HIV VPR alters fat metabolism. Dorothy E Lewis PhD/Ashok Balasubramanyam MD
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1 HIV VPR alters fat metabolism Dorothy E Lewis PhD/Ashok Balasubramanyam MD
2 Old Dogma for HIV associated lipodystrophy Differentiation Block (PI) Lipoatrophy Apoptosis (NRTI) Stem cell Preadipocyte Adipocyte C/EBPβ/δ SREBP1 PPAR-γ Induction LPL, HSL, ap2, of lipogenic perilipin genes BUT, happens without ART and in Elite suppressors COULD HIV CAUSE????
3 Metabolic features of HIV (218) Less lipoatrophy Less severe dyslipidemia More generalized obesity Dysglycemia, CVD, osteoporosis, NAFLD
4 Could there be a direct viral cause? VPR in virally suppressed people VPR is systemic and circulates VPR acts without whole virus
5 Vpr in virally suppressed patients (Immunoaffinity Capillary Electrophoresis) no ART NRTI HAART HAART with undetectable VL N (2) (25) (61) (7) (25) Agarwal et al, Science Transl Med 213
6 Mouse Models to study VPR effects A. Vpr-Transgenic Model Inducible expression of Vpr in liver and fat PEPCK SV4 3 UT Tissue-specific promoter Tetracycline transactivator Tet repressor and DNA binding domain VP16 activation domain (teto) 7 Transgene coding region Vpr SV4 3 UT B. Circulating Vpr Model (svpr treatment) Alzet pump filled with vehicle or svpr Incision made to implant Alzet pump Mouse before and after Alzet implant
7 Vpr mrna Expression in Liver and Adipose Tissues of Mice Liver: Vpr + Fat: Vpr + Vpr Expressed in Liver and Adipose Tissues Also Circulates in the Blood of Mice Serum Vpr (pg/ml) Agarwal et al, Science Transl Med 213
8 Vpr Increases Lipolysis and Blunts Fat Oxidation Increased lipolysis Ra FFA (mmol/kg/h) 5 25 Ra FFAt Ra FFAn Ra FFA (mmol/kg/h) 4 2 Ra FFAt Ra FFAn Vehicle svpr Blunted fat oxidation during fast RER RER Vehicle svpr Agarwal et al, Science Transl Med 213
9 Vpr Decreases Fat Mass Fat weight (% of body wt) svpr-treated Fat weight (% of body wt) IF PGF RPF Total WAT BAT IF PGF RPF Total WAT BAT Vehicle svpr
10 A. Insulin B. AKT-p(S473) / AKT Blood glucose (mg/dl) Vpr Causes Modest Insulin Resistance 12 Wk AKT Min after i.p. glucose injection C. D. Blood glucose (mg/dl) Plasma triglycerides (mg/dl) Wk Min after i.p. glucose injection
11 VPR Mechanisms VPR acts on adipocyte differentiation VPR acts on adipocyte function Lentiviral VPR in cultured adipocytes
12 Early expression of Vpr Vpr Blocks Differentiation of Preadipocytes Preadipocyte proliferation Adipocyte differentiation -2d d 2d 4d 6d 8d 1d 12d Lentiviral infection Doxy induction of viral transgene expression Adipocyte differentiation medium added A 3T3-L1 rtta Vpr Vpr-R8A B. Relative mrna expression Day 1 Day 2 Day 3 Pref1 Relative mrna expression Day2 Day3 Day 4 Day 5 Day 6 PPARγ2 Relative mrna expression Day 6 Day 8 Day 1 Day 12 GLUT4 Control rtta Vpr R8A
13 Vpr Inhibits Preadipocyte Differentiation By blocking Cell Cycle 3T3-L1 rtta Vpr Vpr-R8A
14 Vpr Increases Lipolysis in Mature Adipocytes Late expression of Vpr Preadipocyte proliferation Adipocyte differentiation -2d d 2d 4d 5d 6d 8d 1d 12d Lentiviral infection Adipocyte differentiation medium added Doxy induction of viral transgene expression A. FFA (meq/l).6.3. Basal Stimulated B. Fold enrichment (Input corrected) rtta Vpr Adiponectin ap2 HSL NC Adipo NC ap2 NC HSL Ab PPARγ PPARγ GR PPARγ PPARγ GR Rosi Dexa
15 VPR mechanisms VPR inhibits adipocyte differentiation VPR increases lipolysis in adipocytes What are other consequences????
16 A. Macrophage Infiltration in Fat of Vpr Mice B. C. Vehicle svpr Vehicle svpr D E. F48+ cells / section 1 5 Average fat cell size (µm 2 ) 3 15 F. G. H. F48+ cells / section 16 8 Vehicle svpr Crown like structures /section 6 3 Vehicle svpr Average fat cell size (µm 2 ) 6 3 Vehicle svpr
17 HIV infection in fat HIV/SIV infected immune cells in adipose HIV is infectious but not much transcription HIV VPR could have direct effect in vivo Couturier et al AIDS 215, Couturier et al Retrovirology 216
18 Other consequences of VPR? VPR expressed from transgene in adipose and liver from PEPCK promoter Is there an effect on liver fat metabolism??
19 HIV VPR causes NASH A B Oil Red O liver (% area)
20 VPR increases lipid storage 14 weeks 28 weeks 16 Oil Red O liver (% area) Oil Red O liver (% area)
21 VPR increases liver weight and TGs 5 Std 6. Vehicle svpr Liver weight (% of body weight) TG (relative density units) x1 3 2 Triglycerides (mg / gm liver) Wk 28-Wk 14-Wk Figure 1
22 Liver function enzymes in VPR mice
23 Vpr Increases Hepatic Lipogenesis A. mrna fold difference D. [ 14 C] fatty acids (dpm) / million hepaotocytes / h x B. mrna fold difference mrna fold difference 6 3 E. Dgat Fasn Scd-1 Acc 6 3 svpr mrna fold difference C. 4 2 Srebp1c Chrebp L-pk Dgat Fasn Scd-1 Acc Srebp1c Chrebp L-pk
24 Vpr increases proteins associated with liver lipogenesis SREBP1c Chrebp α-tubulin nsrebp1c nchrebp TBP FASN α-tubulin Acc-p(S79) Acc A. B. Relative protein amount (normalized to α-tubulin) 12 6 SREBP1c ChREBP Relative protein amount (normalized to TBP) 1.5 nsrebp1c nchrebp C. 3 D. FASN/α-tubulin Acc-p(s79) / Acc Vehicle svpr SREBP1c Chrebp α-tubulin nsrebp1c nchrebp TBP FASN α-tubulin Acc-p(S79) Acc E. F. Vehicle svpr Relative protein amount (normalized to α-tubulin) SREBP1c ChREBP Relative protein amount (normalized to TBP) 6 3 Vehicle nsrebp1c svpr nchrebp G. H. FASN / α-tubulin 4 2 pacc / Acc 2 1 Vehicle svpr Vehicle svpr
25 Vpr Blunts Hepatic Fat Oxidation A. B Fatty acid oxidation in liver (dpm/mg/h) RER C. D mrna fold difference.6 mrna fold change 1.5 Pparα PPARa E. F Cpt1 Aox Ehhadh Hsd17b1 acaa2 Lcad Vehicle svpr mrna fold difference.6 mrna fold difference 1.5. Pparα PPARa Cpt1 Aox Ehhadh Hsd17b1 acaa2 Lcad
26 Vpr Inhibits Hepatic VLDL-TG Export A. B. C. MTP BA mrna fold difference.6 MTP expression (normalized to α-tubulin.4 Triglycerides released into plasma (mg/dl) 7 35 min I hr 2 hr D. 2.5 E. Lpl mrna fold difference Heart IF PGF Skeletal Muscle Post heparin Serum LPL mass (pg/ml)
27 Do these Vpr Mouse Models Explain Adipose / Liver Metabolic defects in HIV+ Humans? Lipolysis Fat oxidation Fat mass Fatty liver FGF21 Vpr Mice HIV+ Humans SAT beiging + + Circulating Vpr without intact HIV + + Koch s Postulates?
28 CREDITS Expression / Function Studies: Neeti Agarwal Dinakar Iyer Toni Oplt Sanjeet Patel Jeffrey Kopp Ulrich Schubert Terry Phillips Tomoshige Kino Human Samples: Maria Rodriguez-Barradas Adipose HIV Reservoir Dorothy Lewis Jacob Couturier Sara Cromer Human / Mouse Metabolic Studies: R. V. Sekhar Farook Jahoor Sue Samson Sanjeet Patel Jordan Lake
29 Vpr Associates With Hepatic Genes Involved in Lipid and Sterol Metabolism A. B. C.
30 A. Relative luciferase activity (HepG2) x unrx GW3965 T9137 Vector+LXRE Vpr+LXRE LXR+LXRE Vpr+LXR+LXRE Relative luciferase activit y (Huh7) C. D. Fold enrichment (Input corrected) # # # ### # # ns ## Vpr Coactivates LXR B. Thousands LXR LXR+GW3965 Vpr Vpr+GW3965 Vpr+LXR Vpr+LXR+GW3965 # # # unrx GW3965 T9137 Vector+LXRE Vpr+LXRE LXR+LXRE Vpr+LXR+LXRE GW Vpr LXR LXRE Srebp1c Lxr NC Srebp1c NC Lxr
Supplementary Materials for
www.sciencetranslationalmedicine.org/cgi/content/full/5/213/213ra164/dc1 Supplementary Materials for HIV-1 Vpr Induces Adipose Dysfunction in Vivo Through Reciprocal Effects on PPAR/GR Co-Regulation Neeti
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-. -. SUPPLEMENTARY INFORMATION DOI: 1.1/ncb86 a WAT-1 WAT- BAT-1 BAT- sk-muscle-1 sk-muscle- mir-133b mir-133a mir-6 mir-378 mir-1 mir-85 mir-378 mir-6a mir-18 mir-133a mir- mir- mir-341 mir-196a mir-17