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Alterations in insulin signaling as well as insulin action predispose to infertility as well as adverse pregnancy outcomes; however, little is known about the role of glucagon signaling in reproduction. The glucagon receptor knockout (Gcgr−/−) mouse created by our laboratory was used to define the role of glucagon signaling in maintaining normal reproduction. In this mouse model, lack of glucagon signaling did not alter the hypothalamic-pituitary-ovarian axis. Pregnant Gcgr−/−female mice displayed persistent hypoglycemia and hyperglucagonemia. Gcgr−/−pregnancies were associated with decreased fetal weight, increased late-gestation fetal demise, and significant abnormalities of placentation. Gcgr−/−placentas contained areas of extensive mineralization, fibrinoid necrosis, narrowing of the vascular channels, and a thickened interstitium associated with trophoblast hyperplasia. Absent glucagon signaling did not alter glycogen content in Gcgr−/−placentas but significantly downregulated genes that control growth, adrenergic signaling, vascularization, oxidative stress, and G protein-coupled receptors. Our data suggest that, similarly to insulin, glucagon action contributes to normal female reproductive function.

An oral sulfate salt solution (OSS), under development as a bowel cleansing agent for colonoscopy in humans, is studied in rats and dogs. In rats, amaximumpractical oral OSS dose (5 g/kg/d) is compared with an oral sodium phosphate (OSP) solution, both at about 7 times the clinical dose. OSS induces the intended effects of loose stools and diarrhea. In rats, higher urine sodium and potassium accompany higher clearance rates, considered adaptive to the osmotic load of OSS. OSS for 28 days is well tolerated in rats and dogs. In contrast, OSP causes increased mortality, reduced body weight and food consumption, severe kidney tubular degeneration, and calcium phosphate deposition in rats. These are accompanied by mineralization in the stomach and aorta, along with cardiac and hepatic degeneration and necrosis. The greater safety margin of OSS over OSP at similarmultiples of the clinical dose indicates its suitability for human use.

Two cats with intestinal malabsorption developed a hemorrhagic diathesis. Although unsubstantiated, the probable cause of bleeding was a chronic malabsorption of fat and the fat-soluble vitamin K. When treated with vitamin K1 per os, one cat's clotting times were only partially corrected. Since vitamin K1 is actively absorbed in the proximal small intestine, the incomplete response of this case to orally administered vitamin K1 was predictable. The infrequent occurrence of bleeding in animals with malabsorption is, in part, attributable to the ileal and colonic absorption of bacterially derived vitamin K2. For this reason, nonspecific use of antibiotics in these animals is contraindicated. Since long-chain, polyunsaturated fats impair vitamin K absorption, dietary fat given to animals with malabsorption should be restricted to medium- and short-chain, saturated fats. Vitamin K should be administered subcutaneously to these animals if prolonged clotting times or active bleeding is present, and routinely prior to surgery. Oral supplementation with vitamin K3, which is absorbed in the colon and less lipid soluble than vitamin K1, should be given to animals with malabsorption that are maintained as outpatients. Adequate dosage levels of vitamin K3, however, are yet to be established for the cat, and dose-dependent hemolytic anemia is a probable toxic manifestation.

We present a status report from the NCI Mouse Models of Human Cancers Consortium (MMHCC) Precancers Workshop held November 8 and 9, 2004. An expert panel, the Mouse Models Group (MMG) evaluated the status of mouse models of precancer emphasizing genetically engineered mouse models, especially of lining epithelium and their utilitarian value to human carcinogenesis. An outline of the background for the panel’s considerations is provided with examples of past and current precancerous lesions in mice. The experimental use of oncogenic viruses and chemical carcinogens in mice led to operational definitions of initiation, promotion, and preneoplasia Preneoplastic and precancerous lesions are found in these models. In this precancer concept, most preneoplastic lesions are considered as potentially precancerous or at least an earlier stage in cancer development than typical pre-invasive epithelial lesions, which are often seen in these mouse models. Genetically engineered mice, used to test the oncogenicity of individual genes, develop precancers that are initiated by defined molecular and histopathologic changes. The mouse can be used to isolate and study precancers in detail, thereby providing a level of biological understanding not readily available in clinical disease. These studies suggest that genetically engineered mice are very useful preclinical models for chemoprevention and therapy.

Objectives To perform a comparison to determine which of two methods of partial urethral ligation produces the most consistent outcome and fewest side effects. Such a study has not been previously reported. Partial urethral ligation is a means of causing reproducible bladder outlet obstruction. In the male rat model, partial urethral obstruction can be performed either by perineal incision and bulbous urethral ligation or retropubic incision and midprostatic obstruction. Methods Fifteen male Sprague-Dawley rats were studied. Five were selected for bulbous urethral obstruction through a perineal incision, five for midprostatic obstruction using a retropubic approach, and five for a sham operation through a perineal incision. Results The operative time was shorter and morbidity lower with the perineal approach compared with the retropubic approach. Inflammation or infection, or both, were seen in the prostate, bladder, proximal urethra, ureters, and kidneys in the rats in which a midprostatic obstruction was performed. The proximal urethra and prostate were mildly inflamed in those rats that underwent bulbous obstruction. Sham-operated rats exhibited mild prostatitis only. Conclusions The perineal approach to the bulbous urethra is the method of choice for creating a partial urethral obstruction model of bladder outlet obstruction in the male rat.

Peroxisome proliferator-activated receptor (PPAR) represents a ligand-dependent nuclear receptor family that regulates multiple metabolic processes associated with fatty acid β-oxidation, glucose utilization, and cholesterol transport. These and other receptor-mediated actions pertain to their role in hypolipidemic and antidiabetic therapies and as potential targets for cancer chemopreventive agents. The present study evaluated the chemopreventive activity of two highly potent and selective PPARγ and PPARδ agonists in a progestin- and carcinogen-induced mouse mammary tumorigenesis model. Animals treated with the PPARγ agonist GW7845 exhibited a moderate delay in tumor formation. In contrast, animals treated with the PPARδ agonist GW501516 showed accelerated tumor formation. Significantly, tumors from GW7845-treated mice were predominantly ductal adenocarcinomas, whereas tumors from GW501516-treated animals were adenosquamous and squamous cell carcinomas. Gene expression analysis of tumors arising from GW7845- and GW501516-treated mice identified expression profiles that were distinct from each other and from untreated control tumors of the same histopathology. Only tumors from mice treated with the PPARγ agonist expressed estrogen receptor-α in luminal transit cells, suggesting increased ductal progenitor cell expansion. Tumors from mice treated with the PPARδ agonist exhibited increased PPARδ levels and activated 3-phosphoinositide–dependent protein kinase-1 (PDK1), which co-associated, suggesting a link between the known oncogenic activity of PDK1 in mammary epithelium and PPARδ activation. These results indicate that PPARδ and PPARγ agonists produce diverse, yet profound effects on mammary tumorigenesis that give rise to distinctive histopathologic patterns of tumor differentiation and tumor development.

Caveolae are flask-shaped invaginations of the plasma membrane formed by the oligomerization of caveolins. Because only smooth muscle contains all caveolin (Cav) family members (Cav-1, 2 and 3), we examined the contribution of each caveolin to urogenital smooth muscle structure/function.WT, Cav-1, 2, 3 and -1/3 knockout (KO) mouse bladders were characterized by Western blot, co-immunoprecipitation, immunofluorescence microscopy, electron microscopy, histochemistry and pharmacological techniques. Cystometric analysis was performed in conscious, freely moving mice. Other urogenital organs were investigated by histological analysis.The loss of bladder Cav-1 results in a marked decrease in Cav-2 but not Cav-3 expression. Ablation of Cav-3 fails to alter Cav-1 or Cav-2 expression. Deletion of Cav-1 results in the almost complete loss of caveolae, while Cav-2 KO and Cav-3 KO mouse smooth muscle showed a normal number of caveolae. The loss of Cav-1 generated caveolae led to significant urogenital changes in male mice (most marked by 12 months of age), namely 1) bladder weight-to-body weight ratios were increased, 2) the bladder smooth muscle layer was thickened, 3) the bladders had increased baseline, threshold and spontaneous pressures, 4) bladder strips showed a decreased contractile response to carbachol and KCl, and 5) these smooth muscle changes were accompanied by marked fluid accumulation in the prostate and seminal vesicles, with intracellular vacuolization in the kidneys. As such, male Cav-1 KO mice may be a useful animal model for studying LUTD (lower urinary tract dysfunction) that is so prevalent in aging male patients.The loss of Cav-1 and, thus, of most smooth muscle cell caveolae results in significant bladder dysfunction and urogenital organ changes in aged male mice.

Recently, development of a caveolin-1-deficient (Cav-1 null) mouse model has allowed the detailed analysis of caveolin-1's function in the context of a whole animal. Interestingly, we now report that the hearts of Cav-1 null mice are markedly abnormal, despite the fact that caveolin-1 is not expressed in cardiac myocytes. However, caveolin-1 is abundantly expressed in the nonmyocytic cells of the heart, i.e., cardiac fibroblasts and endothelia. Quantitative imaging studies of Cav-1 null hearts demonstrate a significantly enlarged right ventricular cavity and a thickened left ventricular wall with decreased systolic function. Histological analysis reveals myocyte hypertrophy with interstitial/perivascular fibrosis. Because caveolin-1 is thought to act as a negative regulator of the p42/44 MAP kinase cascade, we performed Western blot analysis with phospho-specific antibodies that only recognize activated ERK1/2. As predicted, the p42/44 MAP kinase cascade is hyperactivated in Cav-1 null heart tissue (i.e., interstitial fibrotic lesions) and isolated cardiac fibroblasts. In addition, endothelial and inducible nitric oxide synthase levels are dramatically upregulated. Thus loss of caveolin-1 expression drives p42/44 MAP kinase activation and cardiac hypertrophy.

It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (−/−) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (−/−) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling.

In human breast carcinomas, overexpression of the macrophage colony–stimulating factor (CSF-1) and its receptor (CSF-1R) correlates with poor prognosis. To establish if there is a causal relationship between CSF-1 and breast cancer progression, we crossed a transgenic mouse susceptible to mammary cancer with mice containing a recessive null mutation in the CSF-1 gene (Csf1op) and followed tumor progression in wild-type and null mutant mice. The absence of CSF-1 affects neither the incidence nor the growth of the primary tumors but delayed their development to invasive, metastatic carcinomas. Transgenic expression of CSF-1 in the mammary epithelium of both Csf1op/Csf1op and wild-type tumor-prone mice led to an acceleration to the late stages of carcinoma and to a significant increase in pulmonary metastasis. This was associated with an enhanced infiltration of macrophages into the primary tumor. These studies demonstrate that the growth of mammary tumors and the development to malignancy are separate processes and that CSF-1 selectively promotes the latter process. CSF-1 may promote metastatic potential by regulating the infiltration and function of tumor-associated macrophages as, at the tumor site, CSF-1R expression was restricted to macrophages. Our data suggest that agents directed at CSF-1/CSF-1R activity could have important therapeutic effects.

Bcl-2 is an anti-apoptotic and anti-proliferative protein over-expressed in several different human cancers including breast. Gain of Bcl-2 function in mammary epithelial cells was superimposed on the WAP-TAg transgenic mouse model of breast cancer progression to determine its effect on epithelial cell survival and proliferation at three key stages in oncogenesis: the initial proliferative process, hyperplasia, and cancer. During the initial proliferative process, Bcl-2 strongly inhibited both apoptosis and mitotic activity. However as tumorigenesis progressed to hyperplasia and adenocarcinoma, the inhibitory effects on mitotic activity were lost. In contrast, anti-apoptotic activity persisted in both hyperplasias and adenocarcinomas. These results demonstrate that the inhibitory effect of Bcl-2 on epithelial cell proliferation and apoptosis can separate during cancer progression. In this model, retention of anti-apoptotic activity with loss of anti-proliferative action resulted in earlier tumor presentation.

Adiponectin is a plasma protein expressed exclusively in adipose tissue. Adiponectin levels are linked to insulin sensitivity, but a direct effect of chronically elevated adiponectin on improved insulin sensitivity has not yet been demonstrated. We identified a dominant mutation in the collagenous domain of adiponectin that elevated circulating adiponectin values in mice by 3-fold. Adiponectinemia raised lipid clearance and lipoprotein lipase activity, and suppressed insulin-mediated endogenous glucose production. The induction of adiponectin during puberty and the sexual dimorphism in adult adiponectin values were preserved in these transgenic animals. As a result of elevated adiponectin, serum PRL values and brown adipose mass both increased. The effects on carbohydrate and lipid metabolism were associated with elevated phosphorylation of 5'-AMP-activated protein kinase in liver and elevated expression of peroxisomal proliferator-activated receptor gamma2, caveolin-1, and mitochondrial markers in white adipose tissue. These studies strongly suggest that increasing endogenous adiponectin levels has direct effects on insulin sensitivity and may induce similar physiological responses as prolonged treatment with peroxisomal proliferator-activated receptor gamma agonists.

Caveolae organelles and caveolin-1 protein expression are most abundant in adipocytes and endothelial cells. Our initial report on mice lacking caveolin-1 (Cav-1) demonstrated a loss of caveolae and perturbations in endothelial cell function. More recently, however, observation of the Cav-1-deficient cohorts into old age revealed significantly lower body weights, as compared with wild-type controls. These results suggest that Cav-1 null mice may have problems with lipid metabolism and/or adipocyte functioning. To test this hypothesis directly, we placed a cohort of wild-type and Cav-1 null mice on a high fat diet. Interestingly, despite being hyperphagic, Cav-1 null mice show overt resistance to diet-induced obesity. As predicted, adipocytes from Cav-1 null null mice lack caveolae membranes. Early on, a lack of caveolin-1 selectively affects only the female mammary gland fat pad and results in a near complete ablation of the hypo-dermal fat layer. There are also indications of generalized adipose tissue pathology. With increasing age, a systemic decompensation in lipid accumulation occurs resulting in dramatically smaller fat pads, histologically reduced adipocyte cell diameter, and a poorly differentiated/hypercellular white adipose parenchyma. To gain mechanistic insights into this phenotype, we show that, although serum insulin, glucose, and cholesterol levels are entirely normal, Cav-1 null mice have severely elevated triglyceride and free fatty acid levels, especially in the post-prandial state. However, this build-up of triglyceride-rich chylomicrons/very low density lipoproteins is not due to perturbed lipoprotein lipase activity, a major culprit of isolated hypertriglyceridemia. The lean body phenotype and metabolic defects observed in Cav-1 null mice are consistent with the previously proposed functions of caveolin-1 and caveolae in adipocytes. Our results show for the first time a clear role for caveolins in systemic lipid homeostasis in vivo and place caveolin-1/caveolae as major factors in hyperlipidemias and obesity.

Barnas, George M., Paul A. Delaney, Ileana Gheorghiu, Srinivas Mandava, Robert G. Russell, Renée Kahn, and Colin F. Mackenzie. Respiratory impedances and acinar gas transfer in a canine model for emphysema. J. Appl. Physiol. 83(1): 179–188, 1997.—We examined how the changes in the acini caused by emphysema affected gas transfer out of the acinus (Taci) and lung and chest wall mechanical properties. Measurements were taken from five dogs before and 3 mo after induction of severe bilateral emphysema by exposure to papain aerosol (170–350 mg/dose) for 4 consecutive wk. With the dogs anesthetized, paralyzed, and mechanically ventilated at 0.2 Hz and 20 ml/kg, we measured Taciby the rate of washout of133Xe from an area of the lung with occluded blood flow. Measurements were repeated at positive end-expiratory pressures (PEEP) of 10, 5, 15, 0, and 20 cmH2O. We also measured dynamic elastances and resistances of the lungs (El and Rl, respectively) and chest wall at the different PEEP and during sinusoidal forcing in the normal range of breathing frequency and tidal volume. After final measurements, tissue sections from five randomly selected areas of the lung each showed indications of emphysema. Taciduring emphysema was similar to that in control dogs. Eldecreased by ∼50% during emphysema ( P < 0.05) but did not change its dependence on frequency or tidal volume. Rl did not change ( P > 0.05) at the lowest frequency studied (0.2 Hz), but in some dogs it increased compared with control at the higher frequencies. Chest wall properties were not changed by emphysema ( P > 0.05). We suggest that although large changes in acinar structure and El occur during uncomplicated bilateral emphysema, secondary complications must be present to cause several of the characteristic dysfunctions seen in patients with emphysema.

To test the effects of three hemoglobin solutions on liver function and hepatic morphology after resuscitation from severe hemorrhagic shock.Prospective study.Laboratory.Thirty-three beagle dogs.Hemorrhagic shock was induced in anesthetized dogs by removal of blood at a rate of 2 mL/kg/min until systolic blood pressure (BP) reached 50 mm Hg. BP was maintained at this level 2 hrs by further withdrawing 5 to 10 mL aliquots whenever BP increased50 mm Hg. Resuscitation was then initiated with autologous whole blood (n = 7), 4% pyridoxalated-hemoglobin-polyoxyethylene conjugate (4% PHP [n = 6]), 8% pyridoxalated-hemoglobin-polyoxyethylene conjugate (8% PHP [n = 9], or 8% stroma-free hemoglobin (n = 7). Four dogs were managed identically but were not resuscitated. Gross necropsy and histologic examination of the liver were performed on all dogs after 7 days, or earlier if death occurred.In vitro interferences of PHP and stroma-free hemoglobin with liver function tests were determined and recommendations for interpretation of results from blood samples containing PHP and stroma-free hemoglobin were made. Blood was collected before, during, and after resuscitation from hemorrhagic shock. The dogs were then awakened and survivors were monitored daily with blood sampling until they were killed and necropsy was performed. After 7 days, the survival rate following hemorrhagic shock was 100% for whole blood and 4% PHP, 86% for stroma-free hemoglobin, and 33% for 8% PHP. Of the resuscitated dogs not surviving 7 days, all but one died within 27 hrs from coagulopathy. All dogs not resuscitated died within 1.75 hrs after 2 hrs of shock. Bilirubin, alkaline phosphatase, and lactic dehydrogenase concentrations could not be measured due to interferences of stroma-free hemoglobin and PHP. Aspartate (AST) and alanine (ALT) aminotransferase concentrations could be measured after dilution to overcome the interferences. Significant increases in AST and ALT values in all groups 24 hrs after resuscitation were attributed to hypoxic hepatocellular damage associated with the severity of the shock model rather than to the resuscitation fluid. Liver histology showed no changes attributed to toxic damage of hepatocytes in dogs resuscitated with stroma-free hemoglobin or PHP. However, changes, were less severe in dogs resuscitated with 4% PHP than in other groups.Morphologic studies at necropsy and liver function tests in dogs receiving hemoglobin solutions, compared with autologous blood, support the conclusion that the PHP and stroma-free hemoglobin solutions tested did not produce hepatic toxicity when used as resuscitation fluids in this model of severe shock.

outbreak investigation: An outbreak of diarrhea, bloody diarrhea, and abdominal cramps occurred among persons undergoing flexable sigmoidoscopy at a branch clinic of a local health center. Illness was associated with use of sigmoidoscopes cleaned by one clinic assistant and appeared to be caused by 2% glutaraldehyde disinfectant solution left in the instruments after cleaning. animal studies: In subsequent animal studies, colonic instillation of 2 % glutaraldehyde solutions caused bloody diarrhea and a distinctive pattern of mucosal damage; similar changes were seen in a review of pathologic samples from other human cases of glutaraldehyde disinfectant-associated diarrhea. conclusion: Our data indicate that improper endoscopic reprocessing can result in serious illness and underscore the importance of adequate training and quality control in this area.

Velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a human disorder characterized by a number of phenotypic features including cardiovascular defects. Most VCFS/DGS patients are hemizygous for a 1.5–3.0 Mb region of 22q11. To investigate the etiology of this disorder, we used a cre-loxP strategy to generate mice that are hemizygous for a 1.5 Mb deletion corresponding to that on 22q11. These mice exhibit significant perinatal lethality and have conotruncal and parathyroid defects. The conotruncal defects can be partially rescued by a human BAC containing the TBX1 gene. Mice heterozygous for a null mutation in Tbx1 develop conotruncal defects. These results together with the expression patterns of Tbx1 suggest a major role for this gene in the molecular etiology of VCFS/DGS.

The aims of diabetic treatment are: correction of hyperglycaemia; control of symptoms; prevention of complications; normal life and good health; and avoidance of any side-effects of treatment.