Week 2 Lecture 4 PDF

Week 2 Lecture 4 By the end of this lecture you should be able to: Understand how animals contribute to the carbon cycle. Explain the difference between Glucose and Fructose. Understand the consumption of sugars and link to metabolic diseases. Understand how pyruvate kinase is regulated t...

Week 2 Lecture 4 By the end of this lecture you should be able to: Understand how animals contribute to the carbon cycle. Explain the difference between Glucose and Fructose. Understand the consumption of sugars and link to metabolic diseases. Understand how pyruvate kinase is regulated through transcriptional regulation and phosphorylation. Role of ChREBP in regulating pyruvate kinase and fat synthesis. Understand how loss of ChREBP leads to metabolic dysfunction. The fixation of carbon dioxide into sugars via photosynthesis forms the basis of Earth’s food web. Heterotrophs cannot synthesize their own sugars; therefore, they evolved to use plant-derived sugars for energetic and synthetic purposes. Sucrose, the predominant circulating sugar in plants, is composed of equal parts of glucose and fructose. Evolutionary pressure favored mechanisms for early mammals to synthesize and store energy in the form of triglycerides when food was abundant as a safeguard against starvation in the face of uncertain food supplies. Some 2.5 million years later, grocery store shelves are reliably stocked with high-sugar and high-fat foods, especially in wealthy countries. It is no coincidence that >65 million Americans are now classified as overweight and obese. Glucose is the major circulating sugar in animals, and the amount of fructose is negligible in comparison. Glucose is a primary energetic and synthetic fuel for most tissues and cell types in the body. In contrast, the fructose component of the ingested sucrose is rapidly cleared by the intestines and liver and is catabolized for energetic purposes, converted to glucose and its polymeric storage form, glycogen, or to fatty acids and is stored as triglycerides. Despite its low levels in the circulation, fructose serves as a signal of the ingested dietary sugar. Sucrose is ingested as a disaccharide and requires cleavage to its monosaccharide constituents, glucose and fructose, prior to absorption.  Estimated intakes of total fructose ( ), free fructose (▴), and high-fructose corn syrup (HFCS, ♦) in relation to trends in the prevalence of overweight (▪) and obesity (x) in the United States. In 1970 HFCS represented < 1% of all caloric sweeteners available for consumption in the United States, but the HFCS portion of the caloric sweetener market jumped rapidly in the 1980s and by 2000 represented 42.0% of all caloric sweeteners Am J Clin Nutr, Volume 79, Issue 4, April 2004, Pages 537–543, https://doi.org/10.1093/ajcn/79.4.537 The content of this slide may be subject to copyright: please see the slide notes for details. In the United States, HFCS is found in almost all foods containing caloric sweeteners. These include most soft drinks and fruit drinks, candied fruits and canned fruits, dairy desserts and flavored yogurts, most baked goods, many cereals, and jellies. Over 60% of the calories in apple juice, which is used as the base for many of the fruit drinks, come from fructose, and thus apple juice is another source of fructose in the diet. High-fructose corn syrup has fructose content similar to that of table sugar (sucrose)—55 and 50 percent, respectively—so their glycemic indices are higher than that of agave. Since the “natural” food craze took hold, agave has been marketed as an alternative to the laboratory evils of HFCS. Pretty much without fail there’s a picture of a plant or Earth on the label, and you can buy yoga mats in an agave palette. https://www.theatlantic.com/health/archive/2014/ 06/sugar-wars/372220/ Table Sugar: sucrose (Glucose-Fructose) According to the 2017-2018 NHANES data, the average American adult eats: 2100 calories a day, 16% of those calories from protein, 47% from carbohydrate 36% from fat. 22% of all calories from added sugars And according to a 2016 study in the BMJ journals, Americans get 58% of all calories from ultra-processed foods. Animals have evolved complex mechanisms to sense sugar and motivate its consumption. Monosaccharides, including glucose and fructose, as well as disaccharides, such as sucrose, potently activate the G protein-coupled receptors Tas1R2 and Tas1R3, which are located on the epithelial cells of the tongue and palate (Nelson et al., 2001; Zhao et al., 2003). Indeed, the sweetness of both caloric and noncaloric sweeteners can induce or increase the consumption of neutral or aversive substances, such as alcohol, in strains of mice that otherwise avoid it (Yoneyama et al., 2008). Utilization of fructose and glucose in the liver. Hepatic fructose metabolism begins with phosphorylation by fructokinase (EC 2.7.1.4). Fructose carbon enters the glycolytic pathway at the triose phosphate level (dihydroxyacetone phosphate and glyceraldehyde-3- phosphate). Thus, fructose bypasses the major control point by which glucose carbon enters glycolysis (phosphofructokinase; EC 2.7.1.11), where glucose metabolism is limited by feedback inhibition by citrate and ATP. This allows fructose to serve as an unregulated source of both glycerol-3- phosphate and acetyl-CoA for hepatic lipogenesis. P, phosphate. Regulation at the transcriptional level by increasing mRNA abundance and subsequent increase in protein expression. Post-transcriptional regulation through phosphorylation The liver (L-PK or PKL) and red blood cell forms (R-PK or PKR) of pyruvate kinase are encoded by the same gene (PKLR) with differences resulting from differential transcriptional initiation. The PKLR gene is located on chromosome 1q22 spanning 9.5 kb and is composed of 13 exons that generate two mRNAs as a result of alternative promoter utilization. The R-PK promoter is active exclusively in erythrocytes due to a strong erythroid cell-specific enhancer element. The L-PK promoter is active in hepatocytes and pancreatic β-cells. Of the 13 exons in the PKLR gene, exons 3-12 encode identical portions of both the liver and erythrocyte mRNAs. The erythrocyte PK mRNA also includes exon 1, whereas exon 2 is included in the liver PK mRNA. The R-PK protein is 574 amino acids in length and the L-PK protein is 543 amino acids. Phosphorylation: A post-translational modification that regulates many aspects of biology. High Carbohydrate Diet Stimulates Pathways that are involved in Glycolysis and Fat Synthesis  Structure of carbohydrate response element binding protein α (ChREBPα). ChREBPα is composed of 864 amino acids and contains several regulatory domains. At the N-terminus the protein contains a glucose-sensing module composed of the low glucose inhibitory domain (LID) and the glucose activated conserved element (GRACE). The protein also contains a polyproline-rich, a bHLH/LZ and a leucine-zipper-like (Zip-like) domain located at the C-terminus. Post-translational modifications are indicated in their respective residues, phosphorylation (red), acetylation (blue) and the recently identified O-GlcNAcylations (green). (B) Gene structure of the ChREBP gene and generation of the two ChREBP isoforms α and β. ChREBPβ is transcribed from an alternative first exon promoter 1b. This transcript is translated from exon 4 generating a shorter protein of 687 amino acids in which the two NES, the NLS and the LID domain are missing. The ChREBPβ isoform has been suggested to be directly regulated by ChREBPα since a ChoRE sequence was identified in the exon promoter 1b. Whether both ChREBP α and β isoforms both bind to the ChoRE is currently not known. Figure adapted from Herman et al. (2012). Transcriptional activation is mediated by chromatin state and transcription factor binding. Densely condensed chromatin (closed) prevents transcription factors and other proteins needed to initiate transcription from binding, thereby inhibiting gene expression. Open chromatin on the other hand is accessible for proteins to bind to promoter and enhancer regions. Transcription factors can either directly guide RNA polymerase II to promoter regions of target genes or work in cooperation with other factors and mediators to assemble a transcription initiation complex. TF, transcription factor; Pol II, RNA polymerase II; CoF, cofactor; M, mediator. Leiz, Janna, Rutkiewicz, Maria, Birchmeier, Carmen, Heinemann, Udo and Schmidt-Ott, Kai M.. "Technologies for profiling the impact of genomic variants on transcription factor binding" Medizinische Genetik, vol. 33, no. 2, 2021, pp. 147-155. https://doi.org/10.1515/medgen-2021-2073 (C) Multi-alignment of ChoRE consensus sequences presents in several ChREBP target gene promoters. Nucleotides-based alignment is presented on the top of the figure together with the consensus sequence ChoRE described in Poungvarin et al. (2015). The logo corresponding to the consensus sequence associated to this particular alignment is also represented. Metabolic contribution to fatty liver Non-alcoholic fatty liver disease is a hallmark of metabolic syndrome, and studies in humans reveal that de novo lipogenesis contributes to about 25% of total liver lipids in patients with NAFLD (Donnelly et al., 2005). In insulin resistant states, hyperglycemia and hyperinsulinemia enhance lipogenesis partly through the activation of ChREBP and SREBP-1c. ChREBP inhibition in liver of obese and insulin resistant ob/ob mice, through RNAi or genetic ablation leads to reversal of hepatic steatosis (Dentin et al., 2006; Iizuka et al., 2006).  Impaired glucose tolerance in mice lacking ChREBP. Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis Katsumi Iizuka, Richard K. Bruick, Guosheng Liang, Jay D. Horton, and Kosaku Uyeda  Impaired glucose tolerance in mice lacking ChREBP. Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis Katsumi Iizuka, Richard K. Bruick, Guosheng Liang, Jay D. Horton, and Kosaku Uyeda  Metabolic Parameters in mice lacking ChREBP. Standard rodent chow High-starch diet Parameter measured WT ChREBP−/− WT ChREBP−/− Body weight, g 22 ± 0.6 22 ± 0.7 26 ± 0.7 26 ± 1.1 Liver weight, g 0.97 ± 0.11 1.01 ± 0.02 1.17 ± 0.04 1.63 ± 0.10* Epididymal fat weight, g 0.37 ± 0.02 0.27 ± 0.02* 0.28 ± 0.03 0.26 ± 0.04 Brown fat weight, g 0.14 ± 0.01 0.08 ± 0.02* ND ND Plasma glucose, mg/dl 157 ± 7 190 ± 6* 271 ± 13 290 ± 13 Plasma insulin, ng/ml 0.84 ± 0.07 1.07 ± 0.2 0.78 ± 0.08 1.34 ± 0.20* Plasma FFA, mM 0.76 ± 0.09 0.40 ± 0.03* 0.45 ± 0.03 0.24 ± 0.02* Plasma triglycerides, mg/dl 63.6 ± 13.5 65.8 ± 8.4 136 ± 18 119 ± 7 Plasma cholesterol, mg/dl ND ND 77 ± 7.6 39 ± 3.7* Liver glycogen, μmol/g 78.8 ± 18.8 214.5 ± 10.7* 91.1 ± 8.3 612.7 ± 46.1* Liver triglyceride, mg/g 7.13 ± 0.92 7.11 ± 0.83 6.11 ± 0.42 2.68 ± 0.28* Muscle glycogen, μmol/g 22.8 ± 2.4 20.6 ± 2.5 23.0 ± 1.9 23.3 ± 2.0 Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis Katsumi Iizuka, Richard K. Bruick, Guosheng Liang, Jay D. Horton, and Kosaku Uyeda  Metabolic Parameters in mice lacking ChREBP. Metabolites in ChREBP−/− and wild-type mouse livers Parameter measured, μmol/g liver Standard diet High-starch diet WT ChREBP−/− WT ChREBP−/− Glucose 4.6 ± 0.3 6.7 ± 0.2 6.0 ± 0.6 7.8 ± 0.5 Glu 6-P 0.13 ± 0.2 0.25 ± 0.03 0.13 ± 0.01 0.31 ± 0.03 PEP 0.11 ± 0.03 0.20 ± 0.08 0.11 ± 0.01 0.29 ± 0.02 Pyruvate 0.13 ± 0.027 0.043 ± 0.003* 0.115 ± 0.012 0.118 ± 0.009 Pyruvate/PEP 1.2 0.22 1.0 0.4 Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis Katsumi Iizuka, Richard K. Bruick, Guosheng Liang, Jay D. Horton, and Kosaku Uyeda  Metabolic Parameters in mice lacking ChREBP. Metabolites in ChREBP−/− and wild-type mouse livers Parameter measured, μmol/g liver Standard diet High-starch diet WT ChREBP−/− WT ChREBP−/− Glucose 4.6 ± 0.3 6.7 ± 0.2 6.0 ± 0.6 7.8 ± 0.5 Glu 6-P 0.13 ± 0.2 0.25 ± 0.03 0.13 ± 0.01 0.31 ± 0.03 PEP 0.11 ± 0.03 0.20 ± 0.08 0.11 ± 0.01 0.29 ± 0.02 Pyruvate 0.13 ± 0.027 0.043 ± 0.003* 0.115 ± 0.012 0.118 ± 0.009 Pyruvate/PEP 1.2 0.22 1.0 0.4 Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis Katsumi Iizuka, Richard K. Bruick, Guosheng Liang, Jay D. Horton, and Kosaku Uyeda  Impaired glucose tolerance in mice lacking ChREBP. ChREBP-/- mice were unable to ingest sucrose and developed hypothermia. Wild-type and ChREBP-/- mice were fed the indicated diet for 7 days, and changes in body temperature were followed daily. ChREBP deficiency caused hypothermia and death in mice because of their inability to metabolize fructose. Data are means of four animals. Sucrose generates glucose and fructose during digestion. Think about which molecule is leading to hypothermia and death in mice. Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis Katsumi Iizuka, Richard K. Bruick, Guosheng Liang, Jay D. Horton, and Kosaku Uyeda  Impaired glucose tolerance in mice lacking ChREBP. Fatty acid synthesis was measure using radioactive isotope. Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis Katsumi Iizuka, Richard K. Bruick, Guosheng Liang, Jay D. Horton, and Kosaku Uyeda