Down regulation of genes controlling fatty acid metabolism and anaerobic respiration in subcutaneous adipose tissue after 18-24 months antiviral treatment with efavirenz-containing regimens Dr Sat Das Consultant Physician, Coventry

Background Effect of antiretroviral therapy on adipose tissue are not same with all regimens The effect changes with duration of therapy Effect of ART on fat cells and fat metabolism are different. The effect changes with duration of treatment It is important to understand the key mechanism responsible the changes

Fat changes with time In a prospective study Mallon et al shown increase in both peripheral and trunk fat after starting HAART, but the peripheral fat drops as the duration of treatment increases. Mallon PW et al. AIDS 2003;17:971-9

Lipid changes: HIV and HAART In the MACS cohort, lipids of all parameter dropped after seroconversion and somewhat correction occurs after starting HAART The effect differs with different agents and a central mechanism involving the nuclear and mitochondrial genes may play important role and can vary with different agents Riddler SA et al. JAMA 2003;289:2978-82

Objective To compare expression of genes involved in: lipid and glucose metabolism adipocyte maturation mitochondrial function in subcutaneous adipose tissue of HIV negative controls with HIV patients before and after 18 months of antiretroviral therapy We aim to compare genes involved in lipid, insulin-glucose metabolism and proliferation and maturation of fat cells and mitochondrial function in HIV patients starting treatment with efavirenz containing regimens comparing them with HIV negative controls

Design ABC/3TC/EFV 18 to 24 months n= 12 This is a prospective randomized collaborative study between University of Birmingham and University of Warwick. 32 treatment naïve HIV patients after initial screening and baseline tests randomized to AZT/3TC/EFV or TDF/FTC/EFV arm. 15 age, sex matched HIV negative controls were compared with the groups A cross-section of 12 patients who started with ABC/3T / EFV regimen were studied at 18-24 months and compared with rest of the groups. ABC/3TC/EFV 18 to 24 months n= 12

Tests at each visit (0, 6 and 18 months) Fat biopsy from iliac crest DEXA CT abdomen to calculate VAT:SAT Total cholesterol, HDL cholesterol, triglyceride, NEFA, lipoprotein-a HOMA Adiponectin and leptin The tests were repeated at 0, 6 and 18 months

Methods Following total RNA extraction, gene expression was profiled using real-time PCR and quantified relative to internal housekeeping gene (18S). Following total RNA extraction, gene expression was profiled using real-time PCR and quantified relative to house keeping gene, 18S Genes included those controlling lipid, glucose metabolism, adipocytokines, fat cell maturation, mitochondrial funtion including Kreb’s cycle

Viral load, log10 copies/ml Baseline Demography Control n=15 AZT N=1n TDF n=17 ABC n=12 Age (range) 37 (25-42) 33  (27-40) 35 (31-42) 39 (32-42) Male sex 7 8 Black Ethnicity     10 13 12 BMI 27.0 (22.8-29.9) 25.8 (23.7-28.2) 24.3 (21.8-28.3) 29 (23.8-30.4) CD4 cells/mm NA 187(153-268) 234 (163-253) 215 (75-306) Viral load, log10 copies/ml 5.1 (4.4-5.4) 4.6 (4.2-5.3) 4.5 (4.4-4.9) The baseline demography including age, sex, ethnicity, BMI, VL, CD-4 count were not different between the groups.

There was increase in VAT in AZT and SAT in TDF arm. Lipids increased in both arms.

Adipocytokines, Insulin resistance and Lipase Control AZT (18- 24m) TDF (18-24 m) ABC (18-24m) p value Adiponectin ug/ml 7.9 (4.8 – 10.3) 8.2 (4.1-15.3) 8.8 (6.7 – 11.6) NS Leptin ng/ml 8.2 (3.9 – 14.2) 7.9 (4.5 – 12.0) 6.8 (4.4 – 15.9) HOMA-IR 2.4 (2.1-2.8) 1.7 (1.5-3.2) 2.1 (1.9-2.4) 2.9 (1.6-3.6) HOMA-B 53 (53.8 -67.8) 40.3 (29.6 -91.2) 41.8 (37.8 – 48.9) 45.1 (30.8 – 53.7) Hepatic lipase nmol/ml/hr 241 (76– 300) 156 964 – 189) 166 (46 – 220) 113 (49 – 203) Lipoprotein lipase nmol/ml/hr 603 (395 – 652) 115 (91 – 280) 151 (212 – 242) 186 (166 – 215) 0.008*, 0.038**, 0.0001*** There was increase in TC, LDL, TG and HDL in both AZT and TDF group compared to pre-treatment level Compared to HIV negative controls, lipoprotein lipase level was higher in all groups and remained g high after 18 months of treatment. But there was no difference in adipocytokines and insulin resistance

Genes profiled FAS PCG-1a ND1 FABP4 NRF- 1 ND4 GR a LPL TFAM CYT- B Cortisol metabolism Fat metabolism Differentiation Glucose Nuclear control of mitochondria Mitochondrial respiratory chain H6PDH FAS PCG-1a ND1 11bHSD-1 FABP4 NRF- 1 ND4 GR a LPL TFAM CYT- B HSL COX-3 ACC1 & 2 ATP5S PPAR-g Glut 4 COX- 4 UCP2 & 3 Genes profiled included We would focus on different colour codings and would compare the fold changes compared to HIV negative controls and pre-treatment levels

Metabolic changes HIV treatment naïve vs controls (=1) Krebs Cycle Steroid This is the comparison between HIV negative and positive groups before treatment There was no definite pattern in the changes as genes responsible for fat cell maturation and proliferation were down regulated, and up-regulation of some others including those responsible for lipid and glucose metabolism Fat and glucose metabolism * * Control = 1 p = 0.028 – 0.01

Genes controlling adipocyte maturation (naïve=1) After 6 months of treatment However, the pattern changes completely after starting treatment. All went into one direction that is up-regulation in all the genes in both groups. This explains what we have seen in MACS cohort and also Mallon’ study AZT TDF Up regulation of genes involved with cortisol, lipid and glucose More marked with AZT Boothby M et al. Antivir Ther. 2009;14(8):1089-100

Genes controlling mitochondrial function (naïve=1) After 6 months of treatment Although similar changes were noticed in mitochondrial genes , but there was down regulation of some genes responsible for oxidative function with a possible compensatory energy utilization genes in mitochondria in AZT arm. AZT TDF AZT (but not TDF) down-regulation of CYT-B and increase in UCP-2 Compensatory increase in nuclear-encoded COX-4

Genes controlling adipocyte maturation (naïve=1) After 18-24 months of treatment AZT (18 – 24 m) vs. naïve TDF (18 – 24 m) vs. naive The picture changes almost upside down after 18 months of treatment and the changes were similar in both arms.

Genes controlling adipocyte maturation (naïve=1) After 18-24 months of treatment AZT (18 – 24 m) vs. naïve TDF (18 – 24 m) vs. naïve ABC (18-24 m) vs. naive The changes were similar in the Abacavir arm

Genes controlling mitochondrial function (naïve=1) After 18-24 months of treatment Almost similar changes in the mitochondrial genes suggesting an anaerobic state in both arms including abacavir arm

Summary Increase in visceral fat (AZT) and subcutaneous fat (TDF) Down regulation of genes involved with adipocyte differentiation Evidence of a switch to anaerobic metabolism in all treatment groups All the groups contain EFV ? ARV encourages adipocyte proliferation rather than maturation There was increase in VAT in AZT, and increase in SAT in TDF and also in ABC arm and also rise lipid levels in all the arms There was down regulation of genes involved in adipocyte maturation or differentiation There was down regulation of mitochondrial genes suggesting a switch to anaerobic metabolism in all treatment groups. The nucleosides were different , but they all contain one thing common which was efavirenz It is possible that ART encourages adipocyte proliferation rather than maturation and differentiation. Further studies should explore the role of efavirenz in the metabolism of fat cells.

Acknowledgement University Hospitals University of Warwick Birmingham FT M Shahmanesh M Boothby University of Birmingham JW Tomlinson LL Gathercole University of Warwick KC McGee KC AL Harte AL P Higgins P CM Kusminski PG McTernan Financial assistance STD Research Foundation Jo Lee Foundation Gilead Upjohn