Well-treated persons with HIV (PWH) develop several adverse metabolic complications including cardiovascular disease, diabetes, and fatty liver disease.1–3 A single mediator of all these metabolic complications could be adipose dysfunction, which provides a rational mechanism for the enhanced risk of metabolic disease among PWH compared with uninfected individuals.
Brown adipose tissue (BAT) is a metabolically favorable and energy efficient depot, which is protective of metabolic dysfunction. Brown adipocytes have unique multilocular fat stores, are rich in mitochondria, and play an important role in dissipating chemical energy as heat through a process called nonshivering thermogenesis. White adipose tissue can undergo a “beiging” process and develop BAT-like functions. Our group has previously demonstrated that the dorsocervical adipose tissue (DCAT) depot is a BAT-like tissue, distinct from traditional abdominal lipohypertrophy or visceral fat accumulation, and is related to reduced brown and beiging capacity of the abdominal subcutaneous adipose tissue (SAT) depot in HIV.4,5 In the current study, we investigated the influence of key HIV-specific parameters in relation to expression of key brown and beige fat genes expressed in the abdominal SAT and to DCAT accumulation.
Eighteen men with HIV were previously recruited for a study assessing adipose dysfunction in lipodystrophy vs. nonlipodystrophy.5 Participants were recruited for ages 18–60 years and body mass index 18–35.0 kg/m2. PWH were required to be on a stable antiretroviral therapy (ART) regimen >12 months. Participants were excluded for hemoglobin <10.0 g/dL; known history of diabetes; abnormal thyroid function; use of glucocorticoids, growth hormone, or other anabolic therapies within 3 months of screening; current substance abuse; active opportunistic infection; or active or serious chronic medical conditions other than HIV. A group of 9 men without HIV who met similar criteria, excluding HIV-specific measures, were also previously recruited as part of the study.5 The study was approved by the Partners Human Research Committee, and all participants provided written informed consent.
Assessment of HIV-Related Parameters
Duration of HIV and ART use and ART use by class was obtained through self-report. T-cell counts were measured by flow cytometry. HIV viral load was quantified by ultrasensitive reverse transcriptase PCR (polymerase chain reaction) (Roche COBAS amplicor; Roche Molecular Systems, Branchburg, NJ).
Assessment of Body Composition
Magnetic resonance imaging of the neck was performed on a Siemens 3T Trio magnetic resonance system using phased array neck and body matrix coils as previously described.5 Axial images at the level of C7 were reconstructed and used for measurement of DCAT area.
Abdominal Fat Biopsy and PCR Analysis
Extraction of subcutaneous abdominal fat was surgically obtained through a 4-mm diameter punch biopsy device under local anesthesia with 1% lidocaine. Specimens were flash-frozen in dry ice/ethanol and transferred to −80°C immediately. Total cellular RNA in the adipose tissue was extracted using an RNeasy minikit (Qiagen) per previous methods.5 cDNA was prepared from 6 ng/µL of RNA via the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). 6 μL (36 ng) of cDNA was used in a 20 μL PCR using TaqMan Gene Expression Assays with a fluorescein dye label. Quantitative RT-PCR assays were run in duplicates and quantitated in the ABI Prism 7700 sequence detection system. Gene expression was performed by a single investigator blinded to group status. The values were normalized to the expression of TATA-binding protein and results were expressed as ratios in arbitrary units. For most adipose genes, mRNA expression was detectable and could be quantified. In a small subset of samples for UCP1, LHX8, PAT2, PRDM16, and TMEM26, mRNA expression was below standard PCR limits, and thus, we imputed the half-minimum value as previously described.6
Normality of distribution was determined using the Shapiro–Wilk test. Data are presented as mean ± SE of mean or median (interquartile range), depending on normality of the distribution. Categorical variables are reported as proportions. Univariate regression was evaluated using Pearson correlation coefficient after variables were logarithmically transformed. Statistical significance was defined as P < 0.05. All statistical analyses were performed using SAS JMP (version 14.0). Data assessing lipodystrophy from this cohort were previously published.5 Presence of lipodystrophy was determined based on clinical exam findings of dorsocervical fullness before study procedures and used in stratified analyses. Additional analyses were performed controlling for age in multivariate modeling exploring independent effects of duration of HIV and ART use on adipose tissue markers to parse out whether these associations were a function of aging or an HIV-related phenomenon. Relationships between HIV parameters and ART use with adipose genes as in the current study were not previously assessed.
Baseline Demographic and Clinical Characteristics
The mean age was 54 ± 2 years. Most participants were Caucasian (72%). The participants had a long-term history of HIV infection (21 ± 2 years) and ART use (16 ± 2 years). Duration of nucleoside/nucleotide reverse transcriptase inhibitors (13 ± 2 years), non-nucleoside reverse-transcriptase inhibitors (10 ± 2 years), and PI (13 ± 2 years) use were similar, whereas duration of integrase inhibitor [integrase strand transfer inhibitor (INSTI)] use (7 ± 2 years) was comparatively less. Overall, the group demonstrated parameters consistent with being well-treated: CD4 T-cell count 582 ± 66 cell/μL and log10 HIV viral load 1.43 ± 0.09. Mean body mass index (29.7 ± 1.0 kg/m2) was in the overweight category (see Table 1, Supplemental Digital Content, http://links.lww.com/QAI/B377).
Relationship of HIV Parameters to Adipose-Related Gene Expression
Duration of HIV was significantly and inversely related to BAT markers [PGC1α (r = −0.47, P = 0.05), P2RX5 (r = −0.57, P = 0.01)], beige fat markers [TMEM26 (r = −0.52, P = 0.03), CD137 (r = −0.51, P = 0.03)], and other metabolic markers [DIO2 (r = −0.55, P = 0.02), HSP60 (r = −0.62, P = 0.006)]. Lower CD4 T-cell count was related to reduced expression of BAT markers [PGC1α (r = 0.57, P = 0.01), ZIC1 (r = 0.49, P = 0.04), PRDM16 (r = 0.61, P = 0.007), PAT2 (r = 0.59, P = 0.01), P2XR5 (r = 0.61, P = 0.007)], beige fat markers [TMEM26 (r = 0.65, P = 0.004), CD137 (r = 0.53, P = 0.02)], and other metabolic markers [DIO2 (r = 0.51, P = 0.03), leptin (r = 0.49, P = 0.04), HSP60 (r = 0.50, P = 0.04)]. Higher CD4 T-cell count was inversely related to DCAT (r = −0.51, P = 0.03). Age and HIV viral load did not correlate with any adipose-related genes (Table 1).
Relationship of Adipose-Related Gene Expression to ART Parameters
Duration of ART use was significantly and negatively associated with all markers: BAT markers, beige fat markers, and other metabolic markers (Table 2). Greater DCAT was also correlated with longer duration of ART use (r = 0.59, P = 0.009). On categorization by class of ART, duration of PI use demonstrated the most significant relationships to adipose-related gene expression compared with duration of nucleoside/nucleotide reverse transcriptase inhibitors or non-nucleoside reverse-transcriptase inhibitors use, and also was significantly correlated with DCAT (r = 0.79, P = 0.01) (Table 2). There was some significant relationship between INSTI use and adipose-related genes; however, these relationships could only be evaluated in very few participants (Table 2).
Exploratory Analyses Assessing Age, Metabolic Parameters, Lipodystrophy, and Specific ART Use
After controlling for age, duration of HIV and ART associations to adipose tissue markers remained (see Table 2, Supplemental Digital Content, http://links.lww.com/QAI/B377). In addition, age did not relate at all to adipose gene expression in a non-HIV group (see Table 3, Supplemental Digital Content, http://links.lww.com/QAI/B377). Stratification by presence of or absence of lipodystrophy are shown in Tables 4A–D, Supplemental Digital Content, http://links.lww.com/QAI/B377. There were no significant associations overall with homeostatic model assessment of insulin resistance and triglycerides (see Table 5, Supplemental Digital Content, http://links.lww.com/QAI/B377); however, in the presence of lipodystrophy, homeostatic model assessment of insulin resistance tended to be related to reduced expression of adipose genes (P2XR5: r = −0.64, P = 0.06; CD137: r = −0.61, P = 0.08; leptin: r = −0.76, P = 0.02), whereas there were no relationships to triglycerides. The relationships of duration of ART use to adipose tissue markers seemed to be more robust among those without previous thymidine analogue exposure (see Tables 6A, B, Supplemental Digital Content, http://links.lww.com/QAI/B377).
In the current study, we show for the first time that key HIV-related parameters reflective of long-term infection (increased duration HIV, duration of ART use) or relatively reduced immunologic function (lower CD4 count) were linked to reduced expression of brown and beige fat gene in the abdominal subcutaneous adipose depot. These parameters were consistently related among several adipogenic genes, suggesting a compelling pattern, and further highlighting a potential link between HIV and adipose biology. Nonacute HIV infection, worse immunologic function, and use of older classes of antiretrovirals may have implications for adipose dysfunction and contribute to cardiometabolic sequalae.
There were no associations between viral load and adipogenic gene expression. To that end, the viral load does not capture long-standing adipocyte-related consequences linked to periods of uncontrolled viremia and poor immunologic reserve,7 presence of viral reservoirs in the fat,8 or adipocyte-specific toxic side effects of antiretrovirals.9 Other markers such as duration of HIV, ART use, and CD4 count may provide a more descriptive perspective on the disease course.
Duration of HIV and ART were both related to reduced adipose gene expression. These may be parallel markers of disease chronicity. Associations of duration of HIV and ART with adipose tissue markers were independent of age, which may suggest relationships with HIV-parameters are not merely a function of aging. Duration of PI use had several associations to HIV-related parameters. PI are well-known to affect the adipose depot, impair mitochondrial function and have direct effects on insulin resistance through impaired GLUT4 translocation. There were also some correlations between the INSTI class and adipogenic gene expression, which is interesting given recent concern that this ART class may have adverse effects on weight gain similar to other classes.10 Only a limited number of individuals were available for analysis who reported INSTI use, and future studies should relate INSTI use to the adipogenic signature in larger populations of PWH.
Among the BAT markers, relationships with PGC1α and P2RX5 were demonstrated across immunologic and ART parameters. PGC1α is a well-known regulator of energy metabolism and mitochondrial biogenesis, is typically highly expressed in BAT, and has a role in lipid and glucose metabolism.11 P2RX5 is an important BAT marker identified to be highly expressed in cervical fat.12 Both TMEM26 and CD137 showed relationships across HIV parameters and are reported to be beige fat-specific markers.13 We have previously reported that DIO2 expression is increased in the DCAT depot and linked to enhanced energy expenditure.4 In line with our hypothesis that the DCAT depot develops as compensatory mechanism in HIV,4,5 DCAT accumulation was related to HIV-related indices in an inverse pattern to adipogenic markers, including DIO2 expression in the SAT.
Leptin is an integral hormone produced by healthy adipocytes. Our data demonstrate reduced leptin in the subcutaneous fat which is associated with longer duration of HIV, lower CD4 count, and longer duration of ART use. In this regard, leptin may serve as a general marker for adipose dysfunction in HIV. The exact mechanism accounting for the evolving spectrum of adipose dysfunction among PWH is not well-understood and may be speculated, for example, to relate to changes in contemporary ART regimens, CD4 recovery,14 composition of gut microbiota and/or factors associated with environment and lifestyle.15
In the contemporary era, attention is focused on the growing trend of obesity (gain of subcutaneous fat and/or gain of visceral fat) among PWH.15,16 Changes in fat redistribution representative of acquired lipodystrophy (loss of subcutaneous fat and/or gain of visceral fat) while still prevalent may occur less often than previously recognized.17 Regardless, obesity and lipodystrophy are both states of adipose dysfunction, and underlying metabolic risk could be attributed to reduced capacity for browning of white adipose tissue or beiging in either clinical presentation among PWH.
There were a few limitations with our study. The study included a small number of participants and was limited to a male group. Information on ART class was obtained through self-report and may not provide a complete account of ART use. These data nonetheless begin to inform us of a critical link between HIV infection and adipose tissue. We were able to obtain adipose tissue biopsies and perform a detailed gene expression analysis focused on relevant brown and beige genes in relation to HIV, which few studies have performed to our knowledge.18,19 Moreover, these studies have been limited and have not investigated the wide range of brown and beige fat markers we were able to assess.
PWH are at an enhanced risk for adipose dysfunction and metabolic complications. These associations highlight the need to further understand whether PWH are at risk for cardiometabolic disease secondary to reduced browning and beiging capacity of the adipose depot and how disease-specific HIV parameters may have unique contribution to adipocyte biology in HIV. In addition, strategies to optimize browning and/or beiging of fat may be useful to reduce metabolic dysfunction in HIV.
The investigators would like to thank the nursing staff on the MGH TCRC for their dedicated patient care, and the volunteers who participated in this study.
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