Suspended Animation Notes
Suspended Animation
Flow of Energy
Energy flow is restricted in a highly coordinated and controlled manner during suspended animation.
A new, stable, but reversible, energetic homeostasis is reached where energy-consuming processes are diminished.
At the cellular level, ATP synthesis must still exceed (though ideally not by much) the reduced ATP demand.
Examples of Suspended Animation
Anhydrobiosis in brine shrimp, Artemia sp.
Extracellular freeze-tolerant Wood frogs, Rana sylvatica.
Anoxic metabolic depression in painted turtles, Chrysemys picta.
Hibernation in Arctic ground squirrels, Urocitellus parryii.
Aestivation in Australian desert frogs, Cyclorana alboguttata.
Anhydrobiosis in Brine Shrimp
In extreme conditions (low oxygen or high salinity), female brine shrimp produce brown eggs with a chorion coating.
These eggs, also known as cysts, are metabolically inactive and can remain in total stasis for two years in dry, oxygen-free conditions, even at temperatures below freezing.
This characteristic is called cryptobiosis, meaning "hidden life."
In cryptobiosis, brine shrimp eggs can survive temperatures of liquid air ( °C or °F) and a small percentage can survive above boiling temperature ( °C or °F) for up to two hours.
Once placed in salt water, the eggs hatch within a few hours.
What is Life?
If an Artemia cyst has no measurable metabolism, can it be considered alive?
What are the biochemical criteria for something to be considered alive?
Freeze-Tolerant Wood Frogs
They are the only frogs that live North of the Arctic circle.
Environmental temperatures can dip to °C.
Antifreeze (urea and glucose) accumulates within cells.
Some extracellular freezing is permitted.
Arctic Ground Squirrels - Hibernation
Weight Gain: Young squirrels leave the burrow in mid-July and weigh as much as adults by October. They gain weight between July and September to prepare for hibernation, storing food and insulating their burrow.
Reproduction: Females bear young in mid-June after a 25-day gestation period.
Entrance into Hibernation: Squirrels enter hibernation in late September or early October, going through stages of torpor and arousal, which gradually lower body temperature.
Deep Hibernation: During deep hibernation:
Breathing rate is approximately three irregular breaths per minute.
Body temperature is near or slightly below freezing.
Heart rate is only three to four beats per minute.
Mating: Mating occurs in mid-May.
Awakening: Awakening takes about three hours.
Weight Loss: About 40% of the squirrel's total body weight is lost during hibernation.
Periodic Arousal: Squirrels may awaken every two to three weeks to move, eat stored food, or venture to the surface.
Fat Deposition Pre-Hibernation
Squirrels eat a lot, creating a caloric surplus.
De novo lipogenesis stores the excess energy as (subcutaneous) fat.
Excess glucose is delivered to the liver and adipose depots via circulation.
Glycolysis and shuttling of pyruvate into the mitochondria proceed as normal.
Pyruvate dehydrogenase (PDH) converts pyruvate to Acetyl CoA within the mitochondria.
Acetyl CoA enters the mitochondrial TCA cycle.
Mitochondrial citrate is exported into the cytosol.
Citrate is broken down to cytoplasmic Acetyl CoA → Malonyl CoA → Fatty Acids.
De Novo Lipogenesis
High glucose levels, high-fat levels, and insulin promote de novo lipogenesis.
Glucose enters the adipocyte via GLUT4 and is metabolized through glycolysis to produce pyruvate.
Pyruvate enters the mitochondria and is converted to Acetyl-CoA.
Citrate, an intermediate of the TCA cycle, is exported to the cytosol.
In the cytosol, citrate is converted back to Acetyl-CoA, which is then carboxylated to form Malonyl-CoA.
Malonyl-CoA is used in fatty acid synthesis, producing palmitate and other fatty acids.
Entrance into Hibernation and Hibernation
The squirrel finds a cozy hibernaculum.
It curls into a tight ball and stops moving.
Oxygen consumption reduces.
Glycolysis, beta-oxidation, and mitochondrial function (TCA cycle and electron transport chain) are all downregulated.
Body temperature reduces from °C to °C due to the effect.
Fat is preferentially combusted over glucose.
The diminished requirement for Acetyl CoA comes from beta-oxidation of fat.
Some protein combustion may occur, leading to muscle atrophy.
Emergence from Hibernation
Rewarming occurs from °C to °C (Q10 effect).
Brown fat (or non-shivering) thermogenesis is utilized.
Brown fat adipocytes are brown colored due to very high mitochondrial content.
The ATP synthase motor is replaced by the UCP1 pore.
Energy from stored TAG is directed towards warming and not ATP synthesis.
Shivering thermogenesis occurs.
Muscle contraction is fueled in the usual manner with glucose from stored glycogen and beta-oxidation of TAG.
Downregulation of ATP Producing Processes During Hibernation
Glycolysis (some ATP from sugar) is most strongly inhibited.
Beta-oxidation (some ATP from fat) is inhibited to a lesser extent.
Mitochondrial combustion (lots of ATP from everything!) is strongly inhibited, including the TCA cycle and electron transport chain.
The Three Stages of Cellular Metabolism
Energy from food (or endogenous stores) is processed through three stages of cellular metabolism.
This series of reactions produces ATP, which is then used to drive biosynthetic reactions and other energy-requiring processes.
Stage 1 occurs outside cells (digestion followed by absorption).
Stage 2 occurs within the cytosol within cells, except for the final step of conversion of pyruvate to acetyl groups on acetyl CoA, which occurs within mitochondria.
Stage 3 occurs within mitochondria within cells.
PFK and (Allosteric) Repression of Glycolysis
Hexokinase traps glucose within cells via phosphorylation, but this step is not unique to glycolysis because glucose-6-phosphate can be used elsewhere.
PFK catalyzes step 3 in glycolysis, which is an early step, a one-way valve (committed step), and the rate-limiting step.
PFK and Allosteric Regulation
Allosteric regulation of enzymes operates on tiny time scales (< 1 second).
Indicators of high energy status (e.g., ATP, citrate, and NADH) provide negative feedback.
Phosphorylation (adding phosphate, catalyzed by kinases) tends to inhibit enzymes (seconds to minutes).
Gene expression of the mRNA encoding the enzyme is also silenced (days to weeks).
Lipolysis and Beta-Oxidation of Fat
IN THE ADIPOCYTE:
Triglycerides are broken down into free fatty acids and glycerol via hormone-sensitive lipase (HSL).
cAMP and protein kinase are involved in the activation of HSL.
IN THE BLOOD:
Epinephrine stimulates the release of free fatty acids.
Free fatty acids are transported by albumin.
IN THE BLOOD(TRANSFER INTO THE MITOCHONDRION):
Acyl-CoA is transferred to carnitine by carnitine palmitoyltransferase I (CPT1).
Acyl-carnitine is shuttled inside by a carnitine-acylcarnitine translocase.
Acyl-carnitine is converted back to acyl-CoA by carnitine palmitoyltransferase II.
METABOLIZING CELL:
Cat is inhibited by Malonyl-CoA.
Beta-oxidation produces FADH2 and NADH2, which contribute to ATP production.
Acetyl-CoA is generated.
Beta-Oxidation of Fat
Free fatty acids must cross the cell membrane through specific transport proteins (e.g., SLC27 family).
Activation of fatty acids involves Long-chain-fatty-acid—CoA ligase, which catalyzes the reaction between a fatty acid with ATP to give a fatty acyl adenylate, plus inorganic pyrophosphate, which then reacts with free coenzyme A to give a fatty acyl-CoA ester and AMP.
For long-chain fatty acyl-CoA, the carnitine shuttle must be utilized:
Acyl-CoA is transferred to the hydroxyl group of carnitine by carnitine palmitoyltransferase I (CPT1), located on the cytosolic faces of the outer and inner mitochondrial membranes.
Acyl-carnitine is shuttled inside by a carnitine-acylcarnitine translocase, as a carnitine is shuttled outside.
Acyl-carnitine is converted back to acyl-CoA by carnitine palmitoyltransferase II, located on the interior face of the inner mitochondrial membrane. The liberated carnitine is shuttled back to the cytosol as an acyl-carnitine is shuttled into the matrix.
Short-chain fatty acyl-CoA can diffuse through the inner mitochondrial membrane.
Decision Time: Fat vs. Sugar Combustion
Metabolic modulators influence the choice between glucose and fatty acid combustion.
Examples include Etomoxir, Oxfenicine, Perhexiline and Trimetazidine, which affect CPT-1 & CPT-2, beta oxidation.
Glucose oxidation yields , while PFA oxidation yields .
Mitochondrial Structure and Function
The electron transport chain (ETC) is located in the inner mitochondrial membrane.
Complexes I, III, and IV pump protons () from the matrix to the intermembrane space, creating an electrochemical gradient.
Cytochrome c (Cyt c) carries electrons between complexes III and IV.
ATP synthase uses the proton gradient to synthesize ATP from ADP and Pi.
The citric acid cycle (TCA cycle) occurs in the matrix, producing NADH and FADH2, which donate electrons to the ETC.
TCA Cycle
Acetyl CoA (2C) combines with oxaloacetate (4C) to form citrate (6C).
The cycle involves a series of steps, regenerating oxaloacetate to continue the cycle.
Net result: One turn of the cycle produces three NADH, one GTP, and one FADH2, and releases two molecules of .
Key enzymes: Citrate synthase (CS) and Isocitrate dehydrogenase (IDH) are considered rate-limiting.
TCA Cycle Continued
It is a cycle because the continual inflow of 2C Acetyl CoA ensures that 6C citrate is repeatedly replenished from the 4C oxaloacetate that is produced.
It is also a roundabout with numerous entrance and exit points.
It is involved in syntheses as well as combustions.
It can ‘back up’ (too much food, too little exercise).
It can flow ‘backwards’ (an adapted ‘reverse TCA’ cycle is observed in some bacteria that create carbon compounds from ).
Krebs Cycle and Electron Transport Chain
NADH and FADH2 donate electrons to the electron transport chain.
Electrons are passed through a series of complexes, pumping protons () into the intermembrane space.
Oxygen is the final electron acceptor, forming water ().
The proton gradient drives ATP synthesis by ATP synthase.
Brown Fat and Futile Cycle
Brown adipose tissue (BAT) makes use of a ‘futile cycle’.
Cold exposure stimulates the sympathetic nervous system (SNS) to release norepinephrine (NE).
NE activates β3-adrenergic receptors (β3-AR) on brown fat cells.
This leads to increased levels of cAMP, activation of protein kinase A (PKA), and lipolysis of triacylglycerols (TAG) into free fatty acids (FFA) and glycerol.
UCP1 (thermogenin) in the inner mitochondrial membrane allows protons to flow back into the matrix without generating ATP, releasing energy as heat.
Futile Cycles
Glycolysis converts glucose into pyruvate with the production of ATP.
Gluconeogenesis converts pyruvate into glucose with the consumption of ATP.
If both reactions run simultaneously at high rates in the same cells, the net result would be a consumption of ATP with little appreciable material benefit (either pyruvate or glucose).
The point is to avoid converting glucose back and forth to pyruvate without a net benefit.
Sensing ATP Levels
AMP kinase is the cellular sensor in hibernation as well as under normal circumstances.
Binding of ‘spent’ ATP in the form of AMP signals low energy status and activates the enzyme complex to drive ATP synthetic combustive pathways.
Biochemical Themes in Hibernation
A new (lower) stable, but reversible homeostasis is reached.
Cellular ATP is still sensed by AMP kinase to ensure ATP supply exceeds ATP demand, but both processes are strategically diminished.
Negative feedback (via ATP and citrate) helps ensure combustive pathways such as glycolysis and beta-oxidation are diminished.
Gene expression is modified (mainly via silencing, although PDK4 is upregulated).
PDH is inhibited (by PDK4) to force Acetyl CoA to come from fat, not sugar.
Cellular/tissue compartmentation ensures fat combustion (mitochondrial) and fat synthesis (cytoplasmic) are kept separate (e.g., liver versus muscle).
Reciprocal regulation ensures combustive and synthetic processes do not run simultaneously, preventing ‘futile cycling’ (e.g., high insulin favors lipogenesis while inhibiting lipolysis).