Tyrphostin AG17 inhibits adipocyte differentiation in vivo and in vitro

Tyrphostin AG17 inhibits adipocyte differentiation in vivo and in vitro Background: Excessive subcutaneous adiposity in obesity is associated to positive white adipocyte tissue (WAT) differentiation (adipogenesis) and WAT expandability. Here, we hypothesized that supplementation with the insulin inhibitor and mitochondrial uncoupler, Tyrphostin (T-AG17), in vitro and in vivo inhibits adipogenesis and adipocyte hypertrophy. Methods: We used a 3T3-L1 proadipocyte cell line to identify the potential effect of T-AG17 on adipocyte differentiation and fat accumulation in vitro. We evaluated the safety of T-AG17 and its effects on physiological and molecular metabolic parameters including hormonal profile, glucose levels, adipogenesis and adipocyte hypertrophy in a diet-induced obesity model using C57BL/6 mice. Results: We found that T-AG17 is effective in preventing adipogenesis and lipid synthesis in the 3T3-L1 cell line, as evidenced by a significant decrease in oil red staining (p < 0.05). In obese C57BL/6 mice, oral administration of T-AG17 (0.175 mg/kg for 2 weeks) lead to decreased fat accumulation and WAT hypertrophy. Further, T-AG17 induced adipocyte apoptosis by activating caspase-3. In the hepatocytes of obese mice, T-AG17 promoted an increase in the size of lipid inclusions, which was accompanied by glycogen accumulation. T-AG17 did not alter serum biochemistry, including glucose, insulin, leptin, free fatty acids, creatinine, and aspartate aminotransferase. Conclusion: T-AG17 promotes adipocyte apoptosis in vivo and is an effective modulator of adipocyte differentiation and WAT hypertrophy in vitro and in vivo. Therefore, T-AG17 may be useful as a pharmacological obesity treatment. Keywords: Tyrphostin, AG17, Adipogenesis, Obesity, Hepatic steatosis, Oxidative phosphorylation, Thermogenesis, Mitochondrial uncoupling, Adipocyte differentiation Background pathways that may contain potential drug targets are the Excessive subcutaneous adiposity and its accumulation white adipose tissue (WAT) differentiation pathway, also into visceral depot during obesity are major risk factors referred to as adipogenesis, and the WAT expandability for developing type 2 diabetes (T2DM) and several other pathway. chronic metabolic disorders [1, 2]. Therefore, identifying During early development, mesenchymal stem cells therapeutic targets to treat the metabolic failures associated differentiate into chondrocytes, osteoblasts, myoblasts with obesity could reduce or prevent the development of and adipocytes [3]. Adipocyte differentiation from mes- these incurable metabolic disorders. Two cellular signaling enchymal stem cells is modulated by signaling cascades involving bone morphogenetic protein-4 and peroxi- * Correspondence: acm590@hotmail.com; alberto.camachomr@uanl.edu.mx; some proliferator-activated receptor (PPAR) β/δ,which rortizlopez@gmail.com 1 support the gene expression of PPARγ [4]. In adults, Departamento de Bioquímica y Medicina Molecular, Facultad de Medicina, Universidad Autónoma de Nuevo León (UANL), Monterrey, Mexico ectopic accumulation of adipocytes might be caused by Unidad de Genómica, UANL, CIDICS, Monterrey, Mexico dysfunction in differentiation pathways, which creates Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 2 of 11 an inability to induce differentiation of adipocyte pre- T-AG17 is also a potent inhibitor of mitochondrial oxphos cursor cells [3, 5] or induce adipocyte de-differentiation and is capable of increasing energy expenditure. How- [6]. On the other hand, once obesity is reached, the ever, the effects of T-AG17 on adipocyte differentiation, number of new adipocytes decreases, and adipocytes adipose tissue hypertrophy and body organ toxicity become hypertrophic, reaching their expandability limit have not been evaluated. until fat accumulates around ectopic organs, resulting Considering the significant suppressive effects of T-AG17 in metabolic complications [7, 8]. on insulin signaling, we hypothesized that T-AG17 might Drugs that act on mitochondria have been used to promote inhibition of adipogenesis and/or adipocyte hyper- combat fat accumulation by forcing cells to use stored trophy. Specifically, we seek to determine if T-AG17: 1) energy. Mitochondrial oxphos uncouplers create a futile decreases lipid accumulation in the 3T3-L1 adipocyte cell cycle of glyceride and fatty acid oxidation without gener- line induced by insulin and 2) promotes adipocyte apop- ating adenosine triphosphate (ATP). 2,4-dinitrophenol tosis in a diet-induced obesity mouse model. (DNP) is one of the best known uncoupler of oxphos widely used as a weight loss agent between 1933 and Methods 1938; however, DNP has been banned due to its high Reagents and antibodies acute toxicity [9]. Further studies on DNP and other re- 3T3-L1 preadipocyte cell line (Cat. CL-173) and newborn lated prodrugs consistently show that the toxic effects calf serum (Cat. 30-2030) were purchased from ATCC, are dose-dependent. Mild mitochondrial uncoupling Inc. Dulbecco’s Modified Eagle’s Medium-high glucose (MMU) with low doses, which seem effective at thermo- (Caisson Labs, Cat.), Penicillin-Streptomycin (Cat. neutrality (30 °C) conditions, may provide a promising P4333), Fetal Bovine Serum (Gibco), Oil Red O (Cat. strategy to reduce body weight with general tolerability O0625), Dimethyl sulfoxide (Cat. D2650), isopropyl [10–18]. Side effects of DNP exposure might be pre- alcohol (Cat. W292907), Formalin solution neutral vented, and MMU might also promote longevity through buffered 10% (Cat. HT501128), Ethyl alcohol (Cat. decreased ROS levels, mitochondrial biogenesis, down- E7023), Harris hematoxylin (Cat. HHS16), Sodium regulation of the mTOR and insulin signaling pathways, citrate (Cat. 1613859), Triton X-100 (Cat. X100), Corn- and upregulation of autophagy [12, 13]. Of interest, ing® cell culture flasks surface area 75 cm2, canted neck, these effects are similar to those seen in caloric restric- cap (vented) (CLS430641) were from SIGMA-ALDRICH. tion [12, 15, 19] and are consistent with the “uncoupling Paraplast® Embedding Media, (Cat. 15159-409, McCormick to survive” hypothesis [20]. These potentially beneficial Scientific), Rabbit polyclonal to Active + pro Caspase 3 effects of MMU may also be induced by niclosamide (Cat. AB13847, ABCAM), Goat polyclonal secondary anti- ethanolamine [21], salsalate [22], TTFB [23], CZ5 [24], body to Rabbit IgG H&L (alexa Fluor® 488) preadsorbed FCCP [6, 17, 25–27], the niclosamide-ethanolamine (Cat. AB150081, ABCAM), and VECTASHIELD Hardset aduct [21] and certain tyrphostins [28]. antifade mounting medium with DAPI (cat no. H-1500 Tyrphostins belong to the benzylidenemalononitrile Vector laboratories). family, which possess a benzene ring pharmacophore, an Adipogenesis Assay Kit for 3T3-L1 preadypocyte differ- exocyclic carbon-carbon double bond, and a cyano group entiation was purchased from Abcam (Cat. Ab133102). (CN) located at the same side of the molecule (cis) as the Also, the primary antibody cleaved Caspase-3 (Asp175) aromatic ring [29, 30]. The tyrphostin T-AG17 is a (Cat. 9661. Cell signaling) and secondary antibody highly selective, reversible inhibitor of epidermal Anti-rabbit IgG (H + L), Alexa Fluor® 488 Conjugate (Cat. growth factor receptor-induced phosphorylation of 4412. Cell signaling) were used. ELISA kits: creatinine tyrosine residues of intracellular proteins [31], and (SIGMA. MAK080), aspartate aminotransferase (SIGMA. its cellular effect is dose-dependent growth inhibition. MAK055), free fatty acids (Roche, 11383175001), insulin T-AG17 has potential therapeutic value for treating (Millipore, Cat. EZRMI-13 k) and leptin (Millipore, Cat. neurodegenerative disorders [28], atherosclerosis [32], dys- EZML-82 K). Acucheck (Cat. 05987270) and glucose lipidemia [33], restenosis [34], and cancer/cell hyperproli- strips (6454011023, Roche). 10% Phosphate-buffered feration [35], partially due to the reduction of free radical saline (PBS) Formalin solution (Cat. SF100–20, Fischer production in mitochondria [28, 36], activation of Nrf2 Scientific), Isopropyl alcohol (Cat. 9084-03 J.T. Baker), transcription factor [37], reduction of CDK2 kinase activity, Ethyl alcohol (Cat. E7023), Trichloromethane (Cat. as well as causing reduced p21 and p16 protein levels [38] 616778, Sigma Aldrich), Acetic acid, glacial (Cat.193829 and decreased in STAT3 phosphorylation [39]. Notably, MP Biomedicals, Inc.), Histological grade xylene (Cat. T-AG17 suppresses insulin-mediated fatty acid synthesis in 534056 Sigma Aldrich), Paraplast® Embedding Media, (Cat. WAT of rats [33], and tyrphostins B46 and A47 (which 15159-409, McCormick Scientific), Eosin–Y 7111 (Richar- bear the same pharmacophore as T-AG17) block GLUT1- d-Allan Scientific), Hematoxylin 7212; (Richard-Allan mediated intracellular glucose transport [40]. Importantly, Scientific), Tissue tek O.C.T. (Cat.4530 Sakura), Oil red Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 3 of 11 (Cat.00625-25g Sigma Aldrich), Schiff ’sreagent (Cat. Animals and housing 3952016 Sigma Aldrich), 99% Periodic Acid (Cat. P7875, All the experiments were performed using 2 month-old Sigma Aldrich). male C57BL/6 mice. Animals were handled according to the NIH guide for the care and use of laboratory animals T-AG17stock solution (NIH Publications No. 80–23, revised in 1996), and The T-AG17 was provided by Ayon Industries (Monterrey, animal protocols were approved by the Local Animal México). T-AG17 was synthesized by reacting (under re- Care Committee. All the animals were housed individu- flux) 4-hydroxy-3,5-di-tert-butylbenzaldehyde (99.1% pure, ally in Plexiglas cages and maintained at 20–23 °C in a purchased from Yongyi Chemicals Group Co., Ltd., temperature-controlled room with a 12-h light/dark Changzhou, Jiangsu, China) with malononitrile in an- cycle. Water and food was available ad libitum in the hydrous ethanol solvent, using ammonium acetate as cata- home cage. lyst. This procedure gives a 95% yield of a light-yellow microcrystalline solid that melts at 141–142 °C, presents Mice long-term feeding and treatments UV absorption maxima at 247 and 365 nm and has an Rf Animals were housed a week before the experiment as value of 0.65 (using benzene as eluent and Merck’sTLC described above. Mice were exposed to either a high-fat silica gel 60 F plastic-backed sheets), with only one spot diet (HFD, 45% kcal from fat; Research Diets, D12451) being observed. or a basic Chow diet (CHOW, 10% kcal from fat; The mixed melting point of this compound and Research Diets, D12450B) for 13 weeks, as described in authentic T-AG17 (acquired from Cayman Chemical) our previous studies [41, 42]. was 141–142 °C, and its spectroscopic and chromato- T-AG17 or vehicle control were administered orally graphic properties were identical to those of the authen- (via gavage) or injected intraperitoneally (i.p.), and body tic product. This compound is stable, with a shelf life of weight, food intake and water consumption were re- over 2 years at 25–35 °C. If necessary it may be recrys- corded every week. Doses of 1.75, 5.5, 17.5, 28, 40 and tallized from ethanol. 55 mg/kg were administered once to determine the Stock solutions were prepared in Dimethyl sulfoxide LD , and doses of 0.175, 0.0175 and 0.00175 mg/kg (DMSO; Sigma-Aldrich, D2650). DMSO was used as the were administered daily for 15 days to determine the vehicle control. therapeutic effects. 3T3-L1 cell line maintenance and treatments Tissue sample collection and histological analysis The 3T3-L1 preadipocyte cell line was expanded in Corn- Mice were sacrificed by cervical dislocation and blood ing® T75 cm flasks with Dulbecco’s modified Eagle’s samples were collected using syringe cardiac punch medium (DMEM, high glucose 4.5 g/l; (Caisson Labs), (22G diameter). Serum was isolated as described below. supplemented with 10% (vol/vol) newborn calf serum, Brain, liver, adipose tissue, pancreas, spleen, gonadal 50 units/ml penicillin, and 50 μg/ml streptomycin in 5% tissue and skeletal muscle were collected and fixed as CO incubator at 37 °C. After confluence, cells were in- described below and stained for: hematoxylin/Eosin (H/E; duced to adipocyte differentiation for 7 days by using Richard-Allan Scientific), oil red, periodic acid–Schiff DMEM supplemented with 10% vol/vol fetal bovine (PAS) and active pro-caspase 3 immunofluorescence. serum, 1 μM dexamethasone, 0.5 mM isobutylmethyl- xanthine, 100 nM insulin, and 50 units/ml penicillin and Serum biochemistry 50 μg/ml streptomycin. The T-AG17 (1 μM) or equivalent Blood samples were collected in a Microtainer and cen- concentration of DMSO (vehicle control) was added be- trifuged at 5000 rpm × 10 min. We determined serum fore (day 0) or after (day 7) adipogenic induction. biochemical composition including glucose levels by glucose strips and insulin, creatinine, aspartate amino- Quantification of lipid accumulation in cells transferase, leptin and free fatty acids were determined Effect of T-AG17 on the accumulation of cellular lipid by Elisa kits according to manufacturers’ instructions: droplets was conducted by comparing T-AG17-treated creatinine, aspartate aminotransferase, free fatty acids, cells to vehicle control-treated cells after 7 days by using insulin and leptin. the oil red solution to stain the cells following manufac- turer’s instructions. Digital images of the cells were taken with a PrimoVert microscope and the AxioCam Hematoxylin and Eosin staining (H&E) ERc5s camera (Zeiss). The stain was extracted from the Samples were fixed in 10% formaldehyde in PBS during cells using 60% isopropyl alcohol for 1 h (10 ml/flask), 24 h, following by automating processing in an auto- and the extract (1 ml) was measured at 510 nm in the mated (Excelsior ES system®, Thermo Scientific. Inc.). iMark Microplate Absorbance Reader (Bio-Rad). Samples were included in paraffin and 4-μm slices were Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 4 of 11 obtained using a microtome (Microm HM355S. Thermo drops were searched in slides stained with red oil and Scientific. Inc.). Finally, samples were stained with H&E. presence of cytoplasmic inclusions of glycogen in slides stained with PAS. Oil red staining for lipid accumulation in tissue samples In brief, samples were fixed in 10% formaldehyde in PBS Confocal microscopy during 24 h, included in “tissue tek” (Tissue-Tek® OCT Active pro-caspase 3-Cy3 stainning in liver and adipose Compound, TEC Pella, Inc) and 9-μm sections were ob- tissue sections were analyzed by confocal microscopy tained using a cryostat (Microm HM 550®, Thermo (Axio imager Z1®. Zeiss, Inc.), using an EC Plan-Neofluar Scientific.Inc). Slides were stained in oil red and counter- (Plan-Neofluar) 40×/1.30 Oil DIC M27; and 488 nm laser. stained with Harris hematoxylin. We scanned for immunoreactive cells using 491/551 nm (Exc/Emi) (LSM 710 scanner, Zeiss, Inc) and ZEN soft- PAS (Periodic Acid Schiff) staining ware (Zeiss 2009) for acquisition of 8-bit images collected Samples were fixed in Carnoy’s solution by 2 h, dehy- 2 over a 45,000 μm surface area. drated with isopropyl alcohol overnight, pre-included in paraffin and cut 4-μm sections were obtained. Statistical analysis For PAS staining samples were reduced in 0.5% peri- The data presented here was analyzed using the Student odic acid solution, placed in Schiff reagent and counter- t-test or analysis of variance (ANOVA) with post-hoc stained in Harris hematoxylin. tests using the program StatView Version 4.5 (Abacus Staining protocols were implemented systematically Concepts, Berkeley, California, United States). For im- using the automated staining equipment, Varistain Gemini munofluorescent semiquantitative analysis, we used the ES® (Thermo Scientific Inc.). ANOVA test followed by Kruskal-Wallis one-way test using Number crunched statistical software (NCSS, LLC, Immunofluorescence staining Utah, United States). The data are presented as mean ± Paraffin slides previously fixed in Carnoy’s solution by SEM, unless otherwise stated; p < 0.05 was considered 2 h, were processed for antigen retrieval system using significant. The significance levels displayed on figures 10 mM sodium citrate buffer, pH 6.0 using an auto- are as follows: * indicates p < 0.05, ** p < 0.001. mated computing assisted Lab Vision™ PT Module (Thermo Fisher Scientific) and blocking of unspecific an- tigens was performed using 5% normal goat serum in Results 0.2% Triton X-100 (Sigma-Aldrich, X100) in PBS 1 mM, T-AG17 blocks adipocyte differentiation in 3T3-L1 cell line pH 7.4, for 1 h. Sections were incubated with active We tested whether T-AG17 prevents the adipocyte dif- pro-caspase 3 primary antibody (1:2500) for 4 h at room ferentiation of the 3T3-L1 cell line. After cells were temperature and incubated with Alexa Fluor® 488 Conju- confluent, adipocyte differentiation was induced for gate and anti-rabbit secondary antibody (1:500) for 1 h 7 days in the presence of T-AG17 or vehicle control at room temperature in the darkness. Entire protocol was (DMSO). Adipocyte differentiation was detected by cel- run into automated immunochemistry computing assisted lular uptake of the oil red stain, which was strikingly in- equipment, Lab Vision™ Autostainer 360® (Thermo duced by treatment with an adipogenic cocktail (insulin Fisher Scientific). Sections were mounted in cover slip stimulation) (Fig. 1a). In the presence of the adipogenic using synthetic mounting medium with DAPI (Vector cocktail with T-AG17 (1 μM), there was a visible reduc- Laboratories, H-1500). tion in the number of positively stained cells and degree of staining per cell (Fig. 1a). Light microscopy In order to quantify the amount of oil red that the Histological slides were observed in a bright field cells absorbed, we extracted the stain and read the microscope (AxioImager Z1®, Zeiss, Inc.) using a 40× absorbance of the extract by a microplate absorbance objective. Five fields from each slide, using axio vision reader (Fig. 1b). The adipogenic cocktain caused a sig- software (ver. 4.8.2) were acquired. Morphological ana- nificant increase in extract absorbance as compared to lysis of slides from visceral fat tissue were performed by the DMEM + 10% FBS control treatment group. No dif- stained with Hematoxylin and eosin stain (H&E), in- ferentiation was observed with the DMEM + 10% FBS, cluded form of adipocytes. Slides stained with PAS were DMEM + T-AG17 and DMEM + 0.5% DMSO control used to search cytoplasmic inclusions reacting with treatment groups as expected. T-AG17 treatment on day 0 PAS. Morphological analysis of slides from liver stained (when the adipogenic cocktail was first added) caused a with H&E included structure of hepatic lobules, the as- significant decrease in absorbance to levels similar to the pect of cytoplasm from hepatocytes, also the form and DMEM + 10% FBS control levels. We found a modest de- aspect of nucleus and it’s chromatin. Presence of oil crease with the vehicle control treatment (adipogenic Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 5 of 11 Fig. 1 T-AG17 inhibits adipocyte differentiation. a 3T3-L1 preadipocyte cells were plated and stimulated with adipogenic cocktail (insulin stimulation). Preadipocytes were incubated with 1μM T-AG17 during 7 days. 3T3-L1 preadypocite lipid droplet staining showed lipid accumulation in cells treated with the adipogenic cocktail. Lipid accumulation was not detected in cells treated with 1μMT-AG17. b Adipocyte differentiation was evaluated by quantifying absorbance at 490 nm. Graphs show mean ± SEM for triplicate experiments and statistical significance after using unpaired Student’s t test. *p <0.05. n = 3. In some experiments, T-AG17 was added after preadipocyte differentiation (1 and 2 μM) cocktail + 0.05% DMSO). This finding suggested that When we looked at behavioral alterations, we observed T-AG17 prevents adipocyte differentiation. that the animals administered with 1.75, 5.5 and 17.5 mg/kg exhibited inhibition in locomotion and Acute doses of T-AG17 and determination of LD piloerection, which lasted up to 30 min after administra- We determined the LD and therapeutic dose for T-AG17 tion. With 28 mg/kg, mice also exhibited increased heart using C57BL/6 mice. We found that oral T-AG17 adminis- rate 5 min after administration and a red appearance of tration caused increased mortality at 28, 40 and 55.5 mg/kg the footpads. One mouse developed prostration and lung doses, with no effect observed for 1.75, 5.5 and 17.5 mg/kg spasms after receiving 28 mg/kg. This mouse was doses. The 55 mg/kg dose killed all animals within 15 min sacrificed by cervical dislocation. Thirty minutes after due to cardiac failure. Administration of 28 mg/kg or T-AG17 administration, mice recovered mobility. At 40 mg/kg doses caused all animals to die after seven and 40 mg/kg, mice showed the same physiological parame- 2 days, respectively. We found that 40 mg/kg administra- ters as the 28 mg/kg dose, and the mice also exhibited tion induced a 60% mortality rate, suggesting that the LD hyperventilation Three out of five mice were sacrificed by value for this compound in mice is 33.3 mg/kg. Animals cervical dislocation due to irreversible negative physio- that survived after 2 weeks post T-AG17 administration logical effects. Two of the mice recovered after 30 min. (5.5, 17.5, 28.5, 40 or 55 mg/kg) did not show altered serum Finally, the 55 mg/kg T-AG17 dose showed exacerbation levels of glucose, insulin and leptin (Fig. 2). of the behavioral and physiological parameters exhibited Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 6 of 11 Fig. 2 Acute T-AG17 administration does not alter body weight, glucose, insulin and leptin serum levels. a Body weight was analyzed every week after 17.5, 28.5, 40 and 55.5 mg/kg oral T-AG17. Changes in body weight are expressed in grams. b-d Serum biochemistry was determined using ELISA kits (insulin and leptin) as described in Methods and glucose levels measurement was determined by Glucose strips. Graphs show the normalized results of mean ± SEM for n =6 at the 40 mg/kg dose. The mice of this group were sacri- cytoplasms and large perinuclear vacuoles. Some areas ficed between 5 and 10 min after administration. had low affinity for staining and others areas had disor- We identify changes in cell morphology of brain, kid- ganized hepatocytes, edema, and the appearance of fat ney, liver, pancreas, adipose tissue and muscle after 17.5, accumulation as evidenced by oil red staining. 28 and 55.5 mg/kg T-AG17 oral administration. The brain showed hyperchromic cytoplasm, perinuclear eo- Chronic doses of T-AG17 administration sinophilic inclusions and dispersed chromatin. The In order to test the potential of T-AG17 oral administration kidneys of these mice showed pale aspects of the glom- to regulate body weight, we selected the lowest dose erular units and proximal tubules. The liver displayed administered in our previous experiment (Fig. 2,oraldose hepatic acinar cells with perinuclear cytoplasmic vacu- AG17 = 1.75 mg/kg). Mice were orally dosed with 0.175, oles and and the acinar unit randomly distributed. The 0.0175 or 0.00175 mg/kg T-AG17 during 2 weeks reaching pancreas displayed exocrine acinar cells with pale ap- 2.45, 0.245 and 0.0245 mg/kg final concentrations after pearance and normal Langerhans islets. White adipose treatment (Fig. 3). We determined plasma biochemistry and tissue exhibited adipocytes with normal aspect and an tested for renal and liver damage using selective markers. apparent decrease in adipocyte cell number intercalated We found that oral 0.175, 0.0175 or 0.00175 mg/kg T-AG17 with major adipocytes evidenced by the red oil stained. doses did not alter plasma biochemistry markers including The skeletal muscle of these mice seemed morphologic- glucose, leptin, insulin and free fatty acids and nor the ally unaltered. creatinine and aspartate aminotransferase (two markers of Mice orally administered 41 mg/kg T-AG17 showed renal and liver damage, respectively) (Fig. 3). neurons displaying eosinophilic and pale cytoplasm and pycnotic nucleus coexisting with normal neurons. Kid- T-AG17 promotes apoptotic cell death of white ney cells seem retracted with pale glomeruli and tubular adipocytes in obese mice cells with poor staining, pycnotic nuclei, and condensed Based on the observation that T-AG17 stimulation chromatin. Hepatocytes from the liver displayed pale promotes apoptotic cell death in 3T3-L1 cell line, we Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 7 of 11 Fig. 3 Chronic T-AG17 does not alter body weight, glucose, insulin, and leptin levels and creatinine and aspartate aminotransferase activity in serum. a Body weight was analyzed every week after 2 weeks of 0.0175, 0.175 and 1.75 mg/kg oral T-AG17 administration. Changes in body weight are expressed in grams. b-e Blood glucose levels were determined using glucose strips and serum biochemistry was determined using ELISA kits as described in Methods. f-g Creatinine and aspartate aminotransferase activity were determined using ELISA kits. Graphs show the normalized results of mean ± SEM for n =6 sought to determine if administration of T-AG17 in size (Fig. 5). Notably, obese mice administered the mice modulates thermogenesis or white adipose tissue T-AG17 showed an apparent decrease in adipocyte fat apoptosis, leading to body weight decrease. Initially, droplet size when compared to Chow and HFD groups we fed mice with Chow or HFD for 18 weeks and (Fig. 5). Also, T-AG17 administration of mice expose to tested the potential of T-AG17 versus dieting. In this Chow diet did not show morphological changes when experiment, we injected T-AG17 intraperitoneally compare to Chow and HFD groups. In addition, we did (grey bar Fig. 4c) or exposing the HFD mice to a nor- not identify positive staining using the PAS protocol for mal Chow diet (red bar Fig. 4c)for 2weeks. As visceral fat tissue in all groups (Fig. 5). T-AG17 induced expected, we found that 18 weeks of HFD intake a significant increase in active pro-caspase 3-Cy3 stain- increased the body weight reaching 45–50 g when ing in obese mice when compared to controls fed with compared to Chow diet values (25–28 g) (Fig. 4a, b). Chow diet (Fig. 5). This effect was replicated in obese No significant changes in food intake were found mice put back to normal Chow diet for 2 weeks (Fig. 5); (Chow = 23.91 ±2.07 g, HFD = 26.83 ±9.72). HFD in- however, positive pro-caspase 3-Cy3 staining of cells was take increased the body weight (black bar vs Chow not significant when compared to obese mice adminis- diet, Fig. 4c); however, we did not find evidence of tered T-AG17 (Fig. 5). weight loss after T-AG17 administration (compare black bar HFD groups vs grey bar Fig. 4c). The latter T-AG17 promotes changes in hepatocytes of obese mice observation correlates which we found when mice We then assessed any morphological changes in the were put back to the Chow diet during 2 weeks livers after T-AG17 or dieting exposure. The livers of (compare black bar HFD groups vs red bar Fig. 4c). mice exposed to HFD showed abnormal retention of Next, we assessed any morphological changes in lipids displaying macrovesicular steatosis (Fig. 6a). Oral WATafter T-AG17 or dieting exposure. WAT of mice T-AG17 administration led to a transition from macro- fed the Chow diet showed typical polyhedral cells with vesicular to microvesicular steatosis, which correlated nuclear location near the plasma membrane; whereas, with an increase in red oil positive inclusions (lipids) cells of mice exposed to HFD reached a higher apparent (Fig. 6a, b). Obese mice put back on Chow diet for Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 8 of 11 Fig. 4 T-AG17 administration and body weight in animals exposed to HFD. a Mice were exposed to HFD (60% kcal from fat) or chow diet during 18 weeks. b Body weight was determined every week. Changes in body weight are expressed in gr. c Body weight change after 2 weeks of oral T-AG17 administration. Graphs show the normalized results of mean ± SEM for n =9–12 and statistical significance after using unpaired Student’s t test. *p < 0.05 2 weeks showed a similar hepatocyte morphology as the Chow diet group and also showed a switch from macrovesicular to microvesicular steatosis. Notably, oral T-AG17 administration promoted a significant increase in caspase-3 activation in adipose tissue but not in liver when compared to HFD or Chow diet (Fig. 6b, c). Finally, obese and normal mice orally administered T-AG17 showed liver positive PAS staining (Fig. 6a), reflecting an increase of cytoplasmic glycogen inclusions. Discussion We have shown that orally administered T-AG17 modu- lates adipocyte differentiation in vitro and induces adi- pocyte apoptosis in vivo. Oral administration of T-AG17 (0.175 mg/kg daily over 2 weeks) decreased fat cell vol- ume in WAT. Incubation with T-AG17 prevents differentiation of 3T3-L1 cells to mature adipocytes, as shown by a decrease in fat droplet formation by oil red staining (Fig. 1a, b). Fat droplet formation is one of the final stages of adipocyte differentiation [43]. This finding is consistent with the previous observations that T-AG17 suppresses insulin-mediated fatty acid synthesis in rat Fig. 5 T-AG17 promotes apoptotic cell death in adipose tissue. Mice white adipocytes [33] and also that Tyrphostin AG490 were exposed to HFD (60% kcal from fat) or Chow diet during (which bears the same pharmacophore as T-AG17) in- 18 weeks and T-AG17(0.175 mg/kg) was orally administered during 2 weeks. Pro caspase 3 activation was evaluated using hibits adipogenesis by disrupting STAT3 signaling in immunohistochemistry. PAS staining was performed to evaluate human adipocytes through inhibition of JAK2 [44]. This glycogen synthesis finding suggests that T-AG17 might be a potential Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 9 of 11 Defects in adipocyte tissue expandability and hyper- trophy lead to ectopic fat accumulation in metabolically relevant organs, including liver [7]. In a fatty liver, trigly- ceride (TG) accumulation, either as small or large lipid deposits, is called micro- or macrovesicular steatosis, respectively [45–47]. Microvesicular steatosis, gradually followed by macrovesicular steatosis, is experimentally induced by 6–10 weeks of HFD feeding [22, 48, 49], which correlates with an elevation in liver enzymes (AST and ALT) [45, 50]. Our results agree with these findings, with mice exhibiting macrovesicular steatosis after 12 weeks of HFD feeding. To the best of our know- ledge, the T-AG17- induced switch from macrovesicular to microvesicular liver steatosis in the HFD group (Fig. 6) is unprecedented. Also, while T-AG17 increases lipid in- clusions in hepatocytes as shown by oil red staining (Fig. 6), we did not find any changes in serum AST levels, suggesting no histological lesions in liver. We hypothesize that this is a transient condition whereby fatty acids are being temporarily exported to the liver from WAT. In any case, our observations parallel those of Dianzani and Scuro [51], who reported increases in hepatic fat droplets in albino rats 48–96 h after injec- tions with DNP, following by glycogen infiltration after Fig. 6 T-AG17 shifts macrovesicular to microvesicular steatosis in liver. a Mice were exposed to HFD or Chow as described and T- 120 h and decreased in neutral fat droplets. T-AG17 ad- AG17(0.175 mg/kg) was orally administered during two weeks. H/E, ministration for 2 weeks results in a microvesicular stea- oil red staining, pro caspase 3 activation and PAS staining were tosis phenotype with normal levels of liver enzymes. performed in adipose tissue (b) or liver (c) to evaluate morphology, Livers from human donors with moderate and severe fat accumulation, apoptosis activation and glycogen synthesis, macrovesicular steatosis are considered unfit for trans- respectively. For immunohistochemistry analysis, we used ANOVA test followed by Kruskal-Wallis test one-way. The data is presented plantation [53]. Therefore, our findings support a role of as mean±SEM unless stated.. *p < 0.05 T-AG17 as a MMU capable of inducing the transition from macrovesicular to microvesicular steatosis in liver and potentially improving the metabolic body profile. effective adipogenesis-blocking agent; therefore, we Finally, our findings show that obese mice treated with tested the tolerance and therapeutic effects in a murine T-AG17 accumulate glycogen in the liver. Previously, in- model of obesity. jection of DNP in albino rats has been shown to also In diet-induced obese C57BL/6 mice, T-AG17 does cause hepatic glycogen infiltration [51], and treatment of not induce weight loss, as we had originally hypothe- obese rats with a controlled-release formulation of DNP sized. Interestingly, the DNP uncoupler does induce led to an 80% increase in liver glycogen content [18], weight loss at thermoneutrality (30 °C) [10], which which is associated with reversal of hypoglycemia. Our differs from our study. However, T-AG17 did promote data are consistent with evidence showing that glycogen an apparent decrease in the size of adipocytes and a accumulates in the liver of rats following prolonged ad- significant activation of caspase 3-dependent apoptosis ministration of DNP, in contrast to other parenchymal or- (Figs. 5, 6). The decrease in adipocyte size might be me- gans [18], and also with the finding that MMU-triggered diated by MMU-induced lipolysis and down regulation by FCCP induces glucose uptake in adipocytes [6]. of lipid synthesis [6]. Tyrphostins, in particular, have been characterized as classical inductors of apoptosis by Conclusions inhibiting protein tyrosine kinase activity [30], suggest- T-AG17 blocks adipocyte differentiation in vivo and in ing that T-AG17 might be a potential inductor of apop- vitro, promotes efficient apoptotic cell death of adipocytes totic cell death in adipocytes. Indeed, T-AG17 has been in vivo, and a switch from macrovesicular to microvesicular shown to inhibit cell growth and induce apoptosis in steatosis during positive energy balance in a diet-induced other cell types [31, 38, 39]. These evidence suggest that obesity mice model. Thus, our data support the develop- T-AG17 activates the apoptotic pathway in adipocytes, ment of T-AG17 as a candidate to pharmacologic preven- although no overall weight loss was observed. tion/treatment of obesity and fatty liver disease. Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 10 of 11 Funding 8. Virtue S, Vidal-Puig A. Adipose tissue expandability, lipotoxicity and the This work was funded by the National Council of Science and Technology in metabolic syndrome-an allostatic perspective. Biochim Biophys Acta-Mol Mexico (CONACYT) (Grant number: 255317) and the Dirección de Cell Biol Lipids. 2010;1801:338–49. Investigación y Desarrollo, Ayon Industries, Monterrey, México. 9. De Pauw A, Tejerina S, Raes M, Keijer J, Arnould T. Mitochondrial (Dys) function in adipocyte (De) differentiation and systemic metabolic Availability of data and materials alterations. Am J Pathol [Internet]. 2009;175:927–39. All data generated or analyzed during this study are included in this 10. Goldgof M, Xiao C, Chanturiya T, Jou W, Gavrilova O, Reitman ML. The published article. chemical uncoupler 2,4-dinitrophenol (DNP) protects against diet-induced obesity and improves energy homeostasis in mice at thermoneutrality. J Authors’ contributions Biol Chem. 2014;289:19341–50. AC and RO contributed to the study design, data interpretation and 11. Schrauwen P, van Marken Lichtenbelt WD. Combatting type 2 diabetes by manuscript writing; JC, AS, JJ, JG, CA and GC performed experiments and turning up the heat. Diabetologia. 2016;59:2269–79. collected data. All authors read and approved the final manuscript. 12. Liu D, Zhang Y, Gharavi R, Park HR, Lee J, Siddiqui S, et al. The mitochondrial uncoupler DNP triggers brain cell mTOR signaling network reprogramming Ethics approval and CREB pathway up-regulation. J Neurochem. 2015;134:677–92. All the experiments were performed using wild-type C57BL/6 male mouse 13. Cerqueira FM, Laurindo FRM, Kowaltowski AJ. Mild mitochondrial 2 months old. Animals were handled according to the NIH guide for the care uncoupling and calorie restriction increase fasting eNOS, Akt and and use of laboratory animals (NIH Publications No. 80–23, revised in 1996), mitochondrial biogenesis. PLoS One. 2011;6:e18433. based on the Basel Declaration to implement the ethical principles of Re- 14. Wu B, Jiang M, Peng Q, Li G, Hou Z, Milne GL, et al. 2,4 DNP improves placement, Reduction and Refinement of experimental animal models and motor function, preserves medium spiny neuronal identity, and reduces with approval of the local Animal Care Committee. All efforts were made to oxidative stress in a mouse model of Huntington’s disease. 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Abstract

Background: Excessive subcutaneous adiposity in obesity is associated to positive white adipocyte tissue (WAT) differentiation (adipogenesis) and WAT expandability. Here, we hypothesized that supplementation with the insulin inhibitor and mitochondrial uncoupler, Tyrphostin (T-AG17), in vitro and in vivo inhibits adipogenesis and adipocyte hypertrophy. Methods: We used a 3T3-L1 proadipocyte cell line to identify the potential effect of T-AG17 on adipocyte differentiation and fat accumulation in vitro. We evaluated the safety of T-AG17 and its effects on physiological and molecular metabolic parameters including hormonal profile, glucose levels, adipogenesis and adipocyte hypertrophy in a diet-induced obesity model using C57BL/6 mice. Results: We found that T-AG17 is effective in preventing adipogenesis and lipid synthesis in the 3T3-L1 cell line, as evidenced by a significant decrease in oil red staining (p < 0.05). In obese C57BL/6 mice, oral administration of T-AG17 (0.175 mg/kg for 2 weeks) lead to decreased fat accumulation and WAT hypertrophy. Further, T-AG17 induced adipocyte apoptosis by activating caspase-3. In the hepatocytes of obese mice, T-AG17 promoted an increase in the size of lipid inclusions, which was accompanied by glycogen accumulation. T-AG17 did not alter serum biochemistry, including glucose, insulin, leptin, free fatty acids, creatinine, and aspartate aminotransferase. Conclusion: T-AG17 promotes adipocyte apoptosis in vivo and is an effective modulator of adipocyte differentiation and WAT hypertrophy in vitro and in vivo. Therefore, T-AG17 may be useful as a pharmacological obesity treatment. Keywords: Tyrphostin, AG17, Adipogenesis, Obesity, Hepatic steatosis, Oxidative phosphorylation, Thermogenesis, Mitochondrial uncoupling, Adipocyte differentiation Background pathways that may contain potential drug targets are the Excessive subcutaneous adiposity and its accumulation white adipose tissue (WAT) differentiation pathway, also into visceral depot during obesity are major risk factors referred to as adipogenesis, and the WAT expandability for developing type 2 diabetes (T2DM) and several other pathway. chronic metabolic disorders [1, 2]. Therefore, identifying During early development, mesenchymal stem cells therapeutic targets to treat the metabolic failures associated differentiate into chondrocytes, osteoblasts, myoblasts with obesity could reduce or prevent the development of and adipocytes [3]. Adipocyte differentiation from mes- these incurable metabolic disorders. Two cellular signaling enchymal stem cells is modulated by signaling cascades involving bone morphogenetic protein-4 and peroxi- * Correspondence: acm590@hotmail.com; alberto.camachomr@uanl.edu.mx; some proliferator-activated receptor (PPAR) β/δ,which rortizlopez@gmail.com 1 support the gene expression of PPARγ [4]. In adults, Departamento de Bioquímica y Medicina Molecular, Facultad de Medicina, Universidad Autónoma de Nuevo León (UANL), Monterrey, Mexico ectopic accumulation of adipocytes might be caused by Unidad de Genómica, UANL, CIDICS, Monterrey, Mexico dysfunction in differentiation pathways, which creates Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 2 of 11 an inability to induce differentiation of adipocyte pre- T-AG17 is also a potent inhibitor of mitochondrial oxphos cursor cells [3, 5] or induce adipocyte de-differentiation and is capable of increasing energy expenditure. How- [6]. On the other hand, once obesity is reached, the ever, the effects of T-AG17 on adipocyte differentiation, number of new adipocytes decreases, and adipocytes adipose tissue hypertrophy and body organ toxicity become hypertrophic, reaching their expandability limit have not been evaluated. until fat accumulates around ectopic organs, resulting Considering the significant suppressive effects of T-AG17 in metabolic complications [7, 8]. on insulin signaling, we hypothesized that T-AG17 might Drugs that act on mitochondria have been used to promote inhibition of adipogenesis and/or adipocyte hyper- combat fat accumulation by forcing cells to use stored trophy. Specifically, we seek to determine if T-AG17: 1) energy. Mitochondrial oxphos uncouplers create a futile decreases lipid accumulation in the 3T3-L1 adipocyte cell cycle of glyceride and fatty acid oxidation without gener- line induced by insulin and 2) promotes adipocyte apop- ating adenosine triphosphate (ATP). 2,4-dinitrophenol tosis in a diet-induced obesity mouse model. (DNP) is one of the best known uncoupler of oxphos widely used as a weight loss agent between 1933 and Methods 1938; however, DNP has been banned due to its high Reagents and antibodies acute toxicity [9]. Further studies on DNP and other re- 3T3-L1 preadipocyte cell line (Cat. CL-173) and newborn lated prodrugs consistently show that the toxic effects calf serum (Cat. 30-2030) were purchased from ATCC, are dose-dependent. Mild mitochondrial uncoupling Inc. Dulbecco’s Modified Eagle’s Medium-high glucose (MMU) with low doses, which seem effective at thermo- (Caisson Labs, Cat.), Penicillin-Streptomycin (Cat. neutrality (30 °C) conditions, may provide a promising P4333), Fetal Bovine Serum (Gibco), Oil Red O (Cat. strategy to reduce body weight with general tolerability O0625), Dimethyl sulfoxide (Cat. D2650), isopropyl [10–18]. Side effects of DNP exposure might be pre- alcohol (Cat. W292907), Formalin solution neutral vented, and MMU might also promote longevity through buffered 10% (Cat. HT501128), Ethyl alcohol (Cat. decreased ROS levels, mitochondrial biogenesis, down- E7023), Harris hematoxylin (Cat. HHS16), Sodium regulation of the mTOR and insulin signaling pathways, citrate (Cat. 1613859), Triton X-100 (Cat. X100), Corn- and upregulation of autophagy [12, 13]. Of interest, ing® cell culture flasks surface area 75 cm2, canted neck, these effects are similar to those seen in caloric restric- cap (vented) (CLS430641) were from SIGMA-ALDRICH. tion [12, 15, 19] and are consistent with the “uncoupling Paraplast® Embedding Media, (Cat. 15159-409, McCormick to survive” hypothesis [20]. These potentially beneficial Scientific), Rabbit polyclonal to Active + pro Caspase 3 effects of MMU may also be induced by niclosamide (Cat. AB13847, ABCAM), Goat polyclonal secondary anti- ethanolamine [21], salsalate [22], TTFB [23], CZ5 [24], body to Rabbit IgG H&L (alexa Fluor® 488) preadsorbed FCCP [6, 17, 25–27], the niclosamide-ethanolamine (Cat. AB150081, ABCAM), and VECTASHIELD Hardset aduct [21] and certain tyrphostins [28]. antifade mounting medium with DAPI (cat no. H-1500 Tyrphostins belong to the benzylidenemalononitrile Vector laboratories). family, which possess a benzene ring pharmacophore, an Adipogenesis Assay Kit for 3T3-L1 preadypocyte differ- exocyclic carbon-carbon double bond, and a cyano group entiation was purchased from Abcam (Cat. Ab133102). (CN) located at the same side of the molecule (cis) as the Also, the primary antibody cleaved Caspase-3 (Asp175) aromatic ring [29, 30]. The tyrphostin T-AG17 is a (Cat. 9661. Cell signaling) and secondary antibody highly selective, reversible inhibitor of epidermal Anti-rabbit IgG (H + L), Alexa Fluor® 488 Conjugate (Cat. growth factor receptor-induced phosphorylation of 4412. Cell signaling) were used. ELISA kits: creatinine tyrosine residues of intracellular proteins [31], and (SIGMA. MAK080), aspartate aminotransferase (SIGMA. its cellular effect is dose-dependent growth inhibition. MAK055), free fatty acids (Roche, 11383175001), insulin T-AG17 has potential therapeutic value for treating (Millipore, Cat. EZRMI-13 k) and leptin (Millipore, Cat. neurodegenerative disorders [28], atherosclerosis [32], dys- EZML-82 K). Acucheck (Cat. 05987270) and glucose lipidemia [33], restenosis [34], and cancer/cell hyperproli- strips (6454011023, Roche). 10% Phosphate-buffered feration [35], partially due to the reduction of free radical saline (PBS) Formalin solution (Cat. SF100–20, Fischer production in mitochondria [28, 36], activation of Nrf2 Scientific), Isopropyl alcohol (Cat. 9084-03 J.T. Baker), transcription factor [37], reduction of CDK2 kinase activity, Ethyl alcohol (Cat. E7023), Trichloromethane (Cat. as well as causing reduced p21 and p16 protein levels [38] 616778, Sigma Aldrich), Acetic acid, glacial (Cat.193829 and decreased in STAT3 phosphorylation [39]. Notably, MP Biomedicals, Inc.), Histological grade xylene (Cat. T-AG17 suppresses insulin-mediated fatty acid synthesis in 534056 Sigma Aldrich), Paraplast® Embedding Media, (Cat. WAT of rats [33], and tyrphostins B46 and A47 (which 15159-409, McCormick Scientific), Eosin–Y 7111 (Richar- bear the same pharmacophore as T-AG17) block GLUT1- d-Allan Scientific), Hematoxylin 7212; (Richard-Allan mediated intracellular glucose transport [40]. Importantly, Scientific), Tissue tek O.C.T. (Cat.4530 Sakura), Oil red Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 3 of 11 (Cat.00625-25g Sigma Aldrich), Schiff ’sreagent (Cat. Animals and housing 3952016 Sigma Aldrich), 99% Periodic Acid (Cat. P7875, All the experiments were performed using 2 month-old Sigma Aldrich). male C57BL/6 mice. Animals were handled according to the NIH guide for the care and use of laboratory animals T-AG17stock solution (NIH Publications No. 80–23, revised in 1996), and The T-AG17 was provided by Ayon Industries (Monterrey, animal protocols were approved by the Local Animal México). T-AG17 was synthesized by reacting (under re- Care Committee. All the animals were housed individu- flux) 4-hydroxy-3,5-di-tert-butylbenzaldehyde (99.1% pure, ally in Plexiglas cages and maintained at 20–23 °C in a purchased from Yongyi Chemicals Group Co., Ltd., temperature-controlled room with a 12-h light/dark Changzhou, Jiangsu, China) with malononitrile in an- cycle. Water and food was available ad libitum in the hydrous ethanol solvent, using ammonium acetate as cata- home cage. lyst. This procedure gives a 95% yield of a light-yellow microcrystalline solid that melts at 141–142 °C, presents Mice long-term feeding and treatments UV absorption maxima at 247 and 365 nm and has an Rf Animals were housed a week before the experiment as value of 0.65 (using benzene as eluent and Merck’sTLC described above. Mice were exposed to either a high-fat silica gel 60 F plastic-backed sheets), with only one spot diet (HFD, 45% kcal from fat; Research Diets, D12451) being observed. or a basic Chow diet (CHOW, 10% kcal from fat; The mixed melting point of this compound and Research Diets, D12450B) for 13 weeks, as described in authentic T-AG17 (acquired from Cayman Chemical) our previous studies [41, 42]. was 141–142 °C, and its spectroscopic and chromato- T-AG17 or vehicle control were administered orally graphic properties were identical to those of the authen- (via gavage) or injected intraperitoneally (i.p.), and body tic product. This compound is stable, with a shelf life of weight, food intake and water consumption were re- over 2 years at 25–35 °C. If necessary it may be recrys- corded every week. Doses of 1.75, 5.5, 17.5, 28, 40 and tallized from ethanol. 55 mg/kg were administered once to determine the Stock solutions were prepared in Dimethyl sulfoxide LD , and doses of 0.175, 0.0175 and 0.00175 mg/kg (DMSO; Sigma-Aldrich, D2650). DMSO was used as the were administered daily for 15 days to determine the vehicle control. therapeutic effects. 3T3-L1 cell line maintenance and treatments Tissue sample collection and histological analysis The 3T3-L1 preadipocyte cell line was expanded in Corn- Mice were sacrificed by cervical dislocation and blood ing® T75 cm flasks with Dulbecco’s modified Eagle’s samples were collected using syringe cardiac punch medium (DMEM, high glucose 4.5 g/l; (Caisson Labs), (22G diameter). Serum was isolated as described below. supplemented with 10% (vol/vol) newborn calf serum, Brain, liver, adipose tissue, pancreas, spleen, gonadal 50 units/ml penicillin, and 50 μg/ml streptomycin in 5% tissue and skeletal muscle were collected and fixed as CO incubator at 37 °C. After confluence, cells were in- described below and stained for: hematoxylin/Eosin (H/E; duced to adipocyte differentiation for 7 days by using Richard-Allan Scientific), oil red, periodic acid–Schiff DMEM supplemented with 10% vol/vol fetal bovine (PAS) and active pro-caspase 3 immunofluorescence. serum, 1 μM dexamethasone, 0.5 mM isobutylmethyl- xanthine, 100 nM insulin, and 50 units/ml penicillin and Serum biochemistry 50 μg/ml streptomycin. The T-AG17 (1 μM) or equivalent Blood samples were collected in a Microtainer and cen- concentration of DMSO (vehicle control) was added be- trifuged at 5000 rpm × 10 min. We determined serum fore (day 0) or after (day 7) adipogenic induction. biochemical composition including glucose levels by glucose strips and insulin, creatinine, aspartate amino- Quantification of lipid accumulation in cells transferase, leptin and free fatty acids were determined Effect of T-AG17 on the accumulation of cellular lipid by Elisa kits according to manufacturers’ instructions: droplets was conducted by comparing T-AG17-treated creatinine, aspartate aminotransferase, free fatty acids, cells to vehicle control-treated cells after 7 days by using insulin and leptin. the oil red solution to stain the cells following manufac- turer’s instructions. Digital images of the cells were taken with a PrimoVert microscope and the AxioCam Hematoxylin and Eosin staining (H&E) ERc5s camera (Zeiss). The stain was extracted from the Samples were fixed in 10% formaldehyde in PBS during cells using 60% isopropyl alcohol for 1 h (10 ml/flask), 24 h, following by automating processing in an auto- and the extract (1 ml) was measured at 510 nm in the mated (Excelsior ES system®, Thermo Scientific. Inc.). iMark Microplate Absorbance Reader (Bio-Rad). Samples were included in paraffin and 4-μm slices were Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 4 of 11 obtained using a microtome (Microm HM355S. Thermo drops were searched in slides stained with red oil and Scientific. Inc.). Finally, samples were stained with H&E. presence of cytoplasmic inclusions of glycogen in slides stained with PAS. Oil red staining for lipid accumulation in tissue samples In brief, samples were fixed in 10% formaldehyde in PBS Confocal microscopy during 24 h, included in “tissue tek” (Tissue-Tek® OCT Active pro-caspase 3-Cy3 stainning in liver and adipose Compound, TEC Pella, Inc) and 9-μm sections were ob- tissue sections were analyzed by confocal microscopy tained using a cryostat (Microm HM 550®, Thermo (Axio imager Z1®. Zeiss, Inc.), using an EC Plan-Neofluar Scientific.Inc). Slides were stained in oil red and counter- (Plan-Neofluar) 40×/1.30 Oil DIC M27; and 488 nm laser. stained with Harris hematoxylin. We scanned for immunoreactive cells using 491/551 nm (Exc/Emi) (LSM 710 scanner, Zeiss, Inc) and ZEN soft- PAS (Periodic Acid Schiff) staining ware (Zeiss 2009) for acquisition of 8-bit images collected Samples were fixed in Carnoy’s solution by 2 h, dehy- 2 over a 45,000 μm surface area. drated with isopropyl alcohol overnight, pre-included in paraffin and cut 4-μm sections were obtained. Statistical analysis For PAS staining samples were reduced in 0.5% peri- The data presented here was analyzed using the Student odic acid solution, placed in Schiff reagent and counter- t-test or analysis of variance (ANOVA) with post-hoc stained in Harris hematoxylin. tests using the program StatView Version 4.5 (Abacus Staining protocols were implemented systematically Concepts, Berkeley, California, United States). For im- using the automated staining equipment, Varistain Gemini munofluorescent semiquantitative analysis, we used the ES® (Thermo Scientific Inc.). ANOVA test followed by Kruskal-Wallis one-way test using Number crunched statistical software (NCSS, LLC, Immunofluorescence staining Utah, United States). The data are presented as mean ± Paraffin slides previously fixed in Carnoy’s solution by SEM, unless otherwise stated; p < 0.05 was considered 2 h, were processed for antigen retrieval system using significant. The significance levels displayed on figures 10 mM sodium citrate buffer, pH 6.0 using an auto- are as follows: * indicates p < 0.05, ** p < 0.001. mated computing assisted Lab Vision™ PT Module (Thermo Fisher Scientific) and blocking of unspecific an- tigens was performed using 5% normal goat serum in Results 0.2% Triton X-100 (Sigma-Aldrich, X100) in PBS 1 mM, T-AG17 blocks adipocyte differentiation in 3T3-L1 cell line pH 7.4, for 1 h. Sections were incubated with active We tested whether T-AG17 prevents the adipocyte dif- pro-caspase 3 primary antibody (1:2500) for 4 h at room ferentiation of the 3T3-L1 cell line. After cells were temperature and incubated with Alexa Fluor® 488 Conju- confluent, adipocyte differentiation was induced for gate and anti-rabbit secondary antibody (1:500) for 1 h 7 days in the presence of T-AG17 or vehicle control at room temperature in the darkness. Entire protocol was (DMSO). Adipocyte differentiation was detected by cel- run into automated immunochemistry computing assisted lular uptake of the oil red stain, which was strikingly in- equipment, Lab Vision™ Autostainer 360® (Thermo duced by treatment with an adipogenic cocktail (insulin Fisher Scientific). Sections were mounted in cover slip stimulation) (Fig. 1a). In the presence of the adipogenic using synthetic mounting medium with DAPI (Vector cocktail with T-AG17 (1 μM), there was a visible reduc- Laboratories, H-1500). tion in the number of positively stained cells and degree of staining per cell (Fig. 1a). Light microscopy In order to quantify the amount of oil red that the Histological slides were observed in a bright field cells absorbed, we extracted the stain and read the microscope (AxioImager Z1®, Zeiss, Inc.) using a 40× absorbance of the extract by a microplate absorbance objective. Five fields from each slide, using axio vision reader (Fig. 1b). The adipogenic cocktain caused a sig- software (ver. 4.8.2) were acquired. Morphological ana- nificant increase in extract absorbance as compared to lysis of slides from visceral fat tissue were performed by the DMEM + 10% FBS control treatment group. No dif- stained with Hematoxylin and eosin stain (H&E), in- ferentiation was observed with the DMEM + 10% FBS, cluded form of adipocytes. Slides stained with PAS were DMEM + T-AG17 and DMEM + 0.5% DMSO control used to search cytoplasmic inclusions reacting with treatment groups as expected. T-AG17 treatment on day 0 PAS. Morphological analysis of slides from liver stained (when the adipogenic cocktail was first added) caused a with H&E included structure of hepatic lobules, the as- significant decrease in absorbance to levels similar to the pect of cytoplasm from hepatocytes, also the form and DMEM + 10% FBS control levels. We found a modest de- aspect of nucleus and it’s chromatin. Presence of oil crease with the vehicle control treatment (adipogenic Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 5 of 11 Fig. 1 T-AG17 inhibits adipocyte differentiation. a 3T3-L1 preadipocyte cells were plated and stimulated with adipogenic cocktail (insulin stimulation). Preadipocytes were incubated with 1μM T-AG17 during 7 days. 3T3-L1 preadypocite lipid droplet staining showed lipid accumulation in cells treated with the adipogenic cocktail. Lipid accumulation was not detected in cells treated with 1μMT-AG17. b Adipocyte differentiation was evaluated by quantifying absorbance at 490 nm. Graphs show mean ± SEM for triplicate experiments and statistical significance after using unpaired Student’s t test. *p <0.05. n = 3. In some experiments, T-AG17 was added after preadipocyte differentiation (1 and 2 μM) cocktail + 0.05% DMSO). This finding suggested that When we looked at behavioral alterations, we observed T-AG17 prevents adipocyte differentiation. that the animals administered with 1.75, 5.5 and 17.5 mg/kg exhibited inhibition in locomotion and Acute doses of T-AG17 and determination of LD piloerection, which lasted up to 30 min after administra- We determined the LD and therapeutic dose for T-AG17 tion. With 28 mg/kg, mice also exhibited increased heart using C57BL/6 mice. We found that oral T-AG17 adminis- rate 5 min after administration and a red appearance of tration caused increased mortality at 28, 40 and 55.5 mg/kg the footpads. One mouse developed prostration and lung doses, with no effect observed for 1.75, 5.5 and 17.5 mg/kg spasms after receiving 28 mg/kg. This mouse was doses. The 55 mg/kg dose killed all animals within 15 min sacrificed by cervical dislocation. Thirty minutes after due to cardiac failure. Administration of 28 mg/kg or T-AG17 administration, mice recovered mobility. At 40 mg/kg doses caused all animals to die after seven and 40 mg/kg, mice showed the same physiological parame- 2 days, respectively. We found that 40 mg/kg administra- ters as the 28 mg/kg dose, and the mice also exhibited tion induced a 60% mortality rate, suggesting that the LD hyperventilation Three out of five mice were sacrificed by value for this compound in mice is 33.3 mg/kg. Animals cervical dislocation due to irreversible negative physio- that survived after 2 weeks post T-AG17 administration logical effects. Two of the mice recovered after 30 min. (5.5, 17.5, 28.5, 40 or 55 mg/kg) did not show altered serum Finally, the 55 mg/kg T-AG17 dose showed exacerbation levels of glucose, insulin and leptin (Fig. 2). of the behavioral and physiological parameters exhibited Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 6 of 11 Fig. 2 Acute T-AG17 administration does not alter body weight, glucose, insulin and leptin serum levels. a Body weight was analyzed every week after 17.5, 28.5, 40 and 55.5 mg/kg oral T-AG17. Changes in body weight are expressed in grams. b-d Serum biochemistry was determined using ELISA kits (insulin and leptin) as described in Methods and glucose levels measurement was determined by Glucose strips. Graphs show the normalized results of mean ± SEM for n =6 at the 40 mg/kg dose. The mice of this group were sacri- cytoplasms and large perinuclear vacuoles. Some areas ficed between 5 and 10 min after administration. had low affinity for staining and others areas had disor- We identify changes in cell morphology of brain, kid- ganized hepatocytes, edema, and the appearance of fat ney, liver, pancreas, adipose tissue and muscle after 17.5, accumulation as evidenced by oil red staining. 28 and 55.5 mg/kg T-AG17 oral administration. The brain showed hyperchromic cytoplasm, perinuclear eo- Chronic doses of T-AG17 administration sinophilic inclusions and dispersed chromatin. The In order to test the potential of T-AG17 oral administration kidneys of these mice showed pale aspects of the glom- to regulate body weight, we selected the lowest dose erular units and proximal tubules. The liver displayed administered in our previous experiment (Fig. 2,oraldose hepatic acinar cells with perinuclear cytoplasmic vacu- AG17 = 1.75 mg/kg). Mice were orally dosed with 0.175, oles and and the acinar unit randomly distributed. The 0.0175 or 0.00175 mg/kg T-AG17 during 2 weeks reaching pancreas displayed exocrine acinar cells with pale ap- 2.45, 0.245 and 0.0245 mg/kg final concentrations after pearance and normal Langerhans islets. White adipose treatment (Fig. 3). We determined plasma biochemistry and tissue exhibited adipocytes with normal aspect and an tested for renal and liver damage using selective markers. apparent decrease in adipocyte cell number intercalated We found that oral 0.175, 0.0175 or 0.00175 mg/kg T-AG17 with major adipocytes evidenced by the red oil stained. doses did not alter plasma biochemistry markers including The skeletal muscle of these mice seemed morphologic- glucose, leptin, insulin and free fatty acids and nor the ally unaltered. creatinine and aspartate aminotransferase (two markers of Mice orally administered 41 mg/kg T-AG17 showed renal and liver damage, respectively) (Fig. 3). neurons displaying eosinophilic and pale cytoplasm and pycnotic nucleus coexisting with normal neurons. Kid- T-AG17 promotes apoptotic cell death of white ney cells seem retracted with pale glomeruli and tubular adipocytes in obese mice cells with poor staining, pycnotic nuclei, and condensed Based on the observation that T-AG17 stimulation chromatin. Hepatocytes from the liver displayed pale promotes apoptotic cell death in 3T3-L1 cell line, we Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 7 of 11 Fig. 3 Chronic T-AG17 does not alter body weight, glucose, insulin, and leptin levels and creatinine and aspartate aminotransferase activity in serum. a Body weight was analyzed every week after 2 weeks of 0.0175, 0.175 and 1.75 mg/kg oral T-AG17 administration. Changes in body weight are expressed in grams. b-e Blood glucose levels were determined using glucose strips and serum biochemistry was determined using ELISA kits as described in Methods. f-g Creatinine and aspartate aminotransferase activity were determined using ELISA kits. Graphs show the normalized results of mean ± SEM for n =6 sought to determine if administration of T-AG17 in size (Fig. 5). Notably, obese mice administered the mice modulates thermogenesis or white adipose tissue T-AG17 showed an apparent decrease in adipocyte fat apoptosis, leading to body weight decrease. Initially, droplet size when compared to Chow and HFD groups we fed mice with Chow or HFD for 18 weeks and (Fig. 5). Also, T-AG17 administration of mice expose to tested the potential of T-AG17 versus dieting. In this Chow diet did not show morphological changes when experiment, we injected T-AG17 intraperitoneally compare to Chow and HFD groups. In addition, we did (grey bar Fig. 4c) or exposing the HFD mice to a nor- not identify positive staining using the PAS protocol for mal Chow diet (red bar Fig. 4c)for 2weeks. As visceral fat tissue in all groups (Fig. 5). T-AG17 induced expected, we found that 18 weeks of HFD intake a significant increase in active pro-caspase 3-Cy3 stain- increased the body weight reaching 45–50 g when ing in obese mice when compared to controls fed with compared to Chow diet values (25–28 g) (Fig. 4a, b). Chow diet (Fig. 5). This effect was replicated in obese No significant changes in food intake were found mice put back to normal Chow diet for 2 weeks (Fig. 5); (Chow = 23.91 ±2.07 g, HFD = 26.83 ±9.72). HFD in- however, positive pro-caspase 3-Cy3 staining of cells was take increased the body weight (black bar vs Chow not significant when compared to obese mice adminis- diet, Fig. 4c); however, we did not find evidence of tered T-AG17 (Fig. 5). weight loss after T-AG17 administration (compare black bar HFD groups vs grey bar Fig. 4c). The latter T-AG17 promotes changes in hepatocytes of obese mice observation correlates which we found when mice We then assessed any morphological changes in the were put back to the Chow diet during 2 weeks livers after T-AG17 or dieting exposure. The livers of (compare black bar HFD groups vs red bar Fig. 4c). mice exposed to HFD showed abnormal retention of Next, we assessed any morphological changes in lipids displaying macrovesicular steatosis (Fig. 6a). Oral WATafter T-AG17 or dieting exposure. WAT of mice T-AG17 administration led to a transition from macro- fed the Chow diet showed typical polyhedral cells with vesicular to microvesicular steatosis, which correlated nuclear location near the plasma membrane; whereas, with an increase in red oil positive inclusions (lipids) cells of mice exposed to HFD reached a higher apparent (Fig. 6a, b). Obese mice put back on Chow diet for Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 8 of 11 Fig. 4 T-AG17 administration and body weight in animals exposed to HFD. a Mice were exposed to HFD (60% kcal from fat) or chow diet during 18 weeks. b Body weight was determined every week. Changes in body weight are expressed in gr. c Body weight change after 2 weeks of oral T-AG17 administration. Graphs show the normalized results of mean ± SEM for n =9–12 and statistical significance after using unpaired Student’s t test. *p < 0.05 2 weeks showed a similar hepatocyte morphology as the Chow diet group and also showed a switch from macrovesicular to microvesicular steatosis. Notably, oral T-AG17 administration promoted a significant increase in caspase-3 activation in adipose tissue but not in liver when compared to HFD or Chow diet (Fig. 6b, c). Finally, obese and normal mice orally administered T-AG17 showed liver positive PAS staining (Fig. 6a), reflecting an increase of cytoplasmic glycogen inclusions. Discussion We have shown that orally administered T-AG17 modu- lates adipocyte differentiation in vitro and induces adi- pocyte apoptosis in vivo. Oral administration of T-AG17 (0.175 mg/kg daily over 2 weeks) decreased fat cell vol- ume in WAT. Incubation with T-AG17 prevents differentiation of 3T3-L1 cells to mature adipocytes, as shown by a decrease in fat droplet formation by oil red staining (Fig. 1a, b). Fat droplet formation is one of the final stages of adipocyte differentiation [43]. This finding is consistent with the previous observations that T-AG17 suppresses insulin-mediated fatty acid synthesis in rat Fig. 5 T-AG17 promotes apoptotic cell death in adipose tissue. Mice white adipocytes [33] and also that Tyrphostin AG490 were exposed to HFD (60% kcal from fat) or Chow diet during (which bears the same pharmacophore as T-AG17) in- 18 weeks and T-AG17(0.175 mg/kg) was orally administered during 2 weeks. Pro caspase 3 activation was evaluated using hibits adipogenesis by disrupting STAT3 signaling in immunohistochemistry. PAS staining was performed to evaluate human adipocytes through inhibition of JAK2 [44]. This glycogen synthesis finding suggests that T-AG17 might be a potential Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 9 of 11 Defects in adipocyte tissue expandability and hyper- trophy lead to ectopic fat accumulation in metabolically relevant organs, including liver [7]. In a fatty liver, trigly- ceride (TG) accumulation, either as small or large lipid deposits, is called micro- or macrovesicular steatosis, respectively [45–47]. Microvesicular steatosis, gradually followed by macrovesicular steatosis, is experimentally induced by 6–10 weeks of HFD feeding [22, 48, 49], which correlates with an elevation in liver enzymes (AST and ALT) [45, 50]. Our results agree with these findings, with mice exhibiting macrovesicular steatosis after 12 weeks of HFD feeding. To the best of our know- ledge, the T-AG17- induced switch from macrovesicular to microvesicular liver steatosis in the HFD group (Fig. 6) is unprecedented. Also, while T-AG17 increases lipid in- clusions in hepatocytes as shown by oil red staining (Fig. 6), we did not find any changes in serum AST levels, suggesting no histological lesions in liver. We hypothesize that this is a transient condition whereby fatty acids are being temporarily exported to the liver from WAT. In any case, our observations parallel those of Dianzani and Scuro [51], who reported increases in hepatic fat droplets in albino rats 48–96 h after injec- tions with DNP, following by glycogen infiltration after Fig. 6 T-AG17 shifts macrovesicular to microvesicular steatosis in liver. a Mice were exposed to HFD or Chow as described and T- 120 h and decreased in neutral fat droplets. T-AG17 ad- AG17(0.175 mg/kg) was orally administered during two weeks. H/E, ministration for 2 weeks results in a microvesicular stea- oil red staining, pro caspase 3 activation and PAS staining were tosis phenotype with normal levels of liver enzymes. performed in adipose tissue (b) or liver (c) to evaluate morphology, Livers from human donors with moderate and severe fat accumulation, apoptosis activation and glycogen synthesis, macrovesicular steatosis are considered unfit for trans- respectively. For immunohistochemistry analysis, we used ANOVA test followed by Kruskal-Wallis test one-way. The data is presented plantation [53]. Therefore, our findings support a role of as mean±SEM unless stated.. *p < 0.05 T-AG17 as a MMU capable of inducing the transition from macrovesicular to microvesicular steatosis in liver and potentially improving the metabolic body profile. effective adipogenesis-blocking agent; therefore, we Finally, our findings show that obese mice treated with tested the tolerance and therapeutic effects in a murine T-AG17 accumulate glycogen in the liver. Previously, in- model of obesity. jection of DNP in albino rats has been shown to also In diet-induced obese C57BL/6 mice, T-AG17 does cause hepatic glycogen infiltration [51], and treatment of not induce weight loss, as we had originally hypothe- obese rats with a controlled-release formulation of DNP sized. Interestingly, the DNP uncoupler does induce led to an 80% increase in liver glycogen content [18], weight loss at thermoneutrality (30 °C) [10], which which is associated with reversal of hypoglycemia. Our differs from our study. However, T-AG17 did promote data are consistent with evidence showing that glycogen an apparent decrease in the size of adipocytes and a accumulates in the liver of rats following prolonged ad- significant activation of caspase 3-dependent apoptosis ministration of DNP, in contrast to other parenchymal or- (Figs. 5, 6). The decrease in adipocyte size might be me- gans [18], and also with the finding that MMU-triggered diated by MMU-induced lipolysis and down regulation by FCCP induces glucose uptake in adipocytes [6]. of lipid synthesis [6]. Tyrphostins, in particular, have been characterized as classical inductors of apoptosis by Conclusions inhibiting protein tyrosine kinase activity [30], suggest- T-AG17 blocks adipocyte differentiation in vivo and in ing that T-AG17 might be a potential inductor of apop- vitro, promotes efficient apoptotic cell death of adipocytes totic cell death in adipocytes. Indeed, T-AG17 has been in vivo, and a switch from macrovesicular to microvesicular shown to inhibit cell growth and induce apoptosis in steatosis during positive energy balance in a diet-induced other cell types [31, 38, 39]. These evidence suggest that obesity mice model. Thus, our data support the develop- T-AG17 activates the apoptotic pathway in adipocytes, ment of T-AG17 as a candidate to pharmacologic preven- although no overall weight loss was observed. tion/treatment of obesity and fatty liver disease. Camacho et al. Lipids in Health and Disease (2018) 17:128 Page 10 of 11 Funding 8. Virtue S, Vidal-Puig A. Adipose tissue expandability, lipotoxicity and the This work was funded by the National Council of Science and Technology in metabolic syndrome-an allostatic perspective. Biochim Biophys Acta-Mol Mexico (CONACYT) (Grant number: 255317) and the Dirección de Cell Biol Lipids. 2010;1801:338–49. Investigación y Desarrollo, Ayon Industries, Monterrey, México. 9. De Pauw A, Tejerina S, Raes M, Keijer J, Arnould T. Mitochondrial (Dys) function in adipocyte (De) differentiation and systemic metabolic Availability of data and materials alterations. Am J Pathol [Internet]. 2009;175:927–39. All data generated or analyzed during this study are included in this 10. Goldgof M, Xiao C, Chanturiya T, Jou W, Gavrilova O, Reitman ML. The published article. chemical uncoupler 2,4-dinitrophenol (DNP) protects against diet-induced obesity and improves energy homeostasis in mice at thermoneutrality. J Authors’ contributions Biol Chem. 2014;289:19341–50. AC and RO contributed to the study design, data interpretation and 11. Schrauwen P, van Marken Lichtenbelt WD. Combatting type 2 diabetes by manuscript writing; JC, AS, JJ, JG, CA and GC performed experiments and turning up the heat. Diabetologia. 2016;59:2269–79. collected data. All authors read and approved the final manuscript. 12. Liu D, Zhang Y, Gharavi R, Park HR, Lee J, Siddiqui S, et al. 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Journal

Lipids in Health and DiseaseSpringer Journals

Published: May 29, 2018

References

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