L8 Metabolism Antioxidants, oxidants and oxidative stress _ Oxidative stress in metabolism _ disease

Overview

This two-part lecture covers the chemistry and biology of reactive oxygen species (ROS), the systems that keep them in check, and what happens when the balance tips. The first half establishes the redox chemistry of oxygen reduction, the endogenous and exogenous sources of ROS, their normal biological roles, and the enzymatic and molecular antioxidant defences (including vitamins E and C). The second half moves from definitions of oxidative stress and oxidative damage (and the difficulty of measuring them) into their role in metabolic disease — particularly how hyperglycaemia drives ROS production via AGEs/RAGE, PKC, and NADPH oxidase, and how this disrupts insulin signalling. It closes with the cell’s transcriptional defence against oxidative stress, the Keap1-Nrf2-ARE pathway, its relevance to diabetes, and existing/emerging Nrf2-targeted drugs, before a final take-home message on lifestyle and oxidative damage.

Transcript flags carried through

Several slides carried a flag from the source transcription: slide 4 (exact source of a before/after leptin-treatment photo not stated), slide 23 (dense ~25-node signalling diagram — some fine connector arrows not fully resolved), slide 30 (a journal excerpt partly obscured by an overlaid text box, though legible), slide 54 (phosphoproteomic heatmap phosphosite labels illegible at render resolution), and slide 72 (no slide title/header was visible in the captured image). Slide 43 was a blank/divider slide and is skipped. Slide 64 prints “Nrf1” where the context (Keap1 regulation of the ARE) indicates this is very likely a typo for Nrf2; this is flagged rather than silently corrected.

Redox chemistry basics and ROS species

• Mnemonic OIL RIG: Oxidation Is Loss of electrons, Reduction Is Gain of electrons.
• Oxidation: loss of electrons, addition of oxygen, removal of hydrogen, increase in oxidation state; carried out by oxidising agents (oxidants). Example: oxidation of glucose during respiration.
• Reduction: gain of electrons, removal of oxygen, addition of hydrogen, decrease in oxidation state; carried out by reducing agents (antioxidants). Example: reduction of CO2 during photosynthesis.
• $O_2$ (molecular oxygen) is itself a free radical, with 2 unpaired electrons. It is essential for life as a substrate for enzymes including cytochrome c oxidase (complex IV, respiration), cytochromes P450 (drug detoxification), D-amino oxidase (neurotransmitter production, host defence), xanthine oxidase (purine breakdown), and proline/lysine hydroxylases (collagen synthesis) — but it is also a toxic molecule and the source of reactive oxygen species (ROS).

Stepwise chemistry of O2 reduction (each step is a one-electron transfer unless noted):

1. Oxygen ($O_2$) -> superoxide radical ($O_2^{\cdot -}$)
2. Superoxide radical -> hydrogen peroxide ($H_2{O}_2$)
3. Hydrogen peroxide -> hydroxyl radical ($H{O}^{\cdot }$)
4. Hydroxyl radical -> water ($H_{2}O$)
   • Hydrogen peroxide can also be reduced directly to water via a two-electron transfer, bypassing the hydroxyl radical intermediate.

Physiological (enzyme-controlled) route of this pathway:

• $O_2\to O_2^{\cdot -}$: catalysed by NADPH oxidases (NOX), or by cytochrome c oxidase in the mitochondrial electron transport chain via a four-electron pathway direct to water.
• $O_2^{\cdot -}\to H_2{O}_2$: catalysed by superoxide dismutase (SOD).
• $H_2{O}_2\to H_{2}O$ (2-electron route): catalysed by peroxidases — catalase, glutathione peroxidases, peroxiredoxins.

Pathological (uncontrolled) route of the same pathway:

• $H_2{O}_2\to H{O}^{\cdot }$: via the non-enzymatic Fenton reaction (iron-catalysed).
• $H{O}^{\cdot }\to H_{2}O$: via reaction with polyunsaturated fatty acids, i.e. lipid peroxidation — this is how the hydroxyl radical causes damage rather than being safely detoxified.

Biologically important ROS, grouped by the slide:

Radicals	Non-radicals
Superoxide, $O_2^{\cdot -}$	Hydrogen peroxide, $H_2{O}_2$
Hydroxyl, $O{H}^{\cdot }$	Peroxynitrite, $ONO{O}^{-}$ (formed from $O_2^{\cdot -}+N{O}^{\cdot }$)
Nitric oxide, $N{O}^{\cdot }$ (produced from arginine by NO synthases, NOS)	Hypochlorous acid, HOCl (formed from $H_2{O}_2$ by myeloperoxidase, MPO)

Reactive oxygen species (ROS) are defined as chemical species that react with targets to oxidise them.

Sources of ROS

Endogenous sources converge on a central intracellular ROS pool from five routes:

1. Mitochondria — the primary intracellular ROS source, via the electron transport chain.
2. Transmembrane NADPH oxidases (NOX).
3. Xanthine oxidoreductase in peroxisomes.
4. Protein disulfide isomerase, involved in the unfolded protein response (UPR) in the endoplasmic reticulum during ER stress.
5. Diverse enzymatic reactions, including prostaglandin synthesis, auto-oxidation of adrenalin, and reduced riboflavin.

Mitochondria as an ROS source:

• Major electron pathway: Complex I oxidises NADH to NAD+ to reduce ubiquinone (Q) to ubiquinol (QH2); Complex II oxidises succinate to fumarate, also producing QH2; Complex III oxidises QH2 to reduce cytochrome c; Complex IV oxidises reduced cytochrome c to reduce $O_2$ to $H_{2}O$. The proton-motive force generated at Complexes I, III and IV drives Complex V (ATP synthase) to make ATP.
• Minor (leak) pathway — the source of mitochondrial ROS: substrate-derived electrons can leak from Complex I, II and III to reduce $O_2$ directly to superoxide ($O_2^{\cdot -}$), instead of completing the normal chain.

NADPH oxidases (NOX) — first identified in neutrophils, where they produce HOCl:

• Pathway: $O_2$ ->(NADPH oxidase)-> superoxide ($O_2^{\cdot -}$) ->(superoxide dismutase)-> $H_2{O}_2$ ->(myeloperoxidase, MPO, + Cl-)-> HOCl.
• Classical NOX complex activation: the resting enzyme has membrane subunits gp91phox (NOX2) and p22phox, while the cytosolic subunits p47phox, p40phox, p67phox and RAC (bound to GDP with Rho GDI) are separate. On activation, the cytosolic subunits translocate to the membrane; RAC exchanges GDP for GTP; p47phox becomes phosphorylated; the assembled complex transfers electrons from NADPH (oxidised to NADP+ + H+ on the cytoplasmic side) through FAD and haems to molecular oxygen, producing superoxide on the extracellular/luminal side.
• NOX isoforms (NOX1, NOX2, NOX4, NOX5) are expressed widely across vascular and immune cell types (endothelial cells, vascular smooth muscle cells, adventitial fibroblasts, monocytes/macrophages, T cells, platelets, mast cells), each with characteristic subcellular locations (endosome, phagosome, mitochondrion, ER, nucleus, cytosol).
• Cardiovascular risk factors (diabetes, hyperlipidaemia, hypertension, obesity, ischaemia/reperfusion, lifestyle) upregulate NOX isoforms in these cells, generating ROS that activate redox-sensitive transcription factors and epigenetic mechanisms, driving vascular inflammation and oxidative stress -> endothelial dysfunction, vascular remodelling, and foam cell formation -> Atherosclerosis.

Nitric oxide (a gaseous ROS):

• Produced by two routes: (1) the L-arginine-nitric oxide pathway — L-arginine + $O_2$ ->(NO synthase)-> NO ->(oxidation)-> $N{O}_3^{-}/N{O}_2^{-}$; and (2) the nitrate-nitrite-nitric oxide pathway — dietary nitrate ($N{O}_3^{-}$) ->(bacterial nitrate reductases, xanthine oxidase)-> nitrite ($N{O}_2^{-}$) ->(deoxygenated haemoglobin/myoglobin, xanthine oxidase, respiratory chain enzymes, protons)-> NO.
• NO reacts fast with other free radicals but slowly with biological molecules, and is a potent vasodilator of smooth muscle.

Exogenous sources/triggers of ROS:

• Ionising radiation (e.g. X-rays, UV)
• Toxic chemicals (e.g. smoking, pollution)
• Alcohol and other drugs (e.g. paracetamol)
• Hyperbaric oxygen treatment (e.g. for premature infants and wound healing)
• Heavy metals

Biological roles of ROS and cell signalling

ROS are not purely damaging; they have essential biological functions:

• Metabolism, e.g. oxidative phosphorylation in mitochondria
• Synthesis of essential compounds, e.g. prostaglandins
• Detoxification of foreign compounds, e.g. cytochrome P450 in the liver
• Immune responses, e.g. phagocytosis
• Cell signalling, e.g. NFkB activation

ROS in cell signalling (cardiovascular example): angiotensin II acting via its AT1 receptor, and platelet-derived growth factor acting via a receptor tyrosine kinase, both signal partly through membrane NOX1/2, generating superoxide that is converted by SOD to $H_2{O}_2$ in the cytoplasm. $H_2{O}_2$ and other redox-modified proteins then act on multiple downstream signalling nodes (ASK1/thioredoxin, PTEN, PI3K/Akt, calcineurin/calmodulin, ryanodine receptor, SERCA, JAK/STAT, PKC/Ras/Raf/MEK/ERK1/2, MEK3/6/p38), converging on transcription factors AP1, NFkB, NFAT and Ets-1, with the outcome of cellular hypertrophy and proliferation. [Some fine connector arrows in this dense diagram were not fully resolved — see flag above.]

Antioxidant defence systems

An antioxidant is defined (Halliwell and Gutteridge, 2015) as “any substance that delays, prevents or removes oxidative damage to a target molecule.” Antioxidants are either synthesised in vivo or obtained from the diet.

Mechanisms of antioxidant defence (categories, as listed):

• Catalytic removal of ROS
• Antioxidant molecules
• Decreasing ROS formation
• Protective proteins (chaperones)
• Synthesis of resistant molecules
• Repair processes/removal
• Sequestration of metal ions

Conceptually, antioxidants act by donating an electron to a free radical (an atom/molecule missing an electron), converting it back to a stable, non-radical species.

Catalytic removal of ROS — the enzymatic network:

1. Enzymatic sources — NOXs, xanthine oxidase (XO), lipoxygenase (LO), cyclooxygenase (COX), and uncoupled eNOS — and non-enzymatic sources — the mitochondrial electron transport chain, endoplasmic reticulum, and peroxisomes — all generate superoxide ($O_2^{\cdot -}$).
2. Superoxide is converted to $H_2{O}_2$ spontaneously or via superoxide dismutases (SODs); NOX-4 and lysyl oxidase (LOX) can also generate $H_2{O}_2$ directly as a by-product.
3. Superoxide can also react rapidly with NO to form peroxynitrite ($ONO{O}^{-}$).
4. $H_2{O}_2$ can be converted to the more reactive hydroxyl radical via the Fenton reaction, or to HOCl via myeloperoxidase (MPO).
5. $H_2{O}_2$ is detoxified to water by three systems: catalase (CAT) directly; the glutathione peroxidase (GPx)/glutathione reductase (GR) system, using the GSH/GSSG cycle; and the thioredoxin (Trx)/peroxiredoxin (PRx) system, using NADPH via thioredoxin reductase (TrxR).

Subcellular compartmentalisation of ROS production and scavenging:

• Extracellular space: superoxide -> (extracellular SOD, EcSOD/SOD3) -> $H_2{O}_2$ -> (glutathione peroxidase 3, Gpx3) -> $H_{2}O$; lipid hydroperoxides are handled by glutathione peroxidase 4 (Gpx4).
• Cytoplasm: superoxide -> (CuZnSOD/SOD1) -> $H_2{O}_2$ -> via peroxiredoxins (Prx), glutathione peroxidase 1 (Gpx1), or catalase -> $H_{2}O$.
• Mitochondrion: superoxide generated at Complex I and Complex III -> (MnSOD/SOD2) -> $H_2{O}_2$ -> via peroxiredoxin 3 (Prx3), thioredoxin 2, and Gpx4 pathways.
• A separate repair pathway, methionine sulfoxide reductase A (MsrA), reduces oxidised methionine (Met=O) back to methionine using thioredoxin 1 (cycling between reduced and oxidised forms), regenerated via thioredoxin reductase 1 and NADPH.

Antioxidant molecules:

• Endogenous: glutathione (GSH), taurine, hypotaurine, polyamines, plasminogens, plasma albumin, coenzyme Q, uric acid.
• Exogenous (dietary): tocopherols (vitamin E), ascorbate (vitamin C), carotenoids, plant phenols (e.g. epigallocatechin gallate, resveratrol, curcumin).

Antioxidant supplement use and public health data (flagged slide 30, partly obscured text but legible)

About 30% of adults in NZ regularly take some kind of supplement (Ministry of Health Nutrition Survey 2008/09). NZ 2021-22 Health Survey: only 10.4% of adults met the vegetable intake recommendation (5-5.5 servings/day); 49.8% of adults met the fruit intake recommendation (2+ servings/day); only 6.4% of children (2-14y) met the vegetable recommendation (2.5-5.5 servings); 74% of children met the fruit recommendation (1-2 servings). A cited review (Gutteridge & Halliwell, 2010) notes there is “an industry-driven public obsession with antioxidants” which are marketed as universally safe/health-giving, but the evidence is mixed and pro-oxidants can sometimes be beneficial.

Vitamin E:

• Not a single chemical but a nutritional term: 8 naturally occurring substances have vitamin E activity (tocopherols and tocotrienols: alpha, beta, gamma, delta forms of each). Alpha-tocopherol is the only bioactive form.
• Alpha-tocopherol is lipid-soluble (found in membranes), transported on lipoproteins, and acts as a scavenger of lipid peroxyl radicals (ROO*), thereby inhibiting lipid peroxidation: alpha-tocopherol + ROO* -> alpha-tocopheroxyl radical + non-radical lipid product.
• High in vegetable oils, nuts, and green vegetables. Deficiency is rare in developed countries and usually associated with fat malabsorption disorders.

Vitamin C (ascorbate):

• Water-soluble; main dietary source is citrus fruits and vegetables. Severe deficiency causes scurvy.
• Effective reducing agent and free-radical scavenger in vitro, but can also generate ROS from copper and iron.
• Regenerates alpha-tocopherol (vitamin E) from the alpha-tocopheroxyl radical (ascorbate <-> ascorbyl radical interconversion).
• Cofactor for at least 15 enzymes: necessary for collagen, carnitine, hormone and amino acid formation, and epigenetic modifications. Example: proline and lysyl hydroxylases use $O_2$ and $F{e}^{2+}/F{e}^{3+}$ to convert proline and lysine to hydroxyproline and hydroxylysine (for collagen), with ascorbic acid/semidehydroascorbic acid cycling as the electron donor/cofactor.
• Absorption is dose-dependent and saturable: plasma ascorbate rises steeply between roughly 50-150 mg/day intake then plateaus at around 65-70 umol/L from ~200 mg/day upward. NZ recommended daily intake is 45 mg/day (WHO guidelines).
• Vitamin C requirement scales with body weight: an additional ~10 mg/day is estimated to be needed per 10 kg increase in body weight, to reach the plasma concentration achieved by a 60 kg person taking ~110 mg/day (the EFSA-recommended intake). As body weight increases, plasma vitamin C concentration falls (shown across three cohort datasets, from ~55-65 umol/L at ~30 kg down to ~20-30 umol/L at ~160 kg). Only about two-thirds of two large cohorts (EPIC-Norfolk, NHANES 2017-18) achieved “adequate” vitamin C status at the RDA.

People with type 2 diabetes have an estimated 1.4-1.6-fold higher vitamin C requirement than people without diabetes -- attributed to increased oxidative stress from abdominal-fat-related inflammation, plus greater renal leakage of vitamin C due to diabetic renal dysfunction. Suggested intake for this group: at least 125 mg/day (vs 45 mg/day standard NZ recommendation), a ~150% increase.

Oxidative stress and oxidative damage: definitions and measurement

• Healthy state: ROS production is approximately balanced by antioxidant defence; ROS levels are controlled, not eliminated, because it is energetically cheaper to repair/replace damage than prevent all of it, some ROS are too reactive to scavenge, and some ROS have essential roles. Oxidative stress/damage arises from a shift in this balance in either direction.
• Oxidative stress (Halliwell & Gutteridge, 2015): a disturbance in the pro-oxidant-antioxidant balance in favour of the pro-oxidant, leading to potential damage.
• Oxidative damage (Halliwell & Gutteridge, 2015): biomolecular damage caused by attack of ROS upon the constituents of living organisms.

Measuring ROS/oxidative damage is difficult:

• Direct method: electron spin resonance (ESR)/electron paramagnetic resonance (EPR), which detects unpaired electrons directly — but this is not useful in vivo.
• Indirect methods: measuring oxidation of detector molecules, or measuring lipid, protein and DNA oxidation products. Biomarkers are grouped by biomolecule: protein (SH oxidation, protein carbonyl formation, oxidised Tyr/Met/Trp, nitrated proteins), lipid/DNA (HNE, MDA, isoprostanes, oxidised bases, nitrated bases), and urine.

Characteristics of a good biomarker of oxidative damage (Giustarini et al 2009):
a) Chemically stable molecule
b) Directly implicated in disease onset/progression
c) Specific for the ROS/RNS in question
d) Non-invasive to assess
e) Low intra- and inter-individual variability in humans
f) Accurate, precise, specific, sensitive, interference-free, validated assay
g) Established reference intervals/values by consensus
h) Established animal models by consensus
i) Demonstrated efficacy of antioxidant supplementation in humans and animal models
j) Use of relevant (physiological) concentrations of ROS/RNS and biomarkers in in vitro/in vivo studies

A key open question raised: what proportion of the total pool of a molecule is actually damaged (e.g. if only 0.1% of total lipid is oxidised, is this functionally significant)? Distinguishing statistically detectable damage from functionally meaningful damage is a recurring problem, as is distinguishing cause from correlation.

The antioxidant paradox and clinical trial evidence

Oxidative stress is implicated in a very wide range of diseases across essentially every organ system (lung, brain, kidney, cardiovascular, joints, immune system, skin, fetal, eyes, and multi-organ conditions including cancer and ageing), reflected in a steadily rising publication count (138,076 PubMed results for “oxidative stress AND disease”).

Despite this, large trials of antioxidant supplementation have not shown benefit and in some cases showed harm:

Cochrane review (Bjelakovic et al, 2012): 56 low-risk-of-bias randomised trials, 244,056 participants. No evidence to support antioxidant supplements for primary or secondary disease prevention. Beta-carotene and vitamin E "seem to increase mortality," and higher doses of vitamin A may also. The authors state antioxidant supplements should be regulated and evaluated as medicinal products before marketing.

• A related commentary (Redberg, 2011, on the Iowa Women’s Health Study) reports 85% of women in that study used supplements; increased mortality was associated with most commonly used vitamin/mineral supplements; the recommendation given was to eat more fruit and vegetables rather than take supplements.
• This gap between expectation and evidence is termed the antioxidant paradox: giving large doses of dietary antioxidant supplements has demonstrated little or no therapeutic effect in humans (Halliwell, 2013).
• A proposed resolution (Gutteridge & Halliwell, 2010): the impact of oxidative stress on disease depends on the stage of tissue injury. Early-stage tissue injury: oxidative stress aggravates disease, and antioxidant intervention has potential therapeutic benefit. Late-stage injury, accompanying cell death: necrotic death spreads damage (e.g. via Fe/Cu/haem-protein release) and triggers inflammation, generating more ROS — but this stage, and the induction of endogenous antioxidant/defence systems (e.g. chaperones, haem oxygenase) after tissue injury, both make “no adverse contribution to disease pathology” and antioxidant interventions here “fail to give benefit and could conceivably be deleterious.”
• A specific example: antioxidant supplementation (vitamin C 1000 mg/day + vitamin E 400 IU/day) was shown to prevent the health-promoting effects of physical exercise on insulin sensitivity in humans (Ristow et al, 2009) — consistent with exercise acting partly as a beneficial, mild pro-oxidant stimulus that antioxidants blunt.

Oxidative stress in metabolic disease: general mechanisms

Oxidative stress is implicated in obesity, type 2 diabetes, and metabolic syndrome via five overlapping mechanisms:

• Mitochondrial dysfunction
• NADPH oxidase activation
• Disruption of metabolism
• Activation of stress-responsive signalling pathways
• Down-regulation of antioxidant systems

General disease mechanism (Sies et al, Nat Rev Mol Cell Biol 2022): endogenous sources (NOXs, peroxisomes, mitochondria, ER) plus exogenous “exposome” factors (nutrition, exercise, toxins, irradiation) generate superoxide, which (with $F{e}^{2+}$) forms $H_2{O}_2$. From here, two branches:

1. Damage to DNA, proteins, and carbohydrates -> lipid peroxidation -> secondary oxidants -> electrophiles -> aberrant signalling -> dysfunction/disease (pathology).
2. Modification of iron-sulfur clusters and cysteine residues on redox-sensitive proteins -> redox signalling (via transcription/regulatory factors including NRF2, NF-kB, HIF, ERR, FOXOs, PGC1alpha, p53, AMPK, mTOR, PPAR, GAPDH, SIRT, UCP) -> physiological signalling (immunity, proliferation, development, ageing, thermogenesis, steroidogenesis, cognition).

Metabolic syndrome and vascular oxidative stress: risk factors of metabolic syndrome (free fatty acids, high glucose, advanced glycation end-products (AGE), pro-inflammatory cytokines, angiotensin II, protein kinase C, and polyol pathway activity) activate ROS/RNS-producing enzymes (NOXs, NOS, COX, xanthine oxidase, uncoupled eNOS, MPO) and increase mitochondrial ROS generation, producing a pool of oxidant/nitrogen species ($O_2^{\cdot -}$, $H_2{O}_2$, $O{H}^{\cdot }$, NO, $ONO{O}^{-}$, $N{O}_2$, $C{O}_3^{\cdot -}$, oxo-ferryl species, HOCl). This down-regulates the antioxidant system (SOD, catalase, GPx, GR, GSH, thioredoxin/TrxR), causing irreversible oxidative modification of targets and impaired redox signalling. The net effect is an altered vascular redox state -> endothelial dysfunction, vascular inflammation, and vascular smooth muscle cell migration/proliferation -> metabolic-syndrome-related cardiovascular disease (coronary atherosclerosis, hypertension, left ventricular hypertrophy, diastolic dysfunction, coronary microvascular disease, autonomic dysfunction). A vicious cycle exists: increased ROS/RNS worsens the disease process that generated it, and also further reduces antioxidant system activity.

Hyperglycaemia-driven pathways: AGEs, RAGE, PKC and NADPH oxidase

Impact of hyperglycaemia — cell death: hyperglycaemia increases methylglyoxal (MGO), advanced glycation end-products (AGEs), and ROS. These converge to increase oxidative stress, which drives apoptosis, autophagy, and necroptosis (programmed necrosis of inflammatory cells), and increases pro-inflammatory cytokines — leading to diabetes complications such as retinopathy and age-related macular degeneration.

Formation of AGEs (glycation, e.g. HbA1c):

1. The carbonyl group of a reducing sugar (glucose) condenses with a protein amine group to form a Schiff base.
2. The Schiff base rearranges (Amadori reaction) into a stable Amadori product.
3. Some Amadori products convert to AGEs via the Hodge pathway; others are oxidised and cleaved to reactive dicarbonyl compounds (glyoxal, methylglyoxal, 3-deoxyglucosone).
4. Reactive dicarbonyls cross-link with proteins to generate AGEs (e.g. CML, pentosidine, attached to lysine residues).
   • Additional routes feeding into the same dicarbonyl/AGE pool: the Wolff pathway (Cu2+/Fe2+ autoxidation of fructose-6-phosphate), the polyol pathway (catalytic reduction of fructose-6-phosphate), the glycolytic pathway (via glyceraldehyde-3-phosphate), and the Namiki pathway.

AGE-RAGE signalling:

• In the extracellular matrix, AGEs form on lipids, collagen, laminin, elastin, and vitronectin, increasing matrix stiffness, and activate the TGF-beta receptor, stimulating cell growth and further ECM production.
• AGEs binding RAGE on the endothelial cell surface activate a signalling cascade: NAD(P)H oxidase activation -> increased ROS, plus activation of p21 RAS and MAPKs (including p38 MAPK, and Rac/Cdc42).
• A key downstream target is NF-kB, which translocates to the nucleus and increases transcription of endothelin-1, ICAM-1, E-selectin, VCAM-1, tissue factor, IL-6, and TNF-alpha.
• AGE and other RAGE ligands (HMGB1, S100 calgranulins) also trigger inflammatory pathways, and AGEs decrease NO availability (via reduced NOS activity and NO quenching).
• Soluble AGEs activate monocytes, increasing expression of macrophage scavenger receptor class A and CD36, leading to increased oxidised-LDL uptake and foam cell formation — linking hyperglycaemia to atherosclerotic change.

PKC activation pathway: high free fatty acids and hyperglycaemia activate diacylglycerol (DAG), which activates protein kinase C (PKC). PKC increases expression of endothelin-1 (ET-1), VCAM, ICAM, NFkB, and NADPH oxidase. NADPH oxidase activation produces superoxide -> oxidative stress -> decreased NO bioavailability.

Oxidative stress, insulin resistance and signalling crosstalk

Oxidative stress and insulin resistance in skeletal muscle: angiotensin II binding its AT1 receptor activates membrane NADPH oxidase, producing superoxide; separately, high glucose and lipid excess increase mitochondrial oxidant production (superoxide, $H_2{O}_2$). Both converge on oxidative stress, which activates a panel of serine kinases (p38 MAPK, JNK, GSK-3beta, IKKbeta, and others). These kinases impair insulin signalling (normally: insulin binds its receptor -> activates signalling -> GLUT4-containing vesicles translocate to the membrane -> glucose uptake), reducing GLUT4 translocation and glucose transport.

Crosstalk between phosphorylation and oxidation in insulin/growth signalling: the insulin/IGF1 receptor -> IRS -> PI3K -> PDK1 -> AKT axis (with PTEN/PIP3 feedback) branches to BAD, FOXO, NFkB, GSK3beta (affecting survival and proliferation), and also intersects with hypoxia signalling (REDD1/2), energy-stress sensing (Sestrin3, AMPK, TSC1/TSC2, Rheb), amino-acid sensing (Rag GTPases) and autophagy (ULK1/ATG13) — all converging on mTORC1 and mTORC2, which drive translation, ribosome biogenesis and cell growth. Large-scale phosphoproteomic data show many sites across the Akt1/2, mTOR and AMPK pathways are regulated by both insulin and oxidation, indicating extensive crosstalk between the two regulatory inputs. [Flag: individual phosphosite labels on the heatmap panels were illegible at render resolution.]

ROS in type 2 diabetes — integrated picture: risk factors (obesity, hypertension, age, genetics, diet, sedentary lifestyle) plus high blood glucose drive ROS production via the polyol pathway, increased AGEs, the DAG-PKC pathway, and the hexosamine pathway. This ROS causes lipid peroxidation, protein modification, and DNA damage -> oxidative stress, which blocks insulin signalling and activates MAPK and other stress signalling, stimulating pro-inflammatory (TNF-alpha, NF-kB) and pro-fibrotic (TGF-beta) factors, further macromolecular damage, and mitochondrial impairment. This contributes to type 2 diabetes and its complications: diabetic retinopathy, stroke, diabetic cardiomyopathy, diabetic nephropathy, diabetic peripheral neuropathy, and diabetic foot ulcers. Proposed therapeutic approaches include pharmacological agents, lifestyle interventions, and antioxidant therapy.

The Keap1-Nrf2-ARE genetic response to oxidative stress

The antioxidant response element (ARE) is a cis-acting enhancer sequence, responsive to a broad range of chemicals, that controls the expression of numerous genes involved in cell protection from, and adaptation to, oxidative stress. Its purpose is expression of defensive genes that promote cell survival by detoxifying chemicals/ROS and by preventing further ROS generation.

• The ARE is recognised by the transcription factor Nrf2 (NF-E2 p45-related factor 2).
• Nrf2 expression is tightly regulated by Keap1 (Kelch-like ECH-associated protein 1), which is cysteine-rich.
• Nrf2 activates expression of more than 300 genes.

Keap1-Nrf2-ARE mechanism, as steps:

1. Under basal conditions, Keap1 (with free thiol groups on its cysteines) binds Nrf2 together with Cul3, Roc1 and an E2 complex, targeting Nrf2 for ubiquitination and proteasomal degradation.
2. A wide range of stressors/inducers act via kinases and directly on Keap1’s cysteine thiols: these include 15d-PGJ2, ER stress, ROS, NO*, diphenols/diamines, Michael acceptors, isothiocyanates, thiocarbamates, dithiolethiones, hydroperoxides, trivalent arsenicals, heavy metals, vicinal dimercaptans, and conjugated polyenes.
3. Oxidation of Keap1’s cysteines prevents it from targeting Nrf2 for degradation [the slide text prints “Nrf1” at this point, which in context appears to be a typo for Nrf2 — flagged rather than corrected].
4. Released, stabilised Nrf2 translocates via importin into the nucleus.
5. Nuclear Nrf2 forms a complex with small Maf proteins and CBP/p300, binds the ARE DNA element, and drives transcription of target genes.
6. Nrf2 target genes fall into functional categories: direct antioxidants, free radical metabolism, electrophile detoxication, glutathione homeostasis, generation of reducing equivalents, solute transport, inhibition of inflammation, DNA damage recognition, and proteasome function — collectively promoting cell survival.
7. Phosphorylation of Nrf2 by a series of kinases also affects its stability and subcellular distribution, and its interaction with other transcription factors/complex proteins allows transactivation of ARE-responsive genes, producing an adaptive response that enhances cellular resistance to electrophiles and free radicals.

Some dietary antioxidants are Nrf2 activators: caffeic acid phenethyl ester (CAPE), CDDO-Im, curcumin, and resveratrol all inhibit NF-kB and trigger Nrf2 nuclear translocation to induce haem oxygenase-1 (HO-1) expression, suppressing oxidative stress. Curcumin and CDDO-Im act partly via MAPK-mediated Nrf2 translocation; resveratrol and CAPE act partly via ERK signalling alongside Nrf2-mediated HO-1 induction.

Nrf2 in diabetes and Nrf2-targeted drugs

Proposed protective role of Nrf2 in diabetes: Nrf2 induction acts across tissues — liver (suppresses gluconeogenesis, activates AMPK, induces FGF21), skeletal muscle (increases glucose uptake and oxygen consumption), adipose tissue (FGF21, anti-obesity effects), and pancreatic beta-cells (antioxidant, detoxification, and anti-inflammatory functions, protecting beta-cells directly). The liver/muscle/adipose effects converge on improved insulin resistance, while the beta-cell effects give direct beta-cell protection; together these are proposed to prevent diabetes mellitus.

A 2022 review (Dodson et al, Diabetes) frames NRF2 as "the good, the bad, and the complex" in diabetes: NRF2 is a critical transcription factor regulating redox, protein and metabolic homeostasis, and proper NRF2 function is confirmed to help prevent diabetic outcomes experimentally -- but the literature is described as contradictory, attributable to differences between models and whether NRF2 activation is acute vs chronic. As of that review, there were no NRF2 activators in clinical trials specifically for diabetes treatment.

• A range of pharmacological Nrf2 inducers have shown antidiabetic effects in different diabetes contexts (examples given): in gestational diabetes, tBHQ improved insulin sensitivity/glucose tolerance; in type 1 diabetes models, sulforaphane/cinnamaldehyde improved wound healing and reduced nephropathy/cardiac dysfunction; in type 2 diabetes models, CDDO-Im, sulforaphane, D3T, MitoQ, resveratrol and others reduced nephropathy, cardiomyopathy, or beta-cell death, and improved insulin secretion/glucose tolerance.
• Ongoing research interest is substantial (3,365 PubMed results for “Nrf2 diabetes”; an active clinical trial, NCT04848792, is testing Nrf2 signalling enhancement in older adults for aging-related problems).

Clinically approved Nrf2 activators (all outside diabetes, for context):

• Tecfidera (dimethyl fumarate) — reduces relapses and slows progression in relapsing-remitting MS, by reducing brain inflammation and protecting myelin, thought to act by activating an antioxidant mechanism that deactivates and removes inflammation-generated free radicals.
• Vumerity — a novel oral fumarate, approved for relapsing forms of MS, converts in the body to monomethyl fumarate (the same active metabolite as dimethyl fumarate).
• Bafiertam — a bioequivalent alternative to Tecfidera, same active ingredient and site of action.
• Skyclarys (omaveloxolone) — approved for Friedreich’s ataxia in adults and adolescents >=16 years; a semi-synthetic terpenoid that directly activates the Nrf2 pathway.

Commercial "Nrf2 activator" supplements (e.g. Protandim, NutriCology Nrf2 Rising, containing sulforaphane, pomegranate, green coffee, olive leaf, green tea, ginkgo) are marketed online, illustrating commercial exploitation of Nrf2 biology; these are dietary supplements, not evaluated or approved as medicines by the FDA, unlike the drugs listed above.

Summary and take-home messages

1. Oxidants/ROS: produced in all cells; highly reactive; short-lived and difficult to measure; can damage lipids, proteins, DNA, and sugars; but also have important biological functions.
2. Antioxidants: act by both direct and indirect mechanisms; some are essential nutrients; different antioxidants have different activities and reactivities.
3. Oxidative damage and stress: arise from an imbalance between oxidant production and antioxidant activity; distinguishing cause from correlation is a recurring difficulty.
4. Oxidative stress in metabolic disease: implicated in many diseases including metabolic disease; hyperglycaemia drives oxidative stress; oxidative stress disrupts metabolic signalling; the benefit of antioxidant supplements remains uncertain.
5. Genetic response to oxidative stress: Nrf2 is a critical mediator of the adaptive response; therapeutic targeting of the Nrf2 pathway may be useful, and several Nrf2-activating drugs are already clinically approved (for MS and Friedreich’s ataxia).

Closing message from the lecture: eat well, including plenty of fruit, grains and vegetables; avoid obesity; don’t smoke; exercise regularly (itself a mild pro-oxidant challenge that triggers a beneficial adaptive response) — this combination is presented as the way to minimise oxidative damage (Halliwell, 2013).

Self-test

1. Using the OIL RIG mnemonic, distinguish oxidation from reduction in terms of electron transfer, oxygen, hydrogen, and oxidation state.
2. Write out, in order, the stepwise chemical reduction of $O_2$ to $H_{2}O$, naming each intermediate ROS.
3. Name the enzyme(s) responsible for each step of the physiological (enzyme-controlled) route of O2 reduction.
4. Contrast the pathological route of O2 reduction with the physiological route — which two reactions differ, and by what mechanism?
5. List the biologically important ROS given in the lecture, divided into radicals and non-radicals.
6. List the five endogenous sources of intracellular ROS shown in the overview diagram.
7. Describe the “minor electron pathway” in mitochondria and explain why it is a source of ROS.
8. Describe the steps of NADPH oxidase (NOX) activation, from resting state to superoxide production.
9. Give the two biosynthetic routes for nitric oxide production described in the lecture.
10. List the exogenous sources/triggers of ROS given in the lecture.
11. List five normal biological functions of ROS, with the example given for each.
12. Define “antioxidant” (Halliwell and Gutteridge, 2015).
13. List the seven mechanisms of antioxidant defence given in the lecture.
14. Describe, as an ordered pathway, how superoxide is generated and then detoxified to water, naming the three enzyme systems that convert H2O2 to water.
15. List the endogenous and exogenous antioxidant molecules given in the lecture.
16. Why is alpha-tocopherol specifically effective against lipid peroxidation, and what is the product of its reaction with a lipid peroxyl radical?
17. Give three functional roles of vitamin C beyond being a “scavenger of free radicals,” including one way it can act as a pro-oxidant.
18. Describe the relationship between vitamin C dose and plasma ascorbate concentration, and explain why body weight affects vitamin C requirements.
19. Define oxidative stress and oxidative damage, and explain how they differ from each other.
20. Why is measuring ROS/oxidative damage directly (e.g. by ESR/EPR) difficult in vivo, and what is used instead?
21. List three criteria that make a good biomarker of oxidative damage.
22. Explain the “antioxidant paradox,” and describe the outcome of the Cochrane review of antioxidant supplement trials.
23. According to Gutteridge & Halliwell’s model, why might antioxidant intervention be beneficial at an early stage of tissue injury but not beneficial (or even harmful) at a late stage?
24. Explain why co-administering vitamin C and vitamin E blunted the beneficial effect of exercise on insulin sensitivity in the Ristow et al (2009) study.
25. List the five general mechanisms by which oxidative stress is implicated in obesity, diabetes, and metabolic syndrome.
26. Describe, as an ordered pathway, the formation of AGEs from a reducing sugar and protein, including the role of the Amadori reaction.
27. Describe the AGE-RAGE signalling cascade in endothelial cells, from ligand binding through to the transcriptional consequence, and its downstream link to foam cell formation.
28. Describe the PKC activation pathway triggered by free fatty acids and hyperglycaemia, and its four downstream effects.
29. Describe how oxidative stress impairs insulin-stimulated glucose transport in skeletal muscle, naming the kinases involved and the transporter affected.
30. Describe the Keap1-Nrf2-ARE pathway as an ordered mechanism, from the resting state through to gene transcription.
31. List the functional categories of Nrf2 target genes.
32. Name two dietary Nrf2 activators and the shared downstream gene (HO-1) they induce.
33. Name four clinically approved Nrf2-activating drugs and the disease each treats.
34. Vignette: A 60 kg woman consumes 110 mg/day of vitamin C and this is judged adequate. Her 90 kg partner also consumes 110 mg/day. Using the relationship given in the lecture, is his intake likely to be adequate, and why?
35. Vignette: A patient with poorly controlled type 2 diabetes and chronically elevated blood glucose is found to have low plasma vitamin C. Using the mechanisms discussed in the lecture (not just “diabetics need more vitamin C”), explain two distinct reasons plasma vitamin C would be reduced in this patient, and state the recommended intake for this population compared with a healthy adult.
36. Integrative question: Trace a single mechanistic path from chronic hyperglycaemia to impaired insulin signalling in skeletal muscle that passes through at least three of the following: AGE formation, NADPH oxidase activation, mitochondrial ROS production, serine/threonine kinase activation, and GLUT4 translocation. Then explain, using the Keap1-Nrf2-ARE pathway, one reason why activating Nrf2 might be expected to interrupt this path, and why the lecture’s review evidence urges caution about doing so pharmacologically or via supplements.

Answers

Reveal answers

1. Oxidation is loss of electrons, addition of oxygen, removal of hydrogen, and an increase in oxidation state (carried out by oxidants). Reduction is gain of electrons, removal of oxygen, addition of hydrogen, and a decrease in oxidation state (carried out by antioxidants/reducing agents).
2. $O_2$ -> superoxide radical ($O_2^{\cdot -}$) -> hydrogen peroxide ($H_2{O}_2$) -> hydroxyl radical ($H{O}^{\cdot }$) -> water ($H_{2}O$); each step is a one-electron transfer (H2O2 can also go directly to water via a 2-electron transfer).
3. $O_2\to O_2^{\cdot -}$: NADPH oxidases (NOX) or cytochrome c oxidase (complex IV, 4-electron route). $O_2^{\cdot -}\to H_2{O}_2$: superoxide dismutase (SOD). $H_2{O}_2\to H_{2}O$: peroxidases — catalase, glutathione peroxidases, peroxiredoxins.
4. The two reactions that differ are H2O2 -> hydroxyl radical and hydroxyl radical -> water. In the pathological route, H2O2 -> hydroxyl radical occurs non-enzymatically via the Fenton reaction (iron-catalysed), and hydroxyl radical -> water occurs via damaging reaction with polyunsaturated fatty acids (lipid peroxidation), rather than via the controlled enzymatic detoxification of the physiological route.
5. Radicals: superoxide (O2*-), hydroxyl (OH*), nitric oxide (NO*). Non-radicals: hydrogen peroxide (H2O2), peroxynitrite (ONOO-), hypochlorous acid (HOCl).
6. Mitochondria (electron transport chain), transmembrane NADPH oxidases, xanthine oxidoreductase in peroxisomes, protein disulfide isomerase/UPR in the ER, and diverse enzymatic reactions (e.g. prostaglandin synthesis, adrenalin auto-oxidation, reduced riboflavin).
7. In the minor pathway, substrate-derived electrons “leak” from Complex I, II or III and reduce O2 directly to superoxide, instead of passing along the normal electron transport chain to Complex IV — this leakage, rather than controlled 4-electron reduction, is the source of mitochondrial ROS.
8. Resting state: membrane subunits gp91phox/NOX2 and p22phox are present in the membrane; cytosolic subunits p47phox, p40phox, p67phox and RAC-GDP (with Rho GDI) are separate in the cytoplasm. On activation: RAC exchanges GDP for GTP, p47phox is phosphorylated, and the cytosolic subunits translocate to and dock with the membrane complex; the assembled enzyme transfers electrons from NADPH (oxidised to NADP+ + H+) through FAD and haems to O2, generating superoxide.
9. (1) The L-arginine-nitric oxide pathway: L-arginine + O2 via NO synthase -> NO. (2) The nitrate-nitrite-nitric oxide pathway: dietary nitrate reduced by bacterial nitrate reductases/xanthine oxidase to nitrite, then reduced to NO by deoxygenated haemoglobin/myoglobin, xanthine oxidase, respiratory chain enzymes, and protons.
10. Ionising radiation (X-rays, UV), toxic chemicals (smoking, pollution), alcohol and other drugs (e.g. paracetamol), hyperbaric oxygen treatment, and heavy metals.
11. Metabolism (oxidative phosphorylation), synthesis of essential compounds (prostaglandins), detoxification of foreign compounds (cytochrome P450 in liver), immune responses (phagocytosis), cell signalling (NFkB activation).
12. “Any substance that delays, prevents or removes oxidative damage to a target molecule” (Halliwell and Gutteridge, 2015).
13. Catalytic removal of ROS; antioxidant molecules; decreasing ROS formation; protective proteins (chaperones); synthesis of resistant molecules; repair processes/removal; sequestration of metal ions.
14. Enzymatic (NOXs, XO, LO, COX, uncoupled eNOS) and non-enzymatic (ER, mitochondrial ETC, peroxisomes) sources generate superoxide; superoxide is converted to H2O2 spontaneously or by SODs (or reacts with NO to form peroxynitrite); H2O2 is detoxified to water by catalase directly, by the glutathione peroxidase/glutathione reductase system (using GSH/GSSG), or by the thioredoxin/peroxiredoxin system (using NADPH via thioredoxin reductase).
15. Endogenous: glutathione (GSH), taurine, hypotaurine, polyamines, plasminogens, plasma albumin, coenzyme Q, uric acid. Exogenous: tocopherols (vitamin E), ascorbate (vitamin C), carotenoids, plant phenols (e.g. EGCG, resveratrol, curcumin).
16. Alpha-tocopherol is lipid-soluble and sits in membranes, where it scavenges lipid peroxyl radicals (ROO*) before they can propagate lipid peroxidation. Reaction: alpha-tocopherol + ROO* -> alpha-tocopheroxyl radical (+ non-radical lipid product).
17. It regenerates alpha-tocopherol from the alpha-tocopheroxyl radical; it is a cofactor for at least 15 enzymes (needed for collagen, carnitine, hormone, and amino acid formation, and epigenetic modifications); it can generate ROS from copper and iron (a pro-oxidant action), despite being an effective reducing agent/scavenger in vitro.
18. Plasma ascorbate rises steeply with dose between roughly 50-150 mg/day, then plateaus around 65-70 umol/L from ~200 mg/day upward (a saturable, sigmoidal dose-response). Heavier individuals show lower plasma vitamin C for a given intake, so vitamin C requirement scales with body weight — roughly an extra 10 mg/day needed per 10 kg of body weight to match the plasma level of a 60 kg person on ~110 mg/day.
19. Oxidative stress is a disturbance in the pro-oxidant-antioxidant balance in favour of the pro-oxidant, leading to potential damage — it describes the imbalance itself. Oxidative damage is the actual biomolecular damage caused by ROS attacking cellular constituents — it describes the consequence. Stress can occur without yet causing (detectable) damage.
20. ESR/EPR detects unpaired electrons directly but is not useful in vivo (requires specialised conditions unsuited to living tissue). Instead, oxidative damage is assessed indirectly, via oxidation of detector molecules or by measuring lipid, protein, and DNA oxidation products (biomarkers).
21. Any three of: chemically stable; directly implicated in disease onset/progression; specific for the ROS/RNS in question; non-invasive; low intra/inter-individual variability; accurate/precise/validated assay; established reference values; established animal models; demonstrated efficacy of antioxidant supplementation; use of physiologically relevant concentrations.
22. The antioxidant paradox is that giving large doses of dietary antioxidant supplements has shown little or no therapeutic effect in humans, despite oxidative stress being implicated in many diseases. The Cochrane review (56 trials, 244,056 participants) found no evidence supporting antioxidant supplements for primary or secondary prevention, and found beta-carotene, vitamin E, and possibly high-dose vitamin A increased mortality.
23. In early-stage tissue injury, oxidative stress actively aggravates the disease process, so reducing it with antioxidants has a plausible therapeutic benefit. In late-stage injury (accompanying cell death), the oxidative stress reflects/accompanies necrotic death spreading damage and triggering inflammation, and coincides with induction of the body’s own defence systems (chaperones, haem oxygenase) — at this stage antioxidant intervention does not contribute usefully to preventing pathology and could be deleterious.
24. The study’s implication (drawn from the lecture’s framing of exercise as a mild, beneficial pro-oxidant challenge that triggers an adaptive response) is that antioxidant supplementation neutralises the ROS signal that exercise normally generates to trigger this beneficial adaptive response, thereby blunting the improvement in insulin sensitivity that exercise alone produces.
25. Mitochondrial dysfunction; NADPH oxidase activation; disruption of metabolism; activation of stress-responsive signalling pathways; down-regulation of antioxidant systems.
26. Reducing sugar’s carbonyl group condenses with a protein amine to form a Schiff base -> rearranges via the Amadori reaction into a stable Amadori product -> some Amadori products convert to AGEs via the Hodge pathway, others are oxidised/cleaved to reactive dicarbonyl compounds (e.g. methylglyoxal) -> dicarbonyls cross-link with proteins to form AGEs (e.g. CML, pentosidine).
27. AGEs (or HMGB1/S100 calgranulins) bind RAGE on the endothelial cell surface -> activates NAD(P)H oxidase (increasing ROS) and p21 RAS/MAPK signalling (including p38, Rac/Cdc42) -> activates NF-kB, which translocates to the nucleus and increases transcription of endothelin-1, ICAM-1, E-selectin, VCAM-1, tissue factor, IL-6, TNF-alpha. Separately, soluble AGEs activate monocytes, increasing macrophage scavenger receptor A and CD36 expression, increasing oxidised-LDL uptake and foam cell formation — linking this pathway to atherosclerotic plaque formation.
28. Free fatty acids and hyperglycaemia activate DAG, which activates PKC. PKC increases expression of ET-1, VCAM/ICAM, NFkB, and NADPH oxidase; NADPH oxidase activation generates superoxide, causing oxidative stress and decreased NO bioavailability.
29. NADPH oxidase (activated via angiotensin II/AT1R) and mitochondria (via high glucose/lipid excess) both generate ROS, causing oxidative stress that activates serine kinases p38 MAPK, JNK, GSK-3beta and IKKbeta (among others). These kinases impair insulin receptor signalling, reducing GLUT4 vesicle translocation to the membrane and thus reducing glucose uptake.
30. Resting state: Keap1 (cysteine thiols reduced) binds Nrf2 with Cul3/Roc1/E2, targeting Nrf2 for ubiquitination and proteasomal degradation. Stress: oxidative/electrophilic stressors oxidise Keap1’s cysteines, preventing Nrf2 degradation; Nrf2 accumulates and translocates via importin to the nucleus; there it complexes with small Maf proteins and CBP/p300, binds the ARE, and drives transcription of ARE target genes.
31. Direct antioxidants; free radical metabolism; electrophile detoxication; glutathione homeostasis; generation of reducing equivalents; solute transport; inhibition of inflammation; DNA damage recognition; proteasome function.
32. Any two of CAPE, CDDO-Im, curcumin, resveratrol — all induce HO-1 (haem oxygenase-1) via Nrf2 nuclear translocation.
33. Tecfidera (dimethyl fumarate) and Vumerity (converts to monomethyl fumarate) — relapsing forms of multiple sclerosis; Bafiertam (bioequivalent to Tecfidera) — relapsing MS; Skyclarys (omaveloxolone) — Friedreich’s ataxia (adults/adolescents >=16y).
34. Likely not adequate on its own: the lecture states requirement rises by roughly 10 mg/day for every additional 10 kg of body weight above the 60 kg reference. At 90 kg (30 kg heavier), the partner would need roughly an extra 30 mg/day (i.e. ~140 mg/day) to reach the same plasma concentration that 110 mg/day achieves in the 60 kg woman — so 110 mg/day for him would likely produce a lower plasma ascorbate level than hers.
35. (1) Enhanced oxidative stress from abdominal-fat-related inflammation increases consumption/depletion of vitamin C. (2) Diabetic renal dysfunction causes increased leakage/loss of vitamin C via the kidneys. The lecture-cited recommendation for people with type 2 diabetes is at least 125 mg/day (a ~150% increase on the standard 45 mg/day NZ recommendation), reflecting an estimated 1.4-1.6-fold higher requirement.
36. Example path: chronic hyperglycaemia -> increased AGE formation (via the Schiff base/Amadori pathway) -> AGE binding RAGE on endothelial/vascular cells -> NAD(P)H oxidase activation -> increased ROS (superoxide/H2O2) -> activation of stress-responsive serine kinases (e.g. p38 MAPK, JNK) -> these kinases impair insulin receptor/IRS signalling -> reduced GLUT4 translocation -> reduced glucose uptake in skeletal muscle (an equally valid alternative path runs through mitochondrial ROS from high glucose/lipid excess rather than AGE-RAGE). Nrf2 activation would be expected to interrupt this path because Nrf2 target genes include direct antioxidants and free-radical-metabolising enzymes that would lower the ROS burden generated by NOX/mitochondria, reducing activation of the stress kinases that impair insulin signalling — consistent with the lecture’s summary that Nrf2 induction is associated with improved insulin resistance and beta-cell protection. However, the lecture’s own evidence (the antioxidant paradox, the Cochrane review showing possible harm from antioxidant supplements, and the “good, the bad, and the complex” framing of NRF2 in diabetes, including contradictory results depending on acute vs chronic activation and model system) argues against assuming pharmacological or supplement-based Nrf2/antioxidant activation will translate into clinical benefit without more definitive trial evidence — and no NRF2 activators were, at the time of the cited review, in clinical trials for diabetes specifically.