High-dose benfotiamine rescues cardiomyocyte contractile dysfunction in streptozotocin-induced diabetes mellitus.

Diabetic cardiomyopathy is characterized by cardiac dysfunction. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse cardiomyocytes. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg ip). Fourteen days later, control and diabetic (fasting plasma glucose > 13.9 mM) mice were put on benfotiamine therapy (100 mg.kg(-1).day(-1) ip) for another 14 days. Mechanical and intracellular Ca2+ properties were evaluated in left ventricular myocytes using an IonOptix MyoCam system. The following indexes were evaluated: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening, resting and rise of intracellular Ca2+ in response to electrical stimulus, sarcoplasmic reticulum (SR) Ca2+ load, and intracellular Ca2+ decay rate (tau). Two- or four-week STZ treatment led to hyperglycemia, prolonged TPS and TR90, reduced SR Ca2+ load, elevated resting intracellular Ca2+ level and prolonged tau associated with normal PS, maximal velocity of shortening/relengthening, and intracellular Ca2+ rise in response to electrical stimulus. Benfotiamine treatment abolished prolongation in TPS, TR90, and tau, as well as reduction in SR Ca2+ load without affecting hyperglycemia and elevated resting intracellular Ca2+. Diabetes triggered oxidative stress, measured by GSH-to-GSSG ratio and formation of advanced glycation end product (AGE) in the hearts. Benfotiamine treatment alleviated oxidative stress without affecting AGE or protein carbonyl formation. Collectively, our results indicated that benfotiamine may rescue STZ-induced cardiomyocyte dysfunction but not AGE formation in short-term diabetes.     

Studies on semicarbazide-sensitive amine oxidase in patients with diabetes mellitus and in transgenic mice

Patients with diabetes mellitus and with vascular complications in particular, exhibit higher plasma activities of semicarbazide-sensitive amine oxidase (SSAO) compared to control subjects. It has been speculated that production of cytotoxic products of SSAO may cause endothelial damage and thus contribute to the development of diabetic vascular complications such as retino-, nephro-, and neuropathies as a result of SSAO activity.In order to explore the possibility that high SSAO activity contributes to the development of vascular complications in diabetes, we have performed two studies in patients with Type-2 diabetes quantifying plasma SSAO activity, HbA(1c), and urinary levels of the SSAO substrate, methylamine. We also examined the prevalence of retinopathy in these patients. Additionally, we have studied a model of transgenic mice expressing human SSAO in smooth muscle cells. The transgenic mice have an increased SSAO activity as well as mRNA expression. Histological studies revealed a specific aorta phenotype with a condensed and rigid vessel wall in some of the transgenic mice. No wild-type animals displayed this phenotype.In conclusion, we suggest that this transgenic mouse model may be of great value for increasing the knowledge about to what extent human SSAO contributes to vascular complications in diabetes, and also to which extent inhibition of SSAO can prevent the development of such complications.     

Changes in brain metallothionein and Zinc during development in transgenic mice

The developmental alterations in metallothionein (MT) proteins and zinc (Zn) were investigated in brains of two transgenic strains of mice. MT protein was measured by a cadmium binding assay and Zn by atomic absorption spectrophotometry. MT proteins were expressed at birth (day 1) both in MT-I overexpressing transgenic mouse (MT-I*) and MT-null (expressing only brain specific isoform, MT-III) transgenic mouse. MT proteins level (mainly MT-I) in MT-I* was 16.1 Μ-g/g at birth, and thereafter increased with age to a maximal adult level of 55.3 Μg/g (day 60). Zn level in MT-I* also increased from 8.43 Μg/g (day 1) to 20.7 Μg/g (day 60) with age. MT protein (MT-III) in MT-null mouse was 9.71 Μg/g at birth and remained relatively unchanged during development. Zn level in MT-null mouse at birth was 9.46 Μg/g and also remained unchanged during development. The similar alterations in MT isoforms and Zn in brain during development suggest that MT isoforms may act as a Zn binding protein.     

Insulin-dependent diabetes mellitus induced in transgenic mice by ectopic expression of class II MHC and interferon-gamma

We have produced transgenic mouse strains harboring class II major histocompatibility complex or interferon-gamma genes linked to the human insulin promoter. These experiments were designed to investigate the consequences of the expression of immunological effector molecules by nonimmunological cells. In both of these studies we observed the disappearance from the pancreas of the insulin-producing beta cells coinciding with the development of insulin-dependent diabetes mellitus. Transgenic mice expressing both chains of the I-A gene showed progressive atrophy of the islets of Langerhans, whereas mice expressing interferon-gamma suffered an inflammatory destruction of the islets.     

The effect of diabetes mellitus on the ventricular epicardial activation and repolarization in mice

Cardiac repolarization is prolonged in diabetes mellitus (DM), however the distribution of repolarization durations in diabetic hearts is unknown. We estimated the ventricular repolarization pattern and its relation to the ECG phenomena in diabetic mice. Potential mapping was performed on the anterior ventricular surface in healthy (n=18) and alloxan-induced diabetic (n=12) mice with the 64-electrode array. Activation times, end of repolarization times, and activation-recovery intervals (ARIs) were recorded along with limb lead ECGs. ARIs were shorter in the left as compared to right ventricular leads (P<0.05). The global dispersion of repolarization, interventricular and apicobasal repolarization gradients were greater in DM than in healthy animals (P<0.03). The increased dispersion of repolarization and apicobasal repolarization gradient in DM correlated with the prolonged QTc and Tpeak-Tend intervals, respectively. The increased ventricular repolarization heterogeneity corresponded to the electrocardiographic markers was demonstrated in DM.     

Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice.

DNA regions of 10 and 7 kb that flank the mouse metallothionein II (MT-II) and MT-I genes, respectively, were combined with a minimally marked MT-I (MT-I*) gene and tested in transgenic mice. This construct resulted in (i) position-independent expression of MT-I* mRNA and copy number-dependent expression, (ii) levels of hepatic MT-I mRNA per cell per transgene that were about half that derived from endogenous MT-I genes, (iii) appropriate regulation by metals and hormones, and (iv) tissue distribution of transgene mRNA that resembled that of endogenous MT-I mRNA. These features were not observed when MT-I* was tested without the flanking regions. These MT-I flanking sequences also improved the expression of rat growth hormone reporter genes, with or without introns, that were under the control of the MT-I promoter. Moreover, they enhanced expression from two of four heterologous promoters/enhancers that were tested. Deletion analysis indicated that regions known to have DNase I-hypersensitive sites were necessary but not sufficient for high-level expression. These data suggest that the DNA regions flanking the mouse MT-I and MT-II genes have functions like the locus control regions described for other genes.     

Male sterility of transgenic mice carrying exogenous mouse interferon-beta gene under the control of the metallothionein enhancer-promoter.

As an approach to elucidating the roles of interferon (IFN) in the normal physiology and diseases of animals, transgenic mice carrying extra mouse IFN-beta genes under the control of a mouse metallothionein I enhancer-promoter were constructed. Upon induction with Cd2+, IFN activity (15-430 IU/ml) was detected in the sera of six out of ten transgenic mouse lines so far obtained. Synthesis of mRNA of the transgene was observed in the liver, the testis and less abundantly in the brain. Interestingly, IFN mRNA was constitutively synthesized in the testis where substantial levels of IFN accumulated without heavy metal induction, whereas synthesis in the liver was mostly dependent on induction by CD2+. Since IFN activity in the serum also depended on heavy metal induction, the IFN in the serum may be produced mainly in the liver. All males expressing the IFN gene in the testis were found to be sterile. Testes were involuted and contained few mature sperm, and degeneration of spermatocytes and spermatids was observed. These findings suggest that high levels of IFN are harmful to spermatogenesis and can cause male sterility.     

Renal function and glucose transport in male and female mice with diet-induced type II diabetes mellitus

The aim of this study was to measure cardiovascular and renal function, including the renal transport capacity for glucose, in male and female C57BL/6J mice with diet-induced Type II diabetes mellitus. Typical of Type II diabetes, mice fed a high-fat, high-simple carbohydrate diet for 3 months were obese (45-65 g), hyperglycemic (138-259 mg%), and hyperinsulinemic (1.8-15.06 ng/ml); significant gender differences were observed in all cases. Based on systolic pressure measurements in conscious mice and arterial blood pressure measurements in anesthetized mice, no diet-induced hypertension was observed in either male or female mice. Urine flow rate, sodium, potassium, osmolar, and protein excretion rates were significantly increased (P < 0.05) in male mice fed the high-fat, high-simple carbohydrate diet compared with female mice fed the same diet. However, no differences in the excretion variables existed between male and female mice fed the control diet. The glomerular filtration rate (ml min-1 g kw-1), determined by FITC-inulin, in male and female mice fed the control diet (0.87 +/- 0.01 and 0.90 +/- 0.1, respectively) and high-fat, high-simple carbohydrate diet (0.96 +/- 0.1 and 0.93 +/- 0.2, respectively) was not different between the groups. These hyperglycemic mice were also not glucosuric. Infusions of progressive amounts of glucose in male mice fed either diet for 3 or 6 months demonstrated that the renal threshold for glucose was 400 mg% for all these mice, well above the fasting plasma glucose concentrations observed in this study. Thus, C57BL/6J mice were valuable tools for studying diet-induced obesity, hyperglycemia, and hyperinsulinemia; however, no hypertension or kidney dysfunction was apparent within the time frame of the current study.     

Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice.

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.     

Virus-induced diabetes mellitus: mengovirus infects pancreatic beta cells in strains of mice resistant to the diabetogenic effect of encephalomyocarditis virus.

In mice, Mengovirus produces a fatal encephalitis. Plaque purification of the virus resulted in the isolation of a clone (Mengo- 2T ), which in addition to encephalitis caused diabetes. Microscopic examination of pancreases from infected mice revealed necrosis in the islets of Langerhans and infiltration of inflammatory cells. By immunofluorescence viral antigens were found in the islets, and radioimmunoassays demonstrated a substantial decrease in pancreatic immunoreactive insulin. Studies on susceptibility among inbred strains of mice showed that whereas the D variant of encephalomyocarditis virus caused diabetes only in SJL/J mice, Mengo- 2T caused diabetes in strains of mice resistant to encephalomyocarditis-induced diabetes (i.e., CBA/J, C3H/HeJ, CE/J, AKR/J, C57BL/6J). The ability of Mengo- 2T to induce diabetes in encephalomyocarditis-resistant mice was found to be due to the greater capacity of Mengo- 2T as compared to the D variant of encephalomyocarditis virus to replicate in and destroy the islets of these animals. Although Mengo- 2T and the D variant of encephalomyocarditis virus are antigenically indistinguishable by hyperimmune sera, our studies show that these viruses have different host ranges and tissue tropisms .     

Variability of the cardiomyocyte ploidy in normal human hearts.

We have performed cytophotometry for DNA in isolated myocytes of the left ventricle from 16 men, aged 19-39 years, who died from various non-cardiac or pulmonary causes. The mean ploidy of myocytes varied from 3.2-3.9 c to 6.6-7.3 c in different layers of the anterior wall of the left ventricle (where c is the haploid DNA content measured by cytophotometry in Feulgen-stained preparations). There was no correlation between the layers. The percentage of binuclear cells varied from 25 to 86% and correlated in every layer with the mean ploidy value of the whole myocyte population. Approximate calculation of total ploidy revealed low values in the ventricles of some individuals, and high values in others. Averaging the values for all the hearts studied obscures this variation. Mean myocyte ploidy in different layers of the anterior wall was similar: in the external layer it was 5.1 +/- 0.3 c, in the middle layer 5.5 +/- 0.3 c and in the inner layer 4.8 +/- 0.4 c. The mean percentage of binuclear myocytes in these three layers was also similar, being 61 +/- 3%, 63 +/- 4% and 54 +/- 5%, respectively. Myocyte ploidy in tissue from the posterior wall of the left ventricle also varied, but was always higher than for the same layer of the anterior wall in the same ventricle. We propose that high or low myocyte ploidy, as well as different proportions of mono- and binucleate cells, can be a factor affecting the course and result of cardiac pathology in the absence of any changes of myocyte genome determined during early ontogenesis and representing a stable characteristic of the individual.     

Hepatic and renal expression of rat apolipoprotein E under control of the metallothionein promoter in transgenic mice

We created three lines of transgenic mice with an integrated rat genomic apolipoprotein E gene fused with the mouse metallothionein I promoter. These lines transcribed rat apoE mRNA in the liver and/or in the kidney and expressed significant amounts of rat apoE in plasma. Enhancement of the plasma level by treatment with Zn ion or Bi ion was observed.     

Aberrant melanogenesis and melanocytic tumour development in transgenic mice that carry a metallothionein/ret fusion gene.

We generated four independent transgenic mouse lines that showed severe melanosis of the whole body by introducing the ret oncogene fused to the mouse metallothionein (MT)-I promoter-enhancer (MT/ret). Whereas melanogenesis was accelerated without distinct proliferative disorders in one line, melanocytic tumours frequently developed in the other three lines. Northern hybridization and in situ hybridization analyses showed that tumour cells and non-tumorous melanin-producing cells expressed the transgene at high levels. The aberrant melanogenesis and tumour development were influenced by genetic and environmental factors. Furthermore, crossbreeding experiments between the transgenic mice and Wv mice suggested that the ret gene product can partially compensate for the defect of melanocyte development in Wv mice. This is a novel mammalian model in which melanosis and melanocytic tumours develop stepwise, triggered by a single transgene.     

Sex difference in the effect of obesity on 24-hour mean serum gonadotropin levels.

To determine the effect of obesity on serum gonadotropin levels and any possible sex difference in the effect, we measured the 24-hour mean serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in 62 healthy men with Body Mass Index (BMI) ranging from 20 - 94 and 61 healthy, regularly cycling women with BMIs ranging from 19 - 76. We also measured free testosterone (T) and estradiol (E2) in these subjects. There was a significant negative correlation between serum FSH and BMI in men: FSH(IU/L) = 49.9 x BMI -0.567; r = - 0.376, p = 0.0026; but a significant positive correlation between serum FSH and BMI in women: FSH(IU/L) =7.66 +/- 0.071 x BMI; r = 0.302, p = 0.018. Serum LH was weight-invariant in both sexes. In men, free T was negatively correlated with BMI: Free T (nmol/L) = 0.74 - 0.0068 x BMI; r = 0.585, p = 0.0381; and free E2 was positively correlated with BMI: Free E2 (pmol/L) = - 1.03 +/- 0.057 x BMI; r = 0.50, p = 0.0014. In obese women as a group, free T was higher than in lean women (33 +/- 6.8 S.E.M. vs. 17.4 +/- 2.0 pmol/L; p < 0.0001), and free E2 was also higher than in lean women: (6.90 +/- 0.80 vs. 4.84 +/- 0.55 pmol/L; p = 0.046). Of the many cases of hypothalamic-pituitary hormonal dysregulation that have been reported in obesity, none has been studied for sex differences. Our results mandate that possible sex differences be investigated in all cases of dysregulation.     

Selective up-regulation of macrophage function in granulocyte-macrophage colony-stimulating factor transgenic mice.

Peritoneal and pleural cells from mice transgenic for GM-CSF were studied with regard to their phenotype and functional capacity, and compared with cells from normal littermates. Transgenic mice showed markedly elevated peritoneal and pleural cell counts compared with littermates, and a significantly higher proportion of cells in the transgenic populations were macrophage in phenotype. Transgenic macrophages were larger than the littermate cells, showing abundant foamy cytoplasm and enhanced spreading on plastic. Analysis by flow cytometry showed a more than sixfold increased expression of the macrophage activation markers MAC-2 and MAC-3, but not other markers, on transgenic macrophages. Superoxide production was measured in whole cell populations, both in their basal state and in response to particulate (zymosan) and soluble (PMA) stimuli. Both basal and stimulated superoxide production were markedly elevated in transgenic mice of 12 wk of age, with the largest differences seen in response to PMA. In younger mice, however, only PMA-stimulated superoxide production was significantly greater in transgenic macrophages than in littermate cells and levels of superoxide were generally lower than those seen in 12-wk-old mice. These findings suggest that the enhanced functional capacity of transgenic cells is a maturation-dependent event. In contrast to these findings, drug-dependent cytotoxicity assays performed on cells from 12-wk-old mice revealed no significant differences in killing capacity between the two mouse strains. Taken together these data indicate a selective rather than uniform functional up-regulation in transgenic macrophages compared with their littermates, with a time scale suggestive of a maturational rather than activation process. These findings may provide an indication of the functional macrophage phenotype resulting from long term exposure to GM-CSF in vivo, and help to explain the macrophage-associated pathology seen in GM-CSF-transgenic mice.