M. Mielenz

Transition period-related changes in the abundance of the mRNAs of adiponectin and its receptors, of visfatin, and of fatty acid binding receptors in adipose tissue of high-yielding dairy cows.

Adipose tissue expresses adipokines, which are involved in regulation of energy expenditure, lipid metabolism, and insulin sensitivity. To adapt for the transition from pregnancy to lactation, particularly in high-yielding dairy cows, adipokines, their receptors, and particular G-protein coupled receptors (GPRs) are of potential importance. Signaling by GPR 41 stimulates leptin release via activation by short-chain fatty acids; GPR 43/109A inhibits lipolysis, and GPR 109A thereby mediates the lipid-lowering effects of nicotinic acid and beta-hydroxybutyrate. The aim of this study was to compare the mRNA expression of adiponectin and visfatin, adiponectin receptors 1 and 2 (AdipoR1/2), leptin receptor (obRb), insulin receptor as of the aforementioned GPRs during the transition period in high-yielding dairy cows. Biopsies from subcutaneous fat and blood samples were obtained from 10 dairy cows 1 week before and 3 weeks after calving. For AdipoR1 and AdipoR2 mRNA abundance as well as for leptin concentrations in plasma, a reduction (P</=.05) was observed postpartum; for visfatin and putative GPR 109A mRNA abundance in adipose tissue, there was a trend (P<.1) for analogous changes. In contrast, the mRNA content of obRb and GPR 41 in adipose tissue was higher (P</=.05) in samples from early lactation than in those from late gestation. Our results indicate decreasing adiponectin sensitivity in adipose tissue after calving, which might be involved in the reduced insulin sensitivity of adipose tissue during early lactation. In addition, visfatin, GPR 41, and GPR 109A may further modulate insulin sensitivity.

Conjugated linoleic acids mediate insulin release through islet G protein coupled receptor FFA1/GPR40

Among dietary components, conjugated linoleic acids (CLAs) have attracted considerable attention as weight loss supplements in the Western world because they reduce fat stores and increase muscle mass. However, a number of adverse effects are also ascribed to the intake of CLAs such as aggravation of insulin resistance and the risk of developing diabetes. However, the mechanisms accounting for the effects of CLAs on glucose homeostasis are incompletely understood. Herein we provide evidence that CLAs specifically activate the cell surface receptor FFA1, an emerging therapeutic target to treat type 2 diabetes. Using different recombinant cellular systems engineered to stably express FFA1 and a set of diverse functional assays including the novel, label-free non-invasive dynamic mass redistribution technology (Corning® Epic® biosensor), both CLA isomers cis-9, trans-11-CLA and trans-10, cis-12-CLA were found to activate FFA1 in vitro at concentrations sufficient to also account for FFA1 activation in vivo. Each CLA isomer markedly increased glucose-stimulated insulin secretion in insulin-producing INS-1E cells that endogenously express FFA1 and in primary pancreatic β-cells of wild type but not FFA1−/− knock-out mice. Our findings establish a clear mechanistic link between CLAs and insulin production and identify the cell surface receptor FFA1 as a molecular target for CLAs, explaining their acute stimulatory effects on insulin secretion in vivo. CLAs are also revealed as insulinotropic components in widely used nutraceuticals, a finding with significant implication for development of FFA1 modulators to treat type 2 diabetes.

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.

Development, validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin

Adiponectin is an adipose tissue-derived glycoprotein circulating as highly abundant multimers. It regulates glucose metabolism and insulin sensitivity. In ruminants, valid data about serum concentrations and tissue-specific protein expression are lacking, and we, therefore, aimed to generate a polyclonal antibody against bovine adiponectin to apply it in immunodetection. The specificity of the purified anti-adiponectin antibody was established by Western blot analysis with the use of reducing and denaturing conditions applied to both the purified protein and the bovine serum samples. Besides bovine serum, the applicability of the antibody for immunodetection of adiponectin was confirmed for the supernatant fluid of in vitro-differentiated bovine adipocytes, for protein extracts from bovine adipose tissue, and also in a multispecies comparison: bands comparable in size with monomeric bovine adiponectin were obtained under denaturing conditions in serum of camel, horse, human, mouse, pig, roe deer, and sheep. In addition, when used in immunohistochemistry on bovine adipose tissue sections, a characteristic adipocyte-specific staining pattern was obtained with this antibody. The antibody was used for establishing a semiquantitative Western blot procedure and the development of an ELISA. Both methods were extensively validated and were first applied to characterize the serum adiponectin concentrations in multiparous dairy cows during the transition from pregnancy to lactation, that is, 3 wk before until 5 wk after calving. With both assays a time effect (P = 0.017, P = 0.026, respectively) with lowest values at the day of parturition was observed. We thus established 2 useful tools to validly assess bovine adiponectin at the protein level.

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.

Putative reference genes for gene expression studies in propionate and β-hydroxybutyrate treated bovine adipose tissue explants.

Accurate gene expression normalization using a stable reference gene (RG) improves the reliability of quantitative real-time PCR (qPCR) results. Therefore, a validation of RGs should be done before their use. Only few studies on RGs have been done in cattle, and none in bovine adipose tissue (AT) explants, therefore, we characterize the effects of an in vitro treatment with propionate and β-hydroxybutyric acid (BHB) on the mRNA content of these RGs comparing the output data from the geNorm™ and the Normfinder(©) program. geNorm™ and Normfinder(©) estimated the most stable RGs in the following sequence for subcutaneous and for retroperitoneal AT explants treated with propionate: low density lipoprotein receptor-related protein 10 (LRP10) > hippocalcin-like 1 (HPCAL1) > glyceraldehyde-phosphate-dehydrogenase (GAPDH) > ribosomal protein S9 (RPS9) > RNA polymerase II (Pol II) > beta2 actin (ACTB) > 18S ribosomal RNA (18S rRNA). BHB treated AT explants yielded a different stability ranking for RGs using geNorm™: HPCAL1, GAPDH > Pol II > LRP10 > ACTB > RPS9 > 18S rRNA. Normfinder(©) estimated a different stability ranking for the RGs as shown in the following sequence for subcutaneous and retroperitoneal AT explants treated with BHB: HPCAL1 > Pol II > GAPDH > ACTB > LRP10 > RPS9 > 18S rRNA. Subsequent pairwise analysis of variation of RGs using geNorm™ suggested that LRP10, HPCAL1 and GAPDH should be used for accurate normalization of subcutaneous and retroperitoneal AT explants treated with propionate, while HPCAL1, GAPDH and Pol II should be used for BHB treatment.

Chlamydophila species in dairy farms: Polymerase chain reaction prevalence, disease association, and risk factors identified in a cross-sectional study in western Germany

The prevalence of Chlamydophila spp. was determined in a cross-sectional study carried out in 2007 using 100 randomly selected dairy herds in the western part of Germany. Ten dairy cows per herd were sampled in herds with fewer than 100 cows; in bigger herds, 10% of the cows were sampled. For the detection of Chlamydophila spp., vaginal swabs from early lactating dairy cows were analyzed using an established highly sensitive genus-specific real-time PCR. In consideration of the discontinuous shedding of the pathogen, a herd was classified as positive if at least 1 animal per herd tested positive for Chlamydophila spp. By use of these methods and definitions, 61% of the dairy herds and 13.5% of the cows were detected as PCR-positive for Chlamydophila spp., which is indicative for ongoing infections. To compare herd health and herd performance between herds testing positive or negative and to identify risk factors for the presence of Chlamydophila spp., a questionnaire was designed to evaluate farm characteristics and management practices. In addition, the performance recordings of the state dairy recording organization were used for these purposes. Milk yield, number of lactations, and calving to first-service interval were lower in herds testing positive for Chlamydophila spp. compared with negative herds. For all these variables, there was no interaction between Chlamydophila status and lactation number. Replacement of animals from outside sources, use of breeding bulls, lack of separate calving pens, and low scores for cleanliness of beddings, walkways, and cows were identified as the main risk factors for Chlamydophila spp.

Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001) and the mRNA abundances of GPR109A (p ≤ 0.05) and chemerin (p ≤ 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.

Differential effects of propionate or β-hydroxybutyrate on genes related to energy balance and insulin sensitivity in bovine white adipose tissue explants from a subcutaneous and a visceral depot1

Ruminants rely on short-chain fatty acids (SCFA) as principal energy source. Herein, we compared the effects of propionate, β-hydroxybutyrate (BHB) and insulin on mRNA abundance of energy balance-related genes by short-term incubation (4 h) in bovine subcutaneous (SC) and retroperitoneal (RP) adipose tissue (AT) explants in vitro. Propionate either significantly (p < 0.05), or as a trend (p ≤ 0.1) affected mRNA abundance of genes such as adiponectin system in both depots in treated samples versus controls. Propionate increased adiponectin receptor 1 (AdipoR1) and AdipoR2 mRNA only in SC AT. β-hydroxybutyrate decreased mRNA abundance of adiponectin and AdipoR1 in SC AT as a trend. The mRNA abundance of free fatty acid receptor 2/3 (FFAR2/3) and other genes of interest (GOI) was increased during differentiation in bovine preadipocyte culture. The mRNA abundance of all the GOI remained unchanged after short-term insulin stimulation. In total, propionate, BHB or insulin during short-term treatment exert divergent effects on the mRNA abundance of GOI in both depots in vitro. Our results indicate that the bovine adiponectin system might be more sensitive to propionate than to BHB. We demonstrated the presence of FFAR2/3 mRNA not only in both AT depots but also in differentiating preadipocytes isolated from bovine SC AT. Therefore, we established that SCFA are able to exert insulin-independent effects on bovine adipose tissue, which might be independent from propionate uptake-related events.