Expression of metabolic sensing receptors in adipose tissues of periparturient dairy cows with differing extent of negative energy balance

We recently showed that the mRNA expression of genes encoding for specific nutrient sensing receptors, namely the free fatty acid receptors (FFAR) 1, 2, 3, and the hydroxycarboxylic acid receptor (HCAR) 2, undergo characteristic changes during the transition from late pregnancy to lactation in certain adipose tissues (AT) of dairy cows. We hypothesised that divergent energy intake achieved by feeding diets with either high or low portions of concentrate (60% v. 30% concentrate on a dry matter basis) will alter the mRNA expression of FFAR 1, 2, 3, as well as HCAR2 in subcutaneous (SCAT) and retroperitoneal AT (RPAT) of dairy cows in the first 3 weeks postpartum (p.p.). For this purpose, 20 multiparous German Holstein cows were allocated to either the high concentrate ration (HC, n=10) or the low concentrate ration (LC, n=10) from day 1 to 21 p.p. Serum samples and biopsies of SCAT (tail head) and RPAT (above the peritoneum) were obtained at day −21, 1 and 21 relative to parturition. The mRNA abundances were measured by quantitative PCR. The concentrations of short-chain fatty acid (SCFA) in serum were measured by gas chromatography-flame ionisation detector. The FFAR1 and FFAR2 mRNA abundance in RPAT was higher at day −21 compared to day 1. At day 21 p.p. the FFAR2 mRNA abundance was 2.5-fold higher in RPAT of the LC animals compared to the HC cows. The FFAR3 mRNA abundance tended to lower values in SCAT of the LC group at day 21. The HCAR2 mRNA abundance was neither affected by time nor by feeding in both AT. On day 21 p.p. the HC group had 1.7-fold greater serum concentrations of propionic acid and lower concentrations of acetic acid (trend: 1.2-fold lower) compared with the LC group. Positive correlations between the mRNA abundance of HCAR2 and peroxisome proliferator-activated receptor γ-2 (PPARG2) indicate a link between HCAR2 and PPARG2 in both AT. We observed an inverse regulation of FFAR2 and FFAR3 expression over time and both receptors also showed an inverse mRNA abundance as induced by different portions of concentrate. Thus, indicating divergent nutrient sensing of both receptors in AT during the transition period. We propose that the different manifestation of negative EB in both groups at day 21 after parturition affect at least FFAR2 expression in RPAT.     

Structural basis for GPCR signaling by small polar versus large lipid metabolites—discovery of non-metabolite ligands

Key metabolites act through specific G protein-coupled receptors (GPCRs) as extracellular signals of fuel availability and metabolic stress. Here, we focus on the succinate receptor SUCNR1/GPR91 and the long chain fatty acid receptor FFAR1/GPR40, for which 3D structural information is available. Like other small polar acidic metabolites, succinate is excreted from the cell by transporter proteins to bind to an extracellular, solvent-exposed pocket in SUCNR1. Non-metabolite pharmacological tool compounds are currently being designed based on the structure of the SUCNR1 binding pocket. In FFAR1, differently signaling lipid mimetics bind in two distinct membrane-exposed sites corresponding to each of the lipid bilayer leaflets. Conceivably endogenous lipid ligands gain access to these sites by way of the membrane and probably occupy both sites under physiological circumstances. Design of polar agonists for a dynamic, solvent-exposed pocket in FFAR1 underlines the possibility of structure-based approaches for development of novel tool compounds even in lipid sensing metabolite GPCRs.     

MicroRNAs in farm animals

MicroRNAs (miRNAs) are a class of ∼22 nucleotide-long small noncoding RNAs that target mRNAs for translational repression or degradation. miRNAs target mRNAs by base-pairing with the 3′-untranslated regions (3′-UTRs) of mRNAs. miRNAs are present in various species, from animals to plants. In this review, we summarize the identification, expression, and function of miRNAs in four important farm animal species: cattle, chicken, pig and sheep. In each of these species, hundreds of miRNAs have been identified through homology search, small RNA cloning and next generation sequencing. Real-time RT-PCR and microarray experiments reveal that many miRNAs are expressed in a tissue-specific or spatiotemporal-specific manner in farm animals. Limited functional studies suggest that miRNAs have important roles in muscle development and hypertrophy, adipose tissue growth, oocyte maturation and early embryonic development in farm animals. Increasing evidence suggests that single-nucleotide polymorphisms in miRNA target sites or miRNA gene promoters may contribute to variation in production or health traits in farm animals.     

A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs

Fatty acids, which are essential nutritional components, are also involved in cardiovascular and metabolic diseases. Here we report a human cell surface receptor that we name free fatty acid receptor (FFAR), because it is specifically activated by medium to long-chain free fatty acids. The receptor belongs to the class of seven-transmembrane, G-protein coupled receptors (GPCRs) and also mediates responses to antidiabetic drugs of the thiazolidinedione type. It is expressed in skeletal muscle, heart, liver, and pancreatic beta-cells. Stimulation of FFAR increases the intracellular calcium concentration in cells expressing the receptor in a native (pancreatic beta-cell line) or in a recombinant form. In view of the nature of the activating substances, their physiological role in the body, and the tissue distribution of FFAR we suggest the term "nutrient sensing receptor" for receptors acting at the interface between dietary components and signalling molecules.     

Differential expression of prostaglandin receptor mRNAs during adipose cell differentiation.

To clarify the molecular basis for the prostaglandin (PG) mediated effects in adipose cells at various stages of their development, expression of mRNAs encoding receptors specific for prostaglandin E2, F2alpha and I2 (i.e. EP, FP, and IP receptors) was investigated in differentiating clonal Ob1771 pre-adipocytes, as well as in mouse primary adipose precursor cells and mature adipocytes. We have further characterized the differential expression of mRNAs encoding three subtypes of the EP receptor, i.e. EP1, EP3, and EP4, and examined the expression of mRNAs encoding the three isoforms (alpha, beta, and gamma) of the EP3 receptor. Altogether the results show that the expression of IP, FP, EP1, and EP4 receptor mRNAs was considerably more pronounced in pre-adipose cells than in adipose cells, mRNAs encoding the alpha, beta, and gamma isoforms of the EP3 receptor were all exclusively expressed in freshly isolated mature adipocytes. These data may indicate that PGI2, PGF2alpha, and PGE2 may interact directly with specific receptors in pre-adipose cells, whose transduction mechanisms are known to affect maturation related changes. In mature adipocytes, however, the equipment of mRNAs encoding the EP3 receptor isoforms is in agreement with the well known effect of PGE2 on adenylate cyclase and lipolysis in mature adipocytes.     

Distribution of prostaglandin E receptors in the rat gastrointestinal tract

: In order to study the role of prostaglandin in the regulation of the gastrointestinal functions, gene expression of prostaglandin receptors along the rat gastrointestinal tracts were investigated.

: Rats were used for the study. The combination of counterflow elutriation separation of mucosal cells and Northern blot analysis was used to detect the gene expression of prostaglandin receptors in gastrointestinal tracts.

: In small intestine and colon, prostaglandin E2 EP1 and EP3 receptor mRNAs were mainly localized in the deeper intestinal wall containing muscle layers. EP4 receptor gene expression, on the other hand, was detected in the intestinal mucosal layer.

In the stomach, EP1 mRNA was detected in gastric muscle layers, whereas EP3 and EN receptor gene expression was mainly present in the gastric mucosal layer containing epithelial cells. In gastric epithelial cells, parietal cells were found to have both EP3 and EP4 receptors. At lower concentrations, prostaglandin E2 inhibited gastric acid secretion by parietal cells probably through EP4 receptors. At higher concentrations, however, it stimulated it. On the other hand, mucous cells possessed only EP4 receptor mRNA.

: Thus, it is suggested that prostaglandin E2 modulates gastrointestinal functions through at least three different prostaglandin receptors (EP1, EP3, and EP4), each of which has a distinct distribution in the gastrointestinal tract.     

游离脂肪酸受体蛋白研究进展

游离脂肪酸不仅是人和动物体的一种重要能量来源,也是一种重要的信号分子。最近研究表明,游离脂肪酸受体蛋白在维持机体内的葡萄糖稳衡、脂肪形成、白细胞功能等生理过程中都有重要的作用,对于调控人或动物的营养代谢及疾病发生具有重要生理意义。     

Expression of adiponectin receptors in pancreatic beta cells

Pancreatic beta cell dysfunction is an early and crucial pathogenic factor in the development of type 2 diabetes. Free fatty acids (FFA) and adipokines released from adipose tissues lead to both the development of insulin resistance and beta cell dysfunction. Adiponectin is a novel adipokine with antidiabetic properties. Its circulating concentrations are reduced in subjects with increased visceral adiposity, insulin resistance, or type 2 diabetes. Very recently, the cloning of two adiponectin receptors AdipoR1 and AdipoR2 was reported. AdipoR1 is abundantly expressed in muscle, while AdipoR2 is predominantly expressed in liver. Here we report the marked expression of mRNAs for the adiponectin receptors AdipoR1 and AdipoR2 in human and rat pancreatic beta cells, at levels similar to liver and greater than muscle. Adiponectin receptor expression is increased by beta cell exposure to the unsaturated FFA oleate, and treatment of insulin-producing cells with globular adiponectin induces lipoprotein lipase expression. Regulated adiponectin receptor expression on pancreatic beta cells might be a novel mechanism modulating the effects of circulating adiponectin.     

Fat metabolism is regulated by altered gene expression of lipogenic enzymes and regulatory factors in liver and adipose tissue but not in semimembranosus muscle of pigs during the fattening period

It has been shown previously that lipid metabolism is regulated by fatty acids (FA) and that thyroid hormones are important regulators of energy metabolism. The effects of weight, dietary fat level and dietary FA profile on thyroid hormone levels and expression of lipogenic genes and tissue FA composition were studied. Sixty-one crossbred gilts weighing 62 ± 5.2 kg BW average were either slaughtered at the beginning of the trial (n = 5) or fed one of seven diets (n = 8 pigs per diet): a semi-synthetic diet formulated to contain a very low level of fat (NF) and six diets based on barley-soybean meal supplemented with approximately 10% fat of different origin and slaughtered at 100 kg BW. The supplemental fats were tallow, high-oleic sunflower oil, sunflower oil (SFO), linseed oil, fat blend (55% tallow, 35% sunflower oil, 10% linseed oil) and fish oil blend (40% fish oil, 60% linseed oil). In general, the dietary FA profiles altered the FA composition of liver, semimembranosus muscle and adipose tissues. Pigs fed the NF diet had the highest free and total triiodothyronine (T3) values followed by pigs fed SFO. Total T3 levels were higher in pigs at 60 kg than in pigs at 100 kg. Correlations between thyroid hormones and genes encoding enzymes of fat synthesis in adipose tissue (acetyl CoA carboxylase (ACACA), fatty acid synthase and stearoyl CoA desaturase (SCD)) and the large differences in expression of lipogenic genes at different weights (60 and 100 kg BW), suggest a role for thyroid hormones and for T3, in particular, in regulating whole animal fat metabolism, with effects brought about by altered expression of lipogenic genes. Liver sterol receptor element binding protein-1 (SREBP1) mRNA content was affected by dietary treatment (P < 0.001) and was correlated with ACACA and SCD, whereas adipose tissue SREBP1 was not correlated with the mRNA abundance of any lipogenic enzyme. Weight and tissue factors showed greater influence on mRNA abundance of genes related with lipid metabolism than diet and tissue FA composition. In the pig, FA synthesis appear to be of greater magnitude in adipose tissue than in the liver as suggested by the higher expression of lipogenic genes in adipose tissue.     

Mapping, expression and regulation of the TRα gene in porcine adipose tissue

Thyroid hormone receptors (TR) are members of the nuclear receptor superfamily. There are at least two TR isoforms, TRα and TRβ. The TRα isoform plays a critical role in mediating the action of thyroid hormone in adipose tissue. We mapped the porcine TRα gene to chromosome 12 p11-p13, by using the ImpRH panel. We examined tissue-localization of TRα and determined expression patterns of TRα in porcine adipose tissue with quantitative real-time PCR. TRα was expressed in all tissues, including heart, liver, spleen, stomach, pancreas, brain, small intestine, skeletal muscle, and subcutaneous adipose tissue. In the adipose tissue, the expression of TRα decreased postnatally. Compared to Yorkshire pigs, Jinhua pigs had significantly lower expression levels of TRα gene in the subcutaneous fat tissue. The expression levels of β2-AR, HSL and ATGL were also significantly lower in Jinhua pigs than in Yorkshire pigs. However, no significant differences in PPARγ and SREBP-1C expression levels were found between Jinhua and Yorkshire pigs. Incubation of porcine adipose tissue explants with high doses of isoproterenol (100 and 1000 nM) significantly increased the expression levels of TRα. We conclude that there is considerable evidence that TRα plays an important role in fat deposition in porcine adipose tissue.     

Expression of TWEAK and its receptor Fn14 in human subcutaneous adipose tissue. Relationship with other inflammatory cytokines in obesity

TWEAK, a cytokine of the TNF family, has been found to be expressed under different inflammatory conditions but no data is available concerning the expression of this cytokine and its receptor (Fn14) in human obesity. In the present work we have evaluated the expression of many pro-inflammatory TNF system cytokines (TNF-alpha, TWEAK and their respective receptors, TNFR1, TNFR2 and Fn14) in human adipose tissue of 84 subjects some with different degree of obesity and type 2 diabetes, and its relation with inflammation by also measuring the expression of macrophage marker CD68. We detected expression of TWEAK and Fn14 in isolated mature adipocytes and in the stromovascular fraction. Additionally, we found that LPS upregulates the expression of both genes on THP-1 human monocytic cell line. TWEAK was expressed in adipose tissue of all studied subjects with no differences between obesity group, and was associated with Fn14 expression in morbid obese, mainly in women with type 2 diabetes. The data obtained here also showed that TNF-alpha and TNFR2 mRNAs were significantly more expressed in subcutaneous adipose tissue of subjects with morbid obesity compared to obese and non-obese subjects. In contrast, TNFR1 gene expression was negatively associated with BMI. Our results suggest that the expression of TNF-derived pro-inflammatory cytokines are increased in severe obesity, where macrophage infiltrate could modulate the inflammatory environment through activation of its receptors.     

Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3-independent mechanisms

Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are metabolites formed by gut microbiota from complex dietary carbohydrates. Butyrate and acetate were reported to protect against diet-induced obesity without causing hypophagia, while propionate was shown to reduce food intake. However, the underlying mechanisms for these effects are unclear. It was suggested that SCFAs may regulate gut hormones via their endogenous receptors Free fatty acid receptors 2 (FFAR2) and 3 (FFAR3), but direct evidence is lacking. We examined the effects of SCFA administration in mice, and show that butyrate, propionate, and acetate all protected against diet-induced obesity and insulin resistance. Butyrate and propionate, but not acetate, induce gut hormones and reduce food intake. As FFAR3 is the common receptor activated by butyrate and propionate, we examined these effects in FFAR3-deficient mice. The effects of butyrate and propionate on body weight and food intake are independent of FFAR3. In addition, FFAR3 plays a minor role in butyrate stimulation of Glucagon-like peptide-1, and is not required for butyrate- and propionate-dependent induction of Glucose-dependent insulinotropic peptide. Finally, FFAR3-deficient mice show normal body weight and glucose homeostasis. Stimulation of gut hormones and food intake inhibition by butyrate and propionate may represent a novel mechanism by which gut microbiota regulates host metabolism. These effects are largely intact in FFAR3-deficient mice, indicating additional mediators are required for these beneficial effects.     

Homology modeling and explicit membrane molecular dynamics simulation to delineate the mode of binding of thiazolidinediones into FFAR1 and the mechanism of receptor activation

Free fatty acid receptor 1 (FFAR1) is a member of a previously characterized cluster of orphan G protein-coupled receptors (GPCRs). Later, this orphan receptor was identified as a target of medium- to long-chain free fatty acids in β-cells of the pancreas. Administration of FFAR1 agonists has been proved to potentiate glucose-stimulated insulin secretion from pancreatic β-cells. It was reported that some thiazolidinediones (TZDs), the best studied PPARγ agonists, are also able to stimulate FFAR1 in a dose-dependent manner. In the present study, a homology model of the human FFAR1 was constructed and inserted into a pre-equilibrated DPPC/TIP3P membrane system. This system was then simulated for 20 ns in complex with the FFAR1 agonist GW9085, as well as rosiglitazone and pioglitazone. We noticed that the salt bridge between Glu172 and Arg258 and the H bond between Glu145 and His153 could be responsible for the stabilization of the receptor in the inactive state. Moreover, we described for the first time the binding mode of TZDs in the binding site of FFAR1. The thiazolidinedione head forms a hydrogen bonding network with the critical polar residues in the binding site, Arg258 and Asn244, while the rest of the molecule is embedded into the receptor hydrophobic pocket. Based on this modeling study, we arrived at a proposal of the pharmacophore required for binding to both PPARγ and FFAR1. Insights gained from this investigation should provide future directions for the design of novel dual acting antidiabetic agents.     

Identification and sequence analysis of chicken Toll-like receptors

Toll-like receptors (TLRs) play an important role in the recognition of microbial components. Only chicken TLR2 and -4 have been reported in the literature. The objectives of this study were to identify new chicken TLRs and to evaluate evolutionary significance of these receptors. Searching chicken genomic databases and DNA sequencing revealed five new TLRs, TLR1 (type 1 and 2), -3, -5, and -7. No chicken orthologues of mammalian TLR8, -9, or -10 were found. As in mammals, all chicken TLRs (chTLRs) share identical protein secondary structure that consists of several leucine-rich domains, a transmembrane domain, and Toll/Interleukin-1 receptor domain(s). Phylogenetic analyses indicate that the identified chTLR genes are the orthologues of TLRs in mammals. Analyses of the number of synonymous substitutions per synonymous site and nonsynonymous substitutions per nonsynonymous site indicate that the nucleotide sequences coding for the leucine-rich repeats of chicken TLR1 type 1 and type 2 were significantly under positive Darwinian selection. In contrast, the sequences of other TLRs were under purifying selection. These results support the hypothesis that one of the major evolutionary strategies of the innate immune system is to recognize a few highly conserved microbial components with several conserved TLRs. The results also indicate that the sequence changes in the ligand-binding domains of TLR1 in chickens provide adaptive advantages during evolution.Nucleotide sequence data reported are available in GenBank database under the accession numbers AY633573–AY633577     

Human olfactory receptor families and their odorants

The human nose detects volatile chemical stimuli by at least three different receptor families: odorant receptors, trace amine-associated receptors, and vomeronasal type-1 receptors. As G protein-coupled receptors, all of the few functionally characterized olfactory receptors share major functional features: when expressed in heterologous cell systems, they 1) respond to odorants of certain chemical groups, e.g., amines, aliphatic carboxylic acids or aldehydes, floral or fruity odorants, including certain key-food odorants, and putative pheromones, and 2) transduce their signals to intracellular cAMP signaling. However, little is known yet about specific differences in the functional designation of the three olfactory receptor families. Recently, two heterologous cell systems expressing olfactory signaling molecules have been developed. Different screening strategies will shed light on the yet sparsely available odorant specificity profiles and structure-function relationships of olfactory receptors, as well as the structure-activity relationships of their odorants.     

Whole body distribution of deuterated linoleic and alpha-linolenic acids and their metabolites in the rat

Little is known about the uptake or metabolism of essential fatty acids (EFAs) in various mammalian organs. Thus, the distribution of deuterated alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) and their metabolites was studied using a stable isotope tracer technique. Rats were orally administered a single dose of a mixture (20 mg each) of ethyl D5-18:3n-3 and D5-18:2n-6, and 25 tissues per animal were analyzed for D5-labeled PUFAs at 4, 8, 24, 96, 168, 240, 360, and 600 h after dosing. Plasma, stomach, and spleen contained the highest concentrations of labeled precursors at the earliest time points, whereas other internal organs and red blood cells reached their maximal concentrations at 8 h. The time-course data were consistent with liver metabolism of EFAs, but local metabolism in other tissues could not be ruled out. Brain, spinal cord, heart, testis, and eye accumulated docosahexaenoic acid with time, whereas skin accumulated mainly 20:4n-6. On average, approximately 16-18% of the D5-18:3n-3 and D5-18:2n-6 initial dosage was eventually accumulated in tissues, principally in adipose, skin, and muscle. Approximately 6.0% of D5-18:3n-3 and 2.6% of D5-18:2n-6 were elongated/desaturated and stored, mainly in muscle, adipose, and the carcass. The remaining 78% of both precursors was apparently catabolized or excreted.