Insulin signaling after exercise in insulin receptor substrate-2-deficient mice

The period immediately after exercise is characterized by enhanced insulin action in skeletal muscle, and on the molecular level, by a marked increase in insulin-stimulated, phosphotyrosine-associated phosphatidylinositol (PI) 3-kinase activity. Because the increase in PI 3-kinase activity cannot be explained by increased insulin receptor substrate (IRS)-1 signaling, the present study examined whether this effect is mediated by enhanced IRS-2 signaling. In wild-type (WT) mice, insulin increased IRS-2 tyrosine phosphorylation (2.5-fold) and IRS-2-associated PI 3-kinase activity (3-fold). Treadmill exercise, per se, had no effect on IRS-2 signaling, but in the period immediately after exercise, there was a further increase in insulin-stimulated IRS-2 tyrosine phosphorylation (3.5-fold) and IRS-2-associated PI 3-kinase activity (5-fold). In IRS-2-deficient (IRS-2-/-) mice, the increase in insulin-stimulated, phosphotyrosine-associated PI 3-kinase activity was attenuated as compared with WT mice. However, in IRS-2-/- mice, the insulin-stimulated, phosphotyrosine-associated PI 3-kinase response after exercise was slightly higher than the insulin-stimulated response alone. In conclusion, IRS-2 tyrosine phosphorylation and associated PI 3-kinase activity are markedly enhanced by insulin in the immediate period after exercise. IRS-2 signaling can partially account for the increase in insulin-stimulated phosphotyrosine-associated PI 3-kinase activity after exercise.

Casitas b-lineage lymphoma-deficient mice are pretected against high-fat diet-induuced obesity and insulin resistance

Casitas b-lineage lymphoma (c-Cbl) is a multiadaptor protein with E3-ubiquitin ligase activity involved in regulating the degradation of receptor tyrosine kinases. We have recently reported that c-Cbl^–/– mice exhibit a lean phenotype and enhanced peripheral insulin action likely due to elevated energy expenditure. In the study reported here, we examined the effect of a high-fat diet on energy homeostasis and glucose metabolism in these animals. When c-Cbl^–/– mice were fed a high-fat diet for 4 weeks, they maintained hyperphagia, higher whole-body oxygen consumption (27%), and greater activity (threefold) compared with wild-type animals fed the same diet. In addition, the activity of several enzymes involved in mitochondrial fat oxidation and the phosphorylation of acetyl CoA carboxylase was significantly increased in muscle of high-fat–fed c-Cbl–deficient mice, indicating a greater capacity for fat oxidation in these animals. As a result of these differences, fat-fed c-Cbl^–/– mice were 30% leaner than wild-type animals and were protected against high-fat diet–induced insulin resistance. These studies are consistent with a role for c-Cbl in regulating nutrient partitioning in skeletal muscle and emphasize the potential of c-Cbl as a therapeutic target in the treatment of obesity and type 2 diabetes.

c-Cbl-deficient mice have reduced adiposity, higher energy expenditure, and improved peripheral insulin action

Casitas b-lineage lymphoma (c-Cbl) is an E3 ubiquitin ligase that has an important role in regulating the degradation of cell surface receptors. In the present study we have examined the role of c-Cbl in whole-body energy homeostasis. c-Cb^-/- mice exhibited a profound increase in whole-body energy expenditure as determined by increased core temperature and whole-body oxygen consumption. As a consequence, these mice displayed a decrease in adiposity, primarily due to a reduction in cell size despite an increase in food intake. These changes were accompanied by a significant
increase in activity (2- to 3-fold). In addition, cc-Cb-/- mice displayed a marked improvement in whole-body insulin action, primarily due to changes in muscle metabolism. We observed increased protein levels of the insulin receptor (4-fold) and uncoupling protein-3 (2-fold) in skeletal muscle and a significant increase in the phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase. These fmdings suggest that c-Cbl plays an integral role in whole-body fuel homeostasis by regulating whole-body energy expenditure and insulin action.

Improved glusoce homeostasis and enhanced insulin signalling in Grb 14-deficient mice

Gene targeting was used to characterize the physiological role of growth factor receptor-bound (Grb)14, an adapter-type signalling protein that associates with the insulin receptor (IR). Adult male Grb14^-/- mice displayed improved glucose tolerance, lower circulating insulin levels, and increased incorporation of glucose into glycogen in the liver and skeletal muscle. In ex vivo studies, insulin-induced 2-deoxyglucose uptake was enhanced in soleus muscle, but not in epididymal adipose tissue. These metabolic effects correlated with tissue-specific alterations in insulin signalling. In the liver, despite lower IR autophosphorylation, enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and activation of protein kinase B (PKB) was observed. In skeletal muscle, IR tyrosine phosphorylation was normal, but signalling via IRS-1 and PKB was increased. Finally, no effect of Grb14 ablation was observed on insulin signalling in white adipose tissue. These findings demonstrate that Grb14 functions in vivo as a tissue-specific modulator of insulin action, most likely via repression of IR-mediated IRS-1 tyrosine phosphorylation, and highlight this protein as a potential target for therapeutic intervention.

Heterozygous tx mice have an increased sensitivity to copper loading: implications for Wilson's Disease carriers

Wilson's disease carriers constitute 1% of the human population. It is unknown whether Wilsons disease carriers are at increased susceptibility to copper overload when exposed to chronically high levels of ingested copper. This study investigated the effect of chronic excess copper in drinking water on the heterozygous form of the Wilson’s disease mouse model – the toxic milk (tx) mouse. Mice were provided with drinking water containing 300 mg/l copper for 4–7, 8–11, 12–15 or 16–20 months. At the completion of the study liver, spleen, kidney and brain tissue were analyzed by atomic absorption spectroscopy to determine copper concentration. Plasma ceruloplasmin oxidase activity and liver histology were also assessed. Chronic copper loading resulted in significantly increased liver copper in both tx heterozygous and tx homozygous mice, while wild type mice were resistant to the effects of copper loading. Copper loading effects were greatest in tx homozygous mice, with increased extrahepatic copper deposition in spleen and kidney – an effect absent in heterozygote and wild type mice. Although liver histology in homozygous mice was markedly abnormal, no histological differences were noted between heterozygous and wild type mice with copper loading. Tx heterozygous mice have a reduced ability to excrete excess copper, indicating that half of the normal liver Atp7b copper transporter activity is insufficient to deal with large copper intakes. Our results suggest that Wilsons disease carriers in the human population may be at increased risk of copper loading if chronically exposed to elevated copper in food or drinking water.

Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Y704, Y729, Y744, Y764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation and cell survival. However, it is unclear whether these tyrosines are equally important under more physiological conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated GCSF- R deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (mO), single tyrosine "add back" mutants or wild type (WT) receptors. G-CSFinduced responses were determined in primary colony assays, serial replatings and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Y764 strongly enhanced proliferativeresponses, which was reverted by inhibition of ERK activitity. Y729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing mO gradually dropped compared to WT. The presence of Y729, but also Y704 and Y744, both involved in activation of STAT3, further reduced replating
efficiencies. Conversely, Y764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a >104–fold increase of colony forming cells over mO after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.

Measuring Ultrasonic Communication Between Mouse Pups and Adult Mother Mice

Measuring ultrasonic communication provides us with a way to study parental influence on animals. In this study I measured the ultrasonic communication between mouse pups and two maternal females, one of which who had given birth to the pups and the other had raised them. I found that there was no significant difference between the amount of noise expressed by pups in response to each the biological mother and foster mother test groups. Mouse pups call to maternal females regardless of genetic relatedness. Communication in mice may be a more complicated model because of their communal nature.

ß1/ß2/ß3-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting

Catecholamines are viewed as major stimulants of diet- and cold-induced thermogenesis and of fasting-induced lipolysis, through the β-adrenoceptors (β1/β2/β3). To test this hypothesis, we generated β1/β2/β3-adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold-intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein-1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, β-adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.

Antitumor activity and underlying mechamisms of ganopoly, the refined polysaccharides extracted from ganodrema lucidum, in mice

Ganopoly is an aqueous polysaccharide fraction extracted from G. lucidum by patented biochemical technique and has been marketed as an over-the-counter product for chronic diseases including cancer and hepatopathy in many Asian countries. This study was undertaken to explore the anti-tumour effect and the underlying mechanisms of Ganopoly in mice and human tumor cell lines. The maximum tolerateddose (MTD) of Ganopoly in mice was estimated to be 100 mg/kg from a pilot study. Treatment of mice with oral Ganopoly for 10 days significantly reduced the tumour weight of sarcoma-180 in a dose-dependent manner, with inhibition rates of 32.3, 48.2 and 84.9% and growth delays of 1.5, 3.5, and 13.1 days at 20, 50, and 100 mg/kg, respectively. Incubation of Ganopoly at 0.05-1.0 mg/ml for 48 hours showed little or negligible cytotoxicity against human tumor CaSki, SiHa, Hep3B, HepG2, HCT116, HT29, and MCF7 cells in vitro. In contrast, 10 mg/ml of Ganopoly caused significant cytotoxicity in all tumour cells tested except MCF7, with marked apoptotic effects observed in CaSki, HepG2, and HCT116 cells, as indicated by nuclear staining and DNA fragmentation. In addition, Ganopoly enhanced concanavalin A-stimulated proliferation of murine splenocytes by 35.3% at 10 mg/ml, and stimulated the production of nitric oxide in thioglycollate-primed murine peritoneal macrophages in a concentration-dependent manner over 0.05-10 mg/ml. Addition of Ganopoly at 1 mg/ml to murine peritoneal macrophages also potentiated lipopolysaccharide-induced nitric oxide production by 64.2%. Treatment of healthy mice or mice bearing sarsoma-180 with oral Ganopoly over 20-100 mg/kg for 7 day significantly increased the expression of both TNF-α and IFN-γ (at both mRNA and protein levels) in splenocytes in a dose-dependent manner. Moreover, treatment of Ganopoly over 20-100 mg/kg significantly increased cytotoxic T lymphocyte cytotoxicity and NK activity in mice. The overall findings indicated that Ganopoly had antitumor activity with a broad spectrum of immuno-modulating activities and may represent a novel promising immunotherapeutic agent in cancer treatment.

The development of a Defense System against Coxsackievirus B5 Infection in Suckling Mice

There are many viruses that are able to infect the alimentary tract of man. Little is known, however, about the mechanism of infection itself or the pathophysiology of the gut during infection. 'The research reported here is concerned with the differences in susceptibility among suckling mice of various ages inoculated by the intraperitoneal and intragastric routes. Since the normal mode of entry of many viruses to the gut is via the oral route, Coxsackievirus B5, a human enterovirus which does attack this way, was utilized. It is a non-tumor producing RNA virus that has been shown to act similarly in the mouse and human. The virus was pooled in HeLa cell cultures and titered by a plaquing assay in the same cell cultures. CD-l mice, 10, 14, 18, and 22 days old , were infected either orally or intraperitoneally with 5.0 x 10^10 (10 day old animals) and 1.0 x10^9 plaque forming units per animal. Dissections were done at 1 and 3 days post infection with samples of the blood, heart, liver, and gut being taken from each animal. Each sample was titered individually and the data presented as an average of six samples. As a result of previous work, it is known that the gut of a newborn mouse isn't able to decrease the concentration of the infecting dose and therefore provides no defense against an enteric infection with Coxsackievirus B5. In contrat, mature mice are able to reduce the amount of viral dissemination across the gut as well as inhibit replication after absorption has occurred. The results of this study indicate that there is a double barrier system developing in suckling mice that is involved with and directly related to the gastrointestinal tract The first part of this defense is the inhibition of penetration of virus across the gut when the primary site of' infection is the intestinal mucosa. This mechanism develops sometime around 20 to 22 days after birth. At about 16-18 days of age, suckling mice that were challenged intragastrically are able to stop active replication and initiate clearance of virus from the systemic circulation. There are many factors that might contribute to the marked decrease in susceptibility with age of suckling mice. Some of these or possibly a combination of these factors might explain the defense mechanisms described above, but to date, the chemistry or mechanical functioning of the gastrointestinal barrier to enteric viral infection is unknown.

Copper transport during lactation in transgenic mice expressing the human ATP7A protein

Both copper transporting ATPases, ATP7A and ATP7B, are expressed in mammary epithelial cells but their role in copper delivery to milk has not been clarified. We investigated the role of ATP7A in delivery of copper to milk using transgenic mice that over-express human ATP7A. In mammary gland of transgenic mice, human ATP7A protein was 10- to 20-fold higher than in control mice, and was localized to the basolateral membrane of mammary epithelial cells in lactating mice. The copper concentration in the mammary gland of transgenic dams and stomach contents of transgenic pups was significantly reduced compared to non-transgenic mice. The mRNA levels of endogenous Atp7a, Atp7b, and Ctr1 copper transporters in the mammary gland were not altered by the expression of the ATP7A transgene, and the protein levels of Atp7b and ceruloplasmin were similar in transgenic and non-transgenic mice. These data suggest that ATP7A plays a role in removing excess copper from the mammary epithelial cells rather than supplying copper to milk.

Mice lacking angiotensin-converting enzyme have increased energy expenditure, with reduced fat mass and improved glucose clearance

In addition to its role in the storage of fat, adipose tissue acts as an endocrine organ, and it contains a functional renin-angiotensin system (RAS). Angiotensin-converting enzyme (ACE) plays a key role in the RAS by converting angiotensin I to the bioactive peptide angiotensin II (Ang II). In the present study, the effect of targeting the RAS in body energy homeostasis and glucose tolerance was determined in homozygous mice in which the gene for ACE had been deleted (ACE^-/-) and compared with wild-type littermates. Compared with wild-type littermates, ACE^-/- mice had lower body weight and a lower proportion of body fat, especially in the abdomen. ACE^-/- mice had greater fed-state total energy expenditure (TEE) and resting energy expenditure (REE) than wild-type littermates. There were pronounced increases in gene expression of enzymes related to lipolysis and fatty acid oxidation (lipoprotein lipase, carnitine palmitoyl transferase, long-chain acetyl CoA dehydrogenase) in the liver of ACE^-/- mice and also lower plasma leptin. In contrast, no differences were detected in daily food intake, activity, fed-state plasma lipids, or proportion of fat excrete in fecal matter. In conclusion, the reduction in ACE activity is associated with a decreased accumulation of body fat, especially in abdominal fat depots. The decreased body fat in ACE^-/- mice is independent of food intake and appears to be due to a high energy expenditure related to increased metabolism of fatty acids in the liver, with the additional effect of increased glucose tolerance.

Bovine milk fat enriched in conjugated linoleic and vaccenic acids attenuates allergic airway disease in mice

Background: It has been argued that a reduction in the Western diet of anti-inflammatory unsaturated lipids, such as n-3 polyunsaturated fatty acids, has contributed to the increase in the frequency and severity of allergic diseases.

Objective: We investigated whether feeding milk fat enriched in conjugated linoleic acid and vaccenic acids (VAs) ('enriched' milk fat), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, will prevent development of allergic airway responses.

Methods: C57BL/6 mice were fed a control diet containing soybean oil and diets supplemented with milk lipids. They were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 14 and 28, and challenged intranasally with OVA on day 42. Bronchoalveolar lavage fluid, lung tissues and serum samples were collected 6 days after the intranasal challenge.

Results: Feeding of enriched milk fat led to marked suppression of airway inflammation as evidenced by reductions in eosinophilia and lymphocytosis in the airways, compared with feeding of normal milk fat and control diet. Enriched milk fat significantly reduced circulating allergen-specific IgE and IgG1 levels, together with reductions in bronchoalveolar lavage fluid of IL-5 and CCL11. Treatment significantly inhibited changes in the airway including airway epithelial cell hypertrophy, goblet cell metaplasia and mucus hypersecretion. The two major components of enriched milk fat, cis-9, trans-11 conjugated linoleic acid and VA, inhibited airway inflammation when fed together to mice, whereas alone they were not effective.

Conclusion: Milk fat enriched in conjugated linoleic and VAs suppresses inflammation and changes to the airways in an animal model of allergic airway disease.

Fatty acid composition in tissues of mice fed diets containing conjugated linolenic acid and conjugated linoleic acid

The influence of 1% alpha-eleostearic acid (α-ESA, cis9,trans11,trans 13-18:3) and 1% punicic acid (PA, cis9,trans11,cis13-18:3) on fatty acid composition in mouse tissues was compared with conjugated linoleic acid (CLA, mixture of primarily cis9,trans11- and trans10,cis12-18:2) in the present study. The content (% total fatty acids) of 18:2n-6 was significantly reduced in the heart and adipose tissues, and total polyunsaturated fatty acids (PUFAs) and n-6 PUFA were significantly reduced in adipose tissue by α-ESA, PA and CLA feeding. The content of 22:6n-3 and total n-3 PUFA were significantly increased in the liver, kidney and heart by PA feeding, but not by α-ESA. In contrast to PA, supplementation with CLA significantly decreased 22:6n-3 in the liver, kidney and heart. The content of 20:4n-6 was significantly decreased in the liver and kidney by CLA feeding, but not by α-ESA and PA. The present results indicate that α-ESA, PA and CLA have differential effects on 22:6n-3 and 20:4n-6 content in mouse tissues.