Dehydroepiandrosterone decreases elevated hepatic glucose production in C57BL/KsJ-db/db mice

Dehydroepiandrosterone (DHEA) is known to improve hyperglycemia in diabetic db/db mice that are obese and insulin resistant. In a previous study, we reported that DHEA suppresses the elevated hepatic gluconeogenic glucose-6-phosphatase (G6Pase) activity and gene expression in C57BL/KsJ-db/db mice. In the present study, we evaluated the total amount of gluconeogenesis using NaH[(14)C]CO(3) and hepatic glucose production using fructose as a substrate in primary cultured hepatocytes. Despite hyperinsulinemia, the glucose production of db/db mice in the total body and hepatocytes was elevated as compared to their heterozygote littermate C57BL/KsJ-db/+m mice. Administration of DHEA significantly decreased the blood glucose level and increased the plasma insulin level in db/db mice. Administration of DHEA decreased the elevated total body and hepatic glucose production in db/db mice. In addition, the glucose production in the primary cultured hepatocytes of db/db mice was decreased significantly by the direct addition of DHEA or DHEA-S to the medium. These results suggest that administration of DHEA suppresses the elevated total body and hepatic glucose production in db/db mice, and this effect on the liver is considered to result from increased plasma insulin and DHEA or DHEA-S itself.     

Dietary flavonoids suppress azoxymethane-induced colonic preneoplastic lesions in male C57BL/KsJ-db/db mice

Obesity is known to be a risk factor for colon carcinogenesis. Although there are several reports on the chemopreventive abilities of dietary flavonoids in chemically induced colon carcinogenesis, those have not been addressed in an obesity-associated carcinogenesis model. In the present study, the effects of 3 flavonoids (chrysin, quercetin and nobiletin) on modulation of the occurrence of putative preneoplastic lesions, aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCACs) in the development of colon cancer were determined in male db/db mice with obesity and diabetic phenotypes. Male db/db mice were given 3 weekly intraperitoneal injections of azoxymethane (AOM) to induce the ACF and BCAC. Each flavonoid (100 ppm), given in the diet throughout the experimental period, significantly reduced the numbers of ACF by 68–91% and BCAC by 64–71%, as well as proliferation activity in the lesions. Clinical chemistry results revealed that the serum levels of leptin and insulin in mice treated with AOM were greater than those in the untreated group. Interestingly, the most pronounced suppression of development of preneoplastic lesions and their proliferation were observed in the quercetin-fed group, in which the serum leptin level was lowered. Furthermore, quercetin-feeding decreased leptin mRNA expression and secretion in differentiated 3T3-L1 mouse adipocytes. These results suggest that the present dietary flavonoids are able to suppress the early phase of colon carcinogenesis in obese mice, partly through inhibition of proliferation activity caused by serum growth factors. Furthermore, they indicate that certain flavonoids may be useful for prevention of colon carcinogenesis in obese humans.     

Effect of mutant genes db(diabetes) and m(misty) on the severity of high-dose streptozotocin diabetes in C57bl/KsJY mice]

It is stated that high-dose streptozotocin diabetes is more serious in gene db-homozygous mice of mutant line C57BL/KsJY than in heterozygotes db +/+ m and mice M+/+m. Manifestation of spontaneous insulin-independent diabetes mellitus in homozygotes db decreases the level of hyperglycemic streptozotocin-induced reactions.     

Genistein and daidzein modulate hepatic glucose and lipid regulating enzyme activities in C57BL/KsJ-db/db mice

This study examines whether anti-diabetic effects of genistein and daidzein are mediated by hepatic glucose and lipid regulating enzyme activities in type 2 diabetic animals. Male C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and age-matched non-diabetic littermates (db/+) were used in this study. The db/db mice were divided into control, genistein (0.02%, w/w) and daidzein (0.02%, w/w) groups. The blood glucose and HbA(1c) levels were significantly lower in the genistein and daidzein groups than in the control group, while glucose tolerance only was significantly improved in the genistein-supplemented group. The plasma insulin and C-peptide levels did not differ significantly between groups, yet the glucagon level was lower in the genistein and daidzein groups compared to that in the control db/db or db/+ group. The genistein and daidzein supplements increased the insulin/glucagon ratio in the type 2 diabetic animals. While the hepatic glucokinase activity was significantly lower in the db/db control group, the glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities were significantly higher in the control group compared to the db/+ group. Interestingly, these hepatic glucose metabolizing enzyme activities were reversed by the genistein and daidzein supplementation in db/db mice compared to the control group. The hepatic fatty acid synthase, beta-oxidation and carnitine palmitoyltransferase activities were all significantly lower in the genistein and daidzein groups than in the control group. The genistein and daidzein supplements also improved the plasma total cholesterol, triglyceride, HDL-cholesterol/total cholesterol, free fatty acid and hepatic triglyceride concentrations in the db/db mice. These results suggest that genistein and daidzein exert anti-diabetic effect in type 2 diabetic conditions by enhancing the glucose and lipid metabolism.     

Effects of 5-campestenone (24-methylcholest-5-en-3-one) on the type 2 diabetes mellitus model animal C57BL/KsJ-db/db mice.

We examined the therapeutic effects of dietary exposure to 5-campestenone (24-methylcholest-5-en-3-one), an enone derivative of campesterol, in C57BL/KsJ-db/db mice, which are an animal model of obese type 2 diabetes. Blood glucose levels of db/db mice linearly increased from 270 to 720 mg/dl in 10 weeks, an approximately 7-fold difference from the levels of db/+m mice. The 0.3 % dietary exposure to 5-campestenone caused a marked reduction in blood glucose levels of 330 mg/dl after 10 weeks of feeding with a concomitant inhibition of glucose excretion in urine. Only slight efficacy was observed with 0.1 % dietary exposure to this chemical in db/db mice. Significant decreases of plasma triglyceride and plasma free fatty acid were also observed in db/db mice at a 0.3 % dose. However, feed efficiency and body-weight gain in db/db mice was improved by 5-campestenone. No obvious anomaly due to consumption of 5-campestenone was detected by necropsy or clinical observation.     

Hepatic phase II biotransformation in C57Bl/KsJ db/db mice: comparison to that in Swiss Webster and 129 REJ mice

1. Cytochrome P-450 concentrations were similar in male and female carrier (db/+) and diabetic (db/db) mice. Benzphetamine N-demethylase and styrene oxide hydrolase activities were 47 and 65% lower in db/+ than in db/db mice. 2. UDP-Glucuronosyltransferase activity toward 1-naphthol, estrone and diethylstilbestrol was not different between db/db and db/+, but was 40% higher in db/db mice toward testosterone. 3. Glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and ethacrynic acid was 47 and 59% lower in db/db mice than in male db/+ mice. Female db/+ mice had similar activities to those found in diabetic animals. 4. The differences in enzyme activity between hyperinsulinemic and normal animals suggest that insulin can influence both phase I and phase II biotransformations. 5. Enzyme activities in db/+ and db/db mice were compared to those in 129 REJ and Swiss Webster mice.     

The effect of irradiation on the development of streptozotocin diabetes in C57BL/KsJY mice heterozygous and homozygous for gene db (diabetes)

In experiments with C57BL/KsJY mice carrying a mutant db gene at the heterozygous and homozygous states it has been found that db+/+db mice are much more sensitive to gamma radiation with respect to the survival rate. In homozygotes, in contrast to heterozygotes, irradiation with a dose of 6 Gy does not reduce the severity of the diabetogenic effect of low doses of streptozotocin.     

MRNA and enzyme activity of hepatic 11beta-hydroxysteroid dehydrogenase type 1 are elevated in C57BL/KsJ-db/db mice.

To evaluate the importance of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in insulin resistant diabetic C57BL/KsJ-db/db mice, we measured the activity and mRNA level of 11beta-HSD1 in the liver of db/db mice and their heterozygote litter mates, db/+m mice. The blood glucose, plasma insulin, and corticosterone levels of db/db mice were significantly higher than those of db/+m mice. Despite hyperinsulinemia, the activity level of this enzyme was significantly higher in db/db mice, and the mRNA level of hepatic 11beta-HSD1 was also significantly higher in db/db mice. Since hepatic 11beta-HSD1 in vivo mainly functions as 11-keto-reductase and does not work as 11beta-oxidase, these results suggest that the rate of hepatic conversion of 11-dehydrocorticosterone to corticosterone is increased in db/db mice, resulting in higher glucocorticoid activity in the liver. The increased hepatic corticosterone concentration due to the elevation of 11beta-HSD1 and high plasma corticosterone concentration may antagonize the action of insulin and cause insulin resistance. These findings have a potentially important implication for relationships between increased hepatic 11beta-HSD1 and insulin resistance in db/db mice. The present paper is the first to demonstrate the increased activities and mRNA level of hepatic 11beta-HSD1 in db/db mice.     

Estrogenic restoration of functional pancreatic islet cytoarchitecture in diabetes (db/db) mutant C57BL/KsJ mice: relationship to estradiol localization,systemic glycemia,and persistent hyperinsulinemia

Cell and Tissue Research - The diabetes (db/db) genotype mutation induces a hyperglycemic–hyperinsulinemic endometabolic state in C57BL/KsJ mice, manifesting a type 2 NIDDM diabetes-obesity...     

Diabetes is not associated with a change in the elemental composition of the pancreatic B cell in diabetic C57BL KsJ-db/db mice

Freeze-dried pancreas sections from 7-, 17-and 27-week-old genetically diabetic (db/db) and normal (±/±) mice were subjected to proton bombardment and the concentrations of 15 elements in B cells and exocrine pancreas were calculated from the characteristic X-rays emitted. In the 7-week-old diabetic animals, B cells contained significantly above-normal levels of Na and S, while exocrine pancreas contained subnormal levels of Ca, and excess Mn. The B cells from the 17-week-old diabetic animals contained subnormal levels of Cu and the exocrine pancreas of the 27-week-old diabetic animals was deficient in Cd. The 7-, 17- and 27-week-old, genetically diabetic (db/db) mice were hyperglycemic, hyperinsulinemic and heavier than age-matched normal (±/±) mice. Although significant changes were found in elemental composition when comparing both B cells and exocrine pancreas at different ages, the changes were not consistent. Therefore, it appears as if the measured elemental changes were random and not related to the onset of diabetes.     

Prenatal expression of N-acetyltransferases in C57Bl/6 mice

Exposure to carcinogens such as 4-aminobiphenyl (4ABP), found in tobacco smoke and other combustion products, results in the formation of detectable levels of 4ABP-hemoglobin adducts in cord blood and 4ABP-DNA adducts in conceptal tissue. The presence of these adducts requires that the parent compound undergo biotransformation. When exposure occurs in utero, the maternal, placental and conceptal tissues are all possible sites for the formation of DNA-reactive products. One step in the activation of 4ABP is catalyzed by N-acetyltransferases (NAT). The expression of NAT was evaluated in gestational day (GD) 10-18 conceptal tissues from C57Bl/6 mice. There was a quantitative increase in NAT1 and NAT2 mRNAs with increasing gestational age that was also reflected in age-related changes in functional protein measured as 4ABP-NAT activity. The ability to acetylate 4ABP increased from GD10 to 18 and was lower in conceptal tissue than in adult liver. The potential toxicologic significance of prenatal NAT expression was assessed by formation of 4ABP-DNA adducts. At GD 15 and 18, 4ABP-DNA adducts were detected by immunohistochemistry 24 h following a single oral dose of 120 mg 4ABP/kg. Based on nuclear fluorescence, conceptual 4ABP-DNA adducts were present at similar levels at GD15 and 18. Levels of 4ABP-DNA adducts were significantly higher in maternal liver compared with the conceptus. Results from this study show that both NAT genes were expressed prenatally and that functional enzymes were present. These data support the possible in situ generation of reactive products by the conceptus. The relative contributions of maternal activation of 4ABP and that by the conceptus remain to be determined.     

Hepatic adrenergic receptors in the genetically diabetic C57 BL/KsJ (db/db) mouse

1. At 30 weeks of age, homozygote diabetic C57 BL KsJ (db/db) mice were grossly obese, lethargic and displayed moderate hair loss relative to heterozygote control C 57 BL KsJ (db/+) mice. 2. In diabetic mice, compared to control, the total body weights, liver weight: body weight ratios, and blood glucose levels were increased 2.3 fold, 20% and 3.1 fold, respectively. 3. Analysis of plasma membranes isolated from control and diabetic mouse liver established that comparable purity levels were achieved since relative specific activities of the plasma membrane markers 5'-nucleotidase and gamma-glutamyltranspeptidase were similar: 10.2 and 11.4 fold with respect to 5'-nucleotidase in control and diabetic states respectively; and 8.0 and 8.3 fold with respect to gamma-glutamyltranspeptidase in control and diabetic states respectively. 4. A select effect of diabetes on gamma-glutamyltranspepetidase, however, was observed. The activity of this enzyme was found to be reduced 16% in diabetic liver compared to control liver. 5. Assessment of [3H]prazosin and [3H]dihydrolalprenolol binding to mouse liver plasma membranes indicated that although there was no difference in beta-adrenergic receptor binding in control and diabetic states, alpha 1-adrenergic receptor binding was found to be reduced 43% in diabetic mouse liver plasma membranes. 6. Scatchard analyses of kinetic studies indicate that the reduction is a reflection of decreases in alpha 1-adrenergic receptor numbers with no change in alpha 1 receptor affinity in the diabetic state: since for diabetic and control liver plasma membranes, Kd values were 3.41 +/- 0.02 nM and 3.40 +/- 0.01 nM respectively; and Bmax were 650.12 +/- 16.44 fmol mg-1 and 380.76 +/- 12.92 fmol mg-1, respectively.     

Increased membrane immunoglobulin capping of B cells from C57Bl/6 lpr/lpr and C57Bl/6 nu/nu mice

When the capping of membrane immunoglobulin on spleen B cells from normal C57Bl/6 mice (B6) is taken as reference, a faster capping rate is found for cells of age-matched B6 mice which are congenic at the lymphoproliferation (lpr) or nude (nu) loci. Though both congenic strains can be characterized by an abnormal T-lineage cell content, the nature of the abnormality itself is very different since B6 nudes lack thymus-processed/influenced lymphocytes whereas B6 mice with the lpr phenotype suffer from an invasion of all lymphoid organs with cells of a particular T-cell subset. Moreover, the more "normal" capping rate of B cells from the double congenic B6 mice (nu/nu, lpr/lpr) is intriguing. Since other mice homozygous at the lpr locus (MRL-1) or at the nu locus (BALB/c nude) also cap faster than their congenic controls (MRL-n and BALB/c, respectively), the observed effects do not appear to depend on a peculiarity of the B6 genetic background. If the faster capping of B cells of nu congenic and of lpr congenic mice had a common origin, it might be that T cells would control in some way the mobility of B-cell membrane immunoglobulins: both congenic mice have in their spleen a very low proportion of mature T cells together with a very high proportion of prethymic/thymic immature T-cell types, either of which might affect B-cell behavioral responses to membrane immunoglobulin clustering.     

Development of nicotine preference in C57Bl/6 and CBA mice

C57BL/6 mice were shown to consume greater amounts of nicotine solution than CBA and random-bred mice. Extensive literature data on C57BL/6 mice preference for ethanol and opiates and the experimental results obtained suggest common genetic mechanisms responsible for the inclination of C57BL/6 mice to toxicomania.     

Morphological characteristics of liver and white adipose tissue during development of melanocortin obesity in C57Bl/6J Agouti yellow mutant mice

The changes of liver and white adipose tissue (WAT) morphology during development of melanocortin obesity in female Agouti yellow (genotype A Y/a) C57Bl/6J mice have been investigated. Mouse strain of a/a genotype was used as a control. Results have been compared with the hormonal and metabolic changes during development of obesity and type 2 diabete in Agouti yellow mice. The tissues obtained from mice of 8-, 11-, 15- and 22-week old have been analyzed. The morphology of liver and WAT of A(Y) and control animals did not differ during 15 weeks of age. 40% of A(Y) mice revealed liver steatosis (fatty liver) at the age of 22 weeks. In addition, elevation in the inflammatory and proliferating processes in the liver and severe inflammation in WAT has been observed in these animals. Since as early as 15 weeks old A(Y) mice demonstrated the appearance of insulin resistance characteristics we conclude that hormonal and metabolic abnormalities could play a role of the primary factor of pathological reorganization of liver and WAT morphology.     

Thymic dysfunction in the mutant diabetic (db/db) mouse

Thymic function has been explored in genetically diabetic homozygous C57BL/KsJ (db/db) mice by evaluating their serum thymic factor (FTS) levels with a rosette assay. As previously reported for other autoimmune mice (NZB or MRL/I mice), the age-dependent decline of FTS levels was significantly accelerated in diabetic mice when compared to heterozygous littermates. Furthermore, FTS inhibitory molecules were detected in db/db mouse sera (as early as 10 wk of age) as evaluated by their ability to absorb in vitro the activity of synthetic FTS in the rosette assay, and in vivo for their capacity to induce the disappearance of endogenous FTS when injected into normal mice. These inhibitors were shown to be immunoglobulins. Histologically, the thymus presented an accelerated involution starting with a cortical lymphocytic depletion and an increased number of Hassall's corpuscles. Ultrastructural studies showed alterations in thymic epithelial cells, mainly represented by an increasing number of cytoplasmic vacuoles. By means of indirect immunofluorescence with anti-FTS monoclonal antibodies, it was shown that the number of FTS+ cells was reduced in db/db mouse thymuses: at the age of 22 wk, diabetic mice had 10 times fewer FTS+ cells than heterozygotes of the same age. Taken together, these results indicate important abnormalities in the thymus of diabetic mice. It is possible that the associated lymphocyte dysfunction plays a role in the pathogenesis of the autoimmune disease presented by db/db mice.     

Effect of androgen insensitivity on diabetogenesis in db/db male mice with testicular feminization (Tfm)

In C3H mice, a major component of susceptibility to the diabetogenic action of an obesity mutation (diabetes, db) is male gender associated. We tested whether increased male susceptibility was an androgen receptor mediated process. C3H.SW/Lt-derived db/db males were rendered androgen-receptor function-deficient by introducing the testicular feminization (Tfm) mutation of the X-linked androgen receptor gene. The db/db Tfm/Y males (phenotypically female in appearance) developed severe diabetes indistinguishable from that observed in standard db/db X + Y males. Castration of standard C3H.SW/Lt-db/db males (producing mutants with normal androgen receptors but reduced serum testosterone) also failed to block the gender-enhanced susceptibility. In contrast, female db/db littermates exhibited a milder hyperglycemia, and were more resistant to pancreatic beta cell necrosis and islet atrophy than any of the groups of db/db males. Although these data indicated that male-enhanced sensitivity to diabetogenic stress was independent of circulating androgens, the possibility that the gender dimorphism is predicated upon tissue ratios of active estrogens to androgens in glucose-producing tissues such as liver is discussed.     

Stress-induced DNA fragmentation in cardiomyocytes of MDX and C57Bl mice

The study deals with apoptosis of cardiomyocytes of adult mdx mice. Our previous investigation of the total DNA from mdx mice myocardium in 0.5% agarose and 5% PAAG revealed middle sized DNA fragments about 64 kb only. The DNA electrophoresis in 5% PAAG after alpha-32P-dATP incorporation into DNA, fragments mediated by terminal transferase, showed no presence of smaller DNA fragments. The low size DNA fragments (0.2, 0.4 and 11.0 kb) were observed only following stress (a 5 min swimming in water bath) by means of alpha-32P-dATP incorporation mediated by TdT reaction. DNA fragmentation lasted for 48 h after the stress. A study of the total DNA extracts of C57B1 mice also demonstrated middle and low molecular sized DNA fragmentation 2 h after the stress under the same condition of the experiment. But during the next 48 h the signs of DNA fragmentation of the total DNA disappeared. The authors conclude that the first stage of apoptosis is a permanent property of cardiomyocytes of mdx mice.