Adipocyte ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/kadi20 Adipose tissue aging partially accounts for fat alterations in HIV lipodystrophy Pere Domingo, Marta Giralt, Aleix Gavaldà-Navarro, Albert Blasco-Roset, Alejandro Delgado-Anglés, José Miguel Gallego-Escuredo, Maria Del Mar Gutiérrez, Maria Gracia Mateo, Rubén Cereijo, Joan Carles Domingo, Francesc Villarroya & Joan Villarroya To cite this article: Pere Domingo, Marta Giralt, Aleix Gavaldà-Navarro, Albert Blasco-Roset, Alejandro Delgado-Anglés, José Miguel Gallego-Escuredo, Maria Del Mar Gutiérrez, Maria Gracia Mateo, Rubén Cereijo, Joan Carles Domingo, Francesc Villarroya & Joan Villarroya (2022) Adipose tissue aging partially accounts for fat alterations in HIV lipodystrophy, Adipocyte, 11:1, 143-152, DOI: 10.1080/21623945.2022.2042962 To link to this article: https://doi.org/10.1080/21623945.2022.2042962 © 2022 The Author(s). Published by Informa View supplementary material UK Limited, trading as Taylor & Francis Group. Published online: 17 Mar 2022. Submit your article to this journal Article views: 1661 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kadi20 ADIPOCYTE 2022, VOL. 11, NO. 01, 143–152 https://doi.org/10.1080/21623945.2022.2042962 BRIEF REPORT Adipose tissue aging partially accounts for fat alterations in HIV lipodystrophy Pere Domingo a*, Marta Giralt b,c,d*, Aleix Gavaldà-Navarro b,c,d, Albert Blasco-Roset b, Alejandro Delgado- Anglés b, José Miguel Gallego-Escuredob, Maria Del Mar Gutiérreza, Maria Gracia Mateoa, Rubén Cereijo a,b,c, Joan Carles Domingo b, Francesc Villarroya b,c,d, and Joan Villarroya b,c,d a Infectious Diseases Unit and Institut de Recerca Hospital de la Santa Creu I Sant Pau, Barcelona, Spain; bDepartament de Bioquímica I Biomedicina Molecular and Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain; cCiber Fisiopatología de la Obesidad Y Nutrición, Barcelona, Spain; dInstitut de Recerca Sant Joan de Déu Barcelona, Spain ABSTRACT ARTICLE HISTORY Lipodystrophy is a major disturbance in people living with HIV-1 (PLWH). Several systemic Received 2 August 2021 alterations in PLWH are reminiscent of those that occur in ageing. It is unknown whether the Revised 4 February 2022 lipodystrophy in PLWH is the consequence of accelerated ageing in adipose tissue. We compared Accepted 11 February 2022 systemic and adipose tissue disturbances in PLWH with those in healthy elderly individuals (~80 KEYWORDS y old). We observed similarly enhanced expression of inflammation-related genes and decreased Lipodystrophy; adipose; HIV; autophagy in adipose tissues from elderly individuals and PLWH. Indications of repressed adipo­ ageing; autophagy; genesis and mitochondrial dysfunction were found specifically in PLWH, whereas reduced telo­ inflammation mere length and signs of senesce were specific to elderly individuals. We conclude that ageing of adipose tissue accounts only partially for the alterations in adipose tissues of PLWH. Introduction Lipodystrophy has been a major disturbance occurring of those that appear progressively with ageing in the in people living with HIV-1 (PLWH) under antiretro­ non-infected population [5]. The concept of ‘acceler­ viral treatment. Lipodystrophy in PLWH is character­ ated ageing’ has been proposed to describe the set of ized by a complex set of alterations in adipose tissue comorbidities appearing in PLWH [6]. Ageing pro­ that involve peripheral lipoatrophy (i.e., loss of subcu­ foundly alters adipose tissue [7,8] and there is taneous adipose tissue in the face, arms, and legs) often a redistribution of fat depots in the elderly, with sub­ associated with visceral fact lipohypertrophy [1]. This is cutaneous depots loss at the expense of increased visc­ associated with enhanced cardiovascular risk and eral fat [9]. Advanced ageing is associated with a loss of altered systemic metabolism. The recent use of less- facial adipose tissue that is reminiscent of the facial toxic antiretroviral drugs (basically withdrawal of thy­ lipoatrophy seen in PLWH [10]. Moreover, adipose midine analogues from the antiretroviral drug patterns tissue may be a site of the so-called ‘inflammaging’ of treatment) has decreased the frequency of overt phenomenon: a mildly increased pro-inflammatory sta­ peripheral lipoatrophy, although signs of fat alterations tus intrinsically associated with ageing [11]. Decreased remain in PLWH [2]. Patients who received thymidine adipogenic capacity, reduced autophagy, and increased analogues may have persistent fat alterations several cellular senescence have been proposed to take place in years after therapy discontinuation [3], and young ageing adipose tissue [12,13]. It has been proposed that adults who were infected with HIV during childhood HIV-associated lipodystrophy may be related to accel­ and exposed to older-generation antiretroviral drugs erated ageing elicited by antiretroviral drugs and HIV appear to be particularly susceptible to persistent lipoa­ infection-associated events [14]. To ascertain whether trophy after discontinuation [4]. accelerated adipose tissue ageing may be a key patho­ Multiple non-infectious comorbidities involving genic component in HIV lipodystrophy, we herein metabolic, neurological, and cardiovascular systems compared alterations in biomarkers of inflammation, occurring in PLWH have been said to be reminiscent adipogenesis, senescence, and autophagy in sera and CONTACT Joan Villarroya jvillarroya@ub.edu Departament de Bioquímica I Departament de Bioquímica I, Facultat de Biologia. Universitat de Barcelona, Barcelona 08028, Spain *These authors have contributed equally to the work. Supplemental data for this article can be accessed here © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 144 P. DOMINGO ET AL. adipose tissue samples obtained from aged individuals Circulating TNFα, IL6, IL8, HGF, NGF, MCP1, with no overt pathology and from PLWH with FGF21 and leptin levels in serum were measured lipodystrophy. using an antibody-linked, fluorescently labelled micro­ sphere bead-based multiplex analysis system (Linco Research/Millipore, Billerica, MA, USA) and quantified Materials and methods using Luminex100ISv2 equipment. Adiponectin levels Three groups were enrolled in this cross-sectional were determined by ELISA (Merck Life Science, study: (a) healthy individuals over 80 y old (n = 28); Madrid, Spain). (b) HIV-infected, antiretroviral-treated patients with Biopsy samples of subcutaneous fat from adults were lipodystrophy (PLWH-LD) (n = 60), (mean age, 46 y) obtained through a small surgical biopsy performed by (see Supplementary Table 1 for treatment); and (c) 34 an 8-mm punch under local anaesthesia with mepiva­ healthy controls age-matched with the PLWH-LD caine. Samples from newborns (n = 5) with a gestational group. An elderly patient was considered healthy age of 25–39 weeks who survived for no more than when there was no evidence of any chronic disease, 24 hours post-partum have been described in detail including inflammatory disease, cardiovascular disease, elsewhere [17]. Tissue samples were immediately frozen hepatitis, liver insufficiency, fever of undetermined ori­ and stored at −80°C. Each sample was homogenized in gin, diabetes mellitus, and cancer. Only arterial hyper­ RA1 buffer, and RNA was isolated using a column- tension and antihypertensive therapy were allowed. affinity-based methodology that included on-column Lipodystrophy in patients was assessed according to DNA digestion (Macherey-Nagel, Düren, Germany). Lichtenstein et al. [15]. Briefly, the degree of lipody­ One microgram of RNA was transcribed to cDNA strophy in PLWH was rated at distinct anatomical sites using MultiScribe reverse transcriptase and random- (face, arms, buttocks, legs, abdomen, neck, and breasts) hexamer primers (TaqMan Reverse Transcription as absent (score of 0), mild (noticeable on close inspec­ Reagents; Applied Biosystems, Foster City, CA, USA). tion; score of 1), moderate (readily noticeable by For quantitative mRNA expression analysis, TaqMan patient and/or physician; score of 2), or severe (readily reverse transcriptase (RT)-polymerase chain reaction noticeable to a casual observer; score of 3). A clinical (PCR) was performed on an ABI PRISM 7700HT diagnosis of lipodystrophy was given to a patient with sequence detection system (Applied Biosystems). The an overall score 17. In addition, patients with severe fat TaqMan RT-PCR reactions were performed in a final changes in one body location were considered to be volume of 25 µl using TaqMan Universal PCR Master lipodystrophic. The overall exclusion criteria included Mix, No AmpErase UNG reagent, and the primer pair body mass index (BMI) >30 kg/m2, anticoagulant treat­ probes listed in Supplementary Table 2. Controls with ment, oral antidiabetic therapy, and hormonal treat­ no RNA, primers, or RT were included in each set of ment. This study was approved by the ethics experiments. Each sample was run in duplicate, and the committee of Hospital de la Santa Creu i Sant Pau, mean value of the duplicate results was used to calcu­ Barcelona, and all patients provided informed written late the mRNA levels for the genes of interest. consent. Expression levels of gene transcripts were considered BMI was calculated, and waist circumference was negligible when, under the above standard RT-PCR measured to the nearest millimetre using anatomical conditions, the cycle threshold was >40. Values were landmarks, as defined by the Third National Health normalized to that of the reference control (18S ribo­ and Nutrition Evaluation Survey. Whole-body, dual- somal RNA) using the comparative 2−ΔCT method, energy X-ray absorptiometry scans (HologicQDR- following the manufacturer’s instructions. Parallel cal­ 4500A; Hologic, Inc., Waltham, MA, USA) were con­ culations performed using the reference gene, PPIA, ducted by a single operator and used to determine the yielded essentially the same results. body fat content. Plasma and serum were obtained For quantification of specific protein levels, adipose from blood drawn from seated patients after a 12- tissue samples were homogenized in cold buffer hour overnight fast and at least 15 minutes after the (10 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM dithio­ placement of a peripheral intravenous catheter. All lipid threitol, 5 mM MgCl2), and a protease inhibitor cock­ measurements were performed using a Hitachi 911 tail (Complete-mini; Roche, Sant Cugat, Spain). For system (Roche Diagnostic Systems, Basel, Western blot analysis, homogenates containing 40 µg Switzerland). Insulin resistance was estimated by the of protein were mixed with equal volumes of 2× homoeostasis model assessment method (HOMA-IR). sodium dodecyl sulphate (SDS) loading buffer, incu­ Laboratory procedures for determination of clinical bated at 90°C for 5 min, and electrophoresed on SDS/ biochemistry have been described elsewhere [16]. polyacrylamide gels. After undergoing transfer to ADIPOCYTE 145 Immobilon-P membranes (Millipore, Billerica, MA, abundance was quantified as the Tel/36B4 ratio using USA), the proteins were probed using a primary anti­ the comparative 2−ΔCT method. body against LC3B (Cell Signalling, Leiden, The For statistical analysis, significance was assessed Netherlands) and monoclonal anti-p53 (Thermo using one-way ANOVA followed by Tukey’s post-hoc Fisher Scientific), detected using a goat anti-rabbit or test, or the two-tailed unpaired Student’s t-test. Data anti-mouse HRP-conjugated secondary antibodies are expressed as means ± standard error of the mean (s. (Santa Cruz Biotechnology, Dallas, TX, USA), and e.m.), and statistical significance was set when P < 0.05. visualized using ECL reagents (Immobilon Western; The GraphPad Prism 6 statistical software (GraphPad, Millipore, Germany). Membranes were stained with San Diego, CA, USA) was used. Discrepancies among Coomassie blue (Sigma-Aldrich, St. Louis, MO, USA) standard deviations of experimental groups were to normalize the amount of protein loaded. The Multi- assessed with the Brown–Forsythe test or F-test as Gauge software (Fujifilm) was used for densitometric appropriate. Welch’s correction was applied whenever analyses (3–6 individual samples were analysed per unequal variances were detected. Outliers were detected group). and removed prior to significance analyses using For telomere length measurements, DNA was iso­ Grubbs’ test. lated from the tissue homogenate used for RNA extraction, dissolved in 70% ethanol, and purified Results by phenol/chloroform extraction. Telomere length was assessed by real-time PCR essentially as Anthropometric, body composition, and metabolic described previously [16,18]. Briefly, PCR was per­ parameters from PLWH patients under antiretroviral formed using 35 ng of total DNA (PCR template) in treatment showing lipodystrophy, including peripheral a reaction containing 10 μL of 2x SYBR Green lipoatrophy (LD group), elderly individuals (mean age, Master Mix reagent (Applied Biosystems) and 80 y) with no overt pathology; and control healthy 100 nM of specific primers (Sigma) for telomere individuals of a similar age range as those in the repeats (Tel) or the single-copy housekeeping gene, PLWH group (C group) are shown in Table 1. BMI 36B4 (acidic ribosomal phosphoprotein P0), in a final was similar in the LD and C groups and significantly volume of 20 μl (See Supplementary Table 3 for increased in the elderly group. The trunk/appendicular primer sequences). CT values were obtained for fat ratio, indicating asymmetry of fat distribution, was both Tel and 36B4 in each sample. Relative telomere highly increased in the LD patients. The elderly group Table 1. Demographic, metabolic, anthropometric and body composition markers. Controls Elderly PLWH-LD (n = 34) (n = 28) (n = 60) P value Sex (no. of men (%)) 25 (73.5) 14 (50) 43 (71.7) Age 40.82 ± 0.52 80.78 ± 1.14*** 46.27 ± 1.05***### <0.0001 Weight 74.34 ± 2.12 73.32 + 2.68 66.77 ± 1.65* 0.01 BMI 24.54 + 0.46 28.35 + 0.76*** 24.10 ± 0.45### <0.0001 Waist to Hip ratio 0.87 ± 0.01 0.92 ± 0.01** 0.95 ± 0.01*** <0.0001 Glucose (mmol/l) 4.84 ± 0.07 5.85 ± 0.21*** 5.66 ± 0.15*** 0.0001 Insulin (pmol/l) 40.28 ± 4.39 211.30 ± 20.51*** 100.10 ± 8.68***### <0.0001 HOMA-r 0.77 ± 0.08 2.61 ± 0.20* 1.98 ± 0.18*** <0.0001 Bilirubin (mmol/l) 8.00 ± 1.32 11.86 ± 0.94 8.46 ± 1.32### 0.04 AST 20.56 ± 1.17 21.47 ± 1.24 31.35 ± 1.9***### <0.0001 ALT 23.76 ± 2.36 18.16 ± 1.00 37.32 ± 3.15**### <0.0001 Triglycerides (mmol/l) 0.89 ± 0.06 1.03 ± 0.07 2.25 ± 0.17***### <0.0001 Cholesterol (mmol/l) 5.16 ± 0.16 5.58 ± 0.17 5.20 ± 0.19 0.30 HDL (mmol/l) 1.48 ± 0.05 1.84 ± 0.06*** 1.23 ± 0.04**### <0.0001 LDL (mmol/l) 3.28 ± 0.15 3.73 ± 0.17 2.94 ± 0.15## 0.002 (n = 9) (n = 14) (n = 20) Total body fat (%) 26.31 ± 2.12 37.87 ± 1.41*** 20.96 ± 1.57### <0.0001 Trunk fat (kg) 9.59 ± 0.89 15.67 ± 1.05** 8.56 ± 1.05### <0.0001 Appendicular fat (kg) Trunk/appendicular fat ratio 6.70 ± 0.62 8.25 ± 0.35 2.51 ± 0.32***### <0.0001 1.48 ± 0.15 2.03 ± 0.69 4.11 ± 0.56**## 0.0005 Parameters are expressed as mean ± SEM unless specified. P values were calculated using one-way analysis of variance and Tukey post-test for parametric data. Comparison with Healthy is P < 0.05; ** when P < 0.01; *** when P< 0.001; # is shown when comparison with Elderly is P < 0.05; ## when P < 0.01; and ### when P < 0.001. BMI, body mass index; HDL, high-density lipoprotein, LDL, low-density lipoprotein, HOMA-r, homoeostasis model assessment of insulin resistance. 146 P. DOMINGO ET AL. showed trunk/appendicular fat ratios and waist-to-hip were similarly increased in adipose tissue from elderly ratios that were intermediate between the C and LD and LD individuals relative to young healthy controls groups. (Figure 2b) whereas the gene expression levels of mur­ Glycaemia, insulin, and the HOMA-IR index were ine double minute-2 (MDM2), a negative regulator of similarly increased in elderly and LD patients relative to p53, were not statistically different in adipose tissue controls (Table 1), indicating that although there was from elderly or LD individuals relative to controls. no overt diabetes in these groups, they showed similar Telomere length tended to be decreased in adipose alterations in glucose/insulin homoeostasis that are tissue from elderly patients relative to young healthy reminiscent of insulin resistance. Biomarkers of hepatic controls, but not to a statistically significant degree. We damage (ALT, AST) and triglyceridemia were increased assessed a set of fat samples from neonates, with the significantly only in the LD group. goal of validating our observations. We found that the The levels of the pro-inflammatory cytokines, telomeres length was significantly shorter in adipose tumour necrosis factor alpha (TNFα), interleukin- tissue from the elderly group relative to neonates, (IL)-6, IL-8, and monocyte chemoattractant protein-1 while that in LD patients was similar to the age- (MCP-1), as well as hepatocyte growth factor (HGF), matched healthy control group (Figure 2c). were increased to similar extents in the LD and elderly We further analysed several parameters related to groups relative to the young healthy controls autophagy, a process known to be altered with the (Figure 1a). Leptin levels were specifically increased in ageing of tissues, including fat [21]. The levels of tran­ the healthy elderly patients, and those of fibroblast scripts encoding proteins involved in autophagy growth factor 21 (FGF21) were significantly increased (ULK1, ATG4A, ATG4D, GARABP) trended lower in in both LD and elderly individuals. The adiponectin/ the elderly and LD groups relative to healthy controls leptin ratio, a proposed index of adipose tissue dysfunc­ (Figure 3a); this down-regulation was systematically tion [19,20], was significantly lower in elderly indivi­ significant in the LD group, but significant only for duals (0.69 ± 0.12, *P = 0.039) than in young controls ULK1 in the elderly group. The transcript level of (1.26 ± 0.30) and LD patients (1.11 ± 0.42). BNIP3 was specifically down-regulated in elderly indi­ Figure 1b shows the expression levels of gene sets viduals. The expression of PARK2, encoding the mito­ related to inflammation, adipose function, and mito­ phagy-related protein parkin, was unaltered. The chondrial function in subcutaneous adipose tissue. For protein levels of LC3B-II, which is used to indicate inflammation, the mRNA levels of TNFα (TNFA) were the autophagy status of a tissue, were down-regulated increased in both elderly and LD individuals. The in elderly and LD patients relative to healthy controls mRNA level of MCP1 (MCP1) showed an increasing (Figure 3b). trend in the elderly and was significantly up-regulated in LD patients. The expression levels of the genes Discussion encoding the master adipogenesis transcription factors, CCAAT-enhancer-binding protein alpha (CEBPA) and Healthy elderly individuals show alteration in the sym­ peroxisome proliferator-activated receptor gamma metry of adipose distribution, with the subcutaneous (PPARG), were specifically reduced in the LD group. depot and central fat being somewhat decreased and A similar expression profile was found for the gene increased, respectively; these changes were milder than encoding adiponectin. Leptin gene expression was sig­ those seen in PLWH, but were reminiscent of lipody­ nificantly increased only in adipose tissue from the strophy, as previously reported in advanced ageing [9]. elderly group. For marker genes of mitochondrial func­ We observed remarkably similar alterations in the cir­ tion, the transcript levels of both cytochrome oxidase culating parameters of cohorts representing PLWH subunit-2 (MT-CO2, mitochondrial genome-encoded) with lipodystrophy and elderly individuals, including and subunit-4 (COX4, nuclear-encoded) were signifi­ mild increases in insulin resistance and increased levels cantly reduced in the LD group. of proinflammatory cytokines and FGF21. These altera­ Next, we analysed several parameters that are tions in systemic parameters had been previously directly indicative of ageing processes in adipose tissue. shown for both groups in independent reports [22– Among biomarkers for senescence, the expression of 27]. Our direct comparative study confirms the pre­ CDKN2A (encoding p16INK4a) was significantly vious findings and shows that there is remarkable simi­ increased only in adipose tissue from elderly indivi­ larity in the extents of these disturbances between duals (Figure 2a). In contrast, the expression of elderly individuals and PLWH with lipodystrophy. CDKN1A (encoding p21WAF−1) was significantly We found mild but concordant increases in pro- decreased only in the LD group. P53 protein levels inflammatory gene expression levels in subcutaneous ADIPOCYTE 147 Figure 1. Circulating and transcript levels of inflammatory and adipose cytokines in elderly healthy and PLWH individuals with lipodystrophy, compared to healthy young controls. (a) Circulating levels of pro-inflammatory and metabolism-related cytokines in plasma from controls (n = 34), elderly (n = 28), and people living with HIV with lipodystrophy (PLWH-LD) (n = 60). (b) Proinflammatory and adipose metabolism-related mRNA levels in adipose tissue samples from the same three groups. The bars represent means ± SEM. Dotted line and grey bar represent mean ± SEM in healthy controls. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with controls; #p < 0.05, ##p < 0.01 comparing PLWH-LD with elderly). 148 P. DOMINGO ET AL. Figure 2. Senescence markers in elderly healthy and PLWH individuals with lipodystrophy, compared to healthy young controls. (a) mRNA levels of CDKN1A, CDKN2A, and MDM2 in adipose tissue samples from controls (n = 10), elderly (n = 12), and PLWH-LD (n = 18). (b) Representative image and quantification of P53 protein levels in control, elderly, and PLWH-LD adipose tissue samples (n = 3–4). C: Coomassie blue staining. (c) Telomere length measurements in adipose tissue samples from the same three groups, also including a subset of neonate samples (n = 5). The bars represent means ± SEM. Dotted line and grey bar represent mean ± SEM in healthy controls. (*p < 0.05, **p < 0.01 compared with controls). fat from PLWH and elderly individuals. However, the adipogenesis was altered only in PLWH seems to sug­ indications of impaired adipogenesis (decreased CEBPA gest that insults other than inflammation may be and PPARG expression) and altered mitochondrial related to lipoatrophy in PLWH. On the other hand, function (decreased cytochrome oxidase subunit gene the known mitochondrial toxicity elicited by antiretro­ expression) found in patients with lipodystrophy, viral drugs, which promotes lipodystrophy [29], may which were consistent with previous reports [24], account for the PLWH-specific alteration in the expres­ were not present in adipose tissue from the elderly. sion of genes encoding mitochondrial proteins. An The induction of a mild systemic and local pro- exception is leptin, whose adipose tissue gene expres­ inflammatory environment has been proposed to sion and circulating levels were increased specifically in account for repressed adipogenesis in subcutaneous the elderly. This was reported previously in aged indi­ fat from PLWH with lipodystrophy [24,28]. The find­ viduals [30] and is likely to be due to the increased ing that pro-inflammatory cytokines were altered simi­ percentage of total adipose tissue body content that is larly between PLWH and elderly individuals but found in the elderly, often at the expense of reduced ADIPOCYTE 149 Figure 3. Autophagy markers in elderly healthy and PLWH individuals with lipodystrophy, compared to healthy young controls. (a) Gene expression levels of autophagy markers in adipose tissue samples from controls (n = 10), elderly (n = 12), and PLWH-LD (n = 18). (b) Representative image and quantification of LC3B-II protein levels in control, elderly, and PLWH-LD adipose tissue samples (n = 3–6). C: Coomassie blue staining. The bars represent means ± SEM. Dotted line and grey bar represent mean ± SEM in healthy controls. (*p < 0.05, **p < 0.01 compared with controls). muscle mass (reference 9 and current results). This PLWH. However, this ratio has not been yet validated phenomenon is not seen in PLWH with lipodystrophy. in relation to ageing and such interpretation should be Because of leptin increase, the adiponectin/leptin ratio, taken with caution. a recently proposed indicator of adipose tissue dysfunc­ Our analysis of parameters strictly related to biolo­ tion [19,20], is specifically decreased in the elderly gical ageing revealed some alterations specifically in individuals. This could indicate the existence of ageing- elderly individuals, whose adipose tissue showed related dysfunction in ageing, potentially related to increased gene expression of CDKN2A (encoding insulin resistance and inflammation, not shared by p16INK4a, a marker of senescence) and reduced 150 P. DOMINGO ET AL. telomere length, consistently with previous reports on steady-state levels of transcripts and proteins. [12,31]. Adipose tissue from PLWH did not show However, the value of the ultimately significant num­ such alterations. In contrast, p53, which is up- ber of individuals enrolled in the healthy elderly regulated in mouse models of adipose tissue ageing group, the determination of DEXA-based adipose [32], is similarly increased in adipose tissues from tissue parameters, and access to adipose tissue sam­ elderly and PLWH individuals and, according to our ples for comparative purposes with LD patients are results, this is not attributable to major alterations in all strengths of the study. the expression of its negative regulator MDM2. This indicates that adipose tissue from PLWH shows some signs of intrinsic biological ageing, but not extensive. Conclusions Increased levels of p16 was reported in adipose tissue Our study identifies several processes that are conco­ from four HIV patients with lipodystrophy treated with mitantly altered in adipose tissue from healthy elderly protease inhibitor-type antiretroviral drugs [33]. Our individuals and PLWH patients with lipodystrophy cohort of PLWH in whom adipose tissue was analysed (inflammation, decreased autophagy) as well as pro­ (18 individuals) included only 10 patients whose anti­ cesses that are not concomitantly altered (repressed retroviral treatment had included protease inhibitors. adipogenesis and mitochondrial dysfunction specific This may explain the discrepancy between our results to PLWH, reduced telomere length specific to the and that previous report. Treatment with protease inhi­ elderly). Given the partial signs of ageing-related altera­ bitors may account for the repression of expression of tions in adipose tissue from PLWH, existing nutritional marker genes of adipogenesis in PLWH, given the and treatment-based procedures to avoid accelerated potential effects of these antiretroviral drugs impairing ageing, although limited, should be considered for adipocyte differentiation [34–36]. However, other drug PLWH affected by lipodystrophy. Further longitudinal components such as reverse transcriptase inhibitors studies in ageing PLWH are warranted to ascertain and even HIV-encoded proteins may also be involved whether their signs of aged adipose tissue (and the in such repression of adipogenesis [37]. The expression related pathogenic consequences) worsen and acceler­ level of CDKN1A (encoding p21WAF−1, a biomarker of ate with ageing, even in the absence of overt cellular senescence in vitro) was unaltered in adipose lipodystrophy. tissue from elderly individuals, in accordance with age­ ing-related findings in other tissues [38]. We found a concordant reduction of autophagy- Authors’ contributions related parameters in adipose tissue from PLWH and elderly individuals. Autophagic activity is known to Conceptualization, P.D., J.C.D., A.F.V., M.G., J.V.; biopsy sampling and analytical procedures, M.G.M, A.G-N, M.M. decrease with ageing in multiple tissues [39], possibly G., A.B-R, M.G-E., R.C.; writing-original draft preparation, P. including adipose tissue [13], and ageing-associated D., F.V., M.G., J.V.; writing-review and editing, P.D., M.G., J. diseases are often ascribed to this decline in autophagy. V.; supervision, P.D., M.G., J.V.; funding acquisition, P.D., Our observations suggest that the adipose tissue dys­ M.G. All authors have read and agreed to the submitted function associated with the ageing-related decline in version of the manuscript autophagy may also be taking place in fat from PLWH developing lipodystrophy. Noteworthy, thymidine analogues are among the antiretroviral drugs that are Ethical approval more prone to induce lipodystrophy, and have been This study was approved by the ethics committee of Hospital reported to repress autophagy in adipocytes de la Santa Creu i Sant Pau, Barcelona, and all patients in vitro [40]. provided informed written consent. There are some limitations to our study. The rela­ tively moderate number of elderly patients, due to the obvious difficulties in recruiting very old healthy Data availability statement individuals volunteering for fat biopsies, ethics con­ The data that support the findings of this study are openly strains as well as the small size of biopsies in this available in Adipoplast website at https://adipoplast.org/ group precluded the study of a more extensive panel datebase/ of parameters. Moreover, this limitation also pre­ cluded performing dynamic studies related to autop­ hagy flux or mitochondrial respiratory activity that Disclosure statement would be more informative than current data based No potential conflict of interest was reported by the author(s). ADIPOCYTE 151 Funding [10] Wollina U, Wetzker R, Abdel-Naser MB, et al. Role of adipose tissue in facial aging. Clin Interv Aging. This research was supported by grants from Fondo de 2017;12:2069–2076. Investigaciones Sanitarias, Instituto de Salud Carlos III, [11] Livshits G, Kalinkovich A. Inflammaging as a common AEI, Spain (grants PI17/00420, PI17/00498, PI20/00106 and ground for the development and maintenance of sar­ PI20/00137) co-financed by the European Regional copenia, obesity, cardiomyopathy and dysbiosis. Development Fund (ERDF). Ageing Res Rev. 2019;56:100980. [12] Tchkonia T, Morbeck DE, Von Zglinicki T, et al. Fat tissue, aging, and cellular senescence. Aging Cell. 2010;9(5):667–684. ORCID [13] Ghosh AK, Mau T, O’Brien M, et al. Impaired autop­ Pere Domingo http://orcid.org/0000-0003-1138-5770 hagy activity is linked to elevated ER-stress and inflam­ Marta Giralt http://orcid.org/0000-0001-7968-4190 mation in aging adipose tissue. Aging (Albany. NY). Aleix Gavaldà-Navarro http://orcid.org/0000-0001-8421- 2016;8(10):2525–2537. 3174 [14] Caron-Debarle M, Lagathu C, Boccara F, et al. HIV- Albert Blasco-Roset http://orcid.org/0000-0002-4686-6075 associated lipodystrophy: from fat injury to premature Alejandro Delgado-Anglés http://orcid.org/0000-0002- agin. Trends Mol Med. 2010;16(5):218–229. 6996-6327 [15] Lichtenstein KA, Ward DJ, Moorman AC, et al. HIV Rubén Cereijo http://orcid.org/0000-0002-1108-230X Outpatient Study Investigators, Clinical assessment of Joan Carles Domingo http://orcid.org/0000-0002-6356- HIV-associated lipodystrophy in an ambulatory 0836 population. AIDS. 2001;15(11):1389–1398. Francesc Villarroya http://orcid.org/0000-0003-1266-9142 [16] Cereijo R, Gallego-Escuredo JM, Moure R, et al. The Joan Villarroya http://orcid.org/0000-0002-7859-1109 molecular signature of HIV-1-associated lipomatosis reveals differential involvement of brown and beige/ brite adipocyte cell lineages. PLoS One. 2015;10(8): e0136571. References [17] Hondares E, Gallego-Escuredo JM, Flachs P, et al. Fibroblast growth factor-21 is expressed in neonatal [1] Domingo P, Estrada V, López-Aldeguer J, et al. Fat and pheochromocytoma-induced adult human brown redistribution syndromes associated with HIV-1 infec­ adipose tissue. Metabolism. 2014;63(3):312–317. tion and combination antiretroviral therapy. AIDS Rev. [18] O’Callaghan NJ, Fenech M. A quantitative PCR 2012;14(2):112–123. method for measuring absolute telomere length. Biol [2] Koethe JR, Lagathu C, Lake JE, et al. HIV and antire­ Proced Online. 2011;13(1):3. troviral therapy-related fat alterations. Nat Rev Dis [19] Frühbeck G, Catalán V, Rodríguez A, et al. Prim. 2020;6(1):48. Adiponectin-leptin ratio is a functional biomarker of [3] Grunfeld C, Saag M, Cofrancesco JJ, et al. Study of fat adipose tissue inflammation. Nutrients. 2019;11(2):454. redistribution and metabolic change in HIV infection [20] Frühbeck G, Catalán V, Rodríguez A, et al. Adiponectin- (FRAM). Regional adipose tissue measured by MRI leptin ratio: a promising index to estimate adipose tissue over 5 years in HIV-infected and control participants dysfunction. Relation with obesity-associated cardiome­ indicates persistence of HIV-associated lipoatrophy. tabolic risk. Adipocyte. 2018;7(1):57–62. AIDS. 2012;24(11):1717–1726. [21] Kitada M, Koya D. Autophagy in metabolic disease and [4] Arrive E, Viard JP, Salanave B, et al. ANRS CO19 ageing. Nat Rev Endocrinol. 2021;17(11):647–661. COVERTE and ENNS study groups. Metabolic risk [22] Domingo P, Cabeza MC, Pruvost A, et al. Relationship factors in young adults infected with HIV since child­ between HIV/highly active antiretroviral therapy hood compared with the general population. PLoS (HAART)-associated lipodystrophy syndrome and One. 2018;13(11):e0206745. stavudine-triphosphate intracellular levels in patients [5] AGEhIV Cohort Study Group, Schouten J, Wit FW, with stavudine-based antiretroviral regimens. Clin Stolte IG, et al. Cross-sectional comparison of the pre­ Infect Dis. 2010;50(7):1033–1040. valence of age-associated comorbidities and their risk [23] Villarroya J, Gallego-Escuredo JM, Delgado-Anglés A, factors between HIV-infected and uninfected indivi­ et al. Aging is associated with increased FGF21 levels duals: the age HIV cohort study. Clin Infect Dis. but unaltered FGF21 responsiveness in adipose tissue. 2014;59(12):1787–1797. Aging Cell. 2018;17(5):e12822. [6] Wing EJ. HIV and aging. Int J Infect Dis. 2016;53:61–68. [7] Stout MB, Justice JN, Nicklas BJ, et al. Physiological [24] Giralt M, Domingo P, Guallar JP, et al. HIV-1 infection aging: links among adipose tissue dysfunction, dia­ alters gene expression in adipose tissue, which contri­ betes, and frailty. Physiology. 2017;32(1):: 09–19. butes to HIV-1/HAART-associated lipodystrophy. [8] Mancuso P, Bouchard B. The impact of aging on adipose Antivir Ther. 2006;11(6):729–740. function and adipokine synthesis. Front Endocrinol [25] Michaud M, Balardy L, Moulis G, et al. (Lausanne). 2019;10(137):eCollection 2019. 10.3389/ Proinflammatory cytokines, aging, and age-related fendo.2019.00137. diseases. J Am Med Dir Assoc. 2013;14(12):877–882. [9] Kuk JL, Saunders TJ, Davidson LE, et al. Age-related [26] Johnson JA, Albu JB, Engelson ES, et al. Increased changes in total and regional fat distribution. Ageing systemic and adipose tissue cytokines in patients with Res Rev. 2009;8(4):339–348. 152 P. DOMINGO ET AL. HIV-associated lipodystrophy. Am J Physiol with prelamin A accumulation, oxidative stress and Endocrinol Metab. 2004;286(2):49–52. premature cellular senescence. Cell Death Differ. [27] Lagathu C, Kim M, Maachi M, et al. HIV antiretroviral 2007;14(10):1759–1767. treatment alters adipokine expression and insulin sen­ [34] Vernochet C, Azoulay S, Duva D, et al. Differential sitivity of adipose tissue in vitro and in vivo. Biochimie. effect of HIV protease inhibitors on adipogenesis: 2005;87(1):65–71. SPEC. ISS. intracellular ritonavir is not sufficient to inhibit [28] Villarroya F, Domingo P, Giralt M. Lipodystrophy in differentiation. AIDS. 2003;17(15):2177–2180. HIV 1-infected patients: lessons for obesity research. [35] Zhang B, MacNaul K, Szalkowski D, et al. Inhibition Int J Obes (London). 2007;31(12):1763–1776 of adipocyte differentiation by HIV protease [29] Villarroya F, Domingo P, Giralt M. Lipodystrophy inhibitors. J Clin Endocrinol Metab. 1999;84 associated with highly active anti-retroviral therapy (11):4274–4277. for HIV infection: the adipocyte as a target of [36] Gallego-Escuredo JM, Gutierrez MM, Diaz-Delfin J, et al. anti-retroviral-induced mitochondrial toxicity. Trends Differential effects of efavirenz and lopinavir/ritonavir on Pharmacol Sci. 2005;26(2):88–93. human adipocyte differentiation, gene expression and [30] Roszkowska-Gancarz M, Jonas M, Owczarz M, et al. release of adipokines and pro-inflammatory cytokines. Age-related changes of leptin and leptin receptor var­ Curr HIV Res. 2010;8(7):545–553. iants in healthy elderly and long-lived adults. Geriatr [37] Giralt M, Domingo P, Villarroya F. Adipose tissue Gerontol Int. 2015;15(3):365–371. biology and HIV-infection. Best Pract Res Clin [31] Lakowa N, Trieu N, Flehmig G, et al. Telomere length Endocrinol Metab. 2011;25(3):487–499. differences between subcutaneous and visceral adipose [38] Hudgins AD, Tazearslan C, Tare A, et al. Age- and tissue in humans. Biochem Biophys Res Commun. tissue-specific expression of senescence biomarkers in 2015;457(3):426–432. mice. Front Genet. 2018eCollection 2018; 91: 10.3389/ [32] Liu Z, Jin L, Yang JK, et al. The dysfunctional fgene.2018.00059 MDM2-p53 axis in adipocytes contributes to [39] Rubinsztein DC, Mariño G, Kroemer G. Autophagy aging-related metabolic complications by induction of and aging. Cell. 2011;146(5):682–695. Cell. lipodystrophy. Diabetes. 2018;67(11):2397–2409. [40] Stankov MV, Panayotova-Dimitrova D, Leverkus M, [33] Caron M, Auclair M, Donadille B, et al. Human lipo­ et al. Thymidine analogues suppress autophagy and dystrophies linked to mutations in A-type lamins and adipogenesis in cultured adipocytes. Antimicrob to HIV protease inhibitor therapy are both associated Agents Chemother. 2013;57(1):543–551.