Expression of inflammation-related genes is associated with adipose tissue location in horses

Background : In humans, adipose tissue (AT) originating from different depots shows varying gene expression profiles. In horses, the risk of certain metabolic disorders may also be influenced by the impact of specific AT depots. Macrophage infiltration in human and rat AT is considered to be a source of inflammatory changes. In horses, this relationship has not been extensively studied yet. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), a useful method to evaluate differences in mRNA expression across different tissues, can be used to evaluate differences between equine AT depots. For a correct interpretation of the RT-qPCR results, expression data have to be normalized by the use of validated reference genes. The main objectives of this study were to compare mRNA expression of inflammation-related genes, as well as adipocyte morphology and number between different equine AT depots; and in addition, to investigate the presence of antigen presenting cells in equine AT and any potential relationship with adipokine mRNA expression. Results : In this study, the mRNA expression of inflammation-related genes (leptin, chemokine ligand 5, interleukin 1β, interleukin 6, interleukin 10, adiponectin, matrix metalloproteinase 2, and superoxide dismutase 2) and candidate reference gene stability was investigated in 8 different AT depots collected from the nuchal, abdominal (mesenteric, retroperitoneal, and peri-renal) and subcutaneous (tail head and loin) AT region. By using GeNorm analysis, HPRT1, RPL32, and GAPDH were found to be the most stable genes in equine AT. The mRNA expression of leptin, chemokine ligand 5, interleukin 10, interleukin 1β, adiponectin, and matrix metalloproteinase 2 significantly differed across AT depots (P 0.05). Adipocyte area and number of antigen presenting cells per adipocyte significantly differed between AT depots (P < 0.05). Conclusions : Adipose tissue location was associated with differences in mRNA expression of inflammation-related genes. This depot-specific difference in mRNA expression suggests that the overall inflammatory status of horses could be partially determined by the relative proportion of the different AT depots

Changes in oxidative stress in response to different levels of energy restriction in obese ponies

The present study evaluated the effect of different levels of energy restriction on metabolic parameters in obese ponies. Relative weight changes, markers of lipid metabolism, and oxidant/antioxidant balance were monitored. Eighteen obese (body condition score≥7/9) Shetland ponies were studied over a 23.5 week trial, divided into 3 periods. First a 4 week adaptation period in which each animal was fed 100% of their maintenance energy requirements needed to maintain stable obese body weight (MERob). Then a 16.5 weeks weight loss period in which ponies were assigned to receive either 100% (control group, CONTROL), 80% (slow weight loss group, SLOW) or 60% (rapid weight loss group, RAPID) of their MERob. During the 3 week end phase period all animals were again fed 100% of their MERob. Relative weight loss was higher in RAPID (P<0.001) compared to SLOW. No linear relationship was found as a doubling in caloric restriction was accompanied with a tripling in weight loss. Relative weight gain afterwards in the end phase period was higher in RAPID (P<0.001) compared to SLOW and CONTROL. During the weight loss period, triacylglycerol and non-esterified fatty acids levels were highest in RAPID, as were α-tocopherol and ferric reducing ability of plasma. After 8 weeks of weight loss, advanced oxidation protein products were higher in RAPID compared to SLOW and CONTROL (P<0.001). In conclusion, the level of energy restriction influences the extent of changes in oxidant/antioxidant balance. Practically, more severe energy restriction regimens may be associated with a greater regain of weight post restriction

Depot-specific mRNA expression by equine adipocytes

Introduction: Obesity in man and horses has been described as a state of low-grade inflammation characterised by elevated concentrations of circulating inflammatory markers. In horses, adipose tissue distributed specifically on the crest of the neck could indicate or contribute to hyperinsulinemia, insulin resistance or risk for laminitis. The purpose of the study was to compare mRNA expression between different equine adipose tissue depots and the influence of body condition score, using a reliable set of reference genes. Materials and methods: From 12 healthy geldings, euthanised for non-research purposes, adipose tissue samples were collected from neck (nuchal), intra-abdominal (mesenterial, right kidney, retroperitoneal) and subcutaneous (loin, tail head) fat depots. The horses were divided into 2 groups (n=6) according to their BCS: normal BCS (4-5/9) or overweight and obese BCS (7-8.5/9). Results and discussion: Reference gene expression stability and optimal number for normalization was evaluated with the geNorm algorithm. The genes ACTB, GAPDH, HPRT, RPL 32 and SDHA were identified as reference genes. No significant effect of body condition was found for any of the parameters evaluated (P>0.05). Leptin mRNA expression was higher in the three neck samples compared to the mesenteric adipose tissue samples (P0.05). Conclusions: Adipose tissue location is associated with differences in mRNA expression of the genes involved in inflammatory processes. These results suggest that individual differences in adipose tissue distribution may affect inflammatory status

Effect of different degrees of caloric restriction on AOPPs and AGEs formation in healthy obese ponies

It is recognized that caloric restriction (CR) decreases oxidative stress1. Since proteins are highly susceptible to oxidative damage by glycoxidation (with the formation of advanced glycation end products (AGEs) like pentosidine (PENT) and carboxymethyllysine (CML)) or oxidation (with the formation of advanced oxidation protein products (AOPPs)2) the analyses of this compounds could be useful to evaluate the cumulative metabolic burden and oxidative stress. We hypothesized that weight loss by CR has an effect on AGEs and AOPPs. Eighteen obese Shetland geldings (BCS:8/9±1) were studied. After a 4 weeks adaption period to maintain stable obese body weight (BW), ponies were randomly divided in three groups for a 16.5 weeks weight loss program (WLP): control group was fed 100% of individual MER to maintain stable obese BW (C, 0.17±0.02 MJ/kgBW), a ‘slow’ weight loss group, 80% of the MER (S, 0.14±0.01 MJ/kgBW) and a ‘rapid’ weight loss group 60% of the MER (R, 0.10±0.01 MJ/kgBW). Ponies received low energy hay (8.08 MJ/kg DM) and an equal amount of a balancer (Spillers Gro’n Win®, MARS Horsecare) to meet protein, mineral and vitamin requirements . Plasma was collected at the beginning (T0), after 8 weeks (T1) and at the end (T2) of WLP to evaluate PENT (HPLC)3, CML and AOPP (immunoassay kit). Statistical analysis was based on a linear model to identify differences between treatment*time periods. During the WLP BW was reduced by 3.10±2.03% for C; 5.10 ±2.01% for S; 12.56±2.06% for R based on initial BW. No significant effect was recorded for CML. For AOPP, significant lower values were recorded for C and S (P=0.04) and C and R (P<0.01) between the T0 and T1. For PENT, a trend for an increased concentration for R compared to C at T2 compared to and T1 was noted (P=0.08). Different levels of energy restriction and consequently weight loss appear to affect the levels of AOPPs; also a trend for an altered AGEs formation was observed. This could be related to the higher oxidative state and production of by-products of oxidative metabolism4. The accumulation of these end products serves as a measure of cumulative metabolic and oxidative stress5

Determination of depot-specific mRNA expression by equine adipocytes

Adipose tissue is a complex, highly active metabolic and endocrine organ that is involved in energy homeostasis and regulation of immune function. The purpose of the study was to establish a reliable set of reference genes and to examine mRNA expression in different equine adipose tissue depots. From twelve geldings, euthanased for non research purposes, adipose tissue samples were collected from neck (nuchal), intra-abdominal (mesenterial, right kidney, retroperitoneal) and subcutaneous (loin, tail head) fat depots. Expression level of mRNA of a set of genes was determined in each sample. Reference gene expression stability and optimal number for normalization was evaluated with the geNorm algorithm. The genes ACTB, GAPDH, HPRT, RPL 32 and SDHA were identified as housekeeping genes. Five of the examined genes showed a significant influence of adipose tissue depot on the mRNA expression (P0.05). Expression of inflammation-related genes in equine adipocytes behaves differently between fat depots. Abbreviations: ACTB (actin, beta); GAPDH (glyceraldehyde phosphodehydrogenase); HPRT (hypoxanthine ribosyl transferase), RPL 32 (ribosomal protein 32), SDHA (succinate dehydrogenase complex, subunit A