Lecture 06: Ca Signalling and Disease -> Cytotoxic Ca2+ Overload

What Diseases Are Associated with Cytotoxic Ca2+ Overload?

• Stroke (involves ischaemic injury)

• Traumatic Brain Injury (involves ischaemic injury)

• Multiple Sclerosis

• Alzheimer’s Disease

• Huntington’s Disease

• Parkinson’s Disease

• Amyotrophic Lateral Sclerosis

• Epilepsy

• Myocardial Infarction (involves ischaemic injury)

  • There are mutations present in key Ca2+ machienery which leads to Ca2+ overload

How Does Agonist Concentration Affect Ca2+ Signalling Via ARC Channels?

• Low [agonist]: low-frequency Ca²⁺ oscillations

• Moderate [agonist]: higher-frequency Ca²⁺ oscillations

• High (supramaximal) [agonist]: sustained increase in cytosolic Ca²⁺ → pathological Ca2+ overload

• ARC channels support oscillatory Ca²⁺ signals at physiological agonist levels

What distinguishes physiological Ca²⁺ oscillations from pathological Ca²⁺ overload?

• Physiological Ca²⁺ signalling: Oscillatory and reversible

  • Occurs at physiological agonist concentrations

• Pathological Ca²⁺ overload: Sustained, irreversible increase in Ca²⁺

  • Occurs at supramaximal agonist concentrations or during cellular stress

  • Leads to cellular dysfunction and can trigger apoptosis or necrosis

• Cellular stress shifts Ca²⁺ signalling from oscillatory (physiological) → sustained (pathological)

How Do TRMP2 and NMDA Receptors Contribute to Excitotoxicity and Neurodegeneration?

• NMDA receptors are glutamate-activated Ca²⁺ channels → Ca²⁺ influx

• TRPM2 channels are Ca²⁺-permeable channel activated by oxidative stress

• Together their combined activity causes Ca²⁺ overload

• Sustained Ca²⁺ elevation → excitotoxicity and neuronal death

How Do Ca2+ Clearance Mechanism Fail in Ca2+ Overload?

• Mitochondria normally buffer Ca²⁺ , but contribute to overload when saturated

• Na-Ca2+ Exchanger (NXC) is invovled in Ca²⁺ efflux in excitable cells, e.g. Cardiomyocytes, but under stress it can reverse

• PMCA & SERCA are ATP-dependent Ca²⁺ pumps → important in cellular and metabolic stress and during ATP depletion

  • ATP depletion → pump failure → worsening Ca²⁺ overload

How Does Mitochondrial Ca2+ Overload Lead To ROS Production and TRPM2 Activation

• Ca²⁺ enters the mitochondria via mitochondrial Ca²⁺ uniporter (MCU)

• Under normal conditions, this physiological Ca2+ entry activates metabolic enzymes and drives metabolism and ATP production → important for stimulus metabolism coupling

  • (ETC, Krebs and ATP synthase are Ca2+ dependent metabolic enzymes)

• High [Ca2+] has detrimental effects on metabolism leading to mitochondrial dysfunction and excessive ROS generation, especially superoxide, produced by ETC Complex II

• This is short-lived and is converted to H₂O₂ by superoxide dismutase

• H₂O₂ diffuses out of mitochondria and can target other signalling pathways → activates TRPM2 channels, promoting further Ca2+ entry

What Can Activate TRPM2 Channels?

• H₂O₂ directly

• Oxidation of the critical cystine residue

• Oxidation of NADH → NAD+

  • NAD+ is converted to cyclic ADP-ribose via PARP

  • cyclic ADP-ribose converted to free ADP-ribose via PARG

  • free ADP ribose can then directly activate the channel

How do ROS and TRPM2 form a positive feedback loop in Ca²⁺ overload?

• ROS can directly regulate Ca release from Ca channels (IP₃Rs and RyRs)

• This inturn can activate TRPM2, causing further Ca²⁺ entry, facilitating further Ca²⁺ release from IP₃Rs and RyRs

• This results in an self-amplifying Ca²⁺ overload

  • ROS also promote ADPR production (via PARP/PARG pathway) → TRPM2 gating → Ca2+ entry

How Does Mitochondrial Ca2+ Overload Lead to Cell Death?

• Excess mitochondrial Ca²⁺ causes mPTP opening and a collapse of mitochondrial membrane potential

• This loss of mitochondrial membrane potential, which normally provides the electrochemical gradient and driving force from the ETC for ATP synthesis via the F₁F₀-ATP synthase, causes ATP synthesis to collapse

• This results in impaired mitochondrial metabolism, leading to ATP depletion and inhibiton of SERCA and PMCA, causing further Ca2+ overload

• ATP depletion also inhibits Na⁺/K⁺-ATPase, which normally maintains membrane potential, leading to membrane depolarisation and activation of VOCCs, and reversal of NCX in cardiac myocytes, which both increase Ca2+ entry further

• This leads to cytochrome C release from the mitochondria via Bax-Bax oligomerisation and subsequent pore formation

• Eventually, there is a complete loss of ion homeostasis and control of cell volume, leading to cell lysis and necrosis or the release of cytochrome C (pro-apoptotic), which activates the executioner caspase cascade, leading to apoptosis

What is the Mitochondrial Permeability Transition Pore?

• A complex of proteins that includes:

  • hexokinase (HK) → glycolytic enzyme

  • cyclophilin-D (CypD) → regulatory component

  • adenine nucleotide transporter (ANT) → Transports ATP and ADP between mitochondria and cytosol

  • voltage-dependent anion channel (VDAC) → undergoes several stages of opening to allow the movement of anions and becomes permeable to most ions when fully open

• Opens in stages; full opening creates a pore permeable to most ions

• Results in Ca²⁺ release from mitochondria which contributes to cytosolic Ca²⁺ overload

• This leads to the collapse of mitochondrial membrane potential and ATP depletion

Why is Ca²⁺ overload often irreversible during neuronal injury?

• Caspases & calpains cleave PMCA and NCX → Persistent cytosolic Ca²⁺ elevation

• This leads to further Ca2+ overload as there is a loss of Ca²+⁺ efflux mechanisms

• This is a point of no return as there no no mechanism of Ca2+ efflux and clearance from the cytosol

• This irreversible loss of ion homeostasis leads to cell death

What is the Ca2+ Overload Response in Excitoxicity and How Does it Cause Neurodegeneration?

• Excitotoxicity is caused by excessive glutamate release

• This glutamate then activates NMDA receptors, causing excessive Ca²⁺ influx

• Sustained Ca²⁺ overload leads to:

  • Opening of the mitochondrial permeability transition pore (mPTP)

  • TRPM2 activation, promoting further Ca²⁺ entry

  • Activation of calpains and caspases, which cleave PMCA and NCX

• Cleavage of PMCA/NCX impairs Ca²⁺ clearance, reinforcing Ca²⁺ overload

• Result: mitochondrial dysfunction, neuronal injury, and neurodegeneration

Where is Ischemia and Activation of mPTP, TRPM2 and Caspase Cleavage Seen?

• In ischaemia and reperfusion injuries including

  • coronary thrombosis,

  • stroke,

  • problems during cardiac surgery

  • kidney and liver problems

• These disease states can result in the activation of mPTP, and calpain and caspase-mediated cleavage of PMCA and NCX leading to Ca2+ overload

How Can the Ca2+ Overload Response Lead to Heart Faliure and Arrhythmias

• Caused by excessive Ca2+ flux due to prolonged AP, leading to enhanced Ca2+ currents

• Leaky RyR channels can lead to Ca2+ sparks, which cause autonomous contraction rather than a regulated sinus rhythm

  • This inefficient uncoordinated pumping leads to heart failure

What is Central Core Disease

• A disease of the skeletal muscle characterised by:

  • Muscle weakness and hypotonia → Muscle cells degrade → leads to muscle cramps due to lactic acid buildup

    • Degredation of central muscle core seen

  • Loss of mitochondrial → caused by mutations in skeletal muscle RyR Type 1 (muscle-specific isoform) → SR becomes leaky

  • Results in high resting [Ca²⁺]_I, due to ‘leaky” RyR and mitochondria Ca2+ overload

• Histological slide: appearance of holes in the membrane → coires emerge in the centre of the cell caused by mitocondrial loss

What is Malignant Hyperthermia

• A disease of the skeletal muscle

• Individual typically asymptomatic → triggered by abnormal reaction to volatile anesthetics (halothane) → generation of muscle cramps

• Caused by a mutation in skeletal muscle RyR1 → hypersensitive to Ca2+, causing excessive Ca2+ induced Ca2+ release during contraction

• This burderns contractile machinery (Actomyosin ATPase) and Ca2+ clearance pathways (SERCA), which have a high ATP demand

• This leads to ATP depletion, inefficient contraction and the generation of heat (due to SERCA & actomyosin ATPase activation)

• Results in metabolic crisis, lactic acid accumulation. heat generation, muscle damage (wasting) and pain → cellular damage

What is Brody’s Disease

• A rare autosomal recessive muscle disease caused by mutations in skeletal muscle SERCA1 (or mutations in accessory protein, sarcolipin?)

• It slows down Ca clearance during excitation-contraction coupling, resulting in delayed muscle relaxation

• This results in

  • Exercise-induced impairment of muscle relaxation, due to inhibition of SERCA and painless muscle cramping (delayed relaxation)

What is Huntingtons Disease?

• An autosomal dominant neurodegenerative disease caused by an elongation mutation in the Huntingtin protein gene (Htt^exp)

  • Hunting is an accessory protein, regulating Ca²⁺ transport protein within different regions of the brain, in cortical and striatum neurons, binding to NDMA and IP₃Rs causing receptor hyperactivation

• It affects neurones in the striatum (movement & coordination) and cerebral cortex (cognition, emotion, memory), leading to progressive abnormalities in movement, emotion, and cognition

How does mutant Huntingtin (Httexp) cause Ca²⁺ overload and neurodegeneration in Huntington’s disease?

• Elongation mutation in huntingtin results in a Mutant Htt^exp, which:

  • sensitises IP₃ receptors to IP₃, increasing the open probability of IP₃Rs.

    • suppourte dby single channel data form planar lipid bilayers

  • Hyperactivates NMDA receptors → excessive Ca²⁺ influx

  • Disrupts mitochondrial function

• This results in a net Excitotoxic effect and cytotoxic Ca2+ overload

• Activation of caspases and calpains leads to apoptotic neuronal death and neurodegeneration

What is Acute Pancreatitis?

• An inflammatory disease of the pancreas and pancreatic acinar cells in which the pancreas digests itself.

• Caused by the premature activation of zymogens inside acinar cells

• Caused by:

  • Excessive alcohol intake,

  • High-fat diet/obesity 

  • Gallstones cause bile acid reflux into the pancreatic duct

    • The gallbladder bile duct + pancreatic duct converge at the ampulla of Vater → Obstruction causes toxic bile acids to reflux up the pancreatic duct into acinar cells·  

How are digestive enzymes safely produced and activated in the pancreas?

• Acinar cells (~95% of pancreatic mass) secrete inactive digestive enzyme precursors (zymogens) into the pancreatic duct

• Ductal cells secrete a bicarbonate-rich fluid that flushes enzymes into the gut

• In the duodenum, enterokinase cleaves the inactive precursors trypsinogen → trypsin

• Trypsin then cleaves and activates all other digestive enzymes, allowing digestion to occur in the gut

How Does Pancreatis Develop at the Cellular Level?

• Alcohol, fatty consumption leads to the accumulation of fatty acids, ethanol metabolites, and bile acids which impair mitochondrial metabolism and normal Ca2+ signalling

• This results in

  • ATP depletion

  • Inhibition of Ca²⁺ pumps (SERCA, PMCA)

  • rreversible Ca²⁺ overload

  • Premature intracellular zymogen activation

  • Acinar cell necrosis and inflammation

• This leads to acinar cell necrotic cell death, self-perpetuating injury and local inflammation

How Are Digestive Enzymes Activated Inside Acinar Cells During Pancreatitis?

• Zymogen-rich secretory granules mislocalise to the basolateral membrane

• Granules fuse with lysosomes containing the enzyme cathepsin

• Cathepsin cleaves trypsinogen → trypsin inside the cell

• Active enzymes are released into the interstitium and lead to autodigestion of the pancreas

• In severe cases these active enzymes enter the circulation, leading to systemic inflammation, multiple organ faliure and sepsis

How does insulin protect against pancreatic cellular injury?

• Insulin protects cells from oxidative stress-induced injury, which mimics pancreatitis.

• Recent studies show insulin mitigates the harmful effects of pancreatitis-inducing agents, such as ethanol and fatty acid metabolites.

How Do Ethanol and Fatty Acids Contibute to Pancreatitis?

• Ethanol and fatty acids combine to produce fatty acid ethyl esters (palmitoleic acid ethyl esters, POAEE), which are hydrolysed into palmitoleic acid (POA), an unsaturated fatty acid by FAEE hydrolase

• POA impairs mitochondrial metabolism, resulting in ATP depletion and inhibition of Ca²⁺ pumps, resulting in cytotoxic Ca2+ overload and necrotic cell death

• This Ca2+ overload, resulting in a loss of apically confined signalling and necrosis, was assessed using Propidium iodide

  • PI enters cells via membrane holes, binds the nucleus, indicating necrosis.

  • Removal of Ca²⁺ shows no recovery, suggesting Ca²⁺ pumps are inhibited.

How do ethanol and fatty acids induce Ca²⁺ overload and pancreatic injury?

• Ethanol and fatty acids combine to form the non-oxidative metabolite fatty acid ethyl ester (FAEE/POAEE), which accumulates in the pancreas.

• POAEE converted to palmitoleic acid (POA) via FAEE hydrolase.

  • Inhibition of FAEE hydrolase or carboxyl ester lipase (CEL) can reduce toxicity caused by POAEE and POA and ethanol, respectively.

• POA impairs mitochondrial function, leading to ATP depletion and inhibition of ATP-dependent Ca²⁺ pumps (SERCA, PMCA).

  • These metabolites may also activate IP3 repceots

• The combination of excessive Ca²⁺ release and Ca2+ pump inhibition leads to ER Ca²⁺ depletion and the activation of SOCE via STIM1 oligomerisation and Orai1.

• POA/FAEE may also activate IP3 receptors, enhancing Ca²⁺ response.

What are potential therapeutic targets in ethanol/fatty acid-induced pancreatitis?

• Orai1 inhibition (e.g., GSK-7975A) can reduce SOCE and FAEE toxicity.

• PMCA activity is crucial for maintaining resting Ca²⁺:

  • PMCA inhibition → irreversible Ca²⁺ overload.

  • Maintaining PMCA activity allows restoration of low Ca²⁺ and activation of protective cellular stress pathways/apoptosis instead of necrosis.

• Targeting FAEE hydrolase or CEL could also mitigate POAEE/POA toxicity.

How does the Orai1-specific inhibitor GSK-7975A affect SOCE in pancreatic acinar cells?

• Evidence for SOCE involvement was shown using the Ca²⁺ overshoot assay:

• Treatment of Thapsigargin and zero external Ca²⁺ followed by the addition of Ca²⁺ activates SOCE.

• GSK-7975A, an Orai1 blocker, reduces Ca²⁺ influx, confirming it inhibits SOCE.

• Store depletion was measured using a low-affinity Ca²⁺ fluorescent dye to show ER Ca²⁺ depletion after thapsigargin application, which coincides with CRAC inward current.

• CRAC current is activated by thapsigargin; addition of GSK-7975A (Orai1 blocker) reverses the current → confirms Orai1/CRAC inhibition.

How Does GSK-7975A Protect Pancreatic Acinar Cells from POAEE-Induced Damage?

• Reduces Ca²⁺ overload and necrosis caused by fatty acid ethyl ester (POAEE).

• Protects against protease activation, shown via fluorescent reporter assays of protease activation.

• Prevents necrotic cell death, measured using propidium iodide (PI).

• This is achieved by blocking Orai1, which inhibits SOCE, preventing Ca²⁺ overload induced by pancreatitis-inducing agents.

What are the Three Main In Vivo Models Used to Represent Pancreatitis?

1. Bile acids (e.g., taurolithocholate sulfate) → injected into the pancreatic duct, mimics bile acid–induced pancreatic injury.

2. Hyperstimulation of the pancreas with CCK analogue (caerulein) → mimics acinar cell Ca²⁺ overload from hyperstimulation.

3. POA + ethanol combination→ mimics the aetiology of alcoholic pancreatitis.

• Collectively they produce the same histological features of

  • Oedema (gaps between cells due to water entry) → swelling and seperation of lobules

  • Necrosis (cells bursting)

  • Infiltration of immune cells (e.g., neutrophils)

What are the Effects of GSK-7975A on Pancreatitis In Vivo?

• The Orai1 inhibitor reduces:

  • Oedema – swelling between pancreatic lobules

  • Inflammatory cell infiltration – e.g., neutrophils

  • Necrosis – cell death

  • Plasma amylase/trypsin – enzymes released in the blood due to pancreatic damage

  • Plasma cytokines – e.g., IL-1β, IL-6, produced by pancreas

  • Tissue myeloperoxidase (MPO) – produced by neutrophils in pancreas and lung

How Do Bile Acids Contribute to Mitochondrial Dysfunction in Pancreatitis, and how is this prevented?

• Bile acids induce mPTP opening, leading to mitochondrial depolarisation & impaired metabolism

  • This impaired metabolism can lead to excessive Ca entry and ROS production, leading to VDAC channel opening and changes in mitochondrial membrane potential

• Depolarisation measured using TMRM dye, which accumulates in polarised mitocondria die to negative ΔΨm

  • High fluorescence = intact ΔΨm

  • Application of bile acids → loss of fluorescence = depolarised mitochondria → mPTP pore opening

• This can be prevented using:

  • Cyclosporin-A (CsA) → inhibits CypD

  • Bongkrekic acid (BA) → inhibits ANT

  • Both block mPTP opening → protect mitochondrial function → preserve TMRM fluorescence

How is the NADH/NAD⁺ ratio used to assess mitochondrial redox state and mPTP activity?

• The NADH/NAD⁺ ratio is used as a marker of mitochondrial redox state:

  • Bile acids or Ca²⁺ overload lead to an increase in NADH accumulation, indicating impaired oxidative phosphorylation

• Fluorescence measurement of NADH, when excited at 350 nm, it emits light at 450 nm → allows measurement of NADH levels in the mitochondria

• MPTP opening disrupts NADH oxidation, leading to elevated NADH fluorescence and a higher NADH:NAD⁺ ratio

• CsA (CypD inhibitor) and bongkrekic acid (ANT inhibitor) prevent this rise in NADH, preserving mitochondrial function and prevents pore opening

How do mPTP inhibitors affect necrotic and apoptotic cell death, and how are these processes measured?

• mPTP inhibitors protect against necrotic cell death, but do not affect apoptosis

• Measured using

  • Fluorescent caspase substrates → indicate apoptosis

  • Propidium iodide (PI) → indicates necrosis

• Data shows that preventing pore opening preserves ATP levels and mitochondrial metabolism → protecting againstnecrosis

  • Necrosis occurs due to ATP depletion

  • Apoptosis requires ATP → if ATP is depleted, apoptosis cannot proceed; necrosis is initiated instead

What is the effect of cyclophilin D (CypD) knockout on bile acid-induced mPTP opening and necrosis in Mice?

• Genetic deletion of Cyclophilin D (CypD KO) mice were protected against:

  • Bile acid-induced mitochondrial membrane depolarisation

  • NADH depletion

• The effect is similar to pharmacological inhibitors (Cyclosporin-A and Bongkrekic Acid)

• Supports idea that mPTP contributes to Ca²⁺ overload and ATP depletion, which drives necrotic cell death

How does cytochrome C release determine apoptosis versus necrosis in pancreatic acinar cells?

• Cytochrome C is released from the mitochondria via Bax/Bak, which can enter the cytosol →and form the apoptosome complex

• The apoptosome complex can then activate caspase-3 & -7 leading to apoptosis

  • This is said to be protective in acute pancreatitis (controlled breakdown of cells)

• Necrosis is a form of uncontrolled cell death, causing the total destruction of pancreatic acinar cells due to explosive cell lysis and the release of active enzymes which triggers inflammation → damaging

What is the Link between Acute Pancreatitis and Diabetes?

• Pancreatitis causes collateral injury to pancreatic beta cells leading to impaire dinsulin secretion and a new onset Type 3C diabetes

• Pre-existing diabetes (Type 1 and Type 2) can increase the risk of Acute Pancreatitis and make it worse

• Mechanism behind this is poorly understood → unclear if this is due to:

  • a lack of insulin secretion/ effectiveness

  • or hyperglycaemia (risk factors for AP)

  • or hyperlipidemia (risk factors for AP)

How Does Insulin Protect Pancreatic Acinar Cells During Acute Pancreatitis?

• Insulin has anti-inflammatory properties and a direct protective effect on acinar cell injury, preserving ATP and maintaining low resting Ca

  • It prevents acinar cell death in Ca overload

• In diabetes and pancreatic beta cell injury (Type 1 or Type 3C), there is a loss of insulin-mediated protection, leading to cellular injury and the escalation to severe pancreatitis and worsening acinar cell injury

What Experimental Models Where Used to Study Acute Pancreatisis in Insulin Deficient Conditions and Why?

• Two models of acute pancreatitis were used:

  • Caerulein-induced hyperstimulation model

  • Palmitoleic acid (POA) / ethanol-induced model

• Experiments performed in the type-1 diabetic Akita mouse

• Akita mice have a spontaneous mutation in the insulin gene, leading to

  • Unfolded protein response & ER stress

  • Impaired protein trafficking

  • β-cell apoptosis

  • Loss of insulin secretion

• This insulin deficiency leads to more severe acute pancreatitis

How was the pancreatic acinar cell insulin receptor knockout (PACIRKO) Mouse Model Generated?

• PACIRKO Mouse: deletion of insulin receptors in pancreatic acinar cells

• Created using Cre-Lox Rrecombinase system

  • Cre recombinase is engineered to express under a pancreatic acinar cell-specific promoter, e.g. elastase

  • Cre is part of a tamoxifen-inducible fusion protein that is inactive until tamoxifen application

• Loss of insulin signalling in acinar cells → mouse develops severe acute pancreatitis

What are the Sources of POA Induced Ca2+ Overload?

• ATP depletion

• SOCE activation

• Toxic (irreversible) Ca overload facilaited by inhibition of PMCA

  • Insulin acts to protect the PMCA

What experimental evidence demonstrates insulin protection in acinar cells during pancreatitis?

• Firefly luciferase luminescence assay used to measure total cellular ATP

  • Incubation of insulin with pancreatitis-inducing agents causes a rightward shift and a prevention of ATP depletion

• The Ca overshoot experiment showed that cyclopiazonic acid (CPA) induces ER Ca²⁺ leak and activates SOCE when Ca²⁺ is high

  • Removal of extracellular Ca²⁺ allows assessment of PMCA-mediated Ca²⁺ clearance

  • The addition of POA inhibits Ca²⁺ clearance

  • Insulin prevents this POA-induced inhibition of Ca²⁺ clearance

• CCK normally induces Ca²⁺ oscillations (physiological Ca²⁺ signalling)

  • Insulin preserves oscillatory Ca²⁺ signalling and prevents sustained Ca²⁺ overload, protecting cells from necrotic cell death

• Protective effects were abolished in PACIRKO mice, confirming insulin receptor dependence

How does insulin alter cellular metabolism to protect pancreatic acinar cells during Ca²⁺ overload?

• Insulin induces a metabolic shift from mitochondrial metabolism to glycolytic metabolism

• Glycolysis provides sufficient ATP to:

  • Fuel PMCA activity

  • Prevent ATP depletion

  • Prevent cytotoxic Ca²⁺ overload

• This metabolic switch protects cells from necrotic cell death during pancreatitis

How was the insulin-induced switch from mitochondrial to glycolytic metabolism measured?

• NADH autofluorescence used as an indirect measure of metabolism →assess metabolic shift

  • Majority of NADH normally derived from the mitochondrial Krebs cycle

  • A smaller contribution arises from glycolysis via GAPDH

• Application of Protonophore and mitochondrial uncoupler (CCCP)

  • Forms holes in the membrane → Causes depolarisation of mitochondria

  • Causes loss of mitochondrial NADH fluorescence

• GAPDH inhibitor (iodoacetate / IAA) causes a further decrease in NADH, confirming glycolytic contribution

• Treatment with insulin causes a reduction in mitochondrial metabolism (and NADH) and an increase in glycolytic metabolism (and NADH) → evidence of a metabolic switch

What is the Molecular Mechanism Behind Insulins Upregulation of Glycolysis?

• Insulin binds the insulin receptor and activates PI3 kinase, which converts PIP₂ → PIP₃

• PIP₃ recruits Akt to the membrane

• Akt is phosphorylated by PDK1 and mTOR

• Activated Akt phosphorylates PFKFB2

• PFKFB2 produces fructose-2,6-bisphosphate, a key metabolite

• Fructose-2,6-bisphosphate allosterically activates PFK1 (rate-limiting enzyme of glycolysis)

  • PFKB2 is a tunable enzyme

  • Akt phosphorylation acts as a volume control for glycolytic flux,

  • Increased glycolysis maintains ATP synthase and PMCA function, preventing Ca overload and ATP depletion (hallmarks of early pancreatitis)

• Results in increased glycolytic flux and ATP maintenance,

• Maintenance of PMCA function, preventing ATP depletion, Cytotoxic Ca²⁺ overload and necrotic cell death in pancreatitis

• Supported by Western Blot: insulin treatment causes phosphorylation of PFKFB2.

Why does type 1 diabetes lead to more severe acute pancreatitis?

• In type 1 diabetes, insulin is absent

• Loss of insulin removes its protective metabolic effect

• Glycolysis is no longer supported/protected, leading to ATP depletion

• ATP depletion impairs Ca²⁺ pumps (PMCA) → results in cytotoxic Ca²⁺ overload

• Causes a shift from mild to severe pancreatitis, with severity depending on collateral β-cell injury and loss of insulin protection

• Prevented through insulin treatments or mimetics → mimic mechanisms driving glycotlytic flux and maintain cellular ATP

What Are They Key Cardinal Events in Acute Pancreatitis

• Impaired metabolism and Ca overload → leads to necrotic cell death

• These are caused by ethanol, fatty acids, bile acids and ROS, which cause mitochondrial depolarisation, Ca uptake into the mitochondria, and mPTP opening, leading to ATP and Ca overload

• ATP depletion and Ca overload lead to necrosis, trypsin activation, inflammation and severe acute pancreatitis

What Are Potenital Theraputic Interventions For Acute Pancreatitis?

• Inhibition of SOCE → supported by clinical data

• Inhibition of mPTP → less successful in clinical trials

• Insulin analogues/ mimetics → mimck insulins regulation of glycolysis, manipulating the system may prove effective