Nutrition Post Lecture Notes 2nd Midterm PDF

Nutrition post lecture notes 2nd midterm THE HYPOTHALAMUS - The master regulator of energy homeostasis - In the early 1900s the role of the hypothalamus in food-seeking behaviour and obesity was suggested due to patients with tumours in the hypothalamo-pitutiary structure - Sub...

Nutrition post lecture notes 2nd midterm THE HYPOTHALAMUS - The master regulator of energy homeostasis - In the early 1900s the role of the hypothalamus in food-seeking behaviour and obesity was suggested due to patients with tumours in the hypothalamo-pitutiary structure - Subsequent chemical and electrical lesion studies in animals confirmed this - The hypothalamus is a central hub that senses inputs and compares them to “set points” for body temperature, sleep-wake cycle, circadian rhythms and energy homeostasis - In response to inputs it will activate autonomic nervous system and endocrine signals to maintain homeostasis MONOGENIC OBESITY - Clinical cases provide important clues about the hypothalamic pathway that controls energy homeostasis ( balance) - The pathway is highly conserved in higher eukaryotes - A pathways with no redundancy ( no back up) Mutations correspond to different “classes” of genes – cause obesity 1. Genes primarily involved in energy balanced ie. Leptin (LEP), leptin receptor , melanocortin-4 receptor (MC4R) , POMC etc. – the more important class of genes that are effected 2. Genes involved in the development of the hypothalamus ie. SIM1 , BNDS – we aren’t going to talk about this in much detail LETS GO OVER THE LEPTIN/MELANOCORTIN PATHWAY Orexigenic means appetite stimulating Anorexigenic means appetite suppressing Leptin is a peptide hormone and is secreted by adipose tissue (endocrine tissue) which is in proportional to overall body fat mass A well-fed state ( high levels of nutrient in the blood) and will lead increased fat which will increase leptin Leptin will then enter the CNS and will bind to the LepR in first order memory and will get into the arcuate nucleus from here the LepR will inhibit POMC which is an appetite suppressor and will cause the release of alpha MSH and beta MSH , these will activate MC4-R which is 2nd order memory and will be in the paraventricular nucleus. MC4-R will then do something to affect SIM1- BDNF/TKRB which will help with the energy balance Going back to the 1st order memory , LepR will also inhibit NPY Insulin will be secreted from the pancreas and will go to the ISR receptor and will also activate POMC and inhibit NPY and follow the same pathway as leptin Ghrelin will be secreted by the stomach and is the hunger hormone – this will in 1st order memory will go the GHR and activate AGRP which causes food and energy seeking. From here is will inhibit MC4-R – SIM1 which will overall affect the energy balance AUTOSOMAL DOMINANT VS RECESSIVE DOMINANT- ONLY NEED TO INHERIT MUTATION FROM ONE PARENT- AND WILL HAVE IT RECESSIVE- NEED TO INHERIT MUTATION FROM BOTH PARENTS AND CAN BE A CARRIER LETS DEEP DIVE INTO THIS SYSTEM LEPTIN - The ob./ob mouse produces no leptin- this will be our genetic model of obesity, will be obese no matter the diet - Leptin is primarily synthesized in subcutaneous adipose tissue - Leptin is regulated at level of gene expression and protein secretion- normally - Signals associated with a positive energy balance ( expansion of adipose tissue) will increase leptin expression – ie, insulin , glucocorticoids, glucose - Signals associated with starvation will decrease the leptin expression- ie. Catecholamines (epi), TNF alpha - In common obesity, the blood leptin levels are strongly and positively correlated with body fat - Obesity is associated with a leptin resistance-leptin is no longer working properly LEPTIN IS MORE THAN JUST AN APPETITE REGULATOR Haemopoiesis- platelet aggregation - High levels of leptin promote platelet aggregation  - Possible link with proliferation and differentiation of haemopoietic precursors ( can provide a link between adipose tissue and risk of CVD?) Reproduction – - Fertility ( hypothalamic, pituitary, gonadal, adipose tissue axis) - Leptin regulates hypothalamic neuronal activity, which activates pathways associated with reproduction and growth - Leptin-deficient mice injected with leptin had improved reproductive success Fetal development - Is leptin a potential signal in maternal-fetal exchanges - The later stages of pregnancy are characterized by a central leptin resistance in the mother, resulting in an increased food intake, and therefore increased availability for the growing fetus LEPTIN LOSS-OF-FUNCTION MUTATIONS IN HUMANS Clinical characteristics - Little to no serum leptin - Severe early onset obesity - Hyperphagic ( increased drive to eat) - Delayed puberty - Thyroid dysfunction - 50% body fat - Congenital leptin deficiency – no leptin LEPTIN (LEP) MUTATIONS IN HUMANS - Congenital leptin deficiency ( ie.no leptin)- this is an autosomal recessive disorder - SNPs in leptin are found to be associated with common obesity but loss-of functions mutations of leptin are VERY RARE Examples of loss of functions mutations are identified as 1. Frameshift mutation- produces a truncated, unsecreted form of leptin (ie. Stuck in the adipose tissue) 2. Missense mutation- a change in the conformation of the leptin protein that prevents its interaction with the leptin receptor LEPTIN THERAPY IN HUMANS - 4 years of daily leptin injections- diagram on slides - The BMI of this study dropped to 24.2 from 43.4 - Body fat drops - Blood triglyceride levels normalized - Increased HDL-C - Normalized insulin  - Improved thyroid function - Improved immunity - We can determine if a patient is leptin deficient due to measuring blood leptin levels - Even if the leptin levels are as expected in a individual with severe obesity , leptin sequencing may still be performed because new mutations can be found that affect the interaction between the leptin and the leptin receptor LEPTIN IN THE HYPOTHALAMIC PATHWAY POMC/CART and NPY/AgRP neurons are considered “first order “ neurons - Neurons that produce peptide products of POMC and cocaine and amphetamine- regulated transcript CART inhibit food intake and increase energy expenditure - Leptin activates POMC/CART neurons - Neurons that produce NPY and AgRP stimulate food intake and decrease energy expenditure - Leptin inhibits NPY/AgRP neurons - Insulin works with leptin LEPTIN RECEPTOR (LEPR) MUTATION IN HUMANS - First reported in 3 sisters from Algerian family- now more subjects identified - Autosomal recessive mutation - Main characteristics are severe early onset obesity in adolescence with no developmental delays and normal leptin levels- normal being proportional to the level of body fat - Complete or partial loss of receptors signalling, despite the fact the leptin levels in the blood are proportional with adiposity - There are 6 isoforms of LEPR in humans - The various isoforms are expressed in different tissues around the body - The long form (full sequence) LEPRb is expressed in hypothalamic neurons - Only LEPRb has the necessary intracellular domains necessary to activate the JAK/STAT signalling pathway in hypothalamic neurons which is needed for appetite regulation - Db/db mouse has a LEPR mutation that eliminates this intracellular domain BUILDING THE HYPOTHALAMIC PATHWAY\ - Leptin binds to the LEPRb dimer- POMC pathway - Dimer activates the Jak2 kinase which allows STAT3 to be activated and intracellular signalling pathway activated - This will also inhibit NPY/AgRP POMC MUTATIONS - Characteristics include severe early onset obesity, hyperphagia (excessive eating) , normal birth weight but rapid weight gain, characterized by red hair, patients have  adrenal insufficiency because they cannot produce ACTH- this product increases cortisol production, therefore the patients display hypercortisolism - This is an autosomal recessive mutation - More of a spike in weight gain in girl pateitns Lecture 2 – human gut biome - The total microbiome has ~150x more genes thar humans- huge genetic potential - Abundance and composition of the microbiome is influenced by diet, medication and the hosts overall metabolic health - The microbiome secretes and alters the production of molecules that affects the hosts health- think of it as an endocrine organ - A healthy microbiome has certain core functions that include the breakdown of dietary fibres, production of short chain fatty acids, and production of vitamins and other co factors MICROBIAL DISTRUBUTION IN THE GUT - Between 500-1000 species live in the human gut- also mouse- several major phyla, with firmicutes, Bacteroidetes, proteobacteria, and actinobacteria are the most prevalent - Aerobic specifc reside in the upper gut, while anaerobic specifc are found in the lower gut ( where there is lower oxygen levels) - Species variability in the human gut is greater than functional variability - Dysbiosis refers to an imbalance in the microbial community – can affect numerous conditions such as psychiatric disorders, metabolic disease and allergies - The gut bacterial community is complex with specific species residing in specific regions of the gut. This contributes to the regional variations in function along said gut STUDYING THE GUT MICROBIOME - Traditional culturing-growing inside a petri dish, but will have challeneges in the o2 rich environment and growing individ apart from community - Classification using SSU rRNA- is present in nearly all specific , and can be obtained from a sample directly from natural environment bypassing the need for traditional culturing - Classification and identification of microbes, functional understanding of microbes and predicting function based on gene content CLASSIFYING AND IDENTIFYING BACTERIA - Can do this by using SSU rRNA- SSU is around 1500 bp in length - Small sequence length is ideal for analysis between different microbes  - 9 variable regions in SSU - Close to 5 million SSU rRNA sequences available in public databases – small compared to bacteria in gut - This info is used to classify bacteria and study bacterial divergence PHYLOGENETIC TREE OF LIFE - Detected in human feces, the larger the leaves of the tree, the greater the diversity and abundance of the groups. CAVEATS WITH THE SSU rRNA APPROACH 1. Most analyses done using fecal samples- this mean the microbes that residues in other regions of the gut such as the SI can be missed 2. Comparisions are always made with “healthy” gut microbial community, but how to define “healthy”?- later on 3. Identification vs function of microbes- SSU rRNA sequencing can tell you that a microbe is present but no info about what it does The healthy gut - The stability of a community reflect the heath of that community where stability can be considered as 1. The ability to resist change in the face of stress 2. The ability to return to equilibrium following a stress induced perturbation - Another way to look at it is that a healthy community has an optimal collection of microbial genes and functions rather than specific microbes - It doesn’t matter what species reside in the gut as long as they provide specific functions - Redundancy in genetic and functional potential exists between microbes - Microbial diversity reflects a healthy population- reduced could become diseases such as obesity and inflammatory bowel disease MICROBIAL DIVERSITY-FUCNTIOANL CONSISTENCY - When studying the composition and functional potential of gut bacteria from fecal samples in 18 people it was shown that the microbial population can be very different between people - But the functional potential of the gut microbiome is very consistent between people IDENTRICIATION VS FUCNTION OF MICROBES - Metagenomics is the application of modern genomics technique to the study of communities of microbial organisms directly in their natural environments, by passing the need for isolation and lab cultivation - There is 2 branches of metagenomics which is determing what the genes are and do - First they will extract all the DNA from microbial community in sampled environment  - Then to determine what genes are – sequenced based metagenomics- identify genes and metabolic pathways, and compare to other communities - Or determine what genes do – function-based metagenomics- screen to identify functions of interest , such as vitamin or antibiotic production and find genes that code of functions of interest SEQUENCE -BASED METAGENOMICS - Provides a snapshot of genetic diversity - Sequence DNA to create a catalogue of genes present in the ecosystem - Investigate the genetic potential of microbes to predict functions - Examine how diversity and genetic potential changes over time in response to stressors FUNCTION-BASED METAGENOMICS - Discover new functions then work backwards to figure out how the genes underlying these functions - DNA is isolated from the microbes in sample (feces) and this DNA is then put into plasmids, and then the plasmids are transfected into E.coli – limitations are because E.coli is only predicted to express 40% of functional potential - Upside- allows new biological activities to be discovered- proof of bio activity - Downside- very labourious and time consuming THE MATERAL MICROBIOME DURING PREGNACY - Vaginal and intestinal microbiome will change in pregnancy – which these are the sites that do microbial transmission from mother to baby in a normal birth which is important - Ie. Late pregnancy is associated with an increase in materal gut bacteria and is very efficient at extracting energy from food – this helps with energy harvest during pregnancy to support growth of fetus and needs for the mother - Therefore changes in mom gut microbiomr are an adaptive response to promote fetal health - How or why remains unclear and possibility linked to changes in immune function in mother - Ie. Increased level of vaginal levels of lactobacilli during pregnancy help maintain a low pH which limits bacterial diversity and reduces the chances of the bacteria ascending to the uterus and causing an infection ESTABLISHING THE HUMA GUT MICROBIOME - The fetal intestine is sterile with colonization beginning at delivery and building throughout the first few years of life - Initial abundance of o2 in the newborn gut influences the first colonizers ( gut environment favours aerobic bacteria - As these initial colonizers grow and expand , the o2 is consumed and now. The gut environment now favours anaerobic bacteria  - Within a few years the anaerobic bacteria vastly outnumber aerobic bacteria - Factors known to have a significant impact on the development of an infant’s gut bacteria include mode of delivery, antibiotic use and diet MODE OF DELIVERY Vaginal vs c section - Differences in microbial specifc between c section and vaginally delivered babies have been seen up to for 7 years after birth - Mothers vaginal microbial community is highly similar to their own baby but different to vaginally and c-sectioned babies from other mother- this shows uniqueness in mother vaginal bacterial community - Mothers skin microbial community were equally similar to that of other children born by c -section- shows a similarity of commonness between mothers skins bacterial community - Tests done in the first 24 hours after birth - Therefore vaginally delivered babies are colonized with bacteria from the vagina and distal gut of the mom, while c-section infant are initially colonized by skin bacteria from the mom which is similar between multiple moms ANTIBIOTICS - Can crush the community and impact diversity and colonization - Use of antibiotics before, during, and after birth alter the microbial colonization which will impact the development of the infant gut bacteria - Alters the microbial diversity and number - Antibiotics have a dramatic effect on bacterial number while use of the treatments antifungal treatments have no affect - Twins have similar gut bacteria mother and transmission are similar NEWBORN DIET Breast milk vs formula feeding - The feeding type influences gut bacteria by providing different substrates that affect the bacterial proliferation and functions as well bacterial species from milk and skin - Breast milk contains bacteria and will have HMOs which is sugars that help promote the growth of specific microbial communitas - The WHO recommends to breast feed for first 6 months and supplement breast feeding upto 2 years because it is really important for the microbial gut community - HMOs encourage the growth of bifidobacterium which helps to inhibit the growth of pathogenic organisms , maintain mucosal barrier function , and regulate inflammatory responses - Formula feeding + use of probiotics has been show to promote a microbial community in infants similar to being breast fed - This can influence the offsprings metabolic health - Breast milk has protective effect of developing obesity in the future CHANGES IN GUT BACTERIA OVER LIFE - Richness and stability always changes - Infancy is a period of rapid colonization that adapts in response to events such as illness and changes in diet, in adult hood gut bacteria is both diverse and stable - Will build over time due to life exposures - It will get stake in adult hood then slowly start to decrease in late senior where disease begins to set in. THE ADULT GUT MICROBIOTA - By increasing diversity it is making the indiv more healthy - Goal of study – to catalogue the content , diversity , and fucntion of gut bacteria in human adults suing sequence0-based metagenomics - In studies including those healthy, obese and IBD it shows there is a core gut microbiome meaning a certain amount is shared, meaning a lot of the gene pool is shared CORE GUT MICROBIOME - The core gut microbiome describes functions shared by the bacteria from ALL PEOPLE - Basic cell functions that regulate RNA/DNA polymerase activity, ATP synthase activity and general secretory apparatus is in bacteria - Energy harvesting ( sugars into SCFA)- eating, a source of energy for both the host and bacteria and influences different metabolic pathways in the host - Vitamin production (biotin, vitamin b12) - Degrades xenobiotics ( a toxic chemical) substances that are foreign to the body- benzoate is a common food supplement that , when degraded by bacteria is converted into pimeloyl CoA which is a precursor for biotin- need bacteria for functions HOST GENETICS SHAPE ADULT GUT MICROBIOME - In a short term of around 60 days there is a little impact in bacterial community when doing temporal changes - A persons gut bacteria will resemble itself more than another person - Similar genotypes (twins) will have more similar bacterial community than related and unrelated individuals - Obesity was associated with a decrease in diversity in gut bacteria, this can affect both type of bacteria present as well as the genetic and functional potential TAKE HOME MESSAGES - SSU rRNA is used to see if bacteria is present while metagenomics is used to study bacterial functions LET’S GO OVER GUT BACTERIA CONTINUED GERM FREE MICE - Deliver baby mice in a germ free environment by caesarean section- rodents eat poop so we need a sterile enviro - Animals are housed in a sterile chamber under positive pressure - All food is sterilized - Germ-free mice have no bacteria STUDY 1 Does the presence of gut bacteria influence mouse body weight Gut microbiota and fat storage - There are 3 groups of adult mice, GF CONV- conventional and CONVD which are raised as GF but then colonized with unfractionated cecal microbiota from CONV-R animals - All animals were housed in the same conditions and ate the same diet - Finding were confirmed using different bacterial strains nad the results were seen in both males and females - Body fat was smaller in GF than the others but CONVD and CONVR we not different from eachother just larger than the GF - Although GF ate more food than the other groups, which is due to the CONVD and CONVR not needing to eat as much food for the same energy , although these groups consumed a lot more O2 and were moving around more - Presence of gut bacteria associated with a higher body fat and larger fat pads, despite consuming less food and having higher oxygen consumption - Predominant microbial phyla- only in CONVD/R firmicutes and Bacteroidetes are the most predominant bacterial phyla and cecum Plasma markers of metabolism - Conventionalized animals had higher levels of key metabolic hormones and glucose; leptin was proportional to body fat levels as expected - Presence of gut bacteria is associated with higher fasting leptin, insulin and glucose levels in the blood Hepatic production of TAGs - Conv-d mice had higher amount of liver triglycerides TAGS than GF mice - Higher expression of key hepatic genes involved in de novo lipogenesis - Presence of gut bacteria associated with more liver TAGS and greater expression of hepatic genes involved in de novolipgenesis Adipocyte lipid storage - Genes involved in adipogenesis not different between the GF and the CONV mice - ACC1 , FAS, and aP2 did not differ - Body weight differences is not due to differences in the adipogenesis - Adipose tissue LPL activity was higher in CONV mice – why? Because LPL hydrolyzes blood TAGS, which allows for fatty acids to be taken up for the adipocytes  - Intestinal FIAF fasting induced adipose factor gene expression was lower in CONV mice vs GF- FIAF inhibits LPL activity - Gut bacteria supress intestinal FIAF production but FIAF inhibits LPL, so by lowering these levels we are increasing LPL activity and that means we are increasing body weight Study conclusions-gut microbes - Increased body fat and epididymal weight - Led to reduction in food intake ( most probably due to a more efficient extraction of dietary energy - Increased oxygen consumption ( measured by indirect calorimetry VO2) - Increased blood leptin, insulin, and glucose levels - Increased hepatic production of TAG - Increased fat storage in adipocytes by increasing LPL activity due to suppression of intestinal FIAF - FIAF is the molecular link between the intestine and adipose tissue that is regulated by gut bacteria STUDY 2 Is obesity associated with changes in the gut microbiota in mice? Gut bacteria in mice - Studied ob/ob , ob/+ , and +/+ WT mice - Ob/ob consumed more food which resulted in a greater body weight and epididymal fat pad weights, but no difference in the ob/+ or +/+ lean mice - DNA extracted from the cecum and SSU rRNA genes were amplified by PCR - The 2 most abundant bacterial phyla were- Bacteroidetes (20-40%) and firmicutes (60-80%) and 75% correspond to clostridium which produces SCFA - All mice were fed the same diet - Obese mice compared to lean mice showed a reduction in Bacteroidetes and increases in firmicutes - Obese mice have a significant difference in the relative proportions of bacterial phyla compared to the lean mice Metagenomic analysis of ob/ob mice - Data was taken by cecal content - The obese microbiome is enriched in genes that encode for enzymes that involve the breakdown of non-digestible dietary carbs - Firmicutes are particulary rich in glycoside hydrolases that digest dietary starch - Firmicutes are also enriched with proteins that import products of glycoside hydrolases (ABC transporters) , metabolize them and generate the end products of fermentations (SCFA) - Bomb calorimetry revealed that ob/ob mice have significantly less energy remaining in their feces to lean mice - The microbiome of obese mice are more efficient at extracting energy from food STUDY 3 Is obesity associated with changes in the gut microbiota in humans ? - Human obesity has similar changes in bacterial phyla. Diet restriction alters the gut microbiota. Changes in bacteria are more related to body weight then diet composition STUDY 4 Can gut microbiota be remodelled by fecal transplants ? - Colonizing germ-free mice with bacteria from an ob/ob obese mouse led to a higher body weight compared to the bacteria from a lean +/+ donor mouse Microbiota transplantation in humans - Obese patients were either given a placebo or a fecal microbiota transplant from a lean healthy individual - No change in BMI after 12 weeks and no change in GLP-1 (incretin/satiety marker) - Microbiome of obese participants shifted towards that of the lean donor including a greater diversity which is determined by the 16S rRNA analysis - Bile acids were reduced in obese partipants who received the transplant and resembled the bile acid profile as the lean donor STUDY 5 Do gut microbiota influence diet-induced obesity? DIO - A western diet of (20% pro, 45% fat and 35% CHO) induces obesity compared to the standard diet ( 15% protein, 9% fat , and 76% CHO)- and have 25% difference in body weight after 9 weeks Role of microbiota in DIO - GF mice are resistant to diet induced obesity even when consuming the western style diet - Therefore considerable interest in understanding the mechanisms underlying the resistance Gf mice are resistant to DIO - After 8 weeks on the western diet CONVD mice gained more weight than the GF - However food consumption and energy content of feces were similar - Examined if fat absorption and clearance were different between the 2 by a bolus of lipid - Absorption is similar but GF has higher levels of blood TAGS - Gut bacteria caused an increase in body weight in animals consuming a western diet, which is not due to any difference of fat absorption - They bith will absorb and clear from circulation therefore they are doing the same amount here, although GF has low levels of LPL and doesent get to the same low TAG blood level as CONVD which is low after 6 hours and is good at deriving lipid from the blood Fat oxidation in the liver and muscle  - Fat oxidation : beta oxidation of FAs occurs in the mitochondria to generate ATP - Fat is a wonderful source of ATP - AMPX is a key enzyme in cellular energy homeostasis - No change in total AMPK between the different groups - But levels of phosphorylated AMPK and ACC are reduced in the CONV which are the active forms of these protein - CPT activity is reduced in the CONV, and this is a rate of limiting step for FFA into mitochondria, meaning less fat is going to the mitochondria and there will be therefore less oxidation - Indicates that the fat oxidation is reduced in animals with gut bacteria by reducing AMPK signaling Putting this all together diagram in lecture notes go back - CONVD will have acetyl coa get created into malonyl CoA by ACC and this will inhibit the CPT which will cause the carrier not to be able to bring as many FA into the mitochondria - GF will have the phosphorylated AMPPK stop this from happening so no malonyl COA is made therefore it will decrease levels as FA go into mitochondria Take away messages - Gut bacteria can cause an increase in body weight independent on food changes - Obese bacteria are more efficient at extracting energy from food - FIAF is an LPL inhibitor , and intestinal FIA is reduced in presence of gut bacteria, thereby leading to the increase TAG uptake in adipose tissue due to more active LPL enzyme - AMPK activation promotes fat oxidation. The presence of gut bacteria is associated with reduced AMPK activity which lowers fat oxidation in the liver and muscle STUDY 6 Do gut microbiota influence energy harvesting from food? SCFAs - The human genome is poor in glycoside hydrolases ( the enzymes needed for the digestion of complex dietary plant polysaccharides)- need gut bacteria to break down this fibre to make them into FAs - microbial genome is rich in these these enzymes- we know this based on sequence based metagenomics - host absorption of SCFAs occurs in both passive and specifc transporter mechanisms – fuel for both host and bacteria - SCFA are signalling molecules and signal through G protein coupled receptors to regulate a number of processes, including their own absorption Gut bacteria and GPR41 - Gut bacteria in GPR41-/- mice do not affect body weight compared to germ free conditions. Mice expressing GPR41 +/+ have greater body weight with gut bacteria  - GPT41 is a receptor in the intestine that transports uptake of SCFA therefore with having the receptor and gut bacteria more of the SCFA are being absorbed from the intestine - GPR41 does not affect energy absorption - Energy content in feces is higher in GPR -/- mice- this shows that GPR41 affects energy absorption - Greater amounts of SCFAs in cecum in animals lacking gpr41- further shows that thid impscts energy absorption - Higher SCFAs in feces- increased fecal energy mice not expressing gpr41 due to higher levels of SCFA - More energy in poop because if there was a transporter the SCFA would be absorbed but instead getting excreted - Therefore no RCX a lot of un absorbed SCFA Microbiota and energy harvesting - Scfa activate GPR41 signalling pathways which can influence energy balance in the host - A defiencieny in this receptor even if the bacteria is present will prevent weight gain compaired to the WT - Serum levels of leptin are lower in GPR41-/- colonized mice verses +/+ - This makes sense as -/- weight less - Authors proposed that microbiota affect host weight gain in part of slowing down food transit times through IT, which will allow for greater production and absorption of SCFAs as well as greater activation of GPR 41 signaling pathways Study 7 Can dietary habits shape human gut bacteria Example 1 - Comparing gut microbiota of children in Africa vs Europe where diets are really different - In Africa Burkina Faso diet is low in fat and animal proteins but rich in starch fibre and plants - In Italy is high in animal protein sugar and starch and fat but is low in fibre - Children were aged matched - Used 16s rRNA gene sequencing – tells us which bacteria is present- more than 94% in both children belonged to the 4 most common bacterial groups - BF children are rich in bacteriodetes while EU is rich in firmicutes, diet is very important for shaping gut bacterial community Energy extraction - SCFA in fecal samples of BF are higher than that of EU children- suggests that energy extraction of BF children is less efficient - BF not breaking down SCFA cause its in poop Example 2  - Goal is to see if dietary interventions change gut microbiome - They fed 10 partipants for 5 days for plant based or animal based - Animal based diet rapidly changed the bacterial community while plant did not - There was a decrease in fibre for animal but increase for plant - A lot of fat in animal, less in plant - Plant diversity is more stable Study 8 Does the gut bacteria influence whole body inflammation - Diet induced or genetic obesity is characterized by low level chronic inflammation, increased expression and secretion of cytokines and chemokines - These cytokines promote crosstalk between tissues leading to insulin resistance - The challenge is to separate the effects of diet, weight gain, and inflammation on insulin resistance - Antibiotic treatment was found partially protect against diabetes in rats Lipopolysaccharide LPS - LPS is a lipid/sugar molecule that is recognized by the immune system - It is continuously produced by lysis of gram – negative bacteria ie. Bacteroidetes - LPS is transported out of the intestine and throughout the body bound to LPS- binding proteins - This will then interact with the receptor CD14 on immune cells increasing the production of pro-inflammatory cytokines High fat diet HFD and inflammation - Mice were fed a high fat diet for 4 weeks compared to the other mice fed a control diet - The HFD increased LPS levels in the blood- which is refered to as metabolic endotoxemia - When an amount of LPS (equiv to the increase seen with HFD) was infused directly into the blood of control-fed animals to achieve metabolic endotoxemia this would cause the mice to increase body weight and fat pads, increase blood glucose, and led to whole body insulin resistance - Inflammation in the absence of higher energy intake can promote weight gain and lead to a higher insulin resistance Inflammatory markers - Low levels of LPS infused directly into animals increase the production of inflammatory markers and is similar to what is seen in a HFD - Therefore when increasing fat you are increasing pro inflammation What happens if LPS has no receptor - CD14 is the receptor - LPS infusion to achieve metabolic endotoxemia - In the WT mice there was an increase in expression of the inflammatory markers, increase in body weight and increase in fat pad weight , increase iun blood glucose levels and accumulation of fat in the liver  - In CD14 -/- mice there was no change in inflammatory markers, no change in body weight m ni change in blood glucose or liver fat - When LPS signalling is prevented there is no change in metabolic health, this work also demonstrates that high fat diet increases in LPS contributes to development of metabolic perturbation Gut bacteria and host metabolism - When increase LPS (inflammation) , increase SCFA (GPR41), and decrease FIAF ( inhibitor of LPL , so we will be increasing LPL- more fat storage) with obesity it will go to… - Liver- increase triglyceride production and storage, decrease fat oxidation and increase inflammation - Sk mus- decrease fat oxidation – AMPK-dependent mechanism and PGC1alpha dependent mechanism and increase inflammation - Adipose tissue- increase LPL activity, increase TAG storage, increase inflammation, and increase leptin but will lead to leptin resistance - Blood- increase glucose and insulin and increase GLP-1 incretin Take home messages - SSU (16s) sequencing and metagenomics provide a powerful approach to the study the gut microbiome - Gut bacteria increase fat mass, blood glucose, insulin and leptin levels - The levels of 2 dominant bacterial phyla are changed with obesity: firicutes and Bacteroidetes - Intestinal FIAF is an important factor that affects adipose tissue TAG uptake in host - AMPK is an important regulator of liver and muscle fat oxidation - GPR41 plays a crucial role in SCFA absorption and subsequent energy absorption by the host - CD14 and LPS represent a link between the gut bacteria , inflammation and metabolic health- also shows a high-fat diet causes increase inflammation INCRETINS How were they found? Intestinal extracts scraped from a pig were given to patients 3x per day – improvements were seen in glucose homestais Incretins are peptide hormones secreted by the gut The incretin effect- oral glucose load that produces a greater insulin response than that of an isoglycemic intravenous glucose infusion- more insulin is secreted from the pancreas when a person consumes glucose compared to when an equivalent amount of glucose is injected directly into the blood The incretin affect explains up to 70% of insulin response to an oral glucose challenge Demonstrating the incretin effect - Comparing oral glucose vs iv infusion - Measured blood glucose and insulin every 2 hours and mice has fat diet or HFD - Oral glucose causes an insulin spike that helps reduce blood glucose levels more effectively than when glucose is infused directly into the blood. - Barely any spike in insulin therefore glucose wont be cleared effectively from blood when inserted therefore incretin help with insulin Incretins GIP and GLP1 - GIP – pro-GIP processed in intestinal cells by prohormone convertase 2 PC2 to form an active GI- active formed stored in vesicles blood levels basal levels = 10 pmol/L and peak is 150-300 pmol/L - GLP-1 – Pro-GLP -1 is activated in intestinal cell by PC1 to form GLP1 – active form sotred in vesicles blood levels basal-5-10 and 25-40 pmol/L Regulation of incretin secretion - Incretin secretion is stimulated by food - Enteroendocrine cells in the intestine are polarized cells that sense the contents in the intestinal lumen - Carbs are potent stimulators of incretins in 2 ways 1. SGLT-1 2. Sweet taste receptors - Both mechanisms lead to the depolarization in the cells, which causes an increase in calcium levels which causes incretin containing vesicles to release their contents into circulation - SGLT1 is necessary for glucose dependent incretin secretion - Glucose bolus causes a larger increase in GIP levels in those with the SGLT1 receptor - The lipid bolus however will stay the same with or without the receptor - Those with the receptor will have stronger increases in blood insulin levels - Therefore a glucose bolus will cause an increase in only in WT has SGLT 1 receptor of GLP-1 - Therefore for both GIP and GLP-1 we need SGLT1 Incretin response to meal - Following consumption of a meal, incretin response is rapid and persists for several hours - Incretins are inactivated by the DPP-4 enzyme Incretin response to different nutritents - Intact is only active form and total is both active and inactive and represents secretion - Consumption of different macronutrients increases total levels ( and active levels ) of GIP and GLP-1 in blood water has no affect on this Incretin secretion and obesity - Obesity doesn’t affect GIP secretion but does reduce active GIP levels  - Obesity reduces both GLP-1 secretion and active levels - Obesity is associated with a general reduction in active incretin levels of circulation Roles of incretins in the pancreas - Incretins bind to a specific receptor in the pancrease - Activation of the incretin receptors lead to the formation of cAMP, which triggers an increase in intracellular calcium levels – depolarization - Depolarization will then cause the exocytosis of insulin granules releasing insulin into the blood - Glucose can also increase intracellular levels of calcium but it’s the combination that gets the massive release of insulin - Incretins also promote insulin transcription and beta cell proliferation and reduce beta cell apoptosis - Incretins decrease glucagon secretion from pancreatic alpha cells Wide ranging effects of incretins - Incretins have a number of effects through the body Incretins as therapeutic targets – trigger such as food - Obesity and T2D are associated with the reduction of incretin - Can use pharma to help target incretins 1. Incretin mimetics- similar structure as incretin but modified to resist DPP-4 inactivation therefore in circulation for longer , essentially giving an incretin a longer half life 2. Incretin enhancers- inhibits the enzyme that inactivates- block DPP-4 enzyme directly, allowing the natural incretin levels to be preserved for a longer time Incretin mimetic – exenatide Will cause a decrease in body weifht , HBAIC will stay the same, ALT change decreases ( when high very unhealthy) therefore all show improvements - Significant reductions in blood TAGS, total cholesteryl, LDL chole bad , and blood pressure therefore better at lipid handling - Significicant increase in HDL cholesterol good - An incretin mimetic improves body weight, and metabolic health, whule reducing CV risk factors in T2D DPP-4 inhibitor (incretin enchancer) - By inhibiting DPP-4 it prevents the inactivation of incretins and leads to a longer half life and will remain active longer - This will increase GLP-1/ GIP actions - MET metformin is a anti diabetic drug which suppreses the hepatic glucose production- controls liver production used for T2D - MET mainrtains HbA1c levels but the addition of DPP-4 inhibitor (LAF) will lower the overall Hba1C levels – short and longer term decrease in HbA1c when using both - Individuals taking the DPP-4 inhibitor in addition to metformin experience a significant improvement in whole body glucose homeostasis Toxicity seen with high incretin levels  - GLP-1 levels achieved with incretin mimetics can cause side effects if increased to much - GLP-1 levels achieved with enhancers only have good side effects - The use of incretin mimetics and enhancers warrants caution high levels can cause nausea , abdominal pain and diarrhoea Lifestyle medication on top of incretin mimetic - A lifestyle modification will reduce body weight but with this ontop can have a additive effect Gut bacteria and insulin - Remember that conventionalized mice had increased levels of fasted insulin and glucose compared to germ-free mice - Therefore higher circulaiyng glu and insulin SCFA regulate incretin production - Gut bacteria increase blood glucose and insulin levels - SCFAs signal through GPR41 and GPR 43- receptors - All mice in thuis study have gut bacteria but some lack the receptors - Animals with both of these receptors will have higher levels of active GLP-1 - Those lacking one of the RC will have lower levels of insulin and higher level of glucose in the blood - SCFAs produced by the gut bacteria have a role via these receptors in regulation of blood insulin levels by influencing the intestinal incretin release - Therefore if u remove 1 rc- affacts on incretins and affect insulin - \ Take home messages - Gut hormones (incretins) have a significant effect on insulin levels and explain upto 70% of insulin response to meal - Incretin secretion is induced by all macronutrients - GIP and GLP-1 released from enteroendocrine cells along the intestinal tract- SGLT1 plays role in regulating glucose mediated incretin release from the gut - Short half life of incretins due to DPP-4 activity - SCFAs produced by gut bacteria influence the production of incretins, producing a partial explanation to the increases in insulin in CONV mice THE ROLE OF LIVER IN THE DEVELOPMENT OF METABOLIC COMPLICATION LOW FATS DIETS INDUCE WEIGHT LOSS - Consuming a lower fat diet without a change in total energy intake for 2 months will show some weight loss therefore by decreasing fat decreasing weight  - Reducing fat intake associated with improvement in parameters of metabolic health Dietary fats - Fats in diet are found in different complexes - TAG – most dominant fat in diet contributing 90-95% of the total energy derived from dietary fat - Is made up of 1 glycerol and 3 FA - Also can be found as PLs, sterols and other lipids TAG DIGESTION - Starts at lingual lipase and will get broken down by targeting the FA on the backbone - Causing it to get into 1 FA and 1 DAG – 2 FA - Then with pancreatic lipase it will be broken down into 2 FA + 1 MAG BILE ACIDS - Effective detergents which promote the solubilisation and absorption of dietary lipids – breaks down fat for enzymes - Primary bild acids ( made in the liver than stored in the gall bladder) such as CA and CDCA - Bile acid conjugation- bile acids are conjugated with taurine or glycine in the liver to increase their hydrophilicity and will then be referred to as bile salts - When CA becomes a salt it will be TCA and CDCA will be GCDCA - Hormones will be released which goes to the gall bladder which will release bile salts - Bile salts are deconjugated by gut bacteria in the SI- necessary to recycle these acids - Bile salts can be further metabolized by gut bacteria in the SI and make secondary bile acids DCA and LCA - Enterohepatic circulation is a very efficient process of recycling bile acids process is greater than 95% efficient some taken up by the body- gut bacteria help cause we make primary they make secondary Microbiota and bile acid recycling - Gut bacteria have an important role regulating the blood lipid and cholesterol levels by influencing bild acid recycling – secondary bild acids produced by the bacteria are more hydrophobic increasing passive uptake in distal intestine - Gut bacteria are responsible for deconjugation – ie. Hydrolysis of the amine bond – BSHs - Why does bacteria deconjugate the bild acids?- deconjugation detoxifies the bile acids , or release atoms that can be used by the microbiota directly Primary bile acid production - ACIDIC pathway- cholesterol is converted into an oxysterol by CYP27A1 , which will travel into the liver and converted mostly into CDCA  - Neutral pathway (primary)- occurs in the liver via CYP7A1 and will produce both CA and CDCA , conjugation occurs in the liver - Brain pathways is not well understood but also will release precusors Endohepatic circulation-look at diagram in slides Low bile acid flux is due to fasting which makes bile acids resins High bile acid flux ( feeding)- bile acid administration First it starts at the gallbladder where when eating it will cause CCK to acitivate the gallbladder to release bile acid which will go into the gut lumen to help digest fats Then the acid can go to ASBT and get taken up by and enterocytes by this transporter which will be sent back to the hepatocyte through circulation Other bile acids will continue through the large I and dthen get to the bacteria get turned into 2nd then gets put into circulatory back into the hepatocyte Once back in the hepatocytes the salt will then go to the FXR rc which is a bile acid sensor and will activate SHP which will inhibit LXR (transc factor) which will surpress CYP7A1 because LXR controls CYP7A1 where the acid will be sent out to the gallbladder Bile acids regulate their own production through hepatic FXR alpha , high bile acid flux activates FXRallpha which then activates SHP ( an inhibitor of LXR alpha ) to supress CYP7A1 BILE ACIDS RESINS AND METABOLIC HEALTH - Bile acids can be used to control blood lipids - Bile acids resins bind bile acids in the digestive tract and prevent enterohepatic circulation therefore FXRalpha is not activate which will lead to the activation of CYP7A1 in the neutral pathway- this causes an increase of conversion of hepatic cholesterol into bile acids - Depletion of heaptic cholesterol levels results in an induced uptake of bl chole into the liver - This will overall result in an decrease of chole and LDL chole in blood Bile acids and glucose metabolism - Bile sequestrants were given to T2D – improve glycemic control and causes a reduction in blood glucose don’t know why - Therefore bild acids decrease blood glucose - Using an FXRalpha negative moose it shows that this regulates gluconeogenic genes and hepatic glucose production HGP TAG ABSORPTION - TAG comes in and will go through digestion wbere pancreatic lipase will ensure the digestion of TAG and break it apart before absorption, once absorbed going into the intestinal EC from lumen through FATP4 and FAT/CD36 fat transpotyers where through MGAT, DGAT2 it will be the fast process where FABP will put it right into the portal vein to the liver, but the glycerol-3-phosphate pathway is slow and will all  bring it to MTP which will create a chylomicron which will be iused in intestinal lymph - Pharma will target MTP to stop creation of chylomicron and this MTP expression is increase with T2D - Numerous points of regulation in the digestion and absorption of dietary tTAG ORLISTAT AND WEIGHT LOSS - An inhibitor that alters fat absorption by inhibiting both pancreatic and gastric lipases at digestive tract - High fats diet assocated with weight gain and diabetes - Orlistat reduces TAG update and increased the excretion in feces - Blocking the intestinal lipid digestion can lead to reductions in body weight and various risk factors Take home messages - Lipid absorption is regulated at multiple stages - Bile acids play a crucial role in fat uptake cycling between the liver and gut as enterohepatic circulation - FXR alpha which is a bile acids sensory serrves as link between the bile acid biosynthesis and glucose homeostasis and lipogenesis -