Developing a Comprehensive Therapeutic Research Strategy for the Converging Epidemics of TB, T2DM, and HIV

NIAID and NIDDK WorkshopMay 10th, 2016

Metabolite-Induced ROS Overproduction Activates the Diverse Biochemical and Molecular Mechanisms Underlying Diabetic

Complications

Michael Brownlee, M.D.Anita and Jack Saltz Chair in Diabetes Research

Associate Director for Biomedical Sciences,Einstein Diabetes Research Center

Professor of Medicine and Pathology Albert Einstein College of Medicine

New York

Physiologic ROS production is essential for normal intracellular signaling and cellular homeostasis

Holmström, K.M. & Finkel, T. Nature Reviews Molecular Cell Biology 15, 411-421, 2014

Current view of ROS stress

Nathan, C., and Cunningham-Bussel, A. Nat Rev Immunol 13:349-361, 2013

The diabetic complications epidemic

DiabeticRetinopathy

Leading causeof blindness

in working ageadults

Stroke

2- to 4- fold increase in stroke and dementia

DiabeticNeuropathy

Leading cause of non-traumatic lower extremity amputations

CardiovascularDisease

CVD: 2/3 of heart attack pts. have diabetes or IGT

Diabetic NephropathyLeading cause of

end-stage renal disease

20-50% of T2D, 33% of T1DIncreased risk of CVD and death

Hyperglycemia- and FFA-induced overproduction of ROS activates all major pathways of diabetic cellular damage

↑ Polyol PW flux

↑PKC ↑Hexosamine PW flux

Cytoplasm

↑ Glucose/FFA

Modified from Brownlee M., Nature, 2001; 4104:813-82

↑Methylglyoxal

↑ NFκB↑RAGE & RAGE ligands

↑ AGE

↑ PARP

Nucleus↓ GAPDH

↑ ROS

Superoxide also inhibits critical endothelial enzymes

Endothelial nitric oxide synthase (eNOS) KD

Zhao, H.J., et al. J. Am. Soc Nephrol 17:2664-2669, 2006 ; Nasu, T., et al., Am J Phys Renal Phys 302:F1616-F1629, 2012

Prostacyclin synthase (PGI2) KD

Recently described mechanisms of diabetic tissue damage activated by metabolite-induced overproduction of ROS

• Nuclear import of NFAT transcription factors• Nuclear translocation of FOXO transcription factors• PPARα nuclear receptor expression• CaMKII activation/increased intracellular Ca++

• Decreased sirtuin 1, PGC1α, AMPK expression• Downregulation and mismatching of specific miRNAs• NRLP3 inflammasome activation• Neutrophil extracellular trap formation/mφ priming

Shah, M.S., and Brownlee, M. Circ Res in press, 2016

Transgenic expression of the antioxidant enzyme superoxide dismutase prevents each

complication in experimental diabetes

Diabetic retinopathy Diabetic nephropathy

Diabetic cardiomyopathyDiabetic neuropathy

Post-translational modifications of histones cause chromatin remodeling and changes in levels of gene expression

Hyperglycemia-induced ROS cause activating modifications of histone 3 lysine 4 and de-repressing modifications of histone 3 lysine 9 at the

NFκB p65 proximal promoter

El-Osta, A., et al. J Exp Med, 2008, 205:2409

In non-diabetic mice, transient hyperglycemia induces a long-term increase in H3K4me1 at the NFκB p65 promoter

by recruiting the histone methyltransferase Set 7

El-Osta, A., et al. J Exp Med, 2008, 205:2409

0

100

200

300

qPCR

for N

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rom

oter

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and

cChI

P by

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HG(6h)+LG(4d) HG(6h)+LG(6d) LG(6d)

* **

*

Treatment Group (clamped mice)

In non-diabetic mice, six hours of transient hyperglycemia causes a six DAY increase in NFκB expression, a master

regulator of inflammatory genes

El-Osta, A., et al. J Exp Med, 2008, 205:2409

000

001

002

003

004

005

006

007

008

009

010

6h 6h 6h+vector 6h+MnSOD 6h+2d 6h+5d

RO

S (n

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g pr

otei

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*

* *

5mM glucose 25mM glucose 25mM glucose + 5mM glucose

After transient hyperglycemia, elevated mitochondrial ROS production persists for days

Ferdinando Giacco et al. Diabetes 2015;64:3273-328

Transient exposure to hyperglycemia above a critical threshold level activates a multi-component positive feedback loop

Ferdinando Giacco et al. Diabetes 2015;64:3273-3284

ROS-induced methylglyoxal adducts accumulate in TB lesions of patients

Rachman H, Kim N, Ulrichs T, Baumann S, Pradl L, et al. (2006) . PLoS ONE 1(1): e29. doi:10.1371/journal.pone.0000029

Van Heijst, H.W.J, and Pamer, E.G. Cell 153:507-508, 2013

TNF-induced mitochondrial ROS in TB-infected macrophages increases killing of mycobacteria, but increases necroptosis and mycobacterial release

into the extracellular milieu and bacterial dissemination

Roca, F.J and Ramakrishnan, L. Cell 153:521-534, 2013;

Colleagues who contributed to the work presented today

CollaboratorsNaila Ahmed Angelika BierhausMark CooperStephanie DimmelerAjit S. DivakaruniAssam El-OstaHans-Peter HammesAnne N.MurphyPeter NawrrothAlessandro PocaiRobert RoederVijay P. SarthyPhilip SchererGuntram SuskePaul ThornalleyCsabo SzaboMark Yorek

Brownlee LabAnna CarratuXueliang DuDiane EdelsteinIda GiardinoFerdinando GiaccoQida JuTakeshi MatsumuraTakeshi NishikawaManasi ShahGuanghi SuiTetsuya Taguchi