Glucose Transport and Glycogen

What are the two types of glucose transporters?

•GLUcose Transporters (GLUT)

•Sodium-Glucose Linked Transporters (SGLT)

GLUT Transporters*

• Operate by facilitated diffusion

  • Glucose binds to receptor of membrane protein

  • Conformation change releases glucose into cytoplasm

  • Does not consume energy

GLUT1*

•Expressed in all cell types

•Basal glucose uptake

•Most highly expressed in erythrocytes, brain, and placenta

•Glucose transport is not insulin-dependent

•Membrane expression increases with reduced blood glucose and decreases with increased glucose levels

GLUT2*

•Bidirectional glucose transport

•Found in hepatocytes, pancreatic β-cells, renal and small intestinal epithelial cells

•Glucose, galactose, and fructose are transported from intestine to portal circulation

•Not insulin-dependent

GLUT3*

•Non insulin-dependent transporter

•Neurons (neuronal GLUT)

•Also found in the embryo, sperm, leukocytes, and some cancer cells

•Higher glucose affinity and transport capacity compared to GLUT1, 2, & 4

GLUT4*

•Key insulin-dependent transporter

•Found in adipocytes, skeletal and cardiac myocytes

•Insulin stimulates translocation of GLUT4 to the cell membrane

Sodium-Dependent Glucose Transporters*

• SGLT1 and SGLT2

• Insulin-independent transporters

• Use Na⁺ gradient to drive transport

  • Intracellular Na⁺ is much lower than extracellular fluid Na⁺

  • Na⁺ gradient is maintained by Na/K ATPase pump

  • Na⁺ and glucose are transported together (symport)

  • Allows glucose to be moved against the concentration gradient

SGLT1 and SGLT2*

• SGLT1 and SGLT2 are expressed in the renal tubular epithelium of the proximal tubule

  • SGLT2 is only present in the kidney

• Resorb glucose from the glomerular filtrate

  • Under normal conditions, virtually all of the glucose is resorbed before urine leaves the kidney

  • At very high blood glucose levels, not all glucose can be resorbed, and glucosuria occurs

• SGLT2 inhibitors (e.g., bexagliflozin) have recently been used therapeutically to decrease blood glucose levels in cats (and humans) with diabetes by “spilling” glucose in the urine

  • Competitive inhibitor of glucose binding site of SGLT2

Why doesn’t glucose diffuse back out of the cells?

• GLUT2 is bidirectional

• Diffusion would cause glucose to move from areas with higher concentration to areas with lower concentrations

Glucose “Trapping”

• Phosphorylation to glucose-6-phosphate (G-6-P)

  • Enzymes hexokinase and glucokinase phosphorylate glucose (converting ATP to ADP)

  • G-6-P is not transported by GLUT2 transporter

Glucose Phosphorylation

•Phosphorylated glucose can be used for synthesis of glycogen

•Also the first step for glycolysis

Hexokinase

a type of Glucose Phosphorylation Enzyme:

• Found in most mammalian cells

• Low Km: high affinity for glucose; operates efficiently at low glucose concentrations

• Strongly inhibited by G-6-P (its own product)

• Will phosphorylate fructose at a slower rate

• Not regulated by insulin

Glucokinase

a type of Glucose Phosphorylation Enzyme:

• Liver (glycogen production)

• Pancreatic β-cells (glucose sensing)

• Lower affinity (weaker attraction; higher concentrations of glucose needed to operate efficiently) for glucose than hexokinase 

• No inhibition by G-6-P: supply-driven reaction (the more glucose, the more phosphorylation)

• Levels rise and fall with blood glucose concentration

• Insulin increases transcription; glucagon decreases transcription

Glucose Phosphorylation Enzymes*

• Hexokinase

• Glucokinase

Glucokinase: Species Variations*

• Hepatic glucokinase is needed to respond to elevated levels of blood glucose (high dietary intake)

• Species that take in low amounts of glucose from their natural diets (starch goes to microbes before getting to abomasum) lack hepatic glucokinase:

  • Ruminants (generate short-chain fatty acids from plant material)

  • Strict carnivores (high protein, low carbohydrate diet)

• These species do have pancreatic glucokinase (glucose sensing)

Glucose-6-Phosphatase (G6Pase)

• Enzyme that removes phosphate from G-6-P*

  • Found in hepatocytes and intestinal & renal cells*

  • Expression is suppressed by insulin (wants to trap glucose in cells)*

• Allows glucose to be released from the hepatocytes via the GLUT2 transporter*

• Muscle cells and adipocytes lack G6Pase (do not export glucose)*

Glycogen

• Storage form of carbohydrates/glucose

• Polymer of glucose

  • Each glycogen molecule can contain up to 30,000 glucose residues

  • Hydrophilic, hydrated (65% water)

• Present in cytosol (myocytes and hepatocytes contain most of the body’s glycogen)

Glycogenesis

formation of glycogen

Glycogenolysis

breakdown of glycogen

Glycogen Synthesis: First Steps

• Phosphorylation of glucose to G-6-P via the enzymes:

  • Hexokinase

  • Glucokinase

• Conversion of G-6-P to G-1-P

  • Enzyme: Phosphoglucomutase

  • Cofactor: Mg²⁺

Glycogen Synthesis: Synthesis of UDP-Glucose

• G-1-P is then converted to uridine diphosphate-glucose (UDP-Glc)

  • Enzyme: UDP-Glc pyrophosphorylase

  • Catalyzes the formation of UDP-glucose from glucose-1-phosphate and UTP.

  • (G-1-P + UTP → UDP-glc)

• Possible Fates of UDP-Glucose

  • Glycogen synthesis

  • Uronic acid pathway

  • Lactose synthesis (mammary gland)

Glycogen Synthesis: Glycogenin*

•Glycogenin is an enzyme that catalyzes the polymerization of the first few glucose molecules forming an oligosaccharide (3-15)

•Protein forms the core of the glycogen complex

Glycogen Synthesis: Elongation and Branching*

• Glycogen synthase:

  • Enzyme that catalyzes the elongation of a chain by addition of glucose molecules in a linear fashion

    - Catalyzes the transfer of the glucosyl residue of UDP-Glc onto glycogen via α-1,4 glycosidic bonds

  • Rate-limiting step of glycogenesis

  • Activated by dephosphorylation (protein phosphatase 1)

• Branching enzyme transfers glucose chains

  • Block of 6-7 units transferred to another chain

Regulation of Glycogen Synthesis

In the fed state (of monogastrics) glucose levels are high, and insulin is secreted

Insulin stimulates glucose transport, utilization, and storage as glycogen

Glycogenolysis

• Mobilization of glucose from glycogen stores

• Not the reverse of glycogenesis: separate pathway

• Glycogen phosphorylase

  • Releases G-1-P (90%)

  • Shortens chains to within 4 glucose molecules from branch

  • Will not break down chains after they reach 4 residues in length

• Debranching enzyme

  • Disassembles branch points

  • Transfers to elongate chain (then elongated chain is broken down by glycogen phosphorylase)

Glycogen Phosphorylase

• Catalyzes rate-limiting step of glycogenolysis*

  • Pyridoxal phosphate as coenzyme

• Cleavage of 1,4 linkages of glycogen to yield G-1-P*

• Muscle form is distinct from liver form (isozymes)

Glycogenolysis: Products*

• G-1-P → G-6-P via phosphoglucomutase (reversible reaction)

• Liver

  • Can convert G-6-P into glucose by glucose-6-phosphatase

    - Export from hepatocytes for blood glucose

  • G-6-P → glycolysis for energy production

• Muscle

  • G-6-P used for glycolysis 

Regulation of Glycogenolysis: Glycogen Phosphorylase

• Glycogen phosphorylase activation:

  • Activated by phosphorylation

    - Inhibited by ATP, G-6-P, glucose (liver)

  • Inactivated by protein phosphatase-1 (dephosphorylation)

    - Protein phosphatase-1 Activated by insulin (wants to keep glucose in cell)

• Glycogen phosphorylase is stimulated by hormones/neurotransmitters:

  • Glucagon

  • Epinephrine

  • Norepinephrine

  • Cortisol

Glycogenolysis Regulation: Glucagon and Glycogen Phosphorylase

• Glucagon (hormone) binds cell membrane receptor

• Adenylate cyclase is activated, cAMP is increased

• Protein kinase A is activated, which then activates phosphorylase kinase

• Phosphorylase kinase

  • Activates glycogen phosphorylase → glycogenolysis

  • Inhibits glycogen synthase

Glycogenolysis Regulation: Ca²⁺ Messaging

• Phosphorylase kinase can also be activated by a calcium second messenger pathway in muscle (and liver)

  • Independent of cAMP

  • Calcium can be increased via nerve impulses, hormones or muscle contraction

  • Synchronizes glycogenolysis with muscle contraction

  • Additive effect with cAMP

Glycogen Storage Diseases

• Inherited disorders of glycogen metabolism caused by deficient or defective enzymes

• Affected animals have hypoglycemia and glycogen accumulation in tissues

  • Defective G6Pase: glycogen can’t be broken down properly

  • Defective branching enzymes: abnormal long chains with low solubility

  • Glycogen precipitates in cells and causes cell injury

• Generally rare diseases in practice

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• Signs become apparent soon after birth (may die in utero)

  • Failure to grow/thrive, anorexia, weakness, abdominal distension, vomiting

  • Fasting hypoglycemia

  • Abnormal glycogen accumulation in tissues (hepatocytes or myocytes)

• Can be identified by genetic testing (carriers)

• Supportive care or gene replacement therapy?

Glycogen Storage Diseases: Classification

What would be the consequences of these mutations?

•Type I: glucose-6-phosphatase mutation (Maltese dog, Border collie)

•Type II: glycogen debranching enzyme (Lapland terriers)

•Type III: glycogen debranching enzyme (German shepherd dog, Akita)

•Type IV: glycogen branching enzyme (Norwegian Forest Cats)

•Type VII: phosphofructokinase (English springer spaniel, American cocker spaniel, whippet, mixed breed)

Glycogen Storage Disease in Cats*

• Type IV (glycogen branching enzyme defect) in Norwegian Forest cats•

• Autosomal recessive genetic defect: Affected kittens may die soon after birth, but some can appear normal until ~5 months of age. Clinical signs are fever and muscle tremors, progressing to generalized muscle atrophy and death.

Glycogen Storage Diseases in Horses

• AKA PolySaccharide Storage Myopathy or PSSM

• Glycogen accumulates in muscle tissue, causes “tying up” syndrome and muscle tremors

• PSSM1 is from a mutation of glycogen synthase 1 (GYS1) gene

  • Quarter horses, draft horses, Appaloosas, and other breeds

• PSSM2 (unknown defect)

  • Arabian/Warmblood, Quarter horse

Clinical Relevance:
Vacuolar Change in Canine Hepatocytes

• Increased glycogen deposition can cause swelling of hepatocytes, visible with light microscopy as diffuse vacuolar change.

  • “Hydropic change” can have a similar appearance

• This can occur with glucocorticoid treatment (e.g., prednisone) or hyperadrenocorticism in dogs, which stimulates gluconeogenesis (leading to increased glycogen synthesis).

  • Glucocorticoids in general have an “anti-insulin” effect.

Blood glucose (“Glycemia”) is highly regulated

• Glucagon (releases stored glucose) vs. Insulin (increases glucose storage)

• Production/release vs. usage/consumption

  • Anabolic vs catabolic states

• Intake vs. storage