Intense and protracted adipose tissue (AT) fat mobilization increases the risk of metabolic and inflammatory periparturient diseases in dairy cows. This vulnerability increases when cows have endotoxemia-common during periparturient diseases such as mastitis, metritis, and pneumonia-but the mechanisms are unknown. Fat mobilization intensity is determined by the balance between lipolysis and lipogenesis. Around parturition, the rate of lipolysis surpasses that of lipogenesis, leading to enhanced free fatty acid release into the circulation. We hypothesized that exposure to endotoxin (ET) increases AT lipolysis by activation of classic and inflammatory lipolytic pathways and reduction of insulin sensitivity. In experiment 1, subcutaneous AT (SCAT) explants were collected from periparturient (n = 12) Holstein cows at 11 ± 3.6 d (mean ± SE) before calving, and 6 ± 1 d and 13 ± 1.4 d after parturition. Explants were treated with the endotoxin lipopolysaccharide (LPS; 20 µg/mL; basal = 0 µg/mL) for 3 h. The effect of LPS on lipolysis was assessed in the presence of the β-adrenergic agonist and promoter of lipolysis isoproterenol (ISO; 1 µM; LPS+ISO). In experiment 2, SCAT explants were harvested from 24 nonlactating, nongestating multiparous Holstein dairy cows and exposed to the same treatments as in experiment 1 for 3 and 7 h. The effect of LPS on the antilipolytic responses induced by insulin (INS = 1 µL/L, LPS+INS) was established during ISO stimulation [ISO+INS, LPS+ISO+INS]. The characterization of lipolysis included the quantification of glycerol release and the assessment of markers of lipase activity [adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and phosphorylated HSL Ser563 (pHSL)], and insulin pathway activation (AKT, pAKT) using capillary electrophoresis. Inflammatory gene networks were evaluated by real-time quantitative PCR. In periparturient cows, LPS increased AT lipolysis by 67 ± 12% at 3 h across all time points compared with basal. In nonlactating cows, LPS was an effective lipolytic agent at 3 h and 7 h, increasing glycerol release by 115 ± 18% and 68.7 ± 16%, respectively, relative to basal. In experiment 2, LPS enhanced ATGL activity with minimal HSL activation at 3 h. In contrast, at 7 h, LPS increased HSL phosphorylation (i.e., HSL activity) by 123 ± 11%. The LPS-induced HSL lipolytic activity at 7 h coincided with the activation of the MEK/ERK inflammatory pathway. In experiment 2, INS reduced the lipolytic effect of ISO (ISO+INS: -63 ± 18%) and LPS (LPS+INS: -45.2 ± 18%) at 3 h. However, the antilipolytic effect of INS was lost in the presence of LPS at 7 h (LPS+INS: -16.3 ± 16%) and LPS+ISO+INS at 3 and 7 h (-3.84 ± 23.6% and -21.2 ± 14.6%). Accordingly, LPS reduced pAKT:AKT (0.11 ± 0.07) compared with basal (0.18 ± 0.05) at 7 h. Our results indicated that exposure to LPS activated the classic and inflammatory lipolytic pathways and reduced insulin sensitivity in SCAT. These data provide evidence that during endotoxemia, dairy cows may be more susceptible to lipolysis dysregulation and loss of adipocyte sensitivity to the antilipolytic action of insulin.

Dairy cows with clinical ketosis (CK) exhibit excessive adipose tissue (AT) lipolysis and systemic inflammation. Lipolysis in cows can be induced by the canonical (hormonally induced) and inflammatory lipolytic pathways. Currently, the most common treatment for CK is oral propylene glycol (PG); however, PG does not reduce lipolysis or inflammation. Niacin (NIA) can reduce the activation of canonical lipolysis, whereas cyclooxygenase inhibitors such as flunixin meglumine (FM) can limit inflammation and inhibit the inflammatory lipolytic pathway. The objective of this study was to determine the effects of including NIA and FM in the standard PG treatment for postpartum CK on AT function. Multiparous Jersey cows [n = 18; 7.1 (SD = 3.8) DIM] were selected from a commercial dairy. Inclusion criteria were CK symptoms (lethargy, depressed appetite, and drop in milk yield) and high blood levels of β-hydroxybutyrate (BHB ≥ 1.2 mmol/L). Cows with CK were randomly assigned to one of 3 treatments: 1) PG: 310 g administered orally once per d for 5 d, 2) PG+NIA: 24 g administered orally oral once per d for 3 d, 3) PG+NIA+FM: 1.1 mg/kg administered IV once per day for 3 d. Healthy cows (HC; n = 6) matched by lactation and DIM (±2 d) were sampled. Subcutaneous AT explants were collected at d 0 (d0) and 7 (d7) relative to enrollment. To assess AT insulin sensitivity, explants were treated with insulin (INS = 1 µL/L) during lipolysis stimulation with a β-adrenergic receptor agonist (isoproterenol, ISO = 1 µM). Lipolysis was quantified by glycerol release in the media. Lipid mobilization and inflammatory gene networks were evaluated using real-time qPCR. Protein biomarkers of lipolysis, insulin signaling, and AT inflammation, including HSL, AKT, and ERK1/2, were quantified by capillary immunoassays. Flow cytometry of AT cellular components was used to characterize macrophage inflammatory phenotypes. Statistical significance was determined by a non-parametric t-test when 2 groups (HC vs CK) were analyzed and an ANOVA test with Tukey adjustment when 3 treatment groups (PG vs PGNIA vs PGNIAFM) were evaluated. At d0, AT from CK cows showed higher mRNA expression of lipolytic enzymes ABHD5, LIPE, and LPL, as well as increased phosphorylation of the lipase HSL (pHSL) compared with HC. At d0, INS reduced lipolysis by 41 ± 8% in AT from HC, while CK cows were unresponsive (-2.9 ± 4%). AT from CK cows exhibited reduced Akt phosphorylation compared with HC. CK had increased AT expression of inflammatory gene markers, including CCL2, IL8, IL10, TLR4, and TNF, along with ERK1/2 phosphorylation. AT from CK cows showed increased macrophage infiltration compared with HC. By d7, AT from PGNIAFM cows had a more robust response to INS, as evidenced by reduced glycerol release (36.5 ± 8% compared with PG, 26.9 ± 7%, and PGNIA, 7.4 ± 8%) and enhanced phosphorylation of Akt. By d7, PGNIAFM cows presented lower inflammatory markers, including ERK1/2 phosphorylation and reduced macrophage infiltration, compared with PG and PGNIA. These data suggest that including NIA and FM in CK treatment improves AT insulin sensitivity and reduces AT inflammation and macrophage infiltration.

During the periparturient period, dairy cows exhibit negative energy balance due to limited appetite and increased energy requirements for lactogenesis. The delicate equilibrium between energy availability and expenditure puts cows in a state of metabolic stress characterized by excessive lipolysis in white adipose tissues (AT), increased production of reactive oxygen species, and immune cell dysfunction. Metabolic stress, especially in AT, increases the risk for metabolic and inflammatory diseases. Around parturition, cows are also susceptible to endotoxemia. Bacterial-derived toxins cause endotoxemia by promoting inflammatory processes and immune cell infiltration in different organs and systems while impacting metabolic function by altering lipolysis, mitochondrial activity, and insulin sensitivity. In dairy cows, endotoxins enter the bloodstream after overcoming the defense mechanisms of the epithelial barriers, particularly during common periparturient conditions such as mastitis, metritis, and pneumonia, or after abrupt changes in the gut microbiome. In the bovine AT, endotoxins induce a pro-inflammatory response and stimulate lipolysis in AT, leading to the release of free fatty acids into the bloodstream. When excessive and protracted, endotoxin-induced lipolysis can impair adipocyte’s insulin signaling pathways and lipid synthesis. Endotoxin exposure can also induce oxidative stress in AT through the production of reactive oxygen species by inflammatory cells and other cellular components. This review provides insights into endotoxins’ impact on AT function, highlighting the gaps in our knowledge of the mechanisms underlying AT dysfunction, its connection with periparturient cows’ disease risk, and the need to develop effective interventions to prevent and treat endotoxemia-related inflammatory conditions in dairy cattle.

Periparturient dairy cows exhibit intense lipolysis driven by reduced DMI, enhanced energy needs, and the loss of adipose tissue (AT) insulin sensitivity. Extended periods of low insulin sensitivity and negative energy balance induce lipolysis dysregulation, leading to increased disease susceptibility and poor lactation performance. Chromium (Cr) supplementation improves systemic insulin sensitivity, whereas palmitic acid (PA) increases energy availability for milk production. However, the effect of supplementing Cr and PA alone or in combination on insulin sensitivity in AT is unknown. A total of 32 multiparous cows were used in a randomized complete block design experiment and randomly assigned to one of 4 diets fed from 1 to 24 DIM: a control diet with no supplementation (CON, n = 8); the Cr diet (Cr propionate at 0.45 mg/kg Cr/kg DM, n = 8); the PA diet (1.5% DM, n = 8); or Cr+PA (n = 8). Plasma samples were collected at -13 ± 5.1 d prepartum (PreP), and at 14.4 ± 1.9 d (PP1) and 21 ± 1.9 d (PP2) after calving for quantification of albumin, BHB, BUN, calcium, cholesterol, glucose, nonesterified fatty acids (NEFA), total protein, iron, transferrin, triglycerides, and oxylipids. Subcutaneous AT (SCAT) explants were collected at PreP, PP1, and PP2 and incubated in the presence of the lipolytic agent isoproterenol (ISO = 1 µM, BAS = 0 µM) for 3 h. The antilipolytic effect of insulin (1 µL/L) on SCAT explants was evaluated during ISO stimulation (ISO+INS). Lipolysis was quantified by glycerol release in the medium (nmol glycerol/mg AT). Macrophage infiltration and adipocyte size were measured using hematoxylin and eosin-stained AT sections and immunohistochemistry. The Cr diet tended to reduce postpartum NEFA concentrations when compared with CON, PA, and Cr+PA. Likewise, Cr increased the percentage of large adipocytes (>9,000 µm²) postpartum compared with other diets. In line with higher lipid content, Cr-fed cows had higher ex vivo BAS lipolysis at PP2 when compared with PA and Cr+PA. Isoproterenol induced higher lipolysis at PP1 and PP2, but it was not affected by Cr and PA. The ISO+INS treatment reduced lipolysis by 29.91% ± 11% in Cr compared with ISO. In contrast, ISO+INS did not affect ISO lipolysis in CON, PA, and Cr+PA. Plasma transferrin was reduced by Cr. At PP2, PA cows had 3.3-fold higher macrophage infiltration in SCAT when compared with CON and Cr. Plasma 9-hydroxyoctadecadienoic acid (HODE) and 9-oxo-octadecadienoic acid (oxoODE) were increased by Cr+PA. Palmitic acid increased plasma 13-oxoODE and Cr increased the ratio of 13-HODE to 13-oxoODE. Palmitic acid increased 5-iso prostaglandin F_2α-VI. Our results demonstrate that supplementing Cr during the immediate postpartum enhances SCAT insulin sensitivity and lipid accumulation. Further studies should determine the effects and mechanisms of action of Cr and PA on AT lipogenesis, adipogenesis, and their impact on lactation performance.

Cardiovascular diseases such as hypertension alter thoracic aorta structure. The role that the outer layer of the aorta, its perivascular adipose tissue (PVAT), plays in the pathogenesis of these alterations is poorly understood. In the descending thoracic aorta, PVAT is organized into three distinct strips: one located anterior to the aorta (AP) and two positioned laterally (LP). Genetic tracing indicates differences in the ontogeny of LP and AP, but the implications of these developmental differences and PVAT distribution on adipocyte development remain unknown. We hypothesize that the anatomical location of adipocyte progenitors influences their adipogenic potential and vasoactive functions. PVAT from LP and AP was collected from male SD rats at 10 wks of age (n = 7) to harvest adipocyte progenitors that were differentiated to adipocytes in adipogenic media. Adipogenesis was evaluated after induction and we performed next-generation RNA-seq on progenitors and adipocytes. We then employed Gene Set Enrichment Analysis for enrichment and network analyses. LP progenitors exhibited a 1.13-fold higher adipogenesis rate compared to those from AP. DEG analysis revealed LP had higher expression of adipogenic regulators and basal collagens Col4a2 and Col4a4. When challenged with angiotensin-II, adipocyte progenitors from LP maintained their adipogenic capacity and adipocytes from the same site maintained their secretion of adiponectin at higher rates than AP cells. However, treatment with a Piezo1 mechanoreceptor agonist reduced LP’s adipogenic capacity and diminished their adiponectin secretion. These findings highlight site-specific differences in adipogenic activity, extracellular matrix composition, and the secretion of the vasoactive adipokine adiponectin between the LP and AP PVAT strips of the thoracic aorta, suggesting potential functional distinctions in vascular health and disease.

Supplementation of lactating dairy cows with n-3 fatty acids (FA) has been shown to modulate inflammatory responses and improve reproductive function. Among the n-3 FA used in dairy diets, docosahexaenoic acid (DHA; C22:6n-3) is a substrate for the biosynthesis of oxylipins (OXL) with pro-resolving and anti-inflammatory functions. These OXL represent a well-documented mechanism by which n-3 supplementation modulates inflammatory responses. However, the magnitude of this response when known amounts of DHA are delivered postruminally remains unknown. Our study's objective was to evaluate the effects of DHA supplementation on the plasma OXL profile in lactating dairy cows. Eight ruminally cannulated multiparous cows (97 ± 37 DIM, 49 ± 3 kg milk/d) were assigned to a 4 × 4 Latin square treatment sequence with 21-d periods consisting of 10 d of washout and 11 d of infusion. Docosahexaenoic acid derived from enriched algal oil (65% DHA) was suspended in ethanol (∼200 g/d) and infused abomasally at 0, 2, 4, or 6 g/d for 11 d. Plasma samples were collected via coccygeal venipuncture on d 11 of each period for targeted lipidomic analysis using HPLC tandem MS. Abomasal infusion of DHA increased plasma concentrations of DHA-derived oxylipins 4-, 14-, and 20-hydroxydocoasahexaenoic acid (HDoHE) and tended to enhance 10-HDoHE in a linear, dose-dependent manner. In contrast, DHA supplementation was inversely correlated with plasma concentrations of the proinflammatory, arachidonic acid-based oxylipins 5S,15S-dihydroxy-eicosatetraenoic acid and 8,9-dihydroxyeicosatrienoic acid. Our results demonstrate that abomasal infusion of DHA increases concentrations of DHA-derived anti-inflammatory and pro-resolving oxylipin species, including HDoHE, which may limit excessive lipolysis and inflammation in dairy cows.