Respective Wild-type Mice Research Articles

Characterization of metabolic phenotypes and distinctive genes in mice with low-weight gain.

Being overweight exacerbates various metabolic diseases, necessitating the identification of target molecules for obesity control. In the current study, we investigated common physiological features related to metabolism in mice with low weight gain: (1) G protein-coupled receptor, family C, group 5, member B-knockout; (2) gastric inhibitory polypeptide receptor-knockout; and (3) Iroquois-related homeobox3-knockout. Moreover, we explored genes involved in metabolism by analyzing differentially expressed genes (DEGs) between low-weight gain mice and the respective wild-type control mice. The common characteristics of the low-weight gain mice were low inguinal white adipose tissue (iWAT) and liver weight despite similar food intake along with lower blood leptin levels and high energy expenditure. The DEGs of iWAT, epididymal (gonadal) WAT, brown adipose tissue, muscle, liver, hypothalamus, and hippocampus common to these low-weight gain mice were designated as candidate genes associated with metabolism. One such gene tetraspanin 7 (Tspan7) from the iWAT was validated using knockout and overexpressing mouse models. Mice with low Tspan7 expression gained more weight, while those with high Tspan7 expression gained less weight, confirming the involvement of the Tspan7 gene in weight regulation. Collectively, these findings suggest that the candidate gene list generated in this study contains potential target molecules for obesity regulation. Further validation and additional data from low-weight gain mice will aid in understanding the molecular mechanisms associated with obesity.

Transcriptomic profiling of the response to excess iodide in Keap1 hypomorphic mice reveals new gene-environment interactions in thyroid homeostasis

Iodide plays a pivotal role in thyroid homeostasis due to its crucial involvement in thyroid hormone biosynthesis. Exposure to pharmacological doses of iodide elicits in the thyroid an autoregulatory response to preserve thyroid function, as well as an antioxidant response that is mediated by the Keap1/Nrf2 signaling pathway. The objective of the present study was to investigate the transcriptional response of the thyroid to excess iodide in a background of enhanced Nrf2 signaling. Keap1 knockdown (Keap1KD) mice that have activated Nrf2 signaling were exposed or not to excess iodide in their drinking water for seven days and compared to respective wild-type mice. RNA-sequencing of individual mouse thyroids identified distinct transcriptomic patterns in response to iodide, with Keap1KD mice showing an attenuated inflammatory response, altered thyroidal autoregulation, and enhanced cell growth/proliferative signaling, as confirmed also by Western blotting for key proteins involved in antioxidant, autoregulatory and proliferative responses. These findings underscore novel gene-environment interactions between the activation status of the Keap1/Nrf2 antioxidant response system and the dietary iodide intake, which may have implications not only for the goiter phenotype of Keap1KD mice but also for humans harboring genetic variations in KEAP1 or NFE2L2 or treated with Nrf2-modulating drugs.

Impaired mitochondrial quality control in skeletal muscles from C57 and D2 mdx model of Duchenne Muscular Dystrophy

Mitochondrial dysfunction is an early pathological feature of Duchenne muscular dystrophy (DMD) and precedes myopathy. Balanced mitochondrial quality control (e.g., fission, fusion, biogenesis, autophagy, and mitophagy) is essential to maintain the overall mitochondrial health and skeletal muscle function. DBA/2J mdx (D2- mdx) has emerged as a more relevant mouse model of DMD as it more closely mirrors early stage of DMD pathology in humans than the C57BL/10 mdx (C57 -mdx) model. The purpose of this study was to determine whether the expression of proteins responsible for mitochondrial quality control were dysregulated in skeletal muscles from these two mouse models of DMD. We hypothesized that the expression of protein markers of mitochondrial fission and mitophagy would be higher, while markers of mitochondrial fusion would be lower in both models of DMD with greater alterations in D2- mdx mice. 8-10-week-old male C57 -mdx and D2- mdx mice, and their respective wildtype (WT) mice were used in this study. Muscle strength was measured using hang wire test. Gastrocnemius muscles were collected for immunoblot analysis to assess mitochondrial quality control proteins. Hang wire impulse, an indicator of muscle strength, was significantly lower in both models of DMD in comparison to their WT controls (main effect of mdx, P = 0.01). Regarding mitochondrial fission, while there was no difference in protein expression and phosphorylation (Ser 616) of Dynamin-Related Protein 1 (Drp1) between any groups, mitochondrial fission 1 protein (Fis1) was markedly higher in skeletal muscles from both models of DMD when compared to WT controls (main effect of mdx, P=0.0001). Greater expression of Fis1 was observed in D2- mdx than C57- mdx when compared to their respective WT controls (~5.7-fold vs. 3.8-fold). Furthermore, mitochondrial fusion marker Mitofusion 1 (Mfn1) was significantly lower in both models of DMD (main effect of mdx, P=0.029), however, it appears to be mainly driven by the reduction in D2-mdx (mdx x strain interaction, P=0.019). No difference of Mfn1 expression was found in C57-mdx mice. Finally, expression of protein markers of mitochondrial biogenesis (PGC1 a) and autophagy (LC3B) were higher in both models of DMD when compared to their respective WT controls (main effect of mdx, P=0.0001 and P=0.001, respectively). In conclusion, these data suggest that there is dysregulated mitochondrial dynamics towards pro-fission state in skeletal muscle from mdx mice with an exacerbated effect in D2- mdx model, which may contribute to the muscle weakness phenotype associated with DMD This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genetic loss of AMPK-glycogen binding destabilises AMPK and disrupts metabolism

ObjectiveGlycogen is a major energy reserve in liver and skeletal muscle. The master metabolic regulator AMP-activated protein kinase (AMPK) associates with glycogen via its regulatory β subunit carbohydrate-binding module (CBM). However, the physiological role of AMPK-glycogen binding in energy homeostasis has not been investigated in vivo. This study aimed to determine the physiological consequences of disrupting AMPK-glycogen interactions. MethodsGlycogen binding was disrupted in mice via whole-body knock-in (KI) mutation of either the AMPK β1 (W100A) or β2 (W98A) isoform CBM. Systematic whole-body, tissue and molecular phenotyping was performed in KI and respective wild-type (WT) mice. ResultsWhile β1 W100A KI did not affect whole-body metabolism or exercise capacity, β2 W98A KI mice displayed increased adiposity and impairments in whole-body glucose handling and maximal exercise capacity relative to WT. These KI mutations resulted in reduced total AMPK protein and kinase activity in liver and skeletal muscle of β1 W100A and β2 W98A, respectively, versus WT mice. β1 W100A mice also displayed loss of fasting-induced liver AMPK total and α-specific kinase activation relative to WT. Destabilisation of AMPK was associated with increased fat deposition in β1 W100A liver and β2 W98A skeletal muscle versus WT. ConclusionsThese results demonstrate that glycogen binding plays critical roles in stabilising AMPK and maintaining cellular, tissue and whole-body energy homeostasis.

Impaired Endothelial Caveolin‐1‐TRPV4 Channel Signaling Contributes to Endothelial Dysfunction in Pulmonary Hypertension

Impaired endothelium‐dependent vasodilation is a key contributor to elevated pulmonary arterial pressure (PAP) in pulmonary hypertension (PH). While the mechanisms of pathogenesis of PH remain elusive, recent studies show that spatially localized Ca2+ influx via endothelial transient receptor potential vanilloid 4 (TRPV4EC) channels promotes vasodilation in resistance‐sized pulmonary arteries (PAs) by activating endothelial nitric oxide synthase (eNOS) and releasing nitric oxide (NO). Caveolin‐1 (Cav‐1EC) is an essential structural protein within the pulmonary endothelium, where it has been found to associate with TRPV4EC channels in cultured endothelial cells. We hypothesized that Cav‐1EC regulates TRPV4EC channel activity and PAP, and that impaired Cav‐1EC‐TRPV4EC signaling contributes to elevated PAP in PH. To test these hypotheses, we used right ventricular systolic pressure (RVSP) and PAP measurements in normoxic or chronic hypoxic Cav‐1EC−/− and TRPV4EC−/− mice, and pressure myography and high‐speed Ca2+ imaging studies in PAs from these mice. TRPV4EC channel activity and TRPV4EC‐mediated vasodilation were significantly impaired in PAs from Cav‐1EC−/− mice, suggesting that Cav‐1EC enhances TRPV4EC channel activity in PAs. Purinergic signaling plays an important role in pulmonary circulation, and we recently identified adenosine triphosphate (ATP) as a novel physiological activator of TRPV4EC channels in PAs. ATP activation of TRPV4EC channels was abolished by protein kinase C (PKC) inhibition, and was absent in the arteries from Cav‐1EC−/− mice, supporting a Cav‐1EC‐PKC‐TRPV4EC signaling that enables ATP activation of TRPV4‐EC channels. Resting RVSP and PAP were elevated in Cav‐1EC−/− and TRPV4EC−/− mice supporting the concept that Cav‐1EC‐TRPV4EC signaling lowers resting PAP. Moreover, exposure to chronic hypoxia (CH, 3 weeks) resulted in higher increases in RVSPs in Cav‐1EC−/− and TRPV4EC−/− mice when compared to the respective wild‐type control mice, suggesting that Cav‐1EC‐TRPV4EC channel signaling is protective against CH‐induced PH. We further used two well‐established models of PH— CH and sugen 5416 injection + CH. TRPV4EC channel activity, and ATP‐ and PKC‐activation of TRPV4EC channels were impaired in PAs from both the models of PH, suggesting that Cav‐1EC‐PKC‐TRPV4EC signaling may be impaired in PH. Taken together, these results suggest that Cav‐1EC‐PKC‐TRPV4EC signaling lowers resting PAP, and is impaired in PH. Deciphering the mechanisms that impair the Cav‐1EC‐PKC‐TRPV4EC signaling in PH may lead to novel therapeutic strategies to restore endothelium‐dependent vasodilation in PH.Support or Funding InformationHL121484‐01, HL‐138496

Impaired muscle spindle function in murine models of muscular dystrophy.

Muscular dystrophy patients suffer from progressive degeneration of skeletal muscle fibres, sudden spontaneous falls, balance problems, as well as gait and posture abnormalities. Dystrophin- and dysferlin-deficient mice, models for different types of muscular dystrophy with different aetiology and molecular basis, were characterized to investigate if muscle spindle structure and function are impaired. The number and morphology of muscle spindles were unaltered in both dystrophic mouse lines but muscle spindle resting discharge and their responses to stretch were altered. In dystrophin-deficient muscle spindles, the expression of the paralogue utrophin was substantially upregulated, potentially compensating for the dystrophin deficiency. The results suggest that muscle spindles might contribute to the motor problems observed in patients with muscular dystrophy. Muscular dystrophies comprise a heterogeneous group of hereditary diseases characterized by progressive degeneration of extrafusal muscle fibres as well as unstable gait and frequent falls. To investigate if muscle spindle function is impaired, we analysed their number, morphology and function in wildtype mice and in murine model systems for two distinct types of muscular dystrophy with very different disease aetiology, i.e. dystrophin- and dysferlin-deficient mice. The total number and the overall structure of muscle spindles in soleus muscles of both dystrophic mouse mutants appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and β-dystroglycan in intrafusal fibres outside the region of contact with the sensory neuron. While utrophin was absent from the central part of intrafusal fibres of wildtype mice, it was substantially upregulated in dystrophin-deficient mice. Single-unit extracellular recordings of sensory afferents from muscle spindles of the extensor digitorum longus muscle revealed that muscle spindles from both dystrophic mouse strains have an increased resting discharge and a higher action potential firing rate during sinusoidal vibrations, particularly at low frequencies. The response to ramp-and-hold stretches appeared unaltered compared to the respective wildtype mice. We observed no exacerbated functional changes in dystrophin and dysferlin double mutant mice compared to the single mutant animals. These results show alterations in muscle spindle afferent responses in both dystrophic mouse lines, which might cause an increased muscle tone, and might contribute to the unstable gait and frequent falls observed in patients with muscular dystrophy.

Hepatocyte peroxisome proliferator-activated receptor α regulates bile acid synthesis and transport

Peroxisome proliferator-activated receptor alpha (PPARα) controls lipid homeostasis through regulation of lipid transport and catabolism. PPARα activators are clinically used for hyperlipidemia treatment. The role of PPARα in bile acid (BA) homeostasis is beginning to emerge. Herein, Ppara-null and hepatocyte-specific Ppara-null (Ppara∆Hep) as well as the respective wild-type mice were treated with the potent PPARα agonist Wy-14,643 (Wy) and global metabolomics performed to clarify the role of hepatocyte PPARα in the regulation of BA homeostasis. Levels of all serum BAs were markedly elevated in Wy-treated wild-type mice but not in Ppara-null and Ppara∆Hep mice. Gene expression analysis showed that PPARα activation (1) down-regulated the expression of sodium-taurocholate acid transporting polypeptide and organic ion transporting polypeptide 1 and 4, responsible for the uptake of BAs into the liver; (2) decreased the expression of bile salt export pump transporting BA from hepatocytes into the bile canaliculus; (3) upregulated the expression of multidrug resistance-associated protein 3 and 4 transporting BA from hepatocytes into the portal vein. Moreover, there was a notable increase in the compositions of serum, hepatic and biliary cholic acid and taurocholic acid following Wy treatment, which correlated with the upregulated expression of the Cyp8b1 gene encoding sterol 12α-hydroxylase. The effects of Wy were identical between the Ppara∆Hep and Ppara-null mice. Hepatocyte PPARα controlled BA synthesis and transport not only via direct transcriptional regulation but also via crosstalk with hepatic farnesoid X receptor signaling. These findings underscore a key role for hepatocyte PPARα in the control of BA homeostasis.

Mispositioned Neurokinin-1 Receptor-Expressing Neurons Underlie Heat Hyperalgesia in Disabled-1 Mutant Mice.

Reelin (Reln) and Disabled-1 (Dab1) participate in the Reln-signaling pathway and when either is deleted, mutant mice have the same spinally mediated behavioral abnormalities, increased sensitivity to noxious heat and a profound loss in mechanical sensitivity. Both Reln and Dab1 are highly expressed in dorsal horn areas that receive and convey nociceptive information, Laminae I-II, lateral Lamina V, and the lateral spinal nucleus (LSN). Lamina I contains both projection neurons and interneurons that express Neurokinin-1 receptors (NK1Rs) and they transmit information about noxious heat both within the dorsal horn and to the brain. Here, we ask whether the increased heat nociception in Reln and dab1 mutants is due to incorrectly positioned dorsal horn neurons that express NK1Rs. We found more NK1R-expressing neurons in Reln-/- and dab1-/- Laminae I-II than in their respective wild-type mice, and some NK1R neurons co-expressed Dab1 and the transcription factor Lmx1b, confirming their excitatory phenotype. Importantly, heat stimulation in dab1-/- mice induced Fos in incorrectly positioned NK1R neurons in Laminae I-II. Next, we asked whether these ectopically placed and noxious-heat responsive NK1R neurons participated in pain behavior. Ablation of the superficial NK1Rs with an intrathecal injection of a substance P analog conjugated to the toxin saporin (SSP-SAP) eliminated the thermal hypersensitivity of dab1-/- mice, without altering their mechanical insensitivity. These results suggest that ectopically positioned NK1R-expressing neurons underlie the heat hyperalgesia of Reelin-signaling pathway mutants, but do not contribute to their profound mechanical insensitivity.

Isoform‐specific Pde3 knockout mice spontaneously develop pulmonary hypertension

Pulmonary hypertension (PH) is associated with vasoconstriction and vascular remodeling characterized by pulmonary artery smooth muscle cell (PASMC) and microvascular endothelial cell (PMVEC) proliferation and dysfunction. Phosphodiesterase 3 (PDE3), of which there are two isoforms (3A and 3B), hydrolyzes cAMP and cGMP, both critical cyclic nucleotides in cellular function. While both isoforms are present in the pulmonary vasculature, their specific contributions to the regulation of pulmonary vascular tone are unclear. Non‐selective PDE3 inhibitors have been used to treat PH for their vasodilatory effect, presumably by increasing cellular cAMP levels. We hypothesize that isoform‐selective knockout of PDE3 may reveal distinct roles in the regulation of pulmonary vascular tone and/or PH pathogenesis. Adult (8–10 weeks) Pde3a−/− (3A‐KO), Pde3b−/− (3B‐KO), and their respective wild type (WT) mice (3A‐WT, 3B‐WT) were exposed to normoxia or normobaric hypoxia (10% O2) for 28 days. Consistent with previous reports, we found that 3A‐WT and 3B‐WT mice exposed to 10% O2 for 28 days had right ventricular (RV) hypertrophy as evidenced by an increased RV/[left ventricle (LV)+septum (S)] weight ratio relative to mice exposed to 21% O2 [p < 0.001; 0.38 ± 0.01 vs 0.25 ± 0.01 (3A) and 0.35 ± 0.01 vs 0.24 ± 0.01 (3B)]. In hypoxia, 3A‐WT and 3B‐WT mice had elevated pulmonary artery pressures estimated by 2D echocardiography measurements of pulmonary acceleration time/ejection time (PAT/PET) (p < 0.0001; 0.21 ± 0.004 vs 0.29 ± 0.01 (3A) and 0.21 ± 0.01 vs 0.31 ± 0.01 (3B) relative to mice exposed to 21% O2. Interestingly, in normoxia both 3A‐KO (p = 0.012; 0.25 ± 0.004 vs 0.29 ± 0.01 and 3B‐KO (p = 0.023; 0.26 ± 0.01 vs 0.31 ± 0.01) mice had evidence of elevated pulmonary artery pressures relative to their respective WT mice, as evidenced by a lower PAT/PET via echocardiography. Consistent with these results, in normoxia RV/(LV+S) was higher in both 3A‐KO (0.29 ± 0.01 vs 0.25 ± 0.01, p = 0.0008) and 3B‐KO (0.31 ± 0.01 vs 0.24 ± 0.01, p < 0.0001) than in their respective normoxiaexposed WT. Normoxia‐exposed 3B‐KO mice also had elevated right ventricular systolic pressure compared to normoxia‐exposed 3B‐WT (28.9 ± 0.7 mmHg vs 24.2 ± 1.2 mmHg, p = 0.0028). In hypoxia, there was no difference in RV/(LV+S) between 3A‐WT and 3A‐KO mice, although lower estimated pulmonary artery pressures were seen in the 3A‐KO mice (p = 0.0043). Conversely, in hypoxia there was a trend towards higher RV/(LV+S) in the 3B‐KO compared to 3B‐WT mice (p = 0.0532; 0.42 ± 0.02 vs 0.35 vs ± 0.01), but no difference was seen in pulmonary artery pressures between hypoxia‐exposed, 3B‐KO and 3B‐WT mice. Our data suggest a complex and isoform‐specific role of the Pde3 genes in the pathogenesis of PH associated with hypoxia. Furthermore, these data suggest a key role for each of the PDE3 isoforms in the maintenance of the low pulmonary vascular resistance found in normal lungs.Support or Funding Information5K08HL105677‐01A1, R01HL136963‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Estrogen and oxytocin involvement in social preference in male mice: a study using a novel long-term social preference paradigm with aromatase, estrogen receptor-α and estrogen receptor-β, oxytocin, and oxytocin receptor knockout male mice.

Certain aspects of social behavior help animals make adaptive decisions during encounters with other animals. When mice choose to approach another conspecific, the motivation and preference behind the interaction is not well understood. Estrogen and oxytocin are known to influence a wide array of social behaviors, including social motivation and social preference. The present study investigated the effects of estrogen and oxytocin on social preference using aromatase (ArKO), estrogen receptor (ER) α (αERKO), ERβ (βERKO), oxytocin (OTKO), oxytocin receptor (OTRKO) knockout and their respective wild-type (WT) male mice. Mice were presented with gonadally-intact versus castrated male (IC), intact male versus ovariectomized female (IF), or intact male versus empty cage (IE) stimuli sets for 5 days. ArWT showed no preference for either stimuli in IC and IF and intact male preference in IE, but ArKO mice preferred a castrated male or an ovariectomized female, or had no preference for either stimulus in IC, IF and IE stimuli sets, respectively, suggesting reduced intact male preference. α and β WT mice preferred a castrated male, showed no preference, and preferred an intact male in IC, IF and IE, respectively. αERKO mice displayed similar modified social preference patterns as ArKO, whereas the social preference of βERKO mice remained similar to βWT. OTWT preferred a castrated male whereas OTKO, OTRWT and OTRKO mice failed to show any preference in IC and none showed preference for either stimuli in IF. Collectively, these findings suggest that estrogen regulates social preference in male mice and that impaired social preference in oxytocin-deficient mice may be due to severe deficits in social recognition.

The impact of chemerin or chemokine-like receptor 1 loss on the mouse gut microbiome

Chemerin is an adipocyte derived signalling molecule (adipokine) that serves as a ligand activator of Chemokine-like receptor 1(CMKLR1). Chemerin/CMKLR1 signalling is well established to regulate fundamental processes in metabolism and inflammation. The composition and function of gut microbiota has also been shown to impact the development of metabolic and inflammatory diseases such as obesity, diabetes and inflammatory bowel disease. In this study, we assessed the microbiome composition of fecal samples isolated from wildtype, chemerin, or CMKLR1 knockout mice using Illumina-based sequencing. Moreover, the knockout mice and respective wildtype mice used in this study were housed at different universities allowing us to compare facility-dependent effects on microbiome composition. While there was no difference in alpha diversity within samples when compared by either facility or genotype, we observed a dramatic difference in the presence and abundance of numerous taxa between facilities. There were minor differences in bacterial abundance between wildtype and chemerin knockout mice, but significantly more differences in taxa abundance between wildtype and CMKLR1 knockout mice. Specifically, CMKLR1 knockout mice exhibited decreased abundance of Akkermansia and Prevotella, which correlated with body weight in CMKLR1 knockout, but not wildtype mice. This is the first study to investigate a linkage between chemerin/CMKLR1 signaling and microbiome composition. The results of our study suggest that chemerin/CMKLR1 signaling influences metabolic processes through effects on the gut microbiome. Furthermore, the dramatic difference in microbiome composition between facilities might contribute to discrepancies in the metabolic phenotype of CMKLR1 knockout mice reported by independent groups. Considered altogether, these findings establish a foundation for future studies to investigate the relationship between chemerin signaling and the gut microbiome on the development and progression of metabolic and inflammatory disease.

MMP‐12 knockout mouse: Is it a relevant animal model to study the role of MMP‐12 in post‐stroke brain damage?

We recently reported for the first time the detrimental role of matrix metalloproteinase (MMP)‐12 on post‐stroke brain damage. We hypothesize that the post‐stroke MMPs dysregulation is similar across species and the genetic deletion of MMP‐12 does not affect the post‐stroke expression of other MMPs. We tested our hypothesis by determining the pre‐ and post‐ischemic expression profile of MMPs in MMP‐12 knockout and respective wild type mice. Focal cerebral ischemia was induced in MMP‐12 knockout and wild type mice by middle cerebral artery occlusion procedure by insertion of a monofilament suture. One hour after ischemia, reperfusion was initiated by removing the monofilament. One‐day after reperfusion, ischemic brain tissues from various groups of mice were collected for real‐time PCR analysis and PCR analysis followed by agarose gel electrophoresis. Although the post‐stroke regulation of several MMPs was altered in wild type mice, huge upregulation of MMP‐12 (~763 fold over sham) was noticed in mice similar to rats as reported earlier by our group. Unlike in rats, MMP‐8 was prominently upregulated (~280 fold over sham) in ischemic mice brains. As expected, MMP‐12 expression is absent in the ischemic brains of MMP‐12 knockout mice. Except the expression of MMP‐9, the expression of several other MMPs such as MMP‐1a, −2, −8, −10, −15, −16, −17, −21 and −28 was altered in the ischemic brain of MMP‐12 knockout mice as compared to wild type mice under similar experimental conditions. In addition, the expression of various MMPs in MMP‐12 knockout mice under non‐ischemic conditions was altered as compared to the wild type mice that did not subject to ischemia. Based on our results, we conclude that the mice with genetic deletion of MMP‐12 are constitutive in nature that warrants the attention of their use in elucidating the role of MMP‐12 in post‐stroke brain damage.Support or Funding InformationThis work was supported in part by the OSF HealthCare Foundation and OSF Illinois Neurological Institute, Peoria, IL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cannabinoid CB2 Agonist GW405833 Suppresses Inflammatory and Neuropathic Pain through a CB1 Mechanism that is Independent of CB2 Receptors in Mice.

GW405833, widely accepted as a cannabinoid receptor 2 (CB2) agonist, suppresses pathologic pain in preclinical models without the unwanted central side effects of cannabinoid receptor 1 (CB1) agonists; however, recent in vitro studies have suggested that GW405833 may also behave as a noncompetitive CB1 antagonist, suggesting that its pharmacology is more complex than initially appreciated. Here, we further investigated the pharmacologic specificity of in vivo antinociceptive actions of GW405833 in models of neuropathic (i.e., partial sciatic nerve ligation model) and inflammatory (i.e., complete Freund's adjuvant model) pain using CB2 and CB1 knockout (KO) mice, their respective wild-type (WT) mice, and both CB2 and CB1 antagonists. GW405833 (3, 10, and 30 mg/kg i.p.) dose dependently reversed established mechanical allodynia in both pain models in WT mice; however, the antiallodynic effects of GW405833 were fully preserved in CB2KO mice and absent in CB1KO mice. Furthermore, the antiallodynic efficacy of GW405833 (30 mg/kg i.p.) was completely blocked by the CB1 antagonist rimonabant (10 mg/kg i.p.) but not by the CB2 antagonist SR144528 (10 mg/kg i.p.). Thus, the antinociceptive properties of GW405833 are dependent on CB1 receptors. GW405833 (30 mg/kg i.p.) was also inactive in a tetrad of tests measuring cardinal signs of CB1 activation. Additionally, unlike rimonabant (10 mg/kg i.p.), GW405833 (10 mg/kg, i.p.) did not act as a CB1 antagonist in vivo to precipitate withdrawal in mice treated chronically with Δ9-tetrahydrocannabinol. The present results suggest that the antiallodynic efficacy of GW405833 is CB1-dependent but does not seem to involve engagement of the CB1 receptor's orthosteric site.

Different Susceptibilities between Apoe- and Ldlr-Deficient Mice to Inflammation-Associated Colorectal Carcinogenesis.

Hypercholesterolemia resulting in atherosclerosis is associated with an increased risk of ischemic heart disease and colorectal cancer (CRC). However, the roles of apoliprotein (Apo) E (Apoe) and low-density lipoprotein (Ldl) receptor (Ldlr) in colorectal carcinogenesis have not yet been investigated. In this study, we examined the susceptibility of Apoe-deficient and Ldlr-deficient mice, which are genetic animal models of atherosclerosis to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. In Experiment 1, male Apoe-deficient (n = 20) and wild type (WT) mice (C57BL/6J, n = 21) were treated with a single intraperitoneal (i.p.) injection of AOM (10 mg/kg body weight) and then given 1.5% DSS in drinking water for seven days. They were maintained up to week 20 and sacrificed for the histopathological examination of colorectal tumors. The mRNA expression of cyclooxygenase (Cox)-2, inducible nitric oxide synthase (Nos2), tumor necrosis factor (Tnf)-α interleukin (Il)-1β, and Il-6 was assayed in the colorectal mucosa. In Experiment 2, male Ldlr-deficient (n = 14) and WT mice (C57BL/6J, n = 10) were given a single i.p. injection of AOM (10 mg/kg body weight) and then given 2% DSS in drinking water for seven days. They were sacrificed at week 20 to evaluate their colorectum histopathologically. In Experiment 1, the multiplicity of CRCs was significantly higher in the Apoe-deficient mice (2.75 ± 1.48) than in the WT mice (0.62 ± 0.67). The serum lipoprotein levels in the Apoe-deficient mice were also significantly higher than in the WT mice. In Experiment 2, the incidence (29%) and multiplicity (0.50 ± 0.94) of CRCs in the Ldlr mice were significantly lower than in the WT mice (80% incidence and 3.10 ± 2.38 multiplicity). The mRNA expression of two inducible enzymes and certain pro-inflammatory cytokines in the colorectum of each genotype was greater than in the respective WT mice. The values in the Apoe-deficient mice were much greater than in the Ldlr mice. These findings suggest that Apoe-deficient mice showed increased susceptibility to inflammation-associated colorectal carcinogenesis due to their high reactivity to inflammatory stimuli.

Interleukin-18 Mediates Immune Responses to Campylobacter jejuni Infection in Gnotobiotic Mice.

BackgroundHuman Campylobacter jejuni infections are progressively rising worldwide. Information about the molecular mechanisms underlying campylobacteriosis, however, are limited. In the present study we investigated whether cytokines such as IL-23, IL-22 and IL-18, which share pivotal functions in host immunity, were involved in mediating intestinal and systemic immunopathological responses upon C. jejuni infection.Methodology/Principal FindingsTo assure stable infection, gnotobiotic (i.e. secondary abiotic) IL-23p19-/-, IL-22-/- and IL-18-/- mice were generated by broad-spectrum antibiotic treatment. Following peroral C. jejuni strain 81–176 infection, mice of all genotypes harbored comparably high pathogenic loads in their intestines. As compared to wildtype controls, however, IL-18-/- mice displayed less distinct C. jejuni induced sequelae as indicated by less pronounced large intestinal shrinkage and lower numbers of apoptotic cells in the colonic epithelial layer at day 8 postinfection (p.i.). Furthermore, lower colonic numbers of adaptive immune cells including regulatory T cells and B lymphocytes were accompanied by less distinct secretion of pro-inflammatory cytokines such as TNF and IFN-γ and lower IL-17A mRNA expression levels in colonic ex vivo biopsies of infected IL-18-/- as compared to wildtype mice. Upon C. jejuni infection, colonic IL-23p19 expression was up-regulated in IL-18-/- mice only, whereas IL-22 mRNA levels were lower in uninfected and infected IL-23p19-/- as well as infected IL-18-/- as compared to respective wildtype control mice. Remarkably, not only intestinal, but also systemic infection-induced immune responses were less pronounced in IL-18-/- mice as indicated by lower TNF, IFN-γ and IL-6 serum levels as compared to wildtype mice.Conclusion/SignificanceWe here show for the first time that IL-18 is essentially involved in mediating C. jejuni infection in the gnotobiotic mouse model. Future studies need to further unravel the underlying regulatory mechanisms orchestrating pathogen-host interaction.

The CRF1 and the CRF2 receptor mediate recognition memory deficits and vulnerability induced by opiate withdrawal

Opiate use disorders are associated with impaired cognitive function and altered stress-responsive systems. The corticotropin-releasing factor (CRF) system mediates stress responses via CRF1 and CRF2 receptors and may be implicated in substance use disorders. However, the specific role for each of the two known CRF receptor subtypes in cognitive impairment induced by opiate administration and withdrawal remains to be elucidated. In the present study, CRF1−/−, CRF2−/− and their respective wild-type mice are injected with escalating doses of morphine and cognitive function assessed by the novel object recognition (NOR) memory task throughout relatively long periods of opiate withdrawal. Early (2 days) phases of opiate withdrawal impair NOR memory in wild-type, CRF1−/− and CRF2−/− mice. However, the duration of opiate withdrawal-induced NOR memory deficits is prolonged in CRF1−/− but shortened in CRF2−/− mice, as compared to their respective wild-type mice, indicating opposite roles for the two CRF receptor subtypes. Nevertheless, following apparent recovery, exposure to an environmental stressor induces the reemergence of NOR memory deficits in long-term opiate-withdrawn wild-type but not CRF1−/− or CRF2−/− mice, indicating an essential role for both CRF receptor subtypes in stress vulnerability. These findings bring initial evidence of a complex physiopathological role for the CRF system in cognitive deficits and the long-lasting vulnerability induced by opiate drugs.

Genetic manipulation of cardiac Hsp72 levels does not alter substrate metabolism but reveals insights into high-fat feeding-induced cardiac insulin resistance.

Heat shock protein 72 (Hsp72) protects cells against a variety of stressors, and multiple studies have suggested that Hsp72 plays a cardioprotective role. As skeletal muscle Hsp72 overexpression can protect against high-fat diet (HFD)-induced insulin resistance, alterations in substrate metabolism may be a mechanism by which Hsp72 is cardioprotective. We investigated the impact of transgenically overexpressing (Hsp72 Tg) or deleting Hsp72 (Hsp72 KO) on various aspects of cardiac metabolism. Mice were fed a normal chow (NC) or HFD for 12weeks from 8weeks of age to examine the impact of diet-induced obesity on metabolic parameters in the heart. The HFD resulted in an increase in cardiac fatty acid oxidation and a decrease in cardiac glucose oxidation and insulin-stimulated cardiac glucose clearance; however, there was no difference in Hsp72 Tg or Hsp72 KO mice in these rates compared with their respective wild-type control mice. Although HFD-induced cardiac insulin resistance was not rescued in the Hsp72 Tg mice, it was preserved in the skeletal muscle, suggesting tissue-specific effects of Hsp72 overexpression on substrate metabolism. Comparison of two different strains of mice (BALB/c vs. C57BL/6J) also identified strain-specific differences in regard to HFD-induced cardiac lipid accumulation and insulin resistance. These strain differences suggest that cardiac lipid accumulation can be dissociated from cardiac insulin resistance. Our study finds that genetic manipulation of Hsp72 does not lead to alterations in metabolic processes in cardiac tissue under resting conditions, but identifies mouse strain-specific differences in cardiac lipid accumulation and insulin-stimulated glucose clearance.

Null mutation of 5α-reductase type I gene alters ethanol consumption patterns in a sex-dependent manner.

The neuroactive steroid allopregnanolone (ALLO) is a positive modulator of GABAA receptors, and manipulation of neuroactive steroid levels via injection of ALLO or the 5α-reductase inhibitor finasteride alters ethanol self-administration patterns in male, but not female, mice. The Srd5a1 gene encodes the enzyme 5α-reductase-1, which is required for the synthesis of ALLO. The current studies investigated the influence of Srd5a1 deletion on voluntary ethanol consumption in male and female wildtype (WT) and knockout (KO) mice. Under a continuous access condition, 6 and 10 % ethanol intake was significantly greater in KO versus WT females, but significantly lower in KO versus WT males. In 2-h limited access sessions, Srd5a1 deletion retarded acquisition of 10 % ethanol intake in female mice, but facilitated it in males, versus respective WT mice. The present findings demonstrate that the Srd5a1 gene modulates ethanol consumption in a sex-dependent manner that is also contingent upon ethanol access condition and concentration.

5-Lypoxygenase products are involved in renal tubulointerstitial injury induced by albumin overload in proximal tubules in mice.

The role of albumin overload in proximal tubules (PT) in the development of tubulointerstitial injury and, consequently, in the progression of renal disease has become more relevant in recent years. Despite the importance of leukotrienes (LTs) in renal disease, little is known about their role in tubulointerstitial injury. The aim of the present work was to investigate the possible role of LTs on tubulointerstitial injury induced by albumin overload. An animal model of tubulointerstitial injury challenged by bovine serum albumin was developed in SV129 mice (wild-type) and 5-lipoxygenase-deficient mice (5-LO–/–). The changes in glomerular morphology and nestin expression observed in wild-type mice subjected to kidney insult were also observed in 5-LO–/– mice. The levels of urinary protein observed in the 5-LO–/– mice subjected or not to kidney insult were lower than those observed in respective wild-type mice. Furthermore, the increase in lactate dehydrogenase activity, a marker of tubule damage, observed in wild-type mice subjected to kidney insult did not occur in 5-LO–/– mice. LTB4 and LTD4, 5-LO products, decreased the uptake of albumin in LLC-PK1 cells, a well-characterized porcine PT cell line. This effect correlated with activation of protein kinase C and inhibition of protein kinase B. The level of proinflammatory cytokines, tumor necrosis factor-α and interleukin (IL)-6, increased in mice subjected to kidney insult but this effect was not modified in 5-LO–/– mice. However, 5-LO–/– mice subjected to kidney insult presented lower macrophage infiltration and higher levels of IL-10 than wild-type mice. Our results reveal that LTs have an important role in tubulointerstitial disease induced by albumin overload.

Toll-like receptor 7-mediated type I interferon signaling prevents cholestasis- and hepatotoxin-induced liver fibrosis

Toll-like receptor 7 (TLR7) signaling predominantly regulates production of type I interferons (IFNs), which has been suggested in clinical studies to be antifibrotic. However, the mechanistic role of the TLR7-type I IFN axis in liver fibrosis has not been elucidated. In the present study, liver fibrosis was induced in wild-type (WT), TLR7-deficient, and IFN-α/β receptor-1 (IFNAR1)-deficient mice and TLR7-mediated signaling was assessed in liver cells isolated from these mice. TLR7-deficient and IFNAR1-deficient mice were more susceptible to liver fibrosis than WT mice, indicating that TLR7-type I IFN signaling exerts a protective effect against liver fibrosis. Notably, the hepatic expression of interleukin-1 receptor antagonist (IL-1ra) was suppressed in TLR7- or IFNAR1-deficient mice compared with respective WT mice, and treatment with recombinant IL-1ra reduced liver fibrosis. In vivo activation of TLR7 significantly increased IFNa4 and IL-1ra expression in the liver. Interestingly, each cytokine had a different cellular source, showing that dendritic cells (DCs) are the responsible cell type for production of type I IFN, while Kupffer cells (KCs) mainly produce IL-1ra in response to type I IFN. Furthermore, TLR7 activation by R848 injection suppressed liver fibrosis and production of proinflammatory cytokines, and these effects were dependent on type I IFN signaling. Consistent with in vivo data, IFN-α significantly induced IL-1ra production in primary KCs. TLR7 signaling activates DCs to produce type I IFN, which in turn induces antifibrogenic IL-1ra production in KCs. Thus, manipulation of the TLR7-type I IFN-IL-1ra axis may be a new therapeutic strategy for the treatment of liver fibrosis.