AJP Gastrointestinal and Liver Physiology

Deans-Fielder, Kali; Wu, Timothy; Nguyen, Thanh; To, Sarah; Huang, Yang-Zhe; Bark, Steven J.; Mills, Jason C.; Shroyer, Noah F.

doi: 10.1152/ajpgi.00126.2023pmid: 38349111

Genotoxic agents like doxorubicin (DXR) can cause damage to the intestines that can be ameliorated by fasting. How fasting is protective and the optimal timing of fasting and refeeding remain unclear. Here, our analysis of fasting/refeeding-induced global intestinal transcriptional changes revealed metabolic shifts and implicated the cellular energetic hub mTORC1 in protecting from DXR-induced DNA damage. Our analysis of specific transcripts and proteins in intestinal tissue and tissue extracts showed that fasting followed by refeeding at the time of DXR administration reduced damage and caused a spike in mTORC1 activity. However, continued fasting after DXR prevented the mTORC1 spike and damage reduction. Surprisingly, the mTORC1 inhibitor, rapamycin, did not block fasting/refeeding-induced reduction in DNA damage, suggesting that increased mTORC1 is dispensable for protection against the initial DNA damage response. In Ddit4-/- mice (DDIT4 functions to regulate mTORC1 activity), fasting reduced DNA damage and increased intestinal crypt viability versus ad libitum-fed Ddit4-/- mice. Fasted/refed Ddit4-/- mice maintained body weight, with increased crypt proliferation by 5 days post-DXR, while ad libitum-fed Ddit4-/- mice continued to lose weight and displayed limited crypt proliferation. Genes encoding epithelial stem cell and DNA repair proteins were elevated in DXR-injured fasted vs ad libitum Ddit4-/- intestines. Thus, fasting strongly reduced intestinal damage when normal dynamic regulation of mTORC1 was lost. Overall, the results confirm that fasting protects the intestines against DXR and suggests that fasting works by pleiotropic - including both mTORC1-dependent and independent - mechanisms across the temporally dynamic injury response.

Bullard, Brooke M.; McDonald, Sierra J.; Cardaci, Thomas D.; VanderVeen, Brandon N.; Mohammed, Ahmed D.; Kubinak, Jason L.; Pierre, Joseph F.; Chatzistamou, Ioulia; Fan, Daping; Hofseth, Lorne J.; Murphy, E. Angela

doi: 10.1152/ajpgi.00004.2024pmid: 38469632

Ulcerative colitis (UC) is an idiopathic inflammatory disease of the large intestine, which impacts millions worldwide. Current interventions aimed at treating UC symptoms can have off-target effects, invoking the need for alternatives that may provide similar benefits with less unintended consequences. This study builds on our initial data, which showed that panaxynol - a novel, potent, bioavailable compound found in American ginseng - can suppress disease severity in murine colitis. Here we explore the underlying mechanisms by which panaxynol improves both chronic and acute murine colitis. 14-week-old C57BL/6 female mice were either given 3 rounds of dextran sulfate sodium (DSS) in drinking water to induce chronic colitis or 1 round to induce acute colitis. Vehicle or panaxynol (2.5 mg/kg) was administered via oral gavage 3x/week for the study duration. Consistent with our previous findings, panaxynol significantly (p<0.05) improved the disease activity index and endoscopic scores in both models. Using the acute model to examine potential mechanisms, we show that panaxynol significantly (p<0.05) reduced DSS-induced crypt distortion, goblet cell loss, and mucus loss in the colon. 16s sequencing revealed panaxynol altered microbial composition to suppress colitis-enriched genera (i.e., Enterococcus, Eubacterium, and Ruminococcus). Additionally, panaxynol significantly (p<0.05) suppressed macrophages and induced regulatory T-cells in the colonic lamina propria. The beneficial effects of panaxynol on mucosal and crypt architecture, combined with its microbial and immune-mediated effects, provide insight into mechanisms by which panaxynol suppresses murine colitis. Overall, this data is promising for the use of panaxynol to improve colitis in the clinic.

Périco, Larissa Lucena; Vegso, Andrew J.; Baggio, Cristiane H.; MacNaughton, Wallace K.

doi: 10.1152/ajpgi.00284.2023pmid: 38440826

The inflamed mucosa contains a complex assortment of proteases which may participate in wound healing or in the development of inflammation-associated colon cancer. We sought to determine the role of protease-activated receptor 2 (PAR2) in epithelial wound healing in both untransformed and transformed colonic epithelial cells. Monolayers of primary epithelial cells derived from organoids cultivated from patient colonic biopsies, and of the T84 colon cancer cell line, were grown to confluence, wounded in the presence of a selective PAR2-activating peptide, and healing visualized by live cell microscopy. Inhibitors of various signaling molecules were used to assess the relevant pathways responsible for wound healing. Activation of PAR2 induced an enhanced wound healing response in T84 but not primary cells. The PAR2-enhanced wound healing response was associated with the development of lamellipodia in cells at the wound edge, consistent with sheet migration. The response to PAR2 activation in T84 cells was completely dependent upon Src kinase activity, and partially dependent on Rac1 activity. The Src-associated signaling molecules, focal adhesion kinase and epidermal growth factor receptor, which typically mediate wound healing responses, were not involved in the PAR2 response. Experiments repeated in the presence of the inflammatory cytokines, TNF and IFNγ, revealed a synergistically enhanced PAR2-wound healing response in T84s, but not primary cells. The epithelial response to proteases may be different between primary and cancer cells and is accentuated in the presence of inflammatory cytokines. Our findings have implications for understanding epithelial restitution in the context of IBD and inflammation-associated colon cancer.

Zhu, Xiaohui; Yang, Yan; Feng, Dongfeng; Wang, Oliver; Chen, Jiaxiang; Wang, Jiale; Wang, Bin; Liu, Yang; Edenfield, Brandy H.; Haddock, Ashley N.; Wang, Ying; Patel, Tushar; Bi, Yan; Ji, Baoan

doi: 10.1152/ajpgi.00263.2023pmid: 38469630

Background and Aims: Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. Methods: A FlpO cDNA was inserted between the last exon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). Results: BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. Conclusions: A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism.

Lu, Yongke; George, Joseph

doi: 10.1152/ajpgi.00281.2023pmid: 38573193

Fatty acid oxidation (FAO) releases the energy stored in fat to maintain basic biological processes. Dehydrogenation is a major way to oxidize fatty acids, which needs NAD+ to accept the released H+ from fatty acids and form NADH, which increases the ratio of NADH/NAD+ and consequently inhibits FAO leading to the deposition of fat in the liver, which is termed fatty liver or steatosis. Consumption of alcohol (ethanol) initiates simple steatosis that progresses to alcoholic steatohepatitis, which constitutes a spectrum of liver disorders called alcohol-associated liver disease (ALD). ALD is linked to ethanol metabolism. Ethanol is metabolized by alcohol dehydrogenase (ADH), microsomal ethanol oxidation system (MEOS), mainly cytochrome P450 2E1 (CYP2E1), and catalase. ADH also requires NAD+ to accept the released H+ from ethanol. Thus, ethanol metabolism by ADH leads to increased ratio of NADH/NAD+, which inhibits FAO and induces steatosis. CYP2E1 directly consumes reducing equivalent NADPH to oxidize ethanol, which generates reactive oxygen species (ROS) that lead to cellular injury. Catalase is mainly present in peroxisomes, where very long-chain fatty acids and branched-chain fatty acids are oxidized, and the resultant short-chain fatty acids will be further oxidized in mitochondria. Peroxisomal FAO generates hydrogen peroxide (H2O2), which is locally decomposed by catalase. When ethanol is present, catalase uses H2O2 to oxidize ethanol. In this review, we introduce FAO (including α-, β-, and ω-oxidation) and ethanol metabolism (by ADH, CYP2E1, and catalase) followed by the interaction between FAO and ethanol metabolism in the liver and its pathophysiological significance.

Schulte, Steven; Decker, Dominique; Nowduri, Bharat; Gries, Manuela; Christmann, Anne; Meyszner, Antoine; Rabe, Holger; Saumer, Monika; Schäfer, Karl-Herbert

doi: 10.1152/ajpgi.00170.2023pmid: 38193168

The enteric nervous system comprises millions of neurons and glia embedded in the wall of the gastrointestinal tract. It not only controls important functions of the gut, but also interacts with the immune system, gut microbiota and the gut-brain-axis, thereby playing a key role in health and disease of the whole organism. Any disturbance of this intricate system is mirrored in an alteration of electrical functionality, making electrophysiological methods important tools for investigating ENS-related disorders. Microelectrode arrays provide an appropriate non-invasive approach of recording signals from multiple neurons or whole networks simultaneously. However, studying isolated cells of the ENS can be challenging, considering the limited time that these cells can be kept vital in vitro. Therefore, we developed an alternative approach cultivating cells on glass samples with spacers (fabricated by photolithography methods). The spacers allow the cells to grow upside-down in a spatially confined environment, while enabling acute consecutive recordings of multiple ENS-cultures on the same MEA. Upside-down culture also shows beneficial effects on growth and behavior of enteric neural cultures. The number of dead cells was significantly decreased, neural networks showed higher resemblance to the myenteric plexus ex vivo, while producing more stable signals than cultures grown in the conventional way. Overall, our results indicate that the upside-down approach not only allows to investigate the impact of neurological diseases in vitro but could also offer insights into growth and development of the ENS under conditions much closer to the in vivo environment.

Huang, I-Hsuan; Schol, Jolien; Calder, Stefan; Gharibans, Armen A.; Van den Houte, Karen; Verheyden, Annelies; Broeders, Bert; Carbone, Florencia; O'Grady, Greg; Tack, Jan

doi: 10.1152/ajpgi.00298.2023pmid: 38375576

Biopsychosocial factors are associated with disorders of gut brain interaction and exacerbate gastrointestinal symptoms. The mechanisms underlying pathophysiological alterations of stress remain unclear. Corticotropin-releasing hormone (CRH) is a central regulator of the hormonal stress response and has diverse impact on different organ systems. The aim of the present study was to investigate the effects of peripheral CRH infusion on meal-related gastrointestinal symptoms, gastric electrical activity and gastric sensorimotor function in healthy volunteers (HVs). In a randomized, double-blinded, placebo-controlled, crossover study, we evaluated the effects of CRH on gastric motility and sensitivity. HVs were randomized to receive either peripheral administered CRH (100 µg bolus + 1 µg/kg/hr) or placebo (saline), followed by at least a 7-day washout period and assignment to the opposite treatment. Tests encompassed saliva samples, gastric emptying (GE) testing, body surface gastric mapping (BSGM, Gastric Alimetry®; Alimetry) to assess gastric myoelectrical activity with real-time symptom profiling, and a gastric barostat study to assess gastric sensitivity to distention and accommodation. 20 HVs (13 women, mean age 29.2±5.3 years, BMI 23.3±3.8 kg/m2) completed GE tests, of which 18 also underwent BSGM measurements during the GE tests. The GE half time decreased significantly after CRH exposure (65.2±17.4 vs 78.8±24.5 minutes, p=0.02) with significantly increased gastric amplitude (49.7 (34.7-55.6) vs 31.7 (25.7-51.0) µV, p<0.01), saliva cortisol levels and postprandial symptom severity. Eleven HVs also underwent gastric barostat studies on a separate day. However, the thresholds for discomfort during isobaric distensions, gastric compliance, and accommodation did not differ between CRH and placebo.

Xu, Binqiang; Fu, Yang; Yin, Nuoming; Qin, Wenfei; Huang, Zehua; Xiao, Wei; Huang, Huizhen; Mei, Qixiang; Fan, Junjie; Zeng, Yue; Huang, Chunlan

doi: 10.1152/ajpgi.00303.2023pmid: 38502145

Fecal microbiota transplantation (FMT) is a promising therapy for inflammatory bowel disease (IBD) via rectifying gut microbiota. The aim of this study was to identify a mechanism of how specific bacteria-associated immune response contributes to alleviated colitis. 40 donors were divided into high (donor-H) and low (donor-L) groups according to diversity and the abundance of Bacteroides and Faecalibacterium by 16S rRNA sequencing. FMT was performed on dextran sulfate sodium (DSS)-induced colitis in mice. Mice with colitis showed significant improvement in intestinal injury and immune imbalance after FMT with group donor-H (p <0.05). Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii were identified as targeted strains in donor feces by real-time PCR and droplet digital PCR. Mice with colitis were treated with mono- or dual-bacterial gavage therapy. Dual-bacterial therapy significantly ameliorated intestinal injury compared with mono-bacterial therapy (p <0.05). Dual-bacterial therapy increased the M2/M1 macrophage polarization and improved the Th17/Treg imbalance and elevated IL-10 production by Tregs compared with the DSS group (p <0.05). Metabolomics showed increased abundance of lecithin in the glycerophospholipid metabolism pathway. In conclusion, B. thetaiotaomicron and F. prausnitzii, as the key bacteria in donor feces, alleviate colitis in mice. The mechanism may involve increasing lecithin and regulating IL-10 production of intestinal Tregs.

King, Andrew J.; Chang, Lin; Li, Qian; Liu, Liansheng; Zhu, Yaohui; Pasricha, Pankaj J.; Wang, Ji; Siegel, Matthew; Caldwell, Jeremy S.; Edelstein, Susan; Rosenbaum, David P.; Kozuka, Kenji

doi: 10.1152/ajpgi.00233.2023pmid: 38252683

The pathogenesis of irritable bowel syndrome (IBS) is multifactorial, characterized in part by increased intestinal permeability and visceral hypersensitivity. Increased permeability is associated with IBS severity and abdominal pain. Tenapanor is FDA-approved for the treatment of IBS with constipation (IBS-C) and has demonstrated improvements in bowel motility and a reduction in IBS-related pain; however, the mechanism by which tenapanor mediates these functions remains unclear. Here, the effects of tenapanor on colonic pain signaling and intestinal permeability were assessed through behavioral, electrophysiological, and cell culture experiments. Intestinal motility studies in rats and humans demonstrated that tenapanor increased luminal sodium and water retention and gastrointestinal transit versus placebo. A significantly reduced visceral motor reflex (VMR) to colonic distension was observed with tenapanor treatment versus vehicle in two rat models of visceral hypersensitivity (neonatal acetic acid sensitization and partial restraint stress; both P < 0.05), returning VMR responses to that of non-sensitized controls. Whole-cell voltage patch-clamp recordings of retrogradely labeled colonic dorsal root ganglia (DRG) neurons from sensitized rats found that tenapanor significantly reduced DRG neuron hyperexcitability to capsaicin versus vehicle (P < 0.05), an effect not mediated by epithelial cell secretions. Tenapanor also attenuated increases in intestinal permeability in human colon monolayer cultures caused by incubation with proinflammatory cytokines (P < 0.001) or fecal supernatants from patients with IBS-C (P < 0.005). These results support a model in which tenapanor reduces IBS-related pain by strengthening the intestinal barrier, thereby decreasing permeability to macromolecules and antigens and reducing DRG-mediated pain signaling.

Schleicher, Eva Maria; Gairing, Simon Johannes; Castven, Darko; Hock, Charlotte Sophie; Dobbermann, Henrike; Heinrich, Sophia; Meineck, Myriam; Kaps, Leonard; Galle, Peter Robert; Weinmann-Menke, Julia; Nguyen-Tat, Marc; Marquardt, Jens U.; Labenz, Christian

doi: 10.1152/ajpgi.00304.2023pmid: