bluuebunny/crossref_metadata_2025 huggingface dataset:
Inheritance of Myxobolus cerebralis Resistance among F1-Generation Crosses of Whirling Disease Resistant and Susceptible Rainbow Trout Strains
Authors: Schisler George J. , Myklebust Karin A. , Hedrick Ronald P.
Published: May 2006

A whirling disease resistant domestic strain of rainbow trout Oncorhynchus mykiss (German (GR)) was crossed with a susceptible wild strain of rainbow trout (Colorado River (CR)). Thirty-two families of F1 crosses, along with five pure GR and two pure CR rainbow trout families, were then evaluated for resistance to whirling disease after experimental exposure to Myxobolus cerebralis. The pure domesticated GR strain was verified to have strong resistance to the parasite. In contrast, wild CR rainbow trout were highly susceptible. Crosses of these two strains resulted in offspring with a range of susceptibility. The resistance to whirling disease in some families was similar to that of pure GR rainbow trout, while other families were as susceptible as pure CR rainbow trout. Infection severity was significantly greater in the CR strain than in the pure GR and the GR × CR strains, as measured by both microscopic pathological scores (histology) and myxospore counts. Infection severity in the reciprocal crosses (CR female × GR male) was not significantly different from that of the pure CR rainbow trout as measured by histology but was significantly different with respect to lower myxospore counts. Future studies will examine the stability of inheritance of whirling disease resistance and the potential use of selective breeding to control whirling disease in free-ranging rainbow trout populations.
Differential expression of cyclin-dependent kinase inhibitors, p27Kip1 and p57Kip2, by corticotropin in rat adrenal cortex
Authors: Kobayashi Hironobu , Kambe Fukushi , Imai Tsuneo , Hibi Yatsuka , Kikumori Toyone , Ohmori Sachiko , Nakao Akimasa , Seo Hisao
Published: May 2006

An important role for the cyclin-dependent kinase inhibitors (CDKIs), p27Kip1 and p57Kip2, in the proliferation and differentiation of adrenal cells has been suggested by their knockout mice, which display adrenal hyperplasia. Adrenal development and function are primarily regulated by ACTH. In the present study, we investigated the effects of ACTH on the expression of p27Kip1, p57Kip2 and proliferating cell nuclear antigen (PCNA) in rat adrenals. Male Wistar rats were treated with dexametha-sone (Dex) to inhibit endogenous ACTH secretion. ACTH was then administered to the rats, and the adrenals were examined by Western blot and immunohisto-chemical analyses. Dex treatment induced shrinkage of adrenals where no PCNA-expressing cells were detected, but most of the cells expressed p27Kip1. Subsequent ACTH treatment resulted in the marked suppression of p27Kip1 expression, specifically in adrenocortical cells at 12 h after the stimulus. At 48 h, the p27Kip1 suppression still continued in the cortex, while the PCNA-expressing cells appeared mainly around the zona glomerulosa and increased at 72 h. At this time, the p27Kip1-expressing cells also appeared in the same zone. In contrast to p27Kip1, the expression of p57Kip2 was not detected in the Dex-treated adrenal. However, its expression was markedly induced by ACTH in the zona glomerulosa at 48 and 72 h. The results demonstrate that the primary site for mitogenic action of ACTH in rat adrenocortex is the zona glomerulosa, and that ACTH modulates proliferation of adrenocortical cells by regulating p27Kip1 and p57Kip2 expression in a time- and site-specific manner.
The expression of atrial natriuretic peptide in the oviduct and its functions in pig spermatozoa
Authors: Zhang Meijia , Hong Haiyan , Zhou Bo , Jin Shiying , Wang Chao , Fu Maoyong , Wang Songbo , Xia Guoliang
Published: May 2006

Locally synthesized atrial natriuretic peptide (ANP) and its receptors have been found in reproductive tissues of various mammals, and play an important role in the acrosome reaction of human sperm. The objective of the present study was to examine the expression of ANP and its receptors in pig spermatozoa and oviduct, and the effect of ANP on pig spermatozoa function. The expression of ANP and its receptors was analyzed by RT-PCR. Only natriuretic peptide receptors-A (NPRA) mRNA was detected in fresh sperm. While the levels of natriuretic peptide receptors-C (NPRC) mRNA were low with no obvious change among different oviductal phases, the levels of ANP mRNA were high in oviduct(OT)1 , OT3 and OT5, but were very low in OT2. On the other hand, the levels of NPRA mRNA were low in OT1 and OT2, increased in OT3 and reached a maximum in OT4 and OT5. Western blot analysis revealed that the level of ANP was high in OT1, decreased in OT2 and OT3, and arrived at the nadir in OT4 and OT5. The effect of ANP on spermatozoa function was studied by the acrosome reaction and IVF. Incubation with ANP for 1 h significantly induced acrosome reaction of preincubated spermatozoa, and maximal response of acrosome reaction (34.1 ± 2.3%) was achieved at 1 nM ANP treatment. Both C-ANP-(4–23), a selective ligand of NPRC, and caffeine had no effect on the acrosome reaction. The stimulatory effect of ANP on acrosome reaction could be mimicked by the permeable cGMP analog, 8-Br-cGMP. ANP and caffeine had a similar effect on improving the oocytes penetration rate, polyspermy rate and the average number of sperm per penetrated oocyte. Also, ANP treatment had a similar effect on cleavage rate, blastocyst formation rate and the number of cells per blastocyst as that of caffeine treatment. The effects of ANP on the acrosome reaction and the parameters of oocyte penetration could be blocked by cGMP-dependent protein kinase (PKG) inhibitors KT5823 and/or Rp-8-pCPT-cGMPS. These results suggest that the expression of ANP in the oviduct may be involved in the regulation of the acrosome reaction and the fertilising ability of pig spermatozoa, and the PKG pathway possibly participates in the process.
Transcriptional regulation of major histocompatibility complex class I gene by insulin and IGF-I in FRTL-5 thyroid cells
Authors: Giuliani C , Saji M , Bucci I , Fiore G , Liberatore M , Singer D S , Monaco F , Kohn L D , Napolitano G
Published: May 2006

Increased major histocompatibility complex (MHC) class I gene expression in nonimmune cell ‘target tissues’ involved in organ-specific diseases may be important in the pathogenesis of autoimmune diseases. This possibility in part evolves from studies of cultured thyrocytes where properties appear relevant to the development of thyroid autoimmune disease. In FRTL-5 rat thyroid cells in continuous culture, hormones and growth factors that regulate cell growth and function specifically decrease MHC class I gene expression. We hypothesized that this could reflect a mechanism to preserve self-tolerance and prevent autoimmune disease. The mechanisms of action of some of these hormones, namely TSH and hydrocortisone, have been already characterized. In this report, we show that IGF-I transcriptionally downregulates MHC class I gene expression and that its action is similar to that of insulin. The two hormones have a complex effect on the promoter of the MHC class I gene, PD1. In fact, they decrease the full promoter activity, but upregulate the activity of deleted mutants that have lost an upstream, tissue-specific regulatory region but still retain the enhancer A region. We show that insulin/IGF-I promotes the interactions of the p50/p65 subunits of NF-κB and AP-1 family members with these two regions, and that the tissue-specific region acts as a dominant silencer element on insulin/IGF-I regulation of promoter activity. These observations may be important to understand how MHC class I gene transcription is regulated in the cells.
Monocarboxylate transporter 8 expression in the human placenta: the effects of severe intrauterine growth restriction
Authors: Chan S-Y , Franklyn J A , Pemberton H N , Bulmer J N , Visser T J , McCabe C J , Kilby M D
Published: May 2006

Thyroid hormones (THs) are essential for normal fetal development, with even mild perturbation in maternal thyroid status in early pregnancy being associated with neurodevelopmental delay in children. Transplacental transfer of maternal THs is critical, with increasing evidence suggesting a role for 3,3′,5-tri-iodothyronine (T3) in development and function of the placenta itself, as well as in development of the central nervous and other organ systems. Intrauterine growth restriction (IUGR) is associated with fetal hypothyroxinaemia, a factor that may contribute to neurodevelopmental delay. The recent description of monocarboxylate transporter 8 (MCT8) as a powerful and specific TH membrane transporter, and the association of MCT8 mutations with profound neurodevelopmental delay, led us to explore MCT8 expression in placenta. We describe the expression of MCT8 in normal human placenta throughout gestation, and in normal third-trimester placenta compared with that associated with IUGR using quantitative reverse transcriptase PCR. MCT8 mRNA was detected in placenta from early first trimester, with a significant increase with advancing gestation (P=0.007). In the early third trimester, MCT8 mRNA was increased in IUGR placenta compared with normal samples matched for gestational age (P<0.05), but there was no difference between IUGR and normal placenta in the late third trimester. Western immunoblotting findings in IUGR and normal placentae were in accord with mRNA data. MCT8 immunostaining was demonstrated in villous cytotrophoblast and syncytiotrophoblast as well as extravillous trophoblast cells from the first trimester onwards with increasingly widespread immunoreactivity seen with advancing gestation. In conclusion, expression of MCT8 in placenta from early gestation is compatible with an important role in TH transport during fetal development and a specific role in placental development. Altered expression in placenta associated with IUGR may reflect a compensatory mechanism attempting to increase T3 uptake by trophoblast cells.
Adenoviral vectors encoding tumor necrosis factor-α and FasL induce apoptosis of normal and tumoral anterior pituitary cells
Authors: Candolfi M , Jaita G , Pisera D , Ferrari L , Barcia C , Liu C , Yu J , Liu G , Castro M G , Seilicovich A
Published: May 2006

Our previous work showed that tumor necrosis factor (TNF)-α and FasL induce apoptosis of anterior pituitary cells. To further analyze the effect of these proapoptotic factors, we infected primary cultures from rat anterior pituitary, GH3 and AtT20 cells with first-generation adenoviral vectors encoding TNF-α, FasL or, as a control, β-galactosidase (β-Gal), under the control of the human cytomegalovirus promoter. Successful expression of the encoded transgenes was determined by immunocytochemistry. Although we observed basal expression of TNF-α and FasL in control cultures of anterior pituitary cells, fluorescence-activated cell sorting (FACS) cell cycle analysis showed that the overexpression of TNF-α or FasL increases the percentage of hypodiploid lactotropes and somatotropes. Nuclear morphology and TUNEL staining revealed that the cells undergo an apoptotic death process. We detected strong immunoreactivity for TNFR1 and Fas in the somatolactotrope cell line GH3. TNF-α, but not FasL, was expressed in control cultures of GH3 cells. The infection of GH3 cells with adenovirus encoding TNF-α or FasL increased the percentages of hypodiploid and TUNEL-positive cells. TNF-α or FasL immunoreactivity was not observed in the corticotrope cell line AtT20. However, adenovirus encoding TNF-α or FasL efficiently transduced these cells and increased the percentages of hypodiploid and TUNEL-positive cells. The expression of β-Gal was detected in all these cultures but did not affect cell viability. In conclusion, these results suggest that death signaling cascades triggered by TNF receptor 1 (TNFR1) and Fas are present in both normal and tumoral pituitary cells. Therefore, overexpression of proapoptotic factors could be a useful tool in the therapy of pituitary adenomas.
Impairment of testicular function in adult male Japanese quail (Coturnix japonica) after a single administration of 3-methyl-4-nitrophenol in diesel exhaust particles
Authors: Li Chun-Mei , Takahashi Shinji , Taneda Shinji , Furuta Chie , Watanabe Gen , Suzuki Akira K , Taya Kazuyoshi
Published: May 2006

The effects of 3-methyl-4-nitrophenol (PNMC), a component of diesel exhaust, on reproductive function were investigated in adult male Japanese quail. The quail were treated with a single i.m. dose of PNMC (78, 103 or 135 mg/kg body weight), and trunk blood and testes were collected 1, 2 or 4 weeks later. Various levels of testicular atrophy were observed in all groups treated with PNMC. Sperm formation, cloacal gland area, and plasma LH and testosterone concentrations were also reduced in birds with testicular atrophy. To determine the acute effect of PNMC on gonadotrophin from the pituitary, adult male quail were administrated a single i.m. injection of PNMC (25 mg/kg), and plasma concentrations of LH were measured at 1, 3 and 6 h. This dose significantly lowered plasma levels of LH at all three time points. These results suggest that PNMC acts on the hypothalamus–pituitary axis, by reducing circulating LH within a few hours of administration and subsequently reducing testosterone secretion. In addition, in order to investigate the direct effects of PNMC on the secretion of testosterone from testicular cells in quail testes, cultured interstitial cells containing Leydig cells were exposed to PNMC (10−6, 10−5 or 10−4 M) for 4, 8 or 24 h. These quantities of PNMC significantly reduced the secretion of testosterone in a time- and dose-dependent manner. The present findings also suggest a direct effect of PNMC on the testis to reduce testosterone secretion. This study clearly indicates that PNMC induces reproductive toxicity at both the central and testicular levels, and disrupts testicular function in adult male quail.
Sex-steroid milieu determines diabetes rescue in pttg-null mice
Authors: Fraenkel M , Caloyeras J , Ren S-G , Melmed S
Published: May 2006

Male mice that are pttg-null develop sexually dimorphic diabetes with hypoinsulinemia secondary to reduced post-natal -cell proliferation and an inability to expand islet cell mass with aging. We therefore examined the effects of sex-steroid manipulation on diabetes development in pttg−/− male mice. Surgical gonadectomy was followed by implantation of 90-day slow-release pellets releasing 17β-estradiol (0.36 mg/pellet), placebo or dihydrotestosterone (DHT; 12.5 mg/pellet). Mean fasting blood sugars at the end of the study were 414 ± 54 mg/dl for pttg−/− controls and 371 ± 14 mg/dl for pttg−/− mice gonad-ectomized and treated with DHT compared with 124 ± 40 and 85 ± 12 mg/dl in gonadectomized pttg−/− males treated with placebo or estradiol, respectively (P < 0.01 compared with control pttg−/−). Gonadectomy with and without estradiol treatment did not increase the very low circulating insulin levels in pttg-null males (fasting insulin 0.44 ± 0.04 ng/ml in pttg−/− controls, 0.47 ± 0.07 and 0.4 ng/ml in pttg−/− gonadectomized males treated with placebo or estradiol, respectively). Gonadectomy increased serum adiponectin levels (4.9 ± 008 μg/ml in pttg−/− controls versus 13 ± 0.08 and 7.5 ± 0.6 μg/ml in pttg−/− gonadectomized males treated with placebo or estradiol, respectively; P < 0.001 and P < 0.05), accompanied by increased insulin sensitivity. The results show that gonadectomy delayed, and gonadectomy with additional estradiol treatment prevented, diabetes development in pttg−/− males, possibly through increased insulin sensitivity mediated by elevated serum adiponectin levels. Male-selective effects of disrupted β-cell proliferation in the absence of pttg are restored by sex-steroid effects on peripheral insulin sensitivity.
Fishing for gene function – endocrine modelling in the zebrafish
Authors: McGonnell I M , Fowkes R C
Published: May 2006

The use of zebrafish (Danio rerio) in scientific research is growing rapidly. It initially became popular as a model of vertebrate development because zebrafish embryos develop rapidly and are transparent. In the past 5 years, the sequencing of the zebrafish genome has increased the profile of zebrafish research even further, expanding into other areas such as pharmacology, cancer research and drug discovery. The use of zebrafish in endocrine research has mainly been confined to the study of the development of endocrine organs. However, it is likely to be a useful model in other areas of endocrinology, as there are a wide variety of both forward and reverse genetic techniques that can be employed in the zebrafish to decipher gene function in disease states. In this review, we compare the endocrine system of the zebrafish to mouse and human, demonstrating that the systems are sufficiently similar for zebrafish to be employed as a model for endocrine research. We subsequently review the repertoire of genetic techniques commonly employed in the zebrafish model to understand gene function in vertebrate development and disease. We anticipate that the use of these techniques will make the zebrafish a prominent model in endocrine research in the coming years.
Changes in mRNAs encoding steroidogenic acute regulatory protein, steroidogenic enzymes and receptors for gonadotropins during spermatogenesis in rainbow trout testes
Authors: Kusakabe M , Nakamura I , Evans J , Swanson P , Young G
Published: May 2006

In vertebrates, sperm development and maturation are directly regulated by gonadal steroid hormone secretion. The relationships among the expression of genes encoding steroidogenic proteins and receptors for gonadotropins, and testicular steroid production have not yet been comprehensively determined in male teleosts. In this study, the changes in levels of mRNAs encoding follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage, 3β-hydroxysteroid dehydrogenase/Δ5–4-isomerase, cytochrome P450 17α-hydroxylase/17,20-lyase, cytochrome P450 11β-hydroxylase, 11β-hydroxysteroid dehydrogenase and 20β-hydroxysteroid dehydrogenase were determined by real-time, quantitative PCR assays and related to changes in serum steroid levels throughout the reproductive cycle in male rainbow trout. Serum 11-ketotestosterone and 17α,20β-dihydroxy-4-pregnen-3-one levels were measured by RIA. Although the pattern of change in the mRNA levels for the enzymes was variable, the increases in steroidogenic enzyme mRNAs started prior to a significant increase of serum steroid levels. The patterns of transcript levels of FSH and LH receptors suggest that changes in StAR and steroidogenic enzyme transcripts are largely mediated by the FSH receptor during early and mid-spermatogenesis and by the LH receptor during late spermatogenesis and spermiation. Levels of StAR (10-fold) and P450 17α-hydroxylase/17,20-lyase (sevenfold) transcripts changed with the greatest magnitude and were closely related to the changes in serum steroids, suggesting that changes in StAR and P450 17α-hydroxylase/17,20-lyase abundance are likely to be the major influences on overall steroidogenic output during the reproductive cycle in male rainbow trout.
Acute exposure of adult male rats to dietary phytoestrogens reduces fecundity and alters epididymal steroid hormone receptor expression
Authors: Glover A , Assinder S J
Published: May 2006

Phytoestrogens are plant-derived compounds with oestrogenic activity. They are common in both human and animal diets, particularly through soy-based foods. This study assessed whether exposure of adult male rats to a high phytoestrogen diet for 3–25 days affected their fertility, and assessed possible mechanisms through which phytoestrogens may disrupt fertility. Adult males, fed a high phytoestrogen diet for 3 days, demonstrated significantly reduced fecundity. This effect was transient, with fecundity returning to control levels by day 12. The expression of oestrogen receptor-α and androgen receptor mRNA was increased in the initial segment of the epididymis, but decreased in the cauda epididymis following 3 days on the high phytoestrogen diet. Epididymal sperm counts cannot account for the reduction in fertility at day 3. However, lipid peroxidation of epididymal sperm was significantly increased in animals fed a high phytoestrogen diet for 3 days. Disruption of the steroid regulation of the epididymis by phytoestrogens may alter its function, resulting in decreased quality of sperm, and thereby reducing fecundity.
Obstetric and neonatal outcomes to recombinant porcine somatotropin administered in the last third of pregnancy to primiparous sows
Authors: Trujillo-Ortega María E , Mota-Rojas Daniel , Hernández-González Rafael , Velázquez-Armenta Elvia Yadira , Nava-Ocampo Alejandro A , Ramírez-Necoechea Ramiro , Becerril-Herrera Marcelino , Alonso-Spilsbury María
Published: May 2006

This study aimed to investigate whether the administration of recombinant porcine somatotropin (rpST) late in gestation is associated with increased rates of obstetric and neonatal complications in primiparous sows. From days 80 to 114 of gestational age, 20 primiparous sows were randomly assigned to receive an intramuscular injection of either saline or 6 mg rpST/day. Throughout pregnancy, sows were fed 2.5 to 3 kg/day of a corn-soybean diet (14 MJ ME/kg). Of 111 piglets born to control sows and 109 piglets born to treated sows, 8.1% and 17.4% piglets respectively died intrapartum (P=0.04). Glucose blood levels in sows and live-born piglets in the rpST-treated group were significantly higher than in their corresponding controls. Birth weight of live-born piglets in the treated group was 1.4 ± 0.1 kg versus 1.3 ± 0.1 kg in the control group (P<0.0001). Birth weight of piglets born dead was also higher in the former than in the latter group (P<0.0001). No evidence of teratogenicity was observed in either of the groups. In conclusion, rpST administration in late pregnancy to primparous sows increased the rate of neonatal deaths and was associated with higher blood glucose levels in both sows and piglets.
Androgen-replacement therapy depresses the ex vivo production of inflammatory cytokines by circulating antigen-presenting cells in aging type-2 diabetic men with partial androgen deficiency
Authors: Corrales J J , Almeida M , Burgo R , Mories M T , Miralles J M , Orfao A
Published: May 2006

Androgens are considered to have immunomodulatory effects but their cellular mechanisms of action remain largely unknown. In the present study we prospectively analyzed the serial effects of androgen-replacement therapy on both the distribution of peripheral blood lymphocytes, monocytes and dendritic cells as well as on the production of interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα) inflammatory cytokines by circulating monocytes and CD33 myeloid, CD16 and plasmacytoid dendritic cell subsets, the most potent antigen-presenting cells (APCs) in type-2 diabetic men with partial androgen deficiency. Analyses were performed before therapy and at 1, 3, 6 and 12 months after treatment with 150 mg testosterone enanthate every 2 weeks in a group of 13 type-2 diabetic men. Our results show for the first time that testosterone-replacement therapy is associated with a reduction or complete abrogation of spontaneous ex vivo production of IL-1β, IL-6 and TNFα by APCs. Meanwhile, the in vitro production of inflammatory cytokines by these cells after stimulation with lipopolysaccharide plus recombinant human interferon-γ remained unchanged, suggesting that APCs preserve their constitutive machinery to produce inflammatory cytokines under androgen treatment. These results confirm and extend previous observations about the anti-inflammatory effects of androgen therapy on APCs in a new, previously unexplored model of androgen deficiency; namely, aging type-2 diabetic men. A decreased production of inflammatory cytokines by APCs might have important consequences for sex differences in susceptibility to autoimmune diseases, inflammatory response to injury and atheromatosis.
Apelin-13 induces a biphasic haemodynamic response and hormonal activation in normal conscious sheep
Authors: Charles Christopher J , Rademaker Miriam T , Richards A Mark
Published: May 2006

Whilst the tissue distribution and range of biological actions reported for apelin suggest a role for the peptide in pressure/volume homeostasis, conflicting reports make the precise role unclear. Furthermore, few integrated studies have been performed and there are no reports of bioactivity of apelin in a large animal model. Accordingly, we have examined the haemodynamic, hormonal and renal effects of apelin in ten normal conscious sheep. Apelin (1 mg i.v. bolus) induced a biphasic haemo-dynamic response characterised by an acute fall in arterial pressure and a rise in heart rate followed immediately by a rise in arterial pressure and a fall in heart rate. The secondary hypertensive phase was associated with a fall in cardiac output (P=0.015) and significant rises in calculated total peripheral resistance (CTPR) (P<0.001) and right atrial pressure (RAP) (P=0.031). Electrocardiogram changes were also observed in four of ten sheep, most notably varying degrees of atrioventricular block. Apelin also induced significant rises in plasma arginine vasopressin (P=0.009), adrenocorticotrophin (P=0.012), aldosterone (P=0.001), cortisol (P=0.014), atrial (P=0.036) and brain (P<0.001) natriuretic peptide, cyclic GMP (P=0.003) and cyclic AMP (P=0.002) levels with no effect on renal indices. In conclusion, high dose administration of apelin to normal conscious sheep induces a significant biphasic response in arterial pressure and heart rate associated with rises in RAP and CTPR and a fall in cardiac output. Apelin also increases circulating levels of a number of vasoactive hormones. Taken together, these results suggest a potential role for apelin in pressure/volume homeostasis.
Evidence for a Mitochondrial Regulatory Pathway Defined by Peroxisome Proliferator–Activated Receptor-γ Coactivator-1α, Estrogen-Related Receptor-α, and Mitofusin 2
Authors: Soriano Francesc X. , Liesa Marc , Bach Daniel , Chan David C. , Palacín Manuel , Zorzano Antonio
Published: May 2006

Mitofusin 2 (Mfn2) is a mitochondrial membrane protein that participates in mitochondrial fusion and regulates mitochondrial metabolism in mammalian cells. Here, we show that Mfn2 gene expression is induced in skeletal muscle and brown adipose tissue by conditions associated with enhanced energy expenditure, such as cold exposure or β3-adrenergic agonist treatment. In keeping with the role of peroxisome proliferator–activated receptor-γ coactivator (PGC)-1α on energy expenditure, we demonstrate a stimulatory effect of PGC-1α on Mfn2 mRNA and protein expression in muscle cells. PGC-1α also stimulated the activity of the Mfn2 promoter, which required the integrity of estrogen-related receptor-α (ERRα)-binding elements located at −413/−398. ERRα also activated the transcriptional activity of the Mfn2 promoter, and the effects were synergic with those of PGC-1α. Mfn2 loss of function reduced the stimulatory effect of PGC-1α on mitochondrial membrane potential. Exposure to cold substantially increased Mfn2 gene expression in skeletal muscle from heterozygous Mfn2 knock-out mice, which occurred in the presence of higher levels of PGC-1α mRNA compared with control mice. Our results indicate the existence of a regulatory pathway involving PGC-1α, ERRα, and Mfn2. Alterations in this regulatory pathway may participate in the pathophysiology of insulin-resistant conditions and type 2 diabetes.
Gene Transfer of an Engineered Transcription Factor Promoting Expression of VEGF-A Protects Against Experimental Diabetic Neuropathy
Authors: Price Sally A. , Dent Carolyn , Duran-Jimenez Beatriz , Liang Yuxin , Zhang Lei , Rebar Edward J. , Case Casey C. , Gregory Philip D. , Martin Tyler J. , Spratt S. Kaye , Tomlinson David R.
Published: May 2006

Peripheral neuropathy is a common, irreversible complication of diabetes. We investigated whether gene transfer of an engineered zinc finger protein transcription factor (ZFP-TF) designed to upregulate expression of the endogenous vascular endothelial growth factor (VEGF)-A gene could protect against experimental diabetic neuropathy. ZFP-TF–driven activation of the endogenous gene results in expression of all of the VEGF-A isoforms, a fact that may be of significance for recapitulation of the proper biological responses stimulated by this potent neuroprotective growth factor. We show here that this engineered ZFP-TF activates VEGF-A in appropriate cells in culture and that the secreted VEGF-A protein induced by the ZFP protects neuroblastoma cell lines from a serum starvation insult in vitro. Importantly, single and repeat intramuscular injections of formulated plasmid DNA encoding the VEGF-A–activating ZFP-TF resulted in protection of both sensory and motor nerve conduction velocities in a streptozotocin-induced rat model of diabetes. These data suggest that VEGF-A–activating ZFP-TFs may ultimately be of clinical utility in the treatment of this disease.
Thiazolidinediones Ameliorate Diabetic Nephropathy via Cell Cycle–Dependent Mechanisms
Authors: Okada Tatsuo , Wada Jun , Hida Kazuyuki , Eguchi Jun , Hashimoto Izumi , Baba Masako , Yasuhara Akihiro , Shikata Kenichi , Makino Hirofumi
Published: May 2006

Thiazolidinediones are ligands for peroxisome proliferator–activated receptor (PPAR)-γ, widely used as insulin sensitizer in type 2 diabetic patients and implicated in apoptosis, cell proliferation, and cell cycle regulation. Here, the effect of thiazolidinediones on G1-phase cell cycle arrest, the hallmark in diabetic nephropathy, was investigated. Eight-week-old male Otsuka Long-Evans Tokushima fatty rats were treated with pioglitazone (1 mg · kg body wt−1 · day−1) until 50 weeks of age and compared with insulin treatment. Although similar HbA1c levels were observed in both groups, pioglitazone significantly inhibited glomerular hypertrophy and mesangial matrix expansion and reduced urinary albumin excretion compared with the insulin-treated group. In addition, pioglitazone significantly reduced the number of glomerular p27Kip1-positive cells. Because prominent expression of PPAR-γ was observed in podocytes in glomeruli and cultured cells, conditionally immortalized mouse podocyte cells were cultured under 5.5 and 25 mmol/l d-glucose supplemented with pioglitazone. Pioglitazone inhibited cell hypertrophy revealed by [3H]thymidine and [3H]proline incorporation, and pioglitazone reversed high glucose–induced G1-phase cell cycle arrest, i.e., an increase in G0/G1 phase and decrease in S and G2 phases. Pioglitazone suppressed high glucose–induced phosphorylation of p44/42 mitogen-activated protein kinase and reduced Bcl-2 and p27Kip1 protein levels. Besides glucose-lowering action, pioglitazone ameliorates diabetic nephropathy via cell cycle–dependent mechanisms.
Exercise-Induced Phosphorylation of the Novel Akt Substrates AS160 and Filamin A in Human Skeletal Muscle
Authors: Deshmukh Atul , Coffey Vernon G. , Zhong Zhihui , Chibalin Alexander V. , Hawley John A. , Zierath Juleen R.
Published: May 2006

Skeletal muscle contraction stimulates multiple signaling cascades that govern a variety of metabolic and transcriptional events. Akt/protein kinase B regulates metabolism and growth/muscle hypertrophy, but contraction effects on this target and its substrates are varied and may depend on the mode of the contractile stimulus. Accordingly, we determined the effects of endurance or resistance exercise on phosphorylation of Akt and downstream substrates in six trained cyclists who performed a single bout of endurance or resistance exercise separated by ∼7 days. Muscle biopsies were taken from the vastus lateralis at rest and immediately after exercise. Akt Ser473 phosphorylation was increased (1.8-fold; P = 0.011) after endurance but was unchanged after resistance exercise. Conversely, Akt Thr308 phosphorylation was unaltered after either bout of exercise. Several exercise-responsive phosphoproteins were detected by immunoblot analysis with a phospho-Akt substrate antibody. pp160 and pp300 were identified as AS160 and filamin A, respectively, with increased phosphorylation (2.0- and 4.9-fold, respectively; P < 0.05) after endurance but not resistance exercise. In conclusion, AS160 and filamin A may provide an important link to mediate endurance exercise–induced bioeffects in skeletal muscle.
Downregulation of Electron Transport Chain Genes in Visceral Adipose Tissue in Type 2 Diabetes Independent of Obesity and Possibly Involving Tumor Necrosis Factor-α
Authors: Dahlman Ingrid , Forsgren Margaretha , Sjögren Annelie , Nordström Elisabet Arvidsson , Kaaman Maria , Näslund Erik , Attersand Anneli , Arner Peter
Published: May 2006

Impaired oxidative phosphorylation is suggested as a factor behind insulin resistance of skeletal muscle in type 2 diabetes. The role of oxidative phosphorylation in adipose tissue was elucidated from results of Affymetrix gene profiling in subcutaneous and visceral adipose tissue of eight nonobese healthy, eight obese healthy, and eight obese type 2 diabetic women. Downregulation of several genes in the electron transport chain was the most prominent finding in visceral fat of type 2 diabetic women independent of obesity, but the gene pattern was distinct from that previously reported in skeletal muscle in type 2 diabetes. A similar but much weaker effect was observed in subcutaneous fat. Tumor necrosis factor-α (TNF-α) is a major factor behind inflammation and insulin resistance in adipose tissue. TNF-α treatment decreased mRNA expression of electron transport chain genes and also inhibited fatty acid oxidation when differentiated human preadipocytes were treated with the cytokine for 48 h. Thus, type 2 diabetes is associated with a tissue- and region-specific downregulation of oxidative phosphorylation genes that is independent of obesity and at least in part mediated by TNF-α, suggesting that impaired oxidative phosphorylation of visceral adipose tissue has pathogenic importance for development of type 2 diabetes.
Diabetic Nephropathy Is Associated With Gene Expression Levels of Oxidative Phosphorylation and Related Pathways
Authors: Huang Chunmei , Kim Youngki , Caramori M. Luiza , Moore Jason H. , Rich Stephen S. , Mychaleckyj Josyf C. , Walker Paul C. , Mauer Michael
Published: May 2006

The in vitro behavior of skin fibroblasts from patients with or without diabetic nephropathy is associated with diabetic nephropathy risk. Here we compared skin fibroblast gene expression profiles from two groups of type 1 diabetic patients: 20 with very fast (“fast-track”) versus 20 with very slow (“slow-track”) rates of development of diabetic nephropathy lesions. Gene expression profiles of skin fibroblasts grown in 25 mmol/l glucose for 36 h were assessed by Affymetrix HG-U133A GeneChips to determine the proportion of genes in a given biological pathway that were directionally consistent in their group differences. Five pathways reached statistical significance. All had significantly greater proportions of genes with higher expression levels in the fast-track group. These pathways, the first four of which are closely related and have overlapping genes, included oxidative phosphorylation (P < 0.001), electron transport system complex III (P = 0.017), citrate cycle (P = 0.037), propanoate metabolism (P = 0.044), and transcription factors (P = 0.046). These results support the concept that oxidative phosphorylation and related upstream pathways may be important in the pathogenesis of diabetic nephropathy. Whether these findings reflect inherent genetic cellular characteristics, “cell memory,” or both requires further study.
Inhibition of Fructose 1,6-Bisphosphatase Reduces Excessive Endogenous Glucose Production and Attenuates Hyperglycemia in Zucker Diabetic Fatty Rats
Authors: van Poelje Paul D. , Potter Scott C. , Chandramouli Visvanathan C. , Landau Bernard R. , Dang Qun , Erion Mark D.
Published: May 2006

Gluconeogenesis is increased in type 2 diabetes and contributes significantly to fasting and postprandial hyperglycemia. We recently reported the discovery of the first potent and selective inhibitors of fructose 1,6-bisphosphatase (FBPase), a rate-controlling enzyme of gluconeogenesis. Herein we describe acute and chronic effects of the lead inhibitor, MB06322 (CS-917), in rodent models of type 2 diabetes. In fasting male ZDF rats with overt diabetes, a single dose of MB06322 inhibited gluconeogenesis by 70% and overall endogenous glucose production by 46%, leading to a reduction in blood glucose of >200 mg/dl. Chronic treatment of freely feeding 6-week-old male Zucker diabetic fatty (ZDF) rats delayed the development of hyperglycemia and preserved pancreatic function. Elevation of lactate (∼1.5-fold) occurred after 4 weeks of treatment, as did the apparent shunting of precursors into triglycerides. Profound glucose lowering (∼44%) and similar metabolic ramifications were associated with 2-week intervention therapy of 10-week-old male ZDF rats. In high-fat diet–fed female ZDF rats, MB06322 treatment for 2 weeks fully attenuated hyperglycemia without evidence of metabolic perturbation other than a modest reduction in glycogen stores (∼20%). The studies confirm that excessive gluconeogenesis plays an integral role in the pathophysiology of type 2 diabetes and suggest that FBPase inhibitors may provide a future treatment option.
Deletion of p66Shc Longevity Gene Protects Against Experimental Diabetic Glomerulopathy by Preventing Diabetes-Induced Oxidative Stress
Authors: Menini Stefano , Amadio Lorena , Oddi Giovanna , Ricci Carlo , Pesce Carlo , Pugliese Francesco , Giorgio Marco , Migliaccio Enrica , Pelicci PierGiuseppe , Iacobini Carla , Pugliese Giuseppe
Published: May 2006

p66Shc regulates both steady-state and environmental stress-dependent reactive oxygen species (ROS) generation. Its deletion was shown to confer resistance to oxidative stress and protect mice from aging-associated vascular disease. This study was aimed at verifying the hypothesis that p66Shc deletion also protects from diabetic glomerulopathy by reducing oxidative stress. Streptozotocin-induced diabetic p66Shc knockout (KO) mice showed less marked changes in renal function and structure, as indicated by the significantly lower levels of proteinuria, albuminuria, glomerular sclerosis index, and glomerular and mesangial areas. Glomerular content of fibronectin and collagen IV was also lower in diabetic KO versus wild-type mice, whereas apoptosis was detected only in diabetic wild-type mice. Serum and renal tissue advanced glycation end products and plasma isoprostane 8-epi-prostaglandin F2α levels and activation of nuclear factor κB (NF-κB) were also lower in diabetic KO than in wild-type mice. Mesangial cells from KO mice grown under high-glucose conditions showed lower cell death rate, matrix production, ROS levels, and activation of NF-κB than those from wild-type mice. These data support a role for oxidative stress in the pathogenesis of diabetic glomerulopathy and indicate that p66Shc is involved in the molecular mechanism(s) underlying diabetes-induced oxidative stress and oxidant-dependent renal injury.
Linkage but Not Association of Calpain-10 to Type 2 Diabetes Replicated in Northern Sweden
Authors: Einarsdottir Elisabet , Mayans Sofia , Ruikka Karin , Escher Stefan A. , Lindgren Petter , Ågren Åsa , Eliasson Mats , Holmberg Dan
Published: May 2006

We present data from a genome-wide scan identifying genetic factors conferring susceptibility to type 2 diabetes. The linkage analysis was based on 59 families from northern Sweden, consisting of a total of 129 cases of type 2 diabetes and 19 individuals with impaired glucose tolerance. Model-free linkage analysis revealed a maximum multipoint logarithm of odds score of 3.19 for D2S2987 at 267.7 cM (P = 0.00058), suggesting that a gene conferring susceptibility to type 2 diabetes in the northern Swedish population resides in the 2q37 region. These data replicate, in a European population, previously identified linkage of marker loci in this region to type 2 diabetes in Mexican Americans. In contrast, no evidence in support of association to the previously identified single nucleotide polymorphisms in the calpain-10 gene was observed in a case-control cohort derived from the same population.
Reduced Access to Insulin-Sensitive Tissues in Dogs With Obesity Secondary to Increased Fat Intake
Authors: Ellmerer Martin , Hamilton-Wessler Marianthe , Kim Stella P. , Huecking Katrin , Kirkman Erlinda , Chiu Jenny , Richey Joyce , Bergman Richard N.
Published: May 2006

Physiological hyperinsulinemia provokes hemodynamic actions and augments access of macromolecules to insulin-sensitive tissues. We investigated whether induction of insulin resistance by a hypercaloric high-fat diet has an effect on the extracellular distribution of macromolecules to insulin-sensitive tissues. Male mongrel dogs were randomly selected into two groups: seven dogs were fed an isocaloric control diet (∼3,900 kcal, 35% from fat), and six dogs were fed a hypercaloric high-fat diet (∼5,300 kcal, 54% from fat) for a period of 12 weeks. During hyperinsulinemic-euglycemic clamps, we determined transport parameters and distribution volumes of [14C]inulin by applying a three-compartment model to the plasma clearance data of intravenously injected [14C]inulin (0.8 μCi/kg). In another study with direct cannulation of the hindlimb skeletal muscle lymphatics, we investigated the effect of physiological hyperinsulinemia on the appearance of intravenously injected [14C]inulin in skeletal muscle interstitial fluid and compared the effect of insulin between control and high-fat diet groups. The hypercaloric high-fat diet resulted in significant weight gain (18%; P < 0.001) associated with marked increases of subcutaneous (140%; P < 0.001) and omental (83%; P < 0.001) fat depots, as well as peripheral insulin resistance, measured as a significant reduction of insulin-stimulated glucose uptake during clamps (−35%; P < 0.05). Concomitantly, we observed a significant reduction of the peripheral distribution volume of [14C]inulin (−26%; P < 0.05), whereas the vascular distribution volume and transport and clearance parameters did not change as a cause of the diet. The second study directly confirmed our findings, suggesting a marked reduction of insulin action to stimulate access of macromolecules to insulin-sensitive tissues (control diet 32%, P < 0.01; high-fat diet 18%, NS). The present results indicate that access of macromolecules to insulin-sensitive tissues is impaired during diet-induced insulin resistance and suggest that the ability of insulin itself to stimulate tissue access is diminished. We speculate that the observed diet-induced defects in stimulation of tissue perfusion contribute to the development of peripheral insulin resistance.
Islet Inflammation and Fibrosis in a Spontaneous Model of Type 2 Diabetes, the GK Rat
Authors: Homo-Delarche Françoise , Calderari Sophie , Irminger Jean-Claude , Gangnerau Marie-Noëlle , Coulaud Josiane , Rickenbach Katharina , Dolz Manuel , Halban Philippe , Portha Bernard , Serradas Patricia
Published: May 2006

The molecular pathways leading to islet fibrosis in diabetes are unknown. Therefore, we studied gene expression in islets of 4-month-old Goto-Kakizaki (GK) and Wistar control rats. Of 71 genes found to be overexpressed in GK islets, 24% belong to extracellular matrix (ECM)/cell adhesion and 34% to inflammatory/immune response families. Based on gene data, we selected several antibodies to study fibrosis development during progression of hyperglycemia by immunohistochemistry. One-month-old GK and Wistar islets appeared to be similar. Two-month-old GK islets were strongly heterogenous in terms of ECM accumulation compared with Wistar islets. GK islet vascularization, labeled by von Willebrand factor, was altered after 1 month of mild hyperglycemia. Numerous macrophages (major histocompatibility complex class II+ and CD68+) and granulocytes were found in/around GK islets. These data demonstrate that marked inflammatory reaction accompanies GK islet fibrosis and suggest that islet alterations in this nonobese model of type 2 diabetes develop in a way reminiscent of microangiopathy.
Hyperglycemia Stimulates Coagulation, Whereas Hyperinsulinemia Impairs Fibrinolysis in Healthy Humans
Authors: Stegenga Michiel E. , van der Crabben Saskia N. , Levi Marcel , de Vos Alex F. , Tanck Michael W. , Sauerwein Hans P. , van der Poll Tom
Published: May 2006

Type 2 diabetes and insulin resistance syndromes are associated with an increased risk for cardiovascular and thrombotic complications. A disturbed balance between coagulation and fibrinolysis has been implicated in the pathogenesis hereof. To determine the selective effects of hyperglycemia and hyperinsulinemia on coagulation and fibrinolysis, six healthy humans were studied on four occasions for 6 h: 1) lower insulinemic-euglycemic clamp, 2) lower insulinemic-hyperglycemic clamp, 3) hyperinsulinemic-euglycemic clamp, and 4) hyperinsulinemic-hyperglycemic clamp. In the hyperglycemic clamps, target levels of plasma glucose were 12 versus 5 mmol/l in the normoglycemic clamps. In the hyperinsulinemic clamps, target plasma insulin levels were 400 versus 100 pmol/l in the lower insulinemic clamps. Hyperglycemia exerted a procoagulant effect irrespective of insulin levels, as reflected by mean twofold rises in thrombin-antithrombin complexes and soluble tissue factor, whereas hyperinsulinemia inhibited fibrinolysis irrespective of glucose levels, as reflected by a decrease in plasminogen activator activity levels due to a mean 2.5-fold rise in plasminogen activator inhibitor type 1. The differential effects of hyperglycemia and hyperinsulinemia suggest that patients with hyperglycemia due to insulin resistance are especially susceptible to thrombotic events by a concurrent insulin-driven impairment of fibrinolysis and a glucose-driven activation of coagulation.
Induction of Tolerance in Type 1 Diabetes via Both CD4+CD25+ T Regulatory Cells and T Regulatory Type 1 Cells
Authors: Battaglia Manuela , Stabilini Angela , Draghici Elena , Migliavacca Barbara , Gregori Silvia , Bonifacio Ezio , Roncarolo Maria-Grazia
Published: May 2006

Success in developing novel therapies to recommence self-tolerance in autoimmunity depends on the induction of T regulatory (Tr) cells. Here, we report that rapamycin combined with interleukin (IL)-10 efficiently blocks type 1 diabetes development and induces long-term immunotolerance in the absence of chronic immunosuppression in nonobese diabetic (NOD) mice. Rapamycin mediates accumulation in the pancreas of suppressive CD4+CD25+FoxP3+ Tr cells, which prevent diabetes. IL-10 induces Tr type 1 (Tr1) cells, which reside in the spleen and prevent migration of diabetogenic T-cells to the draining lymph nodes. These two Tr cell subsets act in concert to control diabetogenic T-cells that are still present in long-term tolerant mice. Rapamycin plus IL-10 treatment, promoting distinct subsets of Tr cells, may constitute a novel and potent tolerance-inducing protocol for immune-mediated diseases.