Effects of dietary protein level on intestinal function and inflammation in nursery pigs.

High crude protein (CP; 21% to 26%) diets fed during the first 21 to 28 d postweaning are viewed negatively because of a perceived increase in the incidence rates of diarrhea due to increased intestinal protein fermentation and/or augmented enteric pathogen burden. This is thought to antagonize nursery pig health and growth performance. Therefore, our objective was to evaluate the impact of low vs. high dietary CP on 21-day postweaned pig intestinal function. Analyzed parameters included ex vivo intestinal barrier integrity (ileum and colon), ileal nutrient transport, tissue inflammation, and fecal DM. One hundred and twenty gilts and barrows (average body weight) were randomly assigned to one of two diets postweaning. Diets were fed for 21 d, in two phases. Phase 1 diets: low CP (17%) with a 1.4% standardized ileal digestible (SID) Lys (LCP), or high CP (24%) with a 1.4% SID Lysine (HCP). Phase 2: LCP (17%) and a 1.35% SID lysine, or HCP (24%) formulated to a 1.35% SID lysine. Pig growth rates, feed intakes, and fecal consistency did not differ (P > 0.05) due to dietary treatment. Six animals per treatment were euthanized for additional analyses. There were no differences in colonic epithelial barrier function as measured by transepithelial electrical resistance (TER) and fluorescein isothiocyanate (FITC)-dextran transport between treatments (P > 0.05). Interleukins (IL)-1α, IL-1ß, IL-1ra, IL-2 IL-4, IL-6, and IL-12 were not different between treatments (P > 0.05). However, IL-8 and IL-18 were higher in HCP- vs. LCP-fed pigs (P < 0.05). There were no differences in fecal dry matter (DM; P > 0.05) between treatments. In the ileum, there was a tendency (P = 0.06) for TER to be higher in HCP-fed pigs, suggesting a more robust barrier. Interestingly, glucose and glutamine transport were decreased in HCP- vs. LCP-fed pigs (P < 0.05). FITC-dextran transport was not different between treatments (P > 0.05). There were also no differences in ileal cytokine concentrations between diets (P > 0.05). Taken together, the data show that low CP does not negatively impact colonic barrier function, fecal DM, or inflammation. In contrast, ileal barrier function and nutrient transport were altered, suggesting a regional effect of diet on overall intestinal function.

High dietary crude protein (CP) is thought to antagonize nursery pig enteric health. Feeding high CP diets to nursery pigs did not exacerbate intestinal health or inflammation, and overall, protein level in the diet has little impact on animal health and performance.

Soluble corn fiber, resistant corn starch, and protected butyrate effects on performance, gastrointestinal volatile fatty acids, and apparent total-tract digestibility of calcium and phosphorus in nursery pigs.

An experiment was conducted to determine how feeding calcium (Ca)-deficient diet would affect gastrointestinal pH and volatile fatty acids (VFAs), Ca digestibility, bone mineral density (BMD), and performance in nursery pigs; and if supplementation of nondigestible oligosaccharides would affect these same parameters. In total, 240 weaned pigs (BW = 7.1 kg) were placed into 80 pens with 3 pigs/pen. The eight dietary treatments consisted of: 1) positive control (PC, 0.83% total Ca), 2) negative control (NC, 0.50% total Ca), 3 and 4) NC + 5% or 7.5% soluble corn fiber (SCF), 5 and 6) NC + 5% or 7.5% resistant corn starch (rCS), 7 and 8) NC + 0.25% or 0.50% fat-protected butyrate (pBRT). Pigs were ad libitum fed the dietary treatments for 21 d to determine average daily gain (ADG), average daily feed intake (ADFI) and gain:feed ratio (GF) with a fecal sample collected from each pen to determine Ca digestibility using acid insoluble ash as the dietary marker, with 1 pig/pen euthanized on d 21 for collection of ileal and colon contents and the left humerus. Pigs fed the NC diet had a lower colonic pH compared with pigs fed the PC (P = 0.06) but no effect on total VFA was observed (P > 0.10). Pigs fed diets containing SCF and rCS had lower colonic pH and total VFA compared to pigs fed the NC diet (P ≤ 0.05). Pigs fed diets containing pBRT had greater colonic total VFA compared to pigs fed the NC diet (P ≤ 0.07), but no difference in colonic pH was observed (P > 0.10). Pigs fed the NC diet had a greater Ca digestibility compared to pigs fed the PC (P ≤ 0.01), with no treatment to the NC having any effect on Ca digestibility compared to pigs fed the NC (P > 0.10). There was no effect of dietary Ca level on BMD and no overall addition of feeding SCF, rCS, or pBRT on BMD compared to pigs fed the NC (P > 0.10). There was no impact on pig ADG, ADFI, or GF by reducing dietary Ca by 40% (i.e., pigs fed the NC) compared to pigs fed the PC (P > 0.10). Relative to pigs fed the NC, there was no overall effect of SCF, rCS, or pBRT on ADG, ADFI, or GF (P > 0.10). In conclusion, feeding young pigs a Ca-deficient diet reduced colonic pH, increased digestibility of Ca, but had no impact on bone mineralization or overall pig performance. Supplementation of nondigestible oligosaccharides pr protected butyrate had either no effect or an inconsistent effect on colonic pH, Ca, or PHOS digestibility, bone mineralization, or overall pig performance.

Calcium (Ca) is a major component of the skeleton in addition to being essential for growth and is imperative for bone mass development. Improvement in Ca absorption in Ca-deficient diets has been shown in human and rodent studies when nondigestible oligosaccharides have been consumed due to a modification of gastrointestinal conditions which increase mineral solubility. Because swine have been shown to be an excellent model for human nutrition research, an experiment was conducted to determine how a moderately Ca-deficient diet would affect gastrointestinal fermentation conditions, Ca and phosphorus (PHOS) digestibility, bone mineralization, and growth performance in nursery pigs; and if supplementation of nondigestible oligosaccharides would affect these same parameters. Results indicate that feeding young pigs a diet below recommended levels of Ca reduced colonic pH, increased apparent total-tract digestibility of Ca and PHOS, but had no impact on bone mineralization or overall pig performance. Supplementation of nondigestible oligosaccharides had inconsistent effects on colonic pH, and did not affect Ca or PHOS digestibility, bone mineralization, or overall pig performance.

Effects of short-chain fatty acids on intestinal function in an enteroid model of hypoxia.

The healthy GI tract is physiologically hypoxic, but this may be perturbed by certain acute and chronic stressors that reduce oxygen availability systemically. Short-chain fatty acids have been shown to have beneficial effects on intestinal barrier function and inflammation. Therefore, our objective was to see whether short-chain fatty acids (SCFA) would improve GI barrier function, reduce production of pro-inflammatory cytokines, and increase the expression of genes regulating GI barrier function in enteroids exposed to hypoxia. Human duodenal enteroid monolayers were placed under hypoxia (1.0% O2) for 72 h with either 24, or 48 h pre-treatment with a high acetate ratio of SCFA's or high butyrate ratio or placed under hypoxia concurrently. Transepithelial electrical resistance (TEER) increased with SCFA pre-treatment, especially 48 h of pre-treatment and this was maintained through the first 48 h of hypoxia while cells saw barrier function dramatically decrease by 72 h of hypoxia exposure. Inflammatory protein secretion largely decreased with exposure to hypoxia, regardless of SCFA pre-treatment. Gene expression of several genes related to barrier function were decreased with exposure to hypoxia, and with concurrent and 24 h SCFA pre-treatment. However, 48 h SCFA pre-treatment with a high butyrate ratio increased expression of several metabolic and differentiation related genes. Overall, pre-treatment or concurrent treatment with SCFA mixtures were not able to overcome the negative impacts of hypoxia on intestinal function and cells ultimately still cannot be sustained under hypoxia for 72 h. However, 48 h pre-treatment maintains TEER for up to 48 h of hypoxia while upregulating several metabolic genes.

Evaluation of digestively resistant or soluble fibers, short- and medium-chain fatty acids, trace minerals, and antibiotics in nonchallenged nursery pigs on performance, digestibility, and intestinal integrity.

Three experiments (EXP) were conducted to determine the effect of feed additives on performance, intestinal integrity, gastrointestinal volatile fatty acids (VFA), and energy and nutrient digestion in nonchallenged nursery pigs. In EXP 1, 480 pigs (6.36-kg body weight, BW) were placed into 96 pens with 5 pigs/pen, and allotted to 1 of 10 dietary treatments: 1) negative control containing no feed additive (NC), 2) NC + 44 mg chlortetracycline and 38.5 mg tiamulin/kg diet (CTsb), 3) NC + 5% resistant potato starch (RSpo), 4) NC + 5% soluble corn fiber (SCF), 5) NC + 5% sugar beet pulp (SBP), 6) NC + 0.30% fatty acid mix (FAM), 7) NC + 0.10% phytogenic blend of essential oils and flavoring compounds (PHY), 8) NC + 50 mg Cu and 1,600 mg zinc oxide/kg diet (CuZn), 9) NC + 5% resistant corn starch (RScn), and 10) NC + 0.05% ß-glucan (BG) for 28 d. There was no impact of dietary treatment on BW gain or feed intake (P ≥ 0.22). Pigs fed diets containing SCF, CTsb, and RSpo resulted in microbial community differences compared to pigs fed the NC (P < 0.05). In EXP 2, 48 barrows (12.8 kg BW) were selected at the end of EXP 1 and fed the same dietary treatments they had previously received: 1) NC, 2) NC + 5% RScn, 3) NC + 5% SCF, and 4) NC + FAM for 8 d. There was no effect of feeding diets containing RScn, SCF, or FAM on in vivo intestinal permeability (P ≤ 0.21). Ileal or colon pH, concentrations of VFA did not differ due to dietary treatment (P ≥ 0.36), but pigs fed diets containing FAM resulted in a greater butyric acid concentration in the cecum compared to pigs fed the NC (P ≤ 0.05). In EXP 3, 156 pigs (6.11 kg BW) were placed into 52 pens with 3 pigs/pen and allotted to 1 of 4 dietary treatments arranged in a factorial manner: 1) NC, 2) NC + 5% RSpo, 3) NC + 0.30% FAM, and 4) NC + 5% RSpo + 0.30% FAM for 24 d. Feeding pigs diets containing RSpo did not affect BW gain (P = 0.91) while pigs fed diets containing FAM grew improved BW gain (P = 0.09). Colonic butyric acid concentrations were greater in pigs fed diets containing RSpo (P = 0.03), while pigs fed diets containing FAM exhibited reduced total VFA concentrations (P = 0.11). The results indicate that supplementing diets with digestively resistant but fermentable fibers, short- and medium-chain fatty acids, or antibiotics do not have a consistent effect, positive or negative, on markers of intestinal integrity or barrier function, intestinal VFA patterns, ATTD of energy and nutrients, or on pig performance.

In-feed antimicrobials have been an important technology in swine production for protecting health and supporting growth. However, with legislative restrictions on the use of most antibiotics for growth promotion, research is needed to evaluate in-feed additives in replacing this growth promoting technology. Thus, strategies to enhance energy and nutrient digestibility, intestinal function and integrity, gastrointestinal volatile fatty acid concentrations, and microbial ecology in nursery pigs are desirable targets. The results of the three experiments conducted herein do not indicate that supplementing diets with digestively resistant but fermentable fibers, short-medium-chain fatty acids, or antibiotics have a consistent positive or negative effect on markers of intestinal integrity or barrier function, VFA patterns (ileal, cecal, or colon), ATTD of energy and nutrients, or pig performance.

Human iPSC colon organoid function is improved by exposure to fecal fermentates.

The host-microbe interaction is critical for intestinal homeostasis. By-products from microbial metabolism of unabsorbed dietary components have been studied increasingly as potential contributors to health and disease. In vitro fermentation systems provide a way to simulate microbial activity and by-product production of the colon using human fecal samples. Objectives of the study were to determine how clarified supernatants from two different fermentation conditions affect markers of cell proliferation, differentiation, barrier function, and immune function in a human-induced pluripotent (iPSC) colon organoid model. SCFA and BCFA's of the supernatants were analyzed and were similar to known in vivo concentrations. Molecular results showed 25% of the clarified supernatant from batch fermentation led to a more physiological intestinal phenotype including increased markers of differentiation, including alkaline phosphatase, chromogranin A, SCFA transport monocarboxylate transporter-1, (6.2-fold, 2.1-fold, and 1.8-fold, respectively; p < 0.05). Mucin production (mucin-2, mucin-4) was increased in cells treated with 25% supernatant, as observed by confocal microscopy. In addition, increased tight junction expression (claudin-3) was noted by immunofluorescence in 25% supernatant- treated cells. A dose-response increase in barrier function was observed over the 72-h time course, with a twofold increase in transepithelial electrical resistance (TER) in the 25% group compared to the control group (p < 0.05). To further investigate host effects, clarified supernatants from a continuous multistage fermentation representing the ascending (AC), transverse (TC), and descending (DC) colonic domains were utilized and some regional differences were observed including increased markers of inflammation (IL-1ß, 6.15 pg/ml; IL-6, 27.58 pg/ml; TNFα, 4.49 pg/ml; p < 0.05) in DC-treated samples only. Overall, clarified supernatants represent a valuable model to examine effects of microbial by-products on host intestinal development and function and future efforts will be designed to further understand microbial communities and metabolites, along with additional host response measures.

Expression of SARS-CoV-2 entry factors, electrolyte, and mineral transporters in different mouse intestinal epithelial cell types.

Angiotensin-converting enzyme 2 (ACE2) and transmembrane proteases (TMPRSS) are multifunctional proteins required for SARS-CoV-2 infection or for amino acid (AA) transport, and are abundantly expressed in mammalian small intestine, but the identity of the intestinal cell type(s) and sites of expression are unclear. Here we determined expression of SARS-CoV-2 entry factors in different cell types and then compared it to that of representative AA, electrolyte, and mineral transporters. We tested the hypothesis that SARS-CoV-2, AA, electrolyte, and mineral transporters are expressed heterogeneously in different intestinal cell types by making mouse enteroids enriched in enterocytes (ENT), goblet (GOB), Paneth (PAN), or stem (ISC) cells. Interestingly, the expression of ACE2 was apical and modestly greater in ENT, the same pattern observed for its associated AA transporters B0 AT1 and SIT1. TMPRSS2 and TMPRSS4 were more highly expressed in crypt-residing ISC. Expression of electrolyte transporters was dramatically heterogeneous. DRA, NBCe1, and NHE3 were greatest in ENT, while those of CFTR and NKCC1 that play important roles in secretory diarrhea, were mainly expressed in ISC and PAN that also displayed immunohistochemically abundant basolateral NKCC1. Intestinal iron transporters were generally expressed higher in ENT and GOB, while calcium transporters were expressed mainly in PAN. Heterogeneous expression of its entry factors suggests that the ability of SARS-CoV-2 to infect the intestine may vary with cell type. Parallel cell-type expression patterns of ACE2 with B0 AT1 and SIT1 provides further evidence of ACE2's multifunctional properties and importance in AA absorption.

Cell-Type-Specific, Ketohexokinase-Dependent Induction by Fructose of Lipogenic Gene Expression in Mouse Small Intestine.

BACKGROUND: High intakes of fructose are associated with metabolic diseases, including hypertriglyceridemia and intestinal tumor growth. Although small intestinal epithelia consist of many different cell types, express lipogenic genes, and convert dietary fructose to fatty acids, there is no information on the identity of the cell type(s) mediating this conversion and on the effects of fructose on lipogenic gene expression. OBJECTIVES: We hypothesized that fructose regulates the intestinal expression of genes involved in lipid and apolipoprotein synthesis, that regulation depends on the fructose transporter solute carrier family 2 member a5 [Slc2a5 (glucose transporter 5)] and on ketohexokinase (Khk), and that regulation occurs only in enterocytes. METHODS: We compared lipogenic gene expression among different organs from wild-type adult male C57BL mice consuming a standard vivarium nonpurified diet. We then gavaged twice daily for 2.5 d fructose or glucose solutions (15%, 0.3 mL per mouse) into wild-type, Slc2a5-knockout (KO), and Khk-KO mice with free access to the nonpurified diet and determined expression of representative lipogenic genes. Finally, from mice fed the nonpurified diet, we made organoids highly enriched in enterocyte, goblet, Paneth, or stem cells and then incubated them overnight in 10 mM fructose or glucose. RESULTS: Most lipogenic genes were significantly expressed in the intestine relative to the kidney, liver, lung, and skeletal muscle. In vivo expression of Srebf1, Acaca, Fasn, Scd1, Dgat1, Gk, Apoa4, and Apob mRNA and of Scd1 protein increased (P < 0.05) by 3- to 20-fold in wild-type, but not in Slc2a5-KO and Khk-KO, mice gavaged with fructose. In vitro, Slc2a5- and Khk-dependent, fructose-induced increases, which ranged from 1.5- to 4-fold (P < 0.05), in mRNA concentrations of all these genes were observed only in organoids enriched in enterocytes. CONCLUSIONS: Fructose specifically stimulates expression of mouse small intestinal genes for lipid and apolipoprotein synthesis. Secretory and stem cells seem incapable of transport- and metabolism-dependent lipogenesis, occurring only in absorptive enterocytes.

Intestinal enteroids recapitulate the effects of short-chain fatty acids on the intestinal epithelium.

Enteroids are cultured primary intestinal epithelial cells that recapitulate epithelial lineage development allowing for a more complex and physiologically relevant model for scientific study. The large presence of intestinal stem cells (ISC) in these enteroids allows for the study of metabolite effects on cellular processes and resulting progeny cells. Short-chain fatty acids (SCFA) such as butyrate (BUT) are bacterial metabolites produced in the gastrointestinal tract that are considered to be beneficial to host cells. Therefore, the objective was to study the effects of SCFAs on biomarkers of ISC activity, differentiation, barrier function and epithelial defense in the intestine using mouse and human enteroid models. Enteroids were treated with two concentrations of acetate (ACET), propionate (PROP), or BUT for 24 h. Enteroids treated with BUT or PROP showed a decrease in proliferation via EdU uptake relative to the controls in both mouse and human models. Gene expression of Lgr5 was shown to decrease with BUT and PROP treatments, but increased with ACET. As a result of BUT and PROP treatments, there was an increase in differentiation markers for enterocyte, Paneth, goblet, and enteroendocrine cells. Gene expression of antimicrobial proteins Reg3ß, Reg3γ, and Defb1 were stimulated by BUT and PROP, but not by ACET which had a greater effect on expression of tight junction genes Cldn3 and Ocln in 3D enteroids. Similar results were obtained with human enteroids treated with 10 mM SCFAs and grown in either 3D or Transwell™ model cultures, although tight junctions were influenced by BUT and PROP, but not ACET in monolayer format. Furthermore, BUT and PROP treatments increased transepithelial electrical resistance after 24 h compared to ACET or control. Overall, individual SCFAs are potent stimulators of cellular gene expression, however, PROP and especially BUT show great efficacy for driving cell differentiation and gene expression.

Intestinal in vitro and ex vivo Models to Study Host-Microbiome Interactions and Acute Stressors.

The gut microbiome is extremely important for maintaining homeostasis with host intestinal epithelial, neuronal, and immune cells and this host-microbe interaction is critical during times of stress or disease. Environmental, nutritional, and cognitive stress are just a few factors known to influence the gut microbiota and are thought to induce microbial dysbiosis. Research on this bidirectional relationship as it pertains to health and disease is extensive and rapidly expanding in both in vivo and in vitro/ex vivo models. However, far less work has been devoted to studying effects of host-microbe interactions on acute stressors and performance, the underlying mechanisms, and the modulatory effects of different stressors on both the host and the microbiome. Additionally, the use of in vitro/ex vivo models to study the gut microbiome and human performance has not been researched extensively nor reviewed. Therefore, this review aims to examine current evidence concerning the current status of in vitro and ex vivo host models, the impact of acute stressors on gut physiology/microbiota as well as potential impacts on human performance and how we can parlay this information for DoD relevance as well as the broader scientific community. Models reviewed include widely utilized intestinal cell models from human and animal models that have been applied in the past for stress or microbiology research as well as ex vivo organ/tissue culture models and new innovative models including organ-on-a-chip and co-culture models.

Effects of Psychological, Environmental and Physical Stressors on the Gut Microbiota.

Stress, a ubiquitous part of daily human life, has varied biological effects which are increasingly recognized as including modulation of commensal microorganisms residing in the gastrointestinal tract, the gut microbiota. In turn, the gut microbiota influences the host stress response and associated sequelae, thereby implicating the gut microbiota as an important mediator of host health. This narrative review aims to summarize evidence concerning the impact of psychological, environmental, and physical stressors on gut microbiota composition and function. The stressors reviewed include psychological stress, circadian disruption, sleep deprivation, environmental extremes (high altitude, heat, and cold), environmental pathogens, toxicants, pollutants, and noise, physical activity, and diet (nutrient composition and food restriction). Stressors were selected for their direct relevance to military personnel, a population that is commonly exposed to these stressors, often at extremes, and in combination. However, the selected stressors are also common, alone or in combination, in some civilian populations. Evidence from preclinical studies collectively indicates that the reviewed stressors alter the composition, function and metabolic activity of the gut microbiota, but that effects vary across stressors, and can include effects that may be beneficial or detrimental to host health. Translation of these findings to humans is largely lacking at present. This gap precludes concluding with certainty that transient or cumulative exposures to psychological, environmental, and physical stressors have any consistent, meaningful impact on the human gut microbiota. However, provocative preclinical evidence highlights a need for translational research aiming to elucidate the impact of stressors on the human gut microbiota, and how the gut microbiota can be manipulated, for example by using nutrition, to mitigate adverse stress responses.

Prolonged environment-induced hyperthermia alters autophagy in oxidative skeletal muscle in Sus scrofa.

Prolonged heat stress represents a continuing threat to human health and agricultural production. Despite the broad, negative impact of prolonged hyperthermia little is known about underlying pathological mechanisms leading to negative health outcomes, which has limited the development of etiological interventions and left clinicians and producers with only cooling and rehydration strategies. The purpose of this investigation was to determine the extent to which prolonged environment-induced hyperthermia altered autophagy in oxidative skeletal muscle in a large animal model, serving the dual purpose of accurately modeling human physiology as well as agricultural production. We hypothesized that prolonged hyperthermia would induce autophagy in skeletal muscle, independent of the accompanying caloric restriction. To test this hypothesis pigs were treated as follows: thermoneutral (20 °C), heat stress (35 °C), or were held under thermoneutral conditions but pair-fed to the heat stress group for seven days. Upon euthanasia the red portion of the semitendinosus was collected. We found that prolonged hyperthermic exposure increased oxidative stress without a corresponding change in antioxidant enzyme activities. Hyperthermia prevented initiation of autophagy despite increased markers of nucleation, elongation and autophagosome formation. However, p62 relative protein abundance, which is inversely correlated with autophagic degradation, was strongly increased suggesting suppressed degradation of autophagosomes. Markers of mitophagy and mitochondrial abundance were largely similar between groups. These data indicate that faulty autophagy plays a key role in hyperthermic muscle dysfunction.

Short-term heat stress results in increased apoptotic signaling and autophagy in oxidative skeletal muscle in Sus scrofa.

Prolonged environment-induced hyperthermia causes morbidities and mortality in humans and animals and appears to cause organ-specific injury and dysfunction. We have previously determined autophagic dysfunction and apoptotic signaling in oxidative skeletal muscle following prolonged hyperthermia. The aim of this investigation was to extend our knowledge regarding the early chronology of heat stress-mediated apoptotic and autophagic signaling in oxidative skeletal muscle. We hypothesized that 2, 4, and 6 h of hyperthermia would increase apoptosis and autophagy in oxidative skeletal muscle compared to thermoneutral (TN) conditions. Pigs were assigned to four groups (n = 8/group) and exposed to environmental heat stress (37 °C) for 0, 2, 4, or 6 h. Immediately following environmental exposure animals were euthanized and the red portion of the semitendinosus was collected. Markers of apoptotic signaling were increased following 2 h of heating but returned to baseline thereafter, while caspase 3 activity remained elevated 2-3 fold (p < .05) throughout the hyperthermic period. Heat stress increased (p < .05) markers of autophagic activation, and nucleation as well as autophagosome formation and degradation linearly throughout the heating intervention. In addition, 6 h of hyperthermia increased (p < .05) markers of mitophagy. These data suggest that apoptotic signaling precedes increased autophagy during acute heat stress in oxidative skeletal muscle.

Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types.

BACKGROUND: Mammalian small intestinal tight junctions (TJ) link epithelial cells to one another and function as a permselective barrier, strictly modulating the passage of ions and macromolecules through the pore and leak pathways, respectively, thereby preventing the absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. Small intestinal epithelial permeability is ascribed primarily to the properties of TJs between adjoining enterocytes (ENTs), because there is almost no information on TJ composition and the paracellular permeability of nonenterocyte cell types that constitute a small but significant fraction of the intestinal epithelia. RESULTS: Here we directed murine intestinal crypts to form specialized organoids highly enriched in intestinal stem cells (ISCs), absorptive ENTs, secretory goblet cells, or Paneth cells. The morphological and morphometric characteristics of these cells in organoids were similar to those in vivo. The expression of certain TJ proteins varied with cell type: occludin and tricellulin levels were high in both ISCs and Paneth cells, while claudin-1, -2, and -7 expression was greatest in Paneth cells, ISCs, and ENTs, respectively. In contrast, the distribution of claudin-15, zonula occludens 1 (ZO-1), and E-cadherin was relatively homogeneous. E-cadherin and claudin-7 marked mainly the basolateral membrane, while claudin-2, ZO-1, and occludin resided in the apical membrane. Remarkably, organoids enriched in ENTs or goblet cells were over threefold more permeable to 4 and 10 kDa dextran compared to those containing stem and Paneth cells. The TJ-regulator larazotide prevented the approximately tenfold increases in dextran flux induced by the TJ-disrupter AT1002 into organoids of different cell types, indicating that this ZO toxin nonselectively increases permeability. Forced dedifferentiation of mature ENTs results in the reacquisition of ISC-like characteristics in TJ composition and dextran permeability, suggesting that the post-differentiation properties of TJs are not hardwired. CONCLUSIONS: Differentiation of adult intestinal stem cells into mature secretory and absorptive cell types causes marked, but potentially reversible, changes in TJ composition, resulting in enhanced macromolecular permeability of the TJ leak pathway between ENTs and between goblet cells. This work advances our understanding of how cell differentiation affects the paracellular pathway of epithelia.

Short-term heat stress altered metabolism and insulin signaling in skeletal muscle.

Heat-related complications continue to be a major health concern for humans and animals and lead to potentially life-threatening conditions. Heat stress (HS) alters metabolic parameters and may alter glucose metabolism and insulin signaling. Therefore, the purpose of this investigation was to determine the extent to which 12 h of HS-altered energetic metabolism in oxidative skeletal muscle. To address this, crossbred gilts (n = 8/group) were assigned to one of three environmental treatments for 12 h: thermoneutral (TN; 21 °C), HS (37 °C), or pair-fed to HS counterparts but housed in TN conditions (PFTN). Following treatment, animals were euthanized and the semitendinosus red (STR) was recovered. Despite increased relative protein abundance of the insulin receptor, insulin receptor substrate (IRS1) phosphorylation was increased (P = 0.0005) at S307, an inhibitory site, and phosphorylated protein kinase B (AKT) (S473) was decreased (P = 0.03) likely serving to impair insulin signaling following 12 h of HS. Further, HS increased phosphorylated protein kinase C (PKC) ζ/λ (P = 0.02) and phosphorylated PKCδ/θ protein abundance (P = 0.02), which are known to regulate inhibitory serine phosphorylation of IRS1 (S307). Sarcolemmal glucose transporter 4 (Glut4) was decreased (P = 0.04) in the membrane fraction of HS skeletal muscle suggesting diminished glucose uptake capacity. HS-mediated increases (P = 0.04) in mechanistic target of rapamycin (mTOR) were not accompanied by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1). HS decreased (P = 0.0006) glycogen synthase (GS) and increased (P = 0.02) phosphorylated GS suggesting impaired glycogen synthesis. In addition, HS altered fatty acid metabolic signaling by increasing (P = 0.02) Acetyl-CoA carboxylase (ACC), decreasing (P = 0.005) phosphorylated ATP-citrate lyase (pATPCL) and fatty acid synthase (P = 0.01) (FAS). These data suggest that 12 h of HS blunted insulin signaling, decreased protein synthesis, and altered glycogen and fatty acid metabolism.

Acute heat stress activated inflammatory signaling in porcine oxidative skeletal muscle.

Despite well-studied clinical manifestations, intracellular mechanisms of prolonged hyperthermic injury remain unclear, especially in skeletal muscle. Given muscle's large potential to impact systemic inflammation and metabolism, the response of muscle cells to heat-mediated injury warrants further investigation. We have previously reported increased activation of NF-κB signaling and increased NF-κB and AP-1-driven transcripts in oxidative skeletal muscle following 12 h of heat stress. The purpose of this investigation was to examine early heat stress-induced inflammatory signaling in skeletal muscle. We hypothesized that heat stress would increase NF-κB and AP-1 signaling in oxidative skeletal muscle. To address this hypothesis, 32 gilts were randomly assigned to one of four treatment groups (n = 8/group): control (0 h: 21°C) or exposed to heat stress conditions (37°C) for 2 h (n = 8), 4 h (n = 8), or 6 h (n = 8). Immediately following environmental exposure pigs were euthanized and the red portion of the semitendinosus muscle (STR) was harvested. We found evidence of NF-κB pathway activation as indicated by increased protein abundance of NF-κB activator IKK-α following 4 h and increased total NF-κB protein abundance following 6 h of heat stress. Heat stress also stimulated AP-1 signaling as AP-1 protein abundance was increased in nuclear fractions following 4 h of heat stress. Interleukin-6 protein abundance and activation of the JAK/STAT pathway were decreased in heat stressed muscle. These data indicate that heat stress activated inflammatory signaling in the porcine STR muscle via the AP-1 pathway and early activation of the NF-κB pathway.

Short-term heat stress alters redox balance in porcine skeletal muscle.

Heat stress contributes to higher morbidity and mortality in humans and animals and is an agricultural economic challenge because it reduces livestock productivity. Redox balance and associated mitochondrial responses appear to play a central role in heat stress-induced skeletal muscle pathology. We have previously reported increased oxidative stress and mitochondrial content in oxidative muscle following 12 h of heat stress. The purposes of this investigation were to characterize heat stress-induced oxidative stress and changes in mitochondrial content and biogenic signaling in oxidative skeletal muscle. Crossbred gilts were randomly assigned to either thermal neutral (21°C; n = 8, control group) or heat stress (37°C) conditions for 2 h (n = 8), 4 h (n = 8), or 6 h (n = 8). At the end, their respective environmental exposure, the red portion of the semitendinosus muscle (STR) was harvested. Heat stress increased concentration of malondialdehyde (MDA) following 2 and 4 h compared to thermal neutral and 6 h, which was similar to thermal neutral, and decreased linearly with time. Protein carbonyl content was not influenced by environment. Catalase activity was increased following 4 h of heat stress and superoxide dismutase activity was decreased following 6 h of heat stress compared to thermal neutral conditions. Heat stress-mediated changes in antioxidant activity were independent of altered protein abundance or transcript expression. Mitochondrial content and mitochondrial biogenic signaling were similar between groups. These data demonstrate that heat stress caused a transient increase in oxidative stress that was countered by a compensatory change in catalase activity. These findings contribute to our growing understanding of the chronology of heat stress-induced intracellular dysfunctions in skeletal muscle.

Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells.

Nutrient sensing triggers responses by the gut-brain axis modulating hormone release, feeding behavior and metabolism that become dysregulated in metabolic syndrome and some cancers. Except for absorptive enterocytes and secretory enteroendocrine cells, the ability of many intestinal cell types to sense nutrients is still unknown; hence we hypothesized that progenitor stem cells (intestinal stem cells, ISC) possess nutrient sensing ability inherited by progenies during differentiation. We directed via modulators of Wnt and Notch signaling differentiation of precursor mouse intestinal crypts into specialized organoids each containing ISC, enterocyte, goblet, or Paneth cells at relative proportions much higher than in situ as determined by mRNA expression and immunocytochemistry of cell type biomarkers. We identified nutrient sensing cell type(s) by increased expression of fructolytic genes in response to a fructose challenge. Organoids comprised primarily of enterocytes, Paneth, or goblet, but not ISC, cells responded specifically to fructose without affecting nonfructolytic genes. Sensing was independent of Wnt and Notch modulators and of glucose concentrations in the medium but required fructose absorption and metabolism. More mature enterocyte- and goblet-enriched organoids exhibited stronger fructose responses. Remarkably, enterocyte organoids, upon forced dedifferentiation to reacquire ISC characteristics, exhibited a markedly extended lifespan and retained fructose sensing ability, mimicking responses of some dedifferentiated cancer cells. Using an innovative approach, we discovered that nutrient sensing is likely repressed in progenitor ISCs then irreversibly derepressed during specification into sensing-competent absorptive or secretory lineages, the surprising capacity of Paneth and goblet cells to detect fructose, and the important role of differentiation in modulating nutrient sensing.NEW & NOTEWORTHY Small intestinal stem cells differentiate into several cell types transiently populating the villi. We used specialized organoid cultures each comprised of a single cell type to demonstrate that 1) differentiation seems required for nutrient sensing, 2) secretory goblet and Paneth cells along with enterocytes sense fructose, suggesting that sensing is acquired after differentiation is triggered but before divergence between absorptive and secretory lineages, and 3) forcibly dedifferentiated enterocytes exhibit fructose sensing and lifespan extension.

Twelve hours of heat stress induces inflammatory signaling in porcine skeletal muscle.

Heat stress causes morbidity and mortality in humans and animals and threatens food security by limiting livestock productivity. Inflammatory signaling may contribute to heat stress-mediated skeletal muscle dysfunction. Previously, we discovered increased circulating endotoxin and intramuscular oxidative stress and TNF-α protein abundance, but not inflammatory signaling following 24 and 72 h of heat stress. Thus the purpose of this investigation was to clarify the role of inflammatory signaling in heat-stressed skeletal muscle. Crossbred gilts (n = 8/group) were assigned to either thermal neutral (24°C), heat stress (37°C), or pair-fed thermal neutral (24°C) conditions for 12 h. Following treatment, animals were euthanized, and the semitendinosus red (STR) and white (STW) were recovered. Heat stress did not alter inflammatory signaling in STW. In STR, relative heat shock protein abundance was similar between groups, as was nuclear content of heat shock factor 1. In whole homogenate, relative abundance of the NF-κB activator inhibitory κB kinase-α was increased by heat stress, although abundance of NF-κB was similar between groups. Relative abundance of phosphorylated NF-κB was increased by heat stress in nuclear fractions. Activator protein-1 (AP-1) signaling was similar between groups. While there were few differences in transcript expression between thermal neutral and heat stress, 80 and 56% of measured transcripts driven by NF-κB or AP-1, respectively, were increased by heat stress compared with pair-fed thermal neutral. Heat stress also caused a reduction in IL-6 transcript and relative protein abundance. These data demonstrate that short-term heat stress causes inflammatory signaling through NF-κB in oxidative, but not glycolytic, skeletal muscle.