21BTC101T - Biochemistry

UNIT - III

Introduction to Metabolism
Glycolysis
Citric acid cycle
Gluconeogenesis
Glycogen metabolism
-Glycogenesis
-Glycogenolysis
-Hormonal regulations - Muscle use of Glycogen
Blood glucose levels regulation by Insulin
-Biochemical aspects of Diabetes Mellitus
 Recap_Introduction to Metabolism
• Complex substances are broken down for energy, required metabolites,
structural components, etc.
• Cells must synthesize new complex substances.
• Thousands of such reactions are occurring simultaneously in a single cell.
• These reactions occur with a minimum of side products, energy loss or
undesired interferences and at reasonable temperatures, pH, and pressure.
• All of these reactions must be controlled or regulated for optimum
efficiency.
• Metabolism = all the
chemical reactions that occur in an
organism
• Catabolism = the
breakdown of complex substances.
• Cellular metabolism
– Cells break down excess carbohydrates
first, then lipids, finally amino acids if
energy needs are not met by
carbohydrates and fat
– Nutrients not used for energy are used
to build up structure, are stored, or
they are excreted
– 40% of the energy released in
catabolism is captured in ATP, the rest
is released as heat
• Anabolism = the synthesis of complex
substances from simpler ones.
– Performance of structural maintenance and repairs
– Support of growth
– Production of secretions
– Building of nutrient reserves
– Starting materials are pyruvate, acetyl CoA, and the intermediates of citric acid cycle
– Dependent on the supply of energy (ATP/GTP) and reducing equivalents (NADPH+,
H+)

NADPH:
• reduced form of
nicotinamide
adenine
dinucleotide
phosphate.
• a coenzyme used in
anabolic reactions,
such as lipid and
nucleic acid
synthesis
Dietary Carbohydrates Are Digested by Alpha-Amylase

• Our primary source of carbohydrates is starch.

• Several enzymes participate in carbohydrate digestion.
• α-Amylase initiates digestion by cleaving α-1,4 bonds but not α-
1,6 bonds.
• Other enzymes, including α-glucosidase and α-dextrinase,
complete the digestion.
• Sucrose and lactose, two common disaccharides, are digested by
sucrase and lactase, respectively.
• Glucose and galactose are transported into the intestine by the
sodium-glucose linked transporter, and the transporter GLUT5
allows entry of fructose.
Diagram of the Digestion of Starch by α-amylase
Diagram of the Uptake of Monosaccharides

Family of Glucose
Transporters
 Carbohydrate Metabolism
• Glucose is the central molecule in carbohydrate metabolism
– Fructose, galactose, and mannose enter the pathways at various points
• All cells can utilize glucose for energy production
– Fasting blood glucose level in normal individuals is 70-100 mg/dl
• Liver is central site for carbohydrate metabolism
– Key monitor and stabilizer of blood glucose levels
• Entry of glucose into cells
– Glucose uptake from blood to cells usually mediated by insulin and
transporters (GLUT-1 to GLUT-5 and GLUT-7)
• GLUT-1 abundant in RBCs and GLUT-4 abundant in skeletal muscle and
adipose tissue
– Glucose uptake independent of insulin in hepatocytes, erythrocytes,
and brain
– Glucose uptake dependent on insulin in muscle and adipose tissue
 Fate of Absorbed Glucose
• 1st Priority: glycogen storage
– Stored in muscle and liver
• 2nd Priority: provide energy
– Oxidized to ATP
• 3rd Priority: stored as fat
– Only excess glucose
– Stored as triglycerides in adipose
 Immediately after eating a meal…

High Blood Glucose

Pancreas

Insulin:
Muscle Glucagon Glycogen

Glucose absorbed Glucose absorbed

Adipose
Cells

Glucose absorbed
 Glycolysis (Embden-Meyerhof pathway)
 What is glycolysis?
• Ten step metabolic pathway to convert glucose (or glycogen) into two
molecules of pyruvate and two molecules each of NADH and ATP.
• All carbohydrates to be catabolized must enter the glycolytic pathway.
– Glycolysis is central in generating both energy and metabolic intermediaries.
• Glycolysis has two stages
– An energy investment phase.
• Reactions, 1-5. Glucose to two glyceraldehyde 3-phosphate molecules. Two
ATPs are invested.
– An energy payoff phase.
• Reactions 6-10. two glyceraldehyde 3-phosphate molecules to two pyruvate
plus four ATP molecules.
• A net of two ATP molecules overall plus two NADH.

NADH: reduced form of Nicotinamide adenine dinucleotide, a coenzyme found in all living
cells
 Salient features of Glycolysis
1. Glycolysis takes place in all cells of the body. Enzymes
are present in the cyotoplasm

2. Glycolysis occurs in the absence of oxygen (anaerobic) or

in the presence of oxygen (aerobic).
– Lactate is the end-product under anaerobic condition
Glucose+2ADP+2Pi → 2 Lactate+2ATP
– Pyruvate formed under aerobic condition is converted to CO2 and
H2O.
Glucose + 2 ADP + 2 Pi→2 Pyruvate + 2 ATP + 2 H2O

3. Major pathway for ATP generation in tissues lacking

mitochondria, e.g., RBCs, Cornea, etc.
 Salient features of Glycolysis (contd…….)
4. Glycolysis is very essential
for brain. Glucose has to Glucose (6C)
undergo glycolysis before it
2 ATP
is oxidized to CO2 and H2O.
4 ADP
2 ADP
5. It is central metabolic
4 ATP
pathway with many
intermediates that are useful 2
for the synthesis of amino NAD
acids and fat.
2 NADH + H
6. Reversal of glycolysis will
result in the synthesis of 2 Pyruvate (3C)
glucose (gluconeogenesis)
 Glucose utilization pathways
that provide or consume ATP

• (A) Schematic of key aspects of the

glycolytic pathway of glucose
utilization for energy metabolism
and major branch points that can
divert carbon for other uses,
including NADPH generation,
storage of glucosyl units in
glycogen, neuromodulator, and
amino acid and nucleotide
biosynthesis.
• (B) Major reactions and net ATP
yields or net ATP consumption of
major pathways derived from the
glycolytic pathway are indicated in
color-coded boxes that correspond
to the color-coded pathways in
panel A.
 Enzyme Classification - Recall
• Dehydrogenase- oxidizes substrate using cofactors as electron
acceptor or donor (pyruvate dehydrogenase)
• Reductase- adds electrons from some reduced cofactor (enoyl
ACP reductase)
• Kinase- phosphorylates substrate (hexokinase)
• Hydrolases - uses water to cleave a molecule
– Phosphatase- hydrolyzes phosphate esters (glucose-6-
phosphatase)
– Esterase (lipase)- hydrolyzes esters (those that act on lipid esters are
lipases) (lipoprotein lipase)
– Thioesterases - hydrolyzes thioesters
• Thiolase- uses thiol to assist in forming thioester (β-
ketothiolase)
• Isomerases- interconversions of isomers (example aldose to
ketose) (triose phosphate isomerase)
 Reactions of Glycolysis
• Sequence of reactions that converts
glucose into pyruvate
– Relatively small amount of energy
produced
– Glycolysis reactions occur in cytoplasm
– Does not require oxygen

• Three distinct phases in the pathway

– Energy investment phase
– Splitting phase
– Energy generation phase

Lactate (anaerobic)

Glucose → 2 Pyruvate
Acetyl-CoA (TCA cycle)
 Energy Investment Phase
1. Glucose is phosphorylated to Glucose 6-
phosphate by hexokinase or glucokinase
(isoenzymes)
– Irreversible reaction
– Dependent on ATP and Mg2+
– Hexokinase is present in all tissues and catalyzes
the phosphorylation of other hexoses, fructose,
mannose, etc. It is inhibited by Glu-6-phos.
– Glucokinase present only in liver catalyzes the
phosphorylation of glucose alone. Not inhibited by
Gluc-6-phos.
Model of Induced Fit in Hexokinase
– Glucose is utilized by hexokinase even at low
concentration whereas glucokinase acts only when
there is a high level of glucose (after a meal)
– Glucose 6-phosphate is impermeable to cell
membrane. Central molecule that can be part of
glycolysis, glycogenesis, gluconeogenesis, and
pentose phosphate pathway
2. Glucose 6-phosphate
undergoes isomerization
to give fructose 6-
phosphate in the presence
of
phosphoglucoisomerase
DID YOU KNOW?
The prefix bis- in bisphosphate means that
two separate monophosphoryl groups are
present, whereas the prefix di- in diphosphate
3. Fructose 6-phosphate is (as in adenosine diphosphate) means that two
phosphorylated to phosphoryl groups are present and are
fructose 1,6-bisphosphate connected by an anhydride linkage.

by phosphofructokinase.
This is a an irreversible
and regulatory step in
glycolysis.
 Splitting phase
4. The 6-carbon fructose 1,6-
bisphosphate is split to two 3-
carbon compounds,
glyceraldehyde 3-phosphate
and dihydroxyacetone
phosphate by the enzyme
aldolase

5. The enzyme triosephosphate

isomerase catalyzes the
reversible conversion of
glyceraldehyde 3-phosphate
and dihydroxyacetone
phosphate. Thus, 2 molecules
of glyceraldehyde 3-
phosphate are obtained from
1 molecule of glucose.
 Energy Generation phase
6. Glyceraldehyde 3-phosphate
dehydrogenase converts
glyceraldehyde 3-phosphate to 1,3-
bisphosphoglycerate. Important step
in which NADH + H+ and a high
energy compound, 1,3-
bisphosphoglycerate is formed. In
aerobic condition, NADH passes
through electron transport chain and 6
ATP (2 X 3 ATP) are synthesized by
oxidative phosphorylation.
7. The enzyme phosphoglycerate kinase
catalyzes the reaction in which 3-
phosphoglycerate is formed.
• Synthesis of ATP occurs
• Substrate level phosphorylation: ATP
synthesized from the substrate without
electron transport chain
• Reversible reaction, a rarity among kinase
reactions.
8. 3-phosphoglycerate is
converted to 2-
phosphoglycerate by
phosphoglycerate mutase.
It is an isomerization
reaction.

9. Enolase converts 2-
phosphoglycerate to the
high energy compound,
phosphoenol pyruvate.
• Enzyme requires Mg2+ Mg2+
• Inhibited by fluoride. In the
lab, fluoride is added to
prevent glycolysis by the
cells so that blood glucose
levels are correctly
estimated.
10.Pyruvate kinase
catalyzes the transfer
of phosphate from
phosphoenol
pyruvate to ADP
Balance sheet for high energy bonds of ATP:
resulting in the 2 ATP expended
4 ATP produced (2 from each of two 3C fragments
synthesis of ATP from glucose)
Net production of 2 ~P bonds of ATP per glucose
• Substrate level
phosphorylation
Glycolysis Pathway (omitting H+):
• Requires K+ and glucose + 2 NAD+ + 2 ADP + 2 Pi →
Mg2+
• Irreversible reaction pyruvate + 2 NADH + 2 ATP
Reactions of Glycolysis - Summary
 Regulation of glycolysis
• Three irreversible kinase reactions primarily
drive glycolysis forward.

 hexokinase or glucokinase
 phosphofructokinase
 pyruvate kinase

• Three of these enzymes regulate glycolysis as

well.
1. HEXOKINASE
• Phosphorylation of glucose.
• Inhibited by its product, glucose 6-phosphate, as a response to slowing of
glycolysis

2. PHOSPHOFRUCTOKINASE
• Major regulatory enzyme, rate limiting for glycolysis
• An allosteric multimeric regulatory enzyme.
Measures adequacy of energy levels.
• Inhibitors: ATP and citrate, high energy
• Activators: ADP, AMP, low energy and fructose 2,6 bisphosphate

3. PYRUVATE KINASE
• An allosteric tetramer
• Inhibitor: ATP, others: acetyl CoA and fatty acids (alternative fuels for TCA
cycle)
• Activator: fructose 1,6-bisphosphate (“feed-forward”)
 Pyruvate Metabolism

• Three fates of pyruvate:

1. Conversion to lactate (anaerobic)
2. Conversion to alanine (amino acid)
3. Entry into the TCA cycle via pyruvate
dehydrogenase pathway
 1. Conversion of pyruvate to lactate
• Under anaerobic condition, pyruvate is
reduced by NADH to lactate by lactate
dehydrogenase (competitive inhibitor)
• NADH utilized in this step is obtained from
the reaction catalyzed by glyceraldehyde 3-
phosphate dehydrogenase.
• Regenerated NAD can be reused by
glyceraldehyde 3-phosphate dehydrogenase
so that glycolysis proceeds even in the
absence of oxygen to supply ATP.
• Occurrence of uninterrupted glycolysis is
important for skeletal muscle during
strenuous exercise where oxygen supply is
limited.
• Glycolysis in the RBCs leads to lactate
production since mitochondria is absent.
Diagram of the Regulation of Glycolysis in Muscle
• Solution:
– Turn NADH back to NAD+ by making lactate (lactic acid)
(reduced) (oxidized)

 Lactate can be transported by blood to liver and used

in gluconeogenesis
 Lactate is converted to pyruvate in the liver
 2. Conversion to alanine (amino acid)
• Convert to alanine and export to blood

Glutamate -Ketoglutarate
COO– COO–
C O HC NH3+
Alanine amino transferase
CH3 (AAT) CH3
Pyruvate Alanine
Keto acid Amino acid
 3. Entry into the TCA cycle via pyruvate
dehydrogenase pathway

• Prepares pyruvate to enter the TCA cycle

Aerobic Conditions
Electron TCA Cycle
Transport
Chain
• Cancer and Glycolysis
– Cancer cells demonstrate increased uptake of glucose and glycolysis.
– Hypoxia due to inadequate supply of oxygen to the tumors results in
anaerobic glycolysis
– Cancer cells get adapted to hypoxic glycolysis through the involvement
of a transcription factor, Hypoxia-Inducible Factor (HIF)
– HIF increases the synthesis of glycolytic enzymes and glucose
transporters.
– For the tumor cells to grow, vascularization is important.
• Pasteur’s Effect
– Inhibition of glycolysis by oxygen is known as Pasteur’s
effect
– In the aerobic condition, the glycolytic intermediates from
fructose 1,6-bisphosphate onwards decreases while the
earlier intermediates accumulate.
– This indicates the Pasteur’s effect is due to the inhibition of
phosphofructokinase.
– The inhibitory effects of ATP and citrate on
phosphofructokinase causes Pasteur’s effect

• Crabtree effect
– Inhibition of oxygen consumption by the addition of
glucose to tissues having high aerobic glycolysis is known
as Crabtree effect
– Opposite to that of Pasteur’s effect
Diagram of the Link between Glycolysis
and the Citric Acid Cycle
 Conversion of pyruvate to acetyl CoA
• Pyruvate converted
to acetyl CoA by
oxidative
decarboxylation
• Irreversible reaction
catalyzed by
multienzyme
complex known as
pyruvate
dehydrogenase
complex (PDH)
• PDH found only in • PDH requires five cofactors (coenzymes):
mitochondria • thiamine pyrophosphate (TPP)
• High activities of • Lipoamide (Lipoic acid linked to e-amino
PDH in kidney, group of lysine)
cardiac muscle • flavin adenine dinucleotide (FAD)
• Coenzyme A
• NAD+
Pyruvate Dehydrogenase Complex of E. coli

Image of the Pyruvate

Dehydrogenase Complex from
B. stearothermophilus
The Synthesis of Acetyl Coenzyme A from Pyruvate Requires
Three Enzymes and Five Coenzymes
 Decarboxylation
The synthesis of acetyl coenzyme A from pyruvate
requires three enzymes and five coenzymes.
1. Decarboxylation:
– Pyruvate dehydrogenase (E1), a component
of the complex, catalyzes the
decarboxylation. Pyruvate combines with
the ionized form of the coenzyme thiamine
pyrophosphate (TPP).
Thiamine Pyrophosphate
The synthesis of acetyl
coenzyme A from pyruvate
requires three enzymes and
five coenzymes.
• Thiamine pyrophosphate,
a coenzyme, is derived
from the vitamin
thiamine.
 Oxidation
The synthesis of acetyl coenzyme A from
pyruvate requires three enzymes and five
coenzymes.
2. Oxidation:
– The hydroxyethyl group attached to
TPP is oxidized to form an acetyl
group while being simultaneously
transferred to lipoamide, a
derivative of lipoic acid. The
disulfide group of lipoamide is
reduced to its disulfhydryl form in
this reaction. The reaction is
catalyzed by E1 and yields acetyl–
lipoamide.
Lipoamide
• Lipoamide, a coenzyme, is formed by the attachment of the
vitamin lipoic acid to a lysine residue in another enzyme in the
complex, dihydrolipoyl transacetylase (E2).
 Formation of Acetyl CoA
The synthesis of acetyl coenzyme A from
pyruvate requires three enzymes and five
coenzymes.
3. Formation of acetyl CoA:
– E2 catalyzes the transfer of the
acetyl group from acetyl–
lipoamide to coenzyme A to form
acetyl CoA.

Dihydrolipoamide Dehydrogenase
The synthesis of acetyl coenzyme A
from pyruvate requires three enzymes
and five coenzymes.
• To participate in another reaction
cycle, dihydrolipoamide must be
reoxidized. This reaction is catalyzed
by dihydrolipoamide dehydrogenase
(E3).
 Diagram of the Reactions of the
Pyruvate Dehydrogenase Complex
• The intermediates of PDH catalyzed reaction
are not free but bound with enzyme complex
• A comparable enzyme of PDH is a-
ketoglutarate dehydrogenase complex of
TCA which catalyzes the formation of Brown rice is milled to remove only the outer

succinyl CoA from a-ketoglutarate. husk. Further milling (polishing) removes the
inner husk also, resulting in white rice

• Arsenite inhibits both the above systems

• PDH is good example of end-product (acetyl
CoA, NADH) inhibition.
– Lack of TPP (deficiency of thiamine) inhibits PDH
leading to accumulation of pyruvate
– In thiamine-deficient alcoholics, pyruvate is
converted to lactate leading to lactic acidosis
– In inherited deficiency of PDH, lactic acidosis is
observed
– PDH inhibited by arsenic and mercuric ions by
binding to –SH groups of lipoic acid
 Acetyl CoA
• Acetyl groups enter TCA as acetyl-coenzyyme A.
• Coenzyme A (CoASH or CoA) consists of a -mercaptoethylamine group bonded
via amide linkage to the vitamin pantothenic acid.
• Pantothenic acid is atttached to a 3’-phosphoadenosine moiety through a
pyrophosphate bridge.
• Acetyl group bonded as a thioester to the sulfhydryl portion of the -
mercaptoethylamine.
• CoA functions as a carrier of acetyl and acyl groups.
• Acetyl-CoA is a “high energy” compound (slightly more exergonic than ATP).

A high energy bond

Acetyl HS-CoA Adenosine 3-phosphate

Diagram of Cellular Respiration
Citric acid cycle (Krebs cycle or Tricarboxylic acid cycle)
• Important pathway for the energy
supply to the body
– About 65-70% of the ATP is synthesized in
Krebs cycle
• Involved oxidation of acetyl CoA to
CO2 and H2O
• Final common oxidative pathway for
carbohydrates, fats, and proteins
– It utilizes 2/3 of total oxygen consumed by
the body
– Provides intermediates for the synthesis of
amino acids, glucose, heme, etc.
• Enzymes are located in mitochondrial
matrix in close proximity to the
electron transport chain
 Overview of Citric acid cycle

• Involves combination of a
2-Carbon acetyl CoA with a
4-Carbon Oxaloacetate to
produce a 6-Carbon
tricarboxylic acid, Citrate.
• In subsequent reactions, the
2-Carbons oxidized to CO2
and Oxaloacetate is
regenerated and recycled.
• Oxaloacetate plays a
catalytic role
 Reactions of Citric acid cycle

1. Formation of citrate: DID YOU KNOW?

A synthase is an enzyme that catalyzes a
Oxaloacetate and acetyl synthetic reaction in which two units are
joined usually without the direct
CoA combine to form participation of ATP (or another nucleoside
triphosphate).
Citrate by the citrate
synthase

2. and 3. Citrate is
isomerized to isocitrate
by the aconitase through
the process of
dehydration followed by
hydration. Cis-aconitate
is formed as an
intermediate.
4. and 5. Formation of a-ketoglutarate:
Isocitrate dehydrogenase catalyzes the
conversion of isocitrate to oxalosuccinate
and then to, a-ketoglutarate. NADH is
formed and CO2 is released.
DID YOU KNOW?
Citric acid is stored in vacuoles in citrus fruits where
it can sometimes reach a concentration of 0.3 M. It
has a tart taste and provides some of the flavor of
citrus drinks.

6. Conversion of a-ketoglutarate to succinyl

CoA through oxidative decarboxylation
by a-ketoglutarate dehydrogenase
complex. Requires five cofactors, TPP,
lipoamide, NAD+, FAD, and CoA.
Second NADH is produced and CO2 is
liberated.

7. Formation of succinate: Succinyl CoA is

converted to succinate by succinyl CoA
synthetase. Involves phosphorylation of
GDP to GTP. This is substrate level
phosphorylation.
8. Conversion of succinate to fumarate:
Succinate is oxidized by succinate
dehydrogenase to fumarate. Results in the
production of FADH2.

9. Formation of malate: Fumarase catalyzes the

conversion of fumarate to malate with the
addition of H2O.

10. Conversion of malate to oxaloacetate:

Malate dehydrogenase oxidizes malate to
oxaloacetate. The third and final NADH is
synthesized at this stage. It combines with
another molecule of acetyl CoA and continues
with the cycle. DID YOU KNOW?
Apples are a rich source of malic acid, which used
to be called “acid of apples.” In fact, the word malic
is derived from the Latin malum, meaning “apple.”
 Overall Reaction of Citric acid cycle

acetyl-CoA + 3NAD+ + FAD + GDP + Pi+2H2O  2CO2 + 3NADH + FADH2 + GTP + 2H+
+ CoA

 high energy compounds made

 four pairs of electrons are made available

to the respiratory chain and oxidative
phosphorylation. These are used to generate
most of the ATP needed.
 Maximum yield of high energy ATP in the aerobic
catabolism of glucose
• Glycolysis:
glucose 2pyruvate + 2NADH+2ATP 8 ATPs
• Pyruvate Dehydrogenase:
2pyruvate  2acetyl CoA + 2NADH 6 ATPs
• TCA cycle:
acetyl CoA2CO2+3NADH+FADH2+GTP 24 ATPs

(6 X 3 ATP= 18 ATP)

(2 X 2 ATP =4 ATP)
• OVERALL yield from glucose 38 ATPs
 Regulation of Citric acid cycle
• Three enzymes, citrate synthase, isocitrate
dehydrogenase, and a-ketoglutarate dehydrogenase,
regulate the cycle

1. CITRATE SYNTHASE
• Inhibited by ATP, NADH, acetyl CoA, and succinyl CoA

2. ISOCITRATE DEHYDROGENASE
• activated by ADP and
• inhibited by ATP, NADH

3. a- KETOGLUTARATE DEHYDROGENASE
• inhibited by products (succinyl-CoA and NADH)
 Requirement of O2 by Citric acid cycle
• No direct participation of oxygen in Krebs cycle

• Cycle operates only under aerobic condition

– NAD and FAD required for the operation of the cycle
can be regenerated in the respiratory chain only in the
presence of O2

• NOTE: Major regulator is intramitochondrial

NAD+/NADH ratio.
 High oxygen level results in an increased ratio
and low level a decreased ratio.
 A measure of oxygen availability
Replacement of Intermediates

• Intermediates of the
citric acid cycle are
precursors to the
synthesis of many
compounds needed for
the body.

• It is both anabolic and

catabolic in nature, and
hence, regarded as
amphibolic
• Oxaloacetate and a-ketoglutarate, respectively, serve as
• Involved in precursors to aspartate and glutamate which are then used
for the synthesis of non-essential AAs, purines and
gluconeogenesis,
pyrimidines
transamination, and • Succinyl CoA for the synthesis of porphyrins and heme
deamination. • Mitrochondrial citrate moves to cytosol where it is cleaved
to acetyl CoA for the biosynthesis of FAs, steroids, etc.
 The Anaplerotic or “Filling Up” Reactions
• Intermediates used for the
synthesis of many compounds,
as described above, need to be
replenished
• Reactions concerned to
replenishment are called
anaplerotic reactions. (Greek:
fill up)
1. Pyruvate carboxylase converts 3. From amino acids
 reversals of transaminations
pyruvate to oxaloacetate in an
-- restores oxaloacetate or
ATP-dependent reaction a-ketoglutarate with abundant
(Gluconeogenesis) asp or glu
2. Pyruvate is converted to malate by  glutamate dehydrogenase
NADP-dependent malate glu + NAD(P)+  a-ketoglutarate
dehydrogenase (malic enzyme) + NAD(P)H + NH4+
 Gluconeogenesis
• The biosynthesis of glucose from non-carbohydrate
compounds
– From pyruvate, lactate, glucogenic amino acids, propionate
and glycerol
Notice glucose cannot be made from acetyl CoA
• Why do we need extra glucose?
– a) Need to maintain glucose levels within a narrow range in
blood especially, during fasting.
– b) Some tissue-- brain, erythrocytes, and muscles in exertion
use glucose at a rapid rate and sometimes require glucose in
addition to dietary glucose.
• The brain uses mostly glucose and erythrocytes can use only glucose
as a source of energy
– c) certain metabolites produced in the tissues accumulate in
the blood, e.g., glycerol, lactate, propionate, etc.
Where is glucose synthesized
during gluconeogenesis?
• Occurs mainly in the cytosol,
although some precursors are
synthesized in the mitochondria
• Mainly takes place in the liver
and to some extent, in the kidney
matrix.

• The gluconeogenesis pathway is

similar to the reverse of
glycolysis but differs at critical
sites.
control of these opposing
pathways is reciprocal so that
physiological conditions
favoring one disfavor the other
and vice versa.
 Reactions of Gluconeogenesis
• The 3 irreversible steps of
glycolysis catalyzed by
hexokinase,
phosphofructokinase, and
pyruvate kinase are bypassed by
alternate enzymes.
– Conversion of pyruvate to
phosphoenolpyruvate by pyruvate
caroboxylase
– Conversion of fructose 1,6-
bisphosphate to fructose 6-
phosphate by fructose 1,6-
bisphosphatase
– Conversion of glucose 6-phosphate
to glucose by glucose 6-phosphatase
 Bypass number 1: Pyruvate to phosphoenolpyruvate

• Pyruvate carboxylase is a biotin-

dependent mitochondrial enzyme
requires ATP, CO2 and Acetyl CoA
– acetyl-CoA is positive modulator
– absolutely required for activity
Pyruvate carboxylase is a
compartmentalized
reaction. Pyruvate is
• The two enzymes that catalyze the converted to oxaloacetate
reactions for bypass of the Pyruvate in the mitochondria.
Because oxaloacetate
Kinase reaction are the following: cannot be transported
• (a) Pyruvate Carboxylase catalyzes: across the mitochondrial
membrane, it must be
pyruvate + HCO3- + ATP → reduced to malate,
oxaloacetate + ADP + Pi transported to the
cytosol, and then
• (b) PEP Carboxykinase catalyzes: oxidized back to
oxaloacetate + GTP → oxaloacetate before
gluconeogenesis can
phosphoenolpyruvate + GDP + CO2 continue.
Bypass number 2: Fructose 1,6-bisphosphate to fructose 6-
phosphate
• PEP undergoes the reversal
of glycolysis until fructose
1,6-bisphosphate is
produced by fructose 1,6-
bisphosphatase
• Fructose 1,6-bisphosphate
+ H2O → Fructose 6-
phosphate + Pi
• A simple hydrolysis,
irreversible
• enzyme is highly regulated
and requires Mg2+
Bypass number 3: Glucose-6-phosphate to glucose
• Glucose 6-phosphatase
catalyzes the conversion of
glucose 6-phosphate to glucose
• Enzyme present in liver and
kidney but absent in muscle,
brain, and adipose tissue
• bypasses hexokinase and
glucokinase
• irreversible
 Net Reaction for gluconeogenesis
• 2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 6 H2O → Glucose
+ 2 NAD+ + 4 ADP + 2 GDP + 6 Pi.
• Glycolysis yields 2 ~P bonds of ATP.
Gluconeogenesis expends 6 ~P bonds of ATP and GTP
• To prevent this waste, Glycolysis and Gluconeogenesis pathways are
reciprocally regulated
• Local Control includes reciprocal allosteric regulation by adenine
nucleotides.
– Phosphofructokinase (Glycolysis) is inhibited by ATP and stimulated
by AMP.
– Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by AMP.
– This insures that when cellular ATP is high (AMP would then be low),
glucose is not degraded to make ATP. When ATP is high it is more useful
to the cell to store glucose as glycogen.
– When ATP is low (AMP would then be high), the cell does not expend
energy in synthesizing glucose.
• Global Control in liver cells includes reciprocal
effects of a cyclic AMP cascade, triggered by the
hormone glucagon (secreted by a-cells of the
pancreatic islets) when blood glucose is low.

• Phosphorylation of enzymes and regulatory proteins

in liver by Protein Kinase A (cAMP-Dependent
Protein Kinase) results in inhibition of glycolysis and
stimulation of gluconeogenesis, making glucose
available for release to the blood.
 Cori Cycle
• Lactate is the primary source for pyruvate.
-- In muscle, lactate is produced in great quantities during exertion.
-- This excess lactate cannot be further oxidized in muscle.
-- Lactate is released from the muscles to the blood and travels to the liver
for conversion to pyruvate and, ultimately to glucose for reuse by non-
hepatic tissues.
-- Note that the gluconeogenic leg of the cycle (on its own) is a net
consumer of energy, costing the body 4 moles of ATP more than are
produced during glycolysis. Therefore, the cycle cannot be sustained
indefinitely.
 Glucose-alanine cycle
• Amino group from excess glutamate
produced in muscle as a result of
amino acid catabolism, is transferred
to pyruvate resulting in the formation
of alanine.
• Alanine is another safe way to
transport ammonia from muscle to
liver via blood.
• In liver alanine aminotransferase
transfers the amino group to glutarate
and pyruvate regenerated is used in
gluconeogenesis.
• Glucose produced by gluconeogenesis
is transported to muscle where it
enters the glycolysis.
• Thus, the excess puruvate and
ammonia generated in muscle are
safely transported to liver.
 Glycogen Metabolism
• Glucose is stored as glycogen (animal Starch) predominantly in
liver and muscle cells.
– Stored in cytosol where the enzymes for synthesis and breakdown are
present
• Liver glycogen maintains blood glucose levels. Muscle
glycogen is fuel reserve during muscle contraction.
• Glycogen is a fuel reserve because
– It can be rapidly mobilized
– It can generate energy in the absence of oxygen
– Brain depends on continuous glucose supply (comes mostly from
glycogen)
• Fat mobilization is slow, needs O2 for energy production, and
can not produce glucose to that extent.
– Fat is considered as Fixed Deposit while
– Glycogen is the Current/Savings Account in a Bank.
 Glycogen structure
• Glycogen is a polymer of
glucose residues linked
mainly by a(1→4)
glycosidic linkages. There
are a(1→6) linkages at
branch points.

• Glucose can be rapidly

delivered to the blood Schematic 2-D
stream when needed cross-sectional view
upondegradation of of glycogen.
glycogen =
At the core of the
glycogenolysis Glycogen particle is
• Enough glucose and a protein enzyme
energy triggers synthesis which starts the
of glycogen = polymerisation
glycogenesis process.
Glycogen Is an Efficient Storage Form of Glucose
• Only two molecules of ATP are required to incorporate
dietary glucose into glycogen.
• The complete oxidation of glucose derived from glycogen
yields 31 molecules of ATP.
 Glycogenesis
STEP 1: Glucose is transported into the liver cell by a specific glucose
transporter and immediately phosphorylated.
• Most of the glucose in a cell is in the form of glucose-6-phosphate.
• Conversion of glucose-6-phosphate to glucose-1-phosphate by
phosphoglucomutase
• Reversible reaction allows G1P conversion to G6P in
glycogenolysis
• A serine hydroxyl at the active site donates and accepts phosphate.
The enzyme-bound bisphosphate intermediate is not released.
STEP 2: Uridine diphosphate glucose (UDP-glucose) is the
immediate precursor for glycogen synthesis.
• As glucose residues are added to glycogen, UDP-glucose is
the substrate and UDP is released as a reaction product.
– Nucleotide diphosphate sugars are precursors also for synthesis of
other complex carbohydrates, including oligosaccharide chains of
glycoproteins, etc.
• UDP-glucose is formed from glucose-1-phosphate and uridine
triphosphate (UTP) by UDP-glucose pyrophosphorylase

• glucose-1-phosphate + UTP → UDP-glucose + PPi

• PPi + H2O → 2 Pi
• Overall: glucose-1-phosphate + UTP → UDP-
glucose + 2 Pi

Spontaneous hydrolysis of the ~ bond in PPi (P~P)

drives the overall reaction.

Cleavage of PPi is the only energy cost

for glycogen synthesis (one ~P bond per
glucose residue).
STEP 3: Glycogen synthase catalyzes elongation of
glycogen chains. Glycogen synthase catalyzes transfer
of the glucose moiety of UDP-glucose to the hydroxyl
at C4 of the terminal residue of a glycogen chain to
form an a(1 4) glycosidic linkage
• glycogen (n residues) + UDP-glucose  glycogen (n +1 residues) + UDP

• A branching enzyme transfers a segment from the end

of a glycogen chain to the C6 hydroxyl of a glucose
residue of glycogen to yield a branch with an a(16)
linkage
 Glycogenenolysis in tissues
• Degradation of stored glycogen in liver and muscle
• Pathways of synthesis and degreadation of glycogen are
not reversible
• There are separate set of enzymes in the cytosol carry
out glycogenolysis
– glycogen broken down at a(14) and a(16)
glycosidic linkages
STEP1: Glycogen phosphorylase catalyzes phosphorolytic
cleavage of the a(14) glycosidic linkages of glycogen,
releasing glucose-1-phosphate as the reaction product.
– Pyridoxal phosphate (PLP), a derivative of vitamin B6, serves
as prosthetic group for Glycogen Phosphorylase
– Glycogen (n residues) + Pi  glycogen (n-1 residues)
+ glucose-1-phosphate
STEP 2: Debranching enzyme has two independent active
sites, consisting of residues in different segments of a single
polypeptide chain, that catalyze a(16) glucosidase and
transferase (transglycosylase) reactions.

• The transferase of the debranching enzyme transfers three

glucose residues from a 4-residue limit branch to the end of
another branch, diminishing the limit branch to a single
glucose residue.
• The a(16) glucosidase moiety of the debranching enzyme
then catalyzes hydrolysis of the a(16) linkage, yielding free
glucose. This is a minor fraction of glucose released from
glycogen. The major product of glycogen breakdown is
glucose-1-phosphate, arising from Phosphorylase activity.
STEP 3: Phosphoglucomutase catalyzes the reversible reaction:
• Glucose-1-phosphate  Glucose-6-phosphate
• The glucose-6-phosphate product may enter Glycolysis or
(mainly in liver) be dephosphorylated for release to the blood.
The liver enzyme Glucose-6-phosphatase catalyzes the following
reaction, essential to the liver's role in maintaining blood glucose:
Glucose-6-phosphate + H2O  glucose + Pi.
• Most other tissues lack this enzyme.
 Hormones regulate Muscle use of Glycogen
• Muscle use of glycogen as
fuel occurs immediately
after onset of exercise and
last for about an hour.
• Glycogenolysis is induced
by three factors in muscle:
– Nerve impulses that activate
muscle contraction act to
release calcium stores from
the sarcoplasmic reticulum.
Ca2+-calmodulin increases
glycogen phosphorylase
--Muscle contraction uses ATP, and
– Epinephrine binding to its cell production of AMP from ADP is another
surface receptor activate the activator of glycogen phosphorylase
AC/cAMP/PKA pathway, --In liver, glycogenolyis is positively
inducing glycogen regulated by glucagon and epinephrine
phosphorylase and negatively regulated by insulin.
Effect of glucagon and epinephrine: promote the
breakdown of both glycogen & fat
on glycogen on fatty acid mobilization.
metabolism.
 Summary of Glycogen Metabolism
• Epinephrine and glucagon
stimulate glycogenolysis and
inhibit glycogenesis via a
cAMP and a phosphorylation
cascade.
 releases glucose
• Glycogenesis is stimulated by
insulin in a pathway ending in
the dephosphorylation of
glycogen synthase.
Glycogenolysis is also nhibited
via dephosphorylation.
 takes up glucose
Cellular locations of major metabolic pathways.
 Biochemical aspects of Diabetes Mellitus

• A group of diseases characterized by

high levels of blood glucose resulting
from defects in insulin production,
insulin action, or both
• Consists of 3 types:
1) Type 1 diabetes
2) Type 2 diabetes
3) Gestational diabetes
¨ Complications :
- Stroke
- Heart attack
Banting, right, and Best, left, with one of the
- Kidney disease diabetic dogs used in experiments with insulin. In
- Eye Disease 1923, the Nobel Committee decided to award
Banting and Macleod the Nobel Prize in
- Nerve Damage Physiology or Medicine
• 1 million microscopic units – the islets of
Langerhans
• 4 most important cell types of the islets
are:
• Β (beta): constitute 70% of the cells and
contain insulin
• A (alpha): 20% of the cells and
elaborate glucagon
• D (delta): secrets somatostatin which
suppresses the insulin and glucagon
secretion
• PP (pancreatic polypeptide): unknown
physiologic function
 Carbohydrate Digestion - Recall
• The complex polysaccharide starch is broken down into glucose by the enzymes
amylase and maltase (secreted by the small intestine).
• CNS/Brain
– Dependent on glucose as primary source of fuel
– Uses ~120g glucose/day of total 160-200 g/d
• RBC
– Dependent on glucose
– Lack mitochondria
 Regulation of blood glucose levels by Insulin
Anabolic in response to hyperglycemia
• Liver: Stimulates glycogen synthesis, glycolysis, and fatty acid
synthesis
• Muscle: Stimulates glycogen synthesis
• Adipose: Stimulates lipoprotein lipase resulting in uptake of fatty
acids from chylomicrons and VLDL
– Stimulates glycolysis for glycerol phosphate synthesis (precurser
to triglycerides)
Diagram of How Insulin Release is Regulated by ATP

Animation showing overview of diabetes:

http://www.healthscout.com/animation/1/34/main.html
Animation showing mechanism of action of insulin:
http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/pancreas/insulin_phys.
html
 Insulin Secretion by b-cells in Pancreas
• Rising blood glucose levels triggers
insulin secretion by b-cells.
• Glucose is uptaken by the GLUT2
transporter and the glycolytic
phosphorylation of glucose causes a rise
in the ATP:ADP ratio.
• Potassium channels are then inactivated
that depolarizes the membrane, causing
the calcium channel to open up allowing
calcium ions to flow inward.
• Rise in calcium leads to the exocytotic
release of insulin from their storage
granule.
• The process by which insulin is released
from b-cells, in response to changes in
blood glucose concentration, is a
complex and interesting mechanism that
illustrates the intricate nature of insulin
regulation.
 Transport of glucose into the cell
• Insulin molecules circulate throughout
the blood stream until they bind to their
associated (insulin) receptors.
• The insulin receptors promote the uptake
of glucose into various tissues (skeletal
muscle and fat tissues) that contain type
4 glucose transporters (GLUT4).
• The initial binding of insulin to its
receptor initiates a signal transduction
cascade that results in the removal of
glucose from blood plasma
• The key step in glucose metabolism is
the immediate activation and increased
levels of GLUT4 glucose transporters.
• By the facilitative transport of glucose
into the cells, the glucose transporters
effectively remove glucose from the
blood stream
 Diabetes Mellitus - DM
• Type 1 Diabetes
- cells that produce insulin are
destroyed
- results in insulin dependence
- commonly detected before 30
• Type 2 Diabetes
- blood glucose levels rise due to
1) Lack of insulin
production
2) Insufficient insulin
action (resistant cells)
- commonly detected after 40
- effects > 90%
- eventually leads to β-cell failure
(resulting in insulin dependence)
Gestational Diabetes
3-5% of pregnant women are prone to
develop gestational diabetes
 Criteria for the Diagnosis of DM

Fasting Plasma Glucose Oral Glucose Tolerance

Test (FPG) - (cheap, Test (OGTT)
fast) *tested for 2 hrs after
*fasting B.G.L. 100-125 glucose - rich drink
mg/dl signals pre- *140-199 mg/dl signals
diabetes pre - diabetes
*>126 mg/dl signals *>200 mg/dl signals
diabetes diabetes

• glucose attaches to the amino group of proteins without the aid of

enzymes.
• nonenzymatic glycosylation of hemoglobin A (HbA) leads to the
formation of HbA1c, which normally constitutes about 4% of
hemoglobin in the red blood cells.
• HbA1c level provides an index of the average blood glucose level over
the preceding 2-4 months
• The results of DCCT trial indicate that a HbA1c value in the 7.2 –
8.0% range (blood glucose concentration of 155 mg/dL) significantly
reduces the appearance and progression of microvascular
complications.
• stimulation of
glycogenolysis, which is
normally inhibited by
insulin and favored by
glucagon. Fasting blood
glucose may reach levels
many times greater than
normal

• exceeds the renal

threshold = glycosuria

• glycosuria induces an
osmotic diuresis and thus
polyuria, causing a
profound loss of water and
electrolytes.
• water loss AND hyperosmolarity = depleted intracellular
water in the osmoreceptors of the thirst in the brain = intense
thirst (polydipsia).

• Through poorly defined pathways, increased appetite

(polyphagia) develops, thus completing the classic triad of
diabetic findings: “polyuria, polydipsia, and polyphagia”.

• With a deficiency of insulin, the scales swing from insulin-

promoted anabolism to catabolism of proteins and fat.

• Two important acute metabolic complications of diabetes are:

ketoacidosis, and non-ketotic hyperosmolar coma.
 Intracellular Hyperglycemia with Disturbance in the Polyol Pathways

• In nerves, lens, kidney and blood vessels, hyperglycemia leads to

increase in intracellular glucose (do not require insulin for glucose
transport)
• polyol pathway - glucose is reduced to sorbitol by the enzyme aldol
reductase
• Sorbitol = tissue toxin and has been implicated in the pathogenesis of
diabetic complications. Sorbitol decreases phosphoinositide
metabolism and signal transduction.
• Aldol reductase inhibition also has been shown to prevent
experimental cataracts and retinopathy.

Cardiovascular disorders: Atherosclerosis Sensory Neuropathy

• In the past it has been common to attribute
– microvascular complications (retinopathy, nephropathy,
neuropathy) to the polyol pathway
– macrovascular complications (stroke, gangrene, myocardial
infarction) to glycation and advanced glycosylation products.
• However both pathways contribute to both sets of complications.

Fovea
A cross-section of the Hemorrhage
kidney showing
ischemic pyramids and
sclerotic arteries and
arterioles.
Albuminuria,
declining GFR, and
hypertension—are the
symptoms of diabetic
nephropathy
Retinopathy
 Who needs insulin?
• Type I (insulin dependent) diabetes patients whose body produces no
insulin.
• Type 2 diabetes patients that do not always produce enough insulin.
• Subcutaneous injection of insulin
Stage 1 Insulin was extracted from the glands of cows and pigs. (1920s)

Stage 2 Convert pig insulin into human insulin by removing the one amino
acid that distinguishes them and replacing it with the human version.

Stage 3 Insert the human insulin gene into E. coli and culture the
recombinant E. coli to produce insulin (trade name = Humulin®). Yeast is
Recombinant DNA technology has also made it possible to
manufacture slightly-modified forms of human insulin that work
faster (Humalog® and NovoLog®) or slower (Lantus®) than
regular human insulin.
 Diabetes – Oral Medications Summary
6 Classes :

• Sulfonylureas [Glucotrol (glipizide), Amaryl (glimepiride)] :

stimulate β cells
• Biguanides [Metformin, Glucophage®, Fortamet®, Riomet®]:
improves insulin’s ability to move glucose
• Sulfonylureas and biguanide combination drugs [Glucovance®
(Glyburide & Metformine HCl)]: BOTH
• Thiazolidinediones [Pioglitazone- Actos®, Avandia®]: cells
more sensitive to insulin
• Alpha-glycosidase inhibitors [Precose ® (acarbose), Glyset ®
(miglitol)]: Block enzymes that help digest starches
• Meglitinides [Prandin ® (repaglinide); -Starlix ® (nateglinide)] :
stimulate β cells (dependant upon glucose conc.)