The Effect of Subclinical Ketosis on the Peripheral Blood Mononuclear Cell Inflammatory Response and Its Crosstalk with Depot-Specific Preadipocyte Function in Dairy Cows.

During the periparturient period, cows undergo heightened energy demands at lactation onset, paired with reduced dry matter intake, leading to negative energy balance (NEB). Excessive lipolysis-driven adipose tissue remodeling, triggered by NEB, significantly contributes to ketosis in periparturient dairy cows. However, the role of peripheral blood mononuclear cells (PBMCs) in the pathogenesis of ketosis and in modulating adipose tissue function remains poorly understood. Here, we investigated how ketosis affects the transcriptional profile and secretome of PBMCs and its influence on preadipocyte function in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT). Twenty-one postpartum Holstein dairy cows were categorized as either subclinical ketosis (SCK; BHB ≥ 1.0 mM) or control (CON; BHB < 0.8 mM) based on blood beta-hydroxybutyrate (BHB) concentration screening. Blood samples were collected intravenously for the isolation of PBMCs and serum metabolic profiling. Ketosis elevated circulating NEFA and BHB levels but reduced total WBC and neutrophil counts. Isolated PBMCs were evaluated for gene expression and used to produce conditioned media (PBMC-CM), during which PBMCs were stimulated with 10 ng/mL LPS. The overall phenotype of PBMCs was largely consistent between SCK and CON cows, with minimal differences detected in immunomodulatory cytokine expression and PBMC-CM composition following stimulation. Preadipocytes isolated from non-ketotic cows were treated with PBMC-CM to assess the effect of PBMC secretomes on adipose cell function. Preadipocytes treated with SCK PBMC-CM showed reduced lipid accumulation compared to those treated with CON PBMC-CM regardless of the depot. SAT preadipocytes had heightened expression of lipid metabolism-related genes, including DGAT1, LIPE, and FASN, compared to VAT when treated with SCK PBMC-CM. Preadipocytes treated with CM from PBMC stimulated by LPS exhibited upregulation in IL1B and IL6 regardless of the depot or source of PBMCs. Together, these results indicate that although PBMC profiles showed minimal differences, preadipocytes treated with PBMC-CM may be influenced by additional factors, leading to altered preadipocyte function and gene expression that may contribute to adipose cellular dysfunction.

White Adipose Tissue Heterogeneity in the Single-Cell Era: From Mice and Humans to Cattle.

Adipose tissue is a major modulator of metabolic function by regulating energy storage and by acting as an endocrine organ through the secretion of adipokines. With the advantage of next-generation sequencing-based single-cell technologies, adipose tissue has been studied at single-cell resolution, thus providing unbiased insight into its molecular composition. Recent single-cell RNA sequencing studies in human and mouse models have dissected the transcriptional cellular heterogeneity of subcutaneous (SAT), visceral (VAT), and intramuscular (IMAT) white adipose tissue depots and revealed unique populations of adipose tissue progenitor cells, mature adipocytes, immune cell, vascular cells, and mesothelial cells that play direct roles on adipose tissue function and the development of metabolic disorders. In livestock species, especially in bovine, significant gaps of knowledge remain in elucidating the roles of adipose tissue cell types and depots on driving the pathogenesis of metabolic disorders and the distinct fat deposition in VAT, SAT, and IMAT in meat animals. This review summarizes the current knowledge on the transcriptional and functional cellular diversity of white adipose tissue revealed by single-cell approaches and highlights the depot-specific function of adipose tissue in different mammalian species, with a particular focus on recent findings and future implications in cattle.

Lumican modulates adipocyte function in obesity-associated type 2 diabetes.

Obesity-associated type 2 diabetes (DM) leads to adipose tissue dysfunction. Lumican is a proteoglycan implicated in obesity, insulin resistance (IR), and adipocyte dysfunction. Using human visceral adipose tissue (VAT) from subjects with and without DM, we studied lumican effects on adipocyte function. Lumican was increased in VAT and adipocytes in DM. Lumican knockdown in adipocytes decreased lipolysis and improved adipogenesis and insulin sensitivity in VAT adipocytes in DM, while treatment with human recombinant lumican increased lipolysis and impaired insulin-sensitivity in an ERK-dependent manner. We demonstrate that lumican impairs adipocyte metabolism, partially via ERK signalling, and is a potential target for developing adipose tissue-targeted therapeutics in DM.

Single-nuclei analysis reveals depot-specific transcriptional heterogeneity and depot-specific cell types in adipose tissue of dairy cows.

Adipose tissue (AT) is an endocrine organ with a central role on whole-body energy metabolism and development of metabolic diseases. Single-cell and single-nuclei RNA sequencing (scRNA-seq and snRNA-seq, respectively) analyses in mice and human AT have revealed vast cell heterogeneity and functionally distinct subtypes that are potential therapeutic targets to metabolic disease. In periparturient dairy cows, AT goes through intensive remodeling and its dysfunction is associated with metabolic disease pathogenesis and decreased productive performance. The contributions of depot-specific cells and subtypes to the development of diseases in dairy cows remain to be studied. Our objective was to elucidate differences in cellular diversity of visceral (VAT) and subcutaneous (SAT) AT in dairy cows at the single-nuclei level. We collected matched SAT and VAT samples from three dairy cows and performed snRNA-seq analysis. We identified distinct cell types including four major mature adipocytes (AD) and three stem and progenitor cells (ASPC) subtypes, along with endothelial cells (EC), mesothelial cells (ME), immune cells, and pericytes and smooth muscle cells. All major cell types were present in both SAT and VAT, although a strong VAT-specificity was observed for ME, which were basically absent in SAT. One ASPC subtype was defined as adipogenic (PPARG+) while the other two had a fibro-adipogenic profile (PDGFRA+). We identified vascular and lymphatic EC subtypes, and different immune cell types and subtypes in both SAT and VAT, i.e., macrophages, monocytes, T cells, and natural killer cells. Not only did VAT show a greater proportion of immune cells, but these visceral immune cells had greater activation of pathways related to immune and inflammatory response, and complement cascade in comparison with SAT. There was a substantial contrast between depots for gene expression of complement cascade, which were greatly expressed by VAT cell subtypes compared to SAT, indicating a pro-inflammatory profile in VAT. Unprecedently, our study demonstrated cell-type and depot-specific heterogeneity in VAT and SAT of dairy cows. A better understanding of depot-specific molecular and cellular features of SAT and VAT will aid in the development of AT-targeted strategies to prevent and treat metabolic disease in dairy cows, especially during the periparturient period.

The human type 2 diabetes-specific visceral adipose tissue proteome and transcriptome in obesity.

Dysfunctional visceral adipose tissue (VAT) in obesity is associated with type 2 diabetes (DM) but underlying mechanisms remain unclear. Our objective in this discovery analysis was to identify genes and proteins regulated by DM to elucidate aberrant cellular metabolic and signaling mediators. We performed label-free proteomics and RNA-sequencing analysis of VAT from female bariatric surgery subjects with DM and without DM (NDM). We quantified 1965 protein groups, 23 proteins, and 372 genes that were differently abundant in DM vs. NDM VAT. Proteins downregulated in DM were related to fatty acid synthesis and mitochondrial function (fatty acid synthase, FASN; dihydrolipoyl dehydrogenase, mitochondrial, E3 component, DLD; succinate dehydrogenase-α, SDHA) while proteins upregulated in DM were associated with innate immunity and transcriptional regulation (vitronectin, VTN; endothelial protein C receptor, EPCR; signal transducer and activator of transcription 5B, STAT5B). Transcriptome indicated defects in innate inflammation, lipid metabolism, and extracellular matrix (ECM) function, and components of complement classical and alternative cascades. The VAT proteome and transcriptome shared 13 biological processes impacted by DM, related to complement activation, cell proliferation and migration, ECM organization, lipid metabolism, and gluconeogenesis. Our data revealed a marked effect of DM in downregulating FASN. We also demonstrate enrichment of complement factor B (CFB), coagulation factor XIII A chain (F13A1), thrombospondin 1 (THBS1), and integrins at mRNA and protein levels, albeit with lower q-values and lack of Western blot or PCR confirmation. Our findings suggest putative mechanisms of VAT dysfunction in DM.

Depot-specific adipocyte-extracellular matrix metabolic crosstalk in murine obesity.

Subcutaneous (SAT) and visceral (VAT) adipose tissues have distinct metabolic phenotypes. We hypothesized that the extracellular matrix (ECM) regulates depot-specific differences in adipocyte metabolic function in murine obesity. VAT and SAT preadipocytes from lean or obese mice were subject to adipogenic differentiation in standard 2D culture on plastic tissue culture plates or in 3D culture in ECM, followed by metabolic profiling. Adipocytes from VAT relative to SAT manifested impaired insulin-stimulated glucose uptake and decreased adipogenic capacity. In 3D-ECM-adipocyte culture, ECM regulated adipocyte metabolism in a depot-specific manner, with SAT ECM rescuing defects in glucose uptake and adipogenic gene expression in VAT adipocytes, while VAT ECM impaired adipogenic gene expression in SAT adipocytes. These findings demonstrate that ECM-adipocyte crosstalk regulates depot-specific differences in adipocyte metabolic dysfunction in murine obesity.

Viscoelastic characterization of diabetic and non-diabetic human adipose tissue.

BACKGROUND: Obesity-induced chronic inflammation and fibrosis in adipose tissue contributes to the progression of type 2 diabetes mellitus (DM). While fibrosis is known to induce mechanical stiffening of numerous tissue types, it is unknown whether DM is associated with alterations in adipose tissue mechanical properties. OBJECTIVE: The purpose of this study was to investigate whether DM is associated with differences in bulk viscoelastic properties of adipose tissue from diabetic (DM) and non-diabetic (NDM) obese subjects. METHODS: Bulk shear rheology was performed on visceral (VAT) and subcutaneous (SAT) adipose tissue, collected from obese subjects undergoing elective bariatric surgery. Rheology was also performed on the remaining extracellular matrix (ECM) from decellularized VAT (VAT ECM). Linear mixed models were used to assess whether correlations existed between adipose tissue mechanical properties and DM status, sex, age, and body mass index (BMI). RESULTS: DM was not associated with significant differences in adipose tissue viscoelastic properties for any of the tissue types investigated. Tissue type dependent differences were however detected, with VAT having significantly lower shear storage and loss moduli than SAT and VAT ECM independent of DM status. CONCLUSION: Although DM is typically associated with adipose tissue fibrosis, it is not associated with differences in macroscopic adipose tissue mechanical properties.

Advanced glycation end-products regulate extracellular matrix-adipocyte metabolic crosstalk in diabetes.

The adipose tissue extracellular matrix (ECM) regulates adipocyte cellular metabolism and is altered in obesity and type 2 diabetes, but mechanisms underlying ECM-adipocyte metabolic crosstalk are poorly defined. Advanced glycation end-product (AGE) formation is increased in diabetes. AGE alter tissue function via direct effects on ECM and by binding scavenger receptors on multiple cell types and signaling through Rho GTPases. Our goal was to determine the role and underlying mechanisms of AGE in regulating human ECM-adipocyte metabolic crosstalk. Visceral adipocytes from diabetic and non-diabetic humans with obesity were studied in 2D and 3D-ECM culture systems. AGE is increased in adipose tissue from diabetic compared to non-diabetic subjects. Glycated collagen 1 and AGE-modified ECM regulate adipocyte glucose uptake and expression of AGE scavenger receptors and Rho signaling mediators, including the DIAPH1 gene, which encodes the human Diaphanous 1 protein (hDia1). Notably, inhibition of hDia1, but not scavenger receptors RAGE or CD36, attenuated AGE-ECM inhibition of adipocyte glucose uptake. These data demonstrate that AGE-modification of ECM contributes to adipocyte insulin resistance in human diabetes, and implicate hDia1 as a potential mediator of AGE-ECM-adipocyte metabolic crosstalk.

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

Fetuin-A modulates lipid mobilization in bovine adipose tissue by enhancing lipogenic activity of adipocytes.

Fetuin-A (FetA) is an adipokine and free fatty acid (FFA) carrier linked to adipose tissue (AT) function in monogastrics and ruminants. In dairy cows, plasma and AT FetA decrease after parturition, coinciding with reduced lipogenesis and increased lipolysis. In monogastrics, FetA enhances lipogenesis, but its role on lipid mobilization of ruminants is unclear. We hypothesized that FetA modulates lipid mobilization in bovine AT by enhancing the lipogenic activity of adipocytes. Our objective was to determine the effects of FetA on lipogenesis and lipolysis in cultured primary adipocytes from dairy cows. Preadipocytes from the tailhead subcutaneous AT depot were induced to differentiate in a 7-d coculture in vitro model. The effects of FetA on lipolytic responses of adipocytes were evaluated after a 2-h ß-adrenergic stimulation with 1 µM isoproterenol (ISO) alone or combined with 0.1 mg/mL of FetA (FetA+ISO), and in cells treated with medium alone (CON) or with 0.1 mg/mL of FetA (FetA). Lipogenic responses of adipocytes treated with CON or FetA from d 5 to 7 of differentiation were assessed by fatty acid (FA) uptake quantification and triacylglycerol (TAG) accumulation, and the gene and protein expression of lipogenic markers. Bovine adipocytes abundantly expressed FetA gene and protein and secreted 48 ± 3.5 ng/DNA relative fluorescence units (RFU). Adrenergic stimulation with ISO increased lipolysis compared with CON, as reflected in the release of glycerol (0.12 ± 0.04 vs. 0.04 ± 0.02 nM/DNA RFU) and FFA (15 ± 13 vs. 6.2 ± 2.4 nM/DNA RFU). Lipolysis induced by ISO was attenuated by the addition of FetA (FetA+ISO) as reflected by lower glycerol (0.06 ± 0.04 nM/DNA RFU) and FFA (5.7 ± 2.7 nM/DNA RFU) release compared with ISO alone. Compared with CON, FetA enhanced lipogenic responses as demonstrated by higher FA uptake and increased accumulation of TAG. Exposure to FetA upregulated 1-acylglycerol-3-phosphate acyltransferase-2 (AGPAT2) gene expression and protein content, as well as its activity. Adipocytes exposed to FetA increased the secretion of the metabolite of AGPAT2, phosphatidic acid. In conclusion, FetA attenuates lipolytic responses and enhances lipogenesis in bovine adipocytes. The upregulation of the rate-limiting lipogenic enzyme AGPAT2 by FetA suggests a potential pathway by which this adipokine promotes TAG synthesis in adipocytes. These findings suggest that FetA is a potential target for lipid mobilization modulation in AT of dairy cows.

Technical note: Bovine adipocyte and preadipocyte co-culture as an efficient adipogenic model.

Reductionist studies of adipose tissue biology require reliable in vitro adipocyte culturing models. Current protocols for adipogenesis induction in stromal vascular fraction-derived preadipocytes require extended culturing periods and have low adipogenic rates. We compared the adipogenic efficiency of a 7-d co-culture model of visceral (VIS) and subcutaneous (SC) stromal vascular fraction-derived preadipocytes with mature adipocytes with a 14-d standard adipocyte differentiation protocol. We obtained preadipocytes and mature adipocytes from SC and VIS adipose tissue of nonlactating, nongestating Holstein cows (n = 6). Adipogenesis induction was performed using a standard protocol for 7 (SD7; control) or 14 d (SD14), and a co-culture model for 7 d (CC7). Culture conditions, including medium composition, were the same for all treatments. For CC7, 900 primary adipocytes/cm2 were placed in 0.4-µm transwell inserts and co-cultured with preadipocytes for adipogenesis induction. Both CC7 and SD14 similarly stimulated gene expression of adipogenic genes such as ADIPOQ, CEBPA, and CEBPB in VIS and SC. The CC7 increased triacylglycerol accumulation compared with SD14 and SD7. CC7 augmented triacylglycerol accumulation by 40- and 16-fold in SC and VIS compared with 22- and 4-fold increment in SD14, respectively. Lipolytic responses to 2-h ß-adrenergic stimulation with 1 µM isoproterenol were higher in CC7 and SD14 than SD7 in SC; CC7 increased glycerol release compared with SD7 in VIS but SD7 and SD14 had similar responses. Overall, CC7 was more efficient in inducing adipogenesis in preadipocytes from VIS and SC than SD14. Furthermore, CC7 stimulated similar lipolysis and lipogenic responses than SD14 but in a shorter time. The adipogenic approach of co-culturing preadipocytes with mature adipocytes will improve the use of reductionist models to study adipocyte physiology in dairy cows and the assessment of pharmacological or nutritional interventions for enhancing dairy cow health and production.

The contribution of hormone sensitive lipase to adipose tissue lipolysis and its regulation by insulin in periparturient dairy cows.

Hormone sensitive lipase (HSL) activation is part of the metabolic adaptations to the negative energy balance common to the mammalian periparturient period. This study determined HSL contribution to adipose tissue (AT) lipolysis and how insulin regulates its activity in periparturient dairy cows. Subcutaneous AT (SCAT) samples were collected at 11 d prepartum (dry) and 11 (fresh) and 24 d (lactation) postpartum. Basal and stimulated lipolysis (ISO) responses were determined using explant cultures. HSL contribution to lipolysis was assessed using an HSL inhibitor (CAY). Basal lipolysis was higher in SCAT at dry compared with fresh. CAY inhibited basal lipolysis negligibly at dry, but at fresh and lactation it reduced basal lipolysis by 36.1 ± 4.51% and 43.1 ± 4.83%, respectively. Insulin inhibited lipolysis more pronouncedly in dry compared to fresh. Results demonstrate that HSL contribution to basal lipolysis is negligible prepartum. However, HSL is a major driver of SCAT lipolytic responses postpartum. Lower basal lipolysis postpartum suggests that reduced lipogenesis is an important contributor to fatty acid release from SCAT. Loss of adipocyte sensitivity to the antilipolytic action of insulin develops in the early lactation period and supports a state of insulin resistance in AT of cows during the first month postpartum.

Short communication: Effects of body fat mobilization on macrophage infiltration in adipose tissue of early lactation dairy cows.

Intense lipolysis triggers an inflammatory response within adipose tissue characterized by adipose tissue macrophage (ATM) infiltration; however, the mechanisms triggering this process are poorly characterized in transition dairy cows. The aim of this study was to determine the association between ATM infiltration and body fat mobilization in the transition period, markers of excessive lipolysis, and adipose tissue expression of genes related to chemotactic and inflammatory responses. Subcutaneous adipose tissue samples were taken from the tailhead of 9 multiparous Holstein cows, 27 ± 2.2 d (far-off) and 10 ± 1.5 d (close-up) before and 9 ± 0.3 d after calving (fresh). Blood samples were collected by coccygeal venipuncture 2 h before adipose sample collections. Body condition score (BCS) was assessed independently by 3 experienced technicians at every time point. Based on BCS loss intensity between the close-up and fresh period, cows were divided into 2 groups: low BCS loss (LBCSL, change in BCS <0.25 units, n = 5) and high BCS loss (HBCSL, change in BCS >0.25 units, n = 4). Although none of the LBCSL cows had a health event, all cows in the HBCSL group suffered from one or more clinical disorder (retained placenta, milk fever, or ketosis) in the transition period. The number of ATM was determined by immunohistochemistry, and expression of selected chemotactic and inflammatory genes was determined by reverse-transcription quantitative real-time PCR in subcutaneous adipose tissue samples. The proportion of ATM in subcutaneous adipose tissue increased in HBCSL during the postpartum period. The proportion of ATM was not associated with serum ß-hydroxybutyrate or free fatty acid concentrations on the day of adipose tissue collection. The ATM infiltration in the fresh period was associated with local expression of the chemotactic genes, C-C motif chemokine ligand 22 (CCL22), osteopontin (SPP1), and the receptor for SPP1, cluster of differentiation 44 (CD44). This supports a potential chemotactic role of CCL22 and SPP1 for ATM in bovine adipose tissue. None of the genes encoding pro- or anti-inflammatory mediators, tumor necrosis factor (TNF), IL6, and IL10 were associated with the proportion of ATM. Our results indicate that ATM infiltration of subcutaneous adipose tissue is associated with body fat mobilization in early-lactation dairy cows and supports a role for ATM in the adaptation of adipose tissues to the metabolic challenges of the transition period.

Fetuin-A: A negative acute-phase protein linked to adipose tissue function in periparturient dairy cows.

Fetuin-A (FetA) is a free fatty acid transporter and an acute-phase protein that enhances cellular lipid uptake and lipogenesis. In nonruminants, FetA is involved in lipid-induced inflammation. Despite FetA importance in lipid metabolism and inflammation, its expression and dynamics in adipose tissue (AT) of dairy cows are unknown. The objectives of this study were to (1) determine serum and AT FetA dynamics over the periparturient period and in mid-lactation cows in negative energy balance (NEB) after a feed restriction protocol and (2) characterize how an inflammatory challenge affects adipocyte FetA expression. Blood and subcutaneous AT were collected from 16 cows with high (≥3.75, n = 8) or moderate (≤3.5, n = 8) body condition score (BCS) at -26 ± 7 d (far off) and -8 ± 5 d (close up) before calving and at 10 ± 2 d after parturition (early lactation) and from 14 nonpregnant mid-lactation cows (>220 d in milk) after a feed restriction protocol. Serum FetA concentrations were 0.89 ± 0.13 mg/mL at far off, 0.96 ± 0.13 mg/mL at close up, and 0.77 ± 0.13 mg/mL at early lactation and were 1.09 ± 0.09 and 1.17 ± 0.09 mg/mL in feed-restricted and control cows, respectively. Serum and AT FetA contents decreased at the onset of lactation when lipolysis was higher. No changes in AT and serum FetA were observed after feed restriction induced NEB in mid-lactation cows. Prepartum BCS had no effect on serum FetA, but AT expression of AHSG, the gene encoding FetA, was reduced in periparturient cows with high BCS at dry-off throughout all time points. Circulating FetA was positively associated with serum albumin and calcium and with BCS variation over the periparturient period. The dynamics of AHSG expression were analogous to the patterns of lipogenic markers ABDH5, ELOVL6, FABP4, FASN, PPARγ, and SCD1. Expression of AHSG and FetA protein in AT was inversely correlated with AT proinflammatory markers CD68, CD44, SPP1, and CCL2. In vitro, bovine adipocytes challenged with lipopolysaccharide downregulated FetA protein expression. Adipocytes treated with FetA had lower CCL2 expression compared with those exposed to lipopolysaccharide. Overall, FetA is a systemic and local AT negative acute-phase protein linked to AT function in periparturient cows. Furthermore, FetA may support physiological adaptations to NEB in periparturient cows.

Symposium review: Modulating adipose tissue lipolysis and remodeling to improve immune function during the transition period and early lactation of dairy cows.

Despite major advances in our understanding of transition and early lactation cow physiology and the use of advanced dietary, medical, and management tools, at least half of early lactation cows are reported to develop disease and over half of cow deaths occur during the first week of lactation. Excessive lipolysis, usually measured as plasma concentrations of free fatty acids (FFA), is a major risk factor for the development of displaced abomasum, ketosis, fatty liver, and metritis, and may also lead to poor lactation performance. Lipolysis triggers adipose tissue (AT) remodeling that is characterized by enhanced humoral and cell-mediated inflammatory responses and changes in its distribution of cellular populations and extracellular matrix composition. Uncontrolled AT inflammation could perpetuate lipolysis, as we have observed in cows with displaced abomasum, especially in those animals with genetic predisposition for excessive lipolysis responses. Efficient transition cow management ensures a moderate rate of lipolysis that is rapidly reduced as lactation progresses. Limiting FFA release from AT benefits immune function as several FFA are known to promote dysregulation of inflammation. Adequate formulation of pre- and postpartum diet reduces the intensity of AT lipolysis. Additionally, supplementation with niacin, monensin, and rumen-protected methyl donors (choline and methionine) during the transition period is reported to minimize FFA release into systemic circulation. Targeted supplementation of energy sources during early lactation improves energy balance and increases insulin concentration, which limits AT lipolytic responses. This review elaborates on the mechanisms by which uncontrolled lipolysis triggers inflammatory disorders. Details on current nutritional and pharmacological interventions that aid the modulation of FFA release from AT and their effect on immune function are provided. Understanding the inherent characteristics of AT biology in transition and early lactation cows will reduce disease incidence and improve lactation performance.

Periparturient lipolysis and oxylipid biosynthesis in bovine adipose tissues.

The periparturient period of dairy cows is characterized by intense lipolysis in adipose tissues (AT), which induces the release of free fatty acids (FFA) into circulation. Among FFA, polyunsaturated fatty acids are susceptible to oxidation and can modulate inflammatory responses during lipolysis within AT. Linoleic and arachidonic acid oxidized products (oxylipids) such as hydroxy-octadecadienoic acids (HODE) and hydroxy-eicosatetraenoic acids (HETE), were recently identified as products of lipolysis that could modulate AT inflammation during lipolysis. However, the effect of lipolysis intensity during the transition from gestation to lactation on fatty acid substrate availability and subsequent AT oxylipid biosynthesis is currently unknown. We hypothesized that in periparturient dairy cows, alterations in AT and plasma fatty acids and oxylipid profiles coincide with changes in lipolysis intensity and stage of lactation. Blood and subcutaneous AT samples were collected from periparturient cows at -27±7 (G1) and -10±5 (G2) d prepartum and at 8±3 d postpartum (PP). Targeted lipidomic analysis was performed on plasma and AT using HPLC-MS/MS. We report that FFA concentrations increased as parturition approached and were highest at PP. Cows exhibiting high lipolysis rate at PP (FFA>1.0 mEq/L) had higher body condition scores at G1 compared to cows with low lipolysis rate (FFA<1.0 mEq/L). Concentrations of plasma linoleic and arachidonic acids were increased at PP. In AT, 13-HODE, and 5-, 11- and 15-HETE were increased at PP compared to G1 and G2. Concentrations of beta hydroxybutyrate were positively correlated with those of 13-HODE and 15-HETE in AT. Plasma concentrations of 5- and 20-HETE were increased at PP. These data demonstrate that prepartum adiposity predisposes cows to intense lipolysis post-partum and may exacerbate AT inflammation because of increased production of pro-inflammatory oxylipids including 5- and 15-HETE and 13-HODE. These results support a role for certain linoleic and arachidonic acid-derived oxylipids as positive and negative modulators of AT inflammation during periparturient lipolysis.

Adipose tissue lipolysis and remodeling during the transition period of dairy cows.

Elevated concentrations of plasma fatty acids in transition dairy cows are significantly associated with increased disease susceptibility and poor lactation performance. The main source of plasma fatty acids throughout the transition period is lipolysis from adipose tissue depots. During this time, plasma fatty acids serve as a source of calories mitigating the negative energy balance prompted by copious milk synthesis and limited dry matter intake. Past research has demonstrated that lipolysis in the adipose organ is a complex process that includes not only the activation of lipolytic pathways in response to neural, hormonal, or paracrine stimuli, but also important changes in the structure and cellular distribution of the tissue in a process known as adipose tissue remodeling. This process involves an inflammatory response with immune cell migration, proliferation of the cellular components of the stromal vascular fraction, and changes in the extracellular matrix. This review summarizes current knowledge on lipolysis in dairy cattle, expands on the new field of adipose tissue remodeling, and discusses how these biological processes affect transition cow health and productivity.

Adipose tissue remodeling in late-lactation dairy cows during feed-restriction-induced negative energy balance.

Excessive rates of demand lipolysis in the adipose tissue (AT) during periods of negative energy balance (NEB) are associated with increased susceptibility to disease and limited lactation performance. Lipolysis induces a remodeling process within AT that is characterized by an inflammatory response, cellular proliferation, and changes in the extracellular matrix (ECMT). The adipose tissue macrophage (ATM) is a key component of the inflammatory response. Infiltration of ATM-forming cellular aggregates was demonstrated in transition cows, suggesting that ATM trafficking and phenotype changes may be associated with disease. However, it is currently unknown if ATM infiltration occurs in dairy cows only during NEB states related to the transition period or also during NEB-induced lipolysis at other stages of lactation. The objective of this study was to evaluate changes in ATM trafficking and inflammatory phenotypes, and the expression of genetic markers of AT remodeling in healthy late-lactation cows during feed restriction-induced NEB. After a 14-d (d -14 to d -1) preliminary period, Holstein cows were randomly assigned to 1 of 2 feeding protocols, ad libitum (AL) or feed restriction (FR), for 4 d (d 1-4). Caloric intake was reduced in FR to achieve a targeted energy balance of -15 Mcal/d of net energy for lactation. Omental and subcutaneous AT samples were collected laparoscopically to harvest stromal vascular fraction (SVF) cells on d -3 and 4. The FR induced a NEB of -14.1±0.62 Mcal/d of net energy for lactation, whereas AL cows remained in positive energy balance (3.2±0.66 Mcal/d of NEL). The FR triggered a lipolytic response reflected in increased plasma nonesterified fatty acids (0.65±0.05 mEq/L on d 4), enhanced phosphorylation of hormone sensitive lipase, and reduced adipocyte diameter. Flow cytometry and immunohistochemistry analysis revealed that on d 4, FR cows had increased numbers of CD172a+, an ATM (M1 and M2) surface marker, cells in SVF that were localized in aggregates. However, FR did not alter the number of SVF cells expressing M1 markers (CD14 and CD11c) or M2 markers (CD11b and CD163). This finding contrasts with the predominately M1 phenotype observed previously in ATM from clinically diseased cows. No changes were observed in the expression of ECMT-related or cell proliferation markers. In summary, an acute 4-d lipolytic stimulus in late-lactation dairy cows led to ATM infiltration with minimal changes in inflammatory phenotype and no changes in ECMT. These results underscore that physiological changes related to parturition, the onset of lactation, extended periods of lipolysis, or a combination of these can induce intense AT remodeling with enhanced ATM inflammatory phenotype expression that may impair the metabolic function of AT in transition dairy cattle.