GLYCOGEN METABOLISM Dr Vivek Joshi, MD

CONTENTS Introduction Biomedical importance Glycogenesis (Glycogen Synthesis) Glycogenolysis (Glycogen Breakdown) Regulation of Glycogen Metabolism Glycogen Storage Disease (GSD)

Glycogen Metabolism Glycogenesis- Synthesis of Glycogen Glycogenolysis- Breakdown of Glycogen Glycogen Carbohydrate Polysaccharide Homopolysaccharide Polymer of Glucose Chain of Glucosyl units linked by α (1 - 4) with α (1 - 6) branch every 8-10 residues.

Glycogen-Ideal storage form Glucose residues in a linear sequence are linked to each other by a 1→4 glycosidic linkage Branched hompolysaccharide of D-glucose Branches every 8-12 residues along the chain, linked as 1→6 linkages Branch

Glycogen –Ideal Storage Form NO OSMOTIC PRESSURE HIGHLY BRANCHED-READILY BROKEN DOWN-HORMONES &ENZYMES WATER SOLUBLE MORE FREE GLUCOSE RESIDUES-NON REDUCING END

Single reducing end bound to Glycogenin Glycogen –Storage Form Single reducing end bound to Glycogenin Nonreducing ends Gn Single reducing end bound to Glycogenin 6

Glycogen breakdown yields Glucose Brain &RBC’S-Dependent on Glucose Replenish Glucose in between meals, sleep, Fasting and Early Starvation Muscle Glycogen-Energy Liver Glycogen-Maintains Blood Glucose(12-14 hrs of Fasting) Source of Glucose in the body: Diet-Inconsistent Gluconeogenesis-SLOW 3. Glycogenolysis-Rapid release of Glucose Liver Weight -1800 g Muscle mass-35 Kg

LIVER Glycogen maintenance of blood glucose levels Stores about 50-100gms of glycogen. Amounts to 200-300gms with water of hydration Glycogen used for maintenance of blood glucose levels Lasts about 12-14 hrs MUSCLE Glycogen as a body energy store Stores about 400-500gm in an average adult Not available for blood glucose maintenance due to absent Glucose 6-phosphatase Provides energy during bursts of muscle activity

Blood Glucose Levels Hyperglycemia – worried about diabetes mellitus Hypoglycemia – can cause death (within minutes)

Source of Glucose in the body Carbohydrates in diet Sporadic Frequency unreliable Glycogen Significant storage Rapidly mobilized - fast response Total hepatic storage-enough to maintain blood glucose levels during a 12-14 hr fast Gluconeogenesis Slow in reacting to a fall in blood glucose levels Advantage of glycogen – it is there and can be quickly used. Enough to last many hours.

Source of Glucose in the body - Exogenous (diet-derived) carbohydrate lasts for only a few hours, - Glycogen for approximately one day. - Gluconeogenesis is required to maintain blood glucose levels during extended carbohydrate starvation The graph, based on observations of a number of individuals, illustrates glucose utilization in a 70kg man who consumed 100g of glucose and then fasted for 40 days ___________________ Hour or two after ingesting food, lots of exogenous glucose is picked up. Glycogen fills the gap before gluconeogenesis kicks in. it can run until the stores run out.

Glycogen synthesis OR Glycogenesis Sites of Synthesis: Most cells of the body significantly in the liver and the muscle Sub cellular site: Cytosol Steps: Glucose uptake -GLUT2 and GLUT4 Conversion of Glucose to Glucose -6-P Conversion of Glucose-6-P to UDP-Glucose Elongation of chain: Glycogen Synthase (Glycogen Fragment/Glycogenin to Initiate Glycogen synthesis) Introduction of branches: Branching enzyme High Carbohydrate meal- Glucose uptake by Glucose transporters

GLUCOSE TRANSPORTERS (GLUT) Name Tissues Km,Glucose Functions GLUT I Most tissues (Brain, Red cells) ~1 mM Basal uptake of glucose GLUT 2 Liver Pancreatic beta cells   ~15 mM Uptake and release of glucose by the liver Beta –cell Glucose sensor GLUT 3 Basal uptake GLUT 4 Skeletal muscle Adipose tissue ~5mM Insulin-stimulated glucose uptake ; Stimulated by exercise in skeletal muscle Normal Blood Glucose Level : 70-100 mg/dl 4-5.5 mmol/L

Glycogen synthesis OR Glycogenesis Branching enzyme [ Amylo -(1 4) (1 6) transglucosidase] transfers 6-8 residues to the neighbouring branch to create a branch point[ α (1 6) linkage] The above processes continues till a highly branched Glycogen is formed. Rate limiting enzyme for synthesis of Glycogen- Glycogen Synthase

Glycogen synthesis OR Glycogenesis Glucose uptake (GLUT2 in the Liver/ GLUT4 in the Muscle) Formation of UDP-Glucose Glucose ATP Hexokinase /Glucokinase ADP Glucose-6-P Phosphoglucomutase Glucose-1-P UDP glucose transfers glucose to the nonreducing end of the growing chain of glycogen UDP Glucose is essential for the synthesis of Glycoconjugates and other sugars GLUT- Glucose Transporters,Facilitative bidirectional transport, Five types - GLUT-1:Brain,Kidney,Placenta-Uptake of Glucose - GLUT-2: Liver,Kidney,Small Intestine,Pancreas- Rapid uptake and release of Glucose - GLUT-3: Brain,Kidney ,placenta- Uptake of Glucose - GLUT-4: Heart,Skeletal muscle,adipose tissue- Insulin stimulated uptake of Glucose -GLUT-5: Small Intestine- Absorption of Glucose Sodium dependent unidirectional transporter -SGLT1&2:Small intestine &kidney-Active uptake of glucose from lumen of intestine and reabsorption of glucose in proximal tubule of kidney against the concentration gradient Glucokinase-High Km, Not Inhibited by Glucose-6-phosphate,Hepatic Tissues Hexokinase-Low Km, Inhibited by Glucose-6-phosphate,Extrahepatic tissues UTP 2Pi PPi Hydrolysis of PPi drives the reaction forwards UDP-Glucose 15

Glycogen synthesis OR Glycogenesis Glycogen Fragment/Glycogenin to Initiate Glycogen synthesis Glycogenin Glucose Glucose Glucose Tyrosine The core sequence is bound to a protein, Glycogenin 16

Elongation- Glycogen Synthase Glycogenin/Pre Existing Glycogen Fragment ? Glycogen Synthase Branching Enzyme UDP UDP -Glucose Branching Enzyme (Glucosyl 4:6 Transferase) Elongation- Glycogen Synthase

Summary of Glycogen Synthesis

Glycogen Breakdown OR Glycogenolysis NOT a reverse of Glycogenesis but a separate pathway Site-Liver, Muscle Glycogen degradation requires the activity of two enzymes Glycogen Phosphorylase Debranching enzyme

Glycogenolysis Glycogen Phosphorylase Removes Glucose residues from the non reducing end of Glycogen . It utilises the cytosolic phosphate for the above process and releases glucose from glycogen as Glucose-1-PO4 The above process continues till 3-4 residues of Glucose remain from the branch point. Debranching enzyme has dual activity- α (1 - 4) , α (1 - 4) glucan transferase activity AND α (1 - 6) transglucosidase activity.

Glycogenolysis Debranching Enzyme Dual Enzyme activity [-(1 4) -(1 4) glucan transferase activity- Transfers 3 glucose residues to the neighbouring branch to expose the branch point [Amylo (1 6) transglucosidase activity breaks the branch point to release free Glucose. 90% of Glucose released from Glycogen as Glucose-1-PO4 and 10% as free Glucose Rate limiting enzyme for Glycogen Degradation- Glycogen Phosphorylase

Glycogenolysis ? GLUCOSE GLYCOGEN PHOSPHORYLASE DEBRANCHING ENZYME Pi Glucose-1-PO4 Glycogenolysis

Regulation of Glycogen Metabolism Allosteric regulation Glycogen Synthesis stimulated at high levels of energy and substrate Glycogen Degradation increased when energy and glucose supplies are low

Allosteric regulation- Glycogen Metabolism in the LIVER Short Term Regulation Allosteric regulation Covalent modification Long Term Regulation: Induction and Repression

Allosteric regulation- Glycogen Metabolism in the MUSCLE

MAJOR HORMONES IN METABOLISM

WATER SOLUBLE HORMONES IN METABOLISM Liver 28

INSULIN-ANABOLIC HORMONE

INSULIN v/s GLUCAGON GLUCAGON INSULIN Phosphorylated form of the enzyme may be active/Inactive depending on the type of hormone example Dephosphorylated form may be active/inactive depending on the type of hormone example INSULIN

Glycogen Phosphorylase Glycogen Phosphorylase GLUCAGON Glycogen Phosphorylase (Glycogen Breakdown) ACTIVE Glycogen Synthase (Glycogen synthesis) LESS ACTIVE Glycogen Phosphorylase (Glycogen Breakdown) LESS ACTIVE Glycogen Synthase (Glycogen synthesis) ACTIVE INSULIN

INSULIN- Dephosphorylates key enzyme Liver and Muscle GLUCAGON Liver GLYCOGEN SYNTHASE INSULIN- Dephosphorylates key enzyme GLUCAGON/EPINEPHRINE- Phosphorylates key enzyme GLYCOGEN PHOSPHORYLASE Glucagon(Glucagon Receptors) and Epinephrine(Beta Adrenergic receptors) Phosphorylates key enzyme GLUCAGON Liver EPINEPHRINE Liver and Muscle Ca++ and AMP Muscle

The muscle does not have receptors for Glucagon Covalent Modification- Glycogen Metabolism The muscle does not have receptors for Glucagon

Covalent Modification- Glycogen Metabolism

Hormone independent Glycogen Phosphorylase activation in the Muscle Ca+2 by nerve stimulation (short bursts of exercise) AMP as a result of rapid ATP consumption

Glycogen Storage Diseases

Glycogen Storage Diseases (GSD) Group of inherited disorders characterized by defective mobilization of NORMAL GLYCOGEN /Deposition of ABNORMAL GLYCOGEN Classification based on the enzyme deficiency and affected tissue. GSD can affect the liver, the muscles or both About Eleven known types of GSD In general, GSDs - Autosomal-recessive conditions EXCEPT, liver phosphorylase kinase deficiency (GSD IX).

GSD-Diagnosis Patient History (Individual's symptoms) Physical Examination Biochemical tests (CK-Level). Occasionally, a muscle or liver biopsy is required to confirm the actual enzyme defect. Keep an eye on what you will biopsy i.e. Mc Ardle’s deficiency – Liver biopsy liver is normal – but needed muscle biopsy.

All recessive Single enzyme deficiencies

GSD-TYPES Primarily Liver involvement- I,IV,VI Primarily Muscle involvement- - II,V Both liver and muscle-III Adult onset-Mc Ardle’s(V) Most common, Clinically significant end-organ GSD with significant morbidity-Type –I* GSD with significant mortality-Type –II* 41

Liver, Skeletal and Cardiac muscle GSD-TYPES Clinicopathologic Category Specific Type Enzyme Deficiency Hepatic Type Type I Von Gierke disease Liver Glucose-6-phosphatase Generalized Type Type II Pompe disease Liver, Skeletal and Cardiac muscle Lysosomal glucosidase (acid maltase) Myopathic Type Type V McArdle Syndrome Skeletal muscle Muscle phosphoylase

Glucose-6-P accumulates- Glycolysis in the muscle - Increase Lactate Glucose uses up phosphate –AMP rise-Breakdown to uric acid-Increase uric acid Hepatomegaly-Glucose-6-P and Glycogen accumulates-Highly osmotic Lactate competes with uric acid for excretion-Increase uric acid Hyperlipidemia

Glycogen Storage Disease Type I (Von Gierke’s disease) Children with GSD I are unable to release glucose from liver glycogen- “FASTING HYPOGLYCEMIA” If untreated this results in prolonged periods when their blood sugar level is too low They present in early childhood with sweating, irritability, poor growth and muscle weakness Liver enlargement -“HEPATOMEGALY” occurs due to excessive accumulation of Glucose-6-P AND glycogen (Cannot be broken down normally).

Treatment Primarily consists of giving glucose drinks frequently during the day and, in most cases, continuously overnight through a tube passed down the nose into the stomach (a nasogastric tube) As children get older, treatment with cornstarch, which releases glucose slowly into the gut, may be very effective. With such intensive treatment most children do well and their symptoms improve as they reach adulthood. Give regulated amount of glucose, not excess Also give something that releases slowly

Can you correlate…….. A 4-year old girl is brought to the hospital OPD with the complaints of sweating,headache,fatigue,nausea and loss of weight with symptoms more severe in the morning before breakfast. On Clinical examination , the child had hepatomegaly with severe fasting hypoglycemia.Further studies reveal inherited enzyme deficiency. Q1.What is your probable diagnosis ? Q2. Which enzyme deficiency is responsible for the above clinical condition? Q3. What is the cause of fasting hypoglycemia? Von Gierke’s disease Glucose -6-phosphatase Glucose -6-phosphatase ,source of Liver Glucose in the body, so its deficiency will reduce glucose levels specially during the sleep .

Glycogen Storage Disease Type II (Alpha Glucosidase -“Acid maltase” deficiency, Pompe's Disease) Some amount of glycogen is continuously degraded by the Lysosomal enzyme, acid maltase Deficiency of the enzyme results in accumulation of glycogen vacuoles in the cytosol (liver, heart, muscle) GSD II usually presents within the first months of life with severe muscle weakness and heart muscle involvement -“CARDIOMEGALY”.

No treatment has been found to prevent the progression of the most severe (infantile) form of this disorder Affected children die from “HEART FAILURE ”, usually before the age of 18 months There are however, milder forms of GSD II in which the heart is not affected and where symptoms do not develop until later in childhood or in adult life and the progression of the illness is slower

Q Which enzyme deficiency leads to the above condition? Correlate…. A 12 month old girl shows slowly progressing muscle weakness involving her arms and legs and developed difficulty in breathing .Liver was enlarged and CT scan revealed CARDIOMEGALY.A muscle biopsy showed muscle degeneration with many enlarged prominent Lysosome filled with clusters of electron dense granules. Her parents were told that without treatment ,the child’s symptom would continue to worsen and likely result in death in 1-2 year. Enzyme replacement therapy was initiated. Acid Maltase (Alpha1-4 Glucosidase) in the Lysosome that degrade Glycogen Q Which enzyme deficiency leads to the above condition?

Glycogen Storage Disease Type III (Debranching enzyme deficiency, Cori disease) Children with GSD III are often first diagnosed with a swollen abdomen due to a very large liver with large stores of normal glycogen “Limit Dextrin” type of Glycogen (Glycogen structure with short outer branch and single glucose residue at alpha 1-6 of outer branch) deposit in the liver &muscle cells Some children have problems with low blood sugars on fasting but this is not as common as in GSD I. Treatment consists of a high protein diet and prevention of prolonged periods of fasting

Glycogen Storage Disease Type V (Muscle glycogen Phosphorylase deficiency, McArdle's disease)

Glycogen Storage Disease Type V (Muscle glycogen Phosphorylase deficiency, McArdle's disease) Patients affected by this rare myopathy are unable to produce Glycogen phosphorylase, the enzyme involved in the cleavage of glycogen to glucose-1- phosphate and glucose during anaerobic exercise The consequence is exercise-induced myalgia and fatigue The disorder affects all skeletal muscle, which results in significant disability.

People with Mc Ardle's disease experience symptoms during anaerobic and sustained exercise. These include fatigue and then pain occurring within a few minutes of exercise, which if continued, leads to muscle spasm known as a contracture. Following severe exercise, muscle damage may occur leading to myoglobinuria. This is a dark discolouration of the urine and may be a warning sign for acute renal failure, which results from excessive muscle breakdown Myoglobinuria, the condition where myoglobin is present in urine, may result from serious damage to the muscles, or rhabdomyolysis, where muscle cells breakdown, sending their contents into the bloodstream.

Diagnosis A simple blood test will usually reveal raised levels of muscle creatine kinase (CK) The diagnosis is confirmed by muscle biopsy, which shows an excess of glycogen and absence of muscle phosphorylase In up to 85% of patients from Northern Europe, an abnormality in the gene encoding for muscle phosphorylase, called the R49X mutation, can be detected on a blood test

Management Regular aerobic exercise would increase the ability to switch to fat as the fuel source for the muscle At the start of exercise, when pain occurs, exercise should be slowed down or stopped until the pain subsides (usually just for 30 seconds), then the exercise tried again Continuing to exercise with intense pain would result in muscle damage and should be avoided.

A sensible diet, rich in protein, to help replace damaged muscle and avoiding excessive weight gain is important Tourniquets should not be used during operative procedures Affected women do not seem to be disadvantaged by pregnancy or childbirth. A normal childbirth is possible in women with McArdle's disease

I NEED YOUR Attention….. A 35 year old man came to the hospital OPD with a history of limited capability to perform strenous exercise .Patient gave a history of muscle cramps and Myoglobinuria after strenuous exercise. Clinically patient was well developed and had no abnormality at rest. On investigation the patient was not hypoglycemic but ECG abnormalities were recorded. This condition is due to the deficiency in the activity of which enzyme? Muscle Phosphorylase

Characterized by Mild Fasting Hypoglycemia with Hepatomegaly Glycogen Storage Disease Type VI (Liver Glycogen Phosphorylase deficiency, Her's disease) Characterized by Mild Fasting Hypoglycemia with Hepatomegaly Von Gierke’s v/s Her’s Disease Breakdown of Glycogen Gluconeogenesis Glucose-1-P Glucose-6-P GLUCOSE