Activation of polyamine catabolism in transgenic rats induces acute pancreatitis

Polyamines are required for optimal growth and function of cells. Regulation of their cellular homeostasis is therefore tightly controlled. The key regulatory enzyme for polyamine catabolism is the spermidine/spermine N(1)-acetyltransferase (SSAT). Depletion of cellular polyamines has been associated with inhibition of growth and programmed cell death. To investigate the physiological function SSAT, we generated a transgenic rat line overexpressing the SSAT gene under the control of the inducible mouse metallothionein I promoter. Administration of zinc resulted in a marked induction of pancreatic SSAT, overaccumulation of putrescine, and appearance of N(1)-acetylspermidine with extensive depletion of spermidine and spermine in transgenic animals. The activation of pancreatic polyamine catabolism resulted in acute pancreatitis. In nontransgenic animals, an equal dose of zinc did not affect pancreatic polyamine pools, nor did it induce pancreatitis. Acetylated polyamines, products of the SSAT-catalyzed reaction, are metabolized further by the polyamine oxidase (PAO) generating hydrogen peroxide, which might cause or contribute to the pancreatic inflammatory process. Administration of specific PAO inhibitor, MDL72527 [N(1),N(2)-bis(2,3-butadienyl)-1,4-butanediamine], however, did not affect the histological score of the pancreatitis. Induction of SSAT by the polyamine analogue N(1),N(11)-diethylnorspermine reduced pancreatic polyamines levels only moderately and without signs of organ inflammation. In contrast, the combination of N(1), N(11)-diethylnorspermine with MDL72527 dramatically activated SSAT, causing profound depletion of pancreatic polyamines and acute pancreatitis. These results demonstrate that acute induction of SSAT leads to pancreatic inflammation, suggesting that sufficient pools of higher polyamine levels are essential to maintain pancreatic integrity. This inflammatory process is independent of the production of hydrogen peroxide by PAO.     

Spermidine/spermine N1-acetyltransferase-mediated polyamine catabolism regulates beige adipocyte biogenesis

ObjectiveCold and β3-adrenergic receptor (AR) agonists activate beige adipocyte biogenesis in white adipose tissue (WAT). The two stimuli also induce expression of inflammatory cytokines in WAT. The low-grade inflammation may further promote WAT browning. However, the mechanisms to reconcile these two biological processes remain to be elucidated. In this study, we aim to investigate the roles of the rate-limiting polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SAT1) in regulating beige adipocyte biogenesis and inflammation.MethodsAdipose-specific SAT1 knockout mice (SAT1-aKO) were generated by crossing adiponectin-cre to SAT1-lox/lox mice. Metabolic phenotype was investigated. Primary pre-adipocytes were isolated from inguinal WAT (iWAT) and differentiated to adipocytes for studying beige adipocyte biogenesis.ResultThe expression and enzymatic activity of SAT1 were up-regulated in iWAT upon cold and β3-AR stimulation. SAT1-aKO mice developed late-onset obesity on a high-fat diet with impaired cold-induced beige adipocyte biogenesis and energy expenditure. RNA-seq analysis of iWAT from cold-challenged SAT1-aKO mice revealed that, in addition to beige adipocyte biogenesis signatures, the immune response markers were highly enriched among reduced genes. In cultured adipocytes, SAT1 overexpression or pharmacological activation with N1, N11-diethylnorspermine (DENSpm) elevated oxygen consumption and increased the expression of beige adipocyte marker UCP1 and PGC-1α. DENSpm treatment of adipocytes also increased the expression of inflammatory genes. SAT1 activation enhanced hydrogen peroxide production in adipocytes. Antioxidant N-acetylcysteine abrogated the elevated UCP1 expression and reversed some inflammatory genes induced by SAT1 activation.ConclusionsSAT1 activation plays a key role in cold and β3-AR agonist-induced beige adipocyte biogenesis and low-grade inflammation.     

Structures of wild-type and mutant human spermidine/spermine N1-acetyltransferase, a potential therapeutic drug target

Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels in human cells, as acetylation of spermidine and spermine triggers export or degradation. Increased intracellular polyamine levels accompany several types of cancers as well as other human diseases, and compounds that affect the expression, activity, or stability of SSAT are being explored as potential therapeutic drugs. We have expressed human SSAT from the cloned cDNA in Escherichia coli and have determined high-resolution structures of wild-type and mutant SSAT, as the free dimer and in binary and ternary complexes with CoA, acetyl-CoA (AcCoA), spermine, and the inhibitor N1,N11bis-(ethyl)-norspermine (BE-3-3-3). These structures show details of binding sites for cofactor, substrates, and inhibitor and provide a framework to understand enzymatic activity, mutations, and the action of potential drugs. Two dimer conformations were observed: a symmetric form with two open surface channels capable of binding substrate or cofactor, and an asymmetric form in which only one of the surface channels appears capable of binding and acetylating polyamines. SSAT was found to self-acetylate lysine-26 in the presence of AcCoA and absence of substrate, a reaction apparently catalzyed by AcCoA bound in the second channel of the asymmetric dimer. These unexpected and intriguing complexities seem likely to have some as yet undefined role in regulating SSAT activity or stability as a part of polyamine homeostasis. Sequence signatures group SSAT with proteins that appear to have thialysine Nepsilon-acetyltransferase activity.     

Estradiol-17 beta modifies the induction of spermidine/spermine N1-acetyltransferase activity in the liver of lipopolysaccharide-treated mice.

In an attempt to elucidate the effects of estrogen on polyamine metabolism in lipopolysaccharide (LPS)-treated mice, we assayed polyamine content and the activity of spermidine/spermine N1-acetyltransferase (SAT) and ornithine decarboxylase (ODC) in some organs. LPS elevated N1-acetylspermidine levels in the liver and lung and putrescine levels in the liver, lung and spleen. LPS increased the activity of ODC at 6 h and that of SAT at 12 h in the liver. When estradiol-17 beta was simultaneously administered with LPS, the maximum increase in hepatic N1-acetylspermidine levels was found 6 h earlier than in the LPS control. Likewise, the peak of the hepatic SAT activity after LPS-treatment was observed 6 h earlier in the estradiol-17 beta-treated mice than in the LPS control. No such effect of estradiol-17 beta was found in the lung and spleen. The LPS-induced ODC activity was not affected by estradiol-17 beta in the liver, lung or spleen. Estrone and 16 beta-ethylestradiol (an anti-estrogen) were also effective in enhancing the LPS-induced elevation of N1-acetyl-spermidine and putrescine in the liver, while both diethylstilbestrol, which has a potent estrogenic activity without steroid structure and estradiol-17 alpha (a non-estrogenic isomer of estradiol-17 beta) were without effect. Tamoxifen (an estrogen receptor antagonist) did not suppress the estrogen-induced increase in hepatic N1-acetylspermidine levels.     

Interleukin-1beta induces elevation of spermidine/spermine N1-acetyltransferase activity and an increase in the amount of putrescine in synovial adherent cells from patients with rheumatoid arthritis

OBJECTIVE: To investigate polyamine metabolism in rheumatoid synovial adherent cells stimulated by interleukin- 1beta (IL-1beta). METHODS: Synovial adherent cells obtained from patients with rheumatoid arthritis (RA) were cultured and incubated in the presence or absence of human recombinant IL-1beta at a concentration of 10 ng/ml for 24 h. The cellular contents of polyamines as well as the activities of spermidine/spermine N1-acetyltransferase (SAT) and ornithine decarboxylase (ODC) were measured. RESULTS: Polyamines in synovial adherent cells decreased significantly after 24 h incubation in the absence of IL-1beta. However, in the presence of IL-Ibeta, putrescine and N'-acetylspermidine increased significantly. No significant difference was observed between the amount of spermidine in synovial adherent cells incubated with and without IL-1beta. Spermine and N8-acetylspermidine in synovial adherent cells incubated with IL-1beta decreased significantly more than in synovial adherent cells incubated without. SAT activity reached a peak 12 h after the addition of IL-1beta and then decreased, while the ODC activity did not increase. SAT activity was elevated by the addition of IL-1beta in a dose dependent manner. CONCLUSION: An increase in the putrescine level in rheumatoid synovial adherent cells as a result of the elevation of SAT activity induced by IL-1beta may play a role in RA.     

Estradiol-17β modifies the induction of spermidine/spermine N1-acetyltransferase activity in the liver of lipopolysaccharide-treated mice

ABSTRACT— In an attempt to elucidate the effects of estrogen on polyamine metabolism in lipopolysaccharide (LPS)-treated mice, we assayed polyamine content and the activity of spermidine/spermine N¹-acetyltransferase (SAT) and ornithine decarboxylase (ODC) in some organs. LPS elevated N'-acetylspermidine levels in the liver and lung and putrescine levels in the liver, lung and spleen. LPS increased the activity of ODC at 6 h and that of SAT at 12 h in the liver. When estradiol-17β was simultaneously administered with LPS, the maximum increase in hepatic N¹-acetylspermidine levels was found 6 h earlier than in the LPS control. Likewise, the peak of the hepatic SAT activity after LPS-treatment was observed 6 h earlier in the estradiol-17β-treated mice than in the LPS control. No such effect of estradiol-17β was found in the lung and spleen. The LPS-induced ODC activity was not affected by estradiol-17β in the liver, lung or spleen. Estrone and 16β-ethylestradiol (an anti-estrogen) were also effective in enhancing the LPS-induced elevation of N¹-acetyl-spermidine and putrescine in the liver, while both diethylstilbestrol, which has a potent estrogenic activity without steroid structure and estradiol-17α (a non-estrogenic isomer of estradiol-17β) were without effect. Tamoxifen (an estrogen receptor antagonist) did not suppress the estrogen-induced increase in hepatic N¹-acetylspermidine levels.     

Putative association between the -1415 T/C polymorphism of spermidine/spermine N1-acetyltransferase (SSAT1) gene and alcohol use disorders in women and men

Background: The activity of N-methyl-D-aspartate (NMDA) glutamate receptor, which responds to the levels of polyamines, modifies the neurotoxicity caused by ethanol. We aimed to investigate if the functionality of the spermidine/spermine N1-acetyltransferase (SSAT1) gene could be associated with a differential risk for alcoholism. Methods: We studied a sample of 586 subjects: 104 alcohol-dependent patients, 273 patients with psychiatric disorders but without substance dependence, and 209 healthy controls. After gender stratification, the allele frequency distribution of the SSAT1 gene was compared between these three groups. Results: In females, the TC genotype was significantly more frequent in alcohol-dependent patients than in non-alcohol-dependent psychiatric controls (χ^2?=?7.509 df?=?2, p?=?0.023). A trend was found when alcohol-dependent females were compared with the healthy control group (χ^2?=?4.897 df?=?2, p?=?0.086). No statistical differences were found among the males. Discussion and conclusion: Gender differences in the regulation of SSAT1 gene expression may possibly be due to gender-specific effects of stress, ethanol toxicity, and/or polyamines levels. Further studies are needed to confirm our findings.     

Hyperthermia induces multiple pancreatic heat shock proteins and protects against subsequent arginine-induced acute pancreatitis in rats

Heat shock proteins (HSPs) which are induced by stress can provide protection against subsequent cellular damage. Whole body hyperthermia in rats leading to induction of HSP70 has been shown to protect against subsequent caerulein-induced acute pancreatitis. We studied the effect of hyperthermia on pancreatic HSP expression and found a significant increase in HSP70 (26.0-fold) and HPS27 (6.0-fold) but no change in HSP60, HSP90 or GRP78. Hyperthermia conferred significant protection against subsequent arginine-induced acute pancreatitis. More specifically, the degradation and disorganization of the actin cytoskeleton, an important early component of acute pancreatitis, was prevented. These results generalize previous work on caerulein-induced pancreatitis to another model of experimental pancreatitis, arginine-induced pancreatitis, and suggest that multiple HSPs may be involved in the cytoprotective effect in rat pancreas.     

Effect of buprenorphine on pancreatic enzyme synthesis and secretion in normal rats and rats with acute edematous pancreatitis

Pancreatic enzyme secretion is inhibited during acute pancreatitis, resulting in an increase in acinar zymogen content. Since the premature activation of zymogens has been assigned a central role in the pathogenesis of acute pancreatitis, minimizing the amount of stored zymogens might lead to less severe acute pancreatitis. Inhibition of enzyme synthesis or stimulation of enzyme secretion would result in reduction of zymogen stores. Opiates have a varying effect on pancreatic secretion, depending on the dosage, site of administration, and presence of pancreatic stimulants. The effect of opiates and acute pancreatitis on individual pancreatic enzyme synthesis is unknown. The following study was undertaken in order to examine the effects of an opiate on pancreatic enzyme secretion and synthesis during experimental acute pancreatitis. Four groups of rats were studied. Group I received cerulein (25 µg/kg); group II received an opiate, buprenorphine (BPN, 0.5 mg/kg); and group III received cerulein and BPN. Drugs were dissolved in gelatin/saline and injected subcutaneously. A control group (group IV) received only gelatin/saline. Rats were sacrificed 4 hr after injection, and pancreatic mass was measured. Pancreatic acini were prepared and assayed for amylase and DNA content. Amylase, trypsinogen, chymotrypsinogen and lipase synthesis, and amylase secretion were measured for 2 hr. Results showed that, compared to controls, acini of rats with AP had increased amylase content, a finding consistent with decreasedin vivo amylase secretion. Total protein and individual enzyme synthesis rates were significantly lower in the acini of the rats with AP than in those of the controls. Negative feedback inhibition of enzyme synthesis due to the increased stores of intracellular enzymes may account for these findings. BPN reduced pancreatic edema in rats with acute pancreatitis (AP). Acinar amylase content of rats with AP treated with BPN was significantly lower than in acini of rats with AP. As amylase secretion was lower in the AP + BPN rats, the reduced acinar amylase content was probably solely due to the reduction in enzyme synthesis observed in the AP + BPN rats. The results suggest that BPN may have a moderating effect on the development of AP.This study was supported by a research grant from the South African Medical Research Council.     

Emodin and baicalein inhibit pancreatic stromal derived factor-1 expression in rats with acute pancreatitis

BACKGROUND: Stromal derived factor-1 (SDF-1) is an efficacious leukocyte chemoattractant, which can attract lymphocytes and mononuclear cells from bloodstream into the site of inflammation. Emodin, an anthraquinone derivative from Radix et Rhizoma Rhei, and baicalein, a flavone from Scutellaria baicalensis Georgi, both have been reported to possess anti-inflammatory activities. The expression pattern of SDF-1 in experimental acute pancreatitis (AP) and the effect of emodin or baicalein on that are not well ...     

白藜芦醇下调线粒体DNA修复酶对重症急性胰腺炎大鼠胰腺腺泡细胞凋亡的影响

目的:观察白藜芦醇对重症急性胰腺炎(SAP)胰腺细胞凋亡的影响,探讨线粒体凋亡通路及线粒体DNA修复酶OGG1与SAP腺泡细胞凋亡的关系.方法:30只SD大鼠随机分为假手术组(Sham组,只行开腹术,n=10),SAP模型组(SAP组,胆管逆行性注射牛黄胆酸钠制备SAP模型,n=10),白藜芦醇治疗组(Res组,SAP模型制作成功后,注射白藜芦醇溶液,n=10).测定各组血清淀粉酶,caspase-3,-9活性,高压液相色谱法测定线粒体DNA 8-氧鸟嘌呤(8-oxodG)含量,罗丹明123法测定线粒体膜电位,流式细胞法检测胰腺腺泡细胞凋亡,Western blot法测定细胞色素C释放及线粒体OGG1蛋白表达,并对胰腺组织进行病理学评分.结果:Res组与SAP组比较,血清胰淀粉酶、线粒体膜电位、线粒体OGG1蛋白表达量及胰腺病理损害评分显著降低(4761.98±501.45 vs7428.91±526.49,18.42±2.04 vs 22.01±2.93,73.97±6.49 vs 159.46±12.85.5.74±0.95 vs12.95±1.54,均P<0.05).胰腺腺泡细胞凋亡指数、线粒体细胞色素C释放、线粒体DNA8-oxodG含量及caspase-3,-9的活性显著升高(19.63±2.07 vs 12.45±1.93,174.31±15.93 vs100±0.00,0.0590±0.074 vs 0.0336±0.0061,2.37±0.35 vs 1.95±0.19.2.07±0.25 vs 1.62±0.15,均P<0.05).结论:白藜芦醇通过下调胰腺腺泡细胞线粒体DNA修复酶OGG1,增加SAP胰腺腺泡细胞凋亡以减轻胰腺组织病理损害,对SAP具有治疗意义.     

硫氧还蛋白-1 在急性坏死性胰腺炎大鼠中的表达及褪黑素对其的影响

目的 观察硫氧还蛋白-1(TRX-1)在急性坏死性胰腺炎(ANP)大鼠胰腺组织的表达和褪黑素干预对其的影响.方法 72只雄性SD大鼠随机分成ANP组、褪黑素组和对照组,每组24只.以腹腔注射6%L-Arginine 25 mL/kg体重3次、每次间隔1 h的方法制备ANP模型.褪黑素组在制模前30min腹腔注射0.25%褪黑素20 ml/kg体重;ANP组和对照组大鼠腹腔注射等容积生理盐水.术后6、12、24 h分批处死大鼠.抽血测定血清淀粉酶含量;取胰腺组织行病理学检查,并评分;检测胰腺组织丙二醛(MDA)、髓过氧化酶(MPO)含量;免疫组化检测胰腺组织TRX-1蛋白表达;RT-PCR检测胰腺组织TRX-1 mRNA的表达.结果 ANP组12 h点血清淀粉酶、胰腺组织MDA和MPO以及TRX-1 mRNA和蛋白表达水平分别为(3 012±1 425)u/L、(4.13±1.85)nmol/mg prot、(7.45±1.26)nmol/mg prot、0.68±0.18、66.8±8.1;褪黑素组分别为(1 835±499)U/L、(3.03±2.12)nmol/mg prot、(5.32±1.06)nmml//mgprot、0.50±0.09、80.29±8.14,两组比较均有显著差异(P<0.05).褪黑素组胰腺TRX-1 mRNA和蛋白表达峰值从ANP组的制模后12 h提前到制模后6 h.结论 ANP大鼠胰腺组织TRX-1 mRNA和蛋白表达增高;褪黑素干预能促进胰腺组织早期表达TRX-1 mRNA和蛋白,减轻胰腺组织的损伤.     

氧化苦参碱诱导跨膜蛋白Claudin-1表达在重症急性胰腺炎大鼠肠黏膜损害中的作用

目的:探讨氧化苦参碱(OM)在重症急性胰腺炎(SAP)肠黏膜屏障损害中的作用及其机制.方法:采用L-精氨酸(1 750 mg/kg)腹腔注射制备SAP大鼠模型.将80只Wistar大鼠随机分为Control组、OM对照组、SAP对照组、OM治疗组1、12、24、36、48 h 5个亚组,每组10只.分时取材,测定血浆内...     

Effects of tetraprenylacetone on pancreatic exocrine secretion and acute pancreatitis in two experimental models in rats

Summary The effects of tetraprenylacetone (TPN), an acyclic polyisoprenoid with antiulcer actions, on pancreatic exocrine secretion, and its preventive and therapeutic effects on acute pancreatitis in two experimental models were studied in rats. Intraduodenal administration of TPN (0, 100, 200 and 400 mg/kg/h) caused dose-dependent increases in pancreatic juice and bicarbonate output without increasing protein output and plasma cholecystokinin (CCK) concentrations. TPN-stimulated pancreatic exocrine secretion was completely abolished by antisecretin serum but it was not by CCK receptor antagonist loxiglumide (50 mg/kg/h). In acute pancreatitis induced by four subcutaneous injections of 20 μg/kg cerulein at hourly intervals over, 3 h, TPN (400 mg/kg) given by an oral route either 1 h before the first cerulein injection or immediately after the last injection significantly reduced the increases in serum amylase and lipase activities and pancreatic wet wt. Pretreatment with TPN caused histologic improvements, whereas posttreatment failed to ameliorate histologic alterations. In severe type of acute pancreatitis induced by retrograde intraductal injection of 1.0 mL/kg of 4% sodium taurocholate, TPN exerted no apparent beneficial effects on biochemical and histologic alterations of acute pancreatitis. It is concluded that TPN given by an oral route stimulates pancreatic exocrine secretion through an increase in endogenous secretin release and causes beneficial effects on the experimental model of mild acute pancreatitis in rats.     

Controlled hypertension induces cerebrovascular and gene alterations in Cyp1a1-Ren2 transgenic rats

Hypertension is a major risk factor for small vessel disease and dementia, but the pathogenic mechanisms are not fully known. This study aimed to assess cerebrovascular alterations in response to different durations (4 or 6 months) of controlled hypertension in an inducible transgenic rat model of hypertension (Cyp1a1-Ren2) as compared with normotensive litter mate controls. After 6 months of hypertension as compared with controls, a significant reduction in vascular width was paralleled by an increase in the protein levels of claudin-5, an endothelial tight junction protein. Notably, vascular alterations were associated with increased microglia, and these changes were preceded by increased eNOS expression. Investigation of global gene expression by microarray analysis indicated alterations in predominantly growth factor related genes. Herein, we show that modest, sustained levels of hypertension are sufficient to cause cerebrovascular alterations accompanied by endothelial and inflammatory changes. These changes are paralleled by alterations in growth factor expression suggestive of a mechanistic role.