Melatonin decreases human adipose tissue insulin sensitivity.

Melatonin is a pineal hormone that modulates the circadian system and exerts soporific and phase-shifting effects. It is also involved in many other physiological processes, such as those implicated in cardiovascular, endocrine, immune, and metabolic functions. However, the role of melatonin in glucose metabolism remains contradictory, and its action on human adipose tissue (AT) explants has not been demonstrated. We aimed to assess whether melatonin (a pharmacological dose) influences insulin sensitivity in human AT. This will help better understand melatonin administration's effect on glucose metabolism. Abdominal AT (subcutaneous and visceral) biopsies were obtained from 19 participants with severe obesity (age: 42.84 ± 12.48 years; body mass index: 43.14 ± 8.26 kg/m2) who underwent a laparoscopic gastric bypass. AT biopsies were exposed to four different treatments: control (C), insulin alone (I) (10 nM), melatonin alone (M) (5000 pg/mL), and insulin plus melatonin combined (I + M). All four conditions were repeated in both subcutaneous and visceral AT, and all were performed in the morning at 8 a.m. (n = 19) and the evening at 8 p.m. (in a subsample of n = 12). We used western blot analysis to determine insulin signaling (using the pAKT/tAKT ratio). Furthermore, RNAseq analyses were performed to better understand the metabolic pathways involved in the effect of melatonin on insulin signaling. As expected, insulin treatment (I) increased the pAKT/tAKT ratio compared with control (p < .0001). Furthermore, the addition of melatonin (I + M) resulted in a decrease in insulin signaling as compared with insulin alone (I); this effect was significant only during the evening time (not in the morning time). Further, RNAseq analyses in visceral AT during the evening condition (at 8 p.m.) showed that melatonin resulted in a prompt transcriptome response (around 1 h after melatonin addition), particularly by downregulating the insulin signaling pathway. Our results show that melatonin reduces insulin sensitivity in human AT during the evening. These results may partly explain the previous studies showing a decrease in glucose tolerance after oral melatonin administration in the evening or when eating late when endogenous melatonin is present.

A perspective current and past modes of inhalation therapy.

Inhalation is the preferred route of delivery for anti-asthma and chronic obstructive pulmonary disease (COPD) drugs. The use of this route has demonstrated efficacy in these and other conditions, it offers rapid onset of action, and is associated with minimal systemic exposure, thereby reducing the risk of adverse effects. Therefore, the current brief covers an interesting collection of inhaler action modes, shedding light on their molecular mechanisms and clinical applications for anti-asthma, COPD and antibacterial inhalation therapy. Hence, not only enriches our understanding of inhalation therapy molecular intricacies but also provides a comprehensive overview of the evolving landscape in clinical and antibacterial inhalation therapy. In doing so, it underscores the pivotal role of microbiology and biotechnology in advancing therapeutic approaches that harness the power of inhalation.

Corrigendum: A standardized extract of Lentinula edodes cultured mycelium inhibits Pseudomonas aeruginosa infectivity mechanisms.

[This corrects the article DOI: 10.3389/fmicb.2022.814448.].

A Standardized Extract of Lentinula edodes Cultured Mycelium Inhibits Pseudomonas aeruginosa Infectivity Mechanisms.

The priority pathogen list of the World Health Organization classified Pseudomonas aeruginosa as the second top critical pathogen. Hence, the development of novel antibacterial strategies to tackle this bacterium is highly necessary. Herein we explore the potential antibacterial effect of a standardized extract of cultured mycelium of Lentinula edodes (AHCC®) on P. aeruginosa. AHCC® was found to inhibit the growth rate and biofilm formation of strain PAO1. No change in swarming was observed, but AHCC® hampered swimming and twitching motility. In accordance, a decreased expression of metabolism, growth, and biofilm formation genes was shown. AHCC® also diminished the levels of exotoxin A and bacteria inside IEC18 cells and the secretion of IL-6, IL-10 and TNF by infected macrophages. This effect was related to a reduced phosphorylation of MAPKs and to bacteria internalization. Taken together, our data suggest that AHCC® has a potential role to prevent P. aeruginosa infections and may lead to the development of new therapies.

Insulin Crystals Grown in Short-Peptide Supramolecular Hydrogels Show Enhanced Thermal Stability and Slower Release Profile.

Protein therapeutics have a major role in medicine in that they are used to treat diverse pathologies. Their three-dimensional structures not only offer higher specificity and lower toxicity than small organic compounds but also make them less stable, limiting their in vivo half-life. Protein analogues obtained by recombinant DNA technology or by chemical modification and/or the use of drug delivery vehicles has been adopted to improve or modulate the in vivo pharmacological activity of proteins. Nevertheless, strategies to improve the shelf-life of protein pharmaceuticals have been less explored, which has challenged the preservation of their activity. Herein, we present a methodology that simultaneously increases the stability of proteins and modulates the release profile, and implement it with human insulin as a proof of concept. Two novel thermally stable insulin composite crystal formulations intended for the therapeutic treatment of diabetes are reported. These composite crystals have been obtained by crystallizing insulin in agarose and fluorenylmethoxycarbonyl-dialanine (Fmoc-AA) hydrogels. This process affords composite crystals, in which hydrogel fibers are occluded. The insulin in both crystalline formulations remains unaltered at 50 °C for 7 days. Differential scanning calorimetry, high-performance liquid chromatography, mass spectrometry, and in vivo studies have shown that insulin does not degrade after the heat treatment. The nature of the hydrogel modifies the physicochemical properties of the crystals. Crystals grown in Fmoc-AA hydrogel are more stable and have a slower dissolution rate than crystals grown in agarose. This methodology paves the way for the development of more stable protein pharmaceuticals overcoming some of the existing limitations.

Deficiency in Tissue Non-Specific Alkaline Phosphatase Leads to Steatohepatitis in Mice Fed a High Fat Diet Similar to That Produced by a Methionine and Choline Deficient Diet.

The liver expresses tissue-nonspecific alkaline phosphatase (TNAP), which may participate in the defense against bacterial components, in cell regulation as part of the purinome or in bile secretion, among other roles. We aimed to study the role of TNAP in the development of hepatosteatosis. TNAP+/- haplodeficient and wild type (WT) mice were fed a control diet (containing 10% fat w/w) or the same diet deficient in methionine and choline (MCD diet). The MCD diet induced substantial weight loss together with hepatic steatosis and increased alanine aminotransferase (ALT) plasma levels, but no differences in IL-6, TNF, insulin or resistin. There were no substantial differences between TNAP+/- and WT mice fed the MCD diet. In turn, TNAP+/- mice receiving the control diet presented hepatic steatosis with alterations in metabolic parameters very similar to those induced by the MCD diet. Nevertheless, no weight loss, increased ALT plasma levels or hypoglycemia were observed. These mice also presented increased levels of liver TNF and systemic resistin and glucagon compared to WT mice. The phenotype of TNAP+/- mice fed a standard diet was normal. In conclusion, TNAP haplodeficiency induces steatosis comparable to that produced by a MCD diet when fed a control diet.

Intestinal epithelial deletion of the glucocorticoid receptor NR3C1 alters expression of inflammatory mediators and barrier function.

Glucocorticoids (GCs) are important hormones involved in the regulation of multiple physiologic functions. GCs are also widely used in anti-inflammatory/immunosuppressant drugs. GCs are synthesized by the adrenal cortex as part of the hypothalamus-pituitary-adrenal axis and also by intestinal epithelial cells, among other peripheral sites. GCs are one of the main therapy choices for the exacerbations of inflammatory bowel disease, but they are not useful to prolong remission, and development of tolerance with secondary treatment failure is frequent. Thus, GC actions at the intestinal epithelial level are of great importance, both physiologically and pharmacologically. We generated a tamoxifen-inducible nuclear receptor subfamily 3 group C member 1 (NR3C1)ΔIEC mouse model to study the effects of GCs on epithelial cells in vivo. Nr3c1 deletion in epithelial cells of the small intestine and colon was associated with limited colonic inflammation at 1 wk postdeletion, involving augmented epithelial proliferation and mucus production, plus local and systemic immune/inflammatory changes. This phenotype regressed substantially, but not completely, after 2 wk. The mechanism may involve augmented inflammatory signaling by epithelial cells or defective barrier function. We conclude that the epithelial GC receptor plays a significant role in colonic homeostasis in basal conditions, but its deficiency can be compensated in the short term. Future studies are required to assess the impact of Nr3c1 deletion in other conditions such as experimental colitis.-Aranda, C. J., Arredondo-Amador, M., Ocón, B., Lavín, J. L., Aransay, A. M., Martínez-Augustin, O., Sánchez de Medina, F. Intestinal epithelial deletion of the glucocorticoid receptor NR3C1 alters expression of inflammatory mediators and barrier function.

Interaction of glucocorticoids with FXR/FGF19/FGF21-mediated ileum-liver crosstalk.

At high doses, glucocorticoids (GC) have been associated with enhanced serum bile acids and liver injury. We have evaluated the effect of GC, in the absence of hepatotoxicity, on FXR/FGF91(Fgf15)/FGF21-mediated ileum-liver crosstalk. Rats and mice (wild type and Fxr-/-, Fgf15-/- and int-Gr-/- strains; the latter with GC receptor (Gr) knockout selective for intestinal epithelial cells), were treated (i.p.) with dexamethasone, prednisolone or budesonide. In both species, high doses of GC caused hepatotoxicity. At a non-hepatotoxic dose, GC induced ileal Fgf15 down-regulation and liver Fgf21 up-regulation, without affecting Fxr expression. Fgf21 mRNA levels correlated with those of several genes involved in glucose and bile acid metabolism. Surprisingly, liver Cyp7a1 was not up-regulated. The expression of factors involved in transcriptional modulation by Fxr and Gr (p300, Drip205, CBP and Smrt) was not affected. Pxr target genes Cyp3a11 and Mrp2 were not up-regulated in liver or intestine. In contrast, the expression of some Pparα target genes in liver (Fgf21, Cyp4a14 and Vanin-1) and intestine (Vanin-1 and Cyp3a11) was altered. In mice with experimental colitis, liver Fgf21 was up-regulated (4.4-fold). HepG2 cells transfection with FGF21 inhibited CYP7A1 promoter (prCYP7A1-Luc2). This was mimicked by pure human FGF21 protein or culture in medium previously conditioned by cells over-expressing FGF21. This response was not abolished by deletion of a putative response element for phosphorylated FGF21 effectors present in prCYP7A1. In conclusion, GC interfere with FXR/FGF19-mediated intestinal control of CYP7A1 expression by the liver and stimulate hepatic secretion of FGF21, which inhibits CYP7A1 promoter through an autocrine mechanism.

Fructooligosaccharides exert intestinal anti-inflammatory activity in the CD4+ CD62L+ T cell transfer model of colitis in C57BL/6J mice.

PURPOSE: Fructooligosaccharides (FOS) are used as functional foods due to their prebiotic effects. Intestinal anti-inflammatory activity has been established in most, but not all, studies in animal models of colitis, using mainly chemically induced inflammation. Our goal was to test the effect of FOS (degree of polymerization 2-8) in the chronic, lymphocyte-driven CD4+ CD62L+ T cell transfer model of colitis. METHODS: Colitis was induced by transfer of CD4+ CD62L+ T cells to C57BL/6J Rag1(-/-) mice. FOS (75 mg day(-1)) was administered by gavage as a post-treatment. Three groups were established: non-colitic (NC), colitic control (C, CD4+ CD62L+ transferred mice treated with vehicle) and colitic+FOS (C+FOS, similar but treated with FOS). Mice were killed after 13 days. RESULTS: Treatment of mice with FOS ameliorated colitis, as evidenced by an increase in body weight, a lesser myeloperoxidase and alkaline phosphatase activities, a lower secretion of proinflammatory cytokines by mesenteric lymph node cells ex vivo (IFN-γ, IL-17, and TNF-α), and a higher colonic expression of occludin (C+FOS vs. C, p < 0.05). Increased relative abundance of lactic acid bacteria was observed in FOS-treated mice (p < 0.05). CONCLUSIONS: FOS exert intestinal anti-inflammatory activity in T lymphocyte-dependent colitis, suggesting it may be useful in the management of inflammatory bowel disease in appropriate conditions.

Proteins and Carbohydrates from Red Seaweeds: Evidence for Beneficial Effects on Gut Function and Microbiota.

Based on their composition, marine algae, and namely red seaweeds, are good potential functional foods. Intestinal mucosal barrier function refers to the capacity of the intestine to provide adequate containment of luminal microorganisms and molecules. Here, we will first outline the component of seaweeds and will summarize the effects of these on the regulation of mucosal barrier function. Special attention will be paid to unique components of red seaweeds: proteins and derived peptides (e.g., phycobiliproteins, glycoproteins that contain "cellulose binding domains", phycolectins and the related mycosporine-like amino acids) together with polysaccharides (e.g., floridean starch and sulfated galactans, such as carrageenans, agarans and "dl-hybrid") and minerals. These compounds have been shown to exert prebiotic effects, to regulate intestinal epithelial cell, macrophage and lymphocyte proliferation and differentiation and to modulate the immune response. Molecular mechanisms of action of peptides and polysaccharides are starting to be elucidated, and evidence indicating the involvement of epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), Toll-like receptors (TLR) and signal transduction pathways mediated by protein kinase B (PKB or AKT), nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) will also be summarized. The need for further research is clear, but in vivo experiments point to an overall antiinflammatory effect of these algae, indicating that they can reinforce membrane barrier function.

Rutin has intestinal antiinflammatory effects in the CD4+ CD62L+ T cell transfer model of colitis.

Rutin, one of the most abundant flavonoids in nature, has been shown to exert intestinal antiinflammatory effects in experimental models of colitis. Our aim was to study the antiinflamatory effect of rutin in the CD4+ CD62L+ T cell transfer model of colitis, one of the closest to the human disease. Colitis was induced by transfer of CD4+ CD62L+ T cells to Rag1(-/-) mice. Rutin was administered by gavage as a postreatment. Treatment with rutin improved colitis at the dose of 57mg/kg/day, while no effect was noted with 28.5mg/kg/day. Therapeutic benefit was evidenced by a reduced disease activity index, weight loss and damage score, plus a 36% lower colonic myeloperoxidase and a 54% lower alkaline phosphatase activity. In addition, a decreased secretion of proinflammatory cytokines (IFNγ and TNFα) by mesenteric lymph node cells was observed ex vivo. The colonic expression of proinflammatory genes, including IFNγ, TNFα, CXCL1, S100A8 and IL-1ß, was significantly reduced by more than 80% with rutin as assessed by RT-qPCR. Flavonoid treated mice exhibited decreased activation of splenic CD4+ cells (STAT4 phosphorylation and IFNγ expression) and reduced plasma cytokine levels. This effect was also apparent in mucosal lymphocytes based on reduced STAT4 phosphorylation. The protective effect was comparable to that of 3mg/kg/day budesonide. Rutin had no effect on splenocytes or murine T cells in vitro, while its aglycone, quercetin, exhibited a concentration dependent inhibition of proinflammatory cytokines, including IFNγ. Rutin but not quercetin showed vectorial basolateral to apical transport in IEC18 cells, associated with reduced biotransformation. We conclude that rutin exerts intestinal antiinflammatory activity in chronic, T lymphocyte dependent colitis via quercetin release and actions involving mucosal and lymph node T cells. Our results suggest that rutin may be useful in the management of inflammatory bowel disease in appropriate dosage conditions.

Prebiotic oligosaccharides directly modulate proinflammatory cytokine production in monocytes via activation of TLR4.

SCOPE: Prebiotic oligosaccharides are currently used in a variety of clinical settings for their effects on intestinal microbiota. Here, we have examined the direct, microbiota independent, effects of prebiotics on monocytes and T lymphocytes in vitro. METHODS AND RESULTS: Prebiotics generally evoked cytokine secretion (TNF-α, IL-6, and IL-10) by mouse splenocytes but inhibited LPS -induced IFN-γ and IL-17 release. Inulin was found to enhance LPS-induced IL-10 secretion. Splenocytes from TLR4(-/-) (where TLR is Toll-like receptor) mice showed a markedly depressed response. Conversely, in both basal and LPS-stimulated conditions, prebiotic inhibition of IFN-γ levels was preserved. These results suggested a predominant effect on monocytes via TLR4 ligation and possible inhibition of T cells. Hence, we studied the modulation of primary rat monocytes and T lymphocytes, focusing on fructooligosaccharides (FOS) and inulin. In monocytes, FOS and inulin induced TNF-α, growth-regulated oncogene α, and IL-10, but not IL-1ß release. The NF-κB inhibitor Bay 11-7082 fully prevented these effects. Pharmacological evidence also indicated a significant involvement of mitogen-activated protein kinase and phosphatidylinositol-3-kinase. There was little effect on T cells. FOS and inulin also generally increased TNF-α, IL-1ß, and IL-10, but not IL-8, in human peripheral blood monocytes. CONCLUSION: We conclude that prebiotics may act as TLR4 ligands or as indirect TLR4 modulators to upregulate cytokine secretion in monocytes.

The nutritional supplement Active Hexose Correlated Compound (AHCC) has direct immunomodulatory actions on intestinal epithelial cells and macrophages involving TLR/MyD88 and NF-κB/MAPK activation.

Active Hexose Correlated Compound (AHCC) is an immunostimulatory nutritional supplement. AHCC effects and mechanism of action on intestinal epithelial cells or monocytes are poorly described. AHCC was added to the culture medium of intestinal epithelial cells (IEC18 and HT29 cells) and monocytes (THP-1 cells) and assessed the secretion of proinflammatory cytokines by ELISA. Inhibitors of NFκB and MAPKs were used to study signal transduction pathways while TLR4 and MyD88 were silenced in IEC18 cells using shRNA. It was found that AHCC induced GROα and MCP1 secretion in IEC18 and IL-8 in HT29 cells. These effects depended on NFκB activation, and partly on MAPKs activation and on the presence of MyD88 and TLR4. In THP-1 cells AHCC evoked IL-8, IL-1ß and TNF-α secretion. The induction of IL-8 depended on JNK and NFκB activation. Therefore, AHCC exerts immunostimulatory effects on intestinal epithelial cells and monocytes involving TLR4/MyD88 and NFκB/MAPK signal transduction pathways.

Dose-dependent antiinflammatory effect of ursodeoxycholic acid in experimental colitis.

The denomination of inflammatory bowel disease comprises a group of chronic inflammatory diseases of the digestive tract, ulcerative colitis and Crohn's disease being the most important conditions. Bile acids may play a role both in etiology and pharmacology of this disease. Thus, although deoxycholic acid is regarded as a proinflammatory agent ursodeoxycholic acid, which is currently being used to treat certain types of cholestasis and primary biliary cirrhosis, because of their choleretic, cytoprotective and immunomodulatory effects, it has been reported to exert an anti-inflammatory activity. We aim to confirm and characterize the intestinal antiinflammatory activity of ursodeoxycholic acid. The experimental model trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats has been used. Animal status was characterized by a number of macroscopic and biochemical parameters. Oral administration of ursodeoxycholic acid was able to ameliorate experimental colonic inflammation. This occurred only at a relatively high dose (50 mg/kg day), whereas ursodeoxycholic acid was without significant effect at doses of 10 and 25 mg/kg day. The therapeutic effect was evidenced, among others, by a higher body weight recovery, a diminished affected to total mucosal area and lower alkaline phosphatase activity in treated vs. control (TNBS treated) animals. These results indicate that, at the appropriate dose, ursodeoxycholic acid is a potentially useful drug to reduce intestinal inflammation and could be envisaged to be incorporated in the treatment of inflammatory bowel diseases.

Immunomodulatory properties of the protein fraction from Phorphyra columbina.

The phycobiliproteins from Rhodophyta , R-phycoerythrin (R-PE) and C-phycocyanin (C-PC), have been shown to exert immunomodulatory effects. This study evaluated the effects of a Phorphyra columbina protein fraction (PF) and R-PE and C-PC on rat primary splenocytes, macrophages, and T-lymphocytes in vitro. PF featured various protein species, including R-PE and C-PC. PF showed mitogenic effects on rat splenocytes and was nontoxic to cells except at 1 g L(-1) protein. IL-10 secretion was enhanced by PF in rat splenocytes, macrophages, and especially T-lymphocytes, whereas it was markedly diminished by R-PE and C-PC. The production of pro-inflammatory cytokines by macrophages was inhibited. The effect of PF on IL-10 was evoked by JNK/p38 MAPK and NF-κB-dependent pathways in macrophages and T-lymphocytes. It was concluded that PF has immunomodulatory effects on macrophages and lymphocytes that appear to be predominantly anti-inflammatory via up-regulated IL-10 production and cannot be accounted for by R-PE and C-PC.

Intestinal inflammation and the enterocyte transportome.

Diarrhoea is a hallmark of intestinal inflammation. The mechanisms operating in acute inflammation of the intestine are well characterized and are related to regulatory changes induced by inflammatory mediators such as prostaglandins, cytokines or reactive oxygen species, along with leakage due to epithelial injury and changes in permeability. In chronic colitis, however, the mechanisms are less well known, but it is generally accepted that both secretory and absorptive processes are inhibited. These disturbances in ionic transport may be viewed as an adaptation to protracted inflammation of the intestine, since prolonged intense secretion may be physiologically unacceptable in the long term. Mechanistically, the changes in transport may be due to adjustments in the regulation of the different processes involved, to broader epithelial alterations or frank damage, or to modulation of the transportome in terms of expression. In the present review, we offer a summary of the existing evidence on the status of the transportome in chronic intestinal inflammation.

New insights into the immunological effects of food bioactive peptides in animal models of intestinal inflammation.

Bioactive peptides have proven to be active in several conditions, including inflammatory bowel disease (IBD). This is a chronic and relapsing condition of unknown aetiology that comprises chiefly ulcerative colitis and Crohn's disease. Although there are treatments for IBD, they have frequent side effects and they are not always effective; therefore there is a need for new therapies that could alleviate this condition. Two bioactive peptides present in milk (transforming growth factor-beta (TGF-beta) and casein macropeptide, also named glycomacropeptide) have been shown to have intestinal anti-inflammatory activities. In fact, TGF-beta is currently added to formulas intended for patients with IBD, and several studies indicate that these formulas could induce clinical remission. In this paper, evidence supporting the anti-inflammatory effect of TGF-beta and bovine glycomacropeptide, as well as their mechanisms of action, is reviewed, focusing on the evidence obtained in animal models.

Reversible Ponceau staining as a loading control alternative to actin in Western blots.

It is becoming standard practice to measure a housekeeping gene, typically actin, in Western blots, as it is the rule in RNA blots. We have applied reversible Ponceau staining to check equal loading of gels and measured actin in parallel under different conditions. Our results show that densitometric analysis is comparable with both techniques. Therefore, routine quantitation of Ponceau staining before antibody probing is validated as an alternative to actin blotting.

The intestinal antiinflammatory agent glycomacropeptide has immunomodulatory actions on rat splenocytes.

Bovine glycomacropeptide (GMP) is an immunologically active milk peptide that is a part of the normal human diet. GMP has therapeutic value in preclinical models of intestinal inflammation, and its mechanism may be related to effects on lymphocytes. This study focuses on the actions of GMP on rat splenocytes in vitro and in vivo. Bovine serum albumin and lactoferrin were used for comparative purposes. GMP (0.01-0.1mgmL(-1)) enhanced Concanavalin A (ConA) evoked but not basal splenocyte proliferation. At 1mgmL(-1) GMP lost this effect but augmented basal TNF-alpha secretion and also iNOS and COX2 expression. IFN-gamma, IL-2 and IL-17 were not affected by GMP in quiescent splenocytes, but IL-10 was augmented at all concentrations tested. On the other hand, GMP produced a marked inhibitory effect (70%) on IFN-gamma secretion and to a lower extent (50%) also on TNF-alpha. GMP was shown to block STAT4 but not IkappaB-alpha phosphorylation. The Treg marker Foxp3 was markedly upregulated by GMP. Bovine serum albumin had some effects on splenocyte function which were of lower magnitude and not entirely coincidental, while lactoferrin had a strong antiproliferative effect, as expected, indicating a specific effect of GMP. When administered for 3 days to normal Wistar rats, GMP reproduced the Foxp3 induction effect observed previously in vitro. This was observed in splenocytes but not in thymocytes, and only when administered by the oral rather than the intraperitoneal route. Thus our results support the hypothesis that GMP may limit intestinal inflammation acting at least in part on lymphocytes.