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Fatty acid handling and complex lipid synthesis are altered in the kidney cortex of diabetic patients. We recently showed that inhibition of the renin-angiotensin system without changes in glycemia can reverse diabetic kidney disease (DKD) and restore the lipid metabolic network in the kidney cortex of diabetic (db/db) mice, raising the possibility that lipid remodeling may play a central role in DKD. However, the roles of specific enzymes involved in lipid remodeling in DKD have not been elucidated. In the present study, we used this diabetic mouse model and a proximal tubule epithelial cell line (HK2) to investigate the potential relationship between long-chain acyl-CoA synthetase 1 (ACSL1) and lipid metabolism in response to fatty acid exposure and inflammatory signals. We found ACSL1 expression was significantly increased in the kidney cortex of db/db mice, and exposure to palmitate or tumor necrosis factor-α significantly increased Acsl1 mRNA expression in HK-2 cells. In addition, palmitate treatment significantly increased the levels of long-chain acylcarnitines and fatty acyl CoAs in HK2 cells, and these increases were abolished in HK2 cell lines with specific deletion of Acsl1(Acsl1KO cells), suggesting a key role for ACSL1 in fatty acid ß-oxidation. In contrast, tumor necrosis factor-α treatment significantly increased the levels of short-chain acylcarnitines and long-chain fatty acyl CoAs in HK2 cells but not in Acsl1KO cells, consistent with fatty acid channeling to complex lipids. Taken together, our data demonstrate a key role for ACSL1 in regulating lipid metabolism, fatty acid partitioning, and inflammation.
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Coenzima A Ligasas , Nefropatías Diabéticas , Ácidos Grasos , Animales , Humanos , Ratones , Coenzima A Ligasas/metabolismo , Diabetes Mellitus/patología , Nefropatías Diabéticas/metabolismo , Ácidos Grasos/metabolismo , Ligasas , Palmitatos , Factor de Necrosis Tumoral alfaRESUMEN
BACKGROUND: Cisplatin is effective against various types of cancers. However, its clinical application is limited owing to its adverse effects, especially acute kidney injury (AKI). Dihydromyricetin (DHM), a flavonoid derived from Ampelopsis grossedentata, has varied pharmacological activities. This research aimed to determine the molecular mechanism for cisplatin-induced AKI. METHODS: A murine model of cisplatin-induced AKI (22 mg/kg, I.P.) and a HK-2 cell model of cisplatin-induced damage (30 µM) were established to evaluate the protective function of DHM. Renal dysfunction markers, renal morphology and potential signaling pathways were investigated. RESULTS: DHM decreased the levels of renal function biomarkers (blood urea nitrogen and serum creatinine), mitigated renal morphological damage, and downregulated the protein levels of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. It upregulated the expression levels of antioxidant enzymes (superoxide dismutase and catalase expression), nuclear factor-erythroid-2-related factor 2 (Nrf2) and its downstream proteins, including heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic (GCLC) and modulatory (GCLM) subunits, thus eventually reducing cisplatin-induced reactive oxygen species (ROS) production. Moreover, DHM partially inhibited the phosphorylation of the active fragments of caspase-8 and -3 and mitogen-activated protein kinase and restored glutathione peroxidase 4 expression, which attenuated renal apoptosis and ferroptosis in cisplatin-treated animals. DHM also mitigated the activation of NLRP3 inflammasome and nuclear factor (NF)-κB, attenuating the inflammatory response. In addition, it reduced cisplatin-induced HK-2 cell apoptosis and ROS production, both of which were blocked by the Nrf2 inhibitor ML385. CONCLUSIONS: DHM suppressed cisplatin-induced oxidative stress, inflammation and ferroptosis probably through regulating of Nrf2/HO-1, MAPK and NF-κB signaling pathways.
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Lesión Renal Aguda , Ferroptosis , Animales , Ratones , Cisplatino/farmacología , Especies Reactivas de Oxígeno/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/prevención & control , Riñón , FN-kappa B/metabolismo , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/prevención & controlRESUMEN
Hypertension is well recognized to be the most important risk factor for cardiovascular diseases, stroke, and end-stage kidney failure. A quarter of the world's adult populations and 46% of the US adults develop hypertension and currently require antihypertensive treatments. Only 50% of hypertensive patients are responsive to current antihypertensive drugs, whereas remaining patients may continue to develop cardiovascular, stroke, and kidney diseases. The mechanisms underlying the poorly controlled hypertension remain incompletely understood. Recently, we have focused our efforts to uncover additional renal mechanisms, pathways, and therapeutic targets of poorly controlled hypertension and target organ injury using novel animal models or innovative experimental approaches. Specifically, we studied and elucidated the important roles of intratubular, intracellular, and mitochondrial angiotensin II (Ang II) system in the development of Ang II-dependent hypertension. The objectives of this invited article are to review and discuss our recent findings that (a) circulating and intratubular Ang II is taken up by the proximal tubules via the (AT1) AT1a receptor-dependent mechanism, (b) intracellular administration of Ang II in proximal tubule cells or adenovirus-mediated overexpression of an intracellular Ang II fusion protein selectively in the mitochonria of the proximal tubules induces blood pressure responses, and (c) genetic deletion of AT1 (AT1a) receptors or the Na+/H+ exchanger 3 selectively in the proximal tubules decreases basal blood pressure and attenuates Ang II-induced hypertension. These studies provide a new perspective into the important roles of the intratubular, intracellular, and mitochondrial angiotensin II/AT1 (AT1a) receptor signaling in Ang II-dependent hypertensive kidney diseases.
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OBJECTIVE: Dyslipidemia is a significant risk factor for progression of diabetic kidney disease (DKD). Determining the changes in individual lipids and lipid networks across a spectrum of DKD severity may identify lipids that are pathogenic to DKD progression. METHODS: We performed untargeted lipidomic analysis of kidney cortex tissue from diabetic db/db and db/db eNOS-/- mice along with non-diabetic littermate controls. A subset of mice were treated with the renin-angiotensin system (RAS) inhibitors, lisinopril and losartan, which improves the DKD phenotype in the db/db eNOS-/- mouse model. RESULTS: Of the three independent variables in this study, diabetes had the largest impact on overall lipid levels in the kidney cortex, while eNOS expression and RAS inhibition had smaller impacts on kidney lipid levels. Kidney lipid network architecture, particularly of networks involving glycerolipids such as triacylglycerols, was substantially disrupted by worsening kidney disease in the db/db eNOS-/- mice compared to the db/db mice, a feature that was reversed with RAS inhibition. This was associated with decreased expression of the stearoyl-CoA desaturases, Scd1 and Scd2, with RAS inhibition. CONCLUSIONS: In addition to the known salutary effect of RAS inhibition on DKD progression, our results suggest a previously unrecognized role for RAS inhibition on the kidney triacylglycerol lipid metabolic network.
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Diabetes Mellitus , Nefropatías Diabéticas , Animales , Antihipertensivos/metabolismo , Diabetes Mellitus/metabolismo , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Riñón/metabolismo , Redes y Vías Metabólicas , Ratones , Sistema Renina-Angiotensina/efectos de los fármacos , Triglicéridos/metabolismoRESUMEN
Increasing evidence indicates that the intra-uterine environment has consequences for later life. However, the mechanisms of this fetal programming remain unclear. We aimed to investigate the impact of diet-induced maternal hypercholesterolemia on the predisposition of offspring to nonalcoholic fatty liver diseases (NAFLD) and metabolic diseases and its underlying mechanisms. Female apolipoprotein (Apo) E-deficient mice were fed a control diet (CD) or high fat/high cholesterol Western-type diet (WD) before and throughout pregnancy and lactation, and their offspring were weaned onto a CD postnatally. Strikingly, male offspring of WD-fed dams developed glucose intolerance and decreased peripheral insulin sensitivity and exhibited hepatic steatosis. Hepatic steatosis could be attributed, at least in part, to increased hepatic lipogenesis in E18.5 embryos and decreased serum VLDL levels in adulthood. In addition, males born to WD-fed dams had lower serum ApoB levels and hepatic ApoB gene expression compared with males born to CD-fed dams. DNA methylation analysis revealed increased methylation of CpG dinucleotides on the promoter region of the ApoB genes in the livers of male offspring of WD-fed dams. Our findings suggest that maternal WD intake can exacerbate the development of NAFLD in male offspring potentially by affecting ApoB gene expression through epigenetic alterations.
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Apolipoproteína B-100/genética , Metilación de ADN , Lipoproteínas VLDL/sangre , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Efectos Tardíos de la Exposición Prenatal , Animales , Glucemia/metabolismo , Islas de CpG , Dieta Occidental , Modelos Animales de Enfermedad , Epigénesis Genética , Femenino , Intolerancia a la Glucosa , Prueba de Tolerancia a la Glucosa , Hipercolesterolemia/metabolismo , Metabolismo de los Lípidos , Lipoproteínas/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Enfermedad del Hígado Graso no Alcohólico/sangre , Fenotipo , Embarazo , PreñezRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.10583.].
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Peroxynitrite (ONOO-) is an important species involved in many physiopathological processes. Progresses have been made in developing novel fluorescent probes to detect peroxynitrite with relatively high sensitivity and specificity. Herein, we report the synthesis, characterization and biological applications of a new boronate-based fluorescent probe, 4-MB. The studies showed that 4-MB exhibits a dual ratiometric and calorimetric response toward peroxynitrite due to ONOO--triggered oxidative reaction. A possible mechanism of the oxidation reaction was proposed and the reaction product was isolated and characterized using different spectroscopic methods. We have thoroughly demonstrated the utility of 4-MB for intracellular peroxynitrite imaging. Further, we showed that 4-MB can be potentially employed to visualize exogenous and endogenous peroxynitrite in RAW264.7 macrophages, EAhy926 cells, zebrafish and in live tissues from a high-fat diet-induced obese mouse model.
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Ácidos Borónicos/química , Colorantes Fluorescentes/química , Imagen Óptica/métodos , Ácido Peroxinitroso/análisis , Animales , Técnicas Biosensibles/métodos , Línea Celular , Macrófagos/química , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/patología , Células RAW 264.7 , Espectrometría de Fluorescencia/métodosRESUMEN
Cellular accumulation of mono(2-ethylhexyl)phthalate (MEHP) has been recently demonstrated to disturb fat cell energy metabolism; however, the underlying mechanism remained unclear. The study aimed to determine how MEHP influenced fat cell transcriptome and how the changes might contribute to bioenergetics. Because of the pivotal role of PPARγ in energy metabolism of fat cells, comparative microarray analysis of gene expression in 3T3-L1 adipocytes treated with both MEHP and rosiglitazone was performed. Pathway enrichment analysis and gene ontology (GO) enrichment analysis revealed that both treatments caused up-regulation of genes involved in PPAR signaling/energy metabolism-related pathways and down-regulation of genes related to adipokine/inflammation signals. MEHP/rosiglitazone-treated adipocytes exhibited increased levels of lipolysis, glucose uptake, and glycolysis; the gene expression profiles provided molecular basis for the functional changes. Moreover, MEHP was shown to induce nuclear translocation and activation of PPARγ. The similarity in gene expression and functional changes in response to MEHP and rosiglitazone suggested that MEHP influenced bioenergetics and adipokine network mainly via PPARγ. Importantly, adipokine levels in C57BL/6J mice with di(2-ethylhexyl)phthalate (DEHP) treatments provided in vivo evidence for microarray results. On the basis of correlation between gene expression and functional assays, possible involvements of genes in bioenergetics of MEHP-treated adipocytes were proposed.
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Adipocitos/efectos de los fármacos , Adipoquinas/metabolismo , Dietilhexil Ftalato/análogos & derivados , Células 3T3-L1 , Adipocitos/metabolismo , Adipoquinas/genética , Animales , Dietilhexil Ftalato/farmacología , Metabolismo Energético/efectos de los fármacos , Ácidos Grasos/metabolismo , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Glucosa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , PPAR gamma/genética , PPAR gamma/metabolismo , Rosiglitazona , Tiazolidinedionas/farmacologíaRESUMEN
Long-standing diabetes or glucose intolerance is recognized as a crucial event in the process of pancreatic cancer. Betatrophin, a novel liver-derived hormone, promotes ß-cell proliferation and improves glucose intolerance. However, the relationship between betatrophin and PDAC-associated diabetes is not fully understood. To evaluate the serum betatrophin levels in PDAC-associated diabetes, a total 105 Taiwanese subjects including 15 healthy subjects, and 12 patients having PDAC with normal glucose tolerance (PDAC-NGT), 12 patients having PC with impaired glucose tolerance (PDAC-IGT), and 66 patients having PC with diabetes mellitus (PDAC-DM) were enrolled for this study. Serum betatrophin and carbohydrate antigen 19-9 (CA19-9) levels were analyzed by enzyme-linked immunosorbent assay (ELISA). Compared to healthy subjects, PDAC patients had higher levels of betatrophin and CA19-9. Consistently, betatrophin protein was significantly expressed in pancreatic ductal of PDAC-associated DM patients using immunohistochemistry (IHC) method. Furthermore, multivariate regression analysis showed the betatrophin was significantly and positively independent with T category (ß= 0.605, P=0.010), serum albumin (ß= 0. 423, P=0.021), lipase (ß= 0.292, P=0.039), and blood urea nitrogen (BUN) (ß= 0.303, P=0.040). Further, the betatrophin was three folds of having PDAC-associated diabetes with the highest odds ratio [OR=3.39; 95% CI (1.20-9.57); P=0.021) and receiver operating characteristic (ROC) curve analysis showed that AUC value of betarophin was 0.853 which is slightly larger than AUC value of CA19-9 (0.792) in PDAC-DM patients. Interestingly, AUC value of betarophin plus CA19-9 was 0.988 in PDAC-DM patients. Therefore, betatrophin combined CA19-9 may serve as a potential biomarker for PDAC-associated diabetes.
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Proteínas Similares a la Angiopoyetina/sangre , Complicaciones de la Diabetes/sangre , Neoplasias Pancreáticas/sangre , Hormonas Peptídicas/sangre , Anciano , Proteína 8 Similar a la Angiopoyetina , Biomarcadores de Tumor/sangre , Antígeno CA-19-9/sangre , Estudios de Casos y Controles , Proliferación Celular , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , TaiwánRESUMEN
BACKGROUND: Betatrophin is a newly identified liver-derived hormone that is associated with glucose homeostasis and lipid metabolism. Although dysregulated lipid metabolism results in diabetic nephropathy (DN) development in patients with type 2 diabetes mellitus (T2DM), it is not understood whether betatrophin is associated with urinary albumin excretion and renal function. METHODS: Based on albumin/creatinine ratio (ACR), 109 T2DM patients were divided into normoalbuminuria (ACR <30 mg/g), microalbuminuria (ACR between 30 and 300 mg/g), and macroalbuminuria (ACR > 300 mg/g). Serum betatrophin levels of 109 T2DM patients and 32 healthy subjects were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum level of betatrophin was significantly increased in T2DM patients with normoalbuminuria, microalbuminuria, and macroalbuminuria as compared with healthy subjects (P < 0.001). Serum betatrophin level was positively correlated with sex, duration of diabetes, systolic blood pressure (SBP), body mass index (BMI), ACR, and triglyceride, whereas it was inversely correlated with estimated glomerular filtration rate (eGFR), total cholesterol, and high-density lipoprotein cholesterol (HDL-C) (P < 0.001). Furthermore, multivariate regression analysis showed the betatrophin was significantly and positively independent with triglyceride and low-density lipoprotein cholesterol (LDL-C) (P < 0.05), whereas it was inversely independent with eGFR, total cholesterol, and low-density lipoprotein cholesterol (HDL-C) (P < 0.05). In addition, the betatrophin had higher odds of having DN [odds ratio (OR) = 5.65, 95 % confidence interval (CI) 2.17-14.57, P < 0.001]. CONCLUSION: Betatrophin is significantly increased in T2DM patients with different stages of albuminuria. Betatrophin may be a novel endocrine regulator involved in DN development.
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Albuminuria/etiología , Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/etiología , Riñón/fisiopatología , Hormonas Peptídicas/sangre , Albuminuria/sangre , Albuminuria/diagnóstico , Albuminuria/fisiopatología , Proteína 8 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Biomarcadores/sangre , Biomarcadores/orina , Presión Sanguínea , Índice de Masa Corporal , Estudios de Casos y Controles , HDL-Colesterol/sangre , LDL-Colesterol , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/fisiopatología , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/diagnóstico , Nefropatías Diabéticas/fisiopatología , Ensayo de Inmunoadsorción Enzimática , Femenino , Tasa de Filtración Glomerular , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Análisis Multivariante , Oportunidad Relativa , Factores de Riesgo , Triglicéridos/sangre , Regulación hacia ArribaRESUMEN
Recent studies have implicated the prorenin receptor (PRR) is associated with pancreatic tumorigenesis. We therefore investigated the role of PRR in pancreatic tumorigenesis and assessed whether PRR can serve as a target for imaging diagnosis at early stages of PDAC. Here we show that aberrant expression of PRR in premalignant PanIN lesions, and human PDAC samples, and PDAC cell lines, particularly in Panc-1 cells. Interestingly, PRR expression was positively associated with PDAC progression. Moreover, overexpression of human PRR resulted in increased cell proliferation and decreased apoptosis, while knockdown of human PRR caused decreased cell proliferation and enhanced apoptosis in pancreatic cancer cells. We also observed that overexpression of human PRR enhanced MAPK and PI3K/Akt signaling pathways in PDAC cells, while knockdown of human PRR suppressed both of pathways. The confocal imaging analysis showed that human PRR was highly expressed in Panc-1, ASPC, and Miapaca cells, whereas BXPC-3, and HPAC cells had a significantly lower fluorescent signals. Consistently, the single-photon emission computed tomography (SPET/CT) showed that the uptake of anti-PRR labelled with 125I was higher in Panc-1 and ASPC tumors-bearing mice after 96 hours injection. Importantly, tumors in pancreas of Pdx1-cre; LSL-KrasG12D mice had a significant increased PRR expression and accumulation of radioactivity at 96 h after injection. These data suggest that 125I-anti-PRR can detect the orthotopic tumors in Pdx1-cre; LSL-KrasG12D mice. Therefore, anti-PRR labelled with 125I is a promising radiotracer for imaging diagnosis at early stages of pancreatic cancer.
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Carcinoma in Situ/diagnóstico , Carcinoma Ductal Pancreático/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Receptores de Superficie Celular/metabolismo , Animales , Apoptosis , Carcinoma in Situ/diagnóstico por imagen , Carcinoma Ductal Pancreático/diagnóstico por imagen , Proliferación Celular , Humanos , Procesamiento de Imagen Asistido por Computador , Radioisótopos de Yodo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Pancreáticas/diagnóstico por imagen , Receptores de Superficie Celular/antagonistas & inhibidores , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor de ProreninaRESUMEN
Polymorphisms in the human endothelial nitric oxide synthase (eNOS) gene (NOS3) have been associated with advanced nephropathy in diabetic patients and with decreased expression in tissue culture. However, direct proof that modest genetic decreases in eNOS expression worsen diabetic nephropathy is lacking. To investigate this effect, we took advantage of the hybrid vigor and genetic uniformity of the F1 progeny (eNOS(+/+), eNOS(+/-), or eNOS(-/-) with or without diabetes) of a cross between heterozygous 129S6/SvEvTac eNOS(+/-) inbred females and heterozygous C57BL/6J eNOS(+/-) inbred males carrying the dominant Akita diabetogenic mutation Ins2(C96Y/+). Whereas all C57BL/6J inbred eNOS(-/-) and eNOS(+/-) diabetic mice died before 5 mo, almost half of the F1 hybrid eNOS(-/-) and eNOS(+/-) diabetic mice lived until killed at 7 mo. Heterozygous eNOS(+/-) diabetic mice expressed â¼35% eNOS mRNA in the kidney and â¼25% glomerular eNOS protein relative to their eNOS(+/+) diabetic littermates. These decreases in eNOS elevated blood pressure (BP) but not blood glucose. Urinary albumin excretion, mesangial expansion, glomerulosclerosis, mesangiolysis, and glomerular filtration rate increased in the order: eNOS(+/+) Akita < eNOS(+/-) Akita < eNOS(-/-) Akita, independently of BP. Glomerular basement membrane thickening depended on increased BP. Renal expression of tissue factor and other inflammatory factors increased with the nephropathy; Nos2 also increased. Surprisingly, however, decreased eNOS expression ameliorated the increases in oxidative stress and tubulointerstitial fibrosis caused by diabetes. Our data demonstrate that a modest decrease in eNOS, comparable to that associated with human NOS3 variants, is sufficient to enhance diabetic nephropathy independently of its effects on BP.
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Nefropatías Diabéticas/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Presión Sanguínea , Nefropatías Diabéticas/fisiopatología , Femenino , Fibrina/metabolismo , Humanos , Riñón/enzimología , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Tromboplastina/metabolismoRESUMEN
The renin angiotensin system (RAS) is important for fluid and blood pressure regulation. Recent studies suggest that an overactive RAS is involved in the metabolic syndrome. This article discusses recent advances on how genetic alteration of the RAS affects cardiovascular and metabolic phenotypes, with a special emphasis on the potential role of angiotensin-independent effects of renin.
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An overactive renin-angiotensin system is associated with obesity and the metabolic syndrome. However, the mechanisms behind it are unclear. Cleaving angiotensinogen to angiotensin I by renin is a rate-limiting step of angiotensin II production, but renin is suggested to have angiotensin-independent effects. We generated mice lacking renin (Ren1c) using embryonic stem cells from C57BL/6 mice, a strain prone to diet-induced obesity. Ren1c(-/-) mice are lean, insulin sensitive, and resistant to diet-induced obesity without changes in food intake and physical activity. The lean phenotype is likely due to a higher metabolic rate and gastrointestinal loss of dietary fat. Most of the metabolic changes in Ren1c(-/-) mice were reversed by angiotensin II administration. These results support a role for angiotensin II in the pathogenesis of diet-induced obesity and insulin resistance.
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Grasas de la Dieta/metabolismo , Metabolismo Energético , Obesidad/prevención & control , Renina/deficiencia , Tejido Adiposo/metabolismo , Angiotensina II/deficiencia , Angiotensina II/farmacología , Animales , Metabolismo Basal , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Grasas de la Dieta/administración & dosificación , Insulina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Obesidad/metabolismo , Fenotipo , Renina/genética , Delgadez/genética , Delgadez/metabolismoRESUMEN
Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. Recent developments indicate that manipulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals. In the present study, a hybrid gene containing bovine alpha-lactalbumin and human FVIII cDNA was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. The alphaLA-hFVIII hybrid gene was confirmed to be successfully integrated and stably germ-line transmitted in 12 (seven females/five males) lines. Western-blot analysis of milk samples obtained from eight of the transgenic founders and F1 offspring indicated that the recombinant hFVIII was secreted into the milk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2 microg/ml, over 35-200-fold higher than that in normal human plasma. Up to 13.4 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.