Role of FFAR3 in ketone body regulated glucagon-like peptide 1 secretion.

Background: Roux-en-Y gastric bypass (RYGB) is an effective treatment for obesity, resulting in long-term weight loss and rapid remission of type 2 diabetes mellitus. Improved glucagon-like peptide 1 (GLP-1) levels is one factor that contributes to the positive effects. Prior to RYGB, GLP-1 response is blunted which can be attributed to intestinal ketogenesis. Intestinal produced ketone bodies inhibit GLP-1 secretion in enteroendocrine cells via an unidentified G-protein coupled receptors (GPCRs). A possible class of GPCRs through which ketone bodies may reach are the free fatty acid receptors (FFARs) located at the basolateral membrane of enteroendocrine cells. Aim: To evaluate FFAR3 expression in enteroendocrine cells of the small intestine under different circumstances, such as diet and bariatric surgery, as well as explore the link between ketone bodies and GLP-1 secretion. Materials and methods: FFAR3 and enteroendocrine cell expression was analyzed using Western blot and immunohistochemistry in biopsies from healthy volunteers, obese patients undergoing RYGB and mice. GLUTag cells were used to study GLP-1 secretion and FFAR3 signaling pathways. Results: The expression of FFAR3 is markedly influenced by diet, especially high fat diet, which increased FFAR3 protein expression. Lack of substrate such as free fatty acids in the alimentary limb after RYGB, downregulate FFAR3 expression. The number of enteroendocrine cells was affected by diet in the normal weight individuals but not in the subjects with obesity. In GLUTag cells, we show that the ketone bodies exert its blocking effect on GLP-1 secretion via the FFAR3, and the Gαi/o signaling pathway. Conclusion: Our findings that ketone bodies via FFAR3 inhibits GLP-1 secretion bring important insight into the pathophysiology of T2D. This highlights the role of FFAR3 as a possible target for future anti-diabetic drugs and treatments.

Intestinal mucosal perfusion and integrity are maintained in hypotensive brain dead mice.

Brain death (BD) leads to complex hemodynamic and inflammatory alterations which may compromise organ perfusion and induce morphologic and functional damage in various organs. The intestine is particularly sensitive to hypoperfusion and donor hypotension usually precludes intestinal donation. Previous studies reported inflammatory intestinal changes following BD but information on mucosal integrity and perfusion are lacking. BD was induced in mice by inflating an epidural balloon catheter. Controls underwent only anesthesia and tracheostomy. Intestinal perfusion was assessed using laser Doppler flowmetry (LDF). Intestinal injury was assessed after 2h of BD by the Chiu-Park score and morphometry. Intestinal tight junction (TJ) proteins (claudin-1, claudin-3, occludin, tricellulin) as well as inflammatory activation (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and interleukin-6) were also analysed and compared with a sham group. Although blood pressure decreased in BD mice, intestinal perfusion remained similar between BD and sham mice. Histologically, mucosal injury was absent/minimal and TJs appeared well maintained in both groups. BD may trigger intrinsic, autoregulatory mechanisms to preserve microvascular tissue perfusion and mucosal integrity in spite of mild hypotension.

Intestinal Ketogenesis and Permeability.

Consumption of a high-fat diet (HFD) has been suggested as a contributing factor behind increased intestinal permeability in obesity, leading to increased plasma levels of microbial endotoxins and, thereby, increased systemic inflammation. We and others have shown that HFD can induce jejunal expression of the ketogenic rate-limiting enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS). HMGCS is activated via the free fatty acid binding nuclear receptor PPAR-α, and it is a key enzyme in ketone body synthesis that was earlier believed to be expressed exclusively in the liver. The function of intestinal ketogenesis is unknown but has been described in suckling rats and mice pups, possibly in order to allow large molecules, such as immunoglobulins, to pass over the intestinal barrier. Therefore, we hypothesized that ketone bodies could regulate intestinal barrier function, e.g., via regulation of tight junction proteins. The primary aim was to compare the effects of HFD that can induce intestinal ketogenesis to an equicaloric carbohydrate diet on inflammatory responses, nutrition sensing, and intestinal permeability in human jejunal mucosa. Fifteen healthy volunteers receiving a 2-week HFD diet compared to a high-carbohydrate diet were compared. Blood samples and mixed meal tests were performed at the end of each dietary period to examine inflammation markers and postprandial endotoxemia. Jejunal biopsies were assessed for protein expression using Western blotting, immunohistochemistry, and morphometric characteristics of tight junctions by electron microscopy. Functional analyses of permeability and ketogenesis were performed in Caco-2 cells, mice, and human enteroids. Ussing chambers were used to analyze permeability. CRP and ALP values were within normal ranges and postprandial endotoxemia levels were low and did not differ between the two diets. The PPARα receptor was ketone body-dependently reduced after HFD. None of the tight junction proteins studied, nor the basal electrical parameters, were different between the two diets. However, the ketone body inhibitor hymeglusin increased resistance in mucosal biopsies. In addition, the tight junction protein claudin-3 was increased by ketone inhibition in human enteroids. The ketone body ß-Hydroxybutyrate (ßHB) did not, however, change the mucosal transition of the large-size molecular FD4-probe or LPS in Caco-2 and mouse experiments. We found that PPARα expression was inhibited by the ketone body ßHB. As PPARα regulates HMGCS expression, the ketone bodies thus exert negative feedback signaling on their own production. Furthermore, ketone bodies were involved in the regulation of permeability on intestinal mucosal cells in vitro and ex vivo. We were not, however, able to reproduce these effects on intestinal permeability in vivo in humans when comparing two weeks of high-fat with high-carbohydrate diet in healthy volunteers. Further, neither the expression of inflammation markers nor the aggregate tight junction proteins were changed. Thus, it seems that not only HFD but also other factors are needed to permit increased intestinal permeability in vivo. This indicates that the healthy gut can adapt to extremes of macro-nutrients and increased levels of intestinally produced ketone bodies, at least during a shorter dietary challenge.

ßHB inhibits glucose-induced GLP-1 secretion in GLUTag and human jejunal enteroids.

Ingestion of nutrients stimulates incretin secretion from enteroendocrine cells (EECs) of the epithelial layer of the gut. Glucagon-like peptide-1 (GLP-1) is one of these incretins that stimulate postprandial insulin release and signal satiety to the brain. Understanding the regulation of incretin secretion might open up new therapeutic options for obesity and type-2 diabetes mellitus. To investigate the inhibitory effect of the ketone body ß-hydroxybutyrate (ßHB) on glucose-induced GLP-1 secretion from EECs, in vitro cultures of murine GLUTag cells and differentiated human jejunal enteroid monolayers were stimulated with glucose to induce GLP-1 secretion. The effect of ßHB on GLP-1 secretion was studied using ELISA and ECLIA methods. GLUTag cells stimulated with glucose and ßHB were analysed using global proteomics focusing on cellular signalling pathways and the results were verified by Western blot. Results demonstrated ßHB had a significant inhibitory effect on glucose-induced GLP-1 secretion at a dose of 100 mM in GLUTag cells. In differentiated human jejunal enteroid monolayers, glucose-induced secretion of GLP-1 was inhibited at a much lower dose of 10 mM ßHB. The addition of ßHB to GLUTag cells resulted in decreased phosphorylation of kinase AKT and transcription factor STAT3 and also influenced the expressions of signalling molecule IRS-2, kinase DGKε and receptor FFAR3. In conclusion, ßHB displays an inhibitory effect on glucose-induced GLP-1 secretion in vitro in GLUTag cells and in differentiated human jejunal enteroid monolayers. This effect may be mediated through multiple downstream mediators of G-protein coupled receptor activation, such as PI3K signalling.

Mucosal Recovery after Intestinal Transplantation in the Rat: A Sequential Histological and Molecular Assessment.

INTRODUCTION: Intestinal cold ischemia and subsequent reperfusion during transplantation result in various degrees of mucosal injury ranging from mild edema to extensive mucosal loss. Mucosal barrier impairment favors bacterial translocation and fluid loss and raises nutritional challenges. The injured intestine also releases proinflammatory mediators and upregulates various epitopes toward an inflammatory phenotype. We studied the process of mucosal injury and repair during the early period after intestinal transplantation from a histological and molecular standpoint. MATERIALS AND METHODS: Adult Sprague-Dawley rats were used as donors and recipients. Donor intestines were perfused and stored in saline for 3 h, then transplanted heterotopically using microvascular anastomoses. Intestinal graft segments were obtained after 20 min, 6 h, 12 h, and 24 h after reperfusion. Histology studies (goblet cell count, morphometry), immunofluorescence, and western blot for several tight junction proteins, apoptosis, and inflammation-related proteins were performed. RESULTS: Cold storage led to extensive epithelial detachment, whereas reperfusion resulted in extensive villus loss (about 60% of the initial length), and goblet cell numbers were drastically reduced. Over the first 24 h, gradual morphologic and molecular recovery was noted, although several molecular alterations persisted (increased apoptosis and inflammation, altered expression of several tight junctions). CONCLUSIONS: The current data suggest that a near-complete morphologic recovery from a moderate mucosal injury occurs within the first 24 h after intestinal transplantation. However, several molecular alterations persist and need to be considered when designing intestinal transplant experiments and choosing sampling and endpoints.

Asymmetric mucosal structure, mesenteric versus antimesenteric, in mouse, rat, and human small intestines.

The morphology of the small intestinal mucosa is reflected by the degree of stimuli. Previous studies have come to different conclusion about whether the mucosa is equally symmetrical. The aim of the study is to investigate whether there are structural differences in the mesenteric versus antimesenteric mucosa in mice, rats, and humans. Jejunal biopsies from mice and rats were saved. Samples from human small intestine were obtained from patients undergoing Roux-en-Y gastric bypass surgery. Fixed samples were used to morphologically evaluate villus height and enlargement factor due to villi. The number of goblet cells, mast cells, enteroendocrine cells, and Paneth cells were histologically analyzed in the villus structure. Cell turnover was analyzed by Ki-67 staining. There was a significant increased villi height and villus enlargement factor antimesenterically in mice, rats, and human small intestines. The distribution of goblet cells, mast cells, and Paneth cells were equal while the number of enteroendocrine cells was increased antimesenteric in the human samples. The crypt mitotic activity was almost 20% higher in the antimesenteric part of jejunum. In summary we found longer villi, greater surface enlargement, and increased number of enteroendocrine cells as well as increased cell turnover antimesenterically. These differences may be of importance in understanding normal gastrointestinal physiology in health and disease.

Morphological Adaptation in the Jejunal Mucosa after Iso-Caloric High-Fat versus High-Carbohydrate Diets in Healthy Volunteers: Data from a Randomized Crossover Study.

BACKGROUND AND AIMS: The conditions for jejunal glucose absorption in healthy subjects have not been thoroughly studied. In this study we investigated differences in the jejunal villi enlargement factor, as well as ultrastructural aspects of the surface enterocytes and mitochondria, comparing 2 weeks of high-carbohydrate (HCD) versus high-fat diets (HFD). We also measured the ketogenesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) in relation to jejunal mitochondria. METHODS: A single-centre, randomized, unblinded crossover study in 15 healthy volunteers ingesting strictly controlled equicaloric diets (either HCD or HFD), with 60% energy from the respective source. An enteroscopy was carried out after 2 weeks of each diet and jejunal mucosal biopsies were acquired. Conventional histology, immunofluorescent staining, transmission electron microscopy and confocal microscopy were used. RESULTS: The villi did not demonstrate any change in the epithelial enlargement factor. Despite an increased mitosis, there were no changes in apoptotic indices. However, the ultrastructural analysis demonstrated a significant increase in the enlargement factor at the bases of the villi. The mitochondria demonstrated increased amounts of cristae after the HFD. The confocal microscopy revealed increased HMGCS2 per mitochondrial marker at the top of the villi after the HFD compared to the HCD. CONCLUSION: There is a morphometric adaption in the jejunal mucosa following the 2-week diets, not only on a histological level, but rather on the ultrastructural level. This study supports the notion that mitochondrial HMGCS2 is regulated by the fat content of the diet and is involved in the expression of monosaccharide transporters.

A Fatty Diet Induces a Jejunal Ketogenesis Which Inhibits Local SGLT1-Based Glucose Transport via an Acetylation Mechanism-Results from a Randomized Cross-Over Study between Iso-Caloric High-Fat versus High-Carbohydrate Diets in Healthy Volunteers.

BACKGROUND AND AIMS: Insights into the nature of gut adaptation after different diets enhance the understanding of how food modifications can be used to treat type 2 diabetes and obesity. The aim was to understand how diets, enriched in fat or carbohydrates, affect glucose absorption in the human healthy jejunum, and what mechanisms are involved. METHODS: Fifteen healthy subjects received, in randomised order and a crossover study design, two weeks of iso-caloric high-fat diet (HFD) and high-carbohydrate diet (HCD). Following each dietary period, jejunal mucosa samples were retrieved and assessed for protein expression using immunofluorescence and western blotting. Functional characterisation of epithelial glucose transport was assessed ex vivo using Ussing chambers. Regulation of SGLT1 through histone acetylation was studied in vitro in Caco-2 and human jejunal enteroid monolayer cultures. RESULTS: HFD, compared to HCD, decreased jejunal Ussing chamber epithelial glucose transport and the expression of apical transporters for glucose (SGLT1) and fructose (GLUT5), while expression of the basolateral glucose transporter GLUT2 was increased. HFD also increased protein expression of the ketogenesis rate-limiting enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) and decreased the acetylation of histone 3 at lysine 9 (H3K9ac). Studies in Caco-2 and human jejunal enteroid monolayer cultures indicated a ketogenesis-induced activation of sirtuins, in turn decreasing SGLT1 expression. CONCLUSION: Jejunal glucose absorption is decreased by a fat-enriched diet, via a ketogenesis-induced alteration of histone acetylation responsible for the silencing of SGLT1 transcription. The work relates to a secondary outcome in ClinicalTrials.gov (NCT02088853).

Glycemic Control and Metabolic Adaptation in Response to High-Fat versus High-Carbohydrate Diets-Data from a Randomized Cross-Over Study in Healthy Subjects.

Granular study of metabolic responses to alterations in the ratio of dietary macro-nutrients can enhance our understanding of how dietary modifications influence patients with impaired glycemic control. In order to study the effect of diets enriched in fat or carbohydrates, fifteen healthy, normal-weight volunteers received, in a cross-over design, and in a randomized unblinded order, two weeks of an iso-caloric high-fat diet (HFD: 60E% from fat) and a high-carbohydrate diet (HCD: 60E% from carbohydrates). A mixed meal test (MMT) was performed at the end of each dietary period to examine glucose clearance kinetics and insulin and incretin hormone levels, as well as plasma metabolomic profiles. The MMT induced almost identical glycemia and insulinemia following the HFD or HCD. GLP-1 levels were higher after the HFD vs. HCD, whereas GIP did not differ. The HFD, compared to the HCD, increased the levels of several metabolomic markers of risk for the development of insulin resistance, e.g., branched-chain amino acid (valine and leucine), creatine and α-hydroxybutyric acid levels. In normal-weight, healthy volunteers, two weeks of the HFD vs. HCD showed similar profiles of meal-induced glycemia and insulinemia. Despite this, the HFD showed a metabolomic pattern implying a risk for a metabolic shift towards impaired insulin sensitivity in the long run.

Intestinal sodium/glucose cotransporter 3 expression is epithelial and downregulated in obesity.

AIM: We aimed to determine whether the sodium/glucose cotransporter family member SGLT3, a proposed glucose sensor, is expressed in the intestine and/or kidney, and if its expression is altered in mouse models of obesity and in humans before and after weight-loss surgery. MAIN METHODS: We used in-situ hybridization and quantitative PCR to determine whether the Sglt3 isoforms 3a and 3b were expressed in the intestine and kidney of C57, leptin-deficient ob/ob, and diabetic BTBR ob/ob mice. Western blotting and immunohistochemistry were also used to assess SGLT3 protein levels in jejunal biopsies from obese patients before and after weight-loss Roux-en-Y gastric bypass surgery (RYGB), and in lean healthy controls. KEY FINDINGS: Sglt3a/3b mRNA was detected in the small intestine (duodenum, jejunum and ileum), but not in the large intestine or kidneys of mice. Both isoforms were detected in epithelial cells (confirmed using intestinal organoids). Expression of Sglt3a/3b mRNA in duodenum and jejunum was significantly lower in ob/ob and BTBR ob/ob mice than in normal-weight littermates. Jejunal SGLT3 protein levels in aged obese patients before RYGB were lower than in lean individuals, but substantially upregulated 6 months post-RYGB. SIGNIFICANCE: Our study shows that Sglt3a/3b is expressed primarily in epithelial cells of the small intestine in mice. Furthermore, we observed an association between intestinal mRNA Sglt3a/3b expression and obesity in mice, and between jejunal SGLT3 protein levels and obesity in humans. Further studies are required to determine the possible role of SGLT3 in obesity.

The Proteomic Signature of Intestinal Acute Rejection in the Mouse.

Intestinal acute rejection (AR) lacks a reliable non-invasive biomarker and AR surveillance is conducted through frequent endoscopic biopsies. Although citrulline and calprotectin have been suggested as AR biomarkers, these have limited clinical value. Using a mouse model of intestinal transplantation (ITx), we performed a proteome-wide analysis and investigated rejection-related proteome changes that may eventually be used as biomarkers. ITx was performed in allogenic (Balb/C to C57Bl) and syngeneic (C57Bl) combinations. Graft samples were obtained three and six days after transplantation (n = 4/time point) and quantitative proteomic analysis with iTRAQ-labeling and mass spectrometry of whole tissue homogenates was performed. Histology showed moderate AR in all allografts post-transplantation at day six. Nine hundred and thirty-eight proteins with at least three unique peptides were identified in the intestinal grafts. Eighty-six proteins varying by >20% between time points and/or groups had an alteration pattern unique to the rejecting allografts: thirty-seven proteins and enzymes (including S100-A8 and IDO-1) were significantly upregulated whereas forty-nine (among other chromogranin, ornithine aminotransferase, and arginase) were downregulated. Numerous proteins showed altered expression during intestinal AR, several of which were previously identified to be involved in acute rejection, although our results also identified previously unreported proteome changes. The metabolites and downstream metabolic pathways of some of these proteins and enzymes may become potential biomarkers for intestinal AR.

Luminal polyethylene glycol solution delays the onset of preservation injury in the human intestine.

The organ damage incurred during the cold storage (CS) of intestinal grafts has short and long-term consequences. Animal studies suggest that additional luminal preservation (LP) with polyethylene glycol (PEG) may alleviate this damage. This study aims to validate these findings using human intestines. Ileal segments, perfused intravascularly with IGL-1 solution, were procured from 32 multiorgan donors and divided into two parts: one containing a PEG 3350-based solution introduced luminally (LP group) and another one without luminal treatment (control). Sampling was performed after 4 h, 8 h, 14 h, and 24 h of CS. Histology was assessed using the Chiu/Park score. Tight junctions (TJ), several inflammatory markers, and transcription factors were examined by immunofluorescence, ddPCR, and western blot. Tissue water content (edema) was also measured. Apoptotic activity was assessed with caspase -2, -3, and -9 assays. LP significantly lowered mucosal injury at all time points. Redistribution of TJ proteins occurred earlier and more severely in the control group. After 24 h of CS, LP intestines showed an emerging unfolding protein response. Increased caspase-3 and -9 activity was found in the control group. The current results indicate that luminal PEG is safe and effective in reducing damage to the intestinal epithelium during CS.

Glycocholic acid and butyrate synergistically increase vitamin D-induced calcium uptake in Caco-2 intestinal epithelial cell monolayers.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) substantially decreases intestinal calcium absorption and may eventually lead to bone resorption. This is likely a consequence of bile diversion from the alimentary limb, as the presence of bile seems necessary for vitamin D-mediated calcium uptake. We recently suggested that the mediating mechanism may be a down-regulation of the vitamin D co-activator heat-shock protein (Hsp)90ß. Recent evidence suggests that vitamin D may have effects on both active and passive calcium absorption. AIM: To identify mechanisms in vitro that may be responsible for the decreased calcium absorption after RYGB. We hypothesized that bile, alone or in concert with nutritional compounds, could be of importance. MATERIAL & METHODS: Caco-2 cells were grown confluent on semi-permeable membranes in a double-chamber setup to mimic small intestinal mucosa. The effect of bile acids chenodeoxycholic, lithocholic, glycocholic and taurocholic acid, with and without the addition of the fatty-acid butyrate, were tested for their effects on Hsp90ß expression and active and passive calcium-flux monitored using radioactive 45Ca. RESULTS: We initially found that whole human bile, but only together with the fatty acid butyrate, potently induced Hsp90ß expression. In line with this, a single bile acid, e.g. glycocholic acid (GCA), in combination with butyrate, increased Hsp90ß expression (40 ± 13% vs. GCA, butyrate or vehicle alone; p < 0,001; n = 14-25). Further, this combination together with vitamin D increased the passive gradient-driven flux of calcium, compared to stimulation with vitamin D alone or in combination with either GCA or butyrate (880 ± 217% vs. vitamin D and GCA or butyrate, or vitamin D only; p = 0,01-0.006; n = 5-11). Surprisingly, this combination had no effect on active calcium transport in the absence of calcium gradient. CONCLUSION: The combination of GCA and butyrate increased gradient-driven calcium uptake up to 9-fold in Caco-2 intestinal epithelial cells, but had no effect on active calcium absorption. This effect was mediated via the vitamin D receptor co-activator Hsp90ß.

The Impact of Age and Luminal Preservation on the Development of Intestinal Preservation Injury in Rats.

BACKGROUND: Organs from older donors are believed to withstand ischemia worse than those from younger donors. The effect of age on the development of intestinal preservation injury (IPI) is unclear. METHODS: We compared the development of IPI in intestines from young (3 mo), adult (14 mo), and old (20 mo) rat donors and assessed if luminal preservation (LP) is effective in delaying IPI. Small intestines were perfused with, and stored in, preservation solution (Custodiol) with or without LP solution (polyethylene glycol 3350). IPI was studied using histology (Chiu score, Alcian blue staining), Western blot, and electrophysiological assessment (Ussing chamber) at 4, 8, and 14 hours. RESULTS: Intestines of old rats did not show major histological alterations, whereas their aortas and kidneys revealed typical age-related changes (arteriosclerosis and glomerulosclerosis). Intestines from old rats fared similarly to their younger counterparts at all time points regarding preservation injury and goblet cells count. Intestines undergoing LP showed fewer histological signs of damage and higher goblet cells count when compared with samples without LP, regardless of donor age. Ussing chamber experiments indicated a time-dependent deterioration of all parameters studied, which was delayed by the use of LP. CONCLUSIONS: Older intestines did not convincingly demonstrate a faster IPI compared with intestines from adult and young donors. The small differences between the age groups were nullified by the use of LP. LP significantly delayed the IPI in all age groups and may allow for longer preservation periods without an increased risk of mucosal damage.

Direct stimulation of angiotensin II type 2 receptor reduces nitric oxide production in lipopolysaccharide treated mouse macrophages.

The angiotensin II type 2 receptor (AT2) is upregulated after tissue damage and mediates protective functions in the renin-angiotensin-aldosterone system (RAAS). One of these is to inhibit inducible nitric oxide synthase (iNOS) in activated macrophages. In the present study, we assessed the effect of AT2 receptor ligands on nitric oxide production in murine macrophages as a potential assay to determine the functional activity of an AT2 receptor ligand. Mouse macrophage J744.2 and RAW264.7 were cultivated in lipopolysaccharide (LPS) to induce M1 differentiation and increase iNOS expression. Using Griess reagent and spectrophotometric analysis, the nitric oxide levels were determined, while employing Western blot and immunocytochemistry to determine basal protein expression. Using the first reported selective non-peptide AT2 receptor agonist, compound C21, we conclude that activation of AT2 receptor reduces nitric oxide production in M1 macrophages. Furthermore, the AT2 receptor selective ligand compound C38, a regioisomer of C21, reported as a selective AT2 receptor antagonist exhibits a similar effect on nitric oxide production. Thus, we propose C38 acts as a partial agonist in the macrophage system. Monitoring nitric oxide attenuation in M1 J744.1 and RAW264.7 macrophages provides a new method for characterizing functional activity of AT2 receptor ligands, foreseen to be valuable in future drug discovery programs.

Suppression of enteroendocrine cell glucagon-like peptide (GLP)-1 release by fat-induced small intestinal ketogenesis: a mechanism targeted by Roux-en-Y gastric bypass surgery but not by preoperative very-low-calorie diet.

OBJECTIVE: Food intake normally stimulates release of satiety and insulin-stimulating intestinal hormones, such as glucagon-like peptide (GLP)-1. This response is blunted in obese insulin resistant subjects, but is rapidly restored following Roux-en-Y gastric bypass (RYGB) surgery. We hypothesised this to be a result of the metabolic changes taking place in the small intestinal mucosa following the anatomical rearrangement after RYGB surgery, and aimed at identifying such mechanisms. DESIGN: Jejunal mucosa biopsies from patients undergoing RYGB surgery were retrieved before and after very-low calorie diet, at time of surgery and 6 months postoperatively. Samples were analysed by global protein expression analysis and Western blotting. Biological functionality of these findings was explored in mice and enteroendocrine cells (EECs) primary mouse jejunal cell cultures. RESULTS: The most prominent change found after RYGB was decreased jejunal expression of the rate-limiting ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGCS), corroborated by decreased ketone body levels. In mice, prolonged high-fat feeding induced the expression of mHMGCS and functional ketogenesis in jejunum. The effect of ketone bodies on gut peptide secretion in EECs showed a ∼40% inhibition of GLP-1 release compared with baseline. CONCLUSION: Intestinal ketogenesis is induced by high-fat diet and inhibited by RYGB surgery. In cell culture, ketone bodies inhibited GLP-1 release from EECs. Thus, we suggest that this may be a mechanism by which RYGB can remove the inhibitory effect of ketone bodies on EECs, thereby restituting the responsiveness of EECs resulting in increased meal-stimulated levels of GLP-1 after surgery.

Intestinal Preservation Injury: A Comparison Between Rat, Porcine and Human Intestines.

Advanced preservation injury (PI) after intestinal transplantation has deleterious short- and long-term effects and constitutes a major research topic. Logistics and costs favor rodent studies, whereas clinical translation mandates studies in larger animals or using human material. Despite diverging reports, no direct comparison between the development of intestinal PI in rats, pigs, and humans is available. We compared the development of PI in rat, porcine, and human intestines. Intestinal procurement and cold storage (CS) using histidine-tryptophan-ketoglutarate solution was performed in rats, pigs, and humans. Tissue samples were obtained after 8, 14, and 24 h of CS), and PI was assessed morphologically and at the molecular level (cleaved caspase-3, zonula occludens, claudin-3 and 4, tricellulin, occludin, cytokeratin-8) using immunohistochemistry and Western blot. Intestinal PI developed slower in pigs compared to rats and humans. Tissue injury and apoptosis were significantly higher in rats. Tight junction proteins showed quantitative and qualitative changes differing between species. Significant interspecies differences exist between rats, pigs, and humans regarding intestinal PI progression at tissue and molecular levels. These differences should be taken into account both with regards to study design and the interpretation of findings when relating them to the clinical setting.

The human duodenal mucosa harbors all components for a local renin angiotensin system.

The renin-angiotensin system (RAS) is present in the gastrointestinal (GI) tract but remains to be fully characterized, particularly in man. The duodenum plays a role in both the upper and lower GI regulation, as well as in distant organs. The present study investigates the presence and functional potential of RAS in the human duodenal mucosa of healthy individuals. Endoscopically acquired mucosal biopsies from healthy volunteers were examined using western blot, immunohistochemistry, and ELISA. Functionality was examined by using Ussing chambers and recording duodenal transmucosal potential difference (PD) and motility in vivo Angiotensinogen, Angiotensin II (AngII) and its receptors (AT1R, AT2R) as well as to the RAS associated enzymes renin, ACE, and neprylisin were detected in all samples of duodenal mucosa. Migrating motility complex induced elevations of transmucosal PD were significantly larger after per-oral administration of the AT1R receptor antagonist candesartan. Fasting duodenal motility per se was not influenced by candesartan. The epithelial current produced by duodenal mucosae mounted in Ussing chambers increased significantly after addition of AngII to specimens where the AT1R was blocked using losartan. The epithelial current also increased after addition of the AT2R-selective agonist C21. Immunostaining and pharmacological data demonstrate the presence of a local RAS in the human duodenal mucosa with capacity to influence epithelial ion transport by way of particulary the AT2R.

Proteomic Approach to the Potential Role of Angiotensin II in Barrett Dysplasia.

BACKGROUND AND AIM: Dysplasia in Barrett's esophagus (BE) is regarded as a preneoplastic lesion. The renin-angiotensin system (RAS), known for its role in electrolyte homeostasis and hemodynamics, has also been shown to have tissue-based features linked to proliferation, inflammation, and cancer. RAS is associated with BE dysplasia. The aim of this study is to investigate possible effects of the RAS in BE dysplasia by using RAS-interfering pharmaceutical agents and by assessment of global protein expression in esophageal mucosal biopsies. METHODS: Endoscopic biopsies are taken from 18 BE in patients with low-grade dysplasia before and after 3 weeks of treatment with either angiotensin-converting enzyme inhibitors (enalapril 5 mg; n = 6) or angiotensin II receptor type 1 blockers (candesartan 8 mg; n = 6), or no treatment (n = 6). A global proteomics analysis by 2D gel electrophoresis and mass spectrometry (MS) is then performed to identify proteins that are regulated after interference with RAS. RESULTS: Three proteins are identified to show significant modulation of expression 60 kDa heat shock protein (downregulated), protein disulfide isomerase A3 (downregulated), and inorganic pyrophosphatase (upregulated). CONCLUSION: Three proteins with no previously known links to esophageal RAS, but with possible relevance for the development of esophageal adenocarcinoma (EAC) are detected. Altered expression by interference with the RAS suggests an involvement of angiotensin II in the development of EAC in BE.

Luminal Polyethylene Glycol Alleviates Intestinal Preservation Injury Irrespective of Molecular Size.

Intestinal preservation injury (IPI) and the resulting mucosa injury raise several serious challenges early after intestinal transplantation. The current clinical approach using only vascular perfusion allows the shortest preservation period among the abdominal organs. The experimental addition of luminal polyethylene glycol (PEG) solutions has been repeatedly suggested to alleviate preservation injury, improve graft quality, and prolong the preservation time. We investigated whether the molecular mass of PEG in solution influences the development of intestinal preservation injury. Small intestines of Sprague-Dawley rats were perfused with University of Wisconsin solution. Group 1 underwent vascular perfusion only (clinical control), group 2 received additional luminal PEG3350 Da, group 3 received luminal PEG10000 Da, and group 4 received luminal PEG20000 Da (n = 8/group). Tissue samples were obtained after 4, 8, and 14 hours. We studied the tissue damage (Chiu/Park score, Goblet cells, apoptosis, tight junctions), activation of c-Jun NH2-terminal kinase (JNK), and p38-mitogen-activated protein kinase (MAPK), and we performed Ussing chamber assessments. Mucosal morphologic and electrophysiologic parameters were significantly improved in the groups receiving luminal PEG. There was significantly less apoptotic activity in groups 2, 3, and 4. Both MAPKs revealed an activation peak after 4 hours with group 3 showing lesser p38-MAPK activation. PEG 20 kDa interfered with protein immunodetection. The results indicate that luminal solutions of PEG of medium and large molecular mass significantly delay the onset and development of IPI, providing further evidence that luminal interventions may allow for longer cold storage intervals of intestinal grafts.