B. Saremi

Technical note: Identification of reference genes for gene expression studies in different bovine tissues focusing on different fat depots

Selection of stable reference genes (REF) is important in real-time PCR data normalization. Bovine tissues such as the mammary gland, liver, muscle, and s.c. fat from the tail head have been thoroughly explored for stable REF, whereas fewer reports exist for other fat depots. Therefore, a suitable combination of REF was tested for different tissues of dairy cattle. Holstein dairy heifers (n = 25) were supplemented (100 g/d) with a control fat (n = 15) without conjugated linoleic acids or with rumen-protected conjugated linoleic acids (n = 10) from the day of calving until slaughter at 1, 42, or 105 d postpartum (n = 5, 10, and 10, respectively). Samples from 6 fat depots (omental, mesenterial, retroperitoneal, s.c. tail head, s.c. withers, and s.c. sternum), liver, semitendinosus muscle, and mammary gland were collected. The REF mRNA were quantified and their stability was analyzed using geNorm(plus). The 3 most stable REF in individual fat tissues and muscle were EMD (emerin), POLR2A (RNA polymerase II), and LRP10 (lipoprotein receptor-related protein 10); in mammary gland were MARVELD1 (marvel domain containing 1), EMD, and LRP10; and in liver were HPCAL1 (hippocalcin-like 1), LRP10, and EIF3K (Eukaryotic translation initiation factor 3). The 3 most stable REF in s.c. fat were EMD, LRP10, and EIF3K; in visceral fat were POLR2A, LRP10, and MARVELD1; and for all 6 adipose tissues were LRP10, EIF3K, and MARVELD1. When the mammary gland was added to the 6 adipose depots, at least 5 REF (LRP10, POLR2A, EIF3K, MARVELD1, and HPCAL1) were needed to reach the threshold of 0.15. Addition of liver to the above-mentioned tissues increased the V value. The data improve the comparison of gene expression between different fat depots. In each case, GAPDH had the lowest stability value.

Longitudinal Profiling of the Tissue-Specific Expression of Genes Related with Insulin Sensitivity in Dairy Cows during Lactation Focusing on Different Fat Depots

In dairy cows the milk associated energy output in early lactation exceeds the input via voluntary feed intake. To spare glucose for mammary lactose synthesis, peripheral insulin sensitivity (IS) is reduced and fat mobilization is stimulated. For these processes a link between IS and the endocrine functions of adipose tissue (AT) is likely; we thus aimed to characterise the mRNA expression from bovine AT derived proteins and receptors that are related to IS according to the literature in metabolically active tissues plus systemic IS throughout lactation. Conjugated linoleic acids (CLA) reduce milk fat thus decreasing the milk drain of energy and potentially dampening lipolysis, but may also affect IS. Subcutaneous (s.c.) AT and liver from pluriparous cows receiving either control fat or CLA supplement (100 g/day from 1 to 182 days in milk each) were biopsied covering week −3 to 36 relative to parturition. In an additional trial with primiparous cows treated analogously and slaughtered on days in milk 1, 42 or 105, samples from liver, udder, skeletal muscle and 3 visceral and 3 s.c. AT were obtained and assayed for mRNA abundance of adiponectin, its receptors, leptin, leptin receptor, PPARγ, PPARγ2, IL-6, and TNF-α. In pluriparous animals, the mRNA abundance of most of the target genes decreased after parturition in s.c. AT but increased in liver. In primiparous cows, AT depot specific differences were mostly related to retroperitoneal AT; adiponectin receptor 1 and TNF-α were affected predominantly. CLA effects in primiparous cows were largely limited to decreased PPARγ2 mRNA abundance in udder tissue. In pluriparous cows, insulin secretion was increased by CLA resulting in decreased systemic IS but without consistent changes in tissue target mRNA abundance. The temporal gene expression profiles from the adipokines and related receptors support their coactive function in adapting to the needs of lactation.

Bovine haptoglobin as an adipokine: Serum concentrations and tissue expression in dairy cows receiving a conjugated linoleic acids supplement throughout lactation

The present study aimed to characterize serum haptoglobin (Hp) concentrations throughout an entire lactation period in both primi- and multiparous cows and to compare them to the Hp mRNA expression in liver and - in view of Hp being potentially an adipokine - also in different subcutaneous (s.c.) and visceral fat depots. In addition, potential anti-inflammatory effects of long-term supplementation with conjugated linoleic acids (CLA) were evaluated by assessing Hp. Trial 1 comprised 33 cows and 16 Holstein heifers from day 21 ante partum until day 252 postpartum. The animals received 100 or 50 g/day CLA or a control fat supplement. Blood samples and biopsy (tail head fat and liver) samples were collected. Trial 2 included 25 Holstein heifers, 5 animals were slaughtered on the day of parturition, the remaining animals were allocated to either CLA (100 g/day, n=10) or control fat supplement (n=10) and slaughtered on days 42 and 105 postpartum, respectively. At slaughter, fat samples were collected from 3 different visceral depots, 3 s.c. depots and from liver tissue. Results indicated no effects of CLA on serum Hp and liver Hp mRNA for both trials and on Hp mRNA in biopsies from s.c. tail head fat. In omental and s.c. withers fat from trial 2, CLA reduced Hp mRNA on both day 42 and day 105. Hp mRNA was detectable in fat tissues from both trials with abundance values being significantly lower than in liver. The Hp mRNA abundance in the s.c. fat depots was generally higher than in the visceral depots. Haptoglobin mRNA abundance in the different tissues from trial 2 was correlated whereby all s.c. depots were interrelated. The evidence of Hp mRNA expression in adipose tissues and the presence of Hp-immune staining in histological fat sections confirm that Hp can be classified as a bovine adipokine. The lack of an evident relationship between circulating Hp concentrations and normal body fat portions in dairy cattle demonstrates that varying degrees of adiposity are not confounding factors when using Hp as inflammatory marker. The physiological changes in serum Hp concentration seem to be limited to parity and parturition. In view of the lack of effects of CLA on serum Hp concentrations, the observed reaction in two out of six different fat depots seems of marginal importance for the organisms as an entity.

Lactation driven dynamics of adiponectin supply from different fat depots to circulation in cows

Adipose tissue (AT) depots are heterogeneous in terms of morphology and adipocyte metabolism. Adiponectin, one of the most abundant adipokines, is known for its insulin sensitizing effects and its role in glucose and lipid metabolism. Little is known about the presence of adiponectin protein in visceral (vc) and subcutaneous (sc) AT depots. We assessed serum adiponectin and adiponectin protein concentrations and the molecular weight forms in vc (mesenterial, omental, and retroperitoneal) and sc (sternum, tail-head, and withers) AT of primiparous dairy cows during early lactation. Primiparous German Holstein cows (n = 25) were divided into a control (CON) and a conjugated linoleic acid (CLA) group. From day 1 of lactation until slaughter, CLA cows were fed 100 g of a CLA supplement/d (approximately 6% of cis-9, trans-11 and trans-10, cis-12 isomers each), whereas the CON cows received 100 g of a fatty acid mixture/d instead of CLA. Blood samples from all animals were collected from 3 wk before calving until slaughter on day 1 (n = 5, CON cows), 42 (n = 5 each of CON and CLA cows), and 105 (n = 5 each of CON and CLA cows) of lactation when samples from different AT depots were obtained. Adiponectin was measured in serum and tissue by ELISA. In all AT depots adiponectin concentrations were lowest on day 1 than on day 42 and day 105, and circulating adiponectin reached a nadir around parturition. Retroperitoneal AT had the lowest adiponectin concentrations; however, when taking total depot mass into consideration, the portion of circulating adiponectin was higher in vc than sc AT. Serum adiponectin was positively correlated with adiponectin protein concentrations but not with the mRNA abundance in all fat depots. The CLA supplementation did not affect adiponectin concentrations in AT depots. Furthermore, inverse associations between circulating adiponectin and measures of body condition (empty body weight, back fat thickness, and vc AT mass) were observed. In all AT depots at each time, adiponectin was present as high (approximately 300 kDa) and medium (approximately 150 kDa) molecular weight complexes similar to that of the blood serum. These data suggest differential contribution of AT depots to circulating adiponectin.

Supplementation with conjugated linoleic acids extends the adiponectin deficit during early lactation in dairy cows

Decreasing insulin sensitivity (IS) in peripheral tissues allows for partitioning nutrients towards the mammary gland. In dairy cows, extensive lipid mobilization and continued insulin resistance (IR) are typical for early lactation. Adiponectin, an adipokine, promotes IS. Supplementation with conjugated linoleic acids (CLA) in rodents and humans reduces fat mass whereby IR and hyperinsulinemia may occur. In dairy cows, CLA reduce milk fat, whereas body fat, serum free fatty acids and leptin are not affected. We aimed to investigate the effects of CLA supplementation on serum and adipose tissue (AT) adiponectin concentrations in dairy cows during the lactation driven and parity modulated changes of metabolism. High yielding cows (n=33) were allocated on day 1 post partum to either 100 g/day of a CLA mixture or a control fat supplement (CON) until day 182 post partum. Blood and subcutaneous (sc) AT (AT) biopsy samples were collected until day 252 post partum to measure adiponectin. Serum adiponectin decreased from day 21 pre partum reaching a nadir at calving and thereafter increased gradually. The distribution of adiponectin molecular weight forms was neither affected by time, parity nor treatment. Cows receiving CLA had decreased serum adiponectin concentrations whereby primiparous cows responded about 4 weeks earlier than multiparous cows. The time course of adiponectin concentrations in sc AT (corrected for residual blood) was similar to serum concentrations, without differences between CLA and CON. CLA supplementation attenuated the post partum increase of circulating adiponectin thus acting towards prolongation of peripartal IR and drain of nutrients towards the mammary gland.

Energy and metabolic sensing G protein–coupled receptors during lactation-induced changes in energy balance

The free fatty acid receptor FFA1, FFA2, and FFA3 and hydroxy-carboxylic acid receptor (HCA2) are G protein-coupled receptors, acting as energy and metabolic sensors. Herein, we characterized the tissue-specific mRNA abundance of genes encoding for these receptors at different stages of lactation. In addition, potential effects of supplementation with or without conjugated linoleic acids (CLA) were tested. Tissues from pluriparous cows (subcutaneous adipose tissue [SAT] and liver) and from primiparous cows (3 SAT locations, 3 visceral adipose tissues, liver, mammary gland, and skeletal muscle) were used from 2 separate trials. In primiparous cows, the mRNA abundance of all receptors (FFA3 was not detectable by the applied protocol in muscle and udder) was lowest in muscle (P < 0.05). With the exception of FFA1, gene expression of the investigated receptors was higher in adipose tissue than in the non-adipose tissue. Expression of FFA1 in liver (P < 0.03), FFAR2 in SAT (P < 0.01), and HCA2 in SAT (P < 0.01) from pluriparous cows changed during the observation period (days 21 to 252 relative to parturition). The correlation between mRNA abundance of HCA2 and peroxisome proliferator-activated receptor gamma (PPARG) and likewise PPARG2 (P < 0.01) in SAT indicates a link between HCA2 and PPARG. Differences in receptor mRNA abundance between the CLA-fed and the control animals were scarce and limited to HCA2 and FFA1 in 1 and 2 time points, respectively (less hepatic HCA2mRNA in CLA-fed pluriparous cows and greater FFA1 mRNA abundance in 2 visceral adipose tissue depots in CLA-treated primiparous cows). In view of the metabolic changes occurring during the different phases of lactation, in particular, the altered concentrations of non-esterified fatty acids and β-hydroxybutyrate acting as receptor ligands, the longitudinal tissue-specific characterization provided herein allows for a first insight into the regulation of these receptors at the gene expression level.

Hepatic and extrahepatic expression of serum amyloid A3 during lactation in dairy cows

Serum amyloid A3 (SAA3) is the predominant SAA isoform secreted by mammary epithelial cells in dairy cows; it is also expressed in bovine adipose tissue (AT). The adipokine SAA3 is linked to obesity and insulin resistance of AT and the respective inflammatory response, at least in mice. Dietary treatment with conjugated linoleic acids (CLA) reportedly also affects insulin sensitivity and inflammatory status in monogastrics. Both SAA3 and CLA thus seem to alter similar functions. Based on changes in insulin sensitivity and the inflammatory status throughout lactation, we hypothesized that the mRNA abundance of SAA3 in various tissues might be regulated as well and that CLA could be a modulator of SAA3 mRNA expression. In 2 trials, 21 pluriparous and 25 primiparous Holstein cows were fed 100g/d of a CLA or a control fat supplement from d 1 to 182 or 105 postpartum, respectively. Biopsies from liver and subcutaneous (s.c.) AT from pluriparous cows and samples from 3 different visceral AT and 3 s.c. AT, muscle, mammary gland, and liver tissue from slaughtered primiparous cows were obtained. In an adipocyte cell culture system, cell samples were collected during differentiation of bovine preadipocytes at d 0, 2, 6, 8, 10, 12, and 13 relative to the onset of differentiation. The SAA3 mRNA abundance in tissues and in differentiating bovine preadipocytes was measured by real-time PCR. The presence of the SAA protein was confirmed by Western blotting. Treatment with CLA yielded only few and inconsistent effects on SAA3 mRNA abundance. In both trials, SAA3 mRNA peaked at d 1 postpartum in all tissues except in mesenteric AT, in which the change was not significant. The highest SAA3 mRNA expression was observed in the mammary gland, followed by omental AT. The SAA protein was present in the visceral and s.c. AT depots investigated. Adipocytes as one source of SAA3 were confirmed by the SAA3 mRNA profile in differentiating adipocytes. The longitudinal changes observed point to SAA3 being involved in the inflammatory situation around parturition.

Plasma amino acids and metabolic profiling of dairy cows in response to a bolus duodenal infusion of leucine

Leucine (Leu), one of the three branch chain amino acids, acts as a signaling molecule in the regulation of overall amino acid (AA) and protein metabolism. Leucine is also considered to be a potent stimulus for the secretion of insulin from pancreatice β-cells. Our objective was to study the effects of a duodenal bolus infusion of Leu on insulin and glucagon secretion, on plasma AA concentrations, and to do a metabolomic profiling of dairy cows as compared to infusions with either glucose or saline. Six duodenum-fistulated Holstein cows were studied in a replicated 3 × 3 Latin square design with 3 periods of 7 days, in which the treatments were applied at the end of each period. The treatments were duodenal bolus infusions of Leu (DIL; 0.15 g/kg body weight), glucose (DIG; at Leu equimolar dosage) or saline (SAL). On the day of infusion, the treatments were duodenally infused after 5 h of fasting. Blood samples were collected at -15, 0, 10, 20, 30, 40, 50, 60, 75, 90, 120, 180, 210, 240 and 300 min relative to the start of infusion. Blood plasma was assayed for concentrations of insulin, glucagon, glucose and AA. The metabolome was also characterized in selected plasma samples (i.e. from 0, 50, and 120 min relative to the infusion). Body weight, feed intake, milk yield and milk composition were recorded throughout the experiment. The Leu infusion resulted in significant increases of Leu in plasma reaching 20 and 15-fold greater values than that in DIG and SAL, respectively. The elevation of plasma Leu concentrations after the infusion led to a significant decrease (P<0.05) in the plasma concentrations of isoleucine, valine, glycine, and alanine. In addition, the mean concentrations of lysine, methionine, phenylalanine, proline, serine, taurine, threonine, and asparagine across all time-points in plasma of DIL cows were reduced (P<0.05) compared with the other groups. In contrast to the working hypothesis about an insulinotropic effect of Leu, the circulating concentrations of insulin were not affected by Leu. In DIG, insulin and glucose concentrations peaked at 30–40 and 40–50 min after the infusion, respectively. Insulin concentrations were greater (P<0.05) from 30–40 min in DIG than DIL and SAL, and glucose was elevated in DIG over DIL and SAL from 30–75 min and 40–50 min, respectively. Multivariate metabolomics data analysis (principal component analysis and partial least squares discriminant analysis) revealed a clear separation when the DIL cows were compared with the DIG and SAL cows at 50 and 120 min after the infusion. By using this analysis, several metabolites, mainly acylcarnitines, methionine sulfoxide and components from the kynurenine pathway were identified as the most relevant for separating the treatment groups. These results suggest that Leu regulates the plasma concentrations of branched-chain AA, and other AA, apparently by stimulating their influx into the cells from the circulation. A single-dose duodenal infusion of Leu did not elicit an apparent insulin response, but affected multiple intermediary metabolic pathways including AA and energy metabolism by mechanisms yet to be elucidated.

Short communication: aquaporin-7 mRNA in adipose depots of primiparous and pluriparous dairy cows: long-term physiological and conjugated linoleic acid-induced changes.

Aquaglyceroporins act as channel proteins and regulate water and glycerol exchange through cell membranes. The aquaglyceroporin aquaporin-7 (AQP7) is abundantly expressed in adipose tissue (AT) and regulates the release of glycerol produced by lipolysis. We aimed to investigate the expression of AQP7 mRNA during lactation in subcutaneous (s.c.) and visceral (v.c.) adipose depots of primiparous and pluriparous dairy cows. In 2 independent experiments, Holstein cows were supplemented with conjugated linoleic acids (CLA) or a control (CON) fat supplement at 100g/d. Pluriparous cows were supplemented starting with the first day in milk (DIM) up to 182 DIM and biopsies from s.c. AT were collected at d -21, 1, 21, 70, 105 182 196, 224, and 252 relative to calving (CLA=11; CON=10). Samples from 3s.c. and v.c. adipose depots were investigated in primiparous cows (n=25) receiving the supplements from 1 DIM until slaughter at 1, 42, or 105 DIM. The AQP7 mRNA abundance decreased from d -21 to 1 in s.c. AT of pluriparous cows without further increase to d 252 of lactation. In primiparous cows of the CON group, the AQP7 mRNA abundance increased from 1 to 105 DIM in s.c. AT from the tail head and in mesenteric AT. In retroperitoneal AT, the only depot for which a significant decrease in mass was observed with DIM, AQP7 mRNA abundance was greater at 42 and 105 than 1 DIM. Comparing the different fat depots, retroperitoneal AT had the highest and mesenterial AT had the lowest AQP7 mRNA abundance, but no general difference was observed between v.c. and s.c. fat depots. The values were not affected by CLA treatment with the exception of mesenteric AT, for which lower AQP7 mRNA abundance values were recorded in the CLA than in the CON group. The longitudinal characterization of the AQP7 mRNA expression profile throughout lactation revealed differences between primiparous and pluriparous cows, with an increase of AQP7 mRNA abundance up to 105 DIM only in the primiparous cows. Due to a lack of CLA effects in pluriparous cows and the limitation to just one fat depot in primiparous cows, a modulatory effect of CLA on AQP7 mRNA abundance in dairy cows is not supported by our study.

Acylcarnitine profiles in serum and muscle of dairy cows receiving conjugated linoleic acids or a control fat supplement during early lactation

Acylcarnitines (ACC) are formed when fatty acid (FA)-coenzyme A enters the mitochondria for β-oxidation and the tricarboxylic acid cycle through the carnitine shuttle. Concentrations of ACC may vary depending on the metabolic conditions, but can accumulate when rates of β-oxidation exceed those of tricarboxylic acid. This study aimed to characterize muscle and blood serum acylcarnitine profiles, to determine the mRNA abundance of muscle carnitine acyltransferases, and to test whether dietary supplementation (from d 1 in milk) with conjugated linoleic acids (CLA; 100 g/d; each 12% of trans-10,cis-12 and cis-9,trans-11 CLA; n = 11) altered these compared with control fat-supplemented cows (CTR; n = 10). Blood samples and biopsies from the semitendinosus musclewere collected on d -21, 1, 21, and 70 relative to parturition. Serum and muscle ACC profiles were quantified using a targeted metabolomics approach. The CLA supplement did not affect the variables examined. The serum concentration of free carnitine decreased with the onset of lactation. The concentrations of acetylcarnitine, hydroxybutyrylcarnitine, and the sum of short-chain ACC in serum were greater from d -21 to 21 than thereafter. The serum concentrations of long-chain ACC tetradecenoylcarnitine (C14:1) and octadecenoylcarnitine (C18:1) concentrations were greater on d 1 and 21 compared with d -21. Muscle carnitine remained unchanged, whereas short- and medium-chain ACC, including propenoylcarnitine (C3:1), hydroxybutyrylcarnitine, hydroxyhexanoylcarnitine, hexenoylcarnitine (C6:1), and pimelylcarnitine were increased on d 21 compared with d -21 and decreased thereafter. In muscle, the concentrations of long-chain ACC (from C14 to C18) were elevated on d 1. The mRNA abundance of carnitine palmitoyltransferase 1, muscle isoform (CPT1B) increased 2.8-fold from d -21 to 1, followed by a decline to nearly prepartum values by d 70, whereas that of CPT2 did not change over time. The majority of serum and muscle short- and long-chain ACC were positively correlated with the FA concentrations in serum, whereas serum carnitine and C5 were negatively correlated with FA. Time-related changes in the serum and muscle ACC profiles were demonstrated that were not affected by the CLA supplement at the dosage used in the present study. The elevated concentrations of long-chain ACC species in muscle and of serum acetylcarnitine around parturition point to incomplete FA oxidation were likely due to insufficient metabolic adaptation in response to the load of FA around parturition.