Metabolic Abnormalities and Mitochondrial Function in Children with Perinatally-Acquired HIV Infection in the Pediatric HIV/AIDS Cohort Study (PHACS) National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute Mariana Gerschenson, Ph.D. Tracie L. Miller MD, Jiajia Wang MS, Denise L. Jacobson PhD, MPH, Jody K. Takemoto PhD, Tanvi Sharma MD, Mitchell Geffner MD, Daniel E. Libutti BS, Susanne Siminski MS, MBA, Gabriel Somarriba DPT, and Patricia Graham MS for the Pediatric HIV/AIDS Cohort Study July 23, 2012 XIX International AIDS Conference

Background Insulin resistance is common among perinatally HIV-infected children (HIV+) Mitochondrial dysfunction, induced by antiretroviral therapy (ARV) or chronic viral infection, is a possible mechanism for metabolic dysfunction National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute

Mitochondrial Function Make ATP – Oxidative phosphorylation  ATP – Citric acid cycle: NADH and FADH  ATP – Fat metabolism: beta oxidation  ATP Responsible for 90% of the oxygen radicals/oxygen stress Mitochondrial DNA (mtDNA) – 16 kb, 2-10 copies/mitochondrion in matrix – Replication: mitochondrial genes The mtDNA polymerase-γ is the principal polymerase required for mtDNA replication – Transcription: mitochondrial proteins Mitochondrial RNA (mtRNA) polymerase and transcription factors Apoptosis Lactic acidosis Adipocyte TEM courtesy of Courtney Kim and Mariana Gerschenson Mitochondria Inner Structure HIV Inner Membrane Outer Membrane Cristae Matrix Adipocyte TEM

Hypothesis: Potential Consequences of Mitochondrial Dysfunction Cardiovascular Disease Lipodystrophy Neuropathies Hepatic Steatosis Myopathy Pancreatitis Lactic Acidosis Metabolic Disease (LD, IR,  TGs) ART Cytokines Day L, et al. Mitochondrion. 2004;4:95–109. AACTG Metabolic Guides: McComsey GA, Morrow JD. J Acquir Immune Defic Syndr. 2003;34:45-9. Dagan T, Sable C, Bray J, Gerschenson M. Mitochondrion. 2002;1: Gerschenson, M. and Brinkman, K. Mitochondrion. 2004;4(5-6): HIV DNA polymerase-  Transcription Uncoupling Transport Oxidative Stress Apoptosis Phosphorylation Proteolytic Processing Glycosylation

Hypothetical Model for HIV Pediatric Metabolic Disease National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute  BMI/ Body Measurements  LDL/HDL/TG/HOMA-IR (Glucose) OXPHOS Krebs  -oxidation  LactateHIV/ART

Objectives To compare mitochondrial function [oxidative phosphorylation (OXPHOS) enzyme activities and lactate levels] of 191 HIV+ and 117 HIV-exposed, uninfected (HEU) children and among HIV+, determine associations with homeostatic model assessment of insulin resistance (IR) (HOMA-IR) and hyperlipidemia In HIV+ children, to determine if mitochondrial function (oxidative phosphorylation [OXPHOS] enzyme activities and lactate and pyruvate levels) are associated with metabolic abnormalities (IR and hyperlipidemia)

Mitochondrial Determinant Component of PHACS The Adolescent Master Protocol (AMP), a part of the Pediatric HIV/AIDS Cohort Study (PHACS), is a prospective cohort study conducted at 12 US sites designed to define the impact of HIV infection and ARV on pre- adolescents and adolescents with perinatal HIV infection. The Mitochondrial Determinants Component (MDC) is a separately funded study (R01 NR12885) that has co-enrolled AMP participants since May The objective of MDC is to determine the influence of mitochondrial abnormalities on metabolic outcomes in HIV+ children who were previously enrolled in AMP from 7 years of age until their 16th birthday. HIV-exposed, uninfected children (HEU) are also enrolled to serve as controls.

Demographics of Cohort by HIV Status National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute CharacteristicHIV+ (191)HEU (117)Total (308)P-value Age, median (yr) <0.001* Male (%)104 (54)60 (51)164 (53)0.64** Race, NHB (%)133 (70)66 (56)199 (65)0.05** Tanner stage 1 (%) Tanner stage 2-3 (%) Tanner stage 5 (%) 9 (5) 68 (36) 83 (43) 14 (12) 72 (63) 19 (16) 23 (7) 140 (45) 102 (33) <0.001** BMI Z-score, median * *Wilcoxon Test ** Fisher’s Exact Test

Clinical Characteristics of HIV+ Children at Study Entry National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute CharacteristicValue CDC Stage, N (%)N/A84 (44) B55 (29) C41 (21) CD4 Count, cell/mm3Median (IQR)624 (463, 859) Viral load, copies/mL (%)> (18) (21) <40061 (11) ARV Regimens (%) HAART with PI111 (58) HAART without PI30 (16) Non-HAART ARV10 (5) No ARV10 (5)

Baseline Metabolic Outcomes in HIV-Infected Children National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute Metabolic OutcomeNAbnormal (%)MedianIQR Fasting Glucose (mg/dL) , 91 Fasting Insulin (  u/mL) , 20.9 HOMA-IR (normal <2.5 for Tanner 1 or <4 for Tanner 2-5) , 4.5 Total cholesterol (mg/dL) , 186 HDL-cholesterol (mg/dL) , 58 LDL-cholesterol (mg/dL) , 112 Triglycerides (mg/dL) , 116

Mitochondrial Function by HIV Status Median OutcomeHIV+HEUP-value POC lactate (mg/dL) [IQR] 1.45 [1.00, 1.90] 1.40 [1.10, 1.90] 0.98 Venous lactate (mg/dL) [IQR] 1.0 [0.78, 1.40] 1.26 [0.89, 1.70] <0.001 Venous pyruvate (mg/dL) [IQR] 0.09 [0.05, 0.11] 0.10 [0.07, 0.13] CI enzyme activity PBMC (OD/min/  g e-6 ) [IQR] [27.73, 58.50] [29.69, 56.87] 0.71 CIV enzyme activity PBMC (OD/min/  g e -6 ) [IQR] [52.13, 83,34] [50.59, 72.21] 0.048

Insulin Resistance is Associated with Increased Venous Lactate and Pyruvate in HIV+ Children National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute IR - YES IR – NO LactatePyruvate

Hypertriglyceridemia is Associated with Point-of-Care (POC) and Venous Lactate in HIV+ Children National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute High TGNormal TGHigh TGNormal TG POC Lactate Venous Lactate P=0.024P<0.001

Low HDL-Cholesterol is Associated with Lower PBMC OXPHOS CI and CIV Enzyme Activities in HIV+ Children National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute Low HDLNormal HDLLow HDLNormal HDL Complex I Complex IV P=0.024 P=0.085

Venous Lactate is Positively Correlated with Total Cholesterol in HIV+ Children P=0.04; r=0.16

Venous Lactate is Positively Correlated with Triglycerides in HIV+ Children P<0.0001; r=0.37

Multivariable Model for HDL-Cholesterol in HIV+Children National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute EffectEstimateSEP-value Gender (male) Age (per year) Viral load (per copy/mL) Venous lactate (mg/dL)

Multivariable Model for Triglycerides in HIV+ Children National Institute on Deafness and Other Communication Disorders National Institute of Allergy and Infectious Diseases National Heart, Lung and Blood Institute EffectEstimateSEP-value Gender (male) Age (per year) PI duration (per year) NNRTI duration (per year) Venous lactate (per mg/dL)

Conclusions in HIV+ Children Insulin resistance is associated with higher lactate and pyruvate levels Hypertriglyceridemia is associated with higher lactate levels Low HDL-cholesterol is associated with lower CI and CIV enzyme activities Venous lactate is independently associated with higher triglyceride levels

Overall Conclusions Mitochondrial dysfunction induced by either HIV or ARV may be responsible for the observed metabolic changes

Acknowledgments We thank the study participants, PHACS Community Advisory Board, Frontier Science Inc., and Westat. PHACS is funded by: Under cooperative agreements with Harvard School of Public Health (HD052102, 3 U01 HD S1, 3 U01 HD S3) and Tulane School of Medicine (HD052104, 3U01HD S1)

The following institutions participated in conducting PHACS in 2011: Baylor College of Medicine Bronx Lebanon Hospital Center Children's Diagnostic & Treatment Center Children’s Hospital, Boston Children’s Memorial Hospital Jacobi Medical Center New York University School of Medicine St. Christopher’s Hospital for Children St. Jude Children's Research Hospital San Juan Hospital/Department of Pediatrics SUNY Downstate Medical Center Tulane University Health Sciences Center University of Alabama, Birmingham University of California, San Diego University of Colorado Health Sciences Center University of Florida/Jacksonville University of Illinois, Chicago University of Medicine and Dentistry of New Jersey University of Miami University of Southern California University of Puerto Rico Medical Center Acknowledgments Funding for this study is from DHHS/NIH/NINR grant R to M. Gerschenson and T.L. Miller. We also acknowledge the support from the John A. Burns School of Medicine, University of Hawaii at Manoa.