Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Large Animal Clinical Sciences
Non Technical Summary
During the periparturient period of dairy cows, adipose tissues (AT) lipolysis fulfills, in part, energy deficits driven by fetal growth and the onset of lactation. Lipolysis induces a remodeling process within AT that is characterized by a moderate inflammatory response with infiltration of macrophages. In periparturient cows that successfully transition into lactation, lipolysis rate decreases and AT inflammation resolves as lactation progresses. However, when lipolysis rate is not reduced, AT inflammation is maintained leading to poor lactation performance and to metabolic and infectious diseases. To make things more complicated, periparturient illnesses occur as complexes that pose severe welfare issues to dairy cows. During lipolysis adipocytes produce hydroxyl-octadecadienoic acids (HODEs) from linoleic acid through enzymatic and non-enzymatic reactions. These lipolytic products are potent modulators of inflammatory responses in AT that may directly increase lipolysis rate by reducing adipocyte response to insulin. An additional periparturient factor that is strongly connected with the presentation of disease complexes is the sharp increase in circulating bacterial endotoxins. Our central hypothesis is that lipolysis dysregulation increases the abundance of a select group of HODEs in AT in a non-enzymatic, ROS dependent manner. This increase enhances ATM infiltration and sustains AT inflammation and this response is exacerbated during exposure to endotoxins. We will test this hypothesis using in vitro and ex-vivo models of AT lipolysis, and in periparturient cows using a nutritional intervention for lipolysis modulation. We will pursue three objectives 1:Determine how HODEs that are synthesized in bovine AT during lipolysis modify the macrophage phenotype and reduce the anti-lipolytic response of adipocytes to insulin. 2: Determine how endotoxins enhance adipocytes' lipolytic responses. Our working hypothesis is that endotoxins trigger lipolysis dysregulation through changes in adipocytes' insulin sensitivity and ATM phenotype. 3: Determine how inhibiting lipolysis in periparturient dairy cows affects the non-enzymatic biosynthesis and profile of HODEs, and the ATM phenotype. Upon completion of this project, we will be able to determine the role of HODEs in the vicious cycle of lipolysis dysregulation and chronic inflammation in AT. These findings will support their use as diagnostic, nutritional, or pharmacological targets in management programs of periparturient dairy cows. In addition, this project will elucidate the mechanisms by which endotoxemia induces lipolysis dysregulation and increases periparturient dairy cows' susceptibility to diseases. Our long-term goal is to develop nutritional and pharmacological tools to prevent lipolysis dysregulation during periods of NEB. We expect these tools will benefit herd health, improve dairy cows' welfare, and increase the economic sustainability of dairy farming.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
In this project, we propose that lipolysis dysregulation and abnormal AT inflammation, including macrophage infiltration and polarization, become connected through the actions of the lipolysis derived linoleic acid metabolites called HODEs (hydroxyl-octadecadienoic acids) and are excacerbated by endotoxin exposure. HODEs are highly bioactive oxylipids that are synthesized enzymatically and nonenzymatically from the linoleic acid released during lipolysis [59]. Our previous research strongly indicate that A) lipolysis is associated with plasma and AT content of HODEs and their substrate, linoleic acid; and B) HODEs are powerful modulators of macrophages' inflammatory phenotype.Our central hypothesis is that lipolysis dysregulation increases the abundance of a select group of HODEs in AT in a non-enzymatic, ROS dependent manner. This increase enhances ATM infiltration and sustains AT inflammation and this response is excacerbated during exposure to endotoxins. We will test this hypothesis using in vitro and ex-vivo models of AT lipolysis, and in periparturient cows using a nutritional intervention for lipolysis modulation. We will pursue three objectives:bjective 1:Determine how HODEs that are synthesized in bovine AT during lipolysis modify the macrophage phenotype and reduce the anti-lipolytic response of adipocytes to insulin.Objective 2: Determine how endotoxins enhance adipocytes' lipolytic responses. Our working hypothesis is that endotoxins trigger lipolysis dysregulation through changes in adipocytes' insulin sensitivity and ATM phenotype.Objective 3: Determine how inhibiting lipolysis in periparturient dairy cows affects the non-enzymatic biosynthesis and profile of HODEs, and the ATM phenotype.
Project Methods
Objective 1: Experiment 1A1: How does hormone-sensitive lipase (HSL) activity affect HODE biosynthesis in adipocytes?We will stimulate differentiated primary adipocytes with 10 µM of isoproterenol (ISO) to induce lipolysis [0 µM as control (CON)]. After 4 h, we will harvest adipocytes and culture media and quantify FA, HODEs, and other lipid mediators of inflammation by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). To further characterize how lipolysis affects adipocyte inflammatory responses, we will evaluate the expression profiles of pro-inflammatory cytokines and enzymes involved in either synthesizing oxylipids or FA metabolism using RT-qPCR, protein blotting, and ELISA. We will evaluate cell viability both before and after treatments. To determine the direct effect of HSL activation on HODE biosynthesis, we will use an inhibitor of HSL [CAY10499, or NIA, niacin 2µM] during the lipolytic stimulus. For this experiment, adipocytes will be exposed to CAY10499 [10 µM] or NIA for 1 h before ISO stimulation. After 4 hours of ISO exposure, we will harvest the adipocytes and media and assess each lipolytic and inflammatory variables.Experiment 1A2. How does HSL activity affect the non-enzymatic biosynthesis of HODEs by ROS-mediated oxidation?We will expose differentiated primary adipocytes to CON or ISO as described above in Experiment 1A1. To determine how ROS, 15LOX, and COX2 affect HODEs biosynthesis during lipolysis, we will treat adipocytes with the following inhibitors: a) a 15LOX inhibitor (PD146176, 2µM); b) a COX2 inhibitor (celecoxib, 5µM); c) an inhibitor of the nicotinamide adenine dinucleotide phosphate oxidase that minimizes ROS production (diphenyliodonium (DPI), 10 µM); d) PD146176+celecoxib, e) PD146176+DPI, f) PD146176+celecoxib+DPI, g) celecoxib+DPI h) a negative control treatment; and i) a positive oxidative control [H2O2, 200 µM]. We will evaluate cell viability both before and after treatments, and collect cells and media as described in experiment 1A1 for HODEs detection and quantification, and gene and protein profiling. We will assess the generation of ROS in adipocytes using: a) the OxiSelect intracellular detection assay, and b) quantifying isoprostanes as a proxy of oxidative stress status.Experiment 1B1. How do HODEs affect markers of bovine macrophage phenotype?We will harvest myeloid-derived monocytes from blood by Ficoll gradient. After they mature into macrophages (M0), we will expose the cells to different concentrations of 9-, 10-, 12-, and 13- HODEs for 24 h (0, 5, 10 and 50 nmol). The doses we selected are based on the plasma concentrations of HODEs that we detected in periparturient cows in our preliminary studies. We will polarize primary bovine macrophages to either the M1 or M2 phenotype for use as positive controls. We will use non-stimulated (M0) macrophages as negative phenotypic controls.Experiment 1B2. What is the direct effect of HODEs on the anti-lipolytic response of adipocytes to insulin?We will expose adipocytes to different concentrations of HODEs for 1 h, and then stimulate lipolysis with 10 μM of ISO with or without insulin (0.2 μg/L). We will test the potency of insulin's anti-lipolytic effect in the adipocytes by measuring a) FA uptake (without ISO stimulation), b) NEFA and glycerol release, c) the expression of genes in the insulin signaling network, and d) the expression/phosphorylation of lipolysis and insulin signaling proteins. We will focus on measuring PKA activity because this kinase phosphorylates HSL, which activates it and induces its translocation to the lipid droplet. It has been suggested that PKA activity is not effectively controlled during lipolysis dysregulation, which could exacerbate the release of NEFA.Objective 2. Determine how endotoxins enhance adipocytes' lipolytic responsesExperiment 2A1 TLR2 and TLR4 activation by LPS and LTA induces lipolysis in bovine adipocytes:We will differentiate primary preadipocytes to adipocytes for 10 d. Then, we will treat the adipocytes for 72h with non-targeting siRNA (Nsp) pools or siRNA ON-TARGET Plus SMARTpool siRNAs targeting TLR2 , TLR4, or both TLR2 and TLR4 according to manufacturer's instructions and using FuGENE transfection reagent. We will optimize the concentration of siRNA prior to the experiments with endotoxin. We will validate TLR2, TLR4, and TLR2/4 knockdown by real-time PCR and western blotting. For all siRNA experiments, cell-viability will be determined with the LIVE/DEAD viability kit. Next, we will treat the adipocytes, including un-transfected and transfection reagent controls, with 1 μM of ISO to induce lipolysis [0 μM as a basal control (BAS)] in the presence or absence of LPS (O55:B5, 20µg/ml) and LTA (Staph aureus 50µg/ml) for 7 h. Thereafter, we will harvest the adipocytes and collect the culture media. We will quantify lipolysis by measuring FFA and glycerol release. We will use an inhibitor of HSL, CAY10499 (10 μM), as a positive control for total inhibition of lipolysis.We will evaluate these two metabolic processes using a systems biology approach. 1) At the transcriptomic level we will use RT-qPCR to determine the effect of endotoxin on gene markers of inflammation, lipogenesis, lipolysis, and insulin signaling. We will perform transcriptomics analyses as described by our group. 2) Certain components of lipolysis and of the inflammatory pathways induced by endotoxin are controlled post-transcriptionally and must be evaluated at the protein level. Therefore, it is necessary to determine not only the protein content, but also the phosphorylation levels of specific peptides that are markers of lipolysis and insulin sensitivity in AT. We will quantify these proteins using traditional western blotting and capillary electrophoresis. 3) Since endotoxins are potent inflammation inducers and lipolysis is an inflammatory event, we will perform a detailed characterization of the chemokine and cytokine profiles of adipocytes and ATM.Experiment 2A2 TLR2 and TLR-4 activation by LPS and LTA trigger the classic and inflammatory lipolytic pathways in bovine adipocytes: To determine how LPS and LTA affect lipolysis, we will use inhibitors to block the following steps of the classical and inflammatory lipolytic pathways: 1) PKA: H-89 (20 µM), 2) PKC: Ro-31-8220 (1 µM), 3) ERK 1/2 : U0126 (2 µM), 4) NFκB: PDTC (80 µM), and 5) TNF-α: CAS 1049741 (22 µM). We will pre-incubate un-transfected, Nsp, and siRNA treated adipocytes with the inhibitors for 1h, and then induce lipolysis with ISO, LPS, and LTA. We will use inhibitors alone (without lipolysis treatment) as negative controls. After 7 h, we will harvest the adipocytes and collect the culture media [90]. We will quantify lipolysis by measuring FFA and glycerol release.Aim 3. Determine how inhibiting lipolysis in periparturient dairy cows affects the biosynthesis and profile of HODEs, ATM phenotype, and AT response to insulin.Experimental Design. All procedures will be approved by the MSU Institutional Animal Care and Use Committee. We will select 40 gestating non-lactating multiparous Holstein cows from the MSU Dairy Field Laboratory 1 week after dry-off. The inclusion criteria are 1) pregnant, 2) 210 to 230 days of gestation, and 3) BCS between 3.25 and 3.75. Cows will be blocked by parity and BCS and then randomly assigned at close-up (d-21) to either the NIA (12g/d NiaShure™) or control (CON) group. At 12 g/d, rumen-protected NIA decreases lipolysis induced by negative energy balance. From dry-off and until ~21 d before expected calving, cows will receive a common dry cow diet. A close-up diet will be fed starting day -21 before expected calving. Cows will move onto a fresh cow diet at calving, and will continue receiving NIA (12g/d) or no NIA (CON) until 21 days after calving