Pyruvate Kinase

  • Last enzyme in glycolysis
  • Irreversibly dephosphorylates phosphoenolpyruvate (PEP) to form pyruvate
  • 1 ATP produced by substrate level phosphorylation
  • Several isozymes: M-type (muscle) and L-type (liver)
  • All isozymes allosterically regulated (L-type also hormonally regulated)

M-TYPE ISOZYMES

Allosteric regulation

Activation

  • AMP: marker of ATP depletion or low energy
  • Fructose 1,6-bisphosphate: product of rate-limiting reaction in glycolysis
    (feed-forward activation: stimulates downstream glycolytic enzymes)

Inhibition

  • ATP: sufficient energy
  • Acetyl CoA: first intermediate of citric acid cycle
  • Alanine: can be produced from pyruvate; sufficient pyruvate in the cell

L-TYPE ISOZYME

  • Allosteric and hormonal regulation (similar to PFK-2)

Hormonal regulation

Activation

  • Insulin activates phosphatases, which remove phosphate from PK
  • Makes PK susceptible to positive allosteric regulators

Inhibition

  • Glucagon promotes phosphorylation of PK via cAMP-dependent pathway
  • Makes PK susceptible to negative allosteric regulators

CLINICAL CORRELATION

Pyruvate kinase deficiency

  • Produce hemolytic anemia (spiculated RBC’s)
  • RBC biconcave shape maintained by sodium-potassium pumps (require ATP)
  • RBC’s do not have mitochondria: rely on glycolysis for ATP

Phosphofructokinase

PHOSPHOFRUCTOKINASE-1 (PFK-1)

  • Catalyzes rate-limiting step in glycolysis
  • Catalyzes irreversible phosphorylation of F6P to F1,6P.
  • Allosterically regulated (hormonally regulated in liver)

PFK-1 INHIBITION

  • Citrate, intermediate of citric acid cycle
  • ATP, final product of glycolysis and cellular respiration
  • H+, symptom of lactic acid buildup in exercising muscle

PFK-1 ACTIVATION

  • AMP, marker of ATP depletion.
  • Fructose-2,6-bisphosphate (special case)

PFK-2/FBP-2 (BIFUNCTIONAL ENZYME)

  • PFK-2: F6P –> F2,6P (activates PFK-1)
  • FBP-2 (fructose-2,6-bisphosphatase): F2,6P –> F6P (deactivates PFK-1)
  • PFK-2/FBP-2 regulated differently in different tissues

Skeletal muscle

  • Feed-forward activation
  • Substrate-level regulation: F6P
  • When F6P HIGH: FBP-2 inactive and PFK-2 active (activate PFK-1)
  • When F6P LOW: FBP-2 active and PFK-2 inactive (inhibits PFK-1 activity)

Liver

  • Hormonal regulation: PFK-2 has phosphorylation site (unlike muscle)
  • PFK-2 is INACTIVE when phosphorylated
  • Glucagon activates protein kinase A (PKA), which phosphorylates PFK-2
  • Insulin activates phosphoprotein phosphatase (PPP), which dephosphorylates PFK-2

FED STATE

  • HIGH blood glucose
  • INSULIN secreted –> PPP activated –> PFK-2 dephosphorylated (ACTIVE)
  • HIGH F2,6P activates PFK-1
  • Promotes glycolysis

FAST

  • LOW blood glucose
  • GLUCAGON secreted –> PKA activated –> PFK-2 phosphorylated (INACTIVE)
  • LOW F2,6P deactivates PFK-1
  • NO glycolysis

Liver responds to entire body’s glucose needs

  • Site of gluconeogenesis: glucose synthesized from non-carbohydrate precursors and released into the bloodstream

Hexokinase

  • Catalyzes phosphorylation of glucose to form glucose-6P
  • Traps glucose inside the cell
  • 1st regulated enzymes in glycolysis – catalyze an irreversible reaction
  • 4 isozymes – I, II, III, and IV (glucokinase)

HEXOKINASE (I, II & III) vs. GLUCOKINASE (IV)

  • Tissue distribution
  • Kinetics (Km and Vmax)
  • Regulation (allosteric vs hormonal)

Hexokinase

  • Ubiquitous in mammals
  • Low Km & low Vmax
  • Allosteric regulation – inhibited by glucose-6P

Glucokinase

  • Liver & pancreatic beta cells
  • High Km & high Vmax: spares glucose for brain, muscle & other tissues (glucose sensor)
  • Hormonal regulation: inhibited by glucagon, activated by insulin
  • Glucokinase regulatory protein (GKRP): nuclear protein that reversibly binds/inactivates glucokinase
  • High [glucose] inhibits GKRP & promotes glucokinase release
  • Fructose-6P (glycolytic intermediate in equilibrium with glucose-6P): promotes GKRP-GK binding
  • Liver glucose-6P shunts into one of three pathways: glycolysis, glycogen or fatty acid synthesis

Glycolysis

  • 1 glucose (6-carbon sugar) breaks down to 2 pyruvates (3-carbon sugar).
  • Net 2 ATP produced: 2 consumed (investment phase) and 4 generated (pay off)
  • 2 NADH produced

REGULATED STEPS

Hexokinase

  • Glucose + ATP Glucose-6-phosphate + ADP

Phosphofructokinase

  • Fructose-6-phosphate + ATP → fructose-1,6-biphosphate + ADP

Pyruvate kinase

  • Phosphoenol pyruvate + ADP → Pyruvate + ATP

ENZYMES OF GLYCOLYSIS
Listed in chronological order (substrate/product in parentheses)

  • Hexokinase (glucose/glucose-6P)
  • Phosphoglucose isomerase (glucose-6P/fructose-6P)
  • Phosphofructokinase (fructose-6P/fructose-1,6P)
  • Aldolase (fructose-1,6P/G3P & DHAP)
  • Triose phosphate isomerase (DHAP/G3P)
  • Glyceraldehyde-3-phosphate dehydrogenase (G3P/1,3-bisphosphoglycerate)
  • Phosphoglycerate kinase (1,3-bisphosphoglycerate /3-phosphoglycerate)
  • Phosphoglycerate mutase (3-phosphoglycerate /2-phosphoglycerate)
  • Enolase (2-phosphoglycerate /phosphoenol pyruvate)
  • Pyruvate kinase (phosphoenol pyruvate /pyruvate)

Glucose Metabolism Overview Part II

GLUCOSE OXIDATION EQUATION
Glucose + 6 O2 –> 6 CO2 + 6 H2O + Energy (ATP + heat)

  • Most energy is generated in mitochondrial matrix

Common Abbreviations:

  • ATP: adenosine triphosphate
  • NADH: nicotinamide adenine dinucleotide
  • FADH2: flavin adenine dinucleotide
  • CoA: Coenzyme A

KEY PROCESSES IN GLUCOSE OXIDATION

  • Glycolysis
  • Pyruvate decarboxylation
  • Citric acid cycle (also known as the Krebs’ cycle and the tri-carboxylic acid (TCA) cycle)
  • Oxidative phosphorylation (electron transport chain & chemiosmosis)

CITRIC ACID CYCLE

  • 1 glucose molecule requires 2 citric acid cycle turns
  • Input for each turn: 1 Acetyl CoA
  • Output for each turn: 3 NADH + 2 CO2 + 1 ATP + 1 FADH2
  • NADH & FADH2: electron transfer molecules for oxidative phosphorylation
  • Occurs in mitochondrial matrix

Substrate level phosphorylation

  • ATP generated from substrates in glycolysis and citric acid cycle
  • NOT from oxidative phosphorylation via NADH or FADH2

OXIDATIVE PHOSPHORYLATION

  • Input: 10 NADH + 2 FADH2 (from glycolysis, pyruvate decarboxylation & CAC)
  • Generates 30-34 molecules of ATP per glucose
  • Main energy-generating process in respiration
  • Comprises electron transport chain and chemiosmosis
  • Occurs on inner mitochondrial membrane

Electron transport chain

  • Series of redox reactions
  • Pumps proton from matrix into intermembrane space
  • Generates electrochemical gradient for ATP synthesis via ATP synthase
  • Contains several protein complexes (I through IV)
  • NADH gives electrons to complex I
  • FADH2 gives electrons to complex II
  • Complex I, III, and IV pump H+ into intermembrane space (Complex II DOES NOT)
  • Complex IV consumes 1 O2 (final e- acceptor) to produce 2 H2O

Chemiosmosis

  • ATP synthase: harnesses energy in electrochemical gradient (generated by ETC) to synthesize ATP from ADP & Pi

Glucose Metabolism Overview Part I

GLUCOSE OXIDATION EQUATION
Glucose + 6 O2 –> 6 CO2 + 6 H2O + Energy (ATP + heat)

  • Most energy is generated in mitochondrial matrix

Common Abbreviations:

  • ATP: adenosine triphosphate
  • NADH: nicotinamide adenine dinucleotide
  • FADH2: flavin adenine dinucleotide
  • CoA: Coenzyme A

KEY PROCESSES IN GLUCOSE OXIDATION

  • Glycolysis
  • Pyruvate decarboxylation
  • Citric acid cycle (also known as the Krebs’ cycle and the tri-carboxylic acid (TCA) cycle)
  • Oxidative phosphorylation (electron transport chain & chemiosmosis)

GLYCOLYSIS

  • 1 glucose –> 2 pyruvate + 2 ATP + 2 NADH
  • Anaerobic reaction: no O2 required
  • Occurs in cytosol

Substrate level phosphorylation

  • ATP generated from substrates in glycolysis and citric acid cycle
  • NOT from oxidative phosphorylation via NADH or FADH2

PYRUVATE DECARBOXYLATION

  • Pyruvate + CoA + NAD+ –> Acetyl-CoA + CO2 + NADH
  • Occurs in mitochondrial matrix

Glucose Metabolism: Energy Yield

GLYCOLYSIS

  • Occurs in the cytosol
  • Anaerobic
  • Inputs: 1 Glucose
  • Outputs: 2 Pyruvates, 2 ATP, 2 NADH

PYRUVATE DECARBOXYLATION

  • Occurs in the matrix
  • Aerobic
  • Input: 2 Pyruvates
  • Outputs: 2 Acetyl CoA, 2 CO2, 2NADH

CITRIC ACID CYCLE

Kreb’s Cycle of Tricarboxylic Acid Cycle

  • Occurs in the mitochondrial matrix
  • Aerobic
  • Input: 2 Acetyl CoA
  • Output: 4CO2, 2ATP, 6NADH, 2FADH2

OXIDATIVE PHOSPHORYLATION

  • Electron transport
  • Chemiosmosis
  • Aerobic
  • Inputs: 10 NADH, 2FADH2
  • Outputs: 30-34 ATP

Total ATP Output: 30-38 ATP

  • Intertextual variation exists: exact calculations beyond the scope of this tutorial.
  • Approximates yield for both eukaryotes & prokaryotes.

Esophagus & Stomach Histology

ESOPHAGUS

Long, muscular tube delivers food from the pharynx to the stomach.

  • Mucosa layer
    • Comprises stratified squamous epithelium; layers of flattened cells provide protection against physical and chemical damage from the foods, liquids, and salivary juices traveling to the stomach.
    • Muscularis mucosae is also visible in our sample.
  • Submucosal layer
  • Submucosal glands
  • Glands of the submucosal and mucosal layers produce protective and lubricating mucous, which contributes to the protective barrier.
  • Submucosa and mucosa form longitudinal folds in the relaxed esophagus; these folds expand to accommodate foods and liquids during swallowing.
  • Muscularis externa layer
    The composition of the muscularis externa layer changes along its length, as follows:
    • The upper 1/3rd comprises skeletal muscle fibers in both the circular and longitudinal layers;
    • The middle 1/3rd comprises a circular layer of skeletal muscle and a longitudinal layer of smooth muscle;
    • Both layers of the lower 1/3rd, which opens to the stomach, contains smooth muscle fibers.
  • The outermost layer of the esophagus is adventitia; once the esophagus passes through the diaphragm, and is no longer anchored to the body wall, it becomes serosa.

STOMACH

Connects with the esophagus, superiorly and the duodenum, inferiorly.
Regions of the stomach:

  • Cardiac, where the esophagus and stomach meet
  • Fundus, in the upper left corner
  • Body, which is the largest region of the stomach
  • Pyloris, which opens to the duodenum
    Gastric Folds, aka, rugae
  • Line the empty stomach; expand to accommodate foods and liquids during gastric filling
    Histological Details:
  • Surface mucous epithelium
    • These cells secrete mucous and form a physical and chemical barrier to protect the underlying stomach wall from stomach contents. In addition to ingested foods and liquids, the stomach wall is potentially vulnerable to the gastric juices it releases as part of digestion.
  • Pits
    • Invaginations of the surface mucosal epithelium form pits, which open to glands deeper within the mucosa; depending on their location in the stomach, the glands produce mucous and/or gastric juices.
    • Lamina propria is visible in our sample between the pits and glands.
    • Slips of muscularis mucosae can be seen extending into the gastric fold.
      Glands:
  • Cardiac and pyloric glands are primarily mucous-secreting; in other words, they do not produce significant quantities of gastric juices.
    • Cardiac glands are highly coiled at their ends, or bases.
    • Pyloric glands are highly branched.
  • Gastric glands of the fundus and body produce both mucous and gastric juices; they are responsible for chemical digestion.
    • Surface epithelial cells line the pit, and secrete surface mucous.
    • Mucous neck cells also secrete mucous.
    • Parietal cells, which appear as round, bulging, and light pink in the histological sample, are found within the neck and base of the gastric gland. These cells, which are also referred to as oxynitic cells, secrete hydrochloric acid and intrinsic factor (required for vitamin B12 absorption).
    • Stem cells are also found within the neck; these cells play important roles in the constant renewal of gastric mucosal cells.
    • Chief cells reside in the base of the gland, and stain darker due to secretory granules containing pepsinogen, which is a precursor to pepsin for protein digestion.
    • Enteroendocrine cells, which is an umbrella term for a variety of cells that secrete peptide hormones. For example, G cells secrete gastrin, and D cells secrete somatostatin.
    • The cells of the gastric gland lie on a basement membrane, which separates them from the surrounding glands and lamina propria.

Early Gut Tube & Mesenteric Attachments

GI & MESENTERY ORGANIZATION

~ Weeks 4 through 6

Three embryologic divisions of the thoracic and abdominal gastrointestinal tube:

The pharyngeal region comprises the cranial-most portion of the GI tube; because it gives rise to the structures of the head and neck, this region is discussed in detail elsewhere.

Foregut

  • Supplied by the celiac artery
  • Gives rise to the:
    • Esophagus
    • Stomach
      – Liver buds, which ultimately form the liver
    • Gallbladder
    • Ventral and dorsal pancreatic buds (aka, diverticula), which will later fuse to form the pancreas
    • Proximal duodenum

Midgut

  • Supplied by the superior mesenteric artery
  • Comprises primary intestinal loop, which connects to the yolk sac via the vitelline duct
  • Gives rise to the:
    • Distal duodenum
    • Jejunum
    • Ileum
    • Ascending colon
    • Proximal 2/3 of the transverse colon

Hindgut

  • Supplied by the inferior mesenteric artery
  • Gives rise to the allantois before ending blindly at the cloaca
  • Gives rise to the:
    • Distal 1/3 of the transverse colon
      – Descending and sigmoid colons
    • The proximal 2/3 of the anorectal canal

The distal 1/3 of the anorectal canal is derived from ectoderm that invaginates the area around the proctodeum (aka, anal pit).

INNERVATION

The enteric nervous system, which is derived from neural crest cells, regulates motility to propel the contents of the GI tract.

MESENTERIES

Mesenteries divide the peritoneal cavity and suspend the gastrointestinal tract.
Additionally, they provide a protective covering for neurovascular structures.

  • The ventral mesentery is derived from the septum transversum, and will give rise to ligaments associated with the liver.
  • The dorsal mesentery secures the gastrointestinal tract to the posterior body wall.
    • The meso-esophagus attaches the esophagus to the dorsal wall
    • The mesogastrium anchors the stomach
    • The mesoduodenum anchors the duodenum
    • The mesentery proper anchors the primary loop of the midgut
    • The dorsal mesocolon anchors the hindgut

Mature Placenta

STRUCTURES AND FUNCTIONS OF THE MATURE PLACENTA, BEGINNING IN WEEK 9

  • The human placenta is hemochorial; that is, it allows direct contact between maternal blood and fetal tissues. As we’ll see, chorionic villi provide the surfaces for exchange of materials, and, therefore, are the functional units of the placenta.
  • Because fetal growth is dependent upon sufficient oxygen, nutrients, and other substances provided in the maternal blood, insufficient vascular remodeling has major clinical consequences, including fetal growth restriction, eclampsia, and even spontaneous abortion.
  • Derived from both maternal and offspring tissues.

Week 9

Differential growth and regression of the chorionic villi initiates formation of the mature placenta.

Key Structures:

  • Embryo is suspended within the amniotic cavity, which is bound by the amniotic membrane.
  • Chorion plate:
    • Chorion Frondosum grows thick with villi at the embryonic pole.
    • Chorion Laeve is the thinner area, where the villi regress.
    • Chorionic Cavity surrounds the amniotic cavity and embryo.
  • Endometrium -> Decidua:
    Its regions are named according to their location in reference to the embryo:
    • Decidua basalis, which comprises the decidua adjacent to the chorion frondosum.
    • Decidua capsularis, which comprises the decidua encapsulating the embryo.
    • Decidua parietalis, which comprises the decidua of the uterine wall opposite the embryo.

Week 20

Expansion of the embryo pushes these layers against each other, leading to fusion and the formation of the mature placenta; a mucous plug blocks the cervical opening.

Embryonic Pole:

  • Placenta comprises fused chorion frondosum and decidua basalis.

Abembryonic Pole:

  • Chorion laeve is greatly reduced, and fused with the amniotic membrane.
  • Decidua capsularis has regressed (due to expansion of the embryo).
  • Decidua parietalis fuses with the chorion laeve.

Placental anatomy

Growth of the fetus is driven by dynamic interactions between the mother and fetus. In healthy pregnancies, both fetal and maternal needs are met by physiological processes that operate for mutual benefit. However, when maternal health is poor, physiological compromises are made that put the mother and/or fetus at greater risk.
The placenta serves as an interface between maternal and fetal environments.

  • Fetal Side
    • Amniotic membrane gives the surface a smooth, shiny appearance.
    • Umbilical cord extends from the placenta to the fetus; holds umbilical arteries and vein.
    • Chorionic plate that gives rise to the chorionic villi (specifically, to the stem villi, which then give rise to branches). “Anchoring villi,” make contact with the decidua; “floating” villi do not.
    • Fetal blood vessels travel through the umbilical cord and within the chorionic plate to reach the villi.
  • Maternal Side
    • Decidual, aka, basal plate forms septa, which demarcate cotyledons; cotyledons give the maternal surface of the placenta a bumpy appearance.
  • Intervillous Space
    -The space between the chorion and decidual plate.
    • Maternal vasculature passes through the decidua and opens to the intervillous space; spiral arteries, which have a cork-screw like appearance, provide the arterial blood supply to the intervillous space.

Recall that the intervillous spaces are derived from connections formed between the synctiotrophoblast and maternal sinusoids during early embryonic development.

  • Transfer of materials occurs across the villous surface:
    Materials leave the maternal blood, enter the intervillous space, cross the villous surface, and then enter the fetal vessels.
    Wastes, etc. exit the fetal blood, cross the villous surface, enter the intervillous space, and enter the maternal veins.

Placental Physiologic Roles

The placenta is a temporary multi-functional organ; it performs many tasks that are taken over by various organ systems after birth.
Exchange of materials occurs in both directions across the placenta:

  • From the maternal side, oxygen, nutrients, hormones, antibodies, and some drugs enter the fetal circulation.
  • From the fetal side, carbon dioxide and other metabolic wastes, hormones, and red blood cell antigens enter the maternal circulation.
    Another important role of the placenta is hormone production, including:
  • Human chorionic growth hormone, estrogen, and progesterone, all of which are necessary for maintenance of the decidua and, therefore, the pregnancy.
  • Placental lactogen and growth hormone, which induce maternal insulin resistance later in pregnancy. By inducing insulin resistance, these hormones ensure that maternal nutrients are readily available for fetal growth.

Placental Blood Barrier

The placenta prohibits passage of many pathogens from the mother to the fetus.

  • However, there are several important exceptions to this rule, which can be summed up with the TORCH rule:
    Toxoplasmosis, Others, Rubella, Cytomegalovirus, and Herpes simplex can all traverse the placenta.
    • TORCHeS
  • The Zika virus is an example of a TORCHeS exception: as you are probably aware, the Zika virus can be transmitted from an infected mother to a developing offspring, resulting in serious birth defects, including microcephaly. Current research is focused on preventing its transmission across the placenta, thereby avoiding fetal infection.