Lecture 24: Other Carbohydrates and Gluconeogenesis

What are the various feeder pathways for glycosis?

• trehalose

• lactose

• sucrose

• mannose

• galactose

• glycogen

• fructose

What is the main dietary source galactose?

lactose

What type of bond does lactose have?

galactosyl B1 → 4 glucose

What does lactase do?

releases galactose from lactose in the small intestine

Is galactose entry into cells insulin dependent or independent?

dependent

What is lactose intolerance due to?

a deficiency in lactase 

True or False: Worldwide, most adults lose this enzyme and become lactose intolerant; dietary lactose is then fermented by gut bacteria—leading to bloating, discomfort, diarrhea, and intestinal gas.

true

What is important to know about the epimerization of galactose to glucose?

• pathway uses three enzymes to change the orientation of the hydroxyl group at carbon 4

• reaction proceeds through a UDP-galactose intermediate

• converts galactose 1-phosphate to glucose 1-phosphate

• NAD⁺ reduces the carbonyl

• NADH + H⁺ reduces the ketone

Can glucose 1-phosphate enter glycolysis?

yes!

What is the epimerization of the galactose to glucose pathway?

1. galactose is converted to galactose 1-phosphate by galactokinase

2. galactokinase is converted to glucose 1-phosphate through galactose 1-phosphate uridyllytransferase

3. UDP-galactose is converted to UDP-glucose through UDP-glucose-4-epimerase

What is important to know about galactokinase deficiency?

• rare autosomal recessive disorder

• causes elevation of galactose in blood (galactosemia) and urine (galactosuria)

• causes galactitol accumulation if galactose is present in the diet

• elevated galactitol can cause cataracts

• treatment is dietary restriction

What is important to know about classic galactosemia?

• galactose 1-phosphate uridyltransferase (GALT) deficiency

• autosomal recessive dirsorder

• causes galactosemia and galactosuria, vomiting, diarrhea, and jaundice

• accumulation of galactose 1-phosphate and galactitol in nerve, lens, liver, and kidney tissue causes liver damage, severe mental retardation, and cataracts

• prenatal diagnosis is possibly by chorionic villus sampling

• therapy: rapid diagnosis and removal of galactose (and therefore lactose) from the diet

  • despite adequate treatment, at risk for development delays, and in females, premature ovarian failure

What is important to know about aldose reductase?

• the enzyme is present in the liver, kidney, retina, lens, nerve tissue, seminal vesicles, and ovaries

• it is physiologically unimportant in galactose metabolism unless galactose levels are high

• elevated galactitol can cause cataracts

What is the sugar in table sugar?

sucrose

What does sucrose digest into?

glucose and fructose

What is fructose found in?

• fruits

• honey

• ½ of the sugar in high-fructose corn syrup

True or False: Sucrose and fructose taste sweeter than glucose. 10% of the daily calorie intake in the US is fructose.

true

Which enzymes phosphorylate fructose? What are their K_ms?

hexokinase (higher K_m) and fructokinase (lower K_m)

Where is fructokinase found?

• liver

• kidney

• small intestine

What does fructokinase produce?

fructose 1-phosphate

True or False: Most dietary fructose is metabolized in the liver via fructokinase.

true

Which enzyme splits fructose 1-phosphate?

fructose 1-phosphate aldolase

What does the splitting of fructose 1-phosphate yield? Where does the reaction go from there?

• produces dihydroxyacetone phosphate and glyceraldehyde

• glyceraldehyde is phosphorylated by triose kinase to produce glyceraldehyde 3-phosphate (way 1) or converted by alcohol dehydrogenase into glycerol and then by glycerol kinase into 3-phosphoglycerol (way 2)

What does fructose catabolism bypass? Why is this so important?

• fructose catabolism bypasses PFK-1, the most important regulatory step in glycolysis

• fructose catabolism by the liver is essentially unregulated

• entry of fructose into cells is not insulin dependent, and fructose doesn’t promote insulin secretion 

What is important to know about hereditary fructose intolerance/fructose poisoning?

• deficiency in aldolase B

• fructose 1-P accumulates

• intracellular P_i and ATP levels fall

• decreased hepatic ATP compromises gluconeogenesis and liver protein production

• AMP accumulates and is degraded—causing hyperuricemia

• can lead to liver failure and death

• autosomal recessive

• causes severe hypoglycemia, vomiting, jaundice, hemorrhage, hepatomegaly, renal dysfunction, hyperuricemia, and lacticaemia

• therapy: rapid detection and removal of fructose and sucrose from the diet

• fructose, sucrose, and sorbitol can cause hepatic failure and death

What is important to know about essential fructosuira?

• benign

• results in fructose accumulation in the urine

• autosomal recessive

• fructokinase deficiency

What is gluconeogenesis?

the biosynthesis of glucose from non-carbohydrate sources such as lactate, glycerol, and certain amino acids

In mammals, where does gluconeogenesis typically occur?

mostly in the liver and, to a lesser extend, the kidney and small intestine

True or False: Gluconeogenesis is the reason there is no dietary requirement for carbohydrate in humans.

true

Where does the first step of gluconeogenesis occur?

the mitochondria of liver cells

In what ways are glycolysis and gluconeogenesis the reverse of each other?

• glycolysis:

  • glucose is catabolized

  • ATP is produced (+2 ATP)

  • NAD⁺ is reduced to generate NADH

• gluconeogenesis

  • glucose is synthesized

  • ATP is consumed (-6 ATP)

  • NADH is oxidized to NAD⁺

Why are the reactions in the gluconeogenesis pathway not the exact reverse of the reactions in the glycolysis pathway?

there is a large overall negative free energy change during glycolysis, and in gluconeogenesis, the pathway must bypass these “kinetically irreversible" steps”

What are the 3 irreversible steps of glycolysis?

1. converting glucose to glucose 6-phosphate

3. converting fructose 6-phosphate to fructose 1,6-biphosphate

10. converting PEP to pyruvate

Gluconeogenesis is an example of what kind of metabolism?

divergent metabolism

Can fatty acids be used to make glucose in animals?

nope!

What is the goal of gluconeogenesis?

to synthesize glucose 6-phosphate, which can be released into the blood as free glucose or used to synthesize other carbohydrates 

What are some entry points of substrates for gluconeogenesis?

• all amino acids except leucine and lysine

• pyruvate

• a-Ketoglutarate (TCA)

• succinyl-CoA (TCA)

• fumarate (TCA)

• lactate

• 3-phosphoglycerate

• triacylglycerols

Where do carbon substrates for gluconeogenesis come from?

• RBCs

• skin cells

• brain cells

• muscle cells

Where does the conversion of lactate to pyruvate occur?

in the cytoplasm of hepatocytes

Which enzyme interconverts lactate to pyruvate?

lactate dehydrogenase

Describe the Cori cycle.

• anaerobic glycolysis

• lactate produced by exercising muscles and cell lacking mitochondria (RBCs and lens of eye) diffuse into the blood and is taken up by the liver where it is converted back to glucose via gluconeogenesis

What is the last resort substrate for gluconeogenesis?

amino acids/muscle proteins

Which enzyme interconverts alanine to pyruvate? What is produced? Where is this done?

• alanine aminotransferase

• a-kg is made when alanine is made

• glutamate is made when pyruvate is made

• occurs in the mitochondria

Where does triacylglycerols come from?

the degradation of triacylglycerides in adipose tissue

What is lipase activated by?

• glucagon

• epinephrine

• cortisol

True or False: Adipose tissue lacks glycerol kinase.

true

What does glycerol need to be converted to for gluconeogenesis? Which enzymes are involved? Where does this occur?

• glycerol is converted to glycerol 3-phosphate by glycerol kinase

• glycerol 3-phosphate is converted to dihydroxyacetone phosphate by glycerol 3-phosphate dehydrogenase 

• occurs at the outer membrane of the mitochondria facing the cytosol

How is pyruvate converted into PEP in gluconeogenesis?

step 1:

• reactant(s): pyruvate

• co-substrate(s): CO₂, ATP, acetyl CoA, Trand biotin

• product(s): ADP + P_i and OAA

• enzyme: pyruvate carboxylase

step 2:

• reactant(s): OAA

• co-substrate(s): CO₂ and GTP

• product(s): GDP and PEP

• enzyme: phosphoenolpyruvate carboxykinase (PEPCK)

True or False: The pairing of carboxylation and decarboxylation helps drive reactions that are otherwise energetically unfavorable such as converting pyruvate to PEP in gluconeogenessis.

true

True or False: The presence of acetyl CoA (from increased B-oxidation due to increased glucagon) is a signal that there is an excess of energy. While acetyl CoA itself cannot be used to generate glucose, its presence activates pyruvate carboxylase which is needed to start gluconeogenesis.

true

What is the processing for generating PEP from gluconeogenic precursors?

1. CO₂ is activated and transferred by pyruvate carboxylase to its biotin prosthetic group.

2. The enzyme then transfers the CO₂ to pyruvate—generating OAA

3. OAA cannot cross the mitochondrial membrane, so it is reduced to malate that can,

4. In the cytosol, malate is oxidized to OAA, which is oxidatively decarboxylated by PEP by PEP carboxykinase.

Describe step 1 of gluconeogenesis.

• substrate(s): bicarbonate and pyruvate

• co-factor(s): ATP and biotin

• enzyme: pyruvate carboxylase

• product(s): OAA and ADP + P_i

• notes: first regulated step of gluconeogenesis, positively activated by acetyl CoA, occurs inside the mitochondria

What is biotin?

vitamin B7 that is a coenzyme used in CO₂ activation and transfer in carboxylation reactions

What is biotin involved in?

• synthesis of fatty acids

• gluconeogenesis

• degradation of valine, isoleucine, and odd-chained fatty acids

What is important to know about multiple carboxylase deficiency?

• a form of metabolic disorder

• results from biotinase deficiency, which cleaves biotin from the lysine

• prevents recycling of biotin, which is lost in the urine as biocytin

• symptoms:

  • muscle pain

  • anorexia

  • depression

• treatment is biotin supplementation

True or False: Humans and animals cannot synthesize biotin.

true

What is biotin covalently attached to in the pyruvate carboxylase reaction?

attached to the pyruvate carboxylase through an amide linkage to the E-amino group of a Lys residue—forming a biotinyl-enzyme

What happens at catalytic site 1 in the pyruvate carboxylase reaction?

• bicarbonate ion is converted to CO₂ at the expense of ATP

• CO₂ reacts with biotin—forming a carboxy-biotinyl-enzyme

What happens at catalytic site 2 in the pyruvate carboxylase reaction?

• the long arm composed of biotin and the Lys side chain to which it is attached carry the CO₂ of the carboxy-biotinyl-enzyme to the catalytic site 2 on the enzyme surface

• CO₂ is released and reacts with pyruvate—forming OAA and regenerates the biotinyl enzyme

What is the pyruvate carboxylase mechanism?

1. bicarbonate is activated by ATP—forming carboxy-phosphate

2. carboxyphosphate breaks down to CO₂

3. CO₂ reacts with biotin to form carboxybiotin

4. biotin transports the CO₂ from one active site to the other

5. biotin is decarboxylated and pyruvate is converted to its enolate form

6. pyruvate enolate reacts with CO₂ to form OAA

7. OAA is released

What is important to know about the mitochondrial shuttle (malate)?

• OAA is converted to malate and shuttled out of the mitochondria to be re-oxidized to OAA

• this also has the effect of transporting reducing equivalents (NADH) to the cytosol

• NADH/NAD⁺ ratio in the cytosol is usually about 8 × 10^-4

• ration in the mitochondria is 10⁵ times higher

• NADH is needed to synthesize glucose

What is step 2 of gluconeogenesis?

• substrate(s): OAA and GTP

• cofactor(s): none

• enzyme: PEP carboxykinase

• product(s): PEP, GDP, and CO₂

• notes: reaction drives forward due to entropic factors

Why does PEPCK deficiency lead to?

fasting-induced hypoglycemia and lactic acidemia

Contrast lactate and pyruvate in gluconeogenesis.

• different pathway for making glucose from lactate

• cytosolic production of NADH via lactate dehydrogenase

• OAA is converted to PEP and then shuttled out of the mitochondrion

• 2 distinct PEP carboxykinase isozymes

Are you familiar with the lactate vs. pyruvate diagram from the lecture slides?

yes!

True or False: The next 6 steps (steps 2-7) of gluconeogenesis are the same as glycolysis in reverse (steps 9-4).

true

Why is NADH needed to synthesize glucose?

GAPDH uses NAD⁺/NADH as a cofactor

How many molecules of pyruvate is needed to generate 1 glucose?

2 pyruvates

What does aldolase do in gluconeogenesis?

makes F-1,6-BP from DHAP and G3P

How does gluconeogenesis bypass the second irreversible step of glycolysis?

F-1,6-BP is converted to F-6-P by F-1,6-biphosphatase

Where does the hydrolysis of glucose 6-P occur?

in the ER lumen

What does glucose 6-phosphatase deficiency cause? What is this disease characterized by?

• Von Gierke’s disease

• characterized by severe hypoglycemia, an enlarged liver, and lactic acidosis

True or False: Glucose 6-phosphatase is only in the liver and kidneys and is highly regulated.

true

What is the energy yield of glycolysis?

glucose + 2 ADP + 2 P_i + 2 NAD⁺ → 2 pyruvate + 2 ATP + 2 NADH

What is the energy cost of gluconeogensis?

• 2 pyruvate + 6 ATP (2 GTP) + 2 NADH → glucose + 6 ADP (2 GDP) + 6 P_i + 2 NAD⁺

• 2 glycerol + 2 ATP + 2 NAD⁺ → glucose + 2 ADP + 2 P_i + 2 NADH

True or False: Glycolysis and gluconeogenesis are reciprocally regulated. If they both proceeded at the same time, we would have futile cycles, waste of chemical energy, and the generation of heat.

true

What does alcohol intoxication due to gluconeogenesis? What is the process?

• alcohol impairs gluconeogenesis because pyruvate is not available as a substrate for glucose production (hypoglycemia)

• alcohol impairs conversion of lactate to pyruvate and increases serum lactic acid levels

• process:

  • the metabolism of ethanol results in a massive increase in the concentration of cytosolic NADH in the liver

  • the ethanol-mediated increase in NADH causes the intermediates of gluconeogenesis to be diverted into alternate reaction pathways—resulting in decreased synthesis of glucose

Why do low carb diets promote weight loss?

due to continuous demand for glucose and the high associated ATP cost that must be met from oxidation of fatty acids