Pathogenic potential of the surviving Salmonella Enteritidis on strawberries after disinfection treatments based on ultraviolet-C light and peracetic acid.

Fresh fruits, especially strawberries, are usually consumed raw without any step to ensure their food safety. Salmonella enterica is one of the most important etiologic agents for foodborne diseases throughout the world and its ability to respond to some stress responses makes it even more dangerous. In the present investigation, we study the survival of S. Enteritidis (CECT-4300) on strawberries after 2-min of various disinfection steps (NaClO (200 ppm), peracetic acid (PAA; 40 ppm), water-assisted UV-C (WUV-C), and the combination WUV-C and 40 ppm of PAA (WUV-C + PAA)) and after 5 days of cold storage (4 °C). Moreover, the pathogenic potential of the surviving bacteria, such as the ability to survive throughout the gastrointestinal tract (GI) and subsequently the capability to adhere to and invade Caco-2 cells, was tested at each sampling point. After 2-min of washing procedures, reductions of S. Enteritidis on strawberries were ≥1.2 log, with no significant differences among treatments. However, the use of WUV-C + PAA treatment achieved the highest reductions in washing water, in which S. Enteritidis was not detected (

Evaluation of a sanitizing washing step with different chemical disinfectants for the strawberry processing industry.

Strawberries are often consumed fresh or only receive minimal processing, inducing a significant health risk to the consumer if contamination occurs anywhere from farm to fork. Outbreaks of foodborne illness associated with strawberries often involve a broad range of microbiological agents, from viruses (human norovirus) to bacteria (Salmonella spp. and Listeria monocytogenes). The addition of sanitizers to water washes is one of the most commonly studied strategies to remove or inactivate pathogens on berries as well as avoid cross contamination due to reuse of process wash water. The risk posed with the safety issues of by-products from chlorine disinfection in the fruit industry has led to a search for alternative sanitizers. We evaluated the applicability of different chemical sanitizers (peracetic acid (PA), hydrogen peroxide (H2O2), citric acid (CA), lactic acid (LA) and acetic acid (AA)) for the inactivation of S. enterica, L. monocytogenes and murine norovirus (MNV-1) on strawberries. A control treatment with chlorine (NaClO) (100 ppm) was included. For each sanitizer, different doses (40, 80 and 120 ppm for PA and 1, 2.5 and 5% for H2O2, LA, AA and CA) and time (2 and 5 min) were studied in order to optimize the decontamination washing step. The best concentrations were 80 ppm for PA, 5% for H2O2 and 2.5% for organic acids (LA, AA and CA) after 2 min treatment. Results indicate that the sanitizers selected may be a feasible alternative to chlorine (100 ppm) for removing selected pathogenic microorganisms (P > 0.05), with reductions about ≥2 log for bacterial strains and ≥ 1.7 log for MNV-1. As the washing water may also increase the microbial counts by cross-contamination, we observed that no pathogenic bacteria were found in wash water after 5% H2O2 and 80 ppm PA after 2 min treatment. On the other hand, we also reported reductions about total aerobic mesophyll (TAM) (0.0-1.4 log CFU/g) and molds and yeasts (M&Y) (0.3-1.8 log CFU/g) with all alternative sanitizers tested. Strawberries treated did not shown significant differences about physio-chemical parameters compared to the untreated samples (initial). For this study, the optimal sanitizer selected was PA, due to the low concentration and cost needed and its microbiocidal effect in wash water and fruit. Notwithstanding the results obtained, the effect of PA in combination with other non-thermal technologies such as water-assisted ultraviolet (UV-C) light should be studied in future research to improve the disinfection of strawberries.

Microbial interaction between Salmonella enterica and main postharvest fungal pathogens on strawberry fruit.

The microbial interaction between Salmonella enterica and the main postharvest fungal pathogens of strawberries was evaluated. Inoculation of fungal suspension was done 2 (D2) and 1 (D1) day(s) before and at the same time (D0) as S. enterica. Fruits were stored at 20 °C and 4 °C. At both temperatures, Botrytis cinerea and Rhizopus stolonifer caused a decrease in S. enterica population. Treatments where the mould was inoculated (D2, D1 and D0) achieved a significant logarithmic reduction (P < 0.05) of S. enterica populations after 48 h (20 °C) and 14 days (4 °C) compared to fungal-uninoculated fruits (CK). Regarding temperature, average reductions were significantly higher at 4 °C (3.38 log10 CFU/wound) than at 20 °C (1.16 log10 CFU/wound) (P < 0.05). Average reductions comprising all treatments were 1.91 and 0.41 log10 CFU/wound for B. cinerea and R. stolonifer at 20 °C, and 3.39 and 3.37 log10 CFU/wound for B. cinerea and R. stolonifer at 4 °C. A linear log10 model was fitted in order to predict the inactivation rate (kmax, log10 CFU/h) of S. enterica. Inactivation rates were higher at 20 °C for D2 treatments than at 4 °C throughout the running time. The main inactivation rate was obtained for B. cinerea at 20 °C (0.160 ±â€¯0.027/h), which was found to have stronger inhibitory activity against S. enterica than R. stolonifer. Univariate analysis ANOVA was carried out to evaluate the effect of different external variables on the inhibition of S. enterica. Results found that single effects were significant (P < 0.05) except for the pH. The inhibitory effect caused by the action of moulds in conjunction with some environmental factors could indicate the potential interactions between strawberry fungal pathogens and S. enterica.

Occurrence of selected viral and bacterial pathogens and microbiological quality of fresh and frozen strawberries sold in Spain.

Strawberry production and exports have been increasing in Spain in recent decades. However, little information is available about their microbiological quality. Due to the growing concern about the microbial safety of these fruits, the objective of this investigation was to study the microbiological quality and the prevalence of the main foodborne pathogens on strawberries sold in Spain. Fresh (n = 152) and frozen (n = 31) samples were obtained from marketplaces and fields in 2017 and 2018. The samples were assayed for total aerobic mesophilic microorganisms (TAM), moulds and yeasts (M&Y), total coliforms (TC), Escherichia coli, Salmonella spp., Listeria monocytogenes as well as Norovirus (NoV) GI and GII. The microbiological counts ranged from <1.70 (detection limit, dl) - 5.89 log10 CFU/g (mean 3.78 log10 CFU/g) for TAM; 2.10-5.86 log10 CFU/g (mean 3.80 log10 CFU/g) for M&Y; and <0.70 (dl) - 4.91 log10 CFU/g (mean 2.15 log10 CFU/g) for TC in fresh strawberries. In frozen strawberries, the counts were <1.70 (dl) - 3.66 log10 CFU/g (mean 2.30 log10 CFU/g) for TAM; <1.70 (dl) - 2.76 log10 CFU/g (mean 1.82 log10 CFU/g) for M&Y; and <0.70(dl) - 1.74 log10 CFU/g (mean 0.77 log10 CFU/g) for TC. All the samples in this study tested negative for Salmonella spp., L. monocytogenes. E. coli and NoV GI and GII genome. A global overview of all the data was executed using Principal Component Analysis (PCA), and the results showed that the scores and loadings according to principal components 1 (PC1) and 2 (PC2) accounted for 75.9% of the total variance, allowing a distinction between fresh and frozen samples. The presence of moulds was significantly higher in the supermarket samples whereas the presence of total coliforms was significantly higher in the field samples (p < 0.05). Although pathogenic microorganisms were not found, preventative measures and prerequisites in the strawberry production chain must be considered in order to avoid possible foodborne diseases related to the microbiological quality of the fruit.

Pancreatic ß-cell imaging in humans: fiction or option?

Diabetes mellitus is a growing worldwide epidemic disease, currently affecting 1 in 12 adults. Treatment of disease complications typically consumes ∼10% of healthcare budgets in developed societies. Whilst immune-mediated destruction of insulin-secreting pancreatic ß cells is responsible for Type 1 diabetes, both the loss and dysfunction of these cells underly the more prevalent Type 2 diabetes. The establishment of robust drug development programmes aimed at ß-cell restoration is still hampered by the absence of means to measure ß-cell mass prospectively in vivo, an approach which would provide new opportunities for understanding disease mechanisms and ultimately assigning personalized treatments. In the present review, we describe the progress towards this goal achieved by the Innovative Medicines Initiative in Diabetes, a collaborative public-private consortium supported by the European Commission and by dedicated resources of pharmaceutical companies. We compare several of the available imaging methods and molecular targets and provide suggestions as to the likeliest to lead to tractable approaches. Furthermore, we discuss the simultaneous development of animal models that can be used to measure subtle changes in ß-cell mass, a prerequisite for validating the clinical potential of the different imaging tracers.

Biopreservative methods to control the growth of foodborne pathogens on fresh-cut lettuce.

Fruits and vegetables can become contaminated by foodborne pathogens such as Escherichia coli O157:H7, Salmonella and Listeria monocytogenes, and it has been demonstrated that current industrial sanitizing treatments do not eliminate the pathogens when present. Chemical control is widely used, but biological control appears to be a better solution, mainly using the native microbiota present on fresh produce. The first objective of this study was to isolate native microbiota from whole and fresh-cut produce and to determine whether these bacteria were antagonistic toward foodborne pathogens. A total of 112 putative antagonist isolates were screened for their ability to inhibit the growth of Salmonella enterica on lettuce disks. Five different genera reduced S. enterica growth more than 1-log unit at 20°C at the end of 3 days. When tested against L. monocytogenes 230/3, only Pseudomonas sp. strain M309 (M309) was able to reduce pathogen counts by more than 1-log unit. Therefore, M309 strain was selected to be tested on lettuce disks at 10°C against S. enterica, E. coli O157:H7 and L. monocytogenes. M309 strain was only able to reduce S. enterica and E. coli O157:H7 populations. The second objective was to test different biopreservative methods including M309 strain, Pseudomonas graminis CPA-7 (CPA-7), bacteriophages (Listex P100 and Salmonelex) and nisin at conditions simulating commercial applications against Salmonella and L. monocytogenes on fresh-cut lettuce. The addition of the biopreservative agents did not result in a significant reduction of Salmonella population. However, CPA-7 strain together with nisin reduced L. monocytogenes numbers after 6 days of storage at 10°C. The cocktail of Salmonella and L. monocytogenes was not markedly inactivated by their respective bacteriophage solutions. This study highlighted the potential of biocontrol, but the combination with other technologies may be required to improve their application on fresh-cut lettuce.

Connexin36 contributes to INS-1E cells survival through modulation of cytokine-induced oxidative stress, ER stress and AMPK activity.

Cell-to-cell communication mediated by gap junctions made of Connexin36 (Cx36) contributes to pancreatic ß-cell function. We have recently demonstrated that Cx36 also supports ß-cell survival by a still unclear mechanism. Using specific Cx36 siRNAs or adenoviral vectors, we now show that Cx36 downregulation promotes apoptosis in INS-1E cells exposed to the pro-inflammatory cytokines (IL-1ß, TNF-α and IFN-γ) involved at the onset of type 1 diabetes, whereas Cx36 overexpression protects against this effect. Cx36 overexpression also protects INS-1E cells against endoplasmic reticulum (ER) stress-mediated apoptosis, and alleviates the cytokine-induced production of reactive oxygen species, the depletion of the ER Ca(2+) stores, the CHOP overexpression and the degradation of the anti-apoptotic protein Bcl-2 and Mcl-1. We further show that cytokines activate the AMP-dependent protein kinase (AMPK) in a NO-dependent and ER-stress-dependent manner and that AMPK inhibits Cx36 expression. Altogether, the data suggest that Cx36 is involved in Ca(2+) homeostasis within the ER and that Cx36 expression is downregulated following ER stress and subsequent AMPK activation. As a result, cytokine-induced Cx36 downregulation elicits a positive feedback loop that amplifies ER stress and AMPK activation, leading to further Cx36 downregulation. The data reveal that Cx36 plays a central role in the oxidative stress and ER stress induced by cytokines and the subsequent regulation of AMPK activity, which in turn controls Cx36 expression and mitochondria-dependent apoptosis of insulin-producing cells.

Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin.

AIMS/HYPOTHESIS: In rodents and humans, the rate of beta cell proliferation declines rapidly after birth; formation of the islets of Langerhans begins perinatally and continues after birth. Here, we tested the hypothesis that increasing levels of E-cadherin during islet formation mediate the decline in beta cell proliferation rate by contributing to a reduction of nuclear ß-catenin and D-cyclins. METHODS: We examined E-cadherin, nuclear ß-catenin, and D-cyclin levels, as well as cell proliferation during in vitro and in vivo formation of islet cell aggregates, using ß-TC6 cells and transgenic mice with green fluorescent protein (GFP)-labelled beta cells, respectively. We tested the role of E-cadherin using antisense-mediated reductions of E-cadherin in ß-TC6 cells, and mice segregating for a beta cell-specific E-cadherin knockout (Ecad [also known as Cdh1] ßKO). RESULTS: In vitro, pseudo-islets of ß-TC6 cells displayed increased E-cadherin but decreased nuclear ß-catenin and cyclin D2, and reduced rates of cell proliferation, compared with monolayers. Antisense knockdown of E-cadherin increased cell proliferation and levels of cyclins D1 and D2. After birth, beta cells showed increased levels of E-cadherin, but decreased levels of D-cyclin, whereas islets of Ecad ßKO mice showed increased levels of D-cyclins and nuclear ß-catenin, as well as increased beta cell proliferation. These islets were significantly larger than those of control mice and displayed reduced levels of connexin 36. These changes correlated with reduced insulin response to ambient glucose, both in vitro and in vivo. CONCLUSIONS/INTERPRETATION: The findings support our hypothesis by indicating an important role of E-cadherin in the control of beta cell mass and function.

Impaired ß-cell-ß-cell coupling mediated by Cx36 gap junctions in prediabetic mice.

Gap junctional intercellular communication between ß-cells is crucial for proper insulin biosynthesis and secretion. The aim of this work was to investigate the expression of connexin (Cx)36 at the protein level as well as the function and structure of gap junctions (GJ) made by this protein in the endocrine pancreas of prediabetic mice. C57BL/6 mice were fed a high-fat (HF) or regular chow diet for 60 days. HF-fed mice became obese and prediabetic, as shown by peripheral insulin resistance, moderate hyperglycemia, hyperinsulinemia, and compensatory increase in endocrine pancreas mass. Compared with control mice, prediabetic animals showed a significant decrease in insulin-secretory response to glucose and displayed a significant reduction in islet Cx36 protein. Ultrastructural analysis further showed that prediabetic mice had GJ plaques about one-half the size of those of the control group. Microinjection of isolated pancreatic islets with ethidium bromide revealed that prediabetic mice featured a ß-cell-ß-cell coupling 30% lower than that of control animals. We conclude that ß-cell-ß-cell coupling mediated by Cx36 made-channels is impaired in prediabetic mice, suggesting a role of Cx36-dependent cell-to-cell communication in the pathogenesis of the early ß-cell dysfunctions that lead to type 2-diabetes.

Deficiency in the NADPH oxidase 4 predisposes towards diet-induced obesity.

OBJECTIVE: NADPH oxidase 4 (NOX4) is a reactive oxygen species (ROS) producing NADPH oxidase that regulates redox homeostasis in diverse insulin-sensitive cell types. In particular, NOX4-derived ROS is a key modulator of adipocyte differentiation and mediates insulin receptor signaling in mature adipocytes in vitro. Our study was aimed at investigating the role of NOX4 in adipose tissue differentiation, whole body metabolic homeostasis and insulin sensitivity in vivo. DESIGN: Mice with genetic ablation of NOX4 (NOX4-deficient mice) were subjected to chow or high-fat-containing diet for 12 weeks. Body weight gain, adiposity, insulin sensitivity, and adipose tissue and liver gene and protein expression were analyzed and compared with similarly treated wild-type mice. RESULTS: Here, we report that NOX4-deficient mice display latent adipose tissue accumulation and are susceptible to diet-induced obesity and early onset insulin resistance. Obesity results from accelerated adipocyte differentiation and hypertrophy, and an increase in whole body energy efficiency. Insulin resistance is associated with increased adipose tissue hypoxia, inflammation and adipocyte apoptosis. In the liver, more severe diet-induced steatosis was observed due to the lack of proper upregulation of mitochondrial fatty acid ß-oxidation. CONCLUSION: These findings identify NOX4 as a regulator of metabolic homeostasis. Moreover, they indicate an anti-adipogenic role for NOX4 in vivo and reveal its function as a protector against the development of diet-induced obesity, insulin resistance and hepatosteatosis.

Obstacles on the way to the clinical visualisation of beta cells: looking for the Aeneas of molecular imaging to navigate between Scylla and Charybdis.

For more than a decade, researchers have been trying to develop non-invasive imaging techniques for the in vivo measurement of viable pancreatic beta cells. However, in spite of intense research efforts, only one tracer for positron emission tomography (PET) imaging is currently under clinical evaluation. To many diabetologists it may remain unclear why the imaging world struggles to develop an effective method for non-invasive beta cell imaging (BCI), which could be useful for both research and clinical purposes. Here, we provide a concise overview of the obstacles and challenges encountered on the way to such BCI, in both native and transplanted islets. We discuss the major difficulties posed by the anatomical and cell biological features of pancreatic islets, as well as the chemical and physical limits of the main imaging modalities, with special focus on PET, SPECT and MRI. We conclude by indicating new avenues for future research in the field, based on several remarkable recent results.

Beta cell coupling and connexin expression change during the functional maturation of rat pancreatic islets.

AIMS/HYPOTHESIS: Cell-cell coupling mediated by gap junctions formed from connexin (CX) contributes to the control of insulin secretion in the endocrine pancreas. We investigated the cellular production and localisation of CX36 and CX43, and gap junction-mediated beta cell coupling in pancreatic islets from rats of different ages, displaying different degrees of maturation of insulin secretion. METHODS: The presence and distribution of islet connexins were assessed by immunoblotting and immunofluorescence. The expression of connexin genes was evaluated by RT-PCR and quantitative real-time PCR. The ultrastructure of gap junctions and the function of connexin channels were assessed by freeze-fracture electron microscopy and tracer microinjection, respectively. RESULTS: Young and adult beta cells, which respond to glucose, expressed significantly higher levels of Cx36 (also known as Gjd2) than fetal and newborn beta cells, which respond poorly to the sugar. Accordingly, adult beta cells also showed a significantly higher membrane density of gap junctions and greater intercellular exchange of ethidium bromide than newborn beta cells. Cx43 (also known as Gja1) was not expressed by beta cells, but was located in various cell types at the periphery of fetal and newborn islets. CONCLUSIONS/INTERPRETATION: These findings show that the pattern of connexins, gap junction membrane density and coupling changes in islets during the functional maturation of beta cells.

[Cellular communication and regulation of insulin in the cell]. / Communications cellulaires et régulation des cellules à insuline.

The appearance of multicellular organisms implicated the development of several mechanisms of communication, which permit the cells to function in coordination. One of the mechanisms found in all tissues of vertebrates is ensured by the proteins of the connexin family. These integral membrane proteins form channels, which allow for the passage ofcytosolic molecules either between adjacent cells or between the cytosol of these cells and the extracellular environment. We have identified connexin 36 (Cx36) as the sole connexin that functionally links ("couples") the beta-cells which produce insulin within pancreatic islets. In vitro and in vivo experiments have shown that Cx36 and/or the intercellular communications to allow play a role in the control of insulin secretion as well as in the resistance of beta-cells against various aggressions, including those induced by the cytokines that are implicated in diabetes. A polymorphism of Cx36 gene is associated to certain forms of human diabetes, opening the possibility that a therapy targeting this protein may be useful in the treatment of diabetic diseases.

In vivo imaging of murine endocrine islets of Langerhans with extended-focus optical coherence microscopy.

AIMS/HYPOTHESIS: Structural and functional imaging of the islets of Langerhans and the insulin-secreting beta cells represents a significant challenge and a long-lasting objective in diabetes research. In vivo microscopy offers a valuable insight into beta cell function but has severe limitations regarding sample labelling, imaging speed and depth, and was primarily performed on isolated islets lacking native innervations and vascularisation. This article introduces extended-focus optical coherence microscopy (xfOCM) to image murine pancreatic islets in their natural environment in situ, i.e. in vivo and in a label-free condition. METHODS: Ex vivo measurements on excised pancreases were performed and validated by standard immunohistochemistry to investigate the structures that can be observed with xfOCM. The influence of streptozotocin on the signature of the islets was investigated in a second step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo. RESULTS: xfOCM circumvents the fundamental physical limit that trades lateral resolution for depth of field, and achieves fast volumetric imaging with high resolution in all three dimensions. It allows label-free visualisation of pancreatic lobules, ducts, blood vessels and individual islets of Langerhans ex vivo and in vivo, and detects streptozotocin-induced islet destruction. CONCLUSIONS/INTERPRETATION: Our results demonstrate the potential value of xfOCM in high-resolution in vivo studies to assess islet structure and function in animal models of diabetes, aiming towards its use in longitudinal studies of diabetes progression and islet transplants.

Functional significance of repressor element 1 silencing transcription factor (REST) target genes in pancreatic beta cells.

AIMS/HYPOTHESIS: The expression of several neuronal genes in pancreatic beta cells is due to the absence of the transcription factor repressor element 1 (RE-1) silencing transcription factor (REST). The identification of these traits and their functional significance in beta cells has only been partly elucidated. Herein, we investigated the biological consequences of a repression of REST target genes by expressing REST in beta cells. METHODS: The effect of REST expression on glucose homeostasis, insulin content and release, and beta cell mass was analysed in transgenic mice selectively expressing REST in beta cells. Relevant target genes were identified in INS-1E and primary beta cells expressing REST. RESULTS: Transgenic mice featuring a beta cell-targeted expression of REST exhibited glucose intolerance and reduced beta cell mass. In primary beta cells, REST repressed several proteins of the exocytotic machinery, including synaptosomal-associated protein (SNAP) 25, synaptotagmin (SYT) IV, SYT VII, SYT IX and complexin II; it impaired first and second phases of insulin secretion. Using RNA interference in INS-1E cells, we showed that SYT IV and SYT VII were implicated in the control of insulin release. CONCLUSIONS/INTERPRETATION: The data document the critical role of REST target genes in pancreatic beta cells. Specifically, we provide evidence that the downregulation of these genes is detrimental for the exocytosis of large dense core vesicles, thus contributing to beta cell dysfunction and impaired glucose homeostasis.

Islet-cell-to-cell communication as basis for normal insulin secretion.

The emergence of pancreatic islets has necessitated the development of a signalling system for the intra- and inter-islet coordination of beta cells. With evolution, this system has evolved into a complex regulatory network of partially cross-talking pathways, whereby individual cells sense the state of activity of their neighbours and, accordingly, regulate their own level of functioning. A consistent feature of this network in vertebrates is the expression of connexin (Cx)-36-made cell-to-cell channels, which cluster at gap junction domains of the cell membrane, and which adjacent beta cells use to share cytoplasmic ions and small metabolites within individual islets. This chapter reviews what is known about Cx36, and the mechanism whereby this beta-cell connexin significantly regulates insulin secretion. It further outlines other less established functions of the protein and evaluates its potential relevance for the development of novel therapeutic approaches to diabetes.

Beta cells preferentially exchange cationic molecules via connexin 36 gap junction channels.

AIMS/HYPOTHESIS: Pancreatic beta cells are connected by gap junction channels made of connexin 36 (Cx36), which permit intercellular exchanges of current-carrying ions (ionic coupling) and other molecules (metabolic coupling). Previous studies have suggested that ionic coupling may extend to larger regions of pancreatic islets than metabolic coupling. The aim of the present study was to investigate whether this apparent discrepancy reflects a difference in the sensitivity of the techniques used to evaluate beta cell communication or a specific characteristic of the Cx36 channels themselves. METHODS: We microinjected several gap junction tracers, differing in size and charge, into individual insulin-producing cells and evaluated their intercellular exchange either within intact islets of control, knockout and transgenic mice featuring beta cells with various levels of Cx36, or in cultures of wild-type and Cx36-transfected MIN6 cells. RESULTS: We found that (1) Cx36 channels favour the exchange of cations and larger positively charged molecules between beta cells at the expense of anionic molecules; (2) this exchange occurs across sizable portions of pancreatic islets; and (3) during glibenclamide (known as glyburide in the USA and Canada) stimulation beta cell coupling increases to an extent that varies for different gap junction-permeant molecules. CONCLUSIONS/INTERPRETATION: The data show that beta cells are extensively coupled within pancreatic islets via exchanges of mostly positively charged molecules across Cx36 channels. These exchanges selectively increase during stimulation of insulin secretion. The identification of this permselectivity is expected to facilitate the identification of endogenous permeant molecules and of the mechanism whereby Cx36 signalling significantly contributes to the modulation of insulin secretion.

Connexin40 regulates renin production and blood pressure.

Renin secretion is regulated by coordinated signaling between the various cells of the juxtaglomerular apparatus. The renin-secreting cells (RSC), which play a major role in the control of blood pressure, are coupled to each other and to endothelial cells by Connexin40 (Cx40)-containing channels. In this study, we show that Cx40 knockout (Cx40-/-) mice, but not their heterozygous littermates, are hypertensive due to the increase in the number of RSC, renin biosynthesis, and plasma renin. Treatment with the angiotensin II receptor AT1 antagonist candesartan or the angiotensin II-converting enzyme inhibitor ramipril reduced the blood pressure of the Cx40-/- mice to the same levels seen in wild-type (WT) mice. The elevated blood pressure of the knockout mice was not affected by clipping one renal artery (2K1C, renin-dependent model of hypertension) or after a high salt diet. Under these conditions, however, Cx40-/- mice showed an altered production and release of renin. The renin mRNA ratio between the clipped and the non-clipped kidney was lower in the knockout than in the WT 2K1C mice. This indicates that the response to a change in blood pressure was altered. The RSC of the Cx40-/- mice did not have a compensatory increase in the levels of either Cx43 or Cx37. Our data show that renin secretion is dependent on Cx40 and suggest the Cx40-/- mice may be a genetic model of renin-dependent hypertension.

Interleukin-1 receptor antagonist is upregulated during diet-induced obesity and regulates insulin sensitivity in rodents.

AIMS/HYPOTHESIS: The IL-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine known to antagonise the actions of IL-1. We have previously shown that IL-1Ra is markedly upregulated in the serum of obese patients, is correlated with BMI and insulin resistance, and is overexpressed in the white adipose tissue (WAT) of obese humans. The aim of this study was to examine the role of IL-1Ra in the regulation of glucose homeostasis in rodents. METHODS: We assessed the expression of genes related to IL-1 signalling in the WAT of mice fed a high-fat diet, as well as the effect of Il1rn (the gene for IL-1Ra) deletion and treatment with IL-1Ra on glucose homeostasis in rodents. RESULTS: We show that the expression of Il1rn and the gene encoding the inhibitory type II IL-1 receptor was upregulated in diet-induced obesity. The blood insulin:glucose ratio was significantly lower in Il1rn ( -/- )animals, which is compatible with an increased sensitivity to insulin, reinforced by the fact that the insulin content and pancreatic islet morphology of Il1rn ( -/- ) animals were normal. In contrast, the administration of IL-1Ra to normal rats for 5 days led to a decrease in the whole-body glucose disposal due to a selective decrease in muscle-specific glucose uptake. CONCLUSIONS/INTERPRETATION: The expression of genes encoding inhibitors of IL-1 signalling is upregulated in the WAT of mice with diet-induced obesity, and IL-1Ra reduces insulin sensitivity in rats through a muscle-specific decrease in glucose uptake. These results suggest that the markedly increased levels of IL-1Ra in human obesity might contribute to the development of insulin resistance.