脂肪组织甘油三酯水解酶参与脂肪分解调控

循环中游离脂肪酸增高与肥胖、胰岛素抵抗和2型糖尿病密切相关,其主要来源于脂肪细胞内甘油三酯水解.调控脂肪分解的脂肪酶主要包括激素敏感脂肪酶(hormone-sensitive lipase,HSL)和最近发现的脂肪组织甘油三酯水解酶(adipose triglyceride lipase,ATGL),后者主要分布在脂肪组织,特异水解甘油三酯为甘油二酯,其转录水平受多种因素调控.CGI-58(属于α/β水解酶家族蛋白),可以活化ATGL,基础条件下该蛋白和脂滴包被蛋白(perilipin)紧密结合于脂滴表面,蛋白激酶A激活刺激脂肪分解时,CGI-58与perilipin分离,进而活化ATGL.     

Differences in the amount of lipolysis induced by atrial natriuretic peptide in small and large adipocytes.

Atrial natriuretic peptide (ANP) is a 28-amino acid polypeptide that is primarily secreted by the heart. ANP is believed to be a hormone that regulates cardiovascular dynamics and renal functions; however, studies conducted in the past few decades revealed that ANP is also a potent lipolytic agent in human adipocytes that functions through the cGMP-dependent pathway. In this study, we separated human adipocytes within the same fat depot into small and large fractions using their floating properties and nylon filters of different pore sizes. Real-time PCR revealed that large adipocytes expressed higher mRNA levels of natriuretic peptide receptor (NPR)-A and hormone sensitive lipase, and binding studies showed that large adipocytes expressed more NPR-A on the membrane than small adipocytes. This finding was confirmed by the increase in the amount of glycerol that was released from adipocytes as the cell size increased. Taken together, these results clearly suggest that adipocyte size is an important determinant of ANP-stimulated lipolysis. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Triphenylmethylphosphonium cation distribution as a measure of hormone-induced alterations in white adipocyte membrane potential

Summary Triphenylmethylphosphonium (TPMP⁺) partitions into the mitochondrial and cytosolic compartments in the rat white adipocyte in a potential-dependent fashion. The relationship between [³H]TPMP⁺ distribution, intracellular cAMP generation and lipolysis in response to hormones and cAMP-mimetic compounds was examined. Half-maximal [³H]TPMP⁺ efflux and glycerol release were produced by 15 and 9nm adrenocorticotropin, 170 and 110nm 1-epinephrine, 70 and 27 m isobutylmethylxanthine and 800 and 750 m dibutyryl cAMP, respectively. Hormone-stimulated cAMP generation was also correlated with [³H]TPMP⁺ efflux and lipolysis in terms of concentration dependency. In kinetic experiments, glycerol release and [³H]TPMP⁺ efflux in response to adrenocorticotropin or cholera toxin proceeded over a similar time course, whereas an earlier rise in cAMP generation was detected.The depolarizing effect of lipolytic compounds was localized to the mitochondrial compartment. When cells were incubated in elevated-[K⁺] _c buffer, the stimulatory effect of dibutyryl cAMP on [³H]TPMP⁺ efflux and lipolysis persisted, suggesting that maintenance of the plasma membrane potential is not critical for demonstration of these responses.When the extracellular concentration of serum albumin, which provides binding sites for free fatty acids, was increased from 1 to 3% an increase in glycerol release and a decrease in [³H]TPMP⁺ efflux was observed. We suggest that intracellular free fatty acid accumulation in response to lipolytic agents causes dissipation of the mitochondrial membrane potential and efflux of [³H]TPMP⁺ from the organelle and cell.     

Effect of endothelin-1 on lipolysis in rat adipocytes

Objective : To explore the role of endothelin‐1 (ET‐1) on lipid metabolism, we examined the effect of ET‐1 on lipolysis in rat adipocytes. Research Methods and Procedure : Adipocytes isolated from male Sprague‐Dawley rats, weighing 400 to 450 grams, were incubated in Krebs‐Ringer buffer with or without 10^?7 M ET‐1 for various times or with various concentrations of ET‐1 for 4 hours; then glycerol release into the incubation medium was measured. In addition, selective ET_AR and ET_BR blockers were used to identify the ET receptor subtype involved. We also explored the involvement of cyclic adenosine monophosphate (cAMP) in ET‐1‐stimulated lipolysis using an adenylyl cyclase inhibitor and by measuring changes in intracellular cAMP levels in response to ET‐1 treatment. To further explore the underlying mechanism of ET‐1 action, we examined the involvement of the extracellular signal‐regulated kinase (ERK)‐mediated pathways. Results : Our results showed that ET‐1 caused lipolysis in rat adipocytes in a time‐ and dose‐dependent manner. BQ610, a selective ET_AR blocker, blocked this effect. The adenylyl cyclase inhibitor, 2′, 5′‐dideoxyadenosine, had no effect on ET‐1‐stimulated lipolysis. ET‐1 did not induce an increase in intracellular cAMP levels. In addition, ET‐1‐induced lipolysis was blocked by inhibition of ERK activation using PD98059. Coincubation of cells with ET‐1 and insulin suppressed ET‐1‐stimulated lipolysis. Discussion : These findings show that ET‐1 stimulates lipolysis in rat adipocytes through the ET_AR and activation of the ERK pathway. The underlying mechanism is cAMP‐independent. However, this non‐conventional lipolytic effect of ET‐1 is inhibited by the anti‐lipolytic effect of insulin.     

Fatty acid binding protein expression in different human adipose tissue depots in relation to rates of lipolysis and insulin concentration in obese individuals

Two fatty acid binding proteins (FABPs) are expressed in adipose tissue, adipocyte lipid binding protein (ALBP) and keratinocyte lipid binding protein (KLBP). This study investigated FABP expression in visceral and subcutaneous human adipose tissue depots and associations with lipolytic differences between the depots and circulating insulin concentrations. ALBP and KLBP (protein and RNA) were quantified in subcutaneous and omental adipose tissue from obese individuals and expressed relative to actin. ALBP RNA and protein expression was significantly higher in subcutaneous compared to omental adipose tissue (both p < 0.05), whereas KLBP RNA and protein expression was no different between the two sites. There were significant inverse correlations between serum insulin concentrations and the ALBP/KLBP RNA ratio in both subcutaneous and omental adipose tissue (both p < 0.02). Basal rates of glycerol and fatty acid release measured in adipocytes isolated from subcutaneous and omental adipose tissue were significantly higher in the former (p 0.02). Therefore the relative ALBP/KLBP content of human adipose tissue is different in different adipose tissue depots and at the RNA level is related to the circulating insulin concentration, at least in obese subjects. The higher rates of basal lipolysis in adipocytes isolated from subcutaneous compared to omental adipose tissue might be related to the increased ALBP content of the former. Therefore adipose tissue FABPs are interesting candidates for investigation to further our understanding of the insulin resistance syndrome and regulation of lipolysis.     

Sensitivity of adipocyte lipolysis to stimulatory and inhibitory agonists in hypothyroidism and starvation.

The responsiveness of lipolysis to the stimulatory agonists noradrenaline, corticotropin and glucagon and to the inhibitory agonists N6-phenylisopropyladenosine, prostaglandin E1 and nicotinic acid was investigated with rat white adipocytes incubated with a high concentration of adenosine deaminase (1 unit/ml). The cells were obtained from fed or 48 h-starved euthyroid animals or from fed or starved animals rendered hypothyroid by 4 weeks of treatment with low-iodine diet and propylthiouracil. Hypothyroidism increased sensitivity to and efficacy of all three inhibitory agonists in their opposition of noradrenaline-stimulated lipolysis. Starvation decreased sensitivity to all three inhibitory agonists when opposing basal lipolysis. Hypothyroidism decreased sensitivity to noradrenaline, glucagon and corticotropin by 37-, 4- and 4-fold respectively and decreased the maximum response to these agonists by approx. 50%, 50% and 75% respectively. Starvation reversed decreases in maximum response to these agonists in hypothyroidism. Starvation in the euthyroid state increased sensitivity to glucagon and noradrenaline, but did not alter sensitivity to corticotropin. Cells from hypothyroid rats were relatively insensitive to Bordetella pertussis toxin, which substantially increased basal lipolysis in the euthyroid state.     

G0/G1 switch gene-2 regulates human adipocyte lipolysis by affecting activity and localization of adipose triglyceride lipase

The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL), which is regulated by two proteins annotated as comparative gene identification-58 (CGI-58) and G0/G1 switch gene-2 (G0S2). CGI-58 activates and G0S2 inhibits ATGL activity. In contrast to mice, the functional role of G0S2 in human adipocyte lipolysis is poorly characterized. Here we show that overexpression or silencing of G0S2 in human SGBS adipocytes decreases and increases lipolysis, respectively. Human G0S2 is upregulated during adipocyte differentiation and inhibits ATGL activity in a dose-dependent manner. Interestingly, C-terminally truncated ATGL mutants, which fail to localize to lipid droplets, translocate to the lipid droplet upon coexpression with G0S2, suggesting that G0S2 anchors ATGL to lipid droplets independent of ATGL''s C-terminal lipid binding domain. Taken together, our results indicate that G0S2 also regulates human lipolysis by affecting enzyme activity and intracellular localization of ATGL. Increased lipolysis is known to contribute to the pathogenesis of insulin resistance, and G0S2 expression has been shown to be reduced in poorly controlled type 2 diabetic patients. Our data indicate that downregulation of G0S2 in adipose tissue could represent one of the underlying causes leading to increased lipolysis in the insulin-resistant state.     

Pertussis toxin-sensitive G proteins mediate the inhibition of basal phosphoinositide metabolism caused by adenosine A1 receptors in rat hippocampal slices

The adenosine A₁ receptor selective agonist, N ⁶-cyclopentyladenosine (CPA, 300 nM) inhibited basal accumulation of [³H]inositol phosphates ([³H]InsPs), but not the total levels of membrane [³H]-phosphoinositides, in rat hippocampal slices. This action of CPA was not significantly modified when synaptic transmission was blocked with tetrodotoxin (TTX, 200 nM) but was prevented in slices pre-incubated with pertussis toxin (PTX, 5 g/mL) for 12-16 hr. Neither PTX nor TTX, when applied in the absence of CPA, influenced basal [³H]InsPs accumulation. It is concluded that the inhibition of the basal phosphatidylinositol metabolism by adenosine A₁ receptor activation is independent of neurotransmission and involves a PTX-sensitive G protein, probably of the G_i/G_o family.     

Soyasapogenols reduce cellular triglyceride levels in 3T3-L1 mouse adipocyte cells by accelerating triglyceride lipolysis

Soyasapogenol is a soyasaponin aglycone, which has been suggested to exert a more potent function than the glycoside form. In this study, the effect of soyasapogenol A and B on cultured adipocyte cell function was investigated using mouse 3T3-L1 adipocyte cells. 3T3-L1 cells were treated with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine for differentiation to adipocytes, and the cells were then cultured in the presence of soyasapogenol A or B (6.25 or 12.5 µM). The media were harvested and refreshed every 2 d. After a 10 d culture, the cells were harvested and the triglyceride content of the cells was determined. The triglyceride content of soyasapogenol B-treated cells was significantly lower than those of vehicle-treated cells. Glycerol and free fatty acid levels in the soyasapogenol-treated cell media were higher than those in vehicle cells. However, there was no difference in the level of adipose triglyceride lipase among soyasapogenol A-, soyasapogenol B-, and vehicle-treated cells. The secreted adiponectin and resistin levels of soyasapogenol-treated cell media were also different compared with those of vehicle-treated cells. Especially, the secreted resistin level in soyasapogenol B-treated cell media was obviously reduced compared with that of vehicle-treated cells. Taken together, these results suggest that soyasapogenol B exerted an anti-obesity and anti-diabetic effect on adipocytes by lowering the cellular triglyceride level by accelerating triglyceride lipolysis with reduced resistin secretion.     

Mutation of the novel acetylation site at K414R of BECN1 is involved in adipocyte differentiation and lipolysis

BECN1, a protein essential for autophagy, is involved in adipocyte differentiation, lipolysis and insulin resistance. The discovery of new mechanisms for modifying BECN1 in adipocytes may provide novel therapeutic targets for obesity. This study aimed to investigate the impact of mutations at the acetylation sites of BECN1 on adipocyte differentiation and lipolysis. We found that Ace-BECN1 levels were increased in 3T3-L1 adipocyte differentiation and isoproterenol-/TNF-α-stimulated lipolysis and in subcutaneous and visceral adipose tissues of high-fat diet mice. K414 was identified as an acetylation site of BECN1, which affects the stability of the BECN1 protein. Mutation at K414 of BECN1 affected autophagy, differentiation and lipolysis in 3T3-L1 adipocytes. These data indicated the potential of BECN1 K414 as a key molecule and a drug target for regulating autophagy and lipid metabolism in adipocytes.     

Tissue-specific knockout of TSHr in white adipose tissue increases adipocyte size and decreases TSH-induced lipolysis

Background

The primary function of TSH is to activate TSH receptors (TSHr) in the thyroid gland and thereby stimulate thyroid hormone synthesis and secretion. TSHr are also expressed in other organs, but their physiological importance is still unclear. We have previously shown that TSHr, expressed in adipocytes, are of potential importance for lipolysis and extrauterine adaptation of the neonate.

Methodology

To further study the role of TSHr in adipocytes we selectively removed the TSHr gene in mice adipocytes by using the Cre-loxP recombination system (B6.Cg-Tg (Fabp4-Cre) 1Rev/J. TSHr knockout (KO) newborn mice were phenotypically characterized. Isolated adipocytes from 8-week-old male mice were studied in term of adipocyte size and metabolism.

Results

Mice lacking TSHr in adipocytes were apparently normal at birth and no differences in thyroid gland function or histology were observed. Sensitivity to TSH-induced lipolysis was ten times lower in adipocytes from targeted animals compared to wild-type. This indicates that adipocytes from targeted animals are refractory to stimulation of physiological concentrations of TSH. Catecholamine-induced lipolysis and insulin-induced inhibition of lipolysis were unaltered. Adipocyte size was increased in the targeted animals. Basal lipolysis was increased as an effect of the increased adipocyte size.

Conclusion

Our results indicate that adipocyte TSHr under normal conditions affects adipocyte growth and development.     

Recombinant acellular pertussis vaccine—from the laboratory to the clinic: improving the quality of the immune response

Abstract Vaccination is the most effective way to prevent infectious diseases. Recombinant DNA technologies have provided powerful new tools to develop vaccines that were previously impossible or difficult to make, and to improve the vaccines that were already available but had been developed using old technology. In the case of whooping cough, an effective vaccine (composed of killed bacterial cells) is available, but its use is controversial because of the many side effects that have been associated with it. An improved vaccine against this disease should contain pertussis toxin, a molecule that needs to be detoxified in order to be included in the vaccine. Classical methods of detoxification, such as formaldehyde treatment have been used to inactivate this toxin. We have used recombinant DNA technologies to clone the pertussis toxin gene, express it in bacteria, map the B and T cell epitopes of the molecule, and to identify the amino acids that are important for enzymatic activity and toxicity. Finally, we have used this information to mutate the gene in the chromosome of Bordetella pertussis in order to obtain a strain that produces a molecule that is already non-toxic. This genetically inactivated pertussis toxin was tested extensively in animal models and clinical trials and was found to induce an immune response that is superior in quality and quantity to that induced by the vaccines produced by conventional technologies.     

Modulatory Role of Adenosine Receptors in Insect Motor Nerve Terminals

The effects of adenosine and ATP were studied on blowfly larvae Calliphora vicina neuromuscular preparation. Adenosine diminished (IC₅₀ = 40 ± 3 M) the amplitude of nerve-evoked postsynaptic currents (EPSCs) and slightly decreased the frequency of spontaneous currents without affecting their amplitude. EPSCs were slightly reduced by ATP, and this effect was prevented by concanavalin A. Presynaptic inhibition by adenosine was temperature-dependent and insensitive to pertussis toxin. A₁ agonists of vertebrate adenosine receptor CPA and NECA failed to reproduce the effect of adenosine, and 2-CADO enhanced the EPSCs. A₁ antagonist DPCPX competitively inhibited adenosine action. A₂ agonist DPMA potentiated EPSCs, and its effect was abolished by A₂ antagonist DMPX. Adenosine and ATP failed to affect the nonquantal release of glutamate. The results show for the first time the presence of presynaptic adenosine receptors regulating transmitter release at insect motor nerve terminals and point to differences in pharmacological properties of adenosine receptor subtypes in insects and vertebrates.     

The hamster adipocyte adenylate cyclase system II. Regulation of enzyme stimulation and inhibition by monovalent cations

In hamster adipocyte ghosts, ACTH and β-adrenergic agonists stimulate adenylate cyclase by a GTP-dependent process; in contrast, inhibition of the enzyme by hormonal factors requires both GTP and sodium ions. The interaction of various monovalent cations and guanine nucleotides was studied on basal, stimulated and inhibited adenylate cyclase activities. In the presence of GTP (0.03–10 μM), which reduced basal activity by up to 90%, monovalent cations (10–500 mM, added as chloride salts) increased the enzyme activity by up to about 8-fold. The potency order obtained was Na⁺>Li⁺>K⁺>choline. The stable GTP analogue, guanylyl-5′-imidodiphosphate, which like GTP was capable of decreasing basal activity, diminished the cation-induced activation. The stimulatory effects of ACTH and isoproterenol on adipocyte adenylate cyclase activity were impaired by the cations in the potency order, Na⁺>Li⁺>K⁺>choline. Additionally, NaCl shifted the concentration-response for ACTH to the right and caused an increase in the maximal activation by the hormone. Similar to basal activity, fluoride-stimulated activity was increased by NaCl, when GTP was present. The inhibitory effect of prostaglandin E₁ on basal adipocyte adenylate cyclase activity was revealed by the cations in the above mentioned potency order by an apparent reversal of the cation-induced activation. In the presence of NaCl, the ACTH- or fluoride-stimulated activities were also reduced by prostaglandin E₁, but the inhibitory hormonal factor did not reverse the NaCl-induced shift in the concentration-response curve for ACTH. Guanylyl-5′-imidodiphosphate completely prevented hormonal inhibition. The data suggest that monovalent cations interact with the guanine nucleotide-binding regulatory component of the adipocyte adenylate cylase system and that this interaction somehow changes the properties of this component, now revealing hormone-induced inhibition partially impairing hormone-induced stimulation.     

Effects of antibodies to the filamentous haemagglutinin and to the pertussis toxin of Bordetella pertussis on adherence and toxic effects to 3T3 cells

Abstract Adherence of B. pertussis to NIH3T3 mouse fibroblasts was efficiently inhibited by a mouse immune serum reacting specifically with the filamentous haemagglutinin (FHA), whereas a mouse immune serum reacting specifically with the pertussis toxin (Ptx) produced partial inhibition only significant after 3 h infection. Protection against cytopathic effects on infected 3T3 cells with anti-FHA antibodies was at least as effective (83.3%± 7.5) as with anti-Ptx antibodies (75%± 4). This suggests that adherence of B. pertussis to eukaryotic receptors is a primary mechanism determining both bacterial proliferation and toxic effects in susceptible cells, and that prevention of B. pertussis attachment to cell receptors might be sufficient to protect against both infectious and toxic processes in whooping cough.     

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E₁, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn²⁺ and Mg²⁺, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn²⁺ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E₁ (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn²⁺, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E_1^?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn²⁺, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn²⁺ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg²⁺, although about 100-fold higher concentrations of Mg²⁺ than of Mn²⁺ were required. The data indicate that Mn²⁺at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn²⁺ on the functions of the guanine nucleotide site(s) in the system.     

Forskolin decreases sensitivity of brain adenylate cyclase to inhibition by 2',5'-dideoxyadenosine

The effects of forskolin on the sensitivity of adenylate cyclase to 'P'-site-mediated inhibition were studied. Stimulation of crude and purified preparations of adenylate cyclase by forskolin led to decreased sensitivity to inhibition by 2',5' dideoxyadenosine with enzyme from rat and bovine brain. This is in contrast with the enhancement of P-site sensitivity induced by calmodulin, divalent cations, and stable GTP analogs and is in contrast with behavior seen with enzyme from liver and S49 cyc membranes. The effect of forskolin on P-site sensitivity of the brain adenylate cyclase was not dependent on the presence of G-proteins or calmodulin. It was not the consequence of proteolysis nor was it due to an obvious artifact in the assay procedures. This distinct behavior of the brain enzyme is most likely due to a structural difference in the catalytic subunit.     

Importance of the degree of antigen polymerization by detoxification in modulating the immunogenicity of acellular pertussis vaccine

For the acellular pertussis vaccine with a high immunogenicity, the concentration, composition and characteristics of acellular pertussis antigens are the crucial points to be considered. Nevertheless, it has not been proved yet whether or not the polymerization degree, one of the characteristics of formalin-detoxified acellular pertussis antigens, has an influence on vaccine potency. Thus, in the present study, the correlations among detoxification conditions of acellular pertussis bulks, their polymerization degrees and their immunogenicities were examined. In addition, the relative importance of pertussis toxoid in vaccine immunogenicity was also investigated. Results show that a lower lysine concentration during detoxification induces highly-polymerized antigens, the immunogenicity has a great dependency on the polymerization degree of antigens, and also pertussis toxoid has a relatively stronger influence on the immunogenicity than other antigens. Accordingly, in the aspect of the potency of detoxified acellular pertussis vaccine, it can be demonstrated that the polymerization of antigens and its degree are the major factors affecting the immunogenicity along with a relatively high content of pertussis toxoid.     

Streptozotocin diabetes results in increased responsiveness of adipocyte lipolysis to glucagon

Adipocytes from streptozotocin-diabetic rats are approximately 50-times more sensitive to the lipolytic action of glucagon. This change is only perceived in the presence of a small quantity of adenosine deaminase which itself has little effect on basal lipolysis. Insulin treatment restores glucagon sensitivity to normal.