Deficiency of exchange protein directly activated by cAMP (EPAC)-1 in mice augments glucose intolerance, inflammation, and gut dysbiosis associated with Western diet.

BACKGROUND: Gut microbiota (GM) dysregulation, known as dysbiosis, has been proposed as a crucial driver of obesity associated with "Western" diet (WD) consumption. Gut dysbiosis is associated with increased gut permeability, inflammation, and insulin resistance. However, host metabolic pathways implicated in the pathophysiology of gut dysbiosis are still elusive. Exchange protein directly activated by cAMP (Epac) plays a critical role in cell-cell junction formation and insulin secretion. Here, we used homozygous Epac1-knockout (Epac1-/-), Epac2-knockout (Epac2-/-), and wild-type (WT) mice to investigate the role of Epac proteins in mediating gut dysbiosis, gut permeability, and inflammation after WD feeding. RESULTS: The 16S rRNA gene sequencing of fecal DNA showed that the baseline GM of Epac2-/-, but not Epac1-/-, mice was represented by a significantly higher Firmicutes to Bacteroidetes ratio and significant alterations in several taxa compared to WT mice, suggesting that Epac2-/- mice had gut dysbiosis under physiological conditions. However, an 8-week WD led to a similar gut microbiome imbalance in mice regardless of genotype. While Epac1 deficiency modestly exacerbated the WD-induced GM dysbiosis, the WD-fed Epac2-/- mice had a more significant increase in gut permeability than corresponding WT mice. After WD feeding, Epac1-/-, but not Epac2-/-, mice had significantly higher mRNA levels of tumor necrosis factor-alpha (TNF-α) and F4/80 in the epididymal white adipose tissue (EWAT), increased circulating lipocalin-2 protein and more severe glucose intolerance, suggesting greater inflammation and insulin resistance in WD-fed Epac1-/- mice than corresponding WT mice. Consistently, Epac1 protein expression was significantly reduced in the EWAT of WD-fed WT and Epac2-/- mice. CONCLUSION: Despite significantly dysregulated baseline GM and a more pronounced increase in gut permeability upon WD feeding, WD-fed Epac2-/- mice did not exhibit more severe inflammation and glucose intolerance than corresponding WT mice. These findings suggest that the role of gut dysbiosis in mediating WD-associated obesity may be context-dependent. On the contrary, we demonstrate that deficiency of host signaling protein, Epac1, drives inflammation and glucose intolerance which are the hallmarks of WD-induced obesity. Video abstract.

Exchange protein directly activated by cAMP (Epac) 1 plays an essential role in stress-induced exercise capacity by regulating PGC-1α and fatty acid metabolism in skeletal muscle.

Exchange protein directly activated by cAMP (Epac) mediates cAMP-mediated cell signal independent of protein kinase A (PKA). Mice lacking Epac1 displayed metabolic defect suggesting possible functional involvement of skeletal muscle and exercise capacity. Epac1 was highly expressed, but not Epac 2, in the extensor digitorum longus (EDL) and soleus muscles. The exercise significantly increased protein expression of Epac 1 in EDL and soleus muscle of wild-type (WT) mice. A global proteomics and pathway analyses revealed that Epac 1 deficiency mainly affected "the energy production and utilization" process in the skeletal muscle. We have tested their forced treadmill exercise tolerance. Epac1-/- mice exhibited significantly reduced exercise capacity in the forced treadmill exercise and lower number of type 1 fibers than WT mice. The basal protein level of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) was reduced in the Epac1-/- mice. Furthermore, increasing expression of PGC-1α by exercise was also significantly attenuated in the skeletal muscle of Epac1-/- mice. The expressions of downstream target genes of PGC-1α, which involved in uptake and oxidation of fatty acids, ERRα and PPARδ, and fatty acid content were lower in muscles of Epac1-/-, suggesting a role of Epac1 in forced treadmill exercise capacity by regulating PGC-1α pathway and lipid metabolism in skeletal muscle. Taken together, Epac1 plays an important role in exercise capacity by regulating PGC-1α and fatty acid metabolism in the skeletal muscle.

Endothelin type B receptor promotes cofilin rod formation and dendritic loss in neurons by inducing oxidative stress and cofilin activation.

Endothelin-1 (ET-1) is a neuroactive peptide produced by neurons, reactive astrocytes, and endothelial cells in the brain. Elevated levels of ET-1 have been detected in the post-mortem brains of individuals with Alzheimer's disease (AD). We have previously demonstrated that overexpression of astrocytic ET-1 exacerbates memory deficits in aged mice or in APPK670/M671 mutant mice. However, the effects of ET-1 on neuronal dysfunction remain elusive. ET-1 has been reported to mediate superoxide formation in the vascular system via NADPH oxidase (NOX) and to regulate the actin cytoskeleton of cancer cell lines via the cofilin pathway. Interestingly, oxidative stress and cofilin activation were both reported to mediate one of the AD histopathologies, cofilin rod formation in neurons. This raises the possibility that ET-1 mediates neurodegeneration via oxidative stress- or cofilin activation-driven cofilin rod formation. Here, we demonstrate that exposure to 100 nm ET-1 or to a selective ET type B receptor (ETB) agonist (IRL1620) induces cofilin rod formation in dendrites of primary hippocampal neurons, accompanied by a loss of distal dendrites and a reduction in dendritic length. The 100 nm IRL1620 exposure induced superoxide formation and cofilin activation, which were abolished by pretreatment with a NOX inhibitor (5 µm VAS2870). Moreover, IRL1620-induced cofilin rod formation was partially abolished by pretreatment with a calcineurin inhibitor (100 nm FK506), which suppressed cofilin activation. In conclusion, our findings suggest a role for ETB in neurodegeneration by promoting cofilin rod formation and dendritic loss via NOX-driven superoxide formation and cofilin activation.

Simple and Rapid Tissue Clearing Method for Three-Dimensional Histology of the Pancreas.

Previously, high-resolution three-dimensional imaging of a whole and intact pancreas was not possible, since light is scattered when it passes through cell compartments with different refractive indices. CLARITY is one of the tissue clearing techniques that has yielded success with the central nervous system. To preserve tissue integrity after delipidation, conventional protocols embed tissue in an acrylamide-based hydrogel, which involves the use of specialized equipment. Recently, we determined that the hydrogel-embedding step could be simplified and replaced by passive tissue fixation in 4% paraformaldehyde (PFA). The whole procedure is less time-consuming and less error-prone, and can be completed within a week, compared to conventional CLARITY protocols that may take weeks to complete. Here, the detailed stepwise procedures involved in the simplified CLARITY workflow are applied to the pancreas of wild-type and gene-knockout 6-week old mice expressing green fluorescent protein (GFP) under the mouse insulin 1 promoter (MIP-GFP). This technique could facilitate high-resolution, three-dimensional imaging of pancreatic islets and comparison between different mouse genotypes under different disease and treatment conditions. © 2017 by John Wiley & Sons, Inc.

Chronic adiponectin deficiency leads to Alzheimer's disease-like cognitive impairments and pathologies through AMPK inactivation and cerebral insulin resistance in aged mice.

BACKGROUND: Insulin resistance is the major pathogenesis underlying type 2 diabetes mellitus (T2DM) and these patients have doubled risk of Alzheimer's disease (AD). Increasing evidence suggests that insulin resistance plays an important role in AD pathogenesis, possibly due to abnormal GSK3ß activation, causing intra- and extracellular amyloid-beta (Aß) accumulation. Adiponectin (APN) is an adipokine with insulin-sensitizing and anti-inflammatory effects. Reduced circulatory APN level is associated with insulin resistance and T2DM. The role of APN in AD has not been elucidated. In this study, we aim to examine if adiponectin deficiency would lead to cerebral insulin resistance, cognitive decline and Alzheimer's-like pathology in mice. METHODS: To study the role of adiponectin in cognitive functions, we employed adiponectin-knockout (APN-KO) mice and demonstrated chronic APN deficiency in their CNS. Behavioral tests were performed to study the cognitions of male APN-KO mice. Brains and tissue lysates were collected to study the pathophysiological and molecular changes in the brain of APN-KO mice. SH-SY5Y neuroblastoma cell line was used to study the molecular mechanism upon APN and insulin treatment. RESULTS: Aged APN-deficient mice displayed spatial memory and learning impairments, fear-conditioned memory deficit as well as anxiety. These mice also developed AD pathologies including increased cerebral Aß42 level, Aß deposition, hyperphosphorylated Tau proteins, microgliosis and astrogliosis with increased cerebral IL-1ß and TNFα levels that associated with increased neuronal apoptosis and reduced synaptic proteins levels, suggesting APN deficiency may lead to neuronal and synaptic loss in the brain. AD pathologies-associated APN-KO mice displayed attenuated AMPK phosphorylation and impaired insulin signaling including decreased Akt induction and increased GSK3ß activation in the hippocampus and frontal cortex. Aged APN-KO mice developed hippocampal insulin resistance with reduced pAkt induction upon intracerebral insulin injection. Consistently, APN treatment in SH-SY5Y cells with insulin resistance and overexpressing Aß induce higher pAkt levels through AdipoR1 upon insulin treatment whereas the induction was blocked by compound C, indicating APN can enhance neuronal insulin sensitivity through AMPK activation. CONCLUSION: Our results indicated that chronic APN deficiency inactivated AMPK causing insulin desensitization and elicited AD-like pathogenesis in aged mice which also developed significant cognitive impairments and psychiatric symptoms.

Epac2-deficiency leads to more severe retinal swelling, glial reactivity and oxidative stress in transient middle cerebral artery occlusion induced ischemic retinopathy.

Ischemia occurs in diabetic retinopathy with neuronal loss, edema, glial cell reactivity and oxidative stress. Epacs, consisting of Epac1 and Epac2, are cAMP mediators playing important roles in maintenance of endothelial barrier and neuronal functions. To investigate the roles of Epacs in the pathogenesis of ischemic retinopathy, transient middle cerebral artery occlusion (tMCAO) was performed on Epac1-deficient (Epac1 (-/-)) mice, Epac2-deficient (Epac2 (-/-)) mice, and their wild type counterparts (Epac1 (+/+) and Epac2 (+/+)). Two-hour occlusion and 22-hour reperfusion were conducted to induce ischemia/reperfusion injury to the retina. After tMCAO, the contralateral retinae displayed similar morphology between different genotypes. Neuronal loss, retinal edema and increase in immunoreactivity for aquaporin 4 (AQP4), glial fibrillary acidic protein (GFAP), peroxiredoxin 6 (Prx6) were observed in ipsilateral retinae. Epac2 (-/-) ipsilateral retinae showed more neuronal loss in retinal ganglion cell layer, increased retinal thickness and stronger immunostaining of AQP4, GFAP, and Prx6 than those of Epac2 (+/+). However, Epac1 (-/-) ipsilateral retinae displayed similar pathology as those in Epac1 (+/+) mice. Our observations suggest that Epac2-deficiency led to more severe ischemic retinopathy after retinal ischemia/reperfusion injury.

Endothelin-1 overexpression exacerbate experimental allergic encephalomyelitis.

BACKGROUND: Multiple sclerosis (MS) is a CNS inflammatory demyelinating disorder. T helper 1 (Th1) and T helper 17 (Th17) cells are important in MS immunopathogenesis. Level of endothelin-1 (ET-1), a potent vasoconstrictor, is increased in sera of MS patients. We studied the role of ET-1 in experimental allergic encephalomyelitis (EAE), a MS animal model. METHODS: EAE is induced in transgenic mice overexpressing endothelial ET-1 (TET-1), transgenic mice overexpressing astrocytic ET-1 (GET-1) and non-transgenic (NTg) mice by immunization with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. EAE scores, spinal cord histology, serum proinflammatory cytokines levels, and proinflammatory cytokines production from splenocytes of ET-1 transgenic and NTg mice with EAE were studied. RESULTS: ET-1 transgenic mice developed more severe EAE than NTg with increased inflammation and demyelination in spinal cord. The mean maximum EAE scores for GET-1, TET-1 and NTg mice with EAE were 4.84, 4.31 and 4.05 respectively (p<0.05). Serum levels of IL-6, IL-17A, IFN-γ and TNF-α were higher in ET-1 transgenic than NTg mice with EAE (p<0.05) while serum IL-4 levels were similar. mRNA levels of IL-6, IL-17A, IFN-γ and TNF-α from cultured splenocytes were higher in ET-1-transgenic than NTg mice with EAE (p<0.05) while IL-4 mRNA levels were similar. Consistently, levels of IL-6, IL-17A, IFN-γ and TNF-α in culture media of splenocytes were higher in ET-1 transgenic than NTg mice with EAE (p<0.05) while IL-4 levels were similar. CONCLUSIONS: Mice with endothelial or astrocytic ET-1 overexpression developed more severe EAE with increased splenic lymphocyte production of Th1 and Th17 proinflammatory cytokines.

Nuclear factor of activated T cells 5 deficiency increases the severity of neuronal cell death in ischemic injury.

Nuclear factor of activated T cells 5 (NFAT5) has been implicated in regulating several genes that are thought to be neuroprotective in ischemic injury. Because of the embryonic lethality of NFAT5 knockout (NFAT5(-/-)) mice, the heterozygous (NFAT5(+/-)) mice were used to study the in vivo role of NFAT5 in hypoxia/ischemia (H/I) condition. The NFAT5(+/-) mice exhibited more severe neurological deficits, larger infarct area and edema formation associated with increased aquaporin 4 expressions in the brain. Under in vitro H/I condition, increased apoptotic cell death was found in NFAT5(-/-) neurons. Moreover, SMIT, a downstream to NFAT5, was upregulated in NFAT5(+/+) neurons, while the SMIT level could not be upregulated in NFAT5(-/-) neurons under H/I condition. The elevation of reactive oxygen species generation in NFAT5(-/-) neurons under H/I condition further confirmed that NFAT5(-/-) neurons were more susceptible to oxidative stress. The present study demonstrated that activation of NFAT5 and its downstream SMIT induction is important in protecting neurons from ischemia-induced oxidative stress.

Embryonic lethality in mice lacking the nuclear factor of activated T cells 5 protein due to impaired cardiac development and function.

Nuclear factor of activated T cells 5 protein (NFAT5) is thought to be important for cellular adaptation to osmotic stress by regulating the transcription of genes responsible for the synthesis or transport of organic osmolytes. It is also thought to play a role in immune function, myogenesis and cancer invasion. To better understand the function of NFAT5, we developed NFAT5 gene knockout mice. Homozygous NFAT5 null (NFAT5(-/-)) mouse embryos failed to develop normally and died after 14.5 days of embryonic development (E14.5). The embryos showed peripheral edema, and abnormal heart development as indicated by thinner ventricular wall and reduced cell density at the compact and trabecular areas of myocardium. This is associated with reduced level of proliferating cell nuclear antigen and increased caspase-3 in these tissues. Cardiomyocytes from E14.5 NFAT5(-/-) embryos showed a significant reduction of beating rate and abnormal Ca(2+) signaling profile as a consequence of reduced sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and ryanodine receptor (RyR) expressions. Expression of NFAT5 target genes, such as HSP 70 and SMIT were reduced in NFAT5(-/-) cardiomyocytes. Our findings demonstrated an essential role of NFAT5 in cardiac development and Ca(2+) signaling. Cardiac failure is most likely responsible for the peripheral edema and death of NFAT5(-/-) embryos at E14.5 days.

Targeted overexpression of endothelin-1 in astrocytes leads to more severe cytotoxic brain edema and higher mortality.

Transgenic mice overexpressing endothelin-1 (ET-1) in astrocytes (GET-1) displayed more severe brain edema and neurologic dysfunction after experimental ischemic stroke. However, it was not clear whether astrocytic ET-1 contributed to cytotoxic or vasogenic edema associated with stroke. In this study, the role of astrocytic ET-1 in cytotoxic edema and brain injury was investigated. Upon acute water intoxication, the GET-1 mice had a lower survival rate and more severe neurologic deficits. Such an exacerbated condition in the GET-1 mice may be a result of a significant increase in cerebral water content and increased expression of the water channel protein, aquaporin 4 (AQP-4). The GET-1 mice treated with OPC-31260, a nonpeptide arginine vasopressin V(2) receptor antagonist, were alleviated from the cerebral water accumulation and neurologic deficit during the early time period after water intoxication. In addition, a significant reduction of AQP-4 expression was observed in astrocytic end-feet AQP-4 in the hippocampus of the GET-1 mice treated with OPC-31260. Therefore, ET-1-induced AQP-4 expression and cerebral water accumulation are the key factors in brain edema associated with acute water intoxication. The V(2) receptor antagonist, OPC-31260, may be one of the effective drugs for the early treatment of ET-1-induced cytotoxic edema and brain injury.

Involvement of calcium mobilization from caffeine-sensitive stores in mechanically induced cell cycle arrest in the dinoflagellate Crypthecodinium cohnii.

Mechanical loads can profoundly alter cell growth and cell proliferation. The dinoflagellates are especially sensitive to mechanical stimulation. Many species will be arrested in cell cycle in response to turbulence or shear stress. We demonstrate here that mechanical shaking and caffeine, the ryanodine-receptor agonist, induced an elevation of cytosolic calcium in the dinoflagellate Crypthecodinium cohnii. Dantrolene, a ryanodine-receptor antagonist, dose-dependently inhibited both shaking-induced and caffeine-induced calcium release. Similar to the effect of mechanical shaking, caffeine alone dose-dependently and reversibly induced cell cycle arrest in dinoflagellates. Prolonged shaking substantially abolished the magnitude of caffeine-induced calcium release and vice-versa, suggesting that both agents released calcium from similar stores through ryanodine receptors. Fluorescence-conjugated ryanodine gave positive labeling, which could be blocked by ryanodine, in the cortice of C. cohnii cells. In addition, caffeine or shaking mobilized intracellular chlortetracycline (CTC)-positive membrane-bound calcium, which could be similarly depleted by t-BuBHQ, a SERCA pump inhibitor. Prior treatment with shaking or caffeine also inhibited the ability of the other agent in mobilizing CTC-positive calcium. CTC-positive microsomal fractions could also be induced to release calcium by caffeine and cADPR, the ryanodinee receptor modulator. t-BuBHQ, but not calcium ionophores, induced cell cycle arrest, and the calcium chelator BAPTA-AM was unable to rescue caffeine-induced cell cycle arrest. These data culminate to suggest that mobilization or depletion of caffeine-sensitive calcium stores, but not calcium elevation per se, is involved in the induction of cell cycle arrest by mechanical stimulation. The present study establishes the role of caffeine-sensitive calcium stores in the regulation of cell cycle progression.