The effect of rosuvastatin in a murine model of influenza A infection

Rationale

HMG-CoA reductase inhibitors such as rosuvastatin may have immunomodulatory and anti-inflammatory effects that may reduce the severity of influenza A infection. We hypothesized that rosuvastatin would decrease viral replication, attenuate lung injury, and improve mortality following influenza A infection in mice.

Methods

C57Bl/6 mice were treated daily with rosuvastatin (10 mg/kg/day) supplemented in chow (or control chow) beginning three days prior to infection with either A//Udorn/72 [H3N2] or A/WSN/33 [H1N1] influenza A virus (1×10⁵ pfu/mouse). Plaque assays were used to examine the effect of rosuvastatin on viral replication in vitro and in the lungs of infected mice. We measured cell count with differential, protein and cytokines in the bronchoalveolar lavage (BAL) fluid, histologic evidence of lung injury, and wet-to-dry ratio on Day 1, 2, 4, and 6. We also recorded daily weights and mortality.

Results

The administration of rosuvastatin had no effect on viral clearance of influenza A after infection. Weight loss, lung inflammation and lung injury severity were similar in the rosuvastatin and control treated mice. In the mice infected with influenza A (A/WSN/33), mortality was unaffected by treatment with rosuvastatin.

Conclusions

Statins did not alter the replication of influenza A in vitro or enhance its clearance from the lung in vivo. Statins neither attenuated the severity of influenza A-induced lung injury nor had an effect on influenza A-related mortality. Our data suggest that the association between HMG CoA reductase inhibitors and improved outcomes in patients with sepsis and pneumonia are not attributable to their effects on influenza A infection.     

Inactivation of Fam20C in Cells Expressing Type I Collagen Causes Periodontal Disease in Mice

Background

FAM20C is a kinase that phosphorylates secretory proteins. Previous studies have shown that FAM20C plays an essential role in the formation and mineralization of bone, dentin and enamel. The present study analyzed the loss-of-function effects of FAM20C on the health of mouse periodontal tissues.

Methods

By crossbreeding 2.3 kb Col 1a1-Cre mice with Fam20C^fl/fl mice, we created 2.3 kb Col 1a1-Cre;Fam20C^fl/fl (cKO) mice, in which Fam20C was inactivated in the cells that express Type I collagen. We analyzed the periodontal tissues in the cKO mice using X-ray radiography, histology, scanning electron microscopy and immunohistochemistry approaches.

Results

The cKO mice underwent a remarkable loss of alveolar bone and cementum, along with inflammation of the periodontal ligament and formation of periodontal pockets. The osteocytes and lacuno-canalicular networks in the alveolar bone of the cKO mice showed dramatic abnormalities. The levels of bone sialoprotein, osteopontin, dentin matrix protein 1 and dentin sialoprotein were reduced in the Fam20C-deficient alveolar bone and/or cementum, while periostin and fibrillin-1 were decreased in the periodontal ligament of the cKO mice.

Conclusion

Loss of Fam20C function leads to periodontal disease in mice. The reduced levels of bone sialoprotein, osteopontin, dentin matrix protein 1, dentin sialoprotein, periostin and fibrillin-1 may contribute to the periodontal defects in the Fam20C-deficient mice.     

Survivin is required for Beta-cell mass expansion in the pancreatic duct-ligated mouse model

Aims/Hypothesis

Pancreatic beta-cell mass expands through adulthood under certain conditions. The related molecular mechanisms are elusive. This study was designed to determine whether surviving (also known as Birc5), which is transiently expressed perinatally in islets, was required for beta-cell mass expansion in the pancreatic duct-ligated mouse model.

Methods

Mice with beta cell–specific deletion of survivin (RIPCre⁺survivin^fl/fl) and their control littermates (RIPCre⁺survivin^+/+) were examined to determine the essential role of survivin in partial pancreatic duct ligation (PDL)-induced beta-cell proliferation, function and survival.

Results

Resurgence of survivin expression occurred as early as day 3 post-PDL. By day 7 post-PDL, control mice showed significant expansion of beta-cell mass and increase in beta-cell proliferation and islet number in the ligated tail of the pancreas. However, mice deficient in beta-cell survivin showed a defect in beta-cell mass expansion and proliferation with a marked attenuation in the increase of total islet number, largely due to an impairment in the increase in number of larger islets while sparing the increase in number of small islets in the ligated tail of pancreas, resulting in insufficient insulin secretion and glucose intolerance. Importantly however, beta cell neogenesis and apoptosis were not affected by the absence of survivin in beta cells after PDL.

Conclusions/Interpretation

Our results indicate that survivin is essential for beta-cell mass expansion after PDL. Survivin appears to exhibit a preferential requirement for proliferation of preexisting beta cells.     

The role of IL-27 in susceptibility to post-influenza Staphylococcus aureus pneumonia

Background

Influenza is a common respiratory virus and Staphylococcus aureus frequently causes secondary pneumonia during influenza infection, leading to increased morbidity and mortality. Influenza has been found to attenuate subsequent Type 17 immunity, enhancing susceptibility to secondary bacterial infections. IL-27 is known to inhibit Type 17 immunity, suggesting a potential critical role for IL-27 in viral and bacterial co-infection.

Methods

A murine model of influenza and Staphylococcus aureus infection was used to mimic human viral, bacterial co-infection. C57BL/6 wild-type, IL-27 receptor α knock-out, and IL-10 knock-out mice were infected with Influenza H1N1 (A/PR/8/34) or vehicle for 6 days followed by challenge with Staphylococcus aureus or vehicle for 24 hours. Lung inflammation, bacterial burden, gene expression, and cytokine production were determined.

Results

IL-27 receptor α knock-out mice challenged with influenza A had increased morbidity compared to controls, but no change in viral burden. IL-27 receptor α knock-out mice infected with influenza displayed significantly decreased IL-10 production compared to wild-type. IL-27 receptor α knock-out mice co-infected with influenza and S. aureus had improved bacterial clearance compared to wild-type controls. Importantly, there were significantly increased Type 17 responses and decreased IL-10 production in IL-27 receptor α knock-out mice. Dual infected IL-10−/− mice had significantly less bacterial burden compared to dual infected WT mice.

Conclusions

These data reveal that IL-27 regulates enhanced susceptibility to S. aureus pneumonia following influenza infection, potentially through the induction of IL-10 and suppression of IL-17.     

Deficiency in type 1 insulin-like growth factor receptor in mice protects against oxygen-induced lung injury

Background

Cellular responses to aging and oxidative stress are regulated by type 1 insulin-like growth factor receptor (IGF-1R). Oxidant injury, which is implicated in the pathophysiology of a number of respiratory diseases, acutely upregulates IGF-1R expression in the lung. This led us to suspect that reduction of IGF-1R levels in lung tissue could prevent deleterious effects of oxygen exposure.

Methods

Since IGF-1R null mutant mice die at birth from respiratory failure, we generated compound heterozygous mice harboring a hypomorphic (Igf-1r^neo) and a knockout (Igf-1r^-) receptor allele. These IGF-1R^neo/- mice, strongly deficient in IGF-1R, were subjected to hyperoxia and analyzed for survival time, ventilatory control, pulmonary histopathology, morphometry, lung edema and vascular permeability.

Results

Strikingly, after 72 h of exposure to 90% O₂, IGF-1R^neo/- mice had a significantly better survival rate during recovery than IGF-1R^+/+ mice (77% versus 53%, P < 0.05). The pulmonary injury was consistently, and significantly, milder in IGF-1R^neo/- mice which developed conspicuously less edema and vascular extravasation than controls. Also, hyperoxia-induced abnormal pattern of breathing which precipitated respiratory failure was elicited less frequently in the IGF-1R^neo/- mice.

Conclusion

Together, these data demonstrate that a decrease in IGF-1R signaling in mice protects against oxidant-induced lung injury.     

Interaction between Age and Obesity on Cardiomyocyte Contractile Function: Role of Leptin and Stress Signaling

Objectives

This study was designed to evaluate the interaction between aging and obesity on cardiac contractile and intracellular Ca²⁺ properties.

Methods

Cardiomyocytes from young (4-mo) and aging (12- and 18-mo) male lean and the leptin deficient ob/ob obese mice were treated with leptin (0.5, 1.0 and 50 nM) for 4 hrs in vitro. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obesity models at young and older age were used for comparison. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ levels and decay. O₂ ⁻ levels were measured by dihydroethidium fluorescence.

Results

Our results revealed reduced survival in ob/ob mice. Aging and obesity reduced PS, ± dL/dt, intracellular Ca²⁺ rise, prolonged TR₉₀ and intracellular Ca²⁺ decay, enhanced O₂ ⁻ production and p ^47phox expression without an additive effect of the two, with the exception of intracellular Ca²⁺ rise. Western blot analysis exhibited reduced Ob-R expression and STAT-3 phosphorylation in both young and aging ob/ob mice, which was restored by leptin. Aging and obesity reduced phosphorylation of Akt, eNOS and p38 while promoting pJNK and pIκB. Low levels of leptin reconciled contractile, intracellular Ca²⁺ and cell signaling defects as well as O₂ ⁻ production and p ^47phox upregulation in young but not aging ob/ob mice. High level of leptin (50 nM) compromised contractile and intracellular Ca²⁺ response as well as O₂ ⁻ production and stress signaling in all groups. High fat diet-induced and db/db obesity displayed somewhat comparable aging-induced mechanical but not leptin response.

Conclusions

Taken together, our data suggest that aging and obesity compromise cardiac contractile function possibly via phosphorylation of Akt, eNOS and stress signaling-associated O₂ ⁻ release.     

Loss of c-Met Disrupts Gene Expression Program Required for G2/M Progression during Liver Regeneration in Mice

Background

Previous work has established that HGF/c-Met signaling plays a pivotal role in regulating the onset of S phase following partial hepatectomy (PH). In this study, we used Met^fl/fl;Alb-Cre^+/− conditional knockout mice to determine the effects of c-Met dysfunction in hepatocytes on kinetics of liver regeneration.

Methodology/Principal Finding

The priming events appeared to be intact in Met^fl/fl;Alb-Cre^+/− livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1 and 6) genes as assessed by RT-qPCR and/or microarray profiling was unchanged. This was consistent with an early induction of MAPK/Erk and STAT3. However, after a successful completion of the first round of DNA replication, c-Met deficient hepatocytes were blocked in early/mid G2 phase as shown by staining with phosphorylated form of histone H3. Furthermore, loss of c-Met in hepatocytes diminished the subsequent G1/S progression and delayed liver recovery after partial hepatectomy. Upstream signaling pathways involved in the blockage of G2/M transition included lack of persistent Erk1/2 activation and inability to up-regulate the levels of Cdk1, Plk1, Aurora A and B, and Mad2 along with a defective histone 3 phosphorylation and lack of chromatin condensation. Continuous supplementation with EGF in vitro increased proliferation of Met^fl/fl;Alb-Cre^+/− primary hepatocytes and partially restored expression levels of mitotic cell cycle regulators albeit to a lesser degree as compared to control cultures.

Conclusion/Significance

In conclusion, our results assign a novel non-redundant function for HGF/c-Met signaling in regulation of G2/M gene expression program via maintaining a persistent Erk1/2 activation throughout liver regeneration.     

Reduced Body Weight and Increased Energy Expenditure in Transgenic Mice Over-Expressing Soluble Leptin Receptor

Background

Soluble leptin receptor (OBRe), one of several leptin receptor isoforms, is the only bona fide leptin binding protein in plasma. Our earlier studies demonstrated that OBRe modulates leptin levels in circulation. Both clinical and in vitro studies have shown that OBRe expression is inversely correlated to body weight and leptin levels. However, it is not clear whether OBRe plays an active role, either in collaboration with leptin or independently, in the maintenance of body weight.

Methodology/Principal Findings

To investigate the function of OBRe in the regulation of energy homeostasis, we generated transgenic mice that express OBRe under the control of human serum amyloid P (hSAP) component gene promoter. The transgene led to approximately doubling of OBRe in circulation in the transgenic mice than in wild type control mice. Transgenic mice exhibited lower body weight at 4 weeks of age, and slower rate of weight gain when compared with control mice. Furthermore, transgenic mice had lower body fat content. Indirect calorimetry revealed that transgenic mice had reduced food intake, increased basal metabolic rate, and increased lipid oxidation, which could account for the differences in body weight and body fat content. Transgenic mice also showed higher total circulating leptin, with the majority of it being in the bound form, while the amount of free leptin is comparable between transgenic and control mice.

Conclusions

These results are consistent with the role of OBRe as a leptin binding protein in regulating leptin''s bioavailability and activity.     

Endothelial Arginine Resynthesis Contributes to the Maintenance of Vasomotor Function in Male Diabetic Mice

Aim

Argininosuccinate synthetase (ASS) is essential for recycling L-citrulline, the by-product of NO synthase (NOS), to the NOS substrate L-arginine. Here, we assessed whether disturbed arginine resynthesis modulates endothelium-dependent vasodilatation in normal and diabetic male mice.

Methods and Results

Endothelium-selective Ass-deficient mice (Ass^fl/fl/Tie2Cre^tg/− = Ass-KO^Tie2) were generated by crossing Ass^fl/fl mice ( = control) with Tie2Cre mice. Gene ablation in endothelial cells was confirmed by immunohistochemistry. Blood pressure (MAP) was recorded in 34-week-old male mice. Vasomotor responses were studied in isolated saphenous arteries of 12- and 34-week-old Ass-KO^Tie2 and control animals. At the age of 10 weeks, diabetes was induced in control and Ass-KO^Tie2 mice by streptozotocin injections. Vasomotor responses of diabetic animals were studied 10 weeks later. MAP was similar in control and Ass-KO^Tie2 mice. Depletion of circulating L-arginine by arginase 1 infusion or inhibition of NOS activity with L-NAME resulted in an increased MAP (10 and 30 mmHg, respectively) in control and Ass-KO^Tie2 mice. Optimal arterial diameter, contractile responses to phenylephrine, and relaxing responses to acetylcholine and sodium nitroprusside were similar in healthy control and Ass-KO^Tie2 mice. However, in diabetic Ass-KO^Tie2 mice, relaxation responses to acetylcholine and endothelium-derived NO (EDNO) were significantly reduced when compared to diabetic control mice.

Conclusions

Absence of endothelial citrulline recycling to arginine did not affect blood pressure and systemic arterial vasomotor responses in healthy mice. EDNO-mediated vasodilatation was significantly more impaired in diabetic Ass-KO^Tie2 than in control mice demonstrating that endothelial arginine recycling becomes a limiting endothelial function in diabetes.     

Absence of Functional Leptin Receptor Isoforms in the POUND (Leprdb/lb) Mouse Is Associated with Muscle Atrophy and Altered Myoblast Proliferation and Differentiation

Objective

Leptin receptors are abundant in human skeletal muscle, but the role of leptin in muscle growth, development and aging is not well understood. Here we utilized a novel mouse model lacking all functional leptin receptor isoforms (POUND mouse, Lepr^db/lb) to determine the role of leptin in skeletal muscle.

Methods and Findings

Skeletal muscle mass and fiber diameters were examined in POUND mice, and primary myoblast cultures were used to determine the effects of altered leptin signaling on myoblast proliferation and differentiation. ELISA assays, integrated pathway analysis of mRNA microarrays, and reverse phase protein analysis were performed to identify signaling pathways impacted by leptin receptor deficiency. Results show that skeletal muscle mass and fiber diameter are reduced 30–40% in POUND mice relative to wild-type controls. Primary myoblast cultures demonstrate decreased proliferation and decreased expression of both MyoD and myogenin in POUND mice compared to normal mice. Leptin treatment increased proliferation in primary myoblasts from muscles of both adult (12 months) and aged (24 months) wild-type mice, and leptin increased expression of MyoD and myogenin in aged primary myoblasts. ELISA assays and protein arrays revealed altered expression of molecules associated with the IGF-1/Akt and MAPK/MEK signaling pathways in muscle from the hindlimbs of mice lacking functional leptin receptors.

Conclusion

These data support the hypothesis that the adipokine leptin is a key factor important for the regulation of skeletal muscle mass, and that leptin can act directly on its receptors in peripheral tissues to regulate cell proliferation and differentiation.     

Impact of interleukin-6 on hypoxia-induced pulmonary hypertension and lung inflammation in mice

Background

Inflammation may contribute to the pathogenesis of various forms of pulmonary hypertension (PH). Recent studies in patients with idiopathic PH or PH associated with underlying diseases suggest a role for interleukin-6 (IL-6).

Methods

To determine whether endogenous IL-6 contributes to mediate hypoxic PH and lung inflammation, we studied IL-6-deficient (IL-6^-/-) and wild-type (IL-6^+/+) mice exposed to hypoxia for 2 weeks.

Results

Right ventricular systolic pressure, right ventricle hypertrophy, and the number and media thickness of muscular pulmonary vessels were decreased in IL-6^-/- mice compared to wild-type controls after 2 weeks'' hypoxia, although the pressure response to acute hypoxia was similar in IL-6^+/+ and IL-6^-/- mice. Hypoxia exposure of IL-6^+/+ mice led to marked increases in IL-6 mRNA and protein levels within the first week, with positive IL-6 immunostaining in the pulmonary vessel walls. Lung IL-6 receptor and gp 130 (the IL-6 signal transducer) mRNA levels increased after 1 and 2 weeks'' hypoxia. In vitro studies of cultured human pulmonary-artery smooth-muscle-cells (PA-SMCs) and microvascular endothelial cells revealed prominent synthesis of IL-6 by PA-SMCs, with further stimulation by hypoxia. IL-6 also markedly stimulated PA-SMC migration without affecting proliferation. Hypoxic IL-6^-/- mice showed less inflammatory cell recruitment in the lungs, compared to hypoxic wild-type mice, as assessed by lung protein levels and immunostaining for the specific macrophage marker F4/80, with no difference in lung expression of adhesion molecules or cytokines.

Conclusion

These data suggest that IL-6 may be actively involved in hypoxia-induced lung inflammation and pulmonary vascular remodeling in mice.     

Leptin resistance in vagal afferent neurons inhibits cholecystokinin signaling and satiation in diet induced obese rats

Background and Aims

The gastrointestinal hormone cholecystokinin (CCK) plays an important role in regulating meal size and duration by activating CCK1 receptors on vagal afferent neurons (VAN). Leptin enhances CCK signaling in VAN via an early growth response 1 (EGR1) dependent pathway thereby increasing their sensitivity to CCK. In response to a chronic ingestion of a high fat diet, VAN develop leptin resistance and the satiating effects of CCK are reduced. We tested the hypothesis that leptin resistance in VAN is responsible for reducing CCK signaling and satiation.

Results

Lean Zucker rats sensitive to leptin signaling, significantly reduced their food intake following administration of CCK8S (0.22 nmol/kg, i.p.), while obese Zucker rats, insensitive to leptin, did not. CCK signaling in VAN of obese Zucker rats was reduced, preventing CCK-induced up-regulation of Y2 receptor and down-regulation of melanin concentrating hormone 1 receptor (MCH1R) and cannabinoid receptor (CB1). In VAN from diet-induced obese (DIO) Sprague Dawley rats, previously shown to become leptin resistant, we demonstrated that the reduction in EGR1 expression resulted in decreased sensitivity of VAN to CCK and reduced CCK-induced inhibition of food intake. The lowered sensitivity of VAN to CCK in DIO rats resulted in a decrease in Y2 expression and increased CB1 and MCH1R expression. These effects coincided with the onset of hyperphagia in DIO rats.

Conclusions

Leptin signaling in VAN is required for appropriate CCK signaling and satiation. In response to high fat feeding, the onset of leptin resistance reduces the sensitivity of VAN to CCK thus reducing the satiating effects of CCK.     

ENU mutagenesis identifies mice with morbid obesity and severe hyperinsulinemia caused by a novel mutation in leptin

Background

Obesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. Leptin is central to the regulation of energy metabolism and control of body weight in mammals.

Methodology/Principal Findings

To better recapitulate the complexity of human obesity syndrome, we applied N-ethyl-N-nitrosourea (ENU) mutagenesis in combination with a set of metabolic assays in screening mice for obesity. Mapping revealed linkage to the chromosome 6 within a region containing mouse Leptin gene. Sequencing on the candidate genes identified a novel T-to-A mutation in the third exon of Leptin gene, which translates to a V145E amino acid exchange in the leptin propeptide. Homozygous Leptin^145E/145E mutant mice exhibited morbid obesity, accompanied by adipose hypertrophy, energy imbalance, and liver steatosis. This was further associated with severe insulin resistance, hyperinsulinemia, dyslipidemia, and hyperleptinemia, characteristics of human obesity syndrome. Hypothalamic leptin actions in inhibition of orexigenic peptides NPY and AgRP and induction of SOCS1 and SOCS3 were attenuated in Leptin^145E/145E mice. Administration of exogenous wild-type leptin attenuated hyperphagia and body weight increase in Leptin^145E/145E mice. However, mutant V145E leptin coimmunoprecipitated with leptin receptor, suggesting that the V145E mutation does not affect the binding of leptin to its receptor. Molecular modeling predicted that the mutated residue would form hydrogen bond with the adjacent residues, potentially affecting the structure and formation of an active complex with leptin receptor within that region.

Conclusions/Significance

Thus, our evolutionary, structural, and in vivo metabolic information suggests the residue 145 as of special function significance. The mouse model harboring leptin V145E mutation will provide new information on the current understanding of leptin biology and novel mouse model for the study of human obesity syndrome.     

Aging exacerbates damage and delays repair of alveolar epithelia following influenza viral pneumonia

Background

Influenza virus infection causes significantly higher levels of morbidity and mortality in the elderly. Studies have shown that impaired immunity in the elderly contributes to the increased susceptibility to influenza virus infection, however, how aging affects the lung tissue damage and repair has not been completely elucidated.

Methods

Aged (16–18 months old) and young (2–3 months old) mice were infected with influenza virus intratracheally. Body weight and mortality were monitored. Different days after infection, lung sections were stained to estimate the overall lung tissue damage and for club cells, pro-SPC⁺ bronchiolar epithelial cells, alveolar type I and II cells to quantify their frequencies using automated image analysis algorithms.

Results

Following influenza infection, aged mice lose more weight and die from otherwise sub-lethal influenza infection in young mice. Although there is no difference in damage and regeneration of club cells between the young and the aged mice, damage to alveolar type I and II cells (AT1s and AT2s) is exacerbated, and regeneration of AT2s and their precursors (pro-SPC-positive bronchiolar epithelial cells) is significantly delayed in the aged mice. We further show that oseltamivir treatment reduces virus load and lung damage, and promotes pulmonary recovery from infection in the aged mice.

Conclusions

These findings show that aging increases susceptibility of the distal lung epithelium to influenza infection and delays the emergence of pro-SPC positive progenitor cells during the repair process. Our findings also shed light on possible approaches to enhance the clinical management of severe influenza pneumonia in the elderly.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0116-z) contains supplementary material, which is available to authorized users.     

Boc5, a non-peptidic glucagon-like Peptide-1 receptor agonist, invokes sustained glycemic control and weight loss in diabetic mice

Background

Our recent discovery of the substituted cyclobutane Boc5, one of the first non-peptidic agonists at glucagon-like peptide-1 receptors, offers the potential of combining oral availability with full agonism capable of eliciting antidiabetic and antiobesity effects. The present study was aimed at determining the in vivo pharmacologic properties of Boc5 in both normal and diabetic mice following chronic administration, with emphasis on glycemic control and weight loss.

Methodology/Principal Findings

C57BL/6J and db/db mice were treated daily with Boc5 for 4 weeks and a range of pharmacologic parameters, including hemoglobin A1c, intraperitoneal glucose tolerance, insulin tolerance, fasting insulin and leptin levels, food intake, body weight and fat mass, were assessed before and after the treatment. Effects on food intake, gastric emptying, and insulinogenic index were also investigated in animals acutely administered with Boc5. Boc5 (3 mg) was able to induce a durable restoration of glycemic control (normalization of both hemoglobin A1c and intraperitoneal glucose tolerance) in db/db mice, following 4 weeks of daily administration. As with peptidic glucagon-like peptide-1 receptor agonists, its glycemic benefit and weight (fat) loss were associated with dose-dependent effects that included reduction in food intake, slowing of gastric emptying (both of which reduce nutrient-drive at β-cells), stimulation of insulin secretion (which was glucose-dependent), and elevation in insulin sensitivity. There was little effect on normal mice treated in the same manner.

Conclusions/Significance

Our findings suggest that Boc5 is the only non-peptidic molecule reported thus far to simultaneously activate this spectrum of antidiabetic effects.     

Positive Regulatory Control Loop between Gut Leptin and Intestinal GLUT2/GLUT5 Transporters Links to Hepatic Metabolic Functions in Rodents

Background and Aims

The small intestine is the major site of absorption of dietary sugars. The rate at which they enter and exit the intestine has a major effect on blood glucose homeostasis. In this study, we determine the effects of luminal leptin on activity/expression of GLUT2 and GLUT5 transporters in response to sugars intake and analyse their physiological consequences.

Methodology

Wistar rats, wild type and AMPKα₂ ^−/− mice were used. In vitro and in vivo isolated jejunal loops were used to quantify transport of fructose and galactose in the absence and the presence of leptin. The effects of fructose and galactose on gastric leptin release were determined. The effects of leptin given orally without or with fructose were determined on the expression of GLUT2/5, on some gluconeogenesis and lipogenic enzymes in the intestine and the liver.

Principal Findings

First, in vitro luminal leptin activating its receptors coupled to PKCβII and AMPKα, increased insertion of GLUT2/5 into the brush-border membrane leading to enhanced galactose and fructose transport. Second in vivo, oral fructose but not galactose induced in mice a rapid and potent release of gastric leptin in gastric juice without significant changes in plasma leptin levels. Moreover, leptin given orally at a dose reproducing comparable levels to those induced by fructose, stimulated GLUT5-fructose transport, and potentiated fructose-induced: i) increase in blood glucose and mRNA levels of key gluconeogenesis enzymes; ii) increase in blood triglycerides and reduction of mRNA levels of intestinal and hepatic Fasting-induced adipocyte factor (Fiaf) and iii) increase in SREBP-1c, ACC-1, FAS mRNA levels and dephosphorylation/activation of ACC-1 in liver.

Conclusion/Significance

These data identify for the first time a positive regulatory control loop between gut leptin and fructose in which fructose triggers release of gastric leptin which, in turn, up-regulates GLUT5 and concurrently modulates metabolic functions in the liver. This loop appears to be a new mechanism (possibly pathogenic) by which fructose consumption rapidly becomes highly lipogenic and deleterious.     

Laninamivir Octanoate and Artificial Surfactant Combination Therapy Significantly Increases Survival of Mice Infected with Lethal Influenza H1N1 Virus

Background

Patients with influenza virus infection can develop severe pneumonia and acute respiratory distress syndrome (ARDS) which have a high mortality. Influenza virus infection is treated worldwide mainly by neuraminidase inhibitors (NAIs). However, monotherapy with NAIs is insufficient for severe pneumonia secondary to influenza virus infection. We previously demonstrated that mice infected with a lethal dose of influenza virus develop diffuse alveolar damage (DAD) with alveolar collapse similar to that seen in ARDS in humans. Additionally, pulmonary surfactant proteins were gradually increased in mouse serum, suggesting a decrease in pulmonary surfactant in the lung. Therefore, the present study examined whether combination therapy of NAI with exogenous artificial surfactant affects mortality of influenza virus-infected mice.

Methodology/Principal Findings

BALB/c mice were inoculated with several viral doses of influenza A/Puerto Rico/8/34 (PR8) virus (H1N1). The mice were additionally administered exogenous artificial surfactant in the presence or absence of a new NAI, laninamivir octanoate. Mouse survival, body weight and general condition were observed for up to 20 days after inoculation. Viral titer and cytokine/chemokine levels in the lungs, lung weight, pathological analysis, and blood O₂ and CO₂ pressures were evaluated. Infected mice treated with combination therapy of laninamivir octanoate with artificial surfactant showed a significantly higher survival rate compared with those that received laninamivir octanoate monotherapy (p = 0.003). However, virus titer, lung weight and cytokine/chemokine responses were not different between the groups. Histopathological examination, a hydrostatic lung test and blood gas analysis showed positive results in the combination therapy group.

Conclusions/Significance

Combination therapy of laninamivir octanoate with artificial surfactant reduces lethality in mice infected with influenza virus, and eventually suppresses DAD formation and preserves lung function. This combination could be effective for prevention of severe pneumonia secondary to influenza virus infection in humans, which is not improved by NAI monotherapy.