Life Sciences 308 (2022) 120948 Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie Differential effects of dolutegravir, bictegravir and raltegravir in adipokines and inflammation markers on human adipocytes Pere Domingo a, b, 1, Tania Quesada-López a, b, c, d, e, f, 1, Joan Villarroya c, d, e, f, Montserrat Cairó c, d, e, f, Maria Del Mar Gutierrez a, b, Maria Gracia Mateo a, b, Isabel Mur a, b, Noemí Corbacho a, b, Joan Carles Domingo c, d, Francesc Villarroya c, d, e, f, Marta Giralt c, d, e, f, * a Department of Infectious Diseases, Hospital de la Santa Creu i Sant Pau, Barcelona, Catalonia, Spain b Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau, Barcelona, Catalonia, Spain c Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Catalonia, Spain d Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Catalonia, Spain e CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain f Institut de Recerca Sant Joan de Déu (IRSJD), Esplugues de Llobregat, Catalonia, Spain A R T I C L E I N F O A B S T R A C T Keywords: Aims: To assess the potential direct effects of the integrase strand-transfer inhibitors (INsTIs) dolutegravir, bic­ Fat tegravir, and raltegravir, drugs used as treatment for people living with human immunodeficiency virus (PLWH), Adipocyte on human adipose cells. Anti-retroviral treatment Main methods: Drugs were added to the differentiation medium of human Simpson-Golabi-Behmel syndrome HIV integrase inhibitors (SGBS) adipose cells and morphological adipogenesis was monitored for 10 days. Also, adipocytes were exposed Adipokine Cytokine to drugs following differentiation (day 14). The gene expression levels of selected adipogenesis markers, adipocyte metabolism markers, adipokines, and cytokines were determined by quantitative-reverse transcription polymerase-chain reaction. The release of adiponectin and leptin into the culture medium was measured using specific enzyme-linked immunosorbent assay, and release of interleukin-6 and chemokine (C–C motif) ligand-2 using Multiplex assays. Key findings: Overall morphological adipogenesis was unaltered by INsTIs. The expression of adipogenesis marker genes (peroxisome proliferator-activated receptor-Ɣ and lipoprotein lipase) was slightly reduced in dolutegravir- treated differentiating adipocytes. Bictegravir repressed gene expression and the release of pro-inflammatory cytokines in differentiating adipocytes. Dolutegravir and raltegravir increased interleukin-6 gene expression, but only dolutegravir increased interleukin-6 release. Dolutegravir repressed adiponectin expression and release in differentiating adipocytes and had a similar but milder effect on leptin. Drug treatment of mature adipocytes reduced adiponectin gene expression in response to dolutegravir. Significance: The INsTIs studied do not have a significant effect on human adipose cell differentiation but exert distinct effects on gene expression and secretion of adipokines and cytokines. These findings will help understand and manage the effects of INsTI-containing treatments on body weight and metabolic dysregulation in PLWH. Abbreviations: INsTIs, integrase strand-transfer inhibitors; HIV, human immunodeficiency virus; PLWH, people living with HIV; SGBS, Simpson-Golabi-Behmel syndrome; PPAR, peroxisome proliferator-activated receptor; LPL, Lipoprotein lipase; TNF, tumor necrosis factor; CCL2, Chemokine (C–C motif) ligand 2; MCP-1, Monocyte chemoattractant protein-1; IL6, Interleukin-6; DTG, dolutegravir; BIC, bictegravir; RAL, raltegravir; FBS, fetal bovine serum; qRT-PCR, Quantitative Reverse Transcription Polymerase-Chain Reaction.. * Corresponding author at: Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Diagonal, 643, 08028 Barcelona, Spain. E-mail address: mgiralt@ub.edu (M. Giralt). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.lfs.2022.120948 Received 30 May 2022; Received in revised form 31 August 2022; Accepted 7 September 2022 Available online 9 September 2022 0024-3205/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). P. Domingo et al. Life Sciences 308 (2022) 120948 1. Introduction [13] and has been extensively used to assess the effects of anti-retroviral drugs on human adipocyte function [14–17]. Human SGBS pre- Weight gain and increased adiposity are increasingly found in people adipocytes were cultured and differentiated into adipocytes, as previ­ living with human immunodeficiency virus, HIV (PLWH), contributing ously reported [14,18]. Briefly, SGBS pre-adipocytes were maintained in to an increased risk of serious medical complications. Recent studies DMEM/F12 containing 10 % FBS. After the cells reached confluency, have raised concerns regarding excess weight gain and metabolic ab­ adipogenic differentiation was initiated by first incubating the cells for normalities specifically associated with the use of integrase strand- seven days in a serum-free medium containing 20 nM insulin, 100 μg/ml transfer inhibitors (INsTIs) for the treatment of PLWH. The first warn­ human apo-transferrin, 0.2 nM triiodothyronine (T3), and 100 nM ing came from observational studies, and connections with INsTI treat­ cortisol, supplemented with 25 nM dexamethasone, 500 μM 3-isobutyl- ments were later confirmed in randomized clinical trials [1–3]. methylxanthine, and, 2 μM rosiglitazone to obtain the fully differenti­ However, the pathophysiological bases of the effects of INsTIs on ated adipocytes or 0.5 μM rosiglitazone (a sub-optimal concentration for adiposity and the exact contribution of each type of INsTI drug remain adipogenic differentiation) for chronic treatments. The cells were sub­ largely unknown [3,4]. sequently switched to an adipogenic differentiation medium containing Increased body weight, as in obesity, is associated with the 20 nM insulin, 100 μg/ml human apo-transferrin, 0.2 nM triiodothyro­ enlargement of adipose tissue. Adipocytes are key sites of fuel meta­ nine (T3), and 100 nM cortisol only and maintained for another three bolism storage (in the form of triglycerides), and energy release in the days (in the case of chronic treatments) or seven days for treatments in form of free fatty acids in periods of energy demand (such as fasting). fully differentiated adipocytes (where, >90 % of the cells had acquired a Additionally, adipocytes are responsible for producing circulating adi­ differentiated adipocyte morphology, as evidenced by lipid droplet pokines that influence systemic metabolism [5]. Increased adiposity is accumulation). Depending on the experimental design, the cells were characterized by the enlargement (hypertrophy) of adipocytes and, treated with drugs throughout the 10-day differentiation process or sometimes, even hyperplasic phenomena. Abnormally enlarged adipose acutely for 24 h once they were fully differentiated (14 days after the tissue in obesity is associated with systemic metabolic alterations, induction of differentiation). mainly hyperglycemia, insulin resistance, and dyslipidemia. Concomi­ tant with the metabolic alterations in the adipose depots, disturbances of 2.3. Assessment of lipid accumulation in adipocytes adipokines (such as leptin, adiponectin) and cytokines (such as pro- inflammatory tumor necrosis factor-alpha (TNFα) and chemokine To assess the effects of these drugs (dolutegravir, raltegravir, and (C–C motif) ligand 2 (CCL2)) secretion by adipose tissue greatly bictegravir) on adipocyte differentiation and lipid accumulation, mi­ contribute to systemic metabolic alterations in overweight and obese crographs of the cell cultures on day 10 after the induction of differen­ people [5]. Such conditions have been increasing in PLWH. tiation were captured using a Motic AE2000 microscope with a Moticam Studies on the effects of anti-retroviral drugs on human adipose cells 5.0 MP camera (Motic Instruments INC, Richmond, BC, Canada). Mi­ can provide pre-clinical information regarding the potential of these crographs, as jpg files, were imported into the ImageJ software (Wayne drugs to alter adipose tissue. Likewise, these pre-clinical approaches Rasband at the National Institutes of Health (NIH)). The image type was have proven useful for identifying molecular targets involved in the processed into an RGB stack (Image > Type > RGB-Stack) and a metabolic side-effects of first-generation anti-retroviral drugs namely montage of the three channels was performed (Image > Stacks > Make protease inhibitors and reverse transcriptase inhibitors [6–12]. The ca­ Montage). Once in the RGB stack, the threshold of the image was pacity of the distinct INsTIs currently used for the treatment of PLWH adjusted (lower threshold of 160 and an upper threshold of 250) (Image suspected to promote adiposity to directly affect human adipose cells is > Adjust > Threshold). Area, area fraction, limit to threshold, and poorly understood. Thus, the goal of the present study was to assess the display label parameters were set for measurement (Analyze > Set potential direct effects of the INsTIs, dolutegravir (DTG), bictegravir Measurements). Image measurements were then performed (Analyze > (BIC), and raltegravir (RAL), on adipogenic differentiation, relevant Measure). The measurements obtained were subsequently used for gene expression, and the release of cytokines and adipokines by human comparison. adipose cells. 2.4. RNA isolation, conventional reverse transcription, and quantitative 2. Materials and methods reverse transcription polymerase-chain reaction (qRT-PCR) 2.1. Reagents Total RNA was purified from cells using an affinity column-based method (Macherey-Nagel; Thermo Fisher Scientific, Waltham, Massa­ Chemicals and reagents for cell culture were purchased from Sigma- chusetts, USA) as previously described [14]. Reverse transcription was Aldrich (St Louis, Missouri, USA). Rosiglitazone which was purchased performed in a total volume of 20 μl using random hexamer primers from Alexis Biochemicals (Enzo Life Sciences, Farmingdale, New York, (Applied Biosystems; Waltham, Massachusetts, USA) and 0.5 μg total USA). DMEM/F12 medium and fetal bovine serum (FBS) were pur­ RNA. PCR was performed on an ABI/Prism 7700 sequence detector chased from Life Technologies (Carlsbad, California, USA). DTG and system using 20 μl of a reaction mixture containing 1 μl of cDNA, 10 μl RAL were obtained from MedChemExpress (Monmouth Junction, NJ TaqMan Universal PCR master mix, 250 nM probes, and 900 nM primers 08852, USA); and BIC was provided by Gilead (Foster City, California, from an Assays-on-Demand gene expression assay mix (TaqMan; USA). For the initial assessments of the effects of anti-retroviral drugs Applied Biosystems; Waltham, Massachusetts, USA). The following before concentration-response analyses, the drugs were used at the Assay-on-Demand probes from Life Technologies were used: Adipo­ highest concentrations that did not cause cytotoxicity in specific cell nectin, Hs00605917; Chemokine (C–C motif) ligand 2 (CCL2), systems. The drugs were dissolved using dimethyl sulfoxide (DMSO) as Hs00234140; Cytochrome-c oxidase subunit IV (COX4I1), Hs00266371; the vehicle. Controls included amounts of DMSO (≤0.1 % DMSO of total Interleukin-6 (IL6), Hs00174131; Leptin, Hs00174877; Lipoprotein cell medium volume) equal to that used in the drug-treated cells. lipase (LPL), Hs00173425; Peroxisome proliferator-activated receptor-Ɣ (PPARƔ), Hs00231674; Peroxisome proliferator-activated receptor 2.2. Human adipocyte cell culture gamma coactivator 1-alpha (PPARGC1A), Hs00173304_m1; Solute carrier family 2, facilitated glucose transporter member 1 (GLUT1, To assess the effects of INsTIs on adipogenesis, we used human Slc2a1), Hs00892681_m1; Solute carrier family 2, facilitated glucose Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocytes. This cell transporter member 4 (GLUT4, Slc2a4), Hs99999901; RPLP0, strain has been validated as a model of human adipogenic differentiation Hs99999902. Controls without RNA, primers, or reverse transcriptase 2 P. Domingo et al. Life Sciences 308 (2022) 120948 Table 1 2.6. Protein analysis Effects of dolutegravir (DTG), raltegravir (RAL) and bictegravir (BIC) on SGBS human adipogenic differentiation. Adiponectin, leptin, IL6, and CCL2 (also referred to as monocyte Drug concentration 0.01 0.1 1 10 chemoattractant protein-1, MCP-1) were quantified using medium (μM) collected from the last five days of adipocyte culture, before harvest. Dolutegravir 99.6 ± 2.3 94.8 ± 3.7 104.8 ± 109.5 ± ELISA kits specific for human adiponectin (EZHADP-61 K, Merck Life 1.3 0.2 Science; Madison, Wisconsin, USA) and leptin (DLP00, R &D Systems; Raltegravir 108.7 ± 100.1 ± 106.8 ± 104.0 ± Minneapolis, Minnesota, USA), were used for quantification. IL6 and 0.6 0.1 3.5 1.8 CCL2 levels were quantified using a multiplex analysis system (Lynco Bictegravir 106.9 ± 101.8 ± 109.4 ± 109.1 ± 0.3 2.6 1.2 0.6 Research/Merck; Darmstadt, Germany) and a Luminex 100ISv2 instru­ ment (Thermo Fisher Scientific, Waltham, Massachusetts, USA). were included in each set of experiments. The relative amounts of in­ dividual mRNAs were calculated using the comparative (2 − ΔCT) 2.7. Statistical analyses method and normalized to that of the reference control gene (RPLP0 mRNA) according to the manufacturer's instructions. Each sample was Statistical analyses were performed using GraphPad Prism 9 Soft­ run in duplicates, and the mean value of duplicates was used in the ware (San Diego, California USA) using one-way ANOVA and Dunnett's calculations. Parallel calculations performed using other reference multiple comparison test. Significance is indicated in the text (the control genes (18S rRNA) yielded essentially the same results. threshold value to determine significance was set to P ≤ 0.05). 2.5. Cytotoxicity assays 3. Results Cytotoxicity was determined using a CytoTox96 kit (Promega; 3.1. Pre-adipocytes treatment with INsTIs does not elicit cytotoxicity Madison, Wisconsin, USA), following the manufacturer's instructions. Briefly, cytotoxicity was calculated as the lactate dehydrogenase activity The effects of DTG, RAL, and BIC on the adipogenic differentiation of present in the cell culture medium relative to the maximum activity human SGBS pre-adipocytes were tested over a concentration range of observed after total cell lysis of cells after exposure to drugs for 24 h. 0.01 to 10 μM, which included the Cmin and Cmax circulating concen­ trations as previously reported in treated patients [19,20]. Differenti­ ating pre-adipocytes were exposed to DTG, BIC, or RAL (0.01 μM–10 μM) for 24 h, and the potential cytotoxicity of these drugs at the different Fig. 1. Effects of dolutegravir, raltegravir and bictegravir on adipogenic differentiation of SGBS human pre-adipocytes in culture. SGBS human pre-adipocytes were differentiated in culture in the presence of the indicated concentrations of drugs. Treatment was initiated on day 0 and continued throughout the differentiation process (10 days). Representative micrographs of adipocyte cell cultures differentiating in the presence of the indicated concentrations of drugs are shown. 3 P. Domingo et al. Life Sciences 308 (2022) 120948 Fig. 2. Effects of dolutegravir (DTG), ral­ Adipogenesis tegravir (RAL), and bictegravir (BIC) on the PPARG mRNA LPL mRNA expression levels of genes related to adipo­ genic function, inflammation, mitochon­ Relative transcript levels Relative transcript levels 1.5 1.5 drial function, and adipokines in SGBS human adipocytes differentiating in culture. 1.0 SGBS human pre-adipocytes were differen­ 1.0 * *** tiated in culture in the presence of the 0.5 0.5 *** indicated concentrations of drugs. Data on mRNA levels are presented as means ± SEM from 3 to 4 independent cell culture exper­ C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 iments, each sample analyzed in duplicate, DTG RAL BIC DTG RAL BIC and are expressed relative to values from untreated control cells (defined as 1). Sta­ tistical analyses were performed using one- Glucose metabolism way ANOVA and Dunnett's multiple com­ SLC2A4 mRNA SLC2A1 mRNA parison test. *P < 0.05, **P < 0.01, ***P < 1.5 Relative transcript levels 0.001, and ****P < 0.0001 for each drug Relative transcript levels 1.5 treatment vs. control. 1.0 1.0 * * 0.5 0.5 C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 DTG RAL BIC DTG RAL BIC Mitochondrial function PPARGC1A mRNA COX4I1 mRNA Relative transcript levels Relative transcript levels 1.5 1.5 * 1.0 1.0 ** * 0.5 0.5 C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 DTG RAL BIC DTG RAL BIC Inflammation CCL2 mRNA IL6 mRNA Relative transcript levels Relative transcript levels 2.5 2.0 ** 2.0 ** ** 1.5 1.5 * 1.0 1.0 **** 0.5 0.5 C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 DTG RAL BIC DTG RAL BIC Adipokines LEP mRNA ADIPOQ mRNA Relative transcript levels Relative transcript levels 1.5 1.5 1.0 1.0 * 0.5 0.5 **** C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 DTG RAL BIC DTG RAL BIC 4 P. Domingo et al. Life Sciences 308 (2022) 120948 Inflammatory cytokines CCL2 (MCP-1) Interleukin-6 500 50 * * 400 40 300 * ng/mL 30 ng/mL 200 20 * 100 10 0 0 C 0,1 1 10 0,1 1 10 0,1 1 10 C 0,1 1 10 0,1 1 10 0,1 1 10 DTG RAL BIC DTG RAL BIC Adipokines Leptin Adiponectin 400 150 300 * 100 pg/mL ng/mL 200 *** * 50 100 **** 0 0 C 0.1 1 10 0.1 1 10 0.1 1 10 C 0.1 1 10 0.1 1 10 0.1 1 10 DTG RAL BIC DTG RAL BIC Fig. 3. Effects of dolutegravir (DTG), raltegravir (RAL), and bictegravir (BIC) on the release of CCL2 (MCP-1), interleukin-6, leptin, and adiponectin to the cell culture medium of differentiating SGBS human adipocytes. SGBS human pre-adipocytes were differentiated in culture in the presence of the indicated concentrations of drugs. Data corresponds to concentrations accumulating in the cell culture medium in the last 5 days of culture. Data are means ± SEM from 3 to 4 independent cell culture experiments, and each sample was analyzed in duplicate. Statistical analyses were performed using one-way ANOVA and Dunnett's multiple comparison test. *P < 0.05, ***P < 0.001, and ****P < 0.0001 for each drug treatment vs. control. concentrations was estimated. No significant cytotoxicity was observed adipogenic conditions, as determined by performing phase-contrast in the cell cultures exposed to any of the tested concentrations of DTG, microscopic imaging and using the Image J software to quantify the BIC, or RAL (Supplemental Table 1). This is consistent with previous cell culture surface occupied by differentiated (lipid droplet-containing) reports, in which DTG and BIC exhibited significant cytotoxicity only at cells. None of the results was significantly different from control values higher concentrations (>10 μM) in other cell types (such as hepatocytes, (one-way ANOVA Dunnett's multiple comparisons test). fibroblasts, human prostate cells, and hepatocarcinoma cells) [21]. RAL Gene expression was quantified after 10 days of the adipogenic dif­ showed no cytotoxicity at concentrations up to 10 μM in SGBS pre- ferentiation induction in the presence of INsTIs (Fig. 2). We observed adipose cells [14,18]. little alteration in response to RAL (only a minor induction of IL6 mRNA levels), consistent with a previous report [14]. BIC did not alter the expression levels of genes encoding adipogenic molecular factors 3.2. INsTIs did not alter overall adipogenesis but have differential effects (peroxisome proliferator-activated receptor-gamma, PPARγ; lipoprotein on adipocyte gene expression lipase, LPL), glucose transporter-4 (SLC2A4), glucose transporter-1 (SLC2A1), or adipokines (leptin, LEP, and adiponectin, ADIPOQ). To study the effects of each drug on adipogenic differentiation, However, BIC significantly reduced the expression levels of genes INsTIs treatments was initiated on day zero and continued throughout encoding the pro-inflammatory cytokines, CCL2 (MCP-1) and the differentiation process (up to 10 days, as described in the previous interleukin-6 (IL6), at the highest concentration tested (10 μM). DTG section). The effects of the drugs on the morphological differentiation of applied at 10 μM significantly reduced the gene expression levels of adipocytes were determined using a phase-contrast microscope and PPARγ (the master gene regulator of adipogenesis), lipoprotein lipase, Image J software to quantify the cell culture surface occupied by leptin, and adiponectin (Fig. 2). Considering the release of leptin and differentiated (lipid droplet-containing) cells (see Materials and adiponectin to the adipocyte culture medium, the highest doses of DTG Methods section). Data are expressed as percentages relative to control mildly reduced the level of released leptin and dramatically decreased cultures (Table 1). None of the tested drugs had a major effect on the that of adiponectin; BIC did not alter the release of leptin or adiponectin; acquisition of an adipocyte morphology, as drug-treated cells consis­ and RAL had minor effects on these parameters (Fig. 3). DTG signifi­ tently exhibited unaltered lipid accumulation relative to the control cantly (P ≤ 0.05) increased the release of CCL2 (MCP-1) and IL6 into the (Fig. 1 and Table 1). cell culture medium, whereas BIC reduced the release of both cytokines Data are expressed as mean ± SEM of the percent of lipid accumu­ in differentiating adipocytes treated at 10 μM (Fig. 3). RAL had minor lation relative to the 100 % adipogenesis elicited by SGBS cultured effects on these parameters. The induction of IL6 mRNA expression was under control (sub-optimal 0.5 μM rosiglitazone-induced adipogenesis) 5 P. Domingo et al. Life Sciences 308 (2022) 120948 Fig. 4. Effects of dolutegravir (DTG), bic­ Adipogenesis tegravir (BIC), and raltegravir (RAL) on the expression levels of genes related to adipo­ PPARG m RNA LPL m RNA genic function, inflammation, mitochondrial 1 .5 2 .5 function, and adipokines in differentiated R e la tiv e t r a n s c r ip t le v e ls SGBS human adipocytes. SGBS human pre- R e la tiv e t r a n s c r ip t le v e ls * 2 .0 adipocytes were differentiated in culture * and, on day 14 of differentiation, treated 1 .0 1 .5 with the indicated concentrations of drugs for 24 h. Data on mRNA levels are presented 1 .0 0 .5 * 0 .5 as means ± SEM from 3 to 4 independent 0 .0 experiments and are expressed relative to values from untreated control cells (defined 0 .0 C 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 C 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 DTG RAL B IC DTG RAL B IC as 1). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 for each drug treatment Glucose metabolism vs. control. SLC 2A 4 m R N A SLC 2A 1 m R N A 1 .5 1 .5 R e la tiv e t r a n s c r ip t le v e ls R e la tiv e t r a n s c r ip t le v e ls 1 .0 1 .0 0 .5 0 .5 0 .0 0 .0 C 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 C 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 DTG RAL B IC DTG RAL B IC Mitochondrial function PPARG C1A m RN A C O X 4 I1 m R N A 2 .0 2 .0 R e la tiv e t r a n s c r ip t le v e ls R e la tiv e t r a n s c r ip t le v e ls 1 .5 1 .5 * * * 1 .0 1 .0 0 .5 0 .5 0 .0 0 .0 C 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 C 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 DTG RAL B IC DTG RAL B IC Inflammation IL 6 m R N A CCL2 m RNA 1 .5 R e la tiv e t r a n s c r ip t le v e ls 1 .5 R e la tiv e t r a n s c r ip t le v e ls 1 .0 1 .0 * 0 .5 0 .5 0 .0 0 .0 C 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 C 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 0 .0 1 0 .1 1 10 DTG RAL B IC DTG RAL B IC Adipokines ADIPOQ mRNA LEP mRNA 1.5 0.8 Relative transcript levels Relative transcript levels 0.6 1.0 * * 0.4 0.5 0.2 0.0 C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.0 C 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 DTG RAL BIC DTG RAL BIC 6 P. Domingo et al. Life Sciences 308 (2022) 120948 not reflected in the altered IL6 secretion (Fig. 3). to translate results obtained in vitro into clinical practice. Moreover, besides adipocytes, INsTIs may affect other cell types that are also pre­ 3.3. Acute effects of INsTIs on human adipocytes sent in adipose tissue depots. For example, it has been reported that DTG reduces inflammation and senescence in endothelial cells, whereas RAL To test the possible acute effects on already differentiated adipo­ has no effect [30]. However, the significance of the drug concentrations cytes, human SGBS pre-adipocytes were cultured and differentiated into used in cell culture studies, even if in the range of circulating levels in adipocytes as previously reported and described in the Materials and treated patients, is not unequivocal. DTG has been reported to penetrate Methods section [18]. When the cells had already acquired an adipose tissue cells much more strongly than nucleoside/nucleotide adipocyte-like morphology after 14 days of inducing adipogenesis, we reverse transcriptase inhibitors [31]. Thus, we speculated that the examined the effects of INsTIs by exposure for 24 h to DTG, BIC, or RAL exposure of fat to DTG may be particularly intense in treated patients. (0.01 μM–10 μM). The tested drugs had minimal effects on the assessed Comparable information on intra-adipose concentrations of RAL and BIC gene expression levels (mild induction of LPL expression by RAL and BIC is lacking. Despite these limitations, our in vitro study allowed us to at the highest concentrations) except adiponectin (ADIPOQ). Adipo­ establish the direct effects of INsTIs on human adipocytes, which could nectin gene expression was significantly decreased by DTG (P ≤ 0.05) not be distinctly assessed in studies in patients. (Fig. 4), although this did not result in a significant change in the levels of adiponectin secreted into the culture medium by the differentiated 5. Conclusions adipocytes (Supplemental Fig. 1). This may reflect the short duration of drug exposure (24 h) used in the experimental setting. In conclusion, our findings show that INsTIs currently used to treat PLWH have differential effects on adipose cells and highlight the po­ 4. Discussion tential of DTG to repress adiponectin expression/release and enhance inflammatory cytokine expression/release. Whereas BIC inhibits pro- Recent evidence suggests an association between anti-retroviral inflammatory cytokines expression and release. These observations regimens containing INsTIs and increased weight gain in PLWH. This may contribute to improving the treatment profiles used in PLWH to weight gain appeared to be greater in DTG and BIC compared to other minimize side effects associated with increased adiposity and metabolic INsTIs. The pathophysiological basis of these effects remains unknown. alterations. Consistent with previous reports, this study confirms that RAL does not exert significant effects on adipogenic differentiation or the levels of CRediT authorship contribution statement the tested genes and secreted proteins in human adipocytes [14,22]. DTG did not markedly affect the acquisition of adipocyte morphology Conceptualization, P.D. and M.G.; methodology, T.Q-L., J.V., M.G., (lipid accumulation), which is also in agreement with a recent report F.V.; experimental procedures and analysis, T.Q-L., J.V., M.C., M.M.G., using omental and subcutaneous pre-adipocytes [23]. However, we M.G.C., I.M., N.C., J.C.D.; writing—original draft preparation, T.Q-L., M. discovered that DTG (but not BIC) at the higher tested concentrations G., F.V.; review and editing, P.D., T.Q-L, F.V., M.G. All authors have read exerted some inhibitory effects on adipocyte gene expression (such as and agreed to the published version of the manuscript. PPARγ and LPL) and markedly reduced adiponectin gene expression and secretion in differentiating adipocytes. Gorwood et al. [24] also reported Declaration of competing interest a decrease in the expression and release of adiponectin and leptin in response to chronic treatment of adipose stem cells (ASCs) with DTG. Dr. Pere Domingo reported receiving honoraria from Merck Sharp & Meanwhile, and valuable in the translatability of these results to pa­ Dohme, Gilead Sciences, ViiV Healthcare, Janssen, Cilag, Thera tech­ tients, González-Cordón et al. [25] have reported hypoadiponectinemia nologies, and Roche. All the other authors declare that they have no in PLWH treated with anti-retroviral patterns that include DTG [25]. known competing financial interests or personal relationships that could The downregulation of adiponectin in treated patients might be have appeared to influence the work reported in this manuscript. The secondary to increased adiposity [26]. However, our observation that authors guarantee that they have the authority to publish the work and DTG reduces adiponectin in a cell-autonomous adipocyte system sug­ that the manuscript and confirm that neither the manuscript nor any gests a potential direct effect of DTG-based regimens in eliciting hypo­ parts of its content are currently under consideration or published in adiponectinemia and subsequent deleterious systemic consequences another journal. (such as insulin resistance and cardiovascular risk). Notably, we found that adiponectin was not significantly altered in adipocytes treated with Acknowledgements BIC, regardless of the differentiation status or concentration, within the tested range. Although some studies have suggested that both DTG- and This research was supported by grants from Fondo de Investigaciones BIC-containing anti-retroviral regimens favor weight gain [3], our data Sanitarias, Instituto de Salud Carlos III, Spain (grants PI17/00420, PI17/ suggest that DTG has a stronger action in disturbing the adiponectin 00498, PI20/00106 and PI20/00137) co-financed by the European system in adipocytes. Interestingly, DTG and BIC exhibited opposite Regional Development Fund (ERDF) and an independent grant from effects (induction and repression, respectively) on the expression and Gilead. This company had no role in the study design, data collection, release of the pro-inflammatory cytokine IL6, and partially on the and interpretation of data or preparation of the manuscript. TQ-L holds a expression and release of CCL2 (MCP-1). As adiponectin represses the Juan de la Cierva – Incorporación grant, IJC2020-043360-I, funded by expression of IL6 in adipocytes [27,28], DTG and BIC may elicit alter­ MCIN/AEI /10.13039/501100011033 and by the European Union ations based on the crosstalk between adiponectin and inflammation in NextGeneration EU/PRTR. adipose tissue. Finally, we found a mild trend for DTG to decrease leptin expression Appendix A. Supplementary data and release. A recent report showed that leptin levels decreased when patients were switched from protease inhibitor-based to INsTI- Supplementary data to this article can be found online at https://doi. containing anti-retroviral regimes [29]. 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