Adipose triglyceride lipase protein abundance and translocation to the lipid droplet increase during leptin-induced lipolysis in bovine adipocytes

Domestic Animal Endocrinology 61 (2017) 62–76

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Adipose triglyceride lipase protein abundance and translocation to the lipid droplet increase during leptin-induced lipolysis in bovine adipocytes D.A. Koltes a, *, y, M.E. Spurlock b, D.M. Spurlock a a b

Department of Animal Science, Iowa State University, Ames, Iowa 50011, USA Department of Food Science and Human Nutrition, Iowa State University, Ames, Iowa 50011, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 January 2017 Received in revised form 30 May 2017 Accepted 2 June 2017

Proper regulation of lipid metabolism is critical for preventing the development of metabolic diseases. It is clear that leptin plays a critical role in the regulation of energy homeostasis by regulating energy intake. However, leptin can also regulate energy homeostasis by inducing lipolysis in adipocytes, but it is unclear how the major lipases are involved in leptin-stimulated lipolysis. Therefore, the objectives of this study were to determine if (1) leptin acts directly to induce lipolysis in bovine adipocytes, (2) the potential lipases involved in leptin-induced lipolysis in bovine adipocytes, and (3) increases translocation of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) during leptin-stimulated lipolysis in bovine stromal vascular cell–derived adipocytes. As hypothesized, leptin induced a lipolytic response (P ¼ 0.02) in isolated adipocytes which was accompanied by an increase in phosphorylation of signal transducer and activator of transcription (STAT)3 (P ¼ 0.03), a well-documented secondary messenger of leptin, and ATGL protein abundance (P < 0.01). Protein abundance of STAT3, perilipin, HSL, and phosphorylation of HSL by PKA and AMPK were not altered during leptin-stimulated lipolysis (P > 0.05). Immunostaining techniques were employed to determine the location of HSL and ATGL. Both lipases translocated to the lipid droplet after 2 h of exposure to isoproterenol (P < 0.02). However, only ATGL was translocated to the lipid droplet during leptin-stimulated lipolysis (P ¼ 0.04), indicating ATGL may be the active lipase in leptin-stimulated lipolysis. In summary, leptin stimulates lipolysis in bovine adipocytes. The lack of phosphorylated HSL and translocation of HSL to the lipid droplet during leptinstimulated lipolysis suggest minimal activity by PKA. Interestingly, leptin-stimulated lipolysis is accompanied by an increase in ATGL protein abundance and translocation to the lipid droplet, indicating its involvement in leptin-stimulated lipolysis either due to an increase in protein abundance or through a novel lipolytic cascade. Ó 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Glycerol Hormone sensitive lipase Signal transducer and activator of transcription 3 Leptin

1. Introduction Lipolysis is a key component of lipid metabolism and energy homeostasis. This is readily apparent in lactating dairy cattle because energy requirements for milk * Corresponding author. Tel.: 479-575-6872; fax: 479-575-3026. E-mail address: [email protected] (D.A. Koltes). y Current address: Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701.

production exceed energy intake, thus necessitating the mobilization of fatty acids from adipose tissue. When lipolysis is acutely upregulated or prolonged for extended periods of time in over-conditioned cows, it increases the risk of ketosis, fatty liver disease, displaced abomasum, dystocia, retained placenta, lower milk production, and reduced reproductive performance [1]. Thus, understanding the regulation of lipolysis in the dairy cow is pivotal to improve her health and productivity.

0739-7240/Ó 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.domaniend.2017.06.001

D.A. Koltes et al. / Domestic Animal Endocrinology 61 (2017) 62–76 Table 1 Antibodies used for semiquantitative Western blotting. Catalog numbera Gel percentage Dilution Size (kDa)

Antibody 705

PSTAT3 Try STAT3 ATGL PHSL Ser563 PHSL Ser565 HSL b-actin

9145 8719 2138 4139 4137 4107 5125

8 8 8 8 8 8 db

1:500 1:500 1:500 1:500 1:500 1:500 1:5000

79,86 86 54 81,83 81,83 81,83 45

Abbreviations: ATGL, adipose triglyceride lipase; HSL, hormone sensitive lipase; PHSL Ser563, phosphorylated HSL at serine 563; PHSL Ser565, phosphorylated HSL at serine 565; PSTAT3 Try705, phosphorylated STAT 3 at tyrosine 705; STAT3, signal transducer and activator of transcription 3. a All antibodies were purchased from Cell Signaling. b b-actin antibody was exposed to all membranes.

Collectively, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) account for approximately 95% of the catecholamine-stimulated lipolytic response in mouse adipocytes [2]. Protein kinase A (PKA) is a major regulator of HSL through its phosphorylation of serine residues 563, 659, and 660 [3,4]. Phosphorylated perilipin via PKA not only allows activated HSL to access the surface of the lipid droplet [5–8] but also activates ATGL via a/b hydroxylase domain containing 5 (ABHD5) [9,10]. In addition, PKA may phosphorylate ATGL at amino acid 406 (murine) [11]. In addition to being regulated by PKA, HSL and ATGL are regulated by adenosine monophosphate–activated protein kinase (AMPK). Phosphorylation of HSL at serine 565 by AMPK is thought to induce a conformational change that precludes phosphorylation by PKA [12]. However, it is unclear if the activation of the AMPK pathway promotes [13], inhibits [12], or has no effect [14] on lipolysis. Phosphorylation of ATGL by AMPK is increased when lipolytic activity is increased in brown adipose tissue [15]. However, it should be noted that this serine residue site (ie, murine amino acid 406) may be phosphorylated by ERK [16] and may indicate a role for ERK in lipolysis. Leptin is a 16 kDa protein secreted from adipose tissue that regulates energy homeostasis through suppression of feed intake and induction of lipolysis. Increasing intracerebral concentrations of leptin reduces feed intake in healthy gonadectomized pigs [17,18], sheep [19–22], and leptinproducing mice [23] through signaling of the janus kinase/ signal transducer and activator of transcription (STAT) pathway as a result of leptin receptors activation in the hypothalamus (reviewed by [24]). In addition to acting centrally, leptin can act locally by increasing lipolysis. In vitro studies in adipocytes from rodents and pigs, leptin induces lipolysis at or above average circulating concentration of leptin via janus kinase/STAT [25–28]. Research in

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ruminants adipose tissue is limited to a single study in sheep that did not observe the activation of STAT3 or STAT5 [29]. Despite the potential ability of leptin to regulate adiposity, the mechanisms by which leptin induce lipolysis remain unclear. The objectives of this study were to determine (1) leptin acts directly to induce lipolysis in bovine adipocytes, (2) the potential lipases involved in leptin induced lipolysis in bovine adipocytes, and (3) increases translocation of ATGL and HSL during leptin-stimulated lipolysis in bovine stromal vascular cell–derived adipocytes. 2. Materials and methods All procedures involving the use of animals were approved by the Iowa State University Institutional Animal Care and Use Committee (Protocol #2-11-7094-B). All cows used in this study were housed at the Iowa State University Dairy Farm located in Ames Iowa. 2.1. Materials Dulbecco’s modified eagle media (DMEM, D5523), 3-isobutyl-1-methylxanthine (IBMX, I5879), dexamethasone (D4902), insulin (I6634), and fatty acid supplement (F7175–5 mL), Free Glycerol Reagent (F6428) were purchased from Sigma Aldrich. Fetal bovine serum (FBS, s11150) was purchased from Atlanta Biologicals; antibioticantimycotic solution (15240) was purchased from Life Technologies; troglitazone (71750) was purchased from Caymen Chemical; and sodium acetate (S209–500g), bicinchoninic acid assay (BCA; 23227), and NEFA-HR(2) (999–34691, 995–34791, 991–34891, 993–35191, 276– 76491) were purchased from Fisher Scientific. Bovine leptin (CYT-502) was purchased from ProSpec and isoproterenol (151358) was purchased from MP Biomedicals. 40 ,6diamidino-2-phenylindole, dihydrochloride (DAPI, IW1404), bodipy (D-2184), and fluorogel with DABCO (17985–01) were purchased from IHC World, Molecular Probes, and Electron Microscopy Sciences, respectively. 2.2. Adipocyte collection Approximately 40 g of subcutaneous adipose tissue were collected under local anesthetic (2% lidocaine) from the tailhead region of 8 lactating Holstein cows (301  15 d in milk). Adipocytes were isolated using a previously described method [30–32]. Briefly, adipose tissue was transported to the laboratory in a warmed saline solution with glucose [0.15 M of sodium chloride, 1-mM HEPES, and

Table 2 Primary and secondary antibodies used for immunofluorescence. Antibody

Company

Catalog number

Secondary antibody

Fluoroform

Catalog numbera

PSTAT3 HSL ATGL ABHD5 G0S2

Cell Signaling Cell Signaling Cell Signaling Santa Cruz Biotechnology Novus Biologicals

9145 8719 2138 Sc-102285 4137

Rabbit IgG Rabbit IgG Rabbit IgG Goat IgG Mouse IgG

FITC Dylight Dylight Dylight Dylight

111-095-003 711-475152 711-475-152 705-495-003 115-515-146

405 405 649 594

Abbreviations: ABHD5, a/b hydroxylase domain containing 5; ATGL, adipose triglyceride lipase; FITC, fluorescein isothiocyanate; G0S2, G0/G1 switch protein 2; HSL, hormone sensitive lipase; PSTAT3, phosphorylated signal transducer and activator of transcription 3 at tyrosine 705. a All secondary antibodies were purchased from Jackson Immunoresearch in West Grove, PA.

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0.1 M of glucose, pH 7.4]. Adipose tissue was minced and digested in cocktail solution containing 20-mM sodium bicarbonate, 20-mM HEPES, 10-mM D-glucose, and 3% fatty acid free bovine serum albumin (w/v), pH 7.4, containing 1 mg/mL (w/v) of collagenase type I (C2674; Sigma; St. Louis, MO, USA). Adipose tissue was digested for 40 min at 37 C. Following an additional collagenase digestion (1.8 mg/mL of collagenase; w/v), adipocytes were collected by filtration, rinsed, and isolated adipocytes were suspended in media [10 g/L low-glucose DMEM, 3% fatty acid free bovine serum albumin (w/v), 6.6-mM sodium bicarbonate, 6.25-mM HEPES, pH 7.2]. Adipocytes were equally aliquoted into plastic scintillation vials, gassed with 5% CO2, and 95% air then allowed to acclimate for 30 min before the initiation of experiments in a shaking incubator at 37 C. 2.3. Treatment of adipocytes Duplicate vials of adipocytes from 8 cows were incubated with 0 (control), 10, 100, or 200 ng/mL of

recombinant bovine leptin. As a positive control for lipolysis, duplicate vials of adipocytes from each cow were treated with 100-nM D-L isoproterenol. Media were aspirated 2 h after treatment and stored at 80 C. Adipocytes were homogenized in a protein homogenization buffer (5% sodium dodecyl sulfate, 50-mM HEPES, 2-mM EDTA, 50mM NaF), 1 mL/mL of broad protease inhibitor cocktail (Sigma, P8340) and 1-mL/mL phosphatase inhibitor cocktail (set II, Calibiochem, 524627) [33,34] and stored at 80 C. Protein concentrations were determined using the BCA protein assay kit (Pierce, 23227), according to manufacturer’s protocol. 2.4. Glycerol and nonesterified fatty acid assays Glycerol (Free Glycerol Reagent) and NEFA (NEFA-HR [2]) in the media were measured in triplicate using colormetric assays in a 96-well plate format, according to manufacturer’s protocol. Data were captured using a Tecan Spectrafluor Plus (Tecan Group Ltd). Glycerol and NEFA

Fig. 1. Demonstration of image assessment. The figure illustrates how translocation was measured for a lipid droplet using the RGB Profiles Plot plug in ImageJ [36]. The white lines represent where lines would have been drawn in ImageJ. The upper inset box is a representation of the data produced by RGB Profiles Plot plug in ImageJ. The intensity values along each pixel in the line were exported, averaged at either 0 and 0.89 mm for the average fluorescence at the lipid droplet, or 2.70 and 3.30 mm for the average fluorescence in the cytosol, and utilized for statistical analysis. Green represents the lipid droplet, blue and red represent ATGL and ABHD5, respectively. ABHD5, a/b hydroxylase domain containing 5; ATGL, adipose triglyceride lipase. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 2. Effects of leptin on lipolysis in bovine adipocytes. N ¼ 8 cows per treatment. (A) Glycerol concentration normalized to protein concentrations. (B) NEFA concentration normalized to protein concentrations. (C and D) Protein abundance normalized to b-actin expressed relative to control abundance. Representative Western blots are shown under the graph for the corresponding antibody. Lane 1: 0 ng/mL of leptin (control), lane 2: 10 ng/mL of leptin, lane 3: 100 ng/mL of leptin, lane 4: 200 ng/mL of leptin. STAT3, signal transducer and activator of transcription 3; HSL, hormone sensitive lipase; PSTAT3 Try705, phosphorylated STAT 3 at tyrosine 705; ATGL, adipose triglyceride lipase; PHSL ser563, phosphorylated HSL at serine 563; PHSL ser565, phosphorylated HSL at serine 565. *Represents P < 0.05 compared with control where the overall effect of leptin was P < 0.05. xRepresents P < 0.05 compared with control where the overall effect of leptin was P < 0.11. Error bars represent SEM.

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Fig. 3. Comparison of control and isoproterenol treated bovine adipocytes from cows in the leptin experiment. N ¼ 8 cows per treatment. (A) Glycerol concentration normalized to protein concentrations. (B) NEFA concentration normalized to protein concentrations. (C) Protein abundance normalized to b-actin and expressed relative to control. Representative Western blots are shown under the graph for the corresponding antibody. Lane 1: Control samples, Lane 2: Adipocytes treated with 100 nM isoproterenol. STAT3, signaling transducer and activator of transcription 3; HSL, hormone sensitive lipase; PSTAT3 Try705, phosphorylated STAT 3 at tyrosine 705; ATGL, adipose triglyceride lipase; PHSL ser563, phosphorylated HSL at serine 563; PHSL ser565, phosphorylated HSL at serine 565. *Represents P < 0.05 compared with control. xRepresents P < 0.1. Error bars represent SEM.

concentrations were normalized to protein abundance, as determined by bicinchoninic acid assay. Duplicate vials of adipocytes were combined before preparation of proteins for Western blotting due to limited sample availability. 2.5. Semiquantitative Western blotting Semiquantitative Western blot assays were performed using previously described methods [33–35]. Antibody companies, catalog numbers, gel percentages, and antibody dilutions can be found in Table 1. All samples were run on duplicate gels. Protein abundance values were normalized to b-actin, and means are presented relative to control samples. 2.6. Culture of stromal vascular cell–derived bovine adipocytes Approximately 40 g of subcutaneous adipose tissue were collected under local anesthetic (2% lidocaine) from

the tailhead region of 2 lactating Jersey cows. Stromal vascular cells were harvested using a previously described method with slight modifications to generate 2 cell lines [31]. Following collagenase digestion of adipose tissue, SVC were pelleted and rinsed 2 times in warmed cocktail media [20-mM sodium bicarbonate, 20-mM HEPES, 10-mM Dglucose, and 3% bovine serum albumin (w/v), pH 7.4]. Viable cells were counted using trypan blue staining, plated at 10,000 cells per cm2 in 6-well plates or 60 mm petri dishes, and grown on glass coverslips in either 6-well plates or 60-mm petri dishes after 2 passages in DMEM containing 5% FBS and 1% antibiotic-antimycotic. Media were changed every 4 d until cells reached 90% confluency. Subsequently, SVCs were cultured in differentiation media [DMEM, 1% antibiotic-antimycotic, 10% FBS, 0.5 mM of IBMX, 1 mM of dexamethasone, and 10 mg/mL of insulin]. The first d of differentiation was denoted as Day 0. After 48 h, differentiation media was change to lipogenic media

D.A. Koltes et al. / Domestic Animal Endocrinology 61 (2017) 62–76 Table 3 List of P-values for the main effects of leptin treated bovine adipocytes. Effect

Leptin treatment

Glycerol NEFA STAT3 PSTAT3 HSL PHSL 563 PHSL 565 ATGL

0.02 0.99 0.64 0.03 0.24 0.07 0.40 <0.01

Abbreviations: ATGL, adipose triglyceride lipase; HSL, hormone sensitive lipase; NEFA, nonesterified fatty acid; PHSL 563, phosphorylation of HSL at serine 563; PHSL 565, phosphorylation of HSL at serine 565; PSTAT3, phosphorylated STAT3 at tyrosine 705; STAT3, signal transducer and activator of transcription 3.

(DMEM, 1% antibiotic-antimycotic, 10% FBS, 10 mM of troglitizone, 10 mg/mL of insulin, 2% v/v of fatty acid supplement [contains 2 mol of linoleic and 1 mol of oleic acid per mole of albumin], 1 mM of sodium acetate), and changed every 2 d until 14 d post differentiation. 2.7. Experimental treatment and immunofluorescent labeling On Day 14, media were changed to DMEM 1 h before the treatment. For the first experiment, SVC-derived adipocytes were exposed to DMEM (control), 100 ng/mL of bovine leptin, or 100 nM of isoproterenol for 2 h. For the second experiment, SVC-derived adipocytes were treated with 100 ng/mL of bovine leptin for 0, 5, 10, 20, 120, or 240 min. Following treatments, SVC-derived adipocytes were rinsed twice with PBS and fixed for 5 min using 4% paraformaldehyde. Cells were rinsed three times using PBS and then permeated for 5 min 0.6% Trition-X 100 in PBS. The SVC-derived adipocytes were blocked for 1 h in PBS containing 5% BSA. Coverslips were then removed and exposed to primary antibodies overnight. Coverslips were rinsed in PBS containing 0.5% Tween-20 and then exposed to fluorescently labeled secondary antibodies and either DAPI to stain nuclei or bodipy to stain lipid droplets.

Table 4 List of P-values for the comparison of control with leptin treated bovine adipocytes. Effect

10 ng/mLa

100 ng/mLa

200 ng/mLa

Isoproterenol treatmentb

Glycerol NEFA STAT3 PSTAT3 HSL PHSL 563 PHSL 565 ATGL

0.46 0.81 0.39 0.01 0.34 0.18 0.62 0.03

0.02 0.92 0.28 0.01 0.20 0.02 0.41 <0.01

0.60 0.96 0.28 0.40 0.05 0.02 0.10 <0.01

<0.01 <0.01 0.70 0.85 0.67 <0.01 0.43 0.08

Abbreviations: ATGL, adipose triglyceride lipase; HSL, hormone sensitive lipase; NEFA, nonesterified fatty acid; PHSL 563, phosphorylation of HSL at serine 563; PHSL 565, phosphorylation of HSL at serine 565; PSTAT3, phosphorylated STAT3 at tyrosine 705; STAT3, signal transducer and activator of transcription 3. a P-values for adipocytes for cows treated with leptin. b P-values for the analyses of control and isoproterenol treated adipocytes run as positive controls with leptin treatment.

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Primary and secondary antibody combinations, company, catalog number, and fluoroform can be found in Table 2. After 1 h of incubation, coverslips were rinsed with PBS containing 0.5% Tween-20 and placed in Fluorogel with DABCO mounting media on microscope slides and sealed with nail polish. 2.8. Analysis of immunofluorescence Fluorescence was captured from dual- and triplelabeled coverslip using a Qimaging QIcam camera (Surrey BC, Canada) accompanied by a Leica DMI3000B microscope (Wetzlar, Germany). Each fluorescent wavelength was individually captured, saved, and then compiled by Qimaging software. Images were captured at 40 magnification with 5 images containing well-formed and welldispersed lipid droplets, or nuclei were captured per coverslip. Images were captured from 2 coverslips per timepoint from a single-cell line for analysis of timedependent translocation of ATGL. All other treatments had one coverslip per treatment for each of the two cows. Fluorescence for each image was measured using ImageJ software in the RGB format [36]. At the time the study was conducted, detecting changes in fluorescence at the lipid droplet was not available. Therefore, to determine if fluorescence at the lipid droplet changed, we determined the difference in fluorescence at the lipid droplet compared with the fluorescence in the cytosol. This was used as a proxy for determining translocation of our protein of interest under the influence of various stimuli, and we will refer to this difference simply as translocation from this point on. To prevent bias during the measurements of fluorescence at the lipid droplet and in the cytosol, fluorescence along a set length (3.30 mm) was taken using the RGB Profiles Plot plug in ImageJ [36]. The straight line was extended from the edge of the lipid droplet (point where the green wavelength fell) into the cytosol. The average fluorescence measured between 0 and 0.89 mm along this line was considered to be fluorescence at the lipid droplet. The average fluorescence between 2.70 and 3.30 mm along this line was considered to be cytosolic fluorescence. This line was arbitrarily drawn at a point around the lipid droplet with care taken to avoiding areas where the line would overlap with other lipid droplets or the edge of the image. To prevent bias from the location of the line, fluorescence was measured at 5 different locations for each lipid droplet with 2 to 3 lipid droplets measured per image (Fig. 1A). We considered lipid droplet as the experimental unit for this experiment. In total, between 25 and 30 lipid droplets were measured for each treatment. The difference in fluorescence was averaged by lipid droplet and used for statistical analysis. To account for background variation, fluorescence was measured at random spots within the background of the image where a line of 3.30 mm in length was measured and the difference in the fluorescence between 2.70 and 3.30 mm and 0 and 0.89 mm was taken. Lipid droplet diameter was measured by drawing a line across the center of the lipid droplet and determining the length using the RGB Profiles Plot plug in ImageJ [36]. Translocation of phosphorylated STAT3 to the nucleus was determined by calculating the colocalization of

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Fig. 4. Translocation of hormone sensitive lipase (HSL) to the lipid droplet after 2 h of exposure. N ¼ 12–15 lipid droplets from 2 cultures per treatment. (A) Representative images of SVC-derived bovine adipocytes treated with media (control), 100 nM of isoproterenol, or 100 ng/mL of leptin. Treatment is listed on the left hand side of the image. In the merged image, HSL is blue and bodipy is green. The white line in the lower right hand image is 10 mm relative to the view. (B) Average translocation of HSL to the lipid droplet SEM. (C) Average lipid droplet diameter size SEM. Differences in subscripts were given when P < 0.05 and indicate pairwise differences of P < 0.05. SEM, standard error of the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

phosphorylated STAT3 (FITC) and nuclei (DAPI) using the Mander’s Coefficient plugin from the ImageJ software [36]. Five images were measured per slide with 1 slide per cow. Views were dropped from the analysis when the colocalization of phosphorylated STAT3 (FITC) and nuclei (DAPI) greatly deviated from nucleus.

2.9. Statistical analysis Western blot, glycerol, and NEFA data were analyzed using mixed model procedures of SAS [37], with experimental treatments fit as fixed effects, and cow modeled as a random effect. Beta-actin was fit as a covariate for all

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Fig. 5. Translocation of adipose triglyceride lipase (ATGL) to the lipid droplet after 2 h of exposure. N ¼ 12–15 lipid droplets from 2 cultures per treatment. (A) Representative images of SVC-derived bovine adipocytes treated with media (control), 100 nM of isoproterenol, or 100 ng/mL of leptin. Treatment is listed on the left hand side of the image. In the merged image, ATGL is blue and bodipy is green. The white line in the lower right hand image is 10 mm relative to the view. (B) Average translocation of ATGL to the lipid droplet SEM. (C) Average lipid droplet diameter size SEM. Differences in subscripts were given when P < 0.05 and indicate pairwise differences of P < 0.05. ATGL, adipose triglyceride lipase; SEM, standard error of the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Western blot analysis, and total protein abundance was fit as a covariate for analyses of phosphoprotein data. If the main treatment effect was significant (P < 0.05), contrasts were defined as comparisons of treatment groups to the control. Residuals were tested for normality and were found to be normally distributed [37]. Variables with non-normally distributed residuals were log transformed before the statistical analysis. These variables were data from the perilipin Western blots for both the leptin and isoproterenol experiments, and data from

the STAT3, both phosphorylated HSL Western blots, and the glycerol and NEFA data from the isoproterenol data. These data were back transform for presentation in the manuscript. Immunofluorescence data were analyzed using mixed model procedures of SAS [37]. Differences in lipid droplet diameters between treatments were analyzed by fitting lipolytic stimulant or time as a fixed effect, and image blocked within slide or cell line as a random effect. Differences in the translocation of HSL, ATGL, ABHD5, and G0S2

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Fig. 6. Colocalization of phosphorylated signal transducer and activator of transcription (STAT) 3 with nuclei. N ¼ 5 images from 2 cultures per treatment. (A) Representative images of SVC-derived bovine adipocytes treated with 100 ng/mL of leptin over a 4-h time course. Time of exposure is listed on the left hand side. In the merged image, blue represents nuclei and green represents phosphorylated STAT3. (B) Average Pearson’s correlation coefficients for the colocalization nuclear DNA and phosphorylated STAT3. Differences in lowercase subscripts were given when P < 0.05 and indicate pairwise differences of P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

to the lipid droplet were analyzed by fitting lipolytic stimulant or time was fit as a fixed effect, lipid droplet diameter and background fluorescence as covariates, and image blocked within slide or cell line as a random effect. Differences in the colocalization of phosphorylated STAT3 and were analyzed by fitting lipolytic stimulant or time was fit as a fixed effect, and cell line as a random effect. For all analysis, pairwise comparisons were conducted using the LSmeans statement in SAS [37]. Significance was set at P < 0.05, and trends at P < 0.1.

3. Results 3.1. Leptin and isoproterenol effect on lipolysis Leptin treatment (100 ng/mL) increased glycerol (P ¼ 0.02, Fig. 2A), but not NEFA concentrations (P ¼ 0.99, Fig. 2B). Phosphorylation of STAT3 Tyr705 increased (P ¼ 0.03) in adipocytes treated with 10- and 100-ng/mL leptin, and abundance of ATGL protein increased (P < 0.01) with 10-, 100-, and 200-ng/mL leptin. Leptin

D.A. Koltes et al. / Domestic Animal Endocrinology 61 (2017) 62–76 Table 5 P-values for pairwise comparisons for the colocalization of phosphorylated STAT3 and the nucleus. Minutes

5a

10

20

120

240

0 5 10 20 120 240

0.02

<0.01 <0.01

0.21 0.07 <0.01

0.02 0.49 <0.01 0.04

<0.01 0.03 0.06 <0.01 0.04

a

Time of leptin treatment in minutes.

treatment tended to alter (P ¼ 0.07) the phosphorylation of HSL Ser563 with increased phosphorylation in adipocytes treated with 100- and 200-ng/mL leptin. Phosphorylation of HSL Ser565 and the abundance of HSL, and STAT3 proteins were not altered with leptin treatment (P > 0.10, Fig. 2C,D). The highly significant lipolytic effect of isoproterenol was confirmed by increased glycerol (P < 0.01) and NEFA (P < 0.01) concentrations (Fig. 3A,B) in media from bovine adipocytes treated with isoproterenol. This lipolytic response was accompanied by increased phosphorylation of HSL Ser563 (P < 0.01). Protein abundance of ATGL tended to increase with isoproterenol treatment (P ¼ 0.08). Protein abundance of HSL and STAT3 was unchanged with isoproterenol treatment (P > 0.05). Phosphorylation of STAT3 Tyr705 and HSL Ser565 was also unresponsive to isoproterenol (P > 0.05). Changes in protein and phosphoprotein abundance are shown in Figure 3C, and P-values for main effects and comparisons with controls are listed in Tables 3 and 4. 3.2. Translocation of lipases to the lipid droplet after 2 h of exposure to isoproterenol and leptin To determine if protein or phosphoprotein abundance corresponds with translocation of proteins to the lipid droplets, bovine SVC-derived adipocytes were cultured for 2 h with either vehicle, 100 nM of isoproterenol, or 100 ng/ mL of bovine leptin before immunostaining. Translocation of HSL was different between treatments (P < 0.01 lipolytic treatment; Fig. 4A,B), where isoproterenol increased the translocation of HSL to the lipid droplet compared with control and leptin-treated SVC-derived bovine adipocytes (P < 0.01, and P < 0.01, respectively). Translocation of ATGL was different between treatments (P ¼ 0.03 lipolytic treatment; Fig. 5A,B) where isoproterenol and leptin increased ATGL translocation to the lipid droplet compared with control SVC-derived bovine adipocytes (P ¼ 0.01 and P ¼ 0.04, respectively). Lipid droplet diameters were similar for all treatments for HSL (P < 0.40; Fig. 4C) and tended to be different between treatments for ATGL (P < 0.08; Fig. 5C). 3.3. Timing phosphorylated STAT3 translocation to the nucleus with leptin treatment

in

To determine the timing of the leptin-signaling cascade bovine SVC-derived adipocytes, colocalization of

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phosphorylated STAT3 and nuclei was determined. Leptin treatment altered the colocalization of phosphorylated STAT3 and nuclei (P ¼ 0.01, Fig. 6). Colocalization between phosphorylated STAT3 and nuclei increased after 5, 10, 120, and 240 min of exposure to leptin compared with 0 min of exposure. Colocalization increased at 10 and 240 min compared with 5 and 120 min, with colocalization tending to be greater at 10 min compared with 240 min. P-values for pairwise comparisons are listed in Table 5. 3.4. Timing of the translocation of adipose triglyceride lipase and its cofactors to the lipid droplet with leptin treatment The lipid droplet diameter for SVC-derived bovine adipocytes were similar for all timepoints (P ¼ 0.21). Translocation of ATGL to the lipid droplet was altered with leptin administration over time (P < 0.01 for leptin treatment, P ¼ 0.80 for background fluorescence, P ¼ 0.82 for lipid droplet diameter; Figs. 7 and 8) with increased translocation of ATGL at 120 min of exposure to bovine leptin compared with all other timepoints. Exposure to leptin did not alter ATGL translocation at 5, 10, and 20 min compared with 0 min of leptin exposure. Translocation of the inhibitor of ATGL, G0S2, to the lipid droplet was not altered with leptin exposure (P ¼ 0.17 for leptin treatment, P ¼ 0.12 for background fluorescence, P ¼ 0.14 for lipid droplet diameter; Fig. 7). Translocation of ABHD5 to the lipid droplet tended to be altered by leptin exposure (P ¼ 0.08 for leptin treatment, P ¼ 0.44 for background fluorescence, P ¼ 0.20 for lipid droplet diameter; Fig. 8). Translocation of ABHD5 increased at 5, 20, and 120 min of leptin exposure compared with control treated SVC-derived bovine adipocytes. P-values for the pairwise comparisons are listed in Tables 6 and 7. 4. Discussion The present study establishes several key principles with respect to the regulation of lipolysis in isolated bovine adipocytes and SVC-derived bovine adipocytes. Although leptin-stimulated lipolysis has been confirmed in human, rodents, and porcine adipocytes, this is the first report in cattle and the first evidence of leptin-stimulated lipolysis in adipocytes in ruminants. Newby et al [29] were unable to detect leptin-stimulated lipolysis in ovine adipose tissue explants or STAT3 and STAT5 activity after the treatment with leptin, despite previous evidence of leptin reducing feed intake when given intracerebrally [19]. Therefore, it was unclear if leptin signaling in ruminant isolated adipocytes were similar to other species. However, the increase in glycerol concentrations, phosphorylation of STAT3, and its translocation to the nuclei with leptin treatment would confirm the ability of isolated bovine adipocytes and SVCderived bovine adipocytes to undergo leptin-stimulated lipolysis. Consistent with rodent and porcine adipocytes, leptin-stimulated lipolysis is observed by an increase in glycerol release, but not NEFA [25,28]. Wang et al [28] observed elevated transcript abundances of important regulators of b-oxidation suggesting that lack of a NEFA response would indicate oxidation of the newly liberated fatty acids by the mitochondria. In pigs, it has been

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Fig. 7. Time course of adipose triglyceride lipase (ATGL) and G0/G1 switch protein 2 (G0S2) translocation. N ¼ 12–15 lipid droplets from 2 cultures per treatment. (A) Representative images of SVC-derived adipocytes treated with 100 ng/mL of leptin over the course of 2 h. Time of exposure is listed on the left hand side. In the merged image, blue represents ATGL, green represents lipid droplets, and red represents G0S2. (B) Average lipid droplet diameter SEM. (C) Average translocation of ATGL and G0S2 to the lipid droplet SEM. The white line in the lower right hand image is 10 mm relative to the view. Differences in subscripts were given when P < 0.05 and indicate pairwise differences of P < 0.05. SEM, standard error of the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

reported that leptin-treated adipocytes will increase glycerol concentration in the media, but not fatty acids into the media [25]. In both rodents and pigs in vivo, there is an increase fatty acid oxidation following administration of leptin [38–40] despite differences in circulating free fatty acids concentrations being decreased in rodent [39] but elevated in pigs [38]. These similarities between in vivo and in vitro models would suggest the leptin can induce a mild lipolytic response in vivo, but other factors may influence circulating factors, such as NEFA.

Given the small increase in lipolysis with high doses of leptin seen in this study and others [26,28,38], it is unlikely that leptin plays a significant role in mobilizing energy when significant energy deficits are encountered. It is more likely that leptin contributes to the regulation of lipid stores and body condition when energy balance is positive. Circulating concentrations of leptin are on average lower (w10 ng/mL [41–44]) than concentrations required to stimulate lipolysis in bovine adipocytes. Slightly higher than circulating concentrations are also required to induce lipolysis by leptin in human [45], pig [25,26], and mice [28].

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Fig. 8. Time course of adipose triglyceride lipase (ATGL) and a/b hydroxylase domain containing 5 (ABHD5) translocation. N ¼ 12–15 lipid droplets from 2 cultures per treatment. (A) Representative images of SVC-derived adipocytes treated with 100 ng/mL of leptin over the course of 2 h. Time of exposure is listed on the left hand side. In the merged image, blue represents ATGL, green represents bodipy, and red represents ABHD5. (B) Average translocation of ATGL to the lipid droplet for both images that contained ABHD5 and G0S2 SEM. The white line in the lower right hand image is 10 mm relative to the view. Differences in subscripts were given when P < 0.05 and indicate pairwise differences of P < 0.05. SEM, standard error of the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

However, if intra-adipose tissue concentrations of leptin exceed circulating concentrations, particularly as expression increases with accumulating body fat, this would support the potential role for leptin in the regulation of adiposity during positive energy balance. To date, little is understood about intra-adipose tissue concentrations of leptin, and it is unclear if concentrations can reach levels required to induce lipolysis in vivo. Mechanisms of leptin-stimulated lipolysis remain unclear at the level of the lipase activity. This study explored

the potential contribution of two known lipolytic cascades, PKA and AMPK. The primary pathway known to initiate lipolysis is the PKA pathway. It is well recognized in many species that the activation of PKA by b-agonists increases lipolysis via phosphorylation of perilipin along with the phosphorylation and translocation of HSL and ATGL [4,8,46]. This study observed many of the characteristic measurements of lipolysis via PKA (increase in phosphorylation of HSL Ser563, increases in protein abundance of ATGL, and the translocation of ATGL and HSL to the lipid

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Table 6 P-values for pairwise comparisons for adipose triglyceride lipase and G0/ G1 switch protein 2 translocation to the lipid droplet. Minutes 0 5 10 20 120

0a

5 0.55

0.96c 0.27 0.09 0.47

b

0.32 0.11 0.47

10

20

120

0.77 0.38

0.37 0.78 0.22

0.01 <0.01 0.01 <0.01

0.51 0.08

0.02

a

Time of leptin treatment in minutes. P-values above the diagonal are pairwise comparison for adipose triglyceride lipase. c P-values below the diagonal are pairwise comparison for G0/G1 switch protein 2. b

droplet) in bovine adipocytes and SVC-derived adipocytes with isoproterenol treatment, suggesting bovine adipocytes use similar a mechanism for lipolysis as human, rodents, and pigs adipocytes. However, when adipocytes were exposed to leptin, there was a lack of phosphorylation of HSL Ser563. In addition, SVC-derived bovine adipocytes lacked the translocation of HSL to the lipid droplet in response to leptin treatment. This is not surprising as leptin has been shown to prevent the activation of the PKA pathway in a breast cancer cell line [47]. Protein abundance of ATGL and the translocation of ATGL to the lipid droplet increased with leptin treatment. The potential activation of ATGL during this mild lipolysis is unlikely to be activated by PKA since other members of the cascade were not activated, and others have shown leptin to inhibit the PKA pathway [47]. Although controversial, AMPK may initiate mild lipolytic activity in adipocytes. Activation of AMPK results from the accumulation of intracellular AMP resulting in the downstream activation of HSL and ATGL through phosphorylation [13,15]. This study confirms the exclusivity of the PKA and AMPK pathway through the lack of phosphorylation of HSL Ser565 during isoproterenol treatment. In addition, treatment of bovine adipocytes with leptin did not increase the phosphorylation of HSL Ser565, suggesting that leptin may not use AMPK to stimulate lipolysis. This study did not look at the phosphorylation of ATGL by AMPK because at the time of this study, a commercial antibody for phosphorylated ATGL was not available, and additional studies are needed to exclude AMPK as a potential downstream messenger during leptinstimulated lipolysis. Granneman et al [46] reported an increase in the abundance of HSL and ATGL at the lipid droplet following treatment with forskolin and IBMX as quickly

Table 7 P-values for pairwise comparisons for a/b hydroxylase domain containing 5 translocation to the lipid droplet. Minutes 0 5 10 20 a

0a

5

10

20

120

0.03

0.18 0.27

0.01 0.73 0.14

0.03 0.63 0.40 0.40

Time of leptin treatment in minutes.

as 8 min following treatment in 3T3-L1 adipocytes. Therefore, this study wanted to determine the timing of translocation to the nuclei or lipid droplet following leptin treatment in SVC-derived bovine adipocytes. In bovine SVC-derived adipocytes, STAT3 translocated to the nucleus following 5 min of exposure to leptin; however, the translocation of ATGL to the lipid droplet did not occur within the first 10 min, as was observed by Granneman et al [46]. Increased time required for translocation of ATGL compared with adipocytes treated with forskolin or for phosphorylated STAT3 to translocate to the nucleus suggests that phosphorylated STAT3 does not directly activate ATGL. Although translocation was increased at 120 min of leptin exposure, translocation could have started anytime between 20 and the 120 min sampling, thus making it possible for lipoltyic pathways to be indirectly activated by STAT3. The lack of consistency between translocation of ABHD5 from the lipid droplet and of HSL translocation indicates different mechanism in catecholamine-induced and leptin-induced lipolysis. Additional studies are needed to determine the potential roles of ERK or AMPK pathways in leptin-induced lipolysis and their contribution to the translocation of ATGL to the lipid droplet. In addition, it is important to determine if STAT3 acts directly to increase the phosphorylation of ATGL, or through increased transcription of ATGL and subsequently translation of ATGL. This increase in transcription of ATGL may occur at a greater rate than that of its inhibitor, G0S2, allowing for lipolysis. In porcine SVCderived adipocytes, ATGL transcript abundances increased with 3 h of leptin treatment [25], suggesting this could be a mechanism by which leptin regulates ATGL. However, they reported decreased protein abundance [25]. In conclusion, this study confirms that leptin elicits a mild lipolytic response in bovine adipocytes characterized by increased release of glycerol, but not NEFA, presumably due to a concomitant increase in b-oxidation of fatty acids. Activation of the leptin pathway was confirmed by increases in phosphorylated STAT3 and its subsequent translocation to the nucleus following leptin treatment. Protein abundance, phosphorylation, and location of known lipolytic proteins were unaltered by leptin exposure, except for ATGL. The increase in protein abundance and translocation to the lipid droplet following leptin treatment would suggest its involvement in leptinstimulated lipolysis. In addition, translocation of ATGL did not occur until 2 h following leptin treatment, well past peak translocation of phosphorylated STAT3 to the nucleus. The increase in ATGL protein abundance, localization of ATGL at the lipid droplet, the lack of involvement of other PKA proteins, and time required for ATGL localization led us to hypothesize that leptin activates STAT3 which activates transcription and subsequent translation of ATGL. Acknowledgments This project was supported by National Research Initiative Competitive grant no. 2009-35206-05222 from

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the USDA Cooperative State Research, Education, and Extension Service. The authors would like to thank J. Selsby and M. Persia at Iowa State University for use of the microscope, and J. Reecy at Iowa State University for scientific discussions.

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