Metformin prevents endothelial oxidative stress and microvascular insulin resistance during obesity development in male rats.

Insulin increases muscle microvascular perfusion, which contributes to its metabolic action in muscle, but this action is impaired in obesity. Metformin improves endothelial function beyond its glucose lowering effects. We aim to examine whether metformin could prevent microvascular insulin resistance and endothelial dysfunction during the development of obesity. Adult male rats were fed a high-fat diet (HFD) with or without simultaneous metformin administration for either 2 or 4 wk. Insulin's metabolic and microvascular actions were determined using a combined euglycemic-hyperinsulinemic clamp and contrast-enhanced ultrasound approach. Compared with chow-fed controls, HFD feeding increased body adiposity without excess body weight gain, and this was associated with a marked decrease in insulin-mediated whole body glucose disposal and abolishment of insulin-induced muscle microvascular recruitment. Simultaneous administration of metformin fully rescued insulin-induced muscle microvascular recruitment as early as 2 wk and normalized insulin-mediated whole body glucose disposal at week 4. The divergent responses between insulin's microvascular and metabolic actions seen at week 2 were accompanied with reduced endothelial oxidative stress and vascular inflammation, and improved endothelial function and vascular insulin signaling in metformin-treated rats. In conclusions, metformin could prevent the development of microvascular insulin resistance and endothelial dysfunction by alleviating endothelial oxidative stress and vascular inflammation during obesity development.NEW & NOTEWORTHY Muscle microvascular insulin action contributes to insulin-mediated glucose use. Microvascular insulin resistance is an early event in diet-induced obesity and is associated with vascular inflammation. Metformin effectively reduces endothelial oxidative stress, improves endothelial function, and prevents microvascular insulin resistance during obesity development. These may contribute to metformin's salutary diabetes prevention and cardiovascular protective actions.

Efficacy of two porcine reproductive and respiratory syndrome (PRRS) modified-live virus (MLV) vaccines against heterologous NADC30-like PRRS virus challenge.

The emergence of novel and variant porcine reproductive and respiratory syndrome virus (PRRSV) strains has made controlling this disease a challenge in China. Several NADC30-like PRRSV outbreaks have occurred in mainland China since 2013. The objective of the present study was to evaluate the cross-protection efficacy of two commercial PRRS modified-live virus (MLV) vaccines, derived from classical PRRSV (VR2332) and highly pathogenic (HP) PRRSV (TJM-F92), against an increasingly circulating NADC30-like lineage in pigs. Thirty-five PRRSV- and antibody-free pigs were randomly divided into the following four groups: strict control (SC), negative control (NC), Boehringer control (BC), and Zoetis control (ZC) groups. The NADC30-like PRRSV used in this study caused fever, clinical respiratory signs, and gross and microscopic lung lesions in inoculated pigs in the NC group. Vaccination with the VR2332 vaccine significantly reduced the percentage of viremic pigs as well as gross lung lesions and improved average daily weight gain compared to the ZC and NC groups, suggesting that this MLV vaccine provides cross-protection against the NADC30-like virus. There were no significant differences in the efficacy of the two MLV vaccines based on clinical scores, immunological responses, or pathological outcomes. This study demonstrated that VR2332 MLV was effective against circulating NADC30-like PRRSV and could be used to control NADC30-like virus infections in the field.

Inhibiting myeloperoxidase prevents onset and reverses established high-fat diet-induced microvascular insulin resistance.

A high-fat diet (HFD) can rapidly recruit neutrophils to insulin target tissues and within days induce microvascular insulin resistance (IR). Myeloperoxidase (MPO) is highly enriched in neutrophils, can inhibit nitric oxide-mediated vasorelaxation in vitro and is associated with increased cardiovascular disease risk. AZD5904 irreversibly inhibits MPO and in human clinical trials. MPO knockout, or chemical inhibition, blunts HFD-induced metabolic IR in mice. Whether MPO affects microvascular IR or muscle metabolic insulin sensitivity in vivo is unknown. We used contrast-enhanced ultrasound and the euglycemic insulin clamp to test whether inhibiting MPO could prevent the development or reverse established HFD-induced metabolic and/or microvascular IR in Sprague-Dawley rats. Two weeks of HFD feeding blocked insulin-mediated skeletal muscle capillary recruitment, inhibited glucose utilization, and insulin signaling to muscle. Continuous subcutaneous AZD5904 infusion during the 2 wk selectively blocked HFD's microvascular effect. Furthermore, AZD5904 infusion during the last 2 of 4 wk of HFD feeding restored microvascular insulin sensitivity but not metabolic IR. We conclude that inhibiting MPO selectively improves vascular IR. This selective microvascular effect may connote a therapeutic potential for MPO inhibition in the prevention of vascular disease/dysfunction seen in IR humans.

GLP-1 and Insulin Recruit Muscle Microvasculature and Dilate Conduit Artery Individually But Not Additively in Healthy Humans.

CONTEXT: Glucagon-like peptide-1 (GLP-1) and insulin increase muscle microvascular perfusion, thereby increasing tissue endothelial surface area and nutrient delivery. OBJECTIVE: To examine whether GLP-1 and insulin act additively on skeletal and cardiac microvasculature and conduit artery. DESIGN: Healthy adults underwent three study protocols in random order. SETTING: Clinical Research Unit at the University of Virginia. METHODS: Overnight-fasted participants received an intravenous infusion of GLP-1 (1.2 pmol/kg/min) or normal saline for 150 minutes with or without a 2-hour euglycemic insulin clamp (1 mU/kg/min) superimposed from 30 minutes onward. Skeletal and cardiac muscle microvascular blood volume (MBV), flow velocity, and flow; brachial artery diameter, flow velocity, and blood flow; and pulse wave velocity (PWV) were measured. RESULTS: GLP-1 significantly increased skeletal and cardiac muscle MBV and microvascular blood flow (MBF) after 30 minutes; these remained elevated at 150 minutes. Insulin also increased skeletal and cardiac muscle MBV and MBF. Addition of insulin to GLP-1 did not further increase skeletal and cardiac muscle MBV and MBF. GLP-1 and insulin increased brachial artery diameter and blood flow, but this effect was not additive. Neither GLP-1, insulin, nor GLP-1 and insulin altered PWV. Combined GLP-1 and insulin infusion did not result in higher whole-body glucose disposal. CONCLUSION: GLP-1 and insulin at physiological concentrations acutely increase skeletal and cardiac muscle microvascular perfusion and dilate conduit artery in healthy adults; these effects are not additive. Thus, GLP-1 and insulin may regulate skeletal and cardiac muscle endothelial surface area and nutrient delivery under physiological conditions.

Enhanced Klebsiella pneumoniae Carbapenemase Expression from a Novel Tn4401 Deletion.

The Klebsiella pneumoniae carbapenemase gene (blaKPC) is typically located within mobile transposon Tn4401 Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of blaKPC Illumina sequences from blaKPC-positive clinical isolates from a single institution were mapped to a Tn4401b reference sequence, which carries no deletions. The novel isoform Tn4401h (188-bp deletion [between istB and blaKPC]) was present in 14% (39/281) of clinical isolates. MICs showed that Escherichia coli strains containing plasmids with Tn4401a and Tn4401h were more resistant to meropenem (≥16 and ≥16, respectively), ertapenem (≥8 and 4, respectively), and cefepime (≥64 and 4, respectively) than E. coli strains with Tn4401b (0.5, ≤0.5, and ≤1, respectively). Quantitative real-time PCR (qRT-PCR) demonstrated that Tn4401a had a 16-fold increase and Tn4401h a 4-fold increase in blaKPC mRNA levels compared to the reference Tn4401b. A lacZ reporter plasmid was used to test the activity of the promoter regions from the different variants, and the results showed that the Tn4401a and Tn4401h promoter sequences generated higher ß-galactosidase activity than the corresponding Tn4401b sequence. Further dissection of the promoter region demonstrated that putative promoter P1 was not functional. The activity of the isolated P2 promoter was greatly enhanced by inclusion of the P1-P2 intervening sequence. These studies indicated that gene expression could be an important consideration in understanding resistance phenotypes predicted by genetic signatures in the context of sequencing-based rapid diagnostics.

Spread from the Sink to the Patient: In Situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs.

There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients.

Chromosomal Integration of the Klebsiella pneumoniae Carbapenemase Gene, blaKPC, in Klebsiella Species Is Elusive but Not Rare.

Carbapenemase genes in Enterobacteriaceae are mostly described as being plasmid associated. However, the genetic context of carbapenemase genes is not always confirmed in epidemiological surveys, and the frequency of their chromosomal integration therefore is unknown. A previously sequenced collection of blaKPC-positive Enterobacteriaceae from a single U.S. institution (2007 to 2012; n = 281 isolates from 182 patients) was analyzed to identify chromosomal insertions of Tn4401, the transposon most frequently harboring blaKPC Using a combination of short- and long-read sequencing, we confirmed five independent chromosomal integration events from 6/182 (3%) patients, corresponding to 15/281 (5%) isolates. Three patients had isolates identified by perirectal screening, and three had infections which were all successfully treated. When a single copy of blaKPC was in the chromosome, one or both of the phenotypic carbapenemase tests were negative. All chromosomally integrated blaKPC genes were from Klebsiella spp., predominantly K. pneumoniae clonal group 258 (CG258), even though these represented only a small proportion of the isolates. Integration occurred via IS15-ΔI-mediated transposition of a larger, composite region encompassing Tn4401 at one locus of chromosomal integration, seen in the same strain (K. pneumoniae ST340) in two patients. In summary, we identified five independent chromosomal integrations of blaKPC in a large outbreak, demonstrating that this is not a rare event. blaKPC was more frequently integrated into the chromosome of epidemic CG258 K. pneumoniae lineages (ST11, ST258, and ST340) and was more difficult to detect by routine phenotypic methods in this context. The presence of chromosomally integrated blaKPC within successful, globally disseminated K. pneumoniae strains therefore is likely underestimated.

Liraglutide prevents microvascular insulin resistance and preserves muscle capillary density in high-fat diet-fed rats.

Muscle microvasculature critically regulates endothelial exchange surface area to facilitate transendothelial delivery of insulin, nutrients, and oxygen to myocytes. Insulin resistance blunts insulin-mediated microvascular recruitment and decreases muscle capillary density; both contribute to lower microvascular blood volume. Glucagon-like peptide 1 (GLP-1) and its analogs are able to dilate blood vessels and stimulate endothelial cell proliferation. In this study, we aim to determine the effects of sustained stimulation of the GLP-1 receptors on insulin-mediated capillary recruitment and metabolic insulin responses, small arterial endothelial function, and muscle capillary density. Rats were fed a high-fat diet (HFD) for 4 wk with or without simultaneous administration of liraglutide and subjected to a euglycemic hyperinsulinemic clamp for 120 min after an overnight fast. Insulin-mediated muscle microvascular recruitment and muscle oxygenation were determined before and during insulin infusion. Muscle capillary density was determined and distal saphenous artery used for determination of endothelial function and insulin-mediated vasodilation. HFD induced muscle microvascular insulin resistance and small arterial vessel endothelial dysfunction and decreased muscle capillary density. Simultaneous treatment of HFD-fed rats with liraglutide prevented all of these changes and improved insulin-stimulated glucose disposal. These were associated with a significantly increased AMPK phosphorylation and the expressions of VEGF and its receptors. We conclude that GLP-1 receptor agonists may exert their salutary glycemic effect via improving microvascular insulin sensitivity and muscle capillary density during the development of insulin resistance, and early use of GLP-1 receptor agonists may attenuate metabolic insulin resistance as well as prevent cardiovascular complications of diabetes.

Elevated dietary zinc oxide levels do not have a substantial effect on porcine reproductive and respiratory syndrome virus (PPRSV) vaccination and infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important infectious agents for the swine industry worldwide. Zinc (Zn) salts, which are widely used as a dietary supplement in swine nutrition, have shown antiviral effects in vitro as well as in vivo. The purpose of this study was to determine the influence of dietary zinc oxide supplementation on vaccination and challenge infection with PRRSV. FINDINGS: The clinical course of PRRS and the success of vaccination with an experimental inactivated vaccine were compared between animals receiving a conventional diet (50 ppm Zn, control group) and diets supplemented with Zn oxide (ZnO) at final Zn concentrations of 150 or 2,500 ppm. Pigs receiving higher dietary Zn levels showed a tendency towards higher neutralizing antibody levels after infection, while dietary Zn levels did not substantially influence the number of antiviral IFN-gamma secreting cells (IFN-gamma-SC) or percentages of blood immune cell subsets after infection. Finally, feeding higher dietary Zn levels reduced neither clinical symptoms nor viral loads. CONCLUSIONS: Our results suggest that higher levels of dietary ZnO do not have the potential to stimulate or modulate systemic immune responses after vaccination and heterologous PRRSV infection to an extent that could improve the clinical and virological outcome.

High-dose dietary zinc oxide mitigates infection with transmissible gastroenteritis virus in piglets.

BACKGROUND: Zinc (Zn) supplementation has been shown to reduce the incidence of diarrhea and to protect animals from intestinal diseases, but the mechanisms of this protective effect against virus infection in vivo have not yet been elucidated. Transmissible gastroenteritis virus (TGEV) causes diarrhea in piglets with an age-dependent decrease of severity. RESULTS: We used 60 weaned piglets that were divided into three groups to evaluate the effect of different Zn levels added to a conventional diet (50 mg Zn/kg diet, Znlow, control group). The other groups received the diet supplemented with ZnO at final concentrations of 150 mg Zn/kg diet (Znmed), or 2,500 mg/kg diet (Znhigh). Oral challenge infection with TGEV was performed when the pigs had been fed for 1 week with the respective diet. Half of the piglets of each group were sacrificed at day 1 and 18 after challenge infection. Fecal consistency was improved and body weights increased in the Znhigh group when compared to the other groups, but no direct effect of Zn concentrations in the diet on fecal TGEV shedding and mucosal immune responses was detectable. However, in the Znhigh group, we found a prevention of villus atrophy and decreased caspase-3-mediated apoptosis of jejunal epithelium. Furthermore, pigs receiving high Zn diet showed a down-regulation of interferon (IFN)-α, oligoadenylate synthetase (OAS), Zn transporter SLC39A4 (ZIP4), but up-regulation of metallothionein-1 (MT1), as well as the Zn transporters SLC30A1 (ZnT1) and SLC30A5 (ZnT5). In addition, forskolin-induced chloride secretion and epithelial resistance were controlled at a physiological level in the Znhigh but not the other groups. Finally, in the Znhigh group, we documented an earlier and higher systemic TGEV-specific serum antibody response. CONCLUSIONS: These results suggest that high dietary Zn could provide enhanced protection in the intestinal tract and stimulate the systemic humoral immune response against TGEV infection.

Glucagon-like peptide 1 recruits muscle microvasculature and improves insulin's metabolic action in the presence of insulin resistance.

Glucagon-like peptide 1 (GLP-1) acutely recruits muscle microvasculature, increases muscle delivery of insulin, and enhances muscle use of glucose, independent of its effect on insulin secretion. To examine whether GLP-1 modulates muscle microvascular and metabolic insulin responses in the setting of insulin resistance, we assessed muscle microvascular blood volume (MBV), flow velocity, and blood flow in control insulin-sensitive rats and rats made insulin-resistant acutely (systemic lipid infusion) or chronically (high-fat diet [HFD]) before and after a euglycemic-hyperinsulinemic clamp (3 mU/kg/min) with or without superimposed systemic GLP-1 infusion. Insulin significantly recruited muscle microvasculature and addition of GLP-1 further expanded muscle MBV and increased insulin-mediated glucose disposal. GLP-1 infusion potently recruited muscle microvasculature in the presence of either acute or chronic insulin resistance by increasing muscle MBV. This was associated with an increased muscle delivery of insulin and muscle interstitial oxygen saturation. Muscle insulin sensitivity was completely restored in the presence of systemic lipid infusion and significantly improved in rats fed an HFD. We conclude that GLP-1 infusion potently expands muscle microvascular surface area and improves insulin's metabolic action in the insulin-resistant states. This may contribute to improved glycemic control seen in diabetic patients receiving incretin-based therapy.

GLP-1 at physiological concentrations recruits skeletal and cardiac muscle microvasculature in healthy humans.

Muscle microvascular surface area determines substrate and hormonal exchanges between plasma and muscle interstitium. GLP-1 (glucagon-like peptide-1) regulates glucose-dependent insulin secretion and has numerous extrapancreatic effects, including a salutary vascular action. To examine whether GLP-1 recruits skeletal and cardiac muscle microvasculature in healthy humans, 26 overnight-fasted healthy adults received a systemic infusion of GLP-1 (1.2 pmol/kg of body mass per min) for 150 min. Skeletal and cardiac muscle MBV (microvascular blood volume), MFV (microvascular flow velocity) and MBF (microvascular blood flow) were determined at baseline and after 30 and 150 min. Brachial artery diameter and mean flow velocity were measured and total blood flow was calculated before and at the end of the GLP-1 infusion. GLP-1 infusion raised plasma GLP-1 concentrations to the postprandial levels and suppressed plasma glucagon concentrations with a transient increase in plasma insulin concentrations. Skeletal and cardiac muscle MBV and MBF increased significantly at both 30 and 150 min (P<0.05). MFV did not change in skeletal muscle, but decreased slightly in cardiac muscle. GLP-1 infusion significantly increased brachial artery diameter (P<0.005) and flow velocity (P=0.05) at 150 min, resulting in a significant increase in total brachial artery blood flow (P<0.005). We conclude that acute GLP-1 infusion significantly recruits skeletal and cardiac muscle microvasculature in addition to relaxing the conduit artery in healthy humans. This could contribute to increased tissue oxygen, nutrient and insulin delivery and exchange and therefore better prandial glycaemic control and tissue function in humans.

Dietary Enterococcus faecium NCIMB 10415 and zinc oxide stimulate immune reactions to trivalent influenza vaccination in pigs but do not affect virological response upon challenge infection.

Swine influenza viruses (SIV) regularly cause significant disease in pigs worldwide. Since there is no causative treatment of SIV, we tested if probiotic Enterococcus (E.) faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements provide beneficial effects upon SIV infection in piglets. Seventy-two weaned piglets were fed three different diets containing either E. faecium or different levels of Zn (2500 ppm, Zn(high); 50 ppm, Zn(low)). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine, while all piglets were then infected intranasally with H3N2 SIV. Significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Zn(high) and E. faecium groups gained weight after infection while those in the control group (Zn(low)) lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Zn(high)+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Using flow cytometry analysis significantly higher activated T helper cells and cytotoxic T lymphocyte percentages in the PBMCs were detected in the Zn(high) and E. faecium groups at single time points after infection compared to the Zn(low) control group, but no prolonged effect was found. In the BAL cells no influence of dietary supplementation on immune cell percentages could be detected. Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology.

Ranolazine recruits muscle microvasculature and enhances insulin action in rats.

Ranolazine, an anti-anginal compound, has been shown to significantly improve glycaemic control in large-scale clinical trials, and short-term ranolazine treatment is associated with an improvement in myocardial blood flow. As microvascular perfusion plays critical roles in insulin delivery and action, we aimed to determine if ranolazine could improve muscle microvascular blood flow, thereby increasing muscle insulin delivery and glucose use. Overnight-fasted, anaesthetized Sprague-Dawley rats were used to determine the effects of ranolazine on microvascular recruitment using contrast-enhanced ultrasound, insulin action with euglycaemic hyperinsulinaemic clamp, and muscle insulin uptake using (125)I-insulin. Ranolazine's effects on endothelial nitric oxide synthase (eNOS) phosphorylation, cAMP generation and endothelial insulin uptake were determined in cultured endothelial cells. Ranolazine-induced myographical changes in tension were determined in isolated distal saphenous artery. Ranolazine at therapeutically effective dose significantly recruited muscle microvasculature by increasing muscle microvascular blood volume (∼2-fold, P < 0.05) and increased insulin-mediated whole body glucose disposal (∼30%, P = 0.02). These were associated with an increased insulin delivery into the muscle (P < 0.04). In cultured endothelial cells, ranolazine increased eNOS phosphorylation and cAMP production without affecting endothelial insulin uptake. In ex vivo studies, ranolazine exerted a potent vasodilatatory effect on phenylephrine pre-constricted arterial rings, which was partially abolished by endothelium denudement. In conclusion, ranolazine treatment vasodilatates pre-capillary arterioles and increases microvascular perfusion, which are partially mediated by endothelium, leading to expanded microvascular endothelial surface area available for nutrient and hormone exchanges and resulting in increased muscle delivery and action of insulin. Whether these actions contribute to improved glycaemic control in patients with insulin resistance warrants further investigation.

Globular adiponectin enhances muscle insulin action via microvascular recruitment and increased insulin delivery.

RATIONALE: Adiponectin enhances insulin action and induces nitric oxide-dependent vasodilatation. Insulin delivery to muscle microcirculation and transendothelial transport are 2 discrete steps that limit insulin's action. We have shown that expansion of muscle microvascular surface area increases muscle insulin delivery and action. OBJECTIVE: To examine whether adiponectin modulates muscle microvascular recruitment thus insulin delivery and action in vivo. METHODS AND RESULTS: Overnight fasted adult male rats were studied. We determined the effects of adiponectin on muscle microvascular recruitment, using contrast-enhanced ultrasound, on insulin-mediated microvascular recruitment and whole-body glucose disposal, using contrast-enhanced ultrasound and insulin clamp, and on muscle insulin clearance and uptake with (125)I-insulin. Globular adiponectin potently increased muscle microvascular blood volume without altering microvascular blood flow velocity, leading to a significantly increased microvascular blood flow. This was paralleled by a ≈30% to 40% increase in muscle insulin uptake and clearance, and ≈30% increase in insulin-stimulated whole-body glucose disposal. Inhibition of endothelial nitric oxide synthase abolished globular adiponectin-mediated muscle microvascular recruitment and insulin uptake. In cultured endothelial cells, globular adiponectin dose-dependently increased endothelial nitric oxide synthase phosphorylation but had no effect on endothelial cell internalization of insulin. CONCLUSIONS: Globular adiponectin increases muscle insulin uptake by recruiting muscle microvasculature, which contributes to its insulin-sensitizing action.

Inhibitory influence of Enterococcus faecium on the propagation of swine influenza A virus in vitro.

The control of infectious diseases such as swine influenza viruses (SwIV) plays an important role in food production both from the animal health and from the public health point of view. Probiotic microorganisms and other health improving food supplements have been given increasing attention in recent years, but, no information on the effects of probiotics on swine influenza virus is available. Here we address this question by assessing the inhibitory potential of the probiotic Enterococcus faecium NCIMB 10415 (E. faecium) on the replication of two porcine strains of influenza virus (H1N1 and H3N2 strain) in a continuous porcine macrophage cell line (3D4/21) and in MDBK cells. Cell cultures were treated with E. faecium at the non-toxic concentration of 1×10(6) CFU/ml in growth medium for 60 to 90 min before, during and after SwIV infection. After further incubation of cultures in probiotic-free growth medium, cell viability and virus propagation were determined at 48 h or 96 h post infection. The results obtained reveal an almost complete recovery of viability of SwIV infected cells and an inhibition of virus multiplication by up to four log units in the E. faecium treated cells. In both 3D4/21- and MDBK-cells a 60 min treatment with E. faecium stimulated nitric oxide (NO) release which is in line with published evidence for an antiviral function of NO. Furthermore, E. faecium caused a modified cellular expression of selected mediators of defence in 3D4-cells: while the expression of TNF-α, TLR-3 and IL-6 were decreased in the SwIV-infected and probiotic treated cells, IL-10 was found to be increased. Since we obtained experimental evidence for the direct adsorptive trapping of SwIV through E. faecium, this probiotic microorganism inhibits influenza viruses by at least two mechanisms, direct physical interaction and strengthening of innate defence at the cellular level.

Losartan increases muscle insulin delivery and rescues insulin's metabolic action during lipid infusion via microvascular recruitment.

Insulin delivery and transendothelial insulin transport are two discrete steps that limit muscle insulin action. Angiotensin II type 1 receptor (AT1R) blockade recruits microvasculature and increases glucose use in muscle. Increased muscle microvascular perfusion is associated with increased muscle delivery and action of insulin. To examine the effect of acute AT1R blockade on muscle insulin uptake and action, rats were studied after an overnight fast to examine the effects of losartan on muscle insulin uptake (protocol 1), microvascular perfusion (protocol 2), and insulin's microvascular and metabolic actions in the state of insulin resistance (protocol 3). Endothelial cell insulin uptake was assessed, using (125)I-insulin as tracer. Systemic lipid infusion was used to induce insulin resistance. Losartan significantly increased muscle insulin uptake (∼60%, P < 0.03), which was associated with a two- to threefold increase in muscle microvascular blood volume (MBV; P = 0.002) and flow (MBF; P = 0.002). Losartan ± angiotensin II had no effect on insulin internalization in cultured endothelial cells. Lipid infusion abolished insulin-mediated increases in muscle MBV and MBF and lowered insulin-stimulated whole body glucose disposal (P = 0.0001), which were reversed by losartan administration. Inhibition of nitric oxide synthase abolished losartan-induced muscle insulin uptake and reversal of lipid-induced metabolic insulin resistance. We conclude that AT1R blockade increases muscle insulin uptake mainly via microvascular recruitment and rescues insulin's metabolic action in the insulin-resistant state. This may contribute to the clinical findings of decreased cardiovascular events and new onset of diabetes in patients receiving AT1R blockers.

Protein kinase A mediates glucagon-like peptide 1-induced nitric oxide production and muscle microvascular recruitment.

Glucagon-like peptide-1 (GLP-1) causes vasodilation and increases muscle glucose uptake independent of insulin. Recently, we have shown that GLP-1 recruits muscle microvasculature and increases muscle glucose use via a nitric oxide (NO)-dependent mechanism. Protein kinase A (PKA) is a major signaling intermediate downstream of GLP-1 receptors. To examine whether PKA mediates GLP-1's microvascular action in muscle, GLP-1 was infused to overnight-fasted male rats for 120 min in the presence or absence of H89, a PKA inhibitor. Hindleg muscle microvascular recruitment and glucose use were determined. GLP-1 infusion acutely increased muscle microvascular blood volume within 30 min without altering microvascular blood flow velocity or blood pressure. This effect persisted throughout the 120-min infusion period, leading to a significant increase in muscle microvascular blood flow. These changes were paralleled with an approximately twofold increase in plasma NO levels and hindleg glucose extraction. Systemic infusion of H89 completely blocked GLP-1-mediated muscle microvascular recruitment and increases in NO production and muscle glucose extraction. In cultured endothelial cells, GLP-1 acutely increased PKA activity and stimulated endothelial NO synthase phosphorylation at Ser(1177) and NO production. PKA inhibition abolished these effects. In ex vivo studies, perfusion of the distal saphenous artery with GLP-1 induced significant vasorelaxation that was also abolished by pretreatment of the vessels with PKA inhibitor H89. We conclude that GLP-1 recruits muscle microvasculature by expanding microvascular volume and increases glucose extraction in muscle via a PKA/NO-dependent pathway in the vascular endothelium. This may contribute to postprandial glycemic control and complication prevention in diabetes.

Antiviral effects of a probiotic Enterococcus faecium strain against transmissible gastroenteritis coronavirus.

The enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV) causes severe disease in young piglets. We have studied the protective effects of the probiotic Enterococcus faecium NCIMB 10415 (E. faecium), which is approved as a feed additive in the European Union, against TGEV infection. E. faecium was added to swine testicle (ST) cells before, concomitantly with, or after TGEV infection. Viability assays revealed that E. faecium led to a dose-dependent rescue of viability of TGEV-infected cells reaching nearly to complete protection. Virus yields of the E. faecium-treated cultures were reduced by up to three log10 units. Western blot analysis of purified TGEV revealed that the levels of all viral structural proteins were reduced after E. faecium treatment. Using transmission electron microscopy, we observed attachment of TGEV particles to the surface of E. faecium which might be a means to trap virus and to prevent infection. Increased production of nitric oxide in the cells treated with E. faecium and elevated expression of interleukin 6 and 8 pointed to stimulated cellular defense as a mechanism to fight TGEV infection.

Regulation of muscle microcirculation in health and diabetes.

Insulin increases microvascular perfusion and substrate exchange surface area in muscle, which is pivotal for hormone action and substrate exchange, by activating insulin signaling cascade in the endothelial cells to produce nitric oxide. This action of insulin is closely coupled with its metabolic action and type 2 diabetes is associated with both metabolic and microvascular insulin resistance. Muscle microvascular perfusion/volume can be assessed by 1-methylxanthine metabolism, contrast-enhanced ultrasound and positron emission tomography. In addition to insulin, several factors have been shown to recruit muscle microvasculature, including exercise or muscle contraction, mixed meals, glucagon-like peptide 1 and angiotensin II type 1 receptor (AT(1)R) blocker. On the other hand, factors that cause metabolic insulin resistance, such as inflammatory cytokines, free fatty acids, and selective activation of the AT(1)R, are capable of causing microvascular insulin resistance. Therapies targeting microvascular insulin resistance may help prevent or control diabetes and decrease the associated cardiovascular morbidity and mortality.