Glycotoxins&Glucotoxicity

김선여 교수
The major precursors for the advanced glycation end-products (AGEs)
identified in vivo; 3DG-H1: 3-Deoxyglucosone-hydroimidazolone 1; CML: Nε-(carboxymethyl)-lysine;
G-H1; Glyoxal-derived hydroimidazolone 1; MG-H1: Methyglyoxal-derived hydroimidazolone 1.
Examples of fluorescent cross-linked
AGEs
Examples of nonfluorescent cross-linked
AGEs
Examples of nonfluorescent non-cross-
linked AGEs
Example of fluorescent non-cross-linked
AGEs
Identification of DNA Adducts of
Methylglyoxal
DNA Advanced Glycation End Products (DNA-AGEs) Are Elevated in
Urine and Tissue in an Animal Model of Type 2 Diabetes
Chem. Res. Toxicol. 2017, 30, 2, 689–698
The main AGEs that have been quantified in vivo are shown.
AGE structures are given as AGE free adducts
Pathways of dicarbonyl detoxification. MG reacts with glutathione to yield hemithioacetal, the substrate for glyoxalase 1 (GLO1). (A) The product, S-
D-lactoylglutathione, is hydrolyzed by glyoxalase 2 (GLO2) to yield D-lactate and reduced glutathione. Glyoxal is also metabolized via this pathway
and results in glycolate production (not shown). Aldose reductases (B) catalyze the NADPH (nicotinamide adenine dinucleotide phosphate)-
dependent reduction of MG which yield hydroxyacetone (major product) and lactaldehyde (minor product) in the absence of reduced glutathione
(GSH). Lactaldehyde may be further reduced to propanediol. In the presence of GSH (C) aldose reductase acts on the hemithioacetal which results
in a shift towards lactaldehyde production. Products of 3-DG and glyoxal metabolism of aldose reductase are 3-deoxyfructose and glycolaldehyde
respectively (not shown). Aldehyde dehydrogenase (D) oxidizes dicarbonyls and is of potential importance for 3-DG metabolism with the resulting
product being 2-keto-3-deoxygluconic acid. Products of MG and glyoxal oxidation are pyruvate and glyoxylate, respectively
Interactions among glucose homeostatic pathways and target cells susceptible to diabetes complications.

 Target cells include endothelial cells, podocytes, proximal

tubular cells, Muller cells, cardiomyocytes, and neuronal

cells.

(Forbes and Cooper 2013)

Type 1 diabetes related to organ and cells

 Type 1 Diabetes is an autoimmune disease that destroys

pancreatic beta cells, resulting in insulin deficiency, which leads

to hypergylcemia and ketoacidosis.

(Mittendorfer and Klein 2014)

Possible molecular mechanism for suppression of insulin biosynthesis in type 2 diabetes

 Under diabetic conditions, hyperglycemia induces oxidative

stress and thereby leads to suppression of insulin biosynthesis

and secretion, which is accompanied by reduction of nuclear

PDX-1 and MafA expression.

 Therefore, it is likely that induction of oxidative stress and

suppression of nuclear PDX-1 and MafA expression are

involved in β-cell glucose toxicity found in type 2 diabetes.

(Kaneto and Matsuoka 2015)

Glucotoxicity targeting

Hypersecretion of insulin to compensate

for insulin resistance

Glucotoxicity Lipotoxicity

Chronic Pancreas High circulating

hyperglycemia Free fatty acids

β-cell (Boden and Shulman 2002)

dysfunction (Kim and Yoon 2011)
Finegood DT & Topp B. Diabetes Obes Metab 2015; S18-S21
Hyperglycemia may contribute to insulin resistance by promoting the formation of AGEs in insulin target cells

 In insulin target cells, high concentration of glucose

leads to the formation of AGEs (advanced

glycosylation end products) by reacting with amino

groups on intracellular and extracellular proteins.

(Lasram, Dhouib et al. 2014)

Multiple pathways and mechanisms through which chronic hyperglycaemia may be impair beta
cell function and cause beta cell apoptosis

• Prolonged exposure to increased glucose concentrations causes gradual loss of insulin gene expression, secondary to the

diminished activity of the key regulators of insulin promoter activity (such as insulin gene transcriptor activators) and other

beta-cell specific genes.

• These processes are mediated at least in part by oxidative stress.

• The oxidative stress activates stress-induced pathways that damage the beta cells by inducing defective insulin

biosynthesis and secretion, and ultimately apoptosis.

Multiple pathways and mechanisms through which chronic hyperglycaemia may be impair beta
cell function and cause beta cell apoptosis

• Long-term sustained hyperglycemia increases the metabolic flux into the mitochondria and induces excessive generation of

reactive oxygen species (ROS) which lead to chronic oxidative stress.

• Elevated ROS disturb the integrity and function of cellular proteins (e.g. enzymes, receptors, transport proteins), lipids and

deoxyribonucleic acid (DNA): they degrade polyunsaturated fatty acids of the membranes, induce lipid peroxidation and

amino acids oxidation and damage purine and pirimidine bases.

AGE-induced pathways involved in cell dysfunction according to.

Membrane depolarization ↓

ATP-K+ channels Ca2+-channels

NO Cyt c
ATP↓ Ca2+↓
oxidase↓ Insulin secretion ↓
↑iNOS

TCA
Mitochondrium
cycle ↓

Nucleus
PDX-1 PDX-1 ↓
Ac
↑FoxO1

FoxO1 Insulin synthesis ↓

AGEs
↓FoxO1
P

Pancreatic beta cell

 Decreased insulin synthesis and reduced insulin secretion are both involved in beta cell failure contributing to hyperglycemia.
 Arrows illustrate direct interactions; dashed arrows illustrate possible targets of AGEs. (Nowotny, Jung et al. 2015)
AGE-induced pathways involved in cell dysfunction according to.

I. AGEs

 FoxO1 (phosphorylation↓), FoxO1 (acetylation↑)

 Induce PDX-1 translocation into the cytoplasm, PDX-1 protein expression↓- Finally affecting insulin gene

transcription and insulin synthesis.

II. Regarding insulin secretion, AGEs cause inhibition by activation of iNOS and consequent blocking of

cytochrome c oxidase activity and ATP depletion.

III. AGEs decrease insulin secretion through alterations in the TCA cycle which limits ATP production.

IV. ATP depletion inhibits closure of ATP-dependent potassium channels which leads to reduced

membrane depolarization and decrease of intracellular calcium concentration inhibiting insulin

secretion.
Schematic illustration of hyperglycemia-induced cell damage

# Diabetes as a trigger of neurodegeneration : From

pathophysiological mechanisms to therapeutic
approaches,Advances in Molecular Mechanisms and
Pharmacology of Diabetic Complications, 2010: 175-198

 These pathways can contribute to overproduction of reactive oxygen species (ROS), which are able to damage mitochondrial and cellular

membranes, ultimately leading to cell degeneration and death.

Mechanisms of hyperglycemia-induced endothelial dysfunction

(Rajasekar, O'Neill et al. 2015)

 Key processes responsible for hyperglycemia-induced endothelial dysfunction include the polyol pathway, reactive oxygen species
(ROS) formation, and advanced glycation end products (AGEs) formation.
Mechanisms of hyperglycemia-induced endothelial dysfunction

 Slide 11

 The excess glucose in endothelial cells enters polyol pathway; the electron donors like reduced nicotinamide adenine

dinucleotide (NADH) and Flavin adenine dinucleotide (FADH2) accumulate in the mitochondria. Thus affecting the electron

transport chain; the excess electrons increase ROS in mitochondria.

→ ROS triggers accumulation of AGEs; ROS and AGEs create mitochondrial DNA damage and mitochondrial dysfunction.

→ Accumulation of AGEs also increases ROS production independent of glucose levels.

 Protein kinase C (PKC) and AGEs mediated activation of nuclear factor kappa B (NF𝜅 B) activate the expression of

inflammation proteins, tumor suppressor p53, and inducible nitric oxide synthase (iNOS); increased nitric oxide (NO) by iNOS

is highly reactive with superoxide anions; the peroxynitrite ONOO-) thus generated acts as a strong oxidant and completes

the vicious cycle of oxidative stress by increasing ROS production.

Hyperglycemia-induced oxidative stress and endothelial dysfunction (possible role of ER stress)

 The excessive ROS so formed can then aggravate cellular injury by

promoting activation of the biochemical pathways (red dotted arrows)

that initiate ROS generation in the first place as a response to

hyperglycemia, thus completing a vicious cycle.

 The possibility that ER stress response also can lead to excessive ROS

formation cannot be ruled out.

(Basha, Samuel et al. 2012)

Hyperglycemia and Diabetes Complications

 In endothelial cells, glucose can pass freely, in an insulin-

independent manner, through the cell membrane.

 Intracellular hyperglycemia induces overproduction of

superoxide at the mitochondrial level.

 This process results in acute endothelial dysfunction in diabetic

blood vessels that contributes to the development of diabetes

complications.
Insulin action in the CNS and PNS

(Kim and Feldman 2012)

 Even though neurons are generally not considered to be insulin-dependent, they are responsive to insulin, and InRs are widely
distributed in both the PNS and CNS.
Model of the development of neuronal IR

(Kim and Feldman 2012)

⒜ Insulin-mediated signaling activates Akt and maintains normal neuronal function.

⒝ In the presence of MetS, as in insulin-dependent tissues, neurons develop IR, and in turn cannot respond to the neurotrophic properties of insulin.
Insulin resistance in the nervous system

 Recently, the study of IR (Insulin resistance) was mainly focused on metabolic tissues such as muscle and adipose tissue; recent

data, however, suggest that IR also develops in the nervous system.

 IR in sensory neurons makes cells respond inappropriately to growth factor signals, and this impairment may contribute to the

development of neurodegeneration and subsequent DN (Diabetic Neuropathy).

 IR in diabetes is tightly correlated with the increased risk of AD by making cortical and hippocampal neurons more vulnerable

to Ab and tau toxicity. In both systems, decreased Akt signaling is the common feature of neuronal dysfunction.

 Therefore, Akt signaling is crucial for cell survival and normal cell function.
Schematic representation of the proposed mechanism by which higher glycolytic rates in astrocytes may provide a
mechanism limiting MG toxicity in neurons

(Allaman, Belanger et al. 2015)

 Glycolysis in both astrocytes and neurons leads to the production of the toxic Methylgloxal (MG) by-product.
 MG is detoxified by both cell types through the glyoxalase system (GLO), producing D-lactate (D-lac).
 Green arrows highlight the prevalent routes of glucose utilization in brain cells.
Damage at each retinal layer. A series of events occur in early DR development. Neurodegeneration of
horizontal, bipolar, amacrine, and ganglion cells. These damages may be determined by proNGF
concentrations as NLRP3 and NLRP1 are related to eye degenerative diseases. NFL: nerve fiber layer; GCL:
ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; ∗OPL: outer plexiform layer; ONL: outer
nuclear layer; PL: photoreceptor laye
Glucose metabolic pathways in the hyperglycemic milieu, oxidative stress in diabetic retinopathy, and
antioxidant targets. In hyperglycemic states, different pathways were activated producing ROS which enhance
inflammatory, apoptotic, and degeneration pathways, ultimately leading to the appearance of diabetic
retinopathy clinical characteristics.
The ROS role in inflammation and pyroptosis. ROS augments NF-κB production which promotes
proinflammatory mediators favoring the expression of VEGF. VEGF translocates NF-κB into the
nucleus, and NF-κB activate NLRP3 with caspase cleavage leading to cytokine release. NLRP3
inflammasome has been associated to diabetic retinopathy by Müller pyroptosis by the caspase-
1/IL-1beta pathway. NF-κB: nuclear factor kappa B; COX-2: cyclooxygenase-2; VEGF: vascular
endothelial growth factor.
The ROS role in neurodegeneration. In physiological conditions, NGF activates VEGF to promote
angiogenesis and protect nerves from hypoxia and ROS inhibits NGF formation from its precursor
which leads to neural apoptosis. ROS activate ZNRF1 that provokes neurodegeneration; at the
same time, TNF-α activates apoptosis via metalloproteinase/caspase pathway. ZNRF1: zinc and ring
finger-1; NGF: nerve growth factor; VEGF: vascular endothelial growth factor; MMP: matrix
metalloproteinases; TNF-α: tumor necrosis factor-α.
Skin aging

 Accumulation of AGEs in the skin has been therefore thoroughly studied and is detected not only in

diabetes as expected but also during chronological aging.

 The appearance of glycated collagen is first observed at the age of 20. It accumulates with a yearly rate

of about 3,7% reaching a 30–50% increase at 80 y of age.

 Accumulation of AGEs was mainly found in sites of solar elastosis in sun-exposed skin, showing that UV

irradiation may also precipitate the formation of AGEs in vivo.

 Smoking, a typical aggravating factor of skin aging, accelerates formation of AGEs and increases their

deposition in various tissues including skin.

Skin aging

 Effects of AGEs on skin

(Gkogkolou and Bohm 2012)

Skin aging

UVs

Biomechanic
Rigidification / Elasticity

Glycation
Chronological Cellular viability
And AGEs
Aging Apoptosis / Senescence
accumulation

Matrix Extracellular balance

Expression of mRNAs
Mechanisms of Glycation production Protein synthesis
Observed tissue and cellular function alteration

(Pageon, Zucchi et al. 2014)

Advanced Glycation End-products (AGEs) - Skin aging

1. When sugar comes in contact with a protein (such as collagen), it immediately reacts. This generates Reactive Oxygen

Species (ROS – free radicals), which leads to a cross-linking of collagen and inflammation.

2. Advanced Glycation End-products (AGEs) are formed, and bond with a Receptor on the cell to form Receptro-AGE (R-

AGEs)

3. This causes inflammation, inhibits skin cell growth and contributes to cross-linking of collagen.
Detected AGEs in skin

(Gkogkolou and Bohm 2012)

 Table lists the most commonly found ones in the skin.

Expression of human RAGE in skin and skin cell

 Keratinocytes, fibroblasts, dendritic cells and to a lesser extent

endothelial cells and lymphocytes express RAGE.

 Not only in vivo, but also in vitro, various skin cells types have

been shown to express RAGE.

(Gkogkolou and Bohm 2012)

Effects of AGEs/RAGE on skin morphology and physiology during aging

(Gkogkolou and Bohm 2012)

 ICAM, intercellular adhesion molecule; MCP-1, monocyte chemotactic protein-1; TIPM, tissue inhibitor of MMP; VCAM, vascular cell adhesion molecule; all

other abbreviations are already explained in the text.

Influence of methylglyoxal-modified collagen on cellular functions relevant to diabetic complications.

(Nowotny, Jung et al. 2015)

 Methylglyoxal-modified collagen reduces cell adhesion and migration of mesangial cells, cardiac fibroblast are less adherent.

 Myofibroblast differentiation is stimulated in cardiac fibroblasts cultured on modified collagen and modifications of basement membrane collagen causes

detachment, cell death and reduced angiogenesis of vascular endothelial cells.

a Melatonin improves diabetic cardiomyopathy through inhibiting mitochondrial fission by activation of SIRT1‐PGC1α pathway
b Melatonin reduces diabetic retinopathy through inhibiting oxidative stress by reduction of ROS, MDA and NO levels and activation of CAT and PI3K/Akt-Nrf2
pathway, improving blood-retinal barrier by reduction of the expression of HIF-1α, VEGF-A and PEDF c Melatonin ameliorates diabetic neuropathy through
inhibiting oxidative stress by increasing CAT, SOD and GPx activity and GSH level and activating Nrf-2-HO-1 pathway, d Melatonin improves diabetic
nephropathy through inhibiting fibrotic process by reduction of the expression of TGF-β, inhibiting oxidative stress by enhancement of mitochondrial complex III,
CAT, SOD, GPx and GST activities and reduction of NOX activity and MDA and NO generation,
References

1. A review on the molecular mechanisms involved in insulin resistance induced by organophosphorus pesticides

2. Absence of leptin triggers type 1 diabetes

3. Advanced Glycation End Products and Oxidative Stress in Type 2 Diabetes Mellitus

4. Advanced glycation end products Key players in skin aging

5. Diabetes as a trigger of neurodegeneration From pathophysiological mechanisms to therapeutic approaches

6. Epigenetic Changes in Endothelial Progenitors as a Possible Cellular Basis for Glycemic Memory in Diabetic Vascular
Complications.

7. Free fatty acids in obesity and type 2 diabetes defining

8. Glucolipotoxicity in Pancreatic β-Cells

9. Insulin resistance in the nervous system

10. Mechanisms of Diabetic Complications

11. Methylglyoxal, the dark side of glycolysis

12. Role of Pancreatic Transcription Factors in Maintenance of Mature β-Cell Function

13. What Causes Skin Aging