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Journal of Animal Science - Animal Nutrition

Changes in lipid metabolism and β-adrenergic response of adipose tissues of periparturient dairy cows affected by an energy-dense diet and nicotinic acid supplementation1

 

This article in JAS

  1. Vol. 93 No. 8, p. 4012-4022
     
    Received: Dec 19, 2014
    Accepted: June 09, 2015
    Published: July 24, 2015


    3 Corresponding author(s): korinna.huber@uni-hohenheim.de
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doi:10.2527/jas.2014-8833
  1. Á. Kenéz*22,
  2. R. Tienken22,
  3. L. Locher,
  4. U. Meyer,
  5. A. Rizk§,
  6. J. Rehage#,
  7. S. Dänicke and
  8. K. Huber 3*
  1. * Institute of Animal Sciences, University of Hohenheim, 70599 Stuttgart, Germany
     Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, 38116 Braunschweig, Germany
     Clinic for Ruminants with Ambulatory and Herd Health Services at the Center of Veterinary Clinical Medicine, LMU Munich, 85764 Oberschleissheim, Germany
    § Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt
    # Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, 30173 Hannover, Germany

Abstract

Dairy cattle will mobilize large amounts of body fat during early lactation as an effect of decreased lipogenesis and increased lipolysis. Regulation of lipid metabolism involves fatty acid synthesis from acetate and β-adrenergic-stimulated phosphorylation of hormone-sensitive lipase (HSL) and perilipin in adipocytes. Although basic mechanisms of mobilizing fat storage in transition cows are understood, we lack a sufficiently detailed understanding to declare the exact regulatory network of these in a broad range of dairy cattle. The objective of the present study was to quantify 1) protein abundance of fatty acid synthase (FAS), 2) extent of phosphorylation of HSL and perilipin in vivo, and 3) β-adrenergic stimulated lipolytic response of adipose tissues in vitro at different stages of the periparturient period. We fed 20 German Holstein cows an energy-dense or an energetically adequate diet prepartum and 0 or 24 g/d nicotinic acid (NA) supplementation. Biopsy samples of subcutaneous and retroperitoneal adipose tissue were obtained at d 42 prepartum (d −42) and at d 1, 21, and 100 postpartum (d +1, d +21, d +100, respectively). To assess β-adrenergic response, tissue samples were incubated with 1 μM isoproterenol for 90 min at 37°C. The NEFA and glycerol release, as well as HSL and perilipin phosphorylation, was measured as indicators of in vitro stimulated lipolysis. In addition, protein expression of FAS and extent of HSL and perilipin phosphorylation were measured in fresh, nonincubated samples. There was no effect of dietary energy density or NA on the observed variables. The extent of HSL and perilipin phosphorylation under isoproterenol stimulation was strongly correlated with the release of NEFA and glycerol, consistent with the functional link between β-adrenergic-stimulated protein phosphorylation and lipolysis. In the nonincubated samples, FAS protein expression was decreased at d +1 and d +21, whereas HSL and perilipin phosphorylation increased from d −42 to d +1 and remained at an increased level throughout the first 100 d of lactation. In vitro lipolytic response was significant in prepartum samples at times when in vivo lipolysis was only minimally activated by phosphorylation. These data extend our understanding of the complex nature of control of lipolysis and lipogenesis in dairy cows and could be useful to the ongoing development of systems biology models of metabolism to help improve our quantitative knowledge of the cow.



INTRODUCTION

Dairy cows mobilize triglycerides (TG) from adipose tissues to overcome energy deficiency during early lactation (McNamara, 1994; Tamminga et al., 1997). Even though excessive rates of lipid mobilization are seen as a major causative factor in the etiology of postpartum metabolic disturbances, we still do not fully understand the extent, timing, and mechanism of control of adipose metabolism in a variety of animals and situations.

The increased lipid mobilization is a function of upregulation of adipose tissue lipolysis as well as downregulation of lipogenesis (Rocco and McNamara, 2013). However, these systems are substantially influenced by a number of nutritional and genetic factors, such as prepartum energy intake and body condition (Kokkonen et al., 2005; McNamara, 2012; Schulz et al., 2014). Our hypothesis was that the metabolic pathways of adipose lipolysis and lipogenesis during early lactation could be influenced by a targeted dietary energy supply, leading to a more severe increase of lipolysis and further downregulation of lipogenesis in cows that were overconditioned prepartum. Additionally, dietary supplemented nicotinic acid (NA), a known antilipolytic agent (Kenéz et al., 2014), could attenuate postpartum lipid mobilization, preventing it from reaching excessive rates, by downregulating the adipose lipolytic pathway.

Therefore, the present study was performed on periparturient cows receiving an energy-dense or an energetically adequate diet with or without NA supplementation. Under these dietary influences, our study aimed to assess 1) in vivo lipogenesis by measuring fatty acid synthase (FAS) protein expression, 2) in vivo lipolysis by measuring hormone-sensitive lipase (HSL) and perilipin protein phosphorylation, and 3) in vitro lipolytic response to a defined β-adrenergic stimulation at 4 different time points in the course of the periparturient period in subcutaneous (SCAT) and retroperitoneal (RPAT) adipose tissues of cows.


MATERIALS AND METHODS

Animals, Feeding, and Sampling

This study was performed on 20 multiparous German Holstein cows in agreement with the German Animal Welfare Act and approved by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES; Oldenburg, Germany). Cows were selected for this study to achieve homogeneity in BW, BCS, and milk yield of previous lactation to attenuate possible effects of different condition and merit. Cows were kept in a free-stall housing system at the Institute of Animal Nutrition, Federal Research Institute for Animal Health (Friedrich-Loeffler-Institut, Braunschweig, Germany), were clinically healthy, and were dried off 8 wk before parturition. The experimental period started when cows reached 42 d prepartum (d −42) and lasted until 100 d postpartum (d +100).

Diets were formulated according to the recommendations of the Society of Nutrition Physiology (Frankfurt am Main, Germany). Before d −42, animals were fed a grass silage–based diet, and thereafter, they were randomly assigned to 1 of 4 treatments arranged in a 2 × 2 factorial design. The experimental factors were low-concentrate (LC) or high-concentrate (HC) diet and NA supplementation (0 or 24 g/d per cow). In the prepartum period, the diet consisted of 30% grain-based concentrate and 70% silage-based roughage mixture for the LC group and 60% concentrate and 40% roughage mixture for the HC group. The roughage mixture was composed of 50% corn silage and 50% grass silage on a DM basis. At parturition, the concentrate proportion was altered to 30% in both groups. Postpartum, concentrate proportion was continuously elevated to 50% within the first 16 d and first 24 d in the LC and HC groups, respectively. Afterward, the concentrate proportion was maintained at 50% until the end of the study. Cows were fed for ad libitum intake and had free access to water. This feeding regimen was designed on the basis of previous studies performed at the same institution, targeting overfeeding in the dry period to promote adipose tissue anabolism (Petzold et al., 2014; Schulz et al., 2014). Nicotinic acid was supplemented from d −42 to d +24 to the respective groups in form of non-rumen-protected NA (Mianyang Vanetta Pharmaceutical Technology Co. Ltd., Sichuan, China) mixed into 1 kg concentrate feed at 24 g/d per cow. The other 2 groups received 1 kg of control concentrate in the same period. This relatively high dose of NA was chosen on the basis of previous feeding trials studying the antilipolytic potential of NA using either a non-rumen-protected or encapsulated form (Schultz, 1971; Fronk and Schultz, 1979; Dufva et al., 1983; Niehoff et al., 2009; Morey et al., 2011; Yuan et al., 2012). Dry matter intake and milk yield were recorded, and NE balance was calculated according to the guidelines of the Society of Nutrition Physiology.

At d −42, d +1, d +21, and d +100, biopsy samples of SCAT and RPAT were collected according to McNamara and Hillers (1986) and Locher et al. (2012). Before biopsy sampling, a blood sample was taken by jugular venipuncture to determine plasma NEFA concentrations. After preparation of the surgical field and local infiltration anesthesia induced with procaine (Procaine 2%; Selectavet Dr. Otto Fischer GmbH, Weyarn-Holzolling, Germany), samples from adipose tissues were collected under aseptic conditions. A 2-cm skin incision was made in the region of the tail head on alternating sides (right and left) to obtain SCAT. For collection of RPAT, a 3- to 5-cm skin incision was made in the angle between the lumbar transversal processes and the iliac bone, muscles were dissected reaching the peritoneum, and adipose tissue samples were taken directly above the peritoneum. Biopsies of RPAT were obtained each time alternating from the left and right flank. Skin incisions were closed with a horizontal interrupted mattress suture pattern (Filovet; Wirtschaftsgenossenschaft Deutscher Tierärzte, Garbsen, Germany). After removal, tissue samples were trimmed of connective tissue and rinsed thoroughly in physiological saline solution to reduce blood contamination.

One part of each tissue sample was immediately snap frozen in liquid nitrogen and stored thereafter at −80°C until Western blot analyses to detect protein expression levels present in vivo. The other part of the tissue samples was used to perform an in vitro lipolysis assay detecting lipolytic response to β-adrenergic stimulation.

Measures of Lipogenesis and Lipolysis in Nonincubated Adipose Tissues

The SCAT and RPAT biopsy samples obtained from all 20 cows at d −42, d +1, d +21, and d +100 were prepared for Western blot analyses. Protein extraction was performed in a lysis buffer containing 50 mM HEPES (Carl Roth GmbH, Karlsruhe, Germany), 4 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (Sigma-Aldrich, St. Louis, MO), 10 mM EDTA (Sigma-Aldrich), 0.1% Triton X-100 (Sigma-Aldrich), 100 mM β-glycerol phosphate (Sigma-Aldrich), 15 mM sodium pyrophosphate (Sigma-Aldrich), 5 mM sodium orthovanadate (Sigma-Aldrich), 2.5 mM sodium fluoride (Sigma-Aldrich), a protease inhibitor cocktail (CompleteMini, Roche Diagnostics GmbH, Mannheim, Germany), and a phosphatase inhibitor cocktail (PhosStop, Roche Diagnostics GmbH). Extracts were centrifuged at 10,000 × g for 10 min at 4°C, and aliquots of the supernatants were stored at −20°C until electrophoresis. Protein concentrations were measured using Bradford reagent (Serva Electrophoresis GmbH, Heidelberg, Germany). Protein extracts were diluted to 0.5 mg/mL in loading buffer (50 mM Tris-HCl [Sigma-Aldrich], 10% glycerol [Sigma-Aldrich], 2% SDS [Serva Electrophoresis GmbH], 0.1% bromophenol blue [Sigma-Aldrich], 2% mercaptoethanol [Sigma-Aldrich]; final concentrations) and heated at 95°C for 5 min. Ten micrograms protein per lane were separated in a Tris-glycine buffer according to Laemmli (1970) by SDS-PAGE on 8.1% handcast gels and transferred to nitrocellulose membranes. Membranes were blocked in a PBS-based solution containing 5% fat-free milk powder (Carl Roth GmbH) and 0.1% Tween 20 (Sigma-Aldrich) for 1 h at room temperature.

As a marker of lipogenesis, the protein abundance of FAS was assessed. Furthermore, 2 markers of adipocyte lipolysis were HSL phosphorylation and perilipin phosphorylation. Also, the total protein abundance of HSL and perilipin was quantified. To detect changes in β2-adrenergic receptor expression and desensitization, the total expression of the receptor protein and its phosphorylation was measured. Technically, blocked membranes were incubated overnight at 4°C with the corresponding primary antibodies: FAS (dilution 1:500; Sigma-Aldrich), total HSL (dilution 1:5,000; Cell Signaling Technology, Danvers, MA), HSL phosphorylated at Ser-563 (dilution 1:1,000; Cell Signaling Technology), total perilipin (dilution 1:2,000; Chemicon International, Temecula, CA), perilipin phosphorylated at Ser-522 (dilution 1:5,000; Vala Sciences, San Diego, CA), total β2-adrenergic receptor (dilution 1:600; Santa Cruz Biotechnology, Santa Cruz, CA), β2-adrenergic receptor phosphorylated at Ser-345/Ser-346 (dilution 1:600; Santa Cruz Biotechnology). Afterward, membranes were incubated with the matching secondary antibody (anti-rabbit IgG, horseradish peroxidase [HRP]-linked, dilution 1:2,500 [Cell Signaling Technology] or anti-mouse IgG, peroxidase conjugate, dilution 1:50,000 [Sigma-Aldrich]) at room temperature for 1 h. Immunodetection was performed by incubating the membranes with LumiGlo reagent (Cell Signaling Technology) or WesternBright Sirius (Advansta Corporation, Menlo Park, CA), and chemiluminescence was detected by a ChemiDoc XRS+ system (Bio-Rad Laboratories GmbH, Munich, Germany). Bands were quantified by densitometry using Image Lab 4.0 software (Bio-Rad Laboratories GmbH). Finally, membranes were india ink stained (Pelikan PBS, Peine, Germany) to control equal loading. Quantities of phospho-HSL, phospho-perilipin, and phospho-β2-adrenergic receptor were normalized for total quantity of the corresponding protein detected in the same sample. These normalized values are referred to as “extent of phosphorylation” throughout.

In Vitro Lipolysis Assay

Subcutaneous adipose tissue and RPAT biopsy samples of all 20 cows at d −42, d +1, d +21, and d +100 were processed in an in vitro lipolysis assay to assess lipolytic response at different stages of lactation. Lipolytic response was triggered with the selective β-adrenergic agonist isoproterenol, according to the method of McNamara and Hillers (1986) with modifications described below. Tissue samples (approximately 100 mg) were preincubated in Dulbeccos’s Modified Eagle’s Medium (DMEM; Life Technologies Corporation, Paisley, UK) containing 2% FA-free BSA (Sigma-Aldrich) for 20 min at 37°C to diminish unspecific release of metabolites due to handling and cutting. Each sample was then incubated separately in 1 mL of freshly prepared DMEM containing 2% FA-free BSA (incubation medium) for 90 min at 37°C with gentle shaking. Incubation medium was supplemented with 0 or 1 μM isoproterenol (Sigma-Aldrich) to establish a basal (control) and a stimulated incubation set, respectively. Incubation sets were performed in triplicate. Following incubation, tissue samples were briefly drained, snap frozen, and weighed and afterward were stored at −80°C until Western blot analyses. Incubation media were stored at −20°C until further biochemical analysis.

To assess lipolytic response of the incubated tissue samples, glycerol concentration (Free glycerol reagent, Sigma-Aldrich) and NEFA concentration (NEFA-HR (2), Wako Chemicals GmbH, Neuss, Germany) in the incubation media were measured. Concentrations of glycerol and NEFA were corrected for wet weight and expressed as micromole of metabolite per gram of tissue per 90 min of incubation. Additionally, to monitor the effect of β-adrenergic stimulation on the activation of lipolytic proteins, the extent of HSL phosphorylation and perilipin phosphorylation were detected in the incubated tissue samples in the same way as in the nontreated tissues (described above).

Experimental Design and Statistical Analysis

The feeding trial was designed as a completely random design with a 2 × 2 arrangement of dietary treatments and with repeated measures. Indicative variables of performance (DMI, NE balance, milk yield, plasma NEFA concentration), data for in vivo and in vitro protein expression and phosphorylation, and data for in vitro glycerol and NEFA release were analyzed by ANOVA. The main effects of tissue (SCAT, RPAT), time related to parturition (d −42, d +1, d +21, d +100), and diet (LC without NA, LC with NA, HC without NA, HC with NA) were evaluated, along with all the corresponding interactions. Effects were declared significant when P values were ≤0.05. Statistical analyses were performed in Prism GraphPad 6.0 (GraphPad Software, Inc., La Jolla, CA). In the case of the glycerol and NEFA release data, the mean of the triplicate measurement was used. Because the proportion of concentrate and NA supplementation were found to have no significant impact on the studied variables, data for the dietary groups were pooled together, and the ANOVA was repeated without these main effects and interactions. Statistical evaluation and P values of this second analysis are indicated throughout the text. Differences between d −42, d +1, d +21, and d +100 were compared with Tukey’s multiple comparisons test.

To confirm the functional link between protein phosphorylation and lipolytic release, the correlation between in vitro HSL phosphorylation, perilipin phosphorylation, NEFA release, and glycerol release was calculated. This was done separately for basal incubation conditions and isoproterenol-stimulated incubation conditions, both for SCAT and for RPAT, collectively for d −42, d +1, d +21, and d +100.


RESULTS

Dietary Effects

Indicative data on DMI, NE balance, milk yield, and plasma NEFA concentration, grouped according to feeding regimens at time points corresponding to biopsy samplings, are shown in Fig. 1. All variables, including lipolytic measures evaluated in the current study, remained unaffected by dietary manipulations. Therefore, dietary groups were pooled together, and further results are presented collectively for all 20 cows.

Figure 1.
Figure 1.

(A) Dry matter intake, (B) NE balance, (C) milk yield, and (D) plasma NEFA concentration as indicative measures of production performance. Data are shown separately for the feeding groups at corresponding time points to biopsy samplings. Variables did not significantly differ among feeding groups. Time-related changes were highly significant in every case (P < 0.001; ANOVA). LC-con = low concentrate level, no nicotinic acid supplementation; LC-nia = low concentrate level, with nicotinic acid supplementation; HC-con = high concentrate level, no nicotinic acid supplementation; HC-nia = high concentrate level, with nicotinic acid supplementation. Shown are means ± SEM, N = 20.

 

Markers of Lipogenesis and Lipolysis in Nonincubated Adipose Tissues

Fatty acid synthase protein expression was decreased at d +1 compared with d −42 and was further decreased at d +21 in both SCAT and RPAT (P < 0.001), as shown in Fig. 2. At d +100, expression returned to the prepartum level.

Figure 2.
Figure 2.

Total expression of fatty acid synthase (FAS) in fresh, nonincubated samples of subcutaneous (SCAT) and retroperitoneal (RPAT) adipose tissue of cows between d 42 prepartum and d 100 postpartum. a–cQuantities of expression were different at days marked with different letters (P < 0.001; Tukey’s posttest). Exemplary Western blot images of an SCAT sample at the 4 different time points are shown at the top. Shown are means ± SEM, N = 20.

 

Total protein expression of HSL and perilipin was maintained at a constant level throughout the experimental period, except for at d +21 in RPAT, when HSL expression was increased (P < 0.05; Fig. 3). The extent of HSL and perilipin phosphorylation varied significantly over time (P < 0.001), as shown in Fig. 3. Phosphorylation was the lowest on d −42 in the case of both proteins in SCAT and in RPAT as well. After parturition, a remarkable increase in the phosphorylation of these proteins was observed.

Figure 3.
Figure 3.

Total expression of (A) hormone-sensitive lipase (HSL) and (C) perilipin and extent of phosphorylation of (B) HSL and (D) perilipin in fresh, nonincubated samples of subcutaneous (SCAT) and retroperitoneal (RPAT) adipose tissue of cows between d 42 prepartum and d 100 postpartum. The extent of phosphorylation displays a ratio of absolute values of phosphoprotein to the total amounts of the protein. a–cQuantities of expression or phosphorylation were different at days marked with different letters (P < 0.05; Tukey’s posttest). Exemplary Western blot images of an SCAT sample at the 4 different time points are shown at the top. Shown are means ± SEM, N = 20.

 

The total protein expression of the β2-aderenergic receptor showed remarkable changes over time (P < 0.001) because it was strongly decreased at d +1. Concurrently, the extent of receptor phosphorylation was the greatest on d +1 (P < 0.001), as shown in Fig. 4.

Figure 4.
Figure 4.

(A) Total expression and (B) extent of phosphorylation of β2-adrenergic receptor in fresh, nonincubated samples of subcutaneous (SCAT) and retroperitoneal (RPAT) adipose tissue of cows between d 42 prepartum and d 100 postpartum. Extent of phosphorylation displays a ratio of absolute values of phosphoprotein to the total amounts of the protein. a,bQuantities of expression or phosphorylation were different at days marked with different letters (P < 0.05; Tukey’s posttest). Exemplary Western blot images of an SCAT sample at the 4 different time points are at the top. Shown are means ± SEM, N = 20.

 

The ANOVA revealed a significant effect of tissue depot in the case of total β2-aderenergic receptor expression (P = 0.04) and in total HSL expression (P = 0.01). With regard to all other expression and phosphorylation data, SCAT and RPAT did not differ significantly.

In Vitro Lipolysis Assay

The in vitro isoproterenol treatment triggered an increase in each of the studied variables (release of NEFA and glycerol, phosphorylation of HSL and perilipin) at all time points compared with the basal incubation conditions.

Basal release of glycerol was continually low during the whole experimental period; however, a significant time effect (P < 0.001) was observed as release rates were greater on d −42 than after parturition (Fig. 5A). Isoproterenol-treated samples released the greatest amount of glycerol on d −42. Besides a significant time effect (P < 0.001), a tissue effect was detected (P = 0.005), and a significant interaction was also found between time and tissue (P = 0.01), indicating that RPAT released more glycerol than SCAT on d −42, but postpartum this difference was not perpetuated.

Figure 5.
Figure 5.

Release of (A) glycerol and (B) NEFA and extent of (C) hormone-sensitive lipase (HSL) phosphorylation and (D) perilipin phosphorylation in in vitro incubated samples of subcutaneous (SCAT) and retroperitoneal (RPAT) adipose tissue of cows between d 42 prepartum and d 100 postpartum. Tissues were incubated for 90 min under basal conditions (circles) and under treatment with 10−6 M isoproterenol (triangles). Extent of phosphorylation displays a ratio of absolute values of phosphoprotein to the total amounts of the protein. a–cQuantities of release or phosphorylation were different at days marked with different letters (P < 0.05; Tukey’s posttest). Exemplary Western blot images of an SCAT sample at the 4 different time points are shown above (C) and (D) (B = basal, I = isoproterenol). Shown are mean ± SEM, N = 20.

 

The NEFA release in the basal incubation sets showed differences over time (P < 0.001) because on d +1 and d +21 release rates were higher than on d −42 and d +100 (Fig. 5B). Isoproterenol treatment triggered the greatest NEFA release at d −42. Besides the time-dependent differences (P < 0.001), a tissue effect was also detected (P < 0.001) as RPAT had greater release rates than SCAT prepartum. Also, a significant interaction between time and tissue was found (P = 0.001), similar to the dynamics seen in glycerol release.

The extent of HSL phosphorylation (Fig. 5C) was continually low in the basal incubation set at all time points; however, it still showed some significant changes over time (P = 0.03). Isoproterenol treatment triggered the greatest extent of HSL phosphorylation on d −42, whereas values of d +1, d +21, and d +100 were considerably lower. Time-specific (P < 0.001) but not tissue-specific differences could be observed in HSL phosphorylation of the isoproterenol-treated tissues.

In case of perilipin, basal incubation did not provide as markedly low phosphorylation as seen in the case of HSL. As shown in Fig. 5D, basal phosphorylation was slightly increased during early lactation, whereas isoproterenol-stimulated phosphorylation was the greatest at d −42 in SCAT. Time-related differences were significant under both basal (P = 0.01) and isoproterenol-treated (P = 0.04) conditions. The latter can be attributed mostly to variation of SCAT because RPAT remained relatively stable. Still, SCAT and RPAT were not found to be statistically different.

Under basal incubation conditions, many of the markers of in vitro lipolysis were correlated with each other, and under isoproterenol treatment, correlations between all studied markers were highly significant (Table 1).


View Full Table | Close Full ViewTable 1.

Correlation coefficients r for the extent of hormone-sensitive lipase (HSL) and perilipin phosphorylation (-P) and NEFA and glycerol release under basal and isoproterenol-stimulated incubation conditions (collectively for d 42 prepartum and d 1, 21, and 100 postpartum)1

 
HSL-P Perilipin-P NEFA Glycerol
Tissues incubated under basal conditions
HSL-P 0.01 0.17 0.46***
Perilipin-P 0.23* 0.15 0.03
NEFA 0.32** 0.31** 0.33**
Glycerol 0.57*** −0.04 0.21
Tissues with induced lipolysis2
HSL-P 0.33** 0.37*** 0.54***
Perilipin-P 0.43*** 0.39*** 0.55***
NEFA 0.53*** 0.47*** 0.66***
Glycerol 0.54*** 0.42*** 0.86***
P < 0.1.
*P < 0.05.
**P < 0.01.
***P < 0.001.
1Coefficients above the diagonal stand for subcutaneous adipose tissue, and those below stand for retroperitoneal adipose tissue.
2Incubation with 1 μM isoproterenol.


DISCUSSION

Lipolysis as an adaptive mechanism to compensate for a negative energy balance is a central pathway within the metabolic network of energy homeostasis. Activation of lipolysis involves phosphorylation of HSL and perilipin as key molecular targets (Garton et al., 1988; McNamara, 1991; Elkins and Spurlock, 2009; Koltes and Spurlock, 2011; Locher et al., 2011; Rocco and McNamara, 2013), which is induced by catecholamines via the β-adrenoceptor– cyclic AMP (cAMP)–protein kinase A (PKA) cellular regulatory axis (for a review, see Holm et al., 2000). Up to now, functional response such as glycerol or NEFA release from bovine adipose tissues or cells to catecholamine stimulation has shown consistent changes in lipolysis during early lactation but with wide variation among animals and studies (Smith and McNamara, 1989; Rukkwamsuk et al., 1998; Sumner and McNamara, 2007; Khan et al., 2013; Kenéz et al., 2014).

The novelty of the current study lies in demonstrating 1) the functional and dynamic link between HSL and perilipin phosphorylation and NEFA and glycerol release using the same adipose tissue explants obtained at different time points during the periparturient period, 2) the relationship between in vitro β-adrenergic response and the concurrent in vivo β-adrenergic receptor expression and the extent of HSL and perilipin phosphorylation, and 3) the coordinative changes in lipogenic protein expression and lipolytic protein phosphorylation in the context of varying energy status at different stages of lactation.

Dietary Influence on Lipid Mobilization

Phosphorylation of HSL and perilipin (as markers of lipolysis) increased in vivo during early lactation as expected based on previous work (McNamara, 1989; Koltes and Spurlock, 2011; Locher et al., 2011; Rocco and McNamara, 2013). However, this increase could not be further enhanced by an energy-dense prepartum diet or attenuated by NA supplementation in this study. Beyond biological reasons, this can also be attributed to a relative inability to detect dietary treatment effects because of the low number of animals per treatment.

Control of Lipid Metabolism in the Course of Transition Period

Adipose tissue lipolysis in cows is regulated primarily at a posttranslational level via catecholamine stimulation (McNamara, 2012) by promoting HSL and perilipin phosphorylation (Sumner and McNamara, 2007; Elkins and Spurlock, 2009; Koltes and Spurlock, 2011; Locher et al., 2011; Rocco and McNamara, 2013). Therefore, the extent of phosphorylation of HSL and perilipin at specific AA residues is considered a relevant indicator of current lipolytic activity under both in vitro and in vivo conditions. Furthermore, the strong correlation found between phosphorylation extent and release rate in incubated tissues confirmed this consideration, in addition to previous work revealing correlations between plasma NEFA and glycerol concentrations and HSL and perilipin phosphorylation levels in untreated bovine SCAT during early lactation (Elkins and Spurlock, 2009). Furthermore, research has consistently shown correlations between HSL activity measured in vivo with milk production, consistent with a connection through endocrine and neurocrine systems (McNamara, 1989; Sumner and McNamara, 2007).

Phosphorylation sites and their physiological meaning have been heavily studied in various species, and among several AA residues of HSL, Ser563 (according to the rat sequence) was identified as the major activator regulatory site (Yeaman, 2004). This phosphorylation site corresponds to Ser552 of the bovine HSL sequence and has been confirmed to be an important regulatory target of lipolysis in periparturient dairy cows (Elkins and Spurlock, 2009; Koltes and Spurlock, 2011; Locher et al., 2011, Kenéz et al., 2014). On the other hand, less is known about the role of perilipin phosphorylation in bovine adipose tissues. The phosphorylation of perilipin A (the major isoform in adipocytes) at Ser522 (according to the human sequence) was found to be the most important PKA-dependent target in mouse adipocytes (the homologous site is Ser517 in the murine and Ser516 in the bovine protein sequence; Miyoshi et al., 2007). In previous studies, the total phosphorylation of perilipin has already been used as an indicator of lipolysis (Elkins and Spurlock, 2009; Koltes and Spurlock, 2011; Faylon et al., 2014).

Of all the proteins involved in adipose lipolytic pathway, HSL was recognized as the rate-limiting key enzyme for TG hydrolysis for a long time; however, recent studies revealed that the involvement of various lipases in TG hydrolysis is altered by differential metabolic conditions (reviewed in Kraemer and Shen, 2006; Lass et al., 2011). Also in the current study, basal incubation conditions were associated with depressed HSL phosphorylation throughout the whole test period. We know now that, in fact, basal or constitutive lipolysis is catalyzed by a different enzyme, adipose triglyceride lipase (ATGL), which is not activated with phosphorylation (Miyoshi et al., 2008; Elkins and Spurlock, 2010; Koltes and Spurlock, 2011). In the present study, treating the tissue samples with 1 μM isoproterenol triggered an approximately 15 to 20 times greater extent of HSL phosphorylation than basal incubation. Thus, there are 2 points of control, basal lipolysis by ATGL action and β-adrenergic-stimulated lipolysis by HSL action. This finding helps explain the different pattern of adaptation of basal and stimulated lipolysis in the current study and also in previous work (McNamara and Hillers, 1986; Khan et al., 2013; Rocco and McNamara, 2013). In contrast to HSL, perilipin phosphorylation was not remarkably reduced during basal incubation, but rather was maintained at a moderate level. This also indicates that lipolysis can be supported even in the absence of β-adrenergic stimuli, particularly through mechanisms controlled by phosphorylation of perilipin, such as increasing accessibility to the lipid droplet and activating ATGL by releasing comparative gene identity-58 (CGI-58; Yamaguchi et al., 2006; Granneman et al., 2009; Koltes and Spurlock, 2011).

The transition period is associated with a marked reduction in transcription of genes coding for lipogenic proteins and in lipogenic activity of adipose tissue (Sumner and McNamara, 2007; Khan et al., 2013; Rocco and McNamara, 2013). In some animals, lipogenesis may rebound from its nadir within 3 to 4 wk of lactation, whereas in others the period of reduced anabolism can extend several weeks (McNamara, 1994; Rocco and McNamara, 2013). As reflected by the present results on FAS protein expression, lipogenesis was clearly downregulated during early lactation as a consequence of the onset of a negative energetic state. As energy intake and NE balance was increasing at d +100, fatty acid synthesis in adipose tissues was already upregulated again, even though phosphorylation of HSL and perilipin was also still high because of the peak milk production. It is clear from this work and past studies that, in fact, lipogenesis is mainly a function of energy intake, whereas the phosphorylation of HSL and perilipin is directly related to the activity of the mammary gland as suggested many years ago (Vernon, 1980) and shown in various studies since (see the summary in McNamara, 2012). It is likely that the latter is a direct effect of increased sympathetic nervous system activity during lactation (McNamara and Murray, 2001).

To extend our understanding of lipolytic control, an in vitro assay was used in the current study to monitor the β-adrenergic response of adipose tissue explants and to track how consistent this is with the lipolytic status in vivo. In fact, the applied β-adrenergic stimulus triggered the highest response at d −42. Postpartum when lipolysis was upregulated, tissue explants had a lower response to the same defined stimulus, which is consistent with the fact that in these tissues samples, HSL and perilipin were already highly activated by phosphorylation. The decrease of β-adrenergic response from d −42 to d +1 is also affected by decreased receptor protein expression and concurrently increased receptor phosphorylation. This is a form of agonist-mediated receptor desensitization, as the activated lipolytic cascade is known to exert a negative feedback effect on the β-receptors (Hausdorff et al., 1990; Lefkowitz et al., 1990). At d +21 and +100 the receptor protein expression was increasing again. Earlier studies showed that β-adrenergic receptor binding capacity increased during lactation (Jaster and Wegner, 1981); also, the mRNA for β2-adrenergic receptors, similar to HSL and perilipin mRNA, was shown to increase postpartum in direct relation to rates of milk production (Sumner and McNamara, 2007).

In previous studies, a greater metabolic flexibility of RPAT over SCAT was suggested (Locher et al., 2011, 2012), and other abdominal adipose tissues of cows were reported to respond to overfeeding by becoming remarkably increased in mass, without any significant increase of BCS (Drackley et al., 2014). However, in the present study, only minor differences were found between SCAT and RPAT, and the issue of whether abdominal adipose tissues could provide more NEFA because of greater lipolytic potential could not be clarified. Further research is warranted on this subject because the commonly used measure of BCS and the associated risk assessment for metabolic disorders in dairy management is based on the evaluation of only the visible SCAT depots. Depot-specific differences are an important aspect from a quantitative biology point of view as well, as subcutaneous and abdominal adipose masses might contribute to the overall energy supply of the animal at different rates. Accordingly, to get a more precise view of energy homeorhesis at an organism level, studies are warranted to assess the metabolic activity, particularly energy efflux rates from various adipose depots.

Conclusions

As noted above, the control of lipolysis is not a simple system, and the control of total adipose carbon flux is not either; it is affected by anabolic and catabolic factors and the balance of lipogenesis, (re)esterification, and lipolysis. The current data on lipolytic proteins and in vitro β-adrenergic response confirm that posttranslational activation of HSL and perilipin via specific phosphorylation sites is a major control mechanism of lipid flux in response to lactation in dairy cattle. To cover the energy need of lactation and maintenance, lipolysis becomes upregulated concurrently with the onset of lactation and is maintained at least up to 100 d postpartum. Lipogenesis is much more a function of energy intake; accordingly, the synthesis of fatty acids reaches its nadir shortly after parturition, and as NE balance increases, lipogenesis becomes active again. Nevertheless, lipolysis and lipogenesis are both embedded in a complex metabolic network, which is why further research and data interpretation are warranted to understand interactions between HSL and perilipin-controlled lipolysis, lipolysis via other lipases (such as ATGL), NEFA reesterification, and lipogenesis under different aspects of energy homeostasis (Faylon et al., 2014).

This work confirms and extends our knowledge of the multifaceted nature of metabolic control in dairy cattle and could also be useful to the ongoing development of systems biology models of metabolism in dairy cattle to help improve our quantitative knowledge of the cow (McNamara, 2012, Huber et al., 2014).

 

References

Footnotes


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