Fatty acid synthetizing enzymes in adipocytes and stromavascular fraction of hyperthyroid rat adipose tissue

In the rat, hyperthyroidism induced from birth has been shown to increase both adipose cell recruitment and de novo lipogenesis at 6 weeks of age. Since preadipose cells are included into the stromavascular fraction (SVF) of adipose tissue, fatty acid synthetizing enzyme activities were estimated separately in adipocytes and SVF cells of adipose tissue of 6 week-old hyperthyroid rats. Fatty acid synthetase (FAS), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities were generally increased in adipocytes and in SVF cells when compared to age-matched controls. Among the enzymes studied, ME activity displayed the highest stimulation in SVF cells and was much more stimulated in these cells than in adipocytes (214% and 73% increase, respectively, above control values, P less than 0.001). These results show that, in vivo, SVF cells and adipocytes can respond differently to an hormonal stimulation and raise the question of the role of ME in the adipose conversion.     

Changes of insulin binding in rat tissues after exposure to stress.

The effects of various stressors on insulin receptors in adipose, liver and skeletal muscle tissues were studied in rats exposed to acute or repeated stress. Adult male rats were exposed to immobilization (IMO) for 2.5 hours daily for 1, 7 and 42 days, or to hypokinesia (HK) for 1, 7 and 21 days. We determined the values of specific insulin binding (SIB) and insulin receptor binding capacity (IR) of plasma cell membranes from adipose, liver and muscle tissue (IMO groups), or insulin binding to isolated adipocytes and hepatocytes (HK groups). A significant decrease of SIB and IR was observed in rats exposed to acute stress (1x IMO) in muscle, adipose and liver tissues. However, in animals exposed to repeated stress (7x and 42x IMO), SIB and IR were diminished in the muscle tissue, whereas no significant changes were noted in the liver and adipose tissue. When tissue samples were collected 3-24 hours after exposure to IMO stress, no changes of SIB and IR were found in liver and adipose tissue, but insulin binding was lowered in skeletal muscles. In animals exposed to HK for one day, a decrease of SIB and IR was found in isolated adipocytes, but no changes in insulin binding were noted in the liver tissue. In rats exposed to HK for 7 and 21 days, values of IR were similar as in control group. Our results indicate a) the different changes of IR in the liver, fat and muscle tissues after exposure to stress situations, b) a long-term decrease of insulin binding in muscles of rats exposed to repeated IMO stress, and c) the return of reduced SIB and IR (induced by acute stress) to control values in the liver and adipose tissue after a short recovery period.     

Strain differences in rat adrenal biosynthetic enzymes and stress-induced increases in plasma catecholamines.

We have examined in two inbred rat strains basal and stress-induced increases in plasma levels of epinephrine (EPI) and norepinephrine (NE) and compared these with activities of the adrenal enzymes involved in the synthesis of catecholamines. There were no differences in basal levels of NE and EPI in plasma of adult male rats of the Wistar-Kyoto (WKY) and Brown-Norway (B-N) strains. However, following 5 min. of intermittent footshock, plasma levels of both catecholamines were twice as high in WKY rats as in B-N rats. In the adrenals of unstressed rats, activities of tyrosine hydroxylase and dopamine-beta-hydroxylase were significantly higher in B-N rats. In addition, the adrenal weights and the contents of NE but not EPI were greater in B-N rats. Thus, in these two rat strains, the capacity of the adrenal gland to synthesize and store catecholamines appeared to be inversely related to plasma levels of NE and EPI after stress. The differences between the strains appeared to be due to differences in the rates of removal of catecholamines from the peripheral circulation as well as to differences in the rate of release of catecholamines from the sympatho-adrenal medullary system. Thus biosynthetic enzyme activities need not be related directly to the capacity to release and elevate plasma levels of catecholamines following stressful stimulation.     

PEX13 is required for thermogenesis of white adipose tissue in cold-exposed mice

Non-shivering thermogenesis (NST) is a heat generating process controlled by the mitochondria of brown adipose tissue (BAT). In the recent decade, ‘functionally’ acting brown adipocytes in white adipose tissue (WAT) has been identified as well: the so-called process of the ‘browning’ of WAT. While the importance of uncoupling protein 1 (UCP1)-oriented mitochondrial activation has been intensely studied, the role of peroxisomes during the browning of white adipocytes is poorly understood. Here, we assess the change in peroxisomal membrane proteins, or peroxins (PEXs), during cold stimulation and importantly, the role of PEX13 in the cold-induced remodeling of white adipocytes. PEX13, a protein that originally functions as a docking factor and is involved in protein import into peroxisome matrix, was highly increased during cold-induced recruitment of beige adipocytes within the inguinal WAT of C57BL/6 mice. Moreover, beige-induced 3 T3-L1 adipocytes and stromal vascular fraction (SVF) cells by exposure to the peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone showed a significant increase in mitochondrial thermogenic factors along with peroxisomal proteins including PEX13, and these were confirmed in SVF cells with the beta 3 adrenergic receptor (β3AR)-selective agonist CL316,243. To verify the relevance of PEX13, we used the RNA silencing method targeting the Pex13 gene and evaluated the subsequent beige development in SVF cells. Interestingly, siPex13 treatment suppressed expression of thermogenic proteins such as UCP1 and PPARγ coactivator 1 alpha (PGC1α). Overall, our data provide evidence supporting the role of peroxisomal proteins, in particular PEX13, during beige remodeling of white adipocytes.     

Inducible beige adipocytes improve impaired glucose metabolism in interscapular BAT-removal mice

Inducible beige adipocytes are emerging as an interesting issue in obesity and metabolism research. There is a neglected possibility that brown adipocytes are equally activated when external stimuli induce the formation of beige adipocytes. Thus, the question is whether beige adipocytes have the same functions as brown adipocytes when brown adipose tissue (BAT) is lacking. This question has not been well studied. Therefore we determine the beneficial effects of beige adipocytes upon cold challenge or CL316243 treatments in animal models of interscapular BAT (iBAT) ablation by surgical denervation. We found that denervated iBAT were activated by cold exposure and CL316243 treatments. The data show that beige adipocytes partly contribute to the improvement of impaired glucose metabolism resulting from denervated iBAT. Thus, we further used iBAT-removal animal models to abolish iBAT functions completely. We found that beige adipocytes upon cold exposure or CL316243 treatments improved impaired glucose metabolism and enhanced glucose uptake in iBAT-removal mice. The insulin signaling was activated in iBAT-removal mice upon cold exposure. Both the activation of insulin signaling and up-regulation of glucose transporter expression were observed in iBAT-removal mice with CL316243 treatments. The data show that inducible beige adipocytes may have different mechanisms to improve impaired glucose metabolism. Inducible beige adipocytes can also enhance energy expenditure and lipolytic activity of white adipose tissues when iBAT is lacking. We provide direct evidences for the beneficial effect of inducible beige adipocytes in glucose metabolism and energy expenditure in the absence of iBAT in vivo.     

Cellular changes during cold acclimatation in adipose tissues

Cold exposure is a well-known physiological stimulus that activates the sympathetic nervous system and induces brown adipose tissue (BAT) hyperplasia. The effects of cold exposure or cold acclimatation have been extensively studied in interscapular BAT (IBAT). However, it has been recently shown that brown adipocytes are present in adipose deposits considered as white fat such as periovarian (PO) fat pad. We have investigated the kinetic of brown precursor recruitment in adipose tissues using DNA measurement and specific marker expression. In IBAT, cold exposure induces proliferation of precursor cells and differentiation into preadipocytes characterized by the expression of A2COL6, a marker specific to early steps of the differentiation process. A chronic stimulation of the tissue is necessary to observe the full effect. In PO fat pad, no proliferation can be detected, whereas differentiation of brown preadipocytes and maybe phenotypic conversion of white adipocytes seems to be promoted. In conclusion, these data demonstrated that 1) the same stimulus (cold exposure) does not induce the same response in terms of preadipocyte proliferation and differentiation in periovarian and brown adipose tissues, although both contain brown adipocytes, and 2) preadipocyte recruitment in adipose tissues after cold exposure depends on the predominant type of fat cells. © 1996 Wiley-Liss, Inc.     

The stromal‐vascular fraction of adipose tissue contributes to major differences between subcutaneous and visceral fat depots

Adipose tissue represents a complex tissue both in terms of its cellular composition, as it includes mature adipocytes and the various cell types comprising the stromal‐vascular fraction (SVF), and in relation to the distinct biochemical, morphological and functional characteristics according to its anatomical location. Herein, we have characterized the proteomic profile of both mature adipocyte and SVF from human visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) fat depots in order to unveil differences in the expression of proteins which may underlie the distinct association of VAT and SAT to several pathologies. Specifically, 24 proteins were observed to be differentially expressed between SAT SVF versus VAT SVF from lean individuals. Immunoblotting and RT‐PCR analysis confirmed the differential regulation of the nuclear envelope proteins lamin A/C, the membrane‐cytoskeletal linker ezrin and the enzyme involved in retinoic acid production, aldehyde dehydrogenase 1A2, in the two fat depots. In sum, the observation that proteins with important cell functions are differentially distributed between VAT and SAT and their characterization as components of SVF or mature adipocytes pave the way for future research on the molecular basis underlying diverse adipose tissue‐related pathologies such as metabolic syndrome or lipodystrophy.     

Plasma catecholamine measurements in resting and stressed conscious rats, using high performance liquid chromatography with electrochemical detection

Plasma epinephrine (EPI) and norepinephrine (NE) were measured in conscious, unrestrained rats at rest, and following exercise, cold stress and hemorrhage. Heart rate and mean arterial pressure were monitored at all points. Minor modifications of a standard, commercially available methodology achieved good chromatographic separation of both EPI and NE. Sensitivity was sufficient for all NE measurements and for EPI measurements in the stress conditions. Estimates of EPI by this procedure were somewhat higher than those in other reports. Absolute values of NE, as well as the selective, qualitative changes found in both EPI and NE under each stress condition, were consistent with those found in other studies which used the radioenzymatic method. Blood withdrawal of two 1.5 ml samples, 3 hours apart, had no detectable effect on EPI, NE, mean arterial pressure or heart rate in otherwise non-stressed rats.     

Caffeine reduces TNFα up-regulation in human adipose tissue primary culture

Adipose tissue secretions play an important role in the development of obesity-related pathologies such as diabetes. Through inflammatory cytokines production, adipose tissue stromavascular fraction cells (SVF), and essentially macrophages, promote adipocyte insulin resistance by a paracrine way. Since xanthine family compounds such as caffeine were shown to decrease inflammatory production by human blood cells, we investigated the possible effect of caffeine on Tumor Necrosis Factor alpha (TNFalpha) and Interleukin-6 (IL-6) expression by human adipose tissue primary culture. For that purpose, human subcutaneous adipose tissue obtained from healthy non-obese women (BMI: 26.7 +/- 2.2 kg/m2) after abdominal dermolipectomy, was split into explants and cultured for 6 hours with or without caffeine. Three different concentrations of caffeine were tested (0.5 microg/mL, 5 microg/mL and 50 microg/mL). After 6 hours of treatment, explants were subjected to collagenase digestion in order to isolate adipocytes and SVF cells. Then, TNFalpha and IL-6 mRNA were analysed by real-time PCR alternatively in adipocytes and SVF cells. In parallel, we checked gene expression of markers involved in adipocyte differenciation and in SVF cells inflammation and proliferation. Our findings show a strong and dose dependent down-regulation of TNF-alpha gene expression in both adipocyte and SVF cells whereas IL-6 was only down regulated in SVF cells. No effect of caffeine was noticed on the other genes studied. Thus, caffeine, by decreasing TNFalpha expression, could improve adipose tissue inflammation during obesity.     

Atypical expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in subcutaneous adipose tissue of male rats.

The mRNAs encoding mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mtHMG-CoA synthase), the rate limiting enzyme in ketone body production, are highly expressed in subcutaneous (SC) and, to a lesser extent, in peri-epididymal (PE) rat adipose tissues. This atypical mtHMG-CoA synthase gene expression is dependent on the age (from 9 weeks of age) and sex (higher in male than in female) of the rats. In contrast, the expression of mtHMG-CoA synthase in SC adipose deposit is independent of the nutritional state (fed versus starved) or of the thermic environment (24 degrees C versus 4 degrees C). The expression of mtHMG-CoA synthase is suppressed in SC fat pads of castrated male rats whereas treatment of castrated rats with testosterone restores a normal level of expression. Moreover, testosterone injection induces the expression mtHMG-CoA synthase in SC adipose tissue of age-matched females. The presence of the mtHMG-CoA synthase immunoreactive protein confers to mitochondria isolated from SC adipose deposits, the capacity to produce ketone bodies at a rate similar to that found in liver mitochondria (SC = 13.7 +/- 0.7, liver = 16.4 +/- 1.4 nmol/min/mg prot). mtHMG-CoA synthase is expressed in the stromal vascular fraction (SVF) whatever the adipose deposit considered. While acetyl-CoA carboxylase (ACC) is only expressed in mature adipocytes, the other lipogenic enzymes, fatty acid synthase (FAS) and citrate cleavage enzyme (CCE), are expressed both in SVF cells and mature adipocytes. The expression of lipogenic enzyme genes is markedly reduced in adipocytes but not in SVF cells isolated from 48-h starved male rats. When SVF is subfractionated, mtHMG-CoA synthase mRNAs are mainly recovered in two fractions containing poorly digested structures such as microcapillaries whereas the lowest expression is found in the pre-adipocyte fraction. Interestingly, FAS and CCE mRNAs co-segregate with mtHMG-CoA synthase mRNA. The possible physiological relevance of such atypical expression of mtHMG-CoA synthase is discussed.     

Carbonic Anhydrase Protects Fatty Liver Grafts against Ischemic Reperfusion Damage

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. CAs are involved in numerous physiological and pathological processes, including acid-base homeostasis, electrolyte balance, oxygen delivery to tissues and nitric oxide generation. Given that these processes are found to be dysregulated during ischemia reperfusion injury (IRI), and taking into account the high vulnerability of steatotic livers to preservation injury, we hypothesized a new role for CA as a pharmacological agent able to protect against ischemic damage. Two different aspects of the role of CA II in fatty liver grafts preservation were evaluated: 1) the effect of its addition to Institut Georges Lopez (IGL-1) storage solution after cold ischemia; 2) and after 24h of cold storage followed by two hours of normothermic ex-vivo perfusion. In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined. In case of the ex-vivo perfusion, we further assessed liver function (bile production, bromosulfophthalein clearance) and Western blot analysis of phosphorylated adenosine monophosphate activated protein kinase (AMPK), mitogen activated protein kinases family (MAPKs) and endoplasmic reticulum stress (ERS) parameters (GRP78, PERK, IRE, eIF2α and ATF6). We found that CA II was downregulated after cold ischemia. The addition of bovine CA II to IGL-1 preservation solution efficiently protected steatotic liver against cold IRI. In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation. Interestingly, CA II supplementation was not associated with enhanced CO₂ hydration. The results suggest that CA II modulation may be a promising target for fatty liver graft preservation.     

免疫细胞内源性儿茶酚胺的免疫调节作用

机体内儿茶酚胺（catecholamines,CAs）包括去甲肾上腺素（norepinephrine,NE）、肾上腺素（epinephrine,E）和多巴胺（dopamine,DA）。CAs由神经元和内分泌细胞合成和分泌,其主要功能是调节心血管、呼吸和消化等内脏活动。近三十年来的研究说明,CAs也参与调控机体的免疫功能,但CAs的这种免疫调节作用一般视为神经和内分泌系统调节的介导作用。然而,近年来的研究发现,免疫细胞也能合成CAs,这是对传统观念的一种补充和提高。免疫细胞内存在经典的CAs代谢途径,既有合成CAs的酪氨酸羟化酶（tyrosine hydroxylase,TH）又有降解CAs的单胺氧化酶（monoamine oxidase,MAO）和儿茶酚氧位甲基移位酶（catechol-O-methyl transferase,COMT）。免疫细胞合成的内源性CAs可以调控细胞的增殖、分化、凋亡和细胞因子生成等多种免疫功能。CAs的这些作用可能主要通过自分泌或旁分泌途径作用于免疫细胞上相应受体和细胞内环磷酸腺苷（cyclicAMP,cAMP）实现。细胞内氧化应激机制可能也参与免疫细胞内源性CAs的免疫调节作用。此外,一些自身免疫性疾病如多发性硬化、风湿性关节炎可能也与免疫细胞内CAs的代谢异常有关。上述发现不仅为免疫系统有可能成为除神经和内分泌系统以外的第三个CA能系统提供了证据,而且为免疫系统内源性CAs的功能意义拓展了认识。     

Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation

Recent research findings postulate that adipocytes and endothelial cells (EC) may share a common progenitor. However, the interlinking pathways between adipose tissue and endothelium, and the differentiation potential of cells to convert from one tissue into the other via progenitor cells have not been elucidated and are therefore the focus of this study. Stromal vascular fraction (SVF) cells were isolated from liposuction aspirates or excised adipose tissue and separated into CD31+ and CD31- populations by magnet-assisted cell sorting. Differentiation to fat tissue was induced in both CD31 fractions after expansion by insulin, dexamethasone, isobutylmethylxanthine, triiodothyronine, pioglitazone, and transferrin. Differentiation was assayed enzymatically and by cell counting. Maturation to endothelium was performed with vascular endothelial growth factor (VEGF), insulin-like growth factor-1 plus 2% fetal calf serum, and confirmed by flow cytometry and tube formation assays on Matrigel. Our results show that the SVF contains a CD31-, S100+ cell type that can differentiate into adipocytes and EC. The SVF also comprises CD31+ cells that, although they have an endothelial phenotype, can be converted into mature adipocytes. These findings demonstrate the potency of SVF cells to perform both adipogenic and endothelial differentiation. Further, they reveal the plasticity of mature cells of mesenchymal origin to undergo conversion from endothelium to adipose tissue and vice versa.     

Invivo β-adrenergic induction of the unmasking of the uncoupling protein in rat brown fat

1. In 28°C adapted rats (WA) both cold stress and norepinephrine (NE) led to a 4-fold increase of uncoupling protein dependent proton conductance which was abolished by propranolol (PRO).2. In 4-day warm re-exposed rats (after 10 days at 5°C) (WR) the same uncoupling by cold stress was observed but the NE effect was lower. Uncoupling by cold stress was not abolished by PRO.3. In WR rats, uncoupling was not due to the involvement of an α-adrenergic pathway.4. Both β-agonist isoproterenol and β-agonists BRL 35135A and ICI D7114 led to high levels of unmasking.5. Interscapular brown adipose tissue surgical denervation, which abolished cold stress unmasking both in WA and WR rats, indicates a mediation by direct sympathetic innervation.6. Depending on the thermal history of the rat, the possibility that unmasking by cold stress could be mediated by different types of β-receptors is discussed.     

Vesicular Monoamine Transporters (VMATs) in Adrenal Chromaffin Cells: Stress-Triggered Induction of VMAT2 and Expression in Epinephrine Synthesizing Cells

Vesicular monoamine transporters (VMATs) mediate transmitter uptake into neurosecretory vesicles. There are two VMAT isoforms, VMAT1 and VMAT2, encoded by separate genes and displaying different cellular distributions and pharmacological properties. We examined the effect of immobilization stress (IMO) on expression of VMATs in the rat adrenal medulla. Under basal conditions, VMAT1 is widely expressed in all adrenal chromaffin cells, while VMAT2 is co-localized with tyrosine hydroxylase (TH) but not phenylethanolamine N-methyltransferase (PNMT), indicating its expression in norepinephrine (NE)-, but not epinephrine (Epi)-synthesizing chromaffin cells. After exposure to IMO, there was no change in levels of VMAT1 mRNA. However, VMAT2 mRNA was elevated after exposure of rats to 2 h IMO once (1× IMO) or daily for 6 days (6× IMO). The changes in VMAT2 mRNA were reflected by increased VMAT2 protein after the repeated IMO. Immunofluorescence revealed an increased number of cells expressing VMAT2 following repeated IMO and its colocalization with PNMT in many chromaffin cells. The findings suggest an adaptive mechanism in chromaffin cells whereby enhanced catecholamine storage capacity facilitates more efficient utilization of the well-characterized heightened catecholamine biosynthesis with repeated IMO stress.     

Adipocytes as a new source of catecholamine production

Catecholamines are an important regulator of lipolysis in adipose tissue. Here we show that rat adipocytes, isolated from mesenteric adipose tissue, express genes of catecholamine biosynthetic enzymes and produce catecholamines de novo. Administration of tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, in vitro significantly reduced concentration of catecholamines in isolated adipocytes. We hypothesize that the sympathetic innervation of adipose tissues is not the only source of catecholamines, since adipocytes also have the capacity to produce both norepinephrine and epinephrine.