Nutritional Biochemistry Exam 1

Glucoamylase

High specificity for ⍺(1,4) glycosidic bonds in oligos 2-9 units in length

Biotin

Water-soluble B vitamin. Bound to a specific lysine residue of the enzyme.

SREBP1c

activates genes for FA and TAG synthesis. Known to become activated by insulin and/or feeding.

SREBP1a

activates genes for cholesterol and FA synthesis.

What does the liver secrete into the GI tract?

bile (containing electrolytes, bile salts

Isomaltase

(aka ⍺-dextrinase) Uniquely capable of cleaving the ⍺(1,6) branch points of ⍺- dextrins. Also capable of cleaving ⍺(1,4) glycosidic bonds of starch oligos

What happens to amino acids in the GI tract?

Amino acids absorbed from the GI tract are released into portal circulation (taking all products of digestion to the liver). Intake/release of amino acids are carried out by distinct proteins with overlapping specificities. Throughout the body, concentrations of localized amino acid pools vary. ◦ Even within individual cells, concentration depends upon the subcellular compartment. ◦ Depending upon cell/tissue type, the concentration among amino acid types will vary. Glutamine approx. 40x greater in muscle cell than plasma. Within cell, Glutamine 200x greater than tyrosine.

What are the two forms of acetyl-coA carboxylase

ACC1: Produces malonyl-CoA in lipogenic tissues (liver, adipose tissue) ACC2: Forms malonyl-CoA in muscle, and other tissues.

Acetyl-coA carboxylase regulation

Acetyl-CoA carboxylase is allosterically activated by citrate, inhibited by fatty acids. In fed state, cytosolic citrate increases and long-chain acyl-CoA is low (increases activity) In starvation state, cytosolic citrate decreases and long-chain acyl-CoA levels increase (decrease activity) Acetyl-CoA carboxylase activity is subject to regulation via phosphorylation. Up to seven serine residues may be phosphorylated. The phosphorylated enzyme is less active, less sensitive to citrate, more sensitive to long-chain acyl-CoA Phosphorylation carried out by several different enzymes. The most important of these is 5'-AMP-activated protein kinase (AMPK). AMPK is allosterically activated by AMP (AMP increases when cell energy is low). Acetyl-CoA carboxylase is phosphorylated and converted to a less active form by AMPK. AMPK is phosphorylated and activated by another kinase.

First step of synthesis of palmitate *Name of enzyme that produces citrate Name of citrate transporter What happens to citrate in the cytosol?

Acetyl-CoA is produced from pyruvate in the mitochondria. Increased acetyl-CoA leads to increased production of citrate by citrate synthase. When citrate levels are elevated, translocation to the cytosol is facilitated by tricarboxylate transporter in exchange for malate. Once citrate enters the cytosol, is it cleaved by ATP-citrate lyase, producing acetyl-CoA and oxaloacetate Cytosolic malate dehydrogenase reduces oxaloacetate to malate, allowing it to be transported across the membrane in exchange for more citrate. Mitochondrial malate dehydrogenase oxidizes malate to re-form oxaloacetate. Energetic cost: 1 mol ATP per mole acetyl-coA transported. Results in transfer of NADH equivalents into matrix for ATP production. Although no shuttle exist to transport acetyl-CoA, this metabolite can be condensed with oxaloacetate to form a compound that can be shuttled across the mitochondrial membrane.

Oxidation of unsaturated fatty acids

Additional reactions are required for the β-oxidation of unsaturated FA. Most naturally occurring double bonds are in the cis conformation.Auxiliary enzymes must rearrange the double bonds into the trans conformation. Separate unsaturated FA into two classes: Double bonds from odd-numbered carbons (ex: ∆9) ß We will discuss this example. Double bonds from even-numbered carbons (ex: ∆12)

Why would a protein turnover be required, even when the body requires no net gain or loss of protein?

Allows the body to replace proteins that have been oxidized, damaged, misfolded, or otherwise non functional. Allows the body to adapt to new physiological and nutritional conditions.

Amino Acids as Signaling Molecules *what body functions do they affect?)

Amino acids play regulatory roles as agents in signaling cascades. ◦ They can affect taste, protein synthesis and degradation, and insulin secretion. Example: Leucine plays a critical role in amino-acid induced insulin secretion.

What are dextrins?

Amylase cleaves much of amylopectin, but the branch points (called dextrins) remain. Dextrins are 5-6 glucose units long, and account for 1/3 of the mass of amylopectin.

Amylose vs amylopectin

Amylose is a linear and unbranched form of starch that contains only ⍺(1,4) glycosidic bonds. Because of this, it is easier to digest. Amylopectin is more complex in structure. It also accounts for approximately 80% of dietary polysaccharides. ◦ Contains glucose units joined by ⍺(1,4) glycosidic bonds. ◦ Contains branch points joined by ⍺(1,6) glycosidic bonds.

HMG-COA reductase *Where is it located? How is it regulated? What is it inhibited by?

Anchored to the ER membrane. Under negative feedback control of mevalonate (immediate product) and cholesterol (downstream product). Main mechanism of regulation is controlling the amount of enzyme present within the cell (transcriptional regulation, and protein stability). Inhibited by statins. Statins are a class of lipid-lowering drugs, and used to treat individuals at high risk of cardiovascular disease, or in early stages of it.

Urea cycle

Aspartate enters the cycle in the cytosol. ATP is required to convert aspartate into arginosuccinate, which is then split into fumarate and arginine. Arginine is hydrolyzed and releases urea. The remaining ornithine is transferred to the mitochondria. Here, it forms citrulline and repeats the cycle.

Insulin signaling cascade

Binding of insulin or IGF-1 (insulin-like growth factor) to its receptor stimulates Tyr phosphorylation of IRS-1. IRS-1 is a signaling adapter protein that plays a role in transmitting the signal from insulin/IGF-1 to MAPKs (PI3K, AKT, etc). IRS-1 activation stimulates PI3K and pAKT. pAKT inactivates FoxO via phosphorylation. When FoxO is phosphorylated, it cannot enter the nucleus. pAKT activates mTOR, promoting protein synthesis

What is cholesterol and what is its function?

Cholesterol is a lipophilic molecule that is essential for animal life. Cholesterol is an important component of the cell membrane, influencing its fluidity. Increases fluidity in colder temperatures.Decreases fluidity in warmer temperatures. Functions as a precursor molecule in the synthesis of vitamin D,steroid hormones (cortisol and aldosterone and adrenal androgens), and sex hormones (testosterone, estrogens, and progesterone). Cholesterol is also a constituent of bile salt. Because cholesterol is mostly lipophilic, it is transported through the blood, along with triglycerides, inside lipoprotein particles (HDL, IDL, LDL, VLDL, and chylomicrons).

Chylomicrons vs VDLs vs HDLs *How do lipoproteins form VLDLs?

Chylomicrons can have molecular weights 100x that of VLDL. Chylomicrons contain approximately 500,000 TAG molecules, 30,000 CE, 45,000 phospholipids, and 25,000 cholesterol. When nascent lipoproteins enter plasma, they pick up additional apolipoproteins from circulating HDLs to become mature chylomicrons and VLDLs. The loss of TAG from the core of the lipoproteins (by actions of lipoprotein lipase), chylomicrons and VLDLs become smaller/denser. Chylomicrons become chylomicron remnants. VLDLs become IDL and LDL. HDLs are thought to be generated in plasma. Apolipoprotein A-1 (secreted by liver) is thought to associate with cell-derived phospholipids to form HDLs, which can then accumulate cholesterol from cells as they circulate.

How/where do cholesterol move throughout the body?

Chylomicrons transport lipids throughout the body. The liver re-packages cholesterol from chylomicrons into VLDL. Tissues remove TAG from VLDL. As this continues, they are converted IDL and LDL. Most tissues take up LDL as a source of cholesterol. HDL takes cholesterol from tissues back to the liver.

Cortisol *Where is it produced? What 3 activities does it influence?

Cortisol is a glucocorticoid hormone produced in the adrenal cortex. ◦ Released in response to low glucose levels, stimulates gluconeogenesis. Decreases protein synthesis, as well as increasing degradation. ◦ Ribosomal S6 kinase activity is downregulated, decreasing ribosomal activation and translation. ◦ Inhibits formation of eIF4 complex (involved in initiating eukaryotic translation). ◦ FoxO translocation to the nucleus increases with glucocorticoid treatment, promoting the ubiquitin- proteasome pathway.

Cytokines *What two factors are associated with protein degradation? What are they indirectly controlled by? acute phase proteins

Cytokines are peptides produced by cells of the immune system. ◦ They are produced in response to injury, infection, or inflammation. ◦ Their activity has a large impact on protein metabolism, but the regulatory mechanisms are not entirely understood. Cytokines interleukin 1β (IL1β) and tumor necrosis factor ⍺ (TNF⍺) are associated with protein degradation. ◦ Their effects are indirectly moderated by the GH/IGF-1 system, and also by glucocorticoid secretion. ◦ Cytokines also reduce the production of GH receptors and IGF-1. However, in liver cells, inflammatory cytokines such as IL1β, IL-6, and TNF⍺ increase synthesis of acute phase proteins. ◦ Acute phase proteins are produced to aid inflammatory response, and promote gluconeogenesis.

Desaturation of fatty acids

Desaturation of saturated FA occurs in the ER, and is carried out by a complex of enzymes. The first double bond introduced into a saturated chain is at the ∆9 position by the ∆9 desaturase complex. This complex uses saturated FA that are 14-18 carbons in length (stearate is most reactive).Uses two electrons and two protons donated by NADH, two electrons and two protons from the fatty acid, and oxygen (as electron acceptor).Generates two water molecules, and a double bond.

Types of amino acid transporters

Di- and tri-peptides also have their own transporters ◦ Ex: Pept1, Pept2. Urea also has two transporters (UT1 and UT2).◦ These transporters are found in many tissues. ◦ Highly expressed in the kidney.

What part of the small intestine does the pancreas and liver secrete into?

Duodenum

Apo A-1

Found only in HDL. Flexible and weakly associated with the surface of HDL. Allows the shape of HDL surface to adapt as lipid core expands. Promotes desorption of free cholesterol from cell membranes. Activates lecithin-cholesterol acyltransferase (LCAT). LCAT is bound to HDL molecules. LCAT produces cholesterol esters that are sequestered inside the core of an HDL particle.*HDL also contains other apolipoprotein types, such as apo-A2, apo-C1, apo-E, etc.

Elongation of fatty acids *Where does it occur? *What is the elongating group? Electron donor? What are the reaction primer? What carries out the reaction steps?

Elongation of FA occurs in the ER, mitochondria, and peroxisomes. The system in the ER is the most active, and most understood. The processes is similar to that of fatty acid synthase. The sequence of reactions is condensation, reduction, dehydration, reduction. Malonyl-CoA is the elongating group, NADPH is the electron donor. The differences are that long-chain acyl-CoAs are the reaction primer (instead of acetyl-CoA), and that the reaction steps are carried out by separate gene products (rather than one multifunctional enzyme).

FoxO *What does it promote the expression of (specific name of enzyme)?

FoxO promotes expression of E3 ubiquitin ligases, and therefore promotes protein breakdown.

GLUT5 *Where is it located? *What kind of affinity does it have?

Fructose transporter Transmembrane protein located on apical side of enterocyte, although some GLUT5 may be present on basolateral side Facilitates movement of the ketohexose fructose into the enterocyte from the intestinal lumen, down the concentration gradient Despite low affinity, it has a relatively high capacity

OXIDATION OF FATTY ACIDS WITH ODD NUMBER OF CARBONS

FA composed of an odd number of carbons are not synthesized in animals, but small amounts may be found in vegetables. In these cases, β-oxidation occurs as it usually would until the final step of the last cycle. Each cycle has produced 1 acetyl-CoA, 1 NADH, and 1 FADH2.However, product of final cleavage is 1 acetyl-CoA and 1 propionyl-CoA. The propionyl-CoA is carboxylated and isomerized to form succinyl-CoA. Succinyl-CoA is a CAC intermediate, and is gluconeogenic.

When is fatty synthase activity high or low? *what enzyme is regulated?

Fatty acid synthase activity is low in fasted state, increases in fed state. This is especially true in a carbohydrate-rich meal. Does not appear to be influenced by allosteric effectors nor phosphorylation. This emphasizes importance of acetyl-CoA carboxylase for immediate regulation of synthesis. Fatty acid synthase is regulated at the level of expression. Insulin and glucagon also influence the synthesis of fatty acids. Insulin, secreted in the fed state, facilitates activation of the pathway. Glucagon does the opposite.

Step 2 of synthesis of palmitate

First committed step in FA synthesis, and highly regulated. This two-step reaction is catalyzed by acetyl-CoA carboxylase. Reaction includes ATP-dependent carboxylation.Dependent upon the cofactor biotin (B vitamin). Step 1: Carboxylation of biotin.ATP hydrolysis provides requisite energy for anabolic reaction. Step 2: Transcarboxylation.Carboxyl group transferred to acetyl-CoA.Malonyl-CoA is produced.Biotin-enzyme regenerated to carry out another reaction.

Oxidation of fatty acids

First step is to transfer FA into the mitochondria. This process is inhibited by malonyl-CoA, which is produced during FA synthesis. Malonyl-CoA specifically inhibits CPT1. Whereas synthesis of FA involves condensation, reduction, dehydration, reduction, β-oxidation of FA involves oxidation, hydration, oxidation, cleavage. The figure on the right demonstrates the oxidation of palmitate. For each cycle of palmitate oxidation 1acetyl-CoA1 NADH1 FADH2 Seven cycles are required 8 acetyl-CoA7 NADH7 FADH2 This results in the production of 129 ATP after complete oxidation.

Step 1 of cholesterol synthesis

First step of cholesterol synthesis: Converting 2-C acetyl group of acetyl-CoA into the 6-C mevalonate. Three acetyl-CoA molecules are condensed to produce the key intermediate 3-hydroxyl-3methylglutaryl-CoA (HMG-CoA). The subsequent step sees the reduction of HMG-CoA to mevalonate by HMG-CoA reductase. HMG-CoA reductase is the rate-limiting enzyme of cholesterol synthesis, and is subject to very important regulatory mechanisms. 2C-->4C-->6C

Oxidation of Oleoyl-CoA (∆9)

First three rounds of β-oxidation occur normally. This intermediate is not a substrate for any enzyme in the pathway. Enoyl CoA isomerase rearranges the double bond, yielding the trans conformation. Also notice that the double bond has shifted up one carbon. The intermediate is now a substrate to undergo the fourth β-oxidation cycle. The first dehydrogenation step in the fourth cycle has been omitted, yielding one less FADH2, and therefore approximately two less ATP.

Lipoproteins *What are on the surfaces of lipoproteins? What are the names from small intestine and those from the liver?

For transport in aqueous plasma, the small intestine and liver package nonpolar lipids (TAG and CE) in the core of large lipoprotein particles. On the surface of lipoproteins are amphipathic cholesterol, phospholipids, and apolipoproteins. The particles from the small intestine are chylomicrons, and those from the liver are VLDLs.

Apo-B

Found in chylomicrons (apo-B48) as well as VLDL, IDL, and LDL (apo-B100).Both B48 and B100 are required for secretion of their lipoproteins. Chylomicrons and VLDL also contain other apolipoproteins that aid in the catabolism of lipids. Apo-B100 produced in the liver.Apo-B100 recognizes and bind to the LDL-receptor, promoting endocytosis. Apo-B48 is made only in intestinal cells. Apo-B48 and B100 are produced by the same gene. Intestine-specific cytosine deaminase converts a cytosine to a uracil, creating a premature stop codon in the mRNA. This results in B48. Apo-B48 cannot initiate endocytosis.Chylomicrons rely on the actions of apo-E to bind to the LDL-Receptor.

Sucrase

High specificity for ⍺(1,4) glycosidic bonds in maltose and maltotriose. Uniquely capable of cleaving sucrose into glucose and fructose.

Tas1R2/Tas1R3 Receptor

G-protein-coupled receptors in taste buds that combine to function as a "sweet" receptor. Evidence suggest that these receptors are also present in the gut. Activated by sugars and sugar substitutes (both mono & disaccharides). Leads to downstream activation of glucagon-like peptide 2 (GLP2), which subsequently activates GLUT2 translocation. Insulin plays a role in the return of GLUT2 back to intracellular storage.

Growth hormone signal transduction pathway

GH binds to its receptor receptor on the cell surface. - Activates JAK2 - Active JAK2 phosphorylates STAT proteins. Phosphorylated STAT proteins dimerize, and enter the nucleus. There they bind to specific DNA sequences, increasing their expression (IGF-1 being one of them). IGF-1 reproduces many of the effects of insulin, but is more potent.

Growth hormone

GH promotes longitudinal growth in children and adolescents. Secreted by the pituitary gland in response to a variety of factors, including exercise, stress, nutrition, age, etc. GH binds to a specific receptor on the surface of target cells. Initiates signal transduction that in turn promotes expression of genes involved in anabolic processes. ◦ Insulin-like growth factor 1 (IGF-1) is one of the proteins activated by GH.

Gastric pits

Gastric pits contain cell types that produce various secretions that aide in digestion. Parietal cells are the source of gastric HCl. These cells contain channels called canaliculi. It is through these channels that HCl is supplied to the lumen. H+ and Cl- are secreted separately into the canaliculi. H+ ions are generated by disassociation with H2O. ◦ The OH- ions combine with CO2 to produce HCO3- (bicarbonate) ◦ HCO3- is transported out of the cell (into blood) in exchange for Cl- ions. Cl- and K+ are transported from the cytoplasm, and into canaliculi. H+ ions are pumped into canaliculi from the cytoplasm in exchange for K+ ions. ◦ This is facilitated by a membrane-bound H+/K+-ATPase (proton pump).

Glucagon

Glucagon promotes gluconeogenesis from amino acids. In all, 13 amino acids can be used to produce glucose. Glucogenic amino acids can be converted into intermediates of the citric acid cycle. Those intermediates can, in turn, be fed into the gluconeogenesis pathway. Glucagon counters the actions of insulin. By directing amino acids towards gluconeogenesis, fewer are being used for protein synthesis.

SGLT1 *What does it transport? What is its Km

Glucose/galactose transporter Transmembrane protein located on apical side of enterocytes Co-transports two sodium ions along with one aldohexose, with high affinity for these two substrates (and therefore a relatively low Km of 0.5mM). This allows the efficient transport of the sugars even at a low luminal concentration secondary active transport Na+ concentrations are higher in the lumen (70mM) than within the enterocyte (12mM). ◦ This concentration gradient is maintained by the Na+/K+-ATPase in basolateral membrane, removing Na+ from the enterocyte.

GLUT2

Glucose/galactose/fructose transporter Low-affinity, high capacity transporter. Km of approximately 20mM. Transports all three major hexoses Always found in the basolateral membrane of the enterocyte. Transports hexoses out of enterocyte, down their concentration gradient. Transiently involved in apical uptake of hexoses from the lumen, and into the enterocyte.

HMG-CoA Reductase Phosphorylation When is it phosphorylated? What phosphorylates it?

HMG-CoA reductase activity can also be influenced by a phosphorylation/dephosphorylation mechanism. In the phosphorylated state, the enzyme is less active. AMPK phosphorylates HMG-CoA reductase.This is the same enzyme that regulates fatty acid synthesis (Inhibits acetyl-CoA carboxylase). Allosterically activated when AMP levels are high (cellular energy is low). Phosphorylates, and therefor decreases activity of HMG-CoA reductase (Prevents anabolic reactions).

HMG-CoA Reductase degredation *Where does Insig bind to? What two processes does the binding trigger?

HMG-CoA reductase contains a sterol-sensing domain that is similar to SCAP. When ER membrane cholesterol level is high, the binding of HMG-CoA reductase to Insig is triggered through its sterol-sensing domain. This binding triggers ubiquitination and proteosomal degradation of HMG-CoA reductase.

Lipoprotein lipase in adipose tissue *When is it down/upregulated? What about its Km?

In fasting/starving state, LPL is downregulated. In fed state, LPL is upregulated Adipose LPL has a high Km, meaning lesser affinity for VLDL and chylomicrons, but never saturated. In fed state, rate of hydrolysis is only dependent upon plasma concentration of lipoproteins.

Synthesis and secretion of VLDL

Precursor particles are assembled with a phospholipid monolayer shell, containing apo B-100, and a core of CE. MTP adds TAG to the precursor molecule. Apo-B proteins (48 and 100) are constantly produced, but rate of degradation depends upon TAG availability. Chylomicrons depend solely upon dietary lipids, whereas VLDL can utilize de novo synthesized TAG.

Lipoprotein lipase in heart and muscle tissue

In fasting state, heart/muscle levels of LPL are maintained. Heart LPL has a lower Km than adipose LPL (higher affinity). Heart LPL is saturated at lower concentrations. Rate of hydrolysis is dependent upon abundance of the enzyme within the tissue.

Protein Balance and Exercise

In response to exercise, work-induced hypertrophy develops. ◦ Involves increases in both synthesis and degradation of protein, but increase in S > increase in D. ◦ Hypertrophy increased by resistance exercise, not by endurance exercise. Resistance exercise increases mTOR signaling to downstream effects on S6 kinase and 4E-BP. ◦ Ribosomal S6 kinase is activated, 4E-BP is inactivated ◦ Both occur via phosphorylation. 4E-BP binds and inhibits eIF4E, preventing activation of the pre-initiation complex. mTOR activation leads to, 4E-BP inhibition facilitating the formation and activation of the pre-initiation complex, and mRNA translation Ribosomal S6 Kinase phosphorylates the S6 subunit of the 40S ribosome. The activation of S6 kinase leads to the formation of an active eIF4 pre-initiation complex, facilitating mRNA translation.

Insulin

Insulin levels increase with feeding and decrease during fasting.◦ This cycle is associated with cyclic responses in carbohydrate, fat, and protein metabolism. ◦ Individuals who lack insulin because of diabetes tend to also be more susceptible to muscle loss. Insulin stimulates protein synthesis, in inhibits protein degradation rates. The mechanism by which insulin acts involves a signaling cascade that results in the activation of mRNA translation.

How is the GI tract regulated? *What does the intrinsic system control? What does the extrinsic system control? What secretes GI hormones?

Intrinsic system: Responsible for neural regulation of GI motility and function. Coordinates movement of food through the GI tract. Extrinsic system: Functions along with intrinsic system. Help to promote digestion/absorption by increasing secretion, muscle tone, relaxing sphincters and blood vessels. GI hormones: Secreted by the enteroendocrine cells (entero- prefix means "intestine"). Located throughout the tract, and exert local effects. Play roles in regulating GI functions, including motility and the secretion of HCl. These hormones may act on other tissues including the brain, pancreas, liver, and gallbladder, and impact the regulation of GI function by those tissues.

Fatty acid synthesis What organ and what part of the cells does this occur?

Primary sites for synthesis are the liver and adipose tissue. The substrates and intermediates in both FA synthesis and oxidative pathways are mainly in the form of thioesters (Fatty Acid + CoA). Synthesis may be suppressed by the common high-fat Western diet. The enzymes carrying out synthesis are located in the cytoplasm, whereas oxidative enzymes are located in the mitochondrial matrix.

Leucine and amino-acid induced insulin secretion

Leucine plays a critical role in amino-acid induced insulin secretion. ◦ Leucine activates glutamate dehydrogenase (GDH) in pancreatic beta cells . ◦ Increased ATP/ADP ratio closes an ATP-gated K+ channel. ◦ Membrane depolarization results in Ca2+ influx. ◦ Ca2+ increase results in insulin secretion. After a meal, concentration of branched-chain amino acids increase. ◦ These amino acids, especially leucine, appear to be anabolic signaling molecules, increasing protein synthesis. (Exact mechanism is not clear) ◦ Leucine leads to activation of mTORC1. ◦ mTORC1 leads to downstream phosphorylation of S6 Kinase (activation), and 4E-BP (inhibition) ◦ Phosphorylation leads to the formation of initiation complex (increasing translation). ◦ Activation of mTORC1 also decreases proteolysis.

Major groups of lipoproteins

Lipoproteins are composed of the types seen in the table on the right, as well as chylomicrons. In fasting state, LDL and HDL are highest in concentration. In general, these lipoproteins are grouped into two classes based upon the presence of an essential apolipoprotein. Apolipoprotein A-1 (Apo A-1) lipoproteins Apolipoprotein B (Apo B) lipoproteins Apolipoproteins are amphipathic, and bind lipids to form lipoproteins. Apo A-1 and B are both characteristic of specific lipoproteins, but lipoproteins contain more than one type of apolipoprotein.

Small Intestine

Location of most digestion and nutrient absorption. Receives secretions from the liver, gallbladder and pancreas, which enter via bile duct. Brunner glands produce an alkaline solution (pH 8.1-9.3), which brings the pH of the chyme to optimal pH for pancreatic digestive enzymes (active at a neutral pH).

Nitrogen Excretion *What are the end products of amino acid catabolism? Why is the net energy gained less than complete protein oxidation?

Major end products of amino acid catabolism ◦ Carbon dioxide, water, urea. ◦ Small amounts of ammonia. The net energy actually gained is less than what would be predicted by the complete oxidation of protein. ◦ Energy is required for urea synthesis.For an adult in nitrogen balance, daily nitrogen excretion as urea approximates the daily intake as protein.◦ Most nitrogen lost as urea, although a small percentage is lost in feces.

During synthesis of palmitate, what happens to malate that isn't transported back into the mitochondrial matrix?

Malate that isn't transported back into the mitochondrial matrix can undergo oxidative decarboxylation, yielding NADPH and CO2 (this reaction is carried out by malic enzyme). Pyruvate subsequently transported back to the matrix, and carboxylated to form oxaloacetate. The NADPH produced from malate oxidation is available for fatty acid synthesis. NADPH is the required electron donor for the reductive step in the pathway.

Regulation of palmitate synthesis via malonyl-coA *What does malonyl-coA inhibit?

Malonyl-CoA is a potent inhibitor of CPT1.CPT1 transfers fatty acids into mitochondria for oxidation.Ensures that FA synthesis and oxidation don't occur simultaneously.

Brief Fasting and Prolonged Starvation *What is the nitrogen balance? What determines the start of the early starvation state? What does the body use for energy then?

Period following a meal (postprandial period), the body is in an absorptive state. As time passes to post-absorptive state, insulin decreases and glucagon increases. ◦ Nitrogen balance is a net negative during this time.As fasting extends, and glycogen levels are spent, the body enters early starvation state. ◦ Body begins to use glycerol and amino acids to meet energy needs, and restore glucose (gluconeogenesis). In a state of longer-term starvation, whole-body protein degradation rates decrease. ◦ Body increases synthesis of ketone bodies from fatty acids. After continued long-term starvation, adipose is depleted. Protein degradation for energy begins again. At this stage, death from starvation is fast approaching.

Triacylglycerol clearance from chylomicrons and VLDL

Most TAG and CE are contained within the core of the particles. Core TAG and CE are in equilibrium with small amount in the surface monolayer. Hydrolysis of TAG by plasma lipases depletes the surface concentration. Surface TAG replenished by the core. Surface TAG hydrolyzed by lipoprotein lipase (LPL). LPL is present on surface of capillary endothelium. Highest concentrations in regions of muscle and adipose tissue.

4 Layers of GI tract (past the oral cavity)

Mucosa: First layer from the lumen. Composed of three layers, including an epithelial layer that comes in direct contact with food particles. A second layer contains small blood & lymphatic vessels. Submucosa: Connective tissue layer. Contains blood and lymphatic vessels, and nerve fibers (controlling secretion). Muscularis Externa: Contains two or three layers of muscle. Their contractions control GI tract motility. Serosa/Adentitia: Serosa is loose connective tissue, and an extension of peritoneal cavity (which holds abdominal organs in place). Adventitia is an outermost connective tissue layer found in regions outside of the peritoneal cavity.

Myostatin

Myostatin is a signaling molecule that inhibits the growth and differentiation of muscle cells. It is an inducer of muscle wasting. ◦ Inhibits insulin and IGF-1 signaling. ◦ Inhibits the effects of mTOR◦ Increases the activity of FoxO.

Mammals and desaturases

Polyunsaturated fatty acids can be produced by adding additional double bonds by other desaturases. Mammalian cells can further introduce double bonds at the ∆5 or ∆6 position by ∆5- or ∆6- desaturases. The mechanism occurs as described for the ∆9 desaturase. Mammals lack desaturases that introduce double bonds beyond the ∆9 position. Due to the lack of desaturases beyond the ∆9 position, some polyunsaturated FA must be obtained through dietary means. Linoleate (18:2, ∆9, ∆12), an n-6 fatty acid⍺-Linolenate (18:3, ∆9, ∆12, ∆15) an n-3 fatty acid These essential PUFA are essential for the syntheses if other PUFA, such as arachidonate (20:4, ∆5, ∆8, ∆11, ∆14) through desaturation and elongation reactions.

Synthesis of cholesterol: Where? What is the substrate and intermediates?

Over half of the cholesterol in the body is synthesized, rather than obtained through diet. The body has the ability to produce all of the cholesterol needed. The liver and intestine are major sites for cholesterol synthesis. Synthesis occurs in other tissues of the body, but not nearly to the same degree. Synthesis occurs in the cytosol, closely associated with the ER. Acetyl-CoA is the substrate for cholesterol synthesis. Mevalonate and squalene are key intermediates in the pathway.

Synthesis of palmitate (overview)

Palmitate is the most common saturated fatty acid in animals, plants, and microorganisms. (C:D ratio of 16:0) Acetyl-CoA is the building block of fatty acids. Acetyl-CoA is formed in the mitochondria from pyruvate. There is no acetyl-coA carrier to shuttle it out of the mitochondria, and the inner mitochondrial membrane is impermeable to it as well.

What does the pancreas secrete?

Pancreas secretes ⍺-amylase, lipase, nucleolytic enzymes

Synthesis and secretion of chylomicrons

Pre-chylomicron formation begins with a TAG-poor monolayer of phospholipids, encapsulating cholesteryl esters. Microsomal triacylglycerol transfer protein (MTP) transfers TAG into the pre-chylomicron. Pre-chylomicron then fuses with a particle containing apo B-48. Pre-chylomicrons are translocated to the Golgi apparatus, where they are processed, and then secreted into the lymph. They eventually enter the venous plasma.

Protein blance and nutrient supply

Protein balance is highly responsive to nutritional status. Food intake stimulates protein production, while incomplete nutrition promotes the opposite. The mechanisms used individual tissues to regulate synthesis and degradation may vary.

Protein turnover equation

S + E = D + I S= synthesis E= oxidation D= degredation I= dietary amino acids In positive nitrogen balance, S > D In negative nitrogen balance, S < D In balance, S = D, does NOT mean that neither is taking place. Rather they occur at equal rates.

Second step of cholesterol synthesis

Second step of cholesterol synthesis: Decarboxylation of 6-C mevalonate to 5-C isoprenoids. This pathway involves three sequential phosphorylations of mevalonate to yield 3-phospho-5-pyrophosphomevalonate. This is a highly reactive intermediate, and is immediately decarboxylated and dephosphorylated. Isopentenyl pyrophosphate can be isomerized to dimethylallyl pyrophosphate by the enzyme isopentenyl pyrophosphate isomerase. Collectively, these are referred to as isoprenoids, and are both used in cholesterol synthesis. 6C-->5C

Step 3 of synthesis of palmitate

Second, and final committed step of FA synthesis Catalyzed by multifunctional enzyme fatty acid synthase. Enzyme catalyzes all reactions shown on the right.To produce one molecule of palmitate, fatty acid synthase requires 7 malonyl-CoA, 1 acetyl-CoA, 14 NADPH, and 14 H+. Reaction produces 1 palmitate, 8 CoA, 7 CO2, 14 NADP+ and 6 H20. Palmitate is built up by the serial addition of two-carbon fragments. After each addition, the added carbons are reduced, dehydrated, and reduced again. Process of condensation, reduction, dehydration, and reduction continue until 16- carbon FA is produced, and released from enzyme.

SREBP2

the main transcription factor involved in responding to cholesterol levels. Activates genes involved in cholesterol synthesis, as well as LDL receptor production.

4th step of cholesterol synthesis

Squalene synthase condenses two 15C farnesyl pyrophosphates in a head-to-tail manner, resulting in complete dephosphorylation. 15C--> 30C Squalene is converted to lanosterol by a series of redox and rearrangement reactions that are dependent upon molecular oxygen and NADPH. The enzyme squalene epoxidase incorporates the oxygen into the lanosterol precursor. Lanosterol cyclase converts the oxidized squalene molecule into a four-ringed structure (Lanosterol). The production of cholesterol requiresmore NADPH, O2, and series of reactions requiring 19 more steps. 3. Three C are removed, and final product is 27-C

Why do we need to be careful about synthesizing and disposing cholesterol?

Synthesis and disposal of cholesterol must be tightly regulated to be sure all needs are met, but also to prevent excess. Deposition of cholesterol and cholesterol-rich lipoproteins in arteries eventually leads to atherosclerosis, contributing to cardiovascular disease.

Third step of cholesterol synthesis

Synthesis of Squalene, Cholesterol and Lanosterol from Isoprendoids. Squalene, and therefore lanosterol & cholesterol, are built up of isoprenoid units. Like with the formation of mevalonate, this portion of the pathway sees the condensation of three units. First isopentenyl pyrophosphate condenses with dimethylallyl pyrophosphate. A third 5-C molecule, another isopentenyl pyrophosphate, condenses to form farnesyl pyrophosphate. 5C-->10C-->15C

Cytokine pathway

TNF⍺ leads to the activation of NF-κB.This induces mRNA expression of genes that promote the ubiquitin-proteasome pathway. Cell differentiation is under the control of NF-κB, among other transcription factors. ◦ElevatedTNF⍺isassociatedwithincreasedlevels of NF-κB activity, resulting in epigenetic silencing of myofibrillar genes. ◦ Myofibrils are rod-like muscle cell units, composed of actin, myosin, and other muscle proteins.

What is the function of secreting HCl?

The accumulation of ions in the canaliculi draws water into the channels from inside the parietal cells, resulting in an HCl-rich secretion. The acidic environment develops in the stomach (ranging from pH 1-3). Chief cells secrete pepsinogen, which becomes activated in the acidic environment. The acidic environment favors the denaturing of proteins. The denaturing of proteins makes the peptide bonds more accessible to proteolytic enzymes, such as pepsin (the activated form of pepsinogen).

Metabolism of NEAA

The carbon chains of NEAA (non-essential amino acids) are synthesized using glucose or glucogenic substrates. 3-C compounds (like pyruvate) are used to make alanine, serine, glycine. 4-C oxaloacetateàAsparagine and Aspartate 5-C ⍺ -ketoglutarate and metabolitesàglutamate, proline, arginine, glutamineNitrogen always come from other amino acids. ◦ Amino acids are the only source of nitrogen for humans. Coordination with other tissues is required. Appropriate enzymes & intermediate transporters need to be present at cell surface for release and uptake of synthesis intermediates The enzymes and transporters will be specific to tissue type.

Regulation of Apical Translocation of GLUT2

Under fasting conditions, GLUT2 localization is restricted to the basolateral membrane. GLUT2 also appears to be restricted to the basolateral membrane after consumption of complex carbohydrates . GLUT2 translocation to the apical membrane appears to be triggered by several factors. ◦ High glucose levels saturating SGLT1 capacity, leading to high intracellular glucose. ◦ High fructose intake activates GLUT2 translocation to the apical membrane. When luminal glucose levels are very high after a sugar-rich meal, GLUT2 is capable of aiding in glucose absorption from through the apical membrane. GLUT2 is shuttled to and from the apical membrane via intracellular vesicles. sugars consumed--> GLP-2 is secreted by the intestinal endocrine cells in small and large intestine.--> GLUT2 to apical vs Insulin is secreted when blood glucose levels are high. (inhibits)

Metabolism of Amino Acid Carbon Chains

The carbon side chains can be used in the metabolically interrelated pathways of ◦ Amino acid oxidation ◦ Gluconeogenesis◦ Ureagenesis The points at which carbon skeletons of various amino acids enter catabolic pathways are shown on the right. Most are converted to CAC intermediates Others are produced as glycolysis end products, or acetyl-CoA. Carbon skeletons from amino acids are needed for gluconeogenesis. Only amino acids that produce CAC intermediates that make oxaloacetate are glucogenic. Gluconeogenesis withdraws oxaloacetate from the cycle, and that intermediate needs to be replenished. Glucogenic amino acids are anaplerotic. They introduce additional intermediates into CAC. ◦ Leucine and Lysine are NOT anaplerotic, and therefore not gluconeogenic. In the liver, amino acid catabolism and ureagenesis go hand-in-hand. Amino acid catabolism and gluconeogenesis both involve the citric acid cycle. Aspartate enters the urea cycle (Others AA can be enter via transamination reactions). Fumarate is a side product of the urea cycle (It is not needed to continue the cycle) Fumarate is a CAC intermediate (and anaplerotic). Once in the cycle, the carbons contributed may be oxidized for ATP, or used to enter gluconeogenesis.

Stomach *What does the stomach secrete?*

The stomach is a site of temporary food storage, before being passed into the intestines. Secretes HCl, enzymes, zymogens (inactive precursor to an enzyme), endocrine hormones that regulate food assimilation, and intrinsic factor (needed for the absorption of vitamin B12). Stomach mixes food/digestive juices, producing a mixture called chyme. The stomach also regulates the rate of chyme entry into the small intestine.

Regulation of cholesterol synthesis

The synthesis of cholesterol is tightly regulated. Synthesis, as well as uptake via the LDL receptor are subjected to negative feedback regulation. The expression of enzymes involved in the synthesis (HMG-CoA reductase, HMG-CoA synthase, squalene synthase, etc.) is coordinated. Primary target of regulation: HMG-CoA reductase.

Anabolic response to eating

To stimulate the translation of mRNA into protein (in response to eating), both insulin and sufficient amino acid supply are needed to have the maximal effect. Two steps are involved in this process: ◦ 1) Phosphorylation of eIF2 is relieved, allowing for the 40s ribosome to form pre-initiation complex. eIF2 complex is mostly responsible for delivering initiator tRNA to initiation complex. When phosphorylated, it is inactive. When active, it can exchange a GDP for GTP, and deliver the initiator tRNA. 2) Formation of mRNA cap-binding eIF4 complex is increased, facilitation mRNA binding to the ribosome. The addition of essential amino acids to a protein meal can promote increase in protein synthesis. ◦ This has been demonstrated with short-term effect.◦ Long-term changes in body composition have not yet yielded consistent results. Protein sources can influence amino acid pools. ◦ 1-2 hours after a meal, whey protein results in higher plasma amino acid levels than other protein sources. ◦ Casein protein, however, maintained elevated plasma amino acids for a longer duration. Food intake is associated with a decreased expression of ubiquitin-proteasome pathway components.

Nitrogen metabolism

Transamination involves moving the ⍺-amino group from one carbon chain to another. This reaction converts an amino and a keto acid into their respective keto and amino acids. These reactions are carried out by enzymes called aminotransferases or transaminases. ◦ Dependent upon pyridoxal phosphate (PLP), which is active form of vitamin B6, as a cofactor. Most have a preferred amino/keto acid substrate. Uses ⍺-ketoglutarate/glutamate as the counter acid. Alanine, aspartate, glutamate, tyrosine, serine, valine, isoleucine, leucine are actively transaminated in humans. In contrast, lysine, proline, tryptophan, and arginine do not participate in transamination directly. ◦ Must be converted to intermediates first. Deamination Removal of amino group.◦ Glutamate dehydrogenase is responsible for this reaction. ◦ Nitrogen is removed in the form of ammonia. Glutamate dehydrogenase (GDH) ◦ Mitochondrial enzyme ◦ Requires NAD/NADH or NADP/NADPH cofactor (B vitamin derivative) ◦ Highly active in liver, kidney cortex, and brain ◦ Fate of products are tissue-specific◦ Liver: ammonia incorporated into urea. ◦ Kidney: N excreted as ammonium (NH4+) GDH only uses glutamate as a substrate.In order to deaminate other amino acids, transamination is required.

System A transport regulation

Transport is subject to short- and long-term regulation. Ex: System A transport regulation ◦ System A transport is responsible for moving several kinds of non-polar amino acids in Na+-dependent mechanism. Short-Term Mechanisms ◦ One amino acid is subject to competitive inhibition by transport of another. ◦ System A activity is increased by glucagon or epidermal growth factor (EGF), and is also sensitive to pH changes. ◦ System A activity can also be induced by insulin in most cells (glucagon and insulin liver cells). System A transporter activity is increased by both glucagon and insulin in the liver. Long-Term Mechanism ◦ Change in expression levels of the protein. EX: Genes coding for System A transporters, as well as others, have been shown to contain amino acid response elements (AARE). ◦ AAREs are responsible for transcriptional upregulation/downregulation of genes under amino acid starvation conditions. (The ATF family are among the transcription factor families that appear to be involved in the binding of AAREs.)

Protein turnover

Turnover is specifically the relationship between synthesis and degradation of protein. Turnover is influenced by a variety of factors such as the rate of oxidation and ingestion/digestion. During the day, humans build and degrade about 300g of protein. Typical Western diet contains about 100g of protein/day. Amino acid breakdown involves the removal of nitrogen from the body in the form of ammonia or urea. The carbon skeletons are used to provide energy either directly (as TCA cycle intermediates), or indirectly (glucose or fatty acid production). Turnover rate must also be considered at the level of the tissue. Proteins produced in some tissues need constant replacement, and for varying reasons.

Sterol Regulatory Element-Binding Protein (SREBP)

Using a mechanism that measures cellular cholesterol content, transcription of enzymes involved in cholesterol homeostasis are regulated. Transcriptional control requires the presence of a sterol regulatory element (SRE). SREs are located in the promoter region of genes. They are bound by transcription factors known as SRE binding protein (SREBP) There are three SREBPs: SREBP1a, SREBP1c (produced by SREBP1 gene via alternative splicing), and SREBP2. SREBPs induce transcription of genes involved in cholesterol metabolism, as well as TAG and FA synthesis. All SREBPs are anchored to the ER by two transmembrane helices.

High cellular cholesterol

When cholesterol levels are high, cholesterol binds direct to SCAP (through SSD). When cholesterol binds to SCAP, it triggers SCAP to bind to an ER-retention protein called Insig (insulin-induced gene). When SCAP and Insig bind, it prevents SCAP from interacting with the COPII proteins. If SCAP doesn't bind to COPII, SCAP/SREBP cannot be sent to the Golgi, and thus active SREBP will not be released.

What happens when there is low cellular cholesterol?

When cholesterol levels in the ER membrane is low, SREBP cleavage-activating protein (SCAP) binds to a component of COPII coat proteins. COPII proteins are involved in vesicle budding from ER. Binding results in of SCAP/SREBP sequestration within vesicles that bud off of the ER and are sent to the Golgi apparatus. In the Golgi, two proteases (S1P and S2P) cleave SREBP proteins, thus releasing the active form. Active SREBPs translocate to the nucleus, where they bind to SREs.

GI tract function and what does it include?

digestion/absorption Contains a muscular tube with a continuous lumen. ◦ Lumen: Inner space of a tubular structure. Includes: Oral cavity, pharynx, esophagus, stomach, small and large intestine, rectum, and accessory glands (salivary glands, pancreas, liver, gallbladder)

Maltase-glucoamylase & Sucrase-Isomaltase What kind of glucosidases are they? What do they digest?

exoglucosidases that cleave one glucose unit at a time. All enzymes are capable of cleaving ⍺(1,4) glycosidic bonds used to complete the digestion of starch. Each enzyme contains two independent active sites, and are named for their main activity group of ⍺-glucosidases work together in a complementary manner to cleave bonds from ⍺-dextrins. This occurs in sequence, beginning from the non-reducing end, releasing monomeric glucose

Catabolic hormones

glucagon, cortisol, myostatin

Anabolic hormones

insulin, growth hormone, IGF-1

Where does digestion begins? *What enzymes are found in saliva?

oral cavity Mechanical digestion: chewing, mixing food with saliva ◦ Saliva contains amylase, resulting in partial digestion of starch.◦ Also found in saliva are bicarbonate and carbonic anhydrase, which neutralize acids.

Where does amylase secretion occur?

salivary/pancreas glands Digestion of starch begins in the mouth, but is halted by the acidic conditions in the stomach (neutral pH required for activity). Pancreatic amylase is secreted in large quantities, and far exceeds the amount needed to cleave the glycosidic bonds. The resulting oligosaccharides are produced while still in the very early region of the small intestine.

What kind of enzymes are amylases?

⍺(1,4) endoglucosidases, only cleaving internal ⍺(1,4) glycosidic bonds. The result of amylase cleavage are glucose oligomers

Maltase

⍺(1,4) glycosidic bonds in both starch oligos and maltose disaccharides