Energy and REDOX Signalling Notes

Explain what is meant by energy state.
Identify the major sources of cellular energy: ATP, Mitochondria, Glycolysis
Describe mechanisms that maintain ATP availability: Phosphocreatine, Adenylate kinase, AMP-activated kinase
Provide specific examples in different tissues.

Cells metabolise substrates to provide energy for cellular processes.
The process involves oxidation/reduction (redox) reactions.
Lack of adequate oxygen or substrate affects the balance of these reactions.
How is production and use of energy (energy state) regulated by the cell according to demand?
What are the causes and consequences of altered cellular energy state?

Balance between production and use of energy sources.
What if energy demand exceeds energy supply (increased activity, hypoxia, metabolic poisoning)?
The cell needs an index to regulate this balance.
ATP is the immediate source of metabolic energy.
Homeostatic mechanisms match ATP availability to ATP utilisation.
Therefore [ATP] may not actually change, so it is a poor indicator of energy state.

Metabolic fuels (glucose, fatty acids, amino-acids)
1. Anaerobic respiration (glycolysis, b-oxidation) to make acetyl CoA (substrate for Krebs’ cycle)
2. Substrates progressively dehydrogenated - H+ and e- transferred to nicotinamide adenosine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD) to form NADH and FADH2; remaining carbon converted to CO2
3. Oxidation of NADH and FADH2 in the electron transport chain releases H+ + e-, final outcome being reduction of O2 to H2O, and production of ATP
Equation: $NADH + H^+ + 3ADP + 3Pi + \frac{1}{2}O<em>2 \rightarrow NAD^+ + 3ATP + H</em>2O$

Anaerobic, membrane-associated
Relatively inefficient (2 ATP)
Generates pyruvate and 2 NADH
Pyruvate utilized by Krebs’ cycle. If oxidative phosphorylation impaired, pyruvate converted to lactate.
May be rate limited by glucose influx. Both glucose uptake and glycolysis increased by hypoxia.
Some tissues and processes have a much higher dependence on glycolysis than others, especially during hypoxia (eg membrane- associated, ATP-dependent processes in smooth muscle)
Inhibited by high end products (eg ATP)
Key Enzymes: Hexokinase, Phosphofructokinase, Glyceraldehyde 3-phosphate dehydrogenase, Phosphoglycerate kinase, Pyruvate kinase, Lactate dehydrogenase
Net: 2 ATP per glucose

Pyruvate dehydrogenase and b-oxidation generate acetyl CoA and NADH, FADH2
Krebs’ cycle generates more NADH and FADH2
These are reoxidized in the ETC producing electrons, and protons (H+) which are extruded across the inner mitochondrial membrane
The H+ gradient drives the F1F0 ATP synthase
Primary regulation of synthase by ADP availability
31 ATP per glucose

Links glycolysis to Krebs’ cycle and transforms pyruvate into acetyl-CoA (pyruvate decarboxylation)
Regulated by specific kinase and phosphatase
Inactive form: PDH-P (phosphorylated PDH)
Active form: PDH (dephosphorylated PDH)
PDH phosphatase is activated by $Ca^{2+}$ , $Mg^{2+}$ .
Reaction: Pyruvate + NAD+ + CoASH → Acetyl-CoA + NADH + CO2
PDH kinase:
- Activated by: ATP, Acetyl-CoA, NADH
- Inhibited by: Pyruvate, NAD+, ADP, $Ca^{2+}$

ATP is normally bound to $Mg^{2+}$ . So, if [ATP] decreases, then free $[Mg^{2+}]$ increases.
More $Mg^{2+}$ → more active PDH → More ATP made
If $[Ca^{2+}]_i$ is high, it needs to be pumped back into the SR or out of the cell. This requires a lot of ATP

ATP can also be regenerated by adenylate kinase, which transfers a high energy phosphate from two ADP to one ATP:
Reaction: [ADP] + [ADP] → [ATP] + [AMP]
Two importance consequences:
- [ATP] can be preserved when utilization is high or production low (e.g. hypoxia)
- The AMP/ATP ratio is approximately proportional to the square of the ADP/ATP ratio. It is therefore a sensitive measure of energy state.

ATP and ADP are large and relatively immobile, and probably highly localized to sites of production and utilization
Creatine kinase in the mitochondrial outer membrane uses ATP to phosphorylate creatine, making PCr, a small, freely diffusible molecule
Reaction: $[ATP] + [Cr] \rightleftharpoons [PCr] + [ADP] + [H^+]$
PCr diffuses to sites of utilisation (low [ATP]), where creatine kinase catalyses the opposite reaction, creating ATP
PCr thus acts as a transport medium, and can effectively buffer [ATP] (i.e. act as a reserve and keep [ATP] constant)

If [ATP] is a poor index of energy state, then the most important index of energy state is probably:
The AMP/ATP ratio
It is increased by various cellular stresses: metabolic poisons, hypoxia, Glucose deprivation, ischaemia

AMP-activated kinase (AMPK)
AMPK is expressed in all tissues
Heterotrimeric serine/threonine (Ser/Thr) kinase composed of a catalytic α- and regulatory b- and γ-subunits
Constitutively active, but activated further by ↑[AMP], ↑[AMP]/[ATP] (ATP competes with AMP), and ↑[ADP]
Also activated by upstream kinases LKB1 and Ca2+-calmodulin-dependent kinase kinase b (CaMKKb)
It has a role in both cellular and whole body energy homeostasis

Brain AMPK á á Appetite á Food intake
Liver AMPK áâ FA synthesis â Cholesterol synthesis â Glycogenesis
Adipose AMPK á â FA synthesis
Muscle AMPK á (catabolic) á FA uptake & b-oxidation á Glucose uptake â Protein synthesis

Muscle AMPK á (anabolic) á Mitochondrial biogenesis á Protein synthesis? á Energy production & utilization
Heart AMPK á á Glucose uptake & glycolysis á FA uptake & b-oxidation â Protein synthesis