Gender Separation Increases Somatic Growth in Females but Does Not Affect Lifespan in Nothobranchius furzeri

According to life history theory, physiological and ecological traits and parameters influence an individual''s life history and thus, ultimately, its lifespan. Mating and reproduction are costly activities, and in a variety of model organisms, a negative correlation of longevity and reproductive effort has been demonstrated. We are employing the annual killifish Nothobranchius furzeri as a vertebrate model for ageing. N. furzeri is the vertebrate displaying the shortest known lifespan in captivity with particular strains living only three to four months under optimal laboratory conditions. The animals show explosive growth, early sexual maturation and age-dependent physiological and behavioural decline. Here, we have used N. furzeri to investigate a potential reproduction-longevity trade-off in both sexes by means of gender separation. Though female reproductive effort and offspring investment were significantly reduced after separation, as investigated by analysis of clutch size, eggs in the ovaries and ovary mass, the energetic surplus was not reallocated towards somatic maintenance. In fact, a significant extension of lifespan could not be observed in either sex. This is despite the fact that separated females, but not males, grew significantly larger and heavier than the respective controls. Therefore, it remains elusive whether lifespan of an annual species evolved in periodically vanishing habitats can be prolonged on the cost of reproduction at all.     

Hepatic Overexpression of Soluble Urokinase Receptor (uPAR) Suppresses Diet-Induced Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient (LDLR-/-) Mice

Objective

Atherosclerosis, a chronic inflammatory disease, arises from metabolic disorders and is driven by inappropriate recruitment and proliferation of monocytes / macrophages and vascular smooth-muscle-cells. The receptor for the urokinase-type plasminogen activator (uPAR, Plaur) regulates the proteolytic activation of plasminogen. It is also a coactivator of integrins and facilitates leukocyte-endothelial interactions and vascular smooth-muscle-cell migration. The role of uPAR in atherogenesis remains elusive.

Methods and Results

We generated C57Bl6/J low-density lipoprotein receptor (LDL) and uPAR double knockout (uPAR^-/-/LDLR^-/-) mice to test the role of uPAR in two distinct atherosclerosis models. In LDLR^-/- mice, hepatic overexpression following hydrodynamic transfection of soluble uPAR that competes with endogenous membrane-bound uPAR was performed as an interventional strategy. Aortic root atherosclerotic lesions induced by feeding a high-fat diet were smaller and comprised less macrophages and vascular smooth-muscle-cells in double knockout mice and animals overexpressing soluble uPAR when compared to controls. In contrast, lesion size, lipid-, macrophage-, and vascular smooth muscle cell content of guide-wire-induced intima lesions in the carotid artery were not affected by uPAR deficiency. Adhesion of uPAR^-/--macrophages to TNFα-stimulated endothelial cells was decreased in vitro accompanied by reduced VCAM-1 expression on primary endothelial cells. Hepatic overexpression of soluble full-length murine uPAR in LDLR^-/- mice led to a reduction of diet-induced atherosclerotic lesion formation and monocyte recruitment into plaques. Ex vivo incubation with soluble uPAR protein also inhibited adhesion of macrophages to TNFα-stimulated endothelial cells in vitro.

Conclusion

uPAR-deficiency as well as competitive soluble uPAR reduced diet-promoted but not guide-wire induced atherosclerotic lesions in mice by preventing monocyte recruitment and vascular smooth-muscle-cell infiltration. Soluble uPAR may represent a therapeutic tool for the modulation of hyperlipidemia-associated atherosclerotic lesion formation.     

Fenofibrate Increases Very Low Density Lipoprotein Triglyceride Production Despite Reducing Plasma Triglyceride Levels in APOE*3-Leiden.CETP Mice

The peroxisome proliferator-activated receptor alpha (PPARα) activator fenofibrate efficiently decreases plasma triglycerides (TG), which is generally attributed to enhanced very low density lipoprotein (VLDL)-TG clearance and decreased VLDL-TG production. However, because data on the effect of fenofibrate on VLDL production are controversial, we aimed to investigate in (more) detail the mechanism underlying the TG-lowering effect by studying VLDL-TG production and clearance using APOE*3-Leiden.CETP mice, a unique mouse model for human-like lipoprotein metabolism. Male mice were fed a Western-type diet for 4 weeks, followed by the same diet without or with fenofibrate (30 mg/kg bodyweight/day) for 4 weeks. Fenofibrate strongly lowered plasma cholesterol (−38%) and TG (−60%) caused by reduction of VLDL. Fenofibrate markedly accelerated VLDL-TG clearance, as judged from a reduced plasma half-life of glycerol tri[³H]oleate-labeled VLDL-like emulsion particles (−68%). This was associated with an increased post-heparin lipoprotein lipase (LPL) activity (+110%) and an increased uptake of VLDL-derived fatty acids by skeletal muscle, white adipose tissue, and liver. Concomitantly, fenofibrate markedly increased the VLDL-TG production rate (+73%) but not the VLDL-apolipoprotein B (apoB) production rate. Kinetic studies using [³H]palmitic acid showed that fenofibrate increased VLDL-TG production by equally increasing incorporation of re-esterified plasma fatty acids and liver TG into VLDL, which was supported by hepatic gene expression profiling data. We conclude that fenofibrate decreases plasma TG by enhancing LPL-mediated VLDL-TG clearance, which results in a compensatory increase in VLDL-TG production by the liver.     

Deficiency of Leishmania Phosphoglycans Influences the Magnitude but Does Not Affect the Quality of Secondary (Memory) Anti-Leishmania Immunity

Despite inducing very low IFN-γ response and highly attenuated in vivo, infection of mice with phosphoglycan (PG) deficient Leishmania major (lpg2-) induces protection against virulent L. major challenge. Here, we show that mice infected with lpg2- L. major generate Leishmania-specific memory T cells. However, in vitro and in vivo proliferation, IL-10 and IFN-γ production by lpg2- induced memory cells were impaired in comparison to those induced by wild type (WT) parasites. Interestingly, TNF recall response was comparable to WT infected mice. Despite the impaired proliferation and IFN-γ response, lpg2- infected mice were protected against virulent L. major challenge and their T cells mediated efficient infection-induced immunity. In vivo depletion and neutralization studies with mAbs demonstrated that lpg2- L. major-induced resistance was strongly dependent on IFN-γ, but independent of TNF and CD8⁺ T cells. Collectively, these data show that the effectiveness of secondary anti-Leishmania immunity depends on the quality (and not the magnitude) of IFN-γ response. These observations provide further support for consideration of lpg2- L. major as a live-attenuated candidate for leishmanization in humans since it protects strongly against virulent challenge, without inducing pathology in infected animals.     

NOS2 Is Critical to the Development of Emphysema in Sftpd Deficient Mice but Does Not Affect Surfactant Homeostasis

Rationale

Surfactant protein D (SP-D) has important immuno-modulatory properties. The absence of SP-D results in an inducible NO synthase (iNOS, coded by NOS2 gene) related chronic inflammation, development of emphysema-like pathophysiology and alterations of surfactant homeostasis.

Objective

In order to test the hypothesis that SP-D deficiency related abnormalities in pulmonary structure and function are a consequence of iNOS induced inflammation, we generated SP-D and iNOS double knockout mice (DiNOS).

Methods

Structural data obtained by design-based stereology to quantify the emphysema-like phenotype and disturbances of the intracellular surfactant were correlated to invasive pulmonary function tests and inflammatory markers including activation markers of alveolar macrophages and compared to SP-D (Sftpd^−/−) and iNOS single knockout mice (NOS2^−/−) as well as wild type (WT) littermates.

Measurements and Results

DiNOS mice had reduced inflammatory cells in BAL and BAL-derived alveolar macrophages showed an increased expression of markers of an alternative activation as well as reduced inflammation. As evidenced by increased alveolar numbers and surface area, emphysematous changes were attenuated in DiNOS while disturbances of the surfactant system remained virtually unchanged. Sftpd^−/− demonstrated alterations of intrinsic mechanical properties of lung parenchyma as shown by reduced stiffness and resistance at its static limits, which could be corrected by additional ablation of NOS2 gene in DiNOS.

Conclusion

iNOS related inflammation in the absence of SP-D is involved in the emphysematous remodeling leading to a loss of alveoli and associated alterations of elastic properties of lung parenchyma while disturbances of surfactant homeostasis are mediated by different mechanisms.     

Repeated Idazoxan Increases Brain Imidazoline Receptors in Normotensive (WKY) but Not in Hypertensive (SHR) Rats

The specific binding of [3H]idazoxan in the presence of 10(-6) M (-)-adrenaline was used to evaluate the density of imidazoline receptors in the brain of spontaneously hypertensive (SHR) rats and sex- and age-matched normotensive Wistar-Kyoto (WKY) rats. In SHR rats the density of imidazoline receptors (cerebral cortex, hypothalamus, and medulla oblongata) was not different from that in normotensive (WKY) rats. However, repeated treatment with idazoxan consistently increased (23-80%) the density of imidazoline receptors in the various brain regions of WKY rats but not in SHR rats. In normotensive Sprague-Dawley rats, repeated treatment with the imidazoline drugs idazoxan and cirazoline also increased (33-37%) the density of imidazoline receptors in the cerebral cortex. The lack of regulation by idazoxan of the density of imidazoline receptors in the brain of SHR rats might reflect the existence of a relevant abnormality of these receptors in this genetic model of hypertension.     

Systemic Central Nervous System (CNS)-targeted Delivery of Neuropeptide Y (NPY) Reduces Neurodegeneration and Increases Neural Precursor Cell Proliferation in a Mouse Model of Alzheimer Disease

Neuropeptide Y (NPY) is one of the most abundant protein transmitters in the central nervous system with roles in a variety of biological functions including: food intake, cardiovascular regulation, cognition, seizure activity, circadian rhythms, and neurogenesis. Reduced NPY and NPY receptor expression is associated with numerous neurodegenerative disorders including Alzheimer disease (AD). To determine whether replacement of NPY could ameliorate some of the neurodegenerative and behavioral pathology associated with AD, we generated a lentiviral vector expressing NPY fused to a brain transport peptide (apoB) for widespread CNS delivery in an APP-transgenic (tg) mouse model of AD. The recombinant NPY-apoB effectively reversed neurodegenerative pathology and behavioral deficits although it had no effect on accumulation of Aβ. The subgranular zone of the hippocampus showed a significant increase in proliferation of neural precursor cells without further differentiation into neurons. The neuroprotective and neurogenic effects of NPY-apoB appeared to involve signaling via ERK and Akt through the NPY R1 and NPY R2 receptors. Thus, widespread CNS-targeted delivery of NPY appears to be effective at reversing the neuronal and glial pathology associated with Aβ accumulation while also increasing NPC proliferation. Overall, increased delivery of NPY to the CNS for AD might be an effective therapy especially if combined with an anti-Aβ therapeutic.     

Deletion of the gabra2 Gene Results in Hypersensitivity to the Acute Effects of Ethanol but Does Not Alter Ethanol Self Administration

Human genetic studies have suggested that polymorphisms of the GABRA2 gene encoding the GABA_A α2-subunit are associated with ethanol dependence. Variations in this gene also convey sensitivity to the subjective effects of ethanol, indicating a role in mediating ethanol-related behaviours. We therefore investigated the consequences of deleting the α2-subunit on the ataxic and rewarding properties of ethanol in mice. Ataxic and sedative effects of ethanol were explored in GABA_A α2-subunit wildtype (WT) and knockout (KO) mice using a Rotarod apparatus, wire hang and the duration of loss of righting reflex. Following training, KO mice showed shorter latencies to fall than WT littermates under ethanol (2 g/kg i.p.) in both Rotarod and wire hang tests. After administration of ethanol (3.5 g/kg i.p.), KO mice took longer to regain the righting reflex than WT mice. To ensure the acute effects are not due to the gabra2 deletion affecting pharmacokinetics, blood ethanol concentrations were measured at 20 minute intervals after acute administration (2 g/kg i.p.), and did not differ between genotypes. To investigate ethanol’s rewarding properties, WT and KO mice were trained to lever press to receive increasing concentrations of ethanol on an FR4 schedule of reinforcement. Both WT and KO mice self-administered ethanol at similar rates, with no differences in the numbers of reinforcers earned. These data indicate a protective role for α2-subunits, against the acute sedative and ataxic effects of ethanol. However, no change was observed in ethanol self administration, suggesting the rewarding effects of ethanol remain unchanged.     

The Neuropeptide Y (NPY) Y2 Receptors Are Largely Dimeric in the Kidney,but Monomeric in the Forebrain

The neuropeptide Y(NPY) Y2 receptors are detected largely as dimers in the clonal expressions in CHO cells and in particulates from rabbit kidney cortex. However, in two areas of the forebrain (rat or rabbit piriform cortex and hypothalamus), these receptors are found mainly as monomers. Evidence is presented that this difference relates to large levels of G proteins containing the Gi α -subunit in the forebrain areas. The predominant monomeric status of these Y2 receptors should also be physiologically linked to large synaptic inputs of the agonist NPY. The rabbit kidney and the human CHO cell-expressed Y2 dimers are converted by agonists to monomers in vitro at a similar rate in the presence of divalent cations.     

The mtDNA nt7778 G/T Polymorphism Augments Formation of Lymphocytic Foci but Does Not Aggravate Cerulein-Induced Acute Pancreatitis in Mice

A polymorphism in the ATP synthase 8 (ATP8) gene of the murine mitochondrial genome, G-to-T transversion at position 7778, has been suggested to increase susceptibility to multiple autoimmune diseases, including autoimmune pancreatitis (AIP). The polymorphism also induces mitochondrial reactive oxygen species generation, secretory dysfunction and β-cell mass adaptation. Here, we have used two conplastic mouse strains, C57BL/6N-mtAKR/J (B6-mtAKR; nt7778 G; control) and C57BL/6N-mtFVB/N (B6-mtFVB; nt7778 T), to address the question if the polymorphism also affects the course of cerulein-induced acute pancreatitis in mice. Therefore, two age groups of mice (3 and 12-month-old, respectively) were subjected to up to 7 injections of the secretagogue cerulein (50 µg/kg body weight) at hourly intervals. Disease severity was assessed at time points from 3 hours to 7 days based on pancreatic histopathology, serum levels of α-amylase and activities of myeloperoxidase (MPO) in lung tissue. A comparison of cerulein-induced pancreatic tissue damage and increases of α-amylase and MPO activities showed no differences between the age-matched groups of both strains. Interestingly, histological evaluation of pancreatic tissue of both untreated and cerulein-treated B6-mtAKR and B6-mtFVB mice also revealed the presence of infiltrates of immune cells surrounding ducts and vessels; a finding that is compatible with an early stage of AIP. After recovery from cerulein-induced pancreatitis (day 7 after the injections), 12-month-old B6-mtFVB mice but not B6-mtAKR mice displayed aggravated lymphocytic lesions. A comparison of 12-month-old mice with other age groups of both strains revealed that lymphocytic foci were largely absent in 3-month-old mice, while 24-month-old mice were more affected. Together, our data suggest that the mtDNA nt7778 G/T polymorphism does not aggravate cerulein-induced acute pancreatitis. Autoimmune-like lesions, however, may progress faster if additional tissue damage occurs.     

Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition,but Causes a Transient Reduction of Articular Cartilage Thickness

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.     

Nonsteroidal Anti-Inflammatory Drugs Increase Tumor Necrosis Factor Production in the Periphery but Not in the Central Nervous System in Mice and Rats

Abstract: Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit prostaglandin (PG) synthesis, augment production of tumor necrosis factor (TNF) in most experimental models. We investigated the effect of two NSAIDs, indomethacin and ibuprofen, on the production of TNF in the CNS induced by intracerebroventricular injection of lipopolysaccharide (LPS). Indomethacin and ibuprofen, administered intraperitoneally, augmented (three- to ninefold) the levels of TNF in serum and peripheral organs of mice injected intraperitoneally with LPS and in rats with adjuvant arthritis (up to a sevenfold increase). However, NSAIDs (intraperitoneally or intracerebroventricularly) did not increase brain TNF production induced by intravenous LPS. In fact, indomethacin decreased (1.4–1.8-fold) TNF levels in the spinal cord of rats with experimental autoimmune encephalomyelitis and in the cortex of rats with focal cerebral ischemia. Systemic administration of iloprost inhibited serum TNF levels after intraperitoneal LPS, whereas intracerebroventricular injection of iloprost or PGE₂ did not inhibit brain TNF induced by intracerebroventricular LPS. Both peripheral and central TNF productions were inhibited by cyclic AMP level-elevating agents or dexamethasone. Thus, a PG-driven negative feedback controls TNF production in the periphery but not in the CNS.     

Portal Hyperperfusion after Extended Hepatectomy Does Not Induce a Hepatic Arterial Buffer Response (HABR) but Impairs Mitochondrial Redox State and Hepatocellular Oxygenation

Background & Aims

Portal hyperperfusion after extended hepatectomy or small-for-size liver transplantation may induce organ dysfunction and failure. The underlying mechanisms, however, are still not completely understood. Herein, we analysed whether hepatectomy-associated portal hyperperfusion induces a hepatic arterial buffer response, i.e., an adaptive hepatic arterial constriction, which may cause hepatocellular hypoxia and organ dysfunction.

Methods

Sprague-Dawley rats underwent 30%, 70% and 90% hepatectomy. Baseline measurements before hepatectomy served as controls. Hepatic arterial and portal venous flows were analysed by ultrasonic flow measurement. Microvascular blood flow and mitochondrial redox state were determined by intravital fluorescence microscopy. Hepatic tissue pO2 was analysed by polarographic techniques. Hepatic function and integrity were studied by bromosulfophthalein bile excretion and liver histology.

Results

Portal blood flow was 2- to 4-fold increased after 70% and 90% hepatectomy. This, however, did not provoke a hepatic arterial buffer response. Nonetheless, portal hyperperfusion and constant hepatic arterial blood flow were associated with a reduced mitochondrial redox state and a decreased hepatic tissue pO2 after 70% and 90% hepatectomy. Microvascular blood flow increased significantly after hepatectomy and functional sinusoidal density was found only slightly reduced. Major hepatectomy further induced a 2- to 3-fold increase of bile flow. This was associated with a 2-fold increase of bromosulfophthalein excretion.

Conclusions

Portal hyperperfusion after extended hepatectomy does not induce a hepatic arterial buffer response but reduces mitochondrial redox state and hepatocellular oxygenation. This is not due to a deterioration of microvascular perfusion, but rather due to a relative hypermetabolism of the remnant liver after major resection.     

Poly(Q) Expansions in ATXN7 Affect Solubility but Not Activity of the SAGA Deubiquitinating Module

Spinocerebellar ataxia type 7 (SCA7) is a debilitating neurodegenerative disease caused by expansion of a polyglutamine [poly(Q)] tract in ATXN7, a subunit of the deubiquitinase (DUB) module (DUBm) in the SAGA complex. The effects of ATXN7-poly(Q) on DUB activity are not known. To address this important question, we reconstituted the DUBm in vitro with either wild-type ATXN7 or a pathogenic form, ATXN7-92Q NT, with 92 Q residues at the N terminus (NT). We found that both forms of ATXN7 greatly enhance DUB activity but that ATXN7-92Q NT is largely insoluble unless it is incorporated into the DUBm. Cooverexpression of DUBm components in human astrocytes also promoted the solubility of ATXN7-92Q, inhibiting its aggregation into nuclear inclusions that sequester DUBm components, leading to global increases in ubiquitinated H2B (H2Bub) levels. Global H2Bub levels were also increased in the cerebellums of mice in a SCA7 mouse model. Our findings indicate that although ATXN7 poly(Q) expansions do not change the enzymatic activity of the DUBm, they likely contribute to SCA7 by initiating aggregates that sequester the DUBm away from its substrates.     

Missense Mutation Lys18Asn in Dystrophin that Triggers X-Linked Dilated Cardiomyopathy Decreases Protein Stability,Increases Protein Unfolding,and Perturbs Protein Structure,but Does Not Affect Protein Function

Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM). However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD). We created and expressed the wild-type (WT) N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein''s overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts), and unfolds faster than the WT (stopped-flow kinetics). Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments). These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.     

The Dual-Specificity Phosphatase 2 (DUSP2) Does Not Regulate Obesity-Associated Inflammation or Insulin Resistance in Mice

Alterations in the immune cell profile and the induction of inflammation within adipose tissue are a hallmark of obesity in mice and humans. Dual-specificity phosphatase 2 (DUSP2) is widely expressed within the immune system and plays a key role promoting immune and inflammatory responses dependent on mitogen-activated protein kinase (MAPK) activity. We hypothesised that the absence of DUSP2 would protect mice against obesity-associated inflammation and insulin resistance. Accordingly, male and female littermate mice that are either wild-type (wt) or homozygous for a germ-line null mutation of the dusp2 gene (dusp2^−/−) were fed either a standard chow diet (SCD) or high fat diet (HFD) for 12 weeks prior to metabolic phenotyping. Compared with mice fed the SCD, all mice consuming the HFD became obese, developed glucose intolerance and insulin resistance, and displayed increased macrophage recruitment and markers of inflammation in epididymal white adipose tissue. The absence of DUSP2, however, had no effect on the development of obesity or adipose tissue inflammation. Whole body insulin sensitivity in male mice was unaffected by an absence of DUSP2 in response to either the SCD or HFD; however, HFD-induced insulin resistance was slightly, but significantly, reduced in female dusp2^−/− mice. In conclusion, DUSP2 plays no role in regulating obesity-associated inflammation and only a minor role in controlling insulin sensitivity following HFD in female, but not male, mice. These data indicate that rather than DUSP2 being a pan regulator of MAPK dependent immune cell mediated inflammation, it appears to differentially regulate inflammatory responses that have a MAPK component.     

3,5 Diiodo-L-Thyronine (T2) Does Not Prevent Hepatic Steatosis or Insulin Resistance in Fat-Fed Sprague Dawley Rats

Thyroid hormone mimetics are alluring potential therapies for diseases like dyslipidemia, nonalcoholic fatty liver disease (NAFLD), and insulin resistance. Though diiodothyronines are thought inactive, pharmacologic treatment with 3,5- Diiodo-L-Thyronine (T2) reportedly reduces hepatic lipid content and improves glucose tolerance in fat-fed male rats. To test this, male Sprague Dawley rats fed a safflower-oil based high-fat diet were treated with T2 (0.25 mg/kg-d) or vehicle. Neither 10 nor 30 days of T2 treatment had an effect on weight, adiposity, plasma fatty acids, or hepatic steatosis. Insulin action was quantified in vivo by a hyperinsulinemic-euglycemic clamp. T2 did not alter fasting plasma glucose or insulin concentration. Basal endogenous glucose production (EGP) rate was unchanged. During the clamp, there was no difference in insulin stimulated whole body glucose disposal. Insulin suppressed EGP by 60% ± 10 in T2-treated rats as compared with 47% ± 4 suppression in the vehicle group (p = 0.32). This was associated with an improvement in hepatic insulin signaling; insulin stimulated Akt phosphorylation was ~2.5 fold greater in the T2-treated group as compared with the vehicle-treated group (p = 0.003). There was no change in expression of genes thought to mediate the effect of T2 on hepatic metabolism, including genes that regulate hepatic lipid oxidation (ppara, carnitine palmitoyltransferase 1a), genes that regulate hepatic fatty acid synthesis (srebp1c, acetyl coa carboxylase, fatty acid synthase), and genes involved in glycolysis and gluconeogenesis (L-pyruvate kinase, glucose 6 phosphatase). Therefore, in contrast with previous reports, in Sprague Dawley rats fed an unsaturated fat diet, T2 administration failed to improve NAFLD or whole body insulin sensitivity. Though there was a modest improvement in hepatic insulin signaling, this was not associated with significant differences in hepatic insulin action. Further study will be necessary before diiodothyronines can be considered an effective treatment for NAFLD and dyslipidemia.     

Ketamine Does Not Produce Relief of Neuropathic Pain in Mice Lacking the β-Common Receptor (CD131)

Neuropathic pain (NP) is a debilitating condition associated with traumatic, metabolic, autoimmune and neurological etiologies. Although the triggers for NP are diverse, there are common underlying pathways, including activation of immune cells in the spinal cord and up-regulation of the N-methyl-D-aspartate receptor (NMDAR). Ketamine, a well-known NDMAR antagonist, reduces neuropathic pain in a sustained manner. Recent study has shown that the novel 11-amino acid peptide erythropoietin derivative ARA290 produces a similar, long-lasting relief of NP. Here, we show that both drugs also have similar effects on the expression of mRNA of the NMDAR, as well as that of microglia, astrocytes and chemokine (C-C motif) ligand 2, all-important contributors to the development of NP. Although the effects of ketamine and ARA 290 on NP and its molecular mediators suggest a common mechanism of action, ARA 290 has no affinity for the NMDAR and acts specifically via the innate repair receptor (IRR) involved in tissue protection. We speculated therefore, that the IRR might be critically involved in the action of ketamine on neuropathic pain. To evaluate this, we studied the effects of ketamine and ARA 290 on acute pain, side effects, and allodynia following a spared nerve injury model in mice lacking the β-common receptor (βcR), a structural component of the IRR. Ketamine (50 mg/kg) and ARA 290 (30 µg/kg) produced divergent effects on acute pain: ketamine produced profound antinociception accompanied with psychomotor side effects, but ARA290 did not, in both normal and knock out mice. In contrast, while both drugs were antiallodynic in WT mice, they had no effect on NP in mice lacking the βcR. Together, these results show that an intact IRR is required for the effective treatment of NP with either ketamine or ARA 290, but is not involved in ketamine’s analgesic and side effects.