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The Influence of Heat Stress on Milk Yield, Gastrointestinal Permeability, and Nutrient Partitioning in Lactating Dairy CattleEllett Jr, Mark David 06 August 2024 (has links)
The US dairy industry loses approximately $1.2 billion due to heat stress related production losses annually. It was formerly believed that heat-stressed lactating dairy cattle produce less milk because they consume less feed. It has since been established that the reduction of feed intake is only responsible for about 50% of the reduced milk yield in HS cows. It is believed that HS increases gastrointestinal permeability (GIP), resulting in microbial components leaking from the lumen of the gastrointestinal tract into underlying tissue and stimulating an immune response. The immune response is suspected to alter overall metabolism, and milk production specifically, by diverting nutrients away from the mammary gland and other non-essential processes to support immune system activation. Topics examined herein focus on identifying markers to assess gastrointestinal permeability and the influence of heat stress on GIP and nutrient metabolism. The first study utilized an in vitro rumen fermentation system to determine if lactulose, sucralose, and D-mannitol could persist in an in vitro rumen culture. Lactulose could not be quantified in the rumen fluid matrix, D-mannitol was rapidly degraded, and sucralose concentrations did not change after 48 h of incubation, establishing sucralose as an indigestible marker in mature ruminants. The second study utilized a pair feeding design to directly assess the effect of HS on GIP, milk yield, and immune activation by lipopolysaccharide (LPS). HS cows (n=7) were exposed to a temperature-humidity index (THI) value of 74-80 for 4 d. The pair-fed thermoneutral cows (PFTN, n=8) were exposed to a constant THI of 64 with their intake matched to the HS cows. HS lowered milk yield without altering GIP, measured using orally dosed sucralose as a permeability marker, or eliciting an LPS related immune response. Jejunal mucosal scrapings were harvested from each cow, tight junction proteins were quantified, and no differences were detected. Lack of treatment responses in GIP marker recovery and tight junction protein abundance indicate that increased GIP may not be a driving force behind production losses in HS dairy cows. The third study focused on energy substrate utilization during HS with the objective of determining if tissue-level energy substrate metabolism could be influencing glucose sparing mechanisms. Metabolic flexibility of skeletal muscle, liver, and mammary tissue was assessed after 4 d of HS. It was determined that HS reduced skeletal muscle metabolic flexibility and did not alter liver and mammary metabolic flexibility. This indicates that skeletal muscle has a greater dependency on glucose as an energy substrate, which may decrease the pool of glucose available for lactose synthesis in lactating cows. Finally, the last study had the objective of assessing branched-chain amino acid (BCAA) requirements during HS. BCAA are oxidized for ATP synthesis in extrahepatic tissues and provide precursors for the biosynthesis of non-essential amino acids. They are also taken up by the mammary gland at a rate greater than what they are used in milk protein. Taken together, it was hypothesized that BCAA requirements may be increased during HS. BCAA entry rates into blood were assessed using a stable isotope approach and a 4-pool model. No differences were detected in daily entry rates or flux rates between pools indicating no change in requirements. When considering the results of all studies, reductions in milk yield are likely a result of altered macronutrient metabolism but further work is needed to confirm that hypothesis. Understanding the physiology behind HS related production losses is the first step in developing mitigation strategies. / Doctor of Philosophy / Heat stress (HS) is a global issue that compromises dairy cattle welfare and reduces milk production. On average the US dairy industry loses approximately $1.2 billion due to heat stress related production losses annually. With the global population expected to exceed 9 billion by 2050, strategies to mitigate HS related production losses are needed. Although cows exposed to HS conditions eat less food, that only explains about 50% of the production losses. It is hypothesized that the other 50% of milk yield losses is at least partially caused by increased gastrointestinal permeability (GIP), which elicits an immune response. Questions examined herein primarily focus on quantification of physiological and metabolic responses to HS. The objective of the first study was to identify a marker to assess GIP we could give orally to cows and detect in their urine. Commonly used GIP markers used in monogastric are carbohydrates, which have the potential to be fermented in the rumen. Sucralose was identified as a suitable marker due to its resistance to degradation in the rumen. The next study focused on measuring physiological responses of lactating dairy cows when exposed to HS conditions. Under the conditions of our experiment, HS decreased dry matter intake and milk yield without increasing GIP or inducing an immune response. It was determined that the reduction in dry matter intake was responsible for 66% of the reduced milk yield with the other 34% being associated with physiological changes other than increased GIP. The next study focused on how HS impacts the ability of skeletal muscle, mammary, and liver tissue to utilize glucose or palmitic acid as an energy substrate. The ability to switch between energy substrates is called metabolic flexibility. It was found that HS lowered the ability of skeletal muscle because it was unable to utilize fat as an energy source. Mammary and liver tissue exhibited no change in metabolic flexibility. The final study focused on how HS changed branched-chain amino acid (BCAA) plasma entry rates into plasma. An in vivo stable isotope method and a 4-pool mathematical model was used to predict how BCAA moved between pools which corresponds to the rate of protein turnover. Under the conditions of this experiment, no differences in BCAA entry rates were observed. Overall, results indicate altered energy substrate metabolism independent of immune activation stemming from altered GIP may be a driving factor in HS related production losses. Overall, this work contributes to understanding of HS biology and questions the established belief that increased GIP resulting in immune activation is responsible for about 50% of production losses.
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Bacterial translocation to adipose tissue in metabolic diseaseMassier, Lucas 23 October 2020 (has links)
Alterations in composition and function of human gut microbiota can affect physiological processes and are known to be associated with many diseases including inflammatory bowel disease, hypertension, asthma and colon cancer. Complex interactions between gut microbiota, environmental toxins, nutrients and host genetics may result in an increased permeability of the gut, which is closely linked to the presence of adverse metabolic conditions. As a consequence, translocation of bacterial DNA into the blood circulation increases in patients with obesity. Obesity is a growing health problem worldwide and often paired with severe secondary complications, such as type 2 diabetes or cardiovascular problems. A main feature of disease progression is a chronic low-grade inflammation of adipose tissue which contributes to the development and aggravation of insulin resistance and many of the underlying mechanisms are still unknown. Although data from mice studies suggest that the presence of bacterial components in adipose tissue can support these processes, human studies on this subject are lacking.
In my thesis entitled ``Bacterial translocation to adipose tissue in metabolic diseas'' I provide evidence supporting the initial hypothesis, namely that bacterial DNA is present in adipose tissue, even after stringent controlling for contaminants. To this end, I established a wet lab routine protocol to eliminate contamination as well as a bioinformatics pipeline accounting for contamination by subtracting negative controls. Briefly, this included the use of lab ware and reagents UV-treated for at least 90 minutes, the use of breath protection, extra-long gloves and single-use lab coats as well as working under a sterile laminar flood hood in a clean lab free of any PCR products. The bioinformatics pipeline employed commonly used 16S rRNA gene analysis tools including qiime2, phyloseq and DESeq2 as well as decontam, a novel tool to extract negative controls. Observed quantity of bacterial DNA was in the range of 1 to 10 pg/µg total isolated genomic DNA, which is equivalent to about 0.01 to 0.7% of bacterial cells per human cell. The highest quantity was present in subcutaneous adipose tissue, followed by mesenteric adipose tissue. Bacterial amount correlated with adipose tissue macrophages and PPARG expression in omental and with IL1B and TNF expression in subcutaneous adipose tissue. Mesenteric adipose tissue showed the highest diversity of the observed genera. The most commonly observed phyla in all tissues were Proteobacteria and Firmicutes, which is in line with previously published data on blood bacterial DNA. Still, many genera were predominantly found in specific tissues, e.g. Enterobacter in subcutaneous and Acinetobacter in omental adipose tissue. I further showed that the distribution of observed features could partially be explained by markers of insulin resistance (HOMA-IR, HbA1c) and inflammation (IL-6, TNFa, macrophages) and that certain genera, such as Rhodoferax or Lactobacillus are associated with type 2 diabetes status. In first functional approaches I demonstrated that concentrations of bacterial DNA in the observed range are sufficient to stimulate an inflammatory response in immortalized subcutaneous adipocytes derived from a healthy donor. The effect was most prominent after four hours of treatment and increased in a dose-dependent matter.
One of the aims in the present study was to determine levels of gut leakage by measuring zonulin, the most commonly used biomarker for intestinal permeability in humans, and analyze possible associations with adipose tissue bacterial signature. As there are few well-conducted studies on circulating zonulin levels in patients with metabolic diseases, I first performed a correlation study in the available and metabolically well-characterized Sorbs cohort. Circulating zonulin correlated significantly positive with BMI, fasting glucose, triglycerides and cholesterol and negatively with HOMA-IS, high density lipoprotein and circulating adiponectin levels. Albeit these strong correlations with markers of glucose and lipid metabolism supported previously reported findings, the results pointed to some inconsistencies. As zonulin is reported to be pre-haptoglobin 2 (preHP2), and about 15% percent of a typical western European population are homozygous for haptoglobin 1, they should not express zonulin at all. I confirmed in the Sorbs cohort previously reported distributions of haptoglobin genotypes and showed that the target of the only commercially available zonulin ELISA kit was not related to haptoglobin genotype, therefore presumably not measuring zonulin/ preHP2. Subsequently, I identified properdin as a possible target by employing mass spectrometry approaches. Properdin is structurally related to haptoglobin, as both proteins belong to the mannose-associated serine protease family, however further experiments are needed to validate a possible functional resemblance.
In regard to bacterial translocation two adipose tissue depots were of notable interest due to their close proximity to the gastrointestinal tract. Mesenteric adipose tissue is located around the small intestine and the adipose tissue of the appendices epiploicae, small chunks of fat also called epiploic adipose tissue, are directly attached to the colon. After a thorough literature research I could also assert that both adipose tissues were rarely analyzed in the context of obesity. Therefore they were extensively investigated by measuring gene expression of adipo(cyto)kines, circulating inflammatory markers and analyzing adipocyte size and adipose tissue macrophages. Furthermore, a ``multiomics'' characterization was conducted and by analyzing transcriptome and methylome data I could identify epiploic adipose tissue as a tissue of interest in regard to type 2 diabetes and insulin resistance, which was further confirmed by untargeted proteomics data. Contrary to initial assumptions, I observed only a slight increase in translocation of bacterial DNA and no increased inflammation, as measured by tissue specific TNF and IL6 expression as well as adipose tissue macrophage infiltration. However, both transcriptome and proteome profiles allowed a clear discrimination of patients with and without insulin resistance which was most distinct in epiploic adipose tissue. Compared to other fat depots, epiploic adipose tissue exhibited a discriminable metabolic profile whereas mesenteric adipose tissue was more similar to omental-visceral adipose tissue. Most strikingly, epiploic adipose tissue showed a strong increase in leptin expression and, in general, the upregulation of various metabolic pathways involved in sugar, amino acid or sphingolipid metabolism. In accord with the leaky gut hypothesis high expression of lipopolysaccharide binding protein and various pathways involved in chemokine signaling were observed. In summary, I did not observe an increase in bacterial DNA or adipose tissue macrophages, but demonstrated elevated inflammatory signals such as increased chemokine or IL-8 signaling which are linked with an overall increase of metabolic processes and an increased expression of various adipokines. Epiploic adipose tissue might have a watch dog function by being the first adipose tissue sensing and forwarding certain (microbial) stimuli from the large intestine to the host.
In the last part of my thesis I addressed a possible role of the HLA genomic region on the development of type 2 diabetes. The influence of HLA genetics on type 2 diabetes has been under debate for several decades, since HLA was recognized to largely contribute to type 1 diabetes heritability. However, studies remained inconclusive due to lacking cohorts with sample sizes providing sufficient statistical power for association analyses. More recently, animal studies suggested MHC class II proteins as crucial factors mediating adipose tissue inflammation and insulin resistance. The sample size of the leaky gut cohort was insufficient to determine any correlation between HLA class II genotypes and the presence or type of bacteria in adipose tissue due to the high variability in the observed genomic region. Yet, I had access to three large population-based cohorts which allowed me to analyze associations between HLA class II alleles and type 2 diabetes. Therefore HLA genotypes of the LIFE-Adult (N=4649), LIFE-Heart (N=4815) and Sorbs (N=949) cohort were imputed from SNP genotyping data and analyzed for association with type 2 diabetes. In a meta-analysis including all three cohorts, I identified a protective effect for the well-established type 1 diabetes protective haplotype DRB1*15:01~DQA1*01:02~DQB1*06:02 and confirmed DRB1*07:01~DQA1*02:01~DQB1*03:03 as a risk haplotype in non-insulin treated diabetes. These results suggest that the genetic foundation of both type 1 and 2 diabetes shares common elements involving the HLA class II locus.
In conclusion, to the best of my knowledge, I provide in my work the first contaminant-aware identification of bacterial DNA in human adipose tissue and highlight the importance of analyzing novel adipose tissue depots by showing that fat of the appendices epiploicae, previously only considered to have a cushioning function, is metabolically active and possibly involved in the development of insulin resistance.
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Utveckling och validering av en qPCR metod för detektion av DNA från tarmbakterier i blod/plasmaJohansson, Kajsa January 2020 (has links)
Enligt "Leaky gut”-hypotesen är ökad translokation av gramnegativa bakterier genom tarmslemhinnan förknippad med neuroimmuna störningar. Denna ökning av permeabiliteten i tarmslemhinnan kan orsakas av störning i tarmfloran efter antibiotikabruk eller sjukdom, vilket kan leda till inflammatoriska processer. Inflammation har sedan tidigare blivit förknippad med allvarlig depressiv störning och självmordsbeteende. Studiens syfte var att utveckla och validera en qPCR-baserad metod för att kunna detektera DNA från tarmbakterier i blod/plasma, som ett tecken på translokering av bakterier. Två primerpar för amplifiering av 16S rDNA utreddes genom observation av PCR-reaktioner med humant och bakteriellt DNA. Det mest optimala primerparets PCR effektiviteten och linjäriteten testades. Metodens funktion kontrollerades sedan med helblod och plasma med tillsats av exogent DNA från E.coli. Den utvecklade qPCR metoden detekterar bakterie DNA i prov med 10 kopior/µl, vilket gör den tillräckligt känslig för detektion av tarmbakterier i blod. / According to the "Leaky gut" hypothesis, increased translocation of gram-negative bacteria through the intestinal mucosa is associated with neuroimmune disorders. The increase of permeability of the intestinal mucosa may be caused by disturbance of the intestinal flora after antibiotic use or disease, which can lead to inflammatory processes. Inflammation has previously been associated with major depressive disorder and suicidal behavior. The purpose of the study was to develop and validate a qPCR-based method for detecting DNA from intestinal bacteria in blod/plasma, as a sign of decreased mucosal integrity. Two different primer pairs, targeting 16S rDNA, were investigated by observing their PCR reactivity with human and bacterial DNA. PCR efficiency and linearity were tested on the most optimal primer pair. The function of the method was then verified with whole blood and plasma with the addition of exogenous DNA from E.coli. The developed qPCR method detects bacterial DNA in samples at 10 copies/µl, making it sufficiently sensitive for detection of intestinal bacterial DNA in blood.
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The role of gut microbiota in systemic lupus erythematosusMu, Qinghui 19 April 2018 (has links)
Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. Despite years of study, the etiology of SLE is still unclear. Both genetic and environmental factors have been implicated in the disease mechanisms. Gut microbiota as an environmental factor and the immune system interact to maintain tissue homeostasis, but whether this interaction is involved in the pathogenesis of SLE is unclear.
In a classical model of lupus nephritis, MRL/lpr, we found decrease of Lactobacillales but increase of Lachnospiraceae in the gut microbiota. Increasing Lactobacillales in the gut by suppling a mixture of 5 Lactobacillus strains improved renal function of these mice and prolonged their survival. Further studies revealed that MRL/lpr mice possessed a "leaky" gut, which was reversed by increased Lactobacillus colonization. Inside the kidney, oral Lactobacillus treatment also skewed the Treg-Th17 balance towards a Treg phenotype.
To remove Lachnospiraceae that was higher in lupus-prone mice than controls, we administered vancomycin orally to MRL/lpr mice after disease onset from 9 to 15 weeks of age. Vancomycin functions by removing Gram-positive bacteria such as Lachnospiraceae but sparing Lactobacillus spp. The treatment during active lupus reshaped the gut microbiota and significantly ameliorated systemic autoimmunity and kidney histopathology at 15 weeks of age. However, when vancomycin treatment was initiated from a very early age, the beneficial effect was not observed. Strikingly, mice given vancomycin only at the young age exhibited an even worse disease outcome. Indeed, regulatory B (Breg) cells were found to be reduced after the vancomycin treatment at young age. Importantly, adoptive transfer of Breg cells at 6-7 weeks of age rescued the beneficial effect, which indicates that Breg cells, inducible by vancomycin-sensitive gut microbiota, plays an important role in suppressing lupus disease initiation and progression. Finally, we demonstrated that bacterial DNA from the gut microbiota might be the inducer of Breg cells, as bacterial DNA administration at young age reproduced the beneficial effect seen in the Breg adoptive transfer experiment. Future studies are required to examine the clinical efficacy of targeting gut microbiota as a novel treatment against SLE. / Ph. D.
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Gut Microbiome, Intestinal Permeability, and Tissue Bacteria in Metabolic Disease: Perpetrators or Bystanders?Chakaroun, Rima M., Massier, Lucas, Kovacs, Peter 20 April 2023 (has links)
The emerging evidence on the interconnectedness between the gut microbiome and host metabolism has led to a paradigm shift in the study of metabolic diseases such as obesity and type 2 diabetes with implications on both underlying pathophysiology and potential treatment. Mounting preclinical and clinical evidence of gut microbiota shifts, increased intestinal permeability in metabolic disease, and the critical positioning of the intestinal barrier at the interface between environment and internal milieu have led to the rekindling of the “leaky gut” concept. Although increased circulation of surrogate markers and directly measurable intestinal permeability have been linked to increased systemic inflammation in metabolic disease, mechanistic models behind this phenomenon are underdeveloped. Given repeated observations of microorganisms in several tissues with congruent phylogenetic findings, we review current evidence on these unanticipated niches, focusing specifically on the interaction between gut permeability and intestinal as well as extra-intestinal bacteria and their joint contributions to systemic inflammation and metabolism. We further address limitations of current studies and suggest strategies drawing on standard techniques for permeability measurement, recent advancements in microbial culture independent techniques and computational methodologies to robustly develop these concepts, which may be of considerable value for the development of prevention and treatment strategies.
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