Chapter 27 Metabolic Integration and Organ Specialization Biochemistry by Reginald Garrett and Charles Grisham What principles underlie the integration of catabolism and energy production with anabolism and energy consumption? How is metabolism integrated in complex organisms with multiple organ systems?

Outline Can systems analysis simplify the complexity of metabolism? What underlying principle relates ATP coupling to the thermodynamics of metabolism? Is there a good index of cellular energy status? How is overall energy balance regulated in cells? How is metabolism integrated in a multicellular organism? What regulates our eating behavior? Can you really live longer by eating less?

27.1 – Can Systems Analysis Simplify the Complexity of Metabolism? The metabolism can be portrayed by a schematic diagram consisting of just three interconnected functional block: Catabolism Anabolism Macromolecular synthesis and growth Catabolic and anabolic pathways, occurring simultaneously, must act as a regulated, orderly, responsive whole

Figure 27.1 Block diagram of intermediary metabolism.

Catabolism: Energy-yield nutrients are oxidized to CO2 and H2O and most of the electrons are passed to O2 via electron-transport pathway coupled with oxidative phosphorylation, resulting in the formation of ATP Some electrons reduce NADP+ to NADPH The intermediates serve as substrates for anabolism Glycolysis The citric acid cycle Electron transport and oxidative phosphorylation Pentose phosphate pathway Fatty acid oxidation

Macromolecular synthesis and growth Anabolism: The biosynthetic reactions Metabolic intermediates in catabolism are the precursor for anabolism NADPH supplies reducing power ATP is the coupling energy Gluconeogenesis Fatty acid biosynthesis Macromolecular synthesis and growth Creating macromolecules Required energy from ATP Macromolecules are the agents of biological function and information Growth can be represented as cellular accumulation of macromolecules

Only a few intermediates interconnect the major metabolic systems Sugar-phosphates (triose-P, tetraose-P, pentose-P, and hexose-P) a-keto acids (pyruvate, oxaloacetate, and a-ketoglutarate) CoA derivatives (acetyl-CoA and suucinyl-CoA) PEP ATP & NADPH couple catabolism & anabolism Phototrophs have an additional metabolic system– the photochemical apparatus

Three types of stoichiometry in biological systems 27.2 – What Underlying Principle Relates ATP Coupling to the Thermodynamics of Metabolism? Three types of stoichiometry in biological systems Reaction stoichiometry - the number of each kind of atom in a reaction Obligate coupling stoichiometry - the required coupling of electron carriers Evolved coupling stoichiometry - the number of ATP molecules that pathways have evolved to consume or produce - a number that is a compromise

1. Reaction stoichiometry The number of each kind of atom in any chemical reaction remains the same, and thus equal numbers must be present on both sides of the equation C6H12O6 + 6 O2  6 CO2 + 6 H2O 6 carbons 12 hydrogens 18 oxygens

2. Obligate coupling stoichiometry Cellular respiration is an oxidation-reduction process, and the oxidation of glucose is coupled to the reduction of NAD+ and FAD (a) C6H12O6 + 10 NAD+ + 2 FAD + 6 H2O  6 CO2 + 10 NADH + 10 H+ + 2 FADH2 (b) 10 NADH + 10 H+ + 2 FADH2 + 6 O2  12 H2O + 10 NAD+ + 2 FAD (24 electrons)

3. Evolved coupling stoichiometry The coupled formation of ATP by oxidative phosphorylation C6H12O6 + 6 O2 + 38 ADP + 38 Pi  6 CO2 + 38 ATP + 44 H2O The value of 38 was established a long time ago in evolution Prokaryotes: 38 ATP Eukaryotes: 32 or 30 ATP

ATP coupling stoichiometry determines the Keq for metabolic sequence The energy release accompanying ATP hydrolysis is transmitted to the unfavorable reaction so that the overall free energy for the coupled process is negative (favorable) DG0’ for ATP hydrolysis is a large negative number ATP changes the Keq by a factor of 108 (p69-70) The involvement of ATP alters the free energy change for a reaction, the role of ATP is to change the equilibrium ratio of [reactants] to [products] for a reaction

The cell maintains a very high [ATP]/([ADP][Pi]) ratio Living cells break down energy-yielding nutrient molecules to generate ATP Glycolysis requires the investment of 2ATP/glucose before any energy yields Fatty acid oxidation depends on fatty acid activation by acyl-CoA synthetase So, ATP hydrolysis can serve as the driving force for virtually all biochemical events

ATP has two metabolic roles ATP is the energy currency of the cells To establish large equilibrium constant for metabolic conversions To render metabolic sequence thermodynamically favorable An important allosteric effector in the kinetic regulation of metabolism PFK in glycolysis FBPase in gluconeogenesis

27.3 – Is there a good index of cellular energy status? Energy transduction and energy storage in the adenylate system – ATP, ADP, and AMP – lie at the very heart of metabolism The metabolic lifetime of an ATP is brief ATP, ADP, and AMP are all important effectors in exerting kinetic control on regulated enzymes The regulation of metabolism by adenylates in turn requires close control of the relative concentrations of ATP, ADP, and AMP

Adenylate Kinase Interconverts ATP, ADP, and AMP Adenylate kinase provides a direct connection among all three members of the adenylate pool ATP + AMP 2 ADP The free energy of hydrolysis of a phosphoanhydride bond is the same in ADP and ATP Adenylate pool: [ATP] + [ADP] + [AMP] The Adenylates system provides phosphoryl groups to drive thermodynamically unfavorable reactions [ATP] [ADP] [AMP] PP

Energy Charge Relates the ATP Levels to the Total Adenine Nucleotide Pool Energy charge (E.C.) is an index of how fully charged adenylates are with phosphoric anhydrides (ATP=2; ADP=1) Energy charge = If all adenylate is [ATP] , E.C.1.0 If [AMP] is the only adenylate form, E.C. 0 2[ATP] + [ADP] 1 2 [ATP] + [ADP] + [AMP]

Figure 27.2 Relative concentrations of AMP, ADP, and ATP as a function of energy charge. (This graph was constructed assuming that the adenylate kinase reaction is at equilibrium and that DG°' for the reaction is -473 J/mol; Keq = 1.2.)

Key enzymes are regulated by Energy charge Regulatory enzymes typically respond in reciprocal fashion to adenine nucleotides For example, phosphofructokinase is stimulated by AMP and inhibited by ATP Regulatory enzymes in energy-producing catabolic pathways show greater activity at low energy charge PFK and pyruvate kinase Regulatory enzymes of anabolic pathways are not very active at low energy charge Acetyl-CoA carboxylase

0.85 - 0.88 Figure 27.3 Responses of regulatory enzymes to variation in energy charge.

27.4 – How is Overall Energy Balance Regulated in Cells? AMP-activated protein kinase (AMPK) is the cellular energy sensor Metabolic inputs to this sensor determine whether its output (protein kinase activity) takes place The competition between ATP (inactivate) and AMP (activate) for binding to the AMPK allosteric sites determines the activity of AMPK When [ATP] is high, AMPK is inactive When [AMP] is high, AMPK is allosterically activated and phosphorylates many targets controlling cellular energy production and consumption

Activation of AMPK Sets in motion catabolic pathways leading to ATP synthesis Shuts down pathways that consume ATP energy, such as biosynthesis and cell growth AMP binding to AMPK increases its protein kinase activity by more than 1000-fold AMP activates AMPK in two ways It is an allosteric activator AMP binding favors phosphorylation of Thr172 within the a-subunit The regulation is reversed if ATP displaces AMP from the allosteric site

The b-subunit has an ag-binding domain that brings a and g together AMPK is an abg heterotrimer; the a-subunit is the catalytic subunit and the g-subunit is regulatory The b-subunit has an ag-binding domain that brings a and g together Figure 27.4 Domain structure of the AMP-activated protein kinase (AMPK) subunits. (CBS: cystathionine-b-synthase)

AMPK targets key enzymes in energy production and consumption Activation of AMPK leads to phosphorylation of many key enzymes in energy metabolism Include phosphorylation of PFK-2 (in liver) → [F-2,6-BP]↑ → stimulates glycolysis glycogen synthase → inhibit glycogen synthesis ACC → inhibit fatty acid biosynthesis HMG-CoA reductase → inhibit cholesterol biosynthesis Phosphorylation of transcription factors diminishes expression of gene encoding biosynthetic enzymes

AMPK controls whole-body energy homeostasis AMPK is activated by hormone such as adiponectin and leptin in sketal muscle. Exercise also activates AMPK Figure 27.6 AMPK regulation of energy production and consumption in mammals.

27.5 – How Is Metabolism Integrated in a Multicellular Organism? In complex multicellular organisms, Organ systems have arisen to carry out specific physiological functions Each organ expresses a repertoire of metabolic pathways Such specialization depends on coordination of metabolic responsibilities among organs so that the organism as a whole may thrive Organs differ in the metabolic fuels they prefer as substrates for energy production (see Figure 27.7)

Figure 27.7 Metabolic relationships among the major human organs.

glycogen > triacylglycerol > protein The major fuel depots in animals are glycogen in live and muscle; triacylglycerols in adipose tissue; and protein, mostly in skeletal muscle The usual order of preference for use of these is glycogen > triacylglycerol > protein The tissues of the body work together to maintain energy homeostasis

The major organ systems have specialized metabolic roles

Brain Brain has two remarkable metabolic features It has a very high respiratory metabolism 20 % of oxygen consumed is used by the brain Only 2% of body mass Oxygen consumption is independent of mental activity, continuing even during sleep It is an organ with no fuel reserves Uses only glucose as a fuel and is dependent on the blood for a continuous, incoming supply (120g per day)

Brain During starvation, the body’s glycogen reserves are depleted, brain can use -hydroxybutyrate -hydroxybutyrate is formed from fatty acids in the liver and converted to acetyl-CoA → enter TCA cycle This allows the brain to use fat as fuel High rate of ATP production are necessary to maintain the membrane potentials essential for transmission of nerve impulses

Figure 27.8 Ketone bodies such as β-hydroxybutyrate provide the brain with a source of acetyl-CoA when glucose is unavailable.

Muscle Skeletal muscles is responsible for about 30% of the O2 consumed by the human body at rest During maximal exertion, skeletal muscle can account for more than 90% of the total metabolism Muscle contraction occurs when a motor never impulse causes Ca+2 release from endomembrane compartments (sarcoplasmic reticulum) The muscle contraction requires hydrolysis of ATP In relaxation, Ca2+ ions are pumped back into the sarcoplamic reticulum. Two Ca2+ ions are translocated per ATP hydrolysis

Creatine Kinase in Muscle Muscle at rest can utilize a variety of fuels --glucose, fatty acids, and ketone bodies Rest muscle contains about 2% glycogen and 0.08% phoshpocreatine by weight When ATP is used to drive muscle contraction, the ADP formed can be reconverted to ATP by creatine kinase at the expense of phosphocreatine Muscle phosphocreatine can generate enough ATP to power about 4 seconds of exertion

Creatine Kinase and Phosphocreatine Provide an Energy Reserve in Muscle Figure 27.9 Phosphocreatine serves as a reservoir of ATP-synthesizing potential.

Creatine Kinase in Muscle During strenuous exertion, once phosphocreatine is depleted, muscle relies solely on its glycogen reserves Glycolysis is capable of explosive bursts of activity The flux of glucose-6-P through glycolysis can increase 2000-fold almost instantaneously The triggers for this activation are Ca2+ and the “fight or flight” hormone epinephine Glycolysis rapidly lowers pH (not lactate accumulation), causing muscle fatigue The conversion of glucose to 2 lactate is accompanied by the release of 2 H+

Muscle Protein Degradation During fasting or excessive activity, muscle protein is degraded to amino acids so that their carbon skeletons can be used as fuel Many amino acids are converted to pyruvate, which can be transaminated to alanine Alanine circulates to liver, where it is converted back to pyruvate – a substrate for gluconeogenesis Muscle protein is a fuel of last resort

Figure 27.10 The transamination of pyruvate to alanine by glutamate:alanine aminotransferase.

Heart The activity of heart muscle is constant and rhythmic The heart functions as a completely aerobic organ and is very rich in mitochondria Prefers fatty acid as fuel Heart tissue has minimal energy reserves: a small amount of phosphocreatine and limited glycogen Continually nourished with oxygen and free fatty acid, glucose, or ketone bodies as fuel

Adipose tissue Amorphous tissue widely distributed about the body Consist of adipocytes Endocrine organ: secrete leptin, adiponectin… ~65% of the weight of adipose tissue is triacylglycerol (TAG) Have a high rate of metabolic activity, synthesizing and breaking down of TAG Free fatty acids are obtained from the liver Lack glycerol kinase; cannot recycle the glycerol of TAG Glucose plays a pivotal role for adipose tissue Glycolysis produces DHAP converted to glycerol-3-P Pentose phosphate pathway provides NAPDH

Brown fat A specialized type of adipose tissue, is found in newborn and hibernating animals Rich in mitochondria (brown color) Thermogenin, uncoupling protein-1, permitting the H+ ions to reenter the mitochondria matrix without generating ATP Is specialized to oxidize fatty acids for heat production rather than ATP synthesis

Liver The major metabolic processing center in vertebrates, except for triacylglycerol Most of the incoming nutrients that pass through the intestines are routed via the portal vein to the liver for processing and distribution Much of the liver’s activity centers around conversions involving glucose-6-phosphate

Figure 27.11 Metabolic conversions of glucose-6-phosphate in the liver.

Cholesterol synthesis Detoxification organ Glucose-6-phosphate From dietary carbohydrate, degradation of glycogen, or muscle lactate Converted to glycogen released as blood glucose, used to generate NADPH and pentoses via the pentose phosphate pathway, catabolized to acetyl-CoA for fatty acid synthesis or for energy production in oxidative phosphorylation Fatty acid turnover Cholesterol synthesis Detoxification organ

27.6 What Regulates Our Eating Behavior? Approximately two-thirds of American are overweight One-third of Americans are clinically obese Obesity is the most important cause of type 2 diabetes Research into the regulatory controls on feeding behavior has become a medical urgency The hormones that control eating behavior come from many different tissues

Eating Behavior The hormones control eating behavior Produced in the stomach, liver, pancreas,... Move to brain and act on neurons, principally on the arcuate nucleus region of the hypothalamus The arcuate nucleus is an anatomically distinct brain area that functions in Homeostasis of body weight Body temperature Blood pressure Other vital functions

Eating Behavior—Are you hungry The hormones can be divided into Short-term regulator: determine individual meal Long-term regulator: act as stabilize the levels of body fat deposit Two subset neurons are involved: NPY/ AgRP-producing neurons – release NPY (neuropeptide Y) stimulating the neurons that trigger eating behavior Melanocortin-producing neurons-- inhibiting the neurons

Figure 27.12 The regulatory pathways that control eating. (-) Figure 27.12 The regulatory pathways that control eating.

AgRP (agouti-related peptide) Melanocortin Block the activity of melanocortin-producing neurons Melanocortin Inhibit the neurons initiating eating behavior Including a- and b-MSH (melanocyte-stimulating hormone) Ghrelin and cholecytokinin are short-term regulators of eating behavior Ghrelin is an appetite-stimulating peptide hormone produced in the stomach Cholecytokinin released from GI tract during eating signals satiety (the sense of fullness) and tends to curtail further eating

PYY3-36 inhibits eating by acting on the NPY/AgRP-producing neurons Insulin and leptin are long-term regulators of eating behavior (Both inhibit eating) Insulin is produced in the b-cells of the pancreas when blood glucose level raise The major role is to stimulate glucose uptake from the blood Insulin also stimulates fat cells to make leptin Leptin is an anorexic (appetite-suppressing) agent and inhibits the release of NPY NPY is a orexic (appetite-stimulating) hormone PYY3-36 inhibits eating by acting on the NPY/AgRP-producing neurons

AMPK mediates many of the hypothalamic responses to these hormones The actions of leptin, gherlin, and NPY converge at AMPK Leptin inhibits AMPK in the arcuate nucleus via MC4R (melanocortin-4-receptor) Gherlin and NPY activate hypothalamic AMPK The effects of AMPK may be mediated through changes in malonyl-CoA levels AMPK phosphorylates ( inhibits) acetyl-CoA carboxylase Malonyl-CoA levels decreased Low [malonyl-CoA] is associated with increased food intake

27.7 Can You Really Live Longer by Eating Less? Caloric restriction leads to longevity For most organisms, caloric restriction results in lower blood glucose levels declines in glycogen and fat stores enhanced responsiveness to insulin lower body temperature diminished reproductive capacity Caloric restriction also diminishes the likelihood for development of many age-related diseases, including cancer, diabetes, and atherosclerosis

Mutations in the SIR2 Gene Decrease Life Span Deletion of a gene termed SIR2 (silent information regulator 2) abolishes the ability of caloric restriction to lengthen life in yeast and roundworms This implicates the SIR2 gene product in longevity Humans have seven genes analogous to SIR2, designed SIRT1 through SIRT7 SIRT is an abbreviation of sirtuin SIRT1 cycles between cytosol and nucleus SIRT3, 4, and 5 confined to the mitochondria

SIRT Sirtuins are NAD+-dependent protein deacetylases The tissue NAD+/NADH ratio controls sirtuin protein deacetylase activity Nicotinamide and NADH are inhibitors of the deacetylase reaction Oxidative metabolism, which drives conversion of NADH to NAD+ NAD+/NADH ratio raise Enhances sirtuin activity CR increases mitochondrial biogenesis and then raises the ratio

Sirtuin-catalyzed removal of acetyl groups from lysine residues of histones Allow the nucleosomes to interact more strongly with DNA, making transcription more difficult Almost all of the enzymes of energy metabolism are acetylated and potentially regulated by SIRT

SIRT1 is a Key Regulator in Caloric Restriction SIRT1 connects nutrient availability to the expression of metabolic genes A striking feature of CR is the loss of fat stores and reduction of WAT (white adipose tissue) SIRT1 participates (prevents) in the transcriptional regulation of adipogenesis through interaction with PPARg (peroxisome proliferator-activator receptor- g) PPARg is a nuclear hormone receptor that activates transcription of genes involved in adipogenesis and fat storage SIRT1 binding to two PPARg corepressors, NCoR and SMRT, prevents transcription of these genes, leading to loss of fat stores.

SIRT1 is a Key Regulator in Caloric Restriction Because adipose tissue functions as an endocrine organ, this loss of fat has significant hormonal consequences for energy metabolism In liver, SIRT1 interacts with and deacetylates PGC-1a (PPAR-g corepressor-1) CR leads to increased transcription of these genes encoding the enzymes of gluconeogenesis and repression of genes encoding glycolytic enzymes SIRT1 connects nutrient availability to the regulation of major pathways of energy storage (glycogen and Fat) and fuel utilization

Resveratrol in Red Wine is a Potent Activator of Sirtuin Activity Is a phytoalexin, is a member of the polyphenol class of natural products Is a free-radical scavenger, which may explain its cancer preventive properties. Is red wine is an excellent source of resveratrol Resveratrol activates SIRT NAD+-dependent deacetylase activity Resveratrol activates AMPK in the brain

Metabolic syndrome High blood pressure Elevated blood triglyceride levels Low blood HDL-cholesterol levels High blood glucose levels Central obesity Caloric restriction reverses all of the symptoms of metabolic syndrome Nutrients are limiting (as in CR) Activates SIRT1 (through deacetylation) and AMPK (phosphorylation) Down-regulates anabolic processes and activates ATP-producing catabolic processes