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

Abstract

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.

Introduction

Haptoglobin (Hp) is a major acute phase protein (APP) in cattle with serum concentrations <20 μg/mL in healthy animals and 100–1000-fold increases in response to immune stimulation (Skinner et al., 1991, Godson et al., 1996). In dairy cows, Hp has been used as a marker for various diseases including typical production diseases such as mastitis and fatty liver syndrome (Eckersall and Bell, 2010). Haptoglobin is produced mainly by the liver, but extrahepatic expression such as mammary gland, leukocytes, forestomach and abomasum has also been demonstrated in cattle (Thielen et al., 2005, Thielen et al., 2007, Dilda et al., 2011). In humans, serum Hp concentrations are increased in obese and diabetic patients (Engstroem et al., 2003), correlate with body fat content in obese states (Chiellini et al., 2004, Doumatey et al., 2009), and white adipose tissue has indeed been identified as a site of Hp expression in several monogastric species, e.g. in mice, dogs and pigs (Friedrichs et al., 1995, Eisele et al., 2005, Ramsay et al., 2010) and in man (Fain et al., 2004). Haptoglobin can thus also be classified as an adipokine in these species. The term adipokine comprises proteins synthesized and secreted from adipose tissue (AT) such as classical cytokines, growth factors, hormones, and also APP. Adipokines exert pleiotropic functions, mainly related to energy metabolism and the immune system. The increased production of inflammatory cytokines and APP by AT in obesity is commonly related to localized events within the expanding fat depots (Trayhurn and Wood, 2004) whereby differences between visceral (v.c.) and s.c. depots exist (Fain et al., 2010). Although obesity rarely occurs in dairy cows it is known that overconditioned cows are more likely to develop reproductive problems (Gearhart et al., 1990) and that massive mobilization of white AT fat mass during the transition from pregnancy to peak lactation is linked to a period of insulin resistance, reduced feed intake, negative energy balance, hypocalcaemia and reduced immune function (LeBlanc, 2010). A direct involvement of AT into immune function in cows is likely since expression of proinflammatory cytokines and of another APP, serum amyloid A3, has recently been demonstrated for both s.c. and mesenterial fat depots together with their in vitro responsiveness toward a proinflammatory stimulus (Mukesh et al., 2010). In view of the established diagnostic relevance of serum Hp concentrations, we aimed to elucidate their relation with body condition and with the potential expression of this APP in different adipose depots, thus pointing out the importance for Hp being a bovine adipokine. We therefore characterized the Hp concentrations throughout an entire lactation period in both primiparous and multiparous cows and compared them to the Hp mRNA expression in liver as well as in six different s.c. and v.c. fat depots. Using immunohistochemistry, we extended the Hp analyzes in AT to the protein level. In addition to the physiological changes during lactation, we also tested the effects of dietary supplementation with conjugated linoleic acids (CLA) on Hp in serum, liver and fat. Supplements containing rumen-protected formulations of CLA mainly contain the cis-9, trans-11 and trans-10, cis-12 CLA isomers (Kramer et al., 2004), and reduce milk fat synthesis whereas the effects on body fat seem to be mostly limited to monogastric species since indicators related to lipolysis and energy homeostasis (non-esterified fatty acids (NEFA), leptin, insulin-like growth factor 1, adrenergic responsiveness) reportedly remain mostly unchanged in CLA supplemented dairy cows (Bauman et al., 2008). However, recent reports indicate that adipocyte size in dairy cows is decreased in response to CLA (Akter et al., 2011) whereas fat mass was only affected in the retroperitoneal depot (von Soosten et al., 2011). Based on results from monogastric species, CLA might also exert immunomodulatory actions by attenuating the proinflammatory state in adipose tissue that predisposes to insulin resistance and by suppressing inflammatory responses as demonstrated in a variety of animal models in an isomer specific manner (Hontecillas et al., 2002, Jaudszus et al., 2008, Moloney et al., 2007). In context with these immunomodulatory actions of CLA, decreased blood concentrations of Hp were demonstrated in obese, insulin-resistant rats (Noto et al., 2007). In our study we aimed (1) to assess whether Hp can be designated as an adipokine in cattle, (2) to compare Hp expression in different fat depots, (3) to test for relations between Hp serum concentrations and Hp expression in liver and adipose tissues and (4) to evaluate potential effects of CLA supplements on circulating Hp concentration and Hp tissue expression.