Adipose tissue angiogenesis

A review of adipose tissue angiogenesis includes the morphological and cytochemical development of adipose tissue vasculature and the concept of primitive fat organs. Spatial and temporal relationships between fetal vascular and fat cell development are discussed, including depot- and genetic-dependent arteriolar differentiation. The relationship between connective tissue deposition and elaboration of adipose tissue vasculature is discussed with respect to regulating adipocyte development in a depot-dependent manner. In vitro studies indicated that depot-dependent vascular traits may be attributable to intrinsic growth characteristics of adipose tissue endothelial cells. These studies indicate that adipogenesis may be regulated by factors that drive angiogenesis. Fundamental aspects of angiogenesis, including basement membrane breakdown, vasculogenesis, angiogenic remodeling, vessel stabilization, and vascular permeability were reviewed. Critical angiogenic factors include vascular endothelial growth factor (VEGF), VEGF receptors, angiopoietins (Ang), ephrins, matrix metalloproteinases, and the plasminogen enzymatic system. Vascular endothelial growth factor is the most critical factor because it initiates the formation of immature vessels and disruption of a single VEGF allele leads to embryonic lethality in mice. Expression of VEGF is influenced by hypoxia, insulin, growth factors, and several cytokines. Angiogenic factors secreted and/or produced by adipocytes or preadipocytes are discussed. Vascular endothelial growth factor expression and secretion by adipocytes is regulated by insulin and hypoxia, and is associated with adipose tissue accretion. Vascular endothelial growth factor accounts for most of the angiogenic activity of adipose tissue. The proposed role of leptin as an adipogenic factor is reviewed with respect to efficacy on various aspects of angiogenesis relative to other angiogenic factors. The VEGF and leptin genes are both hypoxia inducible, but potential links between VEGF and leptin gene expression have not been examined. Finally, several studies including a study of mice treated with antiangiogenic factors indicate that adipose tissue accretion can be controlled through the vasculature per se.     

Spatiotemporal heterogeneity of PPARγ expression in porcine uteroplacenta for regulating of placental angiogenesis through VEGF-mediated signalling

Non-infectious prenatal mortality severely affects the porcine industry, with pathological placentation as a likely key reason. Previous studies have demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) deficiency causes defects in the uteroplacental vasculature and induces embryonic losses in mice. However, its role in porcine placental angiogenesis remains unclear. In the present study, PPARγ expression was investigated in porcine uteroplacental tissues at gestational day (GD) 25, GD40 and GD70 via quantitative polymerase chain reaction (qPCR), Western blot and immunohistochemistry (IHC). Moreover, the roles of PPARγ in porcine placental angiogenesis were investigated using a cell model of porcine umbilical vein endothelial cells (PUVECs) to conduct proliferation, migration and tube formation assays in vitro and a mouse xenograft model to assess capillary formation in vivo. The results showed that PPARγ was mainly located in the glandular epithelium, trophoblast, amniotic chorion epithelium and vascular endothelium, as indicated by the higher expression levels at GD25 and GD40 than at GD70 in endometrium and by higher expression levels at GD40 and GD70 than at GD25 in placenta. Moreover, PPARγ expression was significantly downregulated in placenta with dead foetus. In PUVECs, knocking out PPARγ significantly inhibited proliferation, migration and tube formation in vitro and inhibited capillary formation in mouse xenografts in vivo by blocking S-phase, promoting apoptosis and downregulating the angiogenic factors of VEGF and its receptors. Overall, the spatiotemporal heterogeneity of PPARγ expression in porcine uteroplacental tissue suggests its vital role in endometrial remodelling and placental angiogenesis, and PPARγ regulates placental angiogenesis through VEGF-mediated signalling.     

Fat depot‐specific differences in leptin mRNA expression and its relation to adipocyte size in steers

ABSTRACT This experiment was conducted to investigate leptin mRNA expression, adipocyte size, and their relationship in several adipose tissues of fattening steers. Subcutaneous, perirenal, intermuscular and intramuscular adipose tissues were collected from three crossbred steers (Japanese Black cattle X Holstein) aged 21 months. The mRNA level and adipocyte diameter were determined in these adipose tissues. The intramuscular adipose tissue had a lower leptin mRNA level than the intermuscular and perirenal adipose tissues (P < 0.05). Leptin mRNA level was lower in the subcutaneous depot than in the intermuscular depot (P < 0.05). Adipocyte diameter was larger in the intermuscular adipose tissue than in the subcutaneous and intramuscular adipose tissues (P < 0.05). Leptin mRNA level was positively correlated with adipocyte diameter (r² = 0.81, P < 0.05). These results suggest that the cattle have fat depot‐specific differences in leptin gene expression, which are a result of a difference in adipocyte size.     

猪CRTC3基因表达的品种差异及forskolin对其表达的影响

本试验旨在探究糖脂代谢通路关键基因CRTC3在不同品种猪肌肉和脂肪组织中的表达情况,并通过forskolin处理猪皮下脂肪前体细胞,研究forskolin对脂肪前体细胞分化聚酯和CRTC3基因表达的影响,阐明猪CRTC3基因表达与脂肪沉积的关系。试验选取杜长大猪和莱芜猪各5头,检测肌肉、脂肪组织中CRTC3的mRNA和蛋白表达水平以及脂肪代谢相关基因的mRNA表达水平;选取2头3日龄的杜长大仔猪,分离猪皮下脂肪前体细胞,待完全融合后用MDI诱导培养基诱导4 d,然后用分化培养基继续诱导4 d,完成诱导分化。Forskolin组在诱导分化的第1天即加入forskolin,使其终浓度为10μmol/L,对照组则加入同浓度的二甲基亚砜(DMSO)进行诱导分化。结果表明:在莱芜猪的背最长肌和腰大肌中,CRTC3的蛋白表达水平高于杜长大猪;在莱芜猪的皮下和内脏脂肪组织中,CRTC3及脂肪沉积相关基因过氧化物酶体增殖剂激活受体γ(PPARγ)、脂肪酸结合蛋白4(FABP4)、CCAAT/增强子结合蛋白α(C/EBPα)、围脂滴蛋白(PLIN)和瘦素(LEP)的mRNA表达水平显著或极显著高于杜长大猪(P<0.05或P<0.01),而脂肪棕色化相关基因NF-E2相关因子1(NRF1)、过氧化物酶体增殖物激活受体-γ共激活因子-1α(PGC⁃1α)、PRDM16、解偶联蛋白2(UCP2)、解偶联蛋白3(UCP3)的mRNA表达水平则显著或极显著低于杜长大猪(P<0.05或P<0.01)。进一步的研究发现,猪皮下脂肪前体细胞分化后CRTC3和脂肪沉积相关基因的mRNA表达水平极显著提高(P<0.01),脂肪棕色化相关基因的mRNA表达水平也均极显著升高(P<0.01)。10μmol/L forsko⁃lin处理能抑制猪皮下脂肪前体细胞分化,极显著升高环磷腺苷效应元件结合蛋白(CREB)和脂肪棕色化相关基因的mRNA表达水平(P<0.01),促进CRTC3的进核,极显著降低CRTC3和脂肪沉积相关基因的mRNA表达水平(P<0.01)。上述研究结果表明,CRTC3基因与猪脂肪沉积密切相关,forskolin处理可以调控猪CRTC3及脂质代谢相关基因表达,调控猪皮下脂肪前体细胞分化聚酯。     

Regulation of hepatic peroxisome proliferator‐activated receptor α expression but not adiponectin by dietary protein in finishing pigs

Soy protein regulates adiponectin and peroxisome proliferator‐activated receptor α (PPARα) in some species, but the effect of dietary soy protein on adiponectin and PPARα in the pig has not been studied. Therefore, the objective of this study was to determine whether soya bean meal reduction or replacement influences serum adiponectin, adiponectin mRNA, serum metabolites and the expression of PPARα and other genes involved in lipid deposition. Thirty‐three pigs (11 pigs per treatment) were subjected to one of three dietary treatments: (i) reduced crude protein (CP) diet containing soya bean meal (RCP‐Soy), (ii) high CP diet containing soya bean meal (HCP‐Soy) or (iii) high CP diet with corn gluten meal replacing soya bean meal (HCP‐CGM) for 35 days. Dietary treatment had no effect on overall growth performance, feed intake or measures of body composition. There was no effect of dietary treatment on serum adiponectin or leptin. Dietary treatment did not affect the abundance of the mRNAs for adiponectin, PPARα, PPARγ2, lipoprotein lipase or fatty acid synthase in adipose tissue. The mRNA expression of PPARα, PPARγ2, lipoprotein lipase or fatty acid synthetase in loin muscle was not affected by dietary treatment. In liver tissue, the relative abundance of PPARα mRNA was greater (p < 0.05) in pigs fed the HCP‐Soy diets when compared to pigs fed RCP‐Soy or HCP‐CGM diets. Hepatic mRNA expression of acyl‐CoA oxidase or fatty acid synthase was not affected by dietary treatment. Western blot analysis indicated that hepatic PPARα protein levels were decreased (p < 0.05) in pigs fed the RCP‐Soy diets when compared to pigs fed the HCP‐Soy diets. These data suggest that increasing the soy protein content of swine diets increases hepatic expression of PPARα without associated changes in body composition.     

Leptin mRNA and Protein Immunoreactivity in Adipose Tissue and Liver of Rainbow Trout (Oncorhynchus mykiss) and Immunohistochemical Localization in Liver

Leptin is an important modulator of energy balance and metabolism in mammals, but for evolutionary older vertebrates like fish, the first reports on leptin expression were only recently characterized and the functional role scarcely. In this study, we demonstrated leptin immunoreactivity in liver tissue of rainbow trout (Oncorhynchus mykiss) by immunohistochemistry using three different polyclonal antibodies against mammalian leptin. Immunoreactivity was observed in hepatocytes and also in parts of the biliary system. Using Western blot, we detected an immunoreactive band of about 16 kDa in serum and visceral adipose tissue (AT) of rainbow trout. The presence of leptin in fish AT has been doubted in other studies. Besides the immunoreactivity, leptin mRNA was detected in trout AT albeit not in all animals sampled. Our observations add further evidence to the concept of AT being a source of leptin in trouts. Moreover, the cellular localization of leptin immunoreactivity in liver opens up new vistas for understanding the functional role of leptin in teleosts.     

Growth hormone and lactogenic hormones can reduce the leptin mRNA expression in bovine mammary epithelial cells

Leptin mRNA is expressed in not only adipocytes but also mammary epithelial cells and leptin protein is present in milk. Although milk leptin is thought to influence metabolism or the immune system in neonates, there is little information about the regulation of leptin expression in mammary epithelial cells. We examined the effect of growth hormone (GH) and/or lactogenic hormone complex (DIP; dexamethasone, insulin and prolactin) on leptin mRNA expression in mammary epithelial cells. We used a bovine mammary epithelial cell (BMEC) clonal line, which was established from a 26-day pregnant Holstein heifer. We confirmed that the mRNA was expressed in BMECs and the expression was significantly reduced by GH and/or DIP, when the cells were cultured on both plastic plates and cell culture inserts at days 2 and 7 after stimulation with lactogenic hormones. GH and/or DIP significantly increased level of alpha-casein mRNA in BMECs after 7 days on the cell culture inserts, but no mRNA expression was detected at day 2. GH and DIP significantly stimulated the secretion of alpha-casein from BMEC on cell culture inserts at 3.5 and 7 days. However, neither alpha-casein mRNA expression nor secretion was observed in the BMECs cultured on plastic dishes, even in the presence of GH or/and DIP. These results indicate that GH and DIP can directly reduce leptin mRNA expression in both undifferentiated and functionally differentiated bovine mammary epithelial cell.     

Effect of retinoic acid on gene expression profiles of bovine intramuscular preadipocytes during adipogenesis

To investigate genes involved in intramuscular adipogenesis in ruminants, 16 genes with dramatic variable expression were selected. These were selected from the differentiation‐ and proliferation‐phase libraries of our previous serial analysis of gene expression (SAGE) studies of a clonal bovine intramuscular preadipocyte (BIP) cell line. We harvested the BIP cells over 12 days after adipogenic stimulation with all‐trans retinoic acid (ATRA). Quantitative real‐time PCR confirmed the earlier SAGE study results of the expression patterns of 15 of the genes. On day 6, TG accumulation increased significantly in the BIP cells but was completely inhibited in the 3T3‐L1 cells (the monogastric reference). ATRA enhanced expression levels of six genes whereas it suppressed expression of eight genes on day 3 of adipogenesis in the BIP cells. Forty‐eight hours after transfection, the messenger RNA expression level of the adipose differentiation‐related protein (ADFP), encoded by one of the upregulated genes, in the ADFP small interference RNA (siRNA)‐transfected cells was 3.5% of that in negative control‐transfected cells. Also, 6 days after induction the TG level in the ADFP siRNA‐transfected cells was 21.8% lower than that in negative control‐transfected cells. This analysis of gene expression profiles after ATRA treatment will contribute to our understanding of the molecular mechanisms involved in bovine intramuscular adipogenesis.     

冷应激对猪脂肪、肝脏、心脏与肾脏组织中PPARγ2 mRNA及蛋白表达的影响

为了研究冷应激对机体的影响及过氧化物酶体增殖物激活受体γ2（PPARγ2）在冷应激中的作用,本试验选用30头军牧一号猪,并随机分成5组,包括常温对照组和4个冷应激组。常温组在（21±2）℃饲养,冷应激组的温度设置分别为：（-10±2）、（-5±2）、（0±2）、（5±2）℃。冷应激2h屠宰后分别采集猪腹腔脂肪、颈部皮下脂肪、胸部皮下脂肪、肝脏、心脏与肾脏组织样品提取总RNA与总蛋白。通过荧光定量PCR技术检测了PPARγ2mR-NA的表达量;通过Western blot检测PPARγ2蛋白表达量。结果显示,各温度组猪腹腔脂肪、颈部皮下脂肪、胸部皮下脂肪、肝脏、心脏PPARγ2mRNA及蛋白表达量总体上差异显著（P〈0.01）,肾脏PPARγ2mRNA表达量总体显著升高,但其蛋白表达量总体呈下降趋势。结果表明,PPARγ2对冷刺激非常敏感,它除了可以调节冷应激时的脂肪代谢平衡,还对心脏有较好的保护作用,但是对肾脏的作用有限。本试验显示了冷应激中PPARγ2在脂肪代谢与能量代谢中作用,为研究冷应激对机体的影响及畜禽品质的提高奠定了基础。     

The ontogeny of leptin mRNA expression in growing broilers and its relationship to metabolic body weight.

The polypeptide hormone leptin is produced by both adipose tissue and the liver and has been shown to induce satiety in chickens. In this study we have investigated the developmental regulation of leptin mRNA expression in growing broiler chickens. Leptin expression generally increases in all tissues from 1-12 weeks of age. In the subcutaneous fat depot there is an apparent pattern of increased leptin mRNA expression occurring at 2, 6, and 10 weeks post-hatch. This pattern was not evident in the other tissues surveyed and may relate to the cycle of loading and unloading of adipocytes with lipid. No consistent gender differences in leptin expression patterns were detected in the tissues surveyed, as is often observed in mammals. Positive correlations between metabolic body weight and adipose leptin expression levels were observed. Leptin expression by the liver was highly correlated with metabolic body weight from 1-6 weeks of age, and uncorrelated from 6-12 weeks of age. This pattern of increasing liver leptin expression with increasing body weight during the early rapid growth phase of the bird may be due to limited fat storage during this period, which is followed by rapid body fat accumulation from 6-12 weeks. The characterization and tissue specific distribution of leptin mRNA expression in the growing broiler indicate similar patterns of leptin production to that of growing mammals. Leptin may be involved in lipid flux through the adipocyte as well as the shift in lipid metabolism to increased storage during pre-puberty.     

The brain-pituitary-adipocyte axis: role of leptin in modulating neuroendocrine function.

The recently discovered protein leptin has a molecular mass of 16 kDa, consists of 146 amino acids, and is synthesized and secreted by adipose tissue. Leptin affects feed intake, the neuroendocrine-axis, and immunological processes. The protein was first identified as the gene product that is deficient in the obese ob/ob mouse. Leptin serves as a circulating signal of nutritional status and plays a pivotal role in regulation of body weight, energy expenditure, growth, and reproduction.     

Leptin concentrations in periparturient ewes and their subsequent offspring

Leptin is an adipocyte-derived hormone that suppresses feed intake and increases energy expenditure. Leptin is also involved in regulating body temperature. Thus, the presence of leptin in milk, which can be absorbed through the gut of neonates immediately after birth, may aid in the survival of neonates born in cold weather. Our objectives were to determine the temporal relationship between concentrations of leptin in postpartum ewe blood serum and ewe milk serum, and to determine whether ewe blood and milk serum leptin concentrations were correlated with concentrations of leptin in lamb blood serum in their off-spring. Approximately 1 wk before the expected date of lambing, blood samples, weights, and body condition scores (BCS; 0 to 5 scale) were collected from 27 mixed-parity ewes. Following parturition, ewe blood and milk samples were collected within 2 h of parturition (d 0), 12 h (d 0.5) and 24 h (d 1) after parturition, again on d 5, and weekly thereafter until d 47. Lambs were blood-sampled and weighed within 2 h of parturition (d 0), bled daily until d 5, and bled and weighed weekly thereafter to d 47. Prior to lambing, ewe blood serum leptin was positively correlated with congruent BCS (r2 = 0, 10, P = 0.06), but not weight (P = 0.14). Following parturition, ewe blood serum leptin was positively correlated with BCS, weight, and milk serum leptin (r2 = 0.14, P < 0.0001, r2 = 0.12, P < 0.0001, and r2 = 0.028, P = 0.04). Leptin in milk serum was correlated with ewe weight (r2=0.05, P = 0.007) but not ewe BCS (P = 0.7); however, concentrations of leptin in both ewe blood and milk serum varied with day of lactation (P = 0.0001), being maximal within 24 h of parturition and declining to nadir concentrations by d 5. Leptin in lamb serum was correlated with milk serum leptin, (r2 = -0.05; P = 0.001), but not ewe blood serum leptin (P = 0.5). Concentrations of leptin in lamb serum increased from birth to d 5 and declined thereafter to nadir concentrations by d 19. Elevated concentrations of leptin in milk during the early stages of lactation may provide a mechanism for thermoregulation, satiation, and homeostatic endocrine control in the neonate.     

Effects of acute fasting and age on leptin and peroxisome proliferator‐activated receptor gamma production relative to fat depot in immature and mature pigs

Leptin and peroxisome proliferator‐activated receptor gamma (PPARγ) are adipogenic proteins that are actively involved in metabolic homeostasis of fat. Recently, it was reported that fat tissue in humans and rodents differs in metabolic activity relative to anatomical location of the fat tissue (i.e. depots) and animal age. Hence, we hypothesized that leptin and PPARγ production in various fat depots in female pigs differs in response to acute fasting, and that these responses vary with physiological maturity of the animal. Sixteen intact crossbred immature female pigs [prepubertal (PP); 132.2 ± 4.1 days] and 16 sexually mature female pigs (M; 224 ± 7.4 days) housed in an open‐air, concrete slab, sheltered barn were randomly assigned to either Control or Fasted treatments. Control pigs (PP, n = 8; M, n = 8) had ad libitum access to feed, while Fasted pigs (PP, n = 8; M, n = 8) were denied access to feed from the onset of the study (0 h) to euthanasia at 72 h. Immediately post‐mortem, fat samples were collected from the subcutaneous, pelvic, kidney, and heart (M pigs only) fat depots and analysed for leptin and PPARγ mRNA and protein content. Acute fasting decreased mean leptin mRNA tissue content in a depot specific manner in M pigs (p < 0.01), while mean leptin protein concentrations in fat tissues did not differ with fat depot or age of the pig. Furthermore, acute fasting did not affect mean PPARγ mRNA tissue content in a fat depot or age dependent manner. Mean concentrations of PPARγ protein in fat depots tended to be greater in M vs. PP pigs (p = 0.07). We suggest that these data provide evidence that acute fasting has a greater effect on leptin than PPARγ production in a fat depot dependent manner in M pigs, which may be indicative of changing physiological demands as an animal matures.     

Effects of leptin on the release of luteinizing hormone, growth hormone and prolactin from cultured bovine anterior pituitary cells

The effects of leptin on the release of luteinizing hormone (LH), growth hormone (GH) and prolactin (PRL) were studied in cultured bovine anterior pituitary (AP) cells in vitro. The AP cells were obtained from fully‐fed Japanese Black steers and were incubated for 3 h with 10^?13 to 10^?7 mol/L of leptin after incubating in Dulbecco's modified Eagle's Medium for 3 days. Leptin significantly increased the concentration of LH in the culture medium by 45 and 44% at doses of 10^?8 and 10^?7 mol/L, respectively, compared with the controls (P < 0.05). Leptin significantly increased the concentration of GH in the culture medium by 14 and 12% at doses of 10^?8 and 10^?7 mol/L, respectively (P < 0.05). Leptin also significantly increased the concentration of PRL in the culture medium by 26% compared with the controls at a dose of 10^?7 mol/L (P < 0.05). These results show that leptin stimulates the release of LH, GH and PRL by acting directly on bovine AP cells from fully‐fed steers.     

Blood Levels of Selected Metabolic Factors,Cytokines, and Lymphocyte Subpopulations in Arabian and Thoroughbred Horses During the Longest and Shortest Days of the Year

Day length-related alterations of several metabolic factors (glucose, leptin, insulin, and insulin-like growth factor-1 [IGF-1]), cytokines (interleukin-2 [IL-2], IL-4, IL-6, tumor necrosis factor-alpha [TNF-α], interferon-gamma [IFN-γ], and lymphocyte subpopulations [CD2, CD3, CD4, CD8, CD19, natural killer (NK) cells) were evaluated in Arabian and Thoroughbred horses. Plasma glucose, leptin, IGF-1, insulin, and cytokines levels were measured on the longest day of the breeding season and on the shortest day of the nonbreeding season. Determination of lymphocyte subpopulations was performed by flow cytometry. Glucose and IL-2 levels, CD4:CD8 ratio, and NK cells showed variations that depended on the day length. Mean concentrations of plasma leptin were higher in Arabian horses than in Thoroughbred horses, whereas mean concentrations of IGF-1 and IL-2 were lower in Arabian horses. Day length-by-breed-by-gender interaction was found for insulin, IFN-γ, and IL-4 levels. An interaction was also found between day length and gender for the expressions of CD2, CD3, CD8, and CD19. Correlations were detected between expression of CD8⁺ cells and levels of TNF-α and IFN-γ and between percentages of NK cells and levels of IGF-1, insulin, and glucose. Results suggested that day length and, therefore, season are important determinants or factors in modulating the immune system and could affect lymphocyte subpopulations depending on the sex of the horse. Additionally, it seems that a complex relationship in horses, as in humans and mice, exists between the immune and metabolic system, which changes according to day length, breed, and gender.