huntington's disease (HD) is an inherited neurodegenerative disorder which is caused by a mutation of the huntingtin (HTT) gene. Although the pathogenesis of HD has been associated with inflammatory responses, if and how the immune system contributes to the onset of HD is largely unknown. Invariant natural killer T (iNKT) cells are a group of innate-like regulatory T lymphocytes that can rapidly produce various cytokines such as IFN and IL4 upon stimulation with the glycolipid -galactosylceramide (-GalCer). By employing both R6/2 Tg mice (murine HD model) and J18 KO mice (deficient in iNKT cells), we investigated whether alterations of iNKT cells affect the development of HD in R6/2 Tg mice. We found that J18 KO R6/2 Tg mice showed disease progression comparable to R6/2 Tg mice, indicating that the absence of iNKT cells did not have any significant effects on HD development. However, repeated activation of iNKT cells with -GalCer facilitated HD progression in R6/2 Tg mice, and this was associated with increased infiltration of iNKT cells in the brain. Taken together, our results demonstrate that repeated -GalCer treatment of R6/2 Tg mice accelerates HD progression, suggesting that immune activation can affect the severity of HD pathogenesis.
The NAD+-dependent deacetylase SIRT2 is unique amongst sirtuins as it is effective in the cytosol, as well as the mitochondria. Defining the role of cytosolic acetylation state in specific tissues is difficult since even physiological effects at the whole body level are unknown. We hypothesized that genetic SIRT2 knockout (KO) would lead to impaired insulin action, and that this impairment would be worsened in HF fed mice. Insulin sensitivity was tested using the hyperinsulinemic-euglycemic clamp in SIRT2 KO mice and WT littermates. SIRT2 KO mice exhibited reduced skeletal muscle insulin-induced glucose uptake compared to lean WT mice, and this impairment was exacerbated in HF SIRT2 KO mice. Liver insulin sensitivity was unaffected in lean SIRT2 KO mice. However, the insulin resistance that accompanies HF-feeding was worsened in SIRT2 KO mice. It was notable that the effects of SIRT2 KO were largely disassociated from cytosolic acetylation state, but were closely linked to acetylation state in the mitochondria. SIRT2 KO led to an increase in body weight that was due to increased food intake in HF fed mice. In summary, SIRT2 deletion in vivo reduces muscle insulin sensitivity and contributes to liver insulin resistance by a mechanism that is unrelated to cytosolic acetylation state. Mitochondrial acetylation state and changes in feeding behavior that result in increased body weight correspond to the deleterious effects of SIRT2 KO on insulin action.
BACKGROUND & AIMS - Bile diversion to the ileum (GB-IL) has strikingly similar metabolic and satiating effects to Roux-en-Y gastric bypass (RYGB) in rodent obesity models. The metabolic benefits of these procedures are thought to be mediated by increased bile acids, although parallel changes in body weight and other confounding variables limit this interpretation.
METHODS - Global G protein-coupled bile acid receptor-1 null (Tgr5) and intestinal-specific farnesoid X receptor null (Fxr) mice on high-fat diet as well as wild-type C57BL/6 and glucagon-like polypeptide 1 receptor deficient (Glp-1r) mice on chow diet were characterized following GB-IL.
RESULTS - GB-IL induced weight loss and improved oral glucose tolerance in Tgr5, but not Fxr mice fed a high-fat diet, suggesting a role for intestinal Fxr. GB-IL in wild-type, chow-fed mice prompted weight-independent improvements in glycemia and glucose tolerance secondary to augmented insulin responsiveness. Improvements were concomitant with increased levels of lymphatic GLP-1 in the fasted state and increased levels of intestinal Akkermansia muciniphila. Improvements in fasting glycemia after GB-IL were mitigated with exendin-9, a GLP-1 receptor antagonist, or cholestyramine, a bile acid sequestrant. The glucoregulatory effects of GB-IL were lost in whole-body Glp-1r mice.
CONCLUSIONS - Bile diversion to the ileum improves glucose homeostasis via an intestinal Fxr-Glp-1 axis. Altered intestinal bile acid availability, independent of weight loss, and intestinal Akkermansia muciniphila appear to mediate the metabolic changes observed after bariatric surgery and might be manipulated for treatment of obesity and diabetes.
Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.
Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The 2 major modes of programmed cell death, apoptosis and necroptosis, share molecular machinery but diverge in outcome with important implications for the microenvironment; apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine-directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here, we present a mouse model with increased bone marrow necroptosis. Deletion of the proapoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid (to generate triple-knockout [TKO] mice) leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase (Ripk1). TKO mice display loss of progenitor cells, leading to increased cytokine production and increased stem cell proliferation and exhaustion and culminating in bone marrow failure. Genetically restoring Ripk1 to wild-type levels restores peripheral red cell counts as well as normal cytokine production. TKO bone marrow is hypercellular with abnormal differentiation, resembling the human disorder myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of caspase-8-mediated Ripk1 degradation. Thus, we demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder MDS.
© 2019 by The American Society of Hematology.
Transporting epithelial cells optimize their morphology for solute uptake by building an apical specialization: a dense array of microvilli that serves to increase membrane surface area. In the intestinal tract, individual cells build thousands of microvilli, which pack tightly to form the brush border. Recent studies implicate adhesion molecule CDHR2 in the regulation of microvillar packing via the formation of adhesion complexes between the tips of adjacent protrusions. To gain insight on how CDHR2 contributes to brush border morphogenesis and enterocyte function under native in vivo conditions, we generated mice lacking CDHR2 expression in the intestinal tract. Although CDHR2 knockout (KO) mice are viable, body weight trends lower and careful examination of tissue, cell, and brush border morphology revealed several perturbations that likely contribute to reduced functional capacity of KO intestine. In the absence of CDHR2, microvilli are significantly shorter, and exhibit disordered packing and a 30% decrease in packing density. These structural perturbations are linked to decreased levels of key solute processing and transporting factors in the brush border. Thus, CDHR2 functions to elongate microvilli and maximize their numbers on the apical surface, which together serve to increase the functional capacity of enterocyte.
The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following β-cell loss, 1-2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of β-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive 'brake signals' is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.
Ca/calmodulin-dependent protein kinase II (CaMKII) and metabotropic glutamate receptor 5 (mGlu) are critical signaling molecules in synaptic plasticity and learning/memory. Here, we demonstrate that mGlu is present in CaMKII complexes isolated from mouse forebrain. Further in vitro characterization showed that the membrane-proximal region of the C-terminal domain (CTD) of mGlu directly interacts with purified Thr286-autophosphorylated (activated) CaMKII However, the binding of CaMKII to this CTD fragment is reduced by the addition of excess Ca/calmodulin or by additional CaMKII autophosphorylation at non-Thr286 sites. Furthermore, in vitro binding of CaMKII is dependent on a tribasic residue motif Lys-Arg-Arg (KRR) at residues 866-868 of the mGlu-CTD, and mutation of this motif decreases the coimmunoprecipitation of CaMKII with full-length mGlu expressed in heterologous cells by about 50%. The KRR motif is required for two novel functional effects of coexpressing constitutively active CaMKII with mGlu in heterologous cells. First, cell-surface biotinylation studies showed that CaMKII increases the surface expression of mGlu Second, using Ca fluorimetry and single-cell Ca imaging, we found that CaMKII reduces the initial peak of mGlu-mediated Ca mobilization by about 25% while doubling the relative duration of the Ca signal. These findings provide new insights into the physical and functional coupling of these key regulators of postsynaptic signaling.
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
Solute carrier family 7 member 2 (SLC7A2, also known as CAT2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in wound repair. We have reported that both SLC7A2 expression and L-Arg availability are decreased in colonic tissues from inflammatory bowel disease patients and that mice lacking Slc7a2 exhibit a more severe disease course when exposed to dextran sulfate sodium (DSS) compared to wild-type (WT) mice. Here, we present evidence that SLC7A2 plays a role in modulating colon tumorigenesis in the azoxymethane (AOM)-DSS model of colitis-associated carcinogenesis (CAC). SLC7A2 was localized predominantly to colonic epithelial cells in WT mice. Utilizing the AOM-DSS model, Slc7a2 mice had significantly increased tumor number, burden, and risk of high-grade dysplasia vs. WT mice. Tumors from Slc7a2 mice exhibited significantly increased levels of the proinflammatory cytokines/chemokines IL-1β, CXCL1, CXCL5, IL-3, CXCL2, CCL3, and CCL4, but decreased levels of IL-4, CXCL9, and CXCL10 compared to tumors from WT mice. This was accompanied by a shift toward pro-tumorigenic M2 macrophage activation in Slc7a2-deficient mice, as marked by increased colonic CD11bF4/80ARG1 cells with no alteration in CD11bF4/80NOS2 cells by flow cytometry and immunofluorescence microscopy. The shift toward M2 macrophage activation was confirmed in bone marrow-derived macrophages from Slc7a2 mice. In bone marrow chimeras between Slc7a2 and WT mice, the recipient genotype drove the CAC phenotype, suggesting the importance of epithelial SLC7A2 in abrogating neoplastic risk. These data reveal that SLC7A2 has a significant role in the protection from CAC in the setting of chronic colitis, and suggest that the decreased SLC7A2 in inflammatory bowel disease (IBD) may contribute to CAC risk. Strategies to enhance L-Arg availability by supplementing L-Arg and/or increasing L-Arg uptake could represent a therapeutic approach in IBD to reduce the substantial long-term risk of colorectal carcinoma.
BACKGROUND & AIMS - Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters.
METHODS - We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCre;Myo5b mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry.
RESULTS - Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCre;Myo5b mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCre;Myo5b mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCre;Myo5b mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity.
CONCLUSIONS - Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.
Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.
The gut-to-brain axis exhibits significant control over motivated behavior. However, mechanisms supporting this communication are poorly understood. We reveal that a gut-based bariatric surgery chronically elevates systemic bile acids and attenuates cocaine-induced elevations in accumbal dopamine. Notably, this surgery reduces reward-related behavior and psychomotor sensitization to cocaine. Utilizing a knockout mouse model, we have determined that a main mediator of these post-operative effects is the Takeda G protein-coupled bile acid receptor (TGR5). Viral restoration of TGR5 in the nucleus accumbens of TGR5 knockout animals is sufficient to restore cocaine reward, centrally localizing this TGR5-mediated modulation. These findings define TGR5 and bile acid signaling as pharmacological targets for the treatment of cocaine abuse and reveal a novel mechanism of gut-to-brain communication.