Overexpression of parathyroid hormone-related protein in the pancreatic islets of transgenic mice causes islet hyperplasia, hyperinsulinemia, and hypoglycemia

Parathyroid hormone-related protein (PTHrP) is produced by the pancreatic islet. It also has receptors on islet cells, suggesting that it may serve a paracrine or autocrine role within the islet. We have developed transgenic mice, which overexpress PTHrP in the islet through the use of the rat insulin II promoter (RIP). Glucose homeostasis in these mice is markedly abnormal; RIP-PTHrP mice are hypoglycemic in the postprandial and fasting states and display inappropriate hyperinsulinemia. At the end of a 24-hour fast, blood glucose values are 49 mg/dl in RIP-PTHrP mice, as compared to 77 mg/dl in normal littermates; insulin concentrations at this time are 6.3 and 3.9 ng/ml, respectively. Islet perifusion studies failed to demonstrate abnormalities in insulin secretion. In contrast, quantitative islet histomorphometry demonstrates that the total islet number and total islet mass are 2-fold higher in RIP-PTHrP mice than in their normal littermates. PTHrP very likely plays a normal physiologic role within the pancreatic islet. This role is most likely paracrine or autocrine. PTHrP appears to regulate insulin secretion either directly or indirectly, through developmental or growth effects on islet mass. PTHrP may have a role as an agent that enhances islet mass and/or enhances insulin secretion.     

Overexpression of MnSOD protects against myocardial ischemia/reperfusion injury in transgenic mice

Generation of free radicals upon reperfusion has been cited as one of the major causes of ischaemia/reperfusion injury. The following series of experiments was designed to study the effect of manganese superoxide dismutase (MnSOD) overexpression in transgenic mice on ischemia/reperfusion injury. A species of 1.4 kb human MnSOD mRNA was expressed, and a 325% increase in MnSOD activity was detected in the hearts of transgenic mice with no changes in the other antioxidant enzymes or heat shock proteins. Immunocytochemical study indicated an increased labeling of MnSOD mainly in the heart mitochondria of the transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x left ventricular developed pressure, was 52 +/- 4% in the transgenic hearts as compared to 31 +/- 4% in the non-transgenic hearts. This protection was accompanied by a significant decrease in lactate dehydrogenase release from the transgenic hearts. Overexpression of MnSOD limited the infarct size in vivo in a left coronary artery ligation model. Our results demonstrate that overexpression of MnSOD renders the heart more resistant to ischemia/reperfusion injury.     

Overexpression of nerve growth factor in skin causes preferential increases among innervation to specific sensory targets

The impact of increased levels of skin-derived nerve growth factor (NGF) neurotrophin on sensory and sympathetic innervation to the mouse mystacial pad and postero-orbital vibrissae was determined. Consistent with an approximate doubling of neuron number in trigeminal and superior cervical ganglia, many components of the sensory and sympathetic innervation were substantially enhanced. Although the increased number of neurons raised the possibility that all types of innervation were increased, immunohistochemical analysis indicated that enhanced NGF production had a differential effect upon sensory innervation, primarily increasing unmyelinated innervation. This increased innervation occurred in specific locations known to be innervated by small, unmyelinated fibers, suggesting that NGF modulated sensory innervation density, but not targeting. In contrast, sympathetic innervation was not only increased but also was distributed to some aberrant locations. In the intervibrissal fur of the mystacial pad, both the number of sensory axons and branches appeared increased, whereas in vibrissal follicle sinus complexes, only branching increased. In some areas, sensory ending density was lower than expected based upon the size of the source nerve bundles suggesting that many axons and branches were surviving but failing to form functional endings. Furthermore, the immunochemical profile of innervation was altered in some sensory populations as demonstrated by the coexistence of RT-97 neurofilament labeling in calcitonin gene-related peptide (CGRP) positive axons, by the loss of substance P colocalization in some CGRP axons, and by an absence of neuropeptide Y labeling in tyrosine hydroxylase positive sympathetic axons. Collectively, these results indicate that the NGF mediated increase in neuron number may be selective for particular sets of innervation and that increases among some populations may result from phenotypic switching.     

Ectopic expression of metallothionein-III causes pancreatic acinar cell necrosis in transgenic mice

Mice express four distinct metallothioneins (MTs) that have similar metal-binding properties. MT-I and MT-II are expressed coordinately in most organs, whereas MT-III is expressed predominantly in a subset of neurons and MT-IV is expressed in certain stratified epithelia. The restricted expression of MT-III suggests that it may severe a specialized function. To test this hypothesis, transgenic mice were generated that express MT-III in the wider expression domain of MT-I. Similar transgenic lines expressing extra MT-I under the same regulation were generated as controls for the effect of over-expression of MT. Transgenic mice that express MT-III ectopically frequently die at 2-3 months of age. The pancreata of moribund mice were abnormally small and histological examination, at various ages, revealed a progressive degeneration of the acinar cells. At early stages multifocal acinar cell eosinophilia and swollen nuclei were seen and this pathology progressed to multifocal acinar cell necrosis and fibrosis. The terminal stages were characterized by a loss of the acinar compartment, leaving the islets embedded in a fibrotic remnant. Other organs of these mice were grossly and histologically normal. All organs examined from mice expressing excess MT-I were unremarkable even though expression of either MT-I or MT-III transgenes resulted in similar accumulations of zinc and copper in the pancreata. This study indicates that pancreatic acinar cells are unusually sensitive to chronic expression of MT-III. The mechanism by which MT-III disrupts pancreatic function is unclear, but the results provide further evidence that MT isoforms exhibit distinct properties and probably serve distinct biological functions.     

Noradrenergic and peptidergic innervation of the testis and epididymis in the male pig

Birth-related brachial plexus injury occurs in 0.19-2.5 per 1,000 live births, of which 70-92% improve with conservative management. With the advent of microsurgical techniques, patients who fail expectant treatment may benefit from brachial plexus exploration and reconstruction. From 1991 to 1996, 87 patients were referred to the multidisciplinary brachial plexus clinic at St. Louis Children's Hospital. Twenty patients were selected for surgical management. The average age at surgery was 10.5 months (range 3-35, median = 8), with an average follow-up of 23.9 months (range 7-45, median = 24). Two patients were lost to follow-up. Surgical procedures included neurolysis (n = 8), neurotization (n = 2), nerve grafting (n = 5), and a combination (n = 3) of the above. Two patients underwent exploration without repair. Intercostal nerves, pectoral nerves, and C4 roots were used for neurotizations, and the sural nerve was used for nerve grafting. Results from 18 patients were available for follow-up review. Fifteen patients (83% demonstrated clinical improvement postoperatively. Of the 3 patients without improvement, 2 underwent exploration without repair, and one underwent neurolysis of the axillary nerve. Of patients undergoing reconstruction, 93% had improved strength postoperatively. No subjects had worsening neurologic status, and there were no complications. These results suggest that surgery for birth-related brachial plexus injury may show favorable outcomes if patients are selected appropriately. Patients undergoing neurolysis and nerve grafting had more favorable outcomes than those undergoing neurotization.     

Overexpression of Rab3D enhances regulated amylase secretion from pancreatic acini of transgenic mice

Rab3D, a member of the ras-related GTP-binding protein Rab family, is localized to secretory granules of various exocrine tissues such as acinar cells of the pancreas, chief cells of the stomach, and parotid and lacrimal secretory cells. To elucidate the function of Rab3D in exocytosis, we have generated transgenic mice that over-express Rab3D specifically in pancreatic acinar cells. Hemagglutinin-tagged Rab3D was localized to zymogen granules by immunohistochemistry, and was shown to be present on zymogen granule membranes by Western blotting; both results are similar to previous studies of endogenous Rab3D. Secretion measurements in isolated acinar preparations showed that overexpression of Rab3D enhanced amylase release. Amylase secretion from intact acini of transgenic mice 5 min after 10 pM cholecystokinin octapeptide (CCK) stimulation was enhanced by 160% of control. In streptolysin-O-permeabilized acini of transgenic mice, amylase secretion induced by 100 microM GTP-gamma-S was enhanced by 150%, and 10 microM Ca2+-stimulated amylase secretion was augmented by 206% of that of the control. To further elucidate Rab3D involvement in stimulus-secretion coupling, we examined the effect of CCK on the rate of GTP binding to Rab3D. Stimulation of permeabilized acini with 10 pM CCK increased the incorporation of radiolabeled GTP into HA-tagged Rab3D. These results indicate that overexpression of Rab3D enhances secretagogue-stimulated amylase secretion through both calcium and GTP pathways. We conclude that Rab3D protein on zymogen granules plays a stimulatory role in regulated amylase secretion from pancreatic acini.     

Overexpression of human glutathione peroxidase protects transgenic mice against focal cerebral ischemia/reperfusion damage

As stroke is a major cause of disability and death in the western world, there is great interest in the basic mechanisms by which ischemia/reperfusion (I/R) causes damage. To this end, extensive research has been carried out which identifies reactive oxygen species (ROS) as key participants in brain damage resultant from I/R. Brain tissue is protected from ROS damage by antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GP). Overexpression of SOD in transgenic mice has already been demonstrated to confer protection against I/R damage in murine stroke models. We are using transgenic mice overexpressing the intracellular form of glutathione peroxidase (GP1) to determine the protective capacity of overexpression of this enzyme on stroke damage. 1 h of focal cerebral ischemia followed by 24 h of reperfusion was induced using the intraliminal suture method. Volume of infarction was reduced by 48% in GP1 mice compared to nontransgenic littermates. Brain edema was reduced by 33%. Behavioral deficits agreed with histologic data. Overexpression of glutathione peroxidase confers significant protection against I/R damage in our stroke model possibly through direct scavenging of ROS or through the influencing of signalling mechanisms which lead to tissue damage.     

Treatment with growth hormone and dexamethasone in mice transgenic for human islet amyloid polypeptide causes islet amyloidosis and beta-cell dysfunction

Islet amyloid derived from islet amyloid polypeptide (IAPP) is a well-recognized feature of type II diabetes. However, the mechanism of islet amyloidogenesis is unknown. In vitro studies suggest that amino acid residues 20-29 in human, but not mouse, IAPP confer amyloidogenicity consistent with the absence of spontaneous islet amyloidosis in mice. Several clinical and in vitro studies suggest that increased synthetic rates of IAPP predispose to IAPP-amyloidosis. In the present study, we sought to test the hypothesis that pharmacological induction of insulin resistance in a mouse transgenic (TG) for human IAPP would induce islet amyloid and beta-cell dysfunction. TG and non-transgenic (N-TG) control mice were treated with both rat growth hormone (12 micrograms/day) and dexamethasone (0.24 mg/day) (dex/GH) or received no treatment for 4 weeks, after which animals were killed to examine islet morphology. Treatment with dex/GH caused hyperglycemia (7.3 +/- 0.4 vs. 5.2 +/- 0.1 mmol/l, TG vs. N-TG, P < 0.001) associated with a decreased plasma insulin concentration (595 +/- 51 vs. 996 +/- 100 pmol/l, TG vs. N-TG, P < 0.05) in TG versus control mice. Islet amyloid was induced in treated TG mice but not in control mice. Islet amyloid was identified in both intra- and extracellular deposits, the former being associated with evidence of beta-cell degeneration. We conclude that dex/GH treatment in mice TG for human IAPP induces IAPP-derived islet amyloid, hyperglycemia, and islet dysfunction. The present model recapitulates the islet morphology and phenotype of type II diabetes.     

Elevated luteinizing hormone in prepubertal transgenic mice causes hyperandrogenemia, precocious puberty, and substantial ovarian pathology

In women, chronically elevated androgens have been associated with polycystic ovarian syndrome and infertility. Recently, we described transgenic mice with elevated serum LH secondary to targeted expression of a transgene encoding a chimeric LH beta-subunit. Mature transgenic females exhibit elevated androgens, anovulation, and a range of ovarian phenotypes including cysts, widespread luteinization, and tumors. In the present study we have examined serum levels of LH and testosterone and the concurrent development of the reproductive system in prepubertal mice. Serum LH in prepubertal females was elevated despite increased serum testosterone and estradiol, indicating a relative insensitivity to steroid negative feedback. Elevated serum LH and hyperandrogenemia resulted in accelerated vaginal opening and ovarian follicular development in transgenic females. Precocious antral follicle formation and conspicuous hypertrophy of the theca-interstitium preceded the development of large cysts with marked hemorrhage. Based on these studies we conclude that chronic prepubertal elevation of serum LH results in gonadotropin-dependent hyperandrogenemia, leading to abnormal sexual development and significant ovarian pathology.     

Vascular endothelial cells: targets for studying the activity of hair follicle cell-produced VEGF

Fetal bovine aortic endothelial cells (FBAEC) were exposed to purified fractions of conditioned medium from cultures of hair dermal papilla cells (DPC) to determine the existence of any vascular endothelial growth factor (VEGF)-like paracrine activity of the latter. Such fractions were tested for stimulation of growth and migration of cultured FBAEC. In addition, VEGF secretion by DPC was measured by radioassay of VEGF receptors using FBAEC as target cells. The results showed that stimulation of FBAEC proliferation and migration following exposure to purified conditioned medium was dose-dependent. Radioreceptor assays of recombinant VEGF and purified DPC-conditioned medium showed competitive VEGF binding in FBAEC.     

Overexpression of 2',3'-cyclic nucleotide 3'-phosphodiesterase in transgenic mice alters oligodendrocyte development and produces aberrant myelination

The function of the intracellular protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) of oligodendrocytes (ODC) is unknown. We have now generated several homozygous transgenic mouse lines in which the human CNP gene is overexpressed up to sixfold, revealing new insights into early stages of myelinogenesis. Although no behavioral phenotype is immediately apparent, abnormalities of ODC and their myelin sheaths are striking. These are manifested as redundant myelin membrane and intramyelinic vacuoles, as well as lack of myelin compaction concordant with failure of the cytoplasmic leaflets of compact myelin to fuse. Further, ODC that overexpress CNP appear to mature earlier in development, resulting in earlier maximum gene expression for myelin basic proteins and proteolipid protein. These results indicate that CNP is an early expressed regulator of cellular events that culminate in CNS myelination.     

Overexpression of transforming growth factor !a in transgenic mice alters nonreproductive, sex-related behavioral differences: Interaction with gonadal hormones.

Sexually dimorphic differences in voluntary sodium intake, locomotor activity, immobility in the swim test, and aggressive behavior were found to be altered in transgenic CD-1 mice that overexpressed transforming growth factor α (TGFα). In contrast to nontransgenic CD-1 mice, immobility in the swim test was longer and sodium intake higher in the male TGFα mice than in the female TGFα mice. These findings indicate that the male TGFα mice exhibited feminization of some behaviors. Furthermore, the male TGFα mice were highly aggressive. Castration reversed the behavioral effects in the adult male transgenic mice, but ovariectomy did not reverse the behavioral effects in the adult female transgenic mice. Thus, the feminizing effect of TGFα on some nonreproductive behaviors and increased aggressive behavior in male mice may be mediated through an interaction between this growth factor and gonadal hormones. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Multistage epidermal carcinogenesis in transgenic mice: cooperativity and paradox

Skin cancer is one of the most prevalent forms of human neoplasia with a frequency approaching that of all other neoplasms combined. Given this alarming statistic, which may be further exacerbated by increased ultraviolet B irradiation from ozone depletion, it is vital that realistic, relevant model systems are developed to increase our understanding of the underlying molecular mechanisms of carcinogenesis that result in or evaluate new treatment modalities. Toward this goal, the ability to stably introduce genes into the germline of mice has greatly enhanced prospects for generation of transgenic animal models of multistage molecular carcinogenesis. Moreover, when genes are combined with regulatory sequences that target their expression to specific tissues, investigators are able to study neoplasia both in the context of living organisms and in the tissues suspected of being the targets of these genes. The epidermis is an attractive tissue for targeted gene expression; not only is it a model for epithelial diseases in general, but the accessibility of the epidermis allows easy detection of progressive pathological changes that result from transgene expression and facilitates assessment of the potential role played by environmental factors. We have developed a targeting vector based on the human keratin gene (HK1), which is expressed exclusively in the epidermis of transgenic mice, at a late stage in development and in both basal and differentiated cells. Through the use of this targeting ability, rasHa, fos, and TGF alpha transgenic mice have been developed that exhibit preneoplastic epidermal hyperplasia and hyperkeratosis, and later benign, regression prone papillomas. Together, coexpression of two oncogenes cooperated to give autonomous papillomas, which possessed the phenotypic stability to allow assessment of a third genetic event, namely loss of the p53 tumor suppressor gene, via mating with p53 knockout mice. Loss of p53 expression, however, identified a paradoxical block of papillomatogenesis. This latter result suggests that the epidermis possesses several mechanisms that can effectively compensate for the loss of important tumor suppressor functions and may have evolved to render the skin relatively resistant to the effects of environmental carcinogens.     

Overexpression of natural killer T cells protects Valpha14- Jalpha281 transgenic nonobese diabetic mice against diabetes

Progression to destructive insulitis in nonobese diabetic (NOD) mice is linked to the failure of regulatory cells, possibly involving T helper type 2 (Th2) cells. Natural killer (NK) T cells might be involved in diabetes, given their deficiency in NOD mice and the prevention of diabetes by adoptive transfer of alpha/beta double-negative thymocytes. Here, we evaluated the role of NK T cells in diabetes by using transgenic NOD mice expressing the T cell antigen receptor (TCR) alpha chain Valpha14-Jalpha281 characteristic of NK T cells. Precise identification of NK1.1(+) T cells was based on out-cross with congenic NK1.1 NOD mice. All six transgenic lines showed, to various degrees, elevated numbers of NK1.1(+) T cells, enhanced production of interleukin (IL)-4, and increased levels of serum immunoglobulin E. Only the transgenic lines with the largest numbers of NK T cells and the most vigorous burst of IL-4 production were protected from diabetes. Transfer and cotransfer experiments with transgenic splenocytes demonstrated that Valpha14-Jalpha281 transgenic NOD mice, although protected from overt diabetes, developed a diabetogenic T cell repertoire, and that NK T cells actively inhibited the pathogenic action of T cells. These results indicate that the number of NK T cells strongly influences the development of diabetes.     

Invalid evidence for expectancies as causes: Comment on Kirsch.

Contends that shortcomings in the empirical evidence cited by I. Kirsch (see record 1986-13702-001) in his study of expectancy modification leave open to question whether the cognitive state of response expectancy is causally linked to placebo responses and fear reduction. (32 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)