Glucagon and insulin relationships in genetically diabetic (db/db) and in streptozotocin-induced diabetic mice.

An inappropriate molar ratio of circulating insulin to glucagon is frequently associated with the metabolic alterations accompanying diabetes mellitus. Plasma immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) levels were determined and the IRG:IRI ratio calculated at various intervals in overt diabetes in genetically diabetic (db/db) and in streptozotocin-treated mice. Plasma IRI levels in genetic mutants are elevated at nine weeks of age, but are comparable to values found in lean littermates by 21 weeks. The presence of a prevailing hyperglucagonemia is established for the first time in the intact db/db mice. Streptozotocin diabetics are found to have characteristically low plasma IRI and high plasma IRG values. The hormonal imbalance present in these two experimental animal models is accentuated when the data are expressed as the IRG:IRI ratio, which is seen to increase with the progression of diabetes.     

Adrenalectomy and steroid treatment in obese (ob/ob) and diabetic (db/db) mice

Adrenalectomy in young obese (ob/ob) and the diabetic (db/db) mouse slowed body weight gain. Treatment of adrenalectomized ob/ob mice with cortisone or deoxycorticosterone acetate (DOCA) significantly increased weight gain in a dose-related manner. Cortisone had no effect on weight gain on lean mice and treatment with dehydroepiandrosterone sulfate was without effect on either ob/ob or lean mice. The increment in body weight of adrenalectomized ob/ob mice treated with corticosterone and DOCA was associated with an increase in body weight and an increase in food intake. When adrenalectomy was performed at twenty-three days of age (five days before weaning), animals carrying the (db/db) genotype remained lighter than their normal littermates. These data document the importance of the adrenal gland and its steroids for the development and maintenance of many features of the obese or diabetes mouse.     

Effect of immunodeficiency on diabetogenesis in genetically diabetic (db/db) mice

The pathogenesis of diabetes in C57BL/KsJ-db/db mice has been proposed to entail autoimmune mechanisms. We have combined immunodeficiency genes with the db mutation to determine whether beta cell necrosis and establishment of severe diabetes would occur in the absence of normal T and/or B lymphocyte functions. Inbred mice carrying the recessive mutations, severe combined immunodeficiency (scid), X-linked immunodeficiency (xid), nude (nu), and the Y-linked autoimmune accelerator (Yaa), were crossed with strains congenic for the db mutation. The diabetes syndrome was studied in double homozygotes produced in the F2 generation. In another experiment, C57BL/KsJ-db/db males were made T cell function deficient by adolescent thymectomy followed by lethal irradiation and bone marrow reconstitution. None of these manipulations served to prevent the induction of a severe diabetes syndrome in any of the model systems analyzed. Thus, diabetogenesis characterized by massive necrosis of the pancreatic beta cells and atrophy of the pancreatic islets was observed in both the absence of normal T cell function (as assessed by absence of T cell mitogen response) and humoral autoimmunity against beta cell antigens (insulin, retroviral p73). In conclusion, our data indicate that anti-beta cell autoimmunity is not a primary event in the etiopathogenesis of diabetes in the db/db mouse.     

Pulmonary administered palmitic-acid modified exendin-4 peptide prolongs hypoglycemia in type 2 diabetic db/db mice

Hypoglycemia caused by palmitic-acid modified exendin-4 (Pal-Ex4) administered via the pulmonary route was evaluated and compared with that caused by native Ex4. Pal-Ex4 and Ex4 in solution (each 50 μl) were administered using a microsprayer directly into the trachea of type 2 diabetic db/db mice at 75 or 150 nmol/kg. The lung depositions of Cy5.5-labeled Ex4 or Pal-Ex4 were monitored using an infrared imaging system after administration. The hypoglycemia caused by Pal-Ex4 was found to be 3.4 and 2.3 times greater than that caused by native Ex4 at 75 and 150 nmol/kg, respectively. Furthermore, time to blood glucose level (BGL) rebound to >150 mg/dl for Pal-Ex4 was 3.5 times greater than that of Ex4 (18.1 h vs. 5.2 h at 150 nmol/kg). In particular, the time taken for Pal-Ex4 to reach a BGL nadir was significantly greater than that of Ex4 (~8 h versus 4 h). Furthermore, lung deposition images clearly showed that Pal-Ex4 was slowly absorbed from lungs and barely distributed into kidneys until 8 h post-administration. It is likely that the prolonged hypoglycemia exhibited by Pal-Ex4 was due to; (i) delayed absorption in the lungs and (ii) albumin-binding in the circulation. The study demonstrates that palmitic acid-modified exendin-4 should be viewed as a long-acting inhalation candidate for the treatment of type 2 diabetes.     

Sleep-wake regulation is altered in leptin-resistant (db/db) genetically obese and diabetic mice

Recent epidemiological and clinical studies indicate that the control of sleep-wake states may be an important factor in the regulation of energy metabolism. Leptin is a peripherally synthesized hormone that has critical signaling properties in the brain for the control of long-term energy homeostasis. In this study, we examined the hypothesis that leptin signaling exerts a role in sleep-wake regulation and that leptin may represent an important mechanistic link in the coordination of sleep-wake states and metabolism. Sleep-wake patterns were recorded in a genetic mouse model of obesity and diabetes, the db/db mouse, which harbors a mutation in a particular isoform of the leptin receptor (long form, LRb). We found that db/db mice exhibit a variety of alterations in sleep regulation, including an increase in overall sleep time, a dramatic increase in sleep fragmentation, attenuated diurnal rhythmicity in rapid eye movement sleep and non-rapid eye movement EEG delta power (a measure of sleep homeostatic drive), and a decrease in the compensatory response to acute (i.e., 6 h) sleep deprivation. The db/db mice also generated low amounts of locomotor activity and a reduction in the diurnal rhythm of activity. These results indicate that impaired leptin signaling has deleterious effects on the regulation of sleep amount, sleep architecture, and temporal consolidation of these arousal states. In summary, leptin may represent an important molecular component in the integration of sleep, circadian rhythms, and energy metabolism.     

Impaired response of UCP family to cold exposure in diabetic (db/db) mice

Impaired activity of the uncoupling protein (UCP) family has been proposed to promote obesity development. The present study examined differences in UCP responses to cold exposure between leptin-resistance obese (db/db) mice and their lean (C57Ksj) littermates. Basal UCP1 and UCP3 mRNA expression in brown adipose tissue was lower in obese mice compared with lean mice, but UCP2 expression in white adipose tissue (WAT) was higher. Basal skeletal muscle UCP3 did not change remarkably. The UCP family mRNAs, which were upregulated 12 and 24 h after cold exposure (4 degrees C), were returned to prior levels 12 h after rewarming exposure (21 degrees C) in lean mice. The accelerating effects of cold exposure on the UCP family were impaired in db/db obese mice. Together with these changes, WAT lipoprotein lipase mRNA was downregulated, and the concentration of serum free fatty acid was increased in response to cold exposure in the lean mice but not in db/db obese littermates. The impaired function of the UCP family and diminished lipolysis in response to cold exposure indicate that the reduced lipolytic activity may contribute to the inactivation of the UCP family in db/db obese mice.     

Glucosyl and galactosyl transferase activities of diabetic (db/db) and obese (ob/ob) mice kidneys

• 1.1. Glucosyl and galactosyl activities were determined in kidney cortex tissue prepared from two strains of mice, genetically diabetic and obese mice.
• 2.2. These activities were measured as a function of ageing between 6 weeks and 13 months.
• 3.3. For both strains glucosyl transferase activity was shown to increase with respect to ageing whereas galactosyl transferase activity decreased at the same time.
• 4.4. These changes of enzymatic activities would suggest that a smaller increase of hydroxylysine-linked glycans than expected was observed under these pathological conditions.

     

Lipogenesis in genetically diabetic (db/db) mice: developmental changes in brown adipose tissue, white adipose tissue and the liver

Developmental changes in lipogenesis have been examined in interscapular brown adipose tissue (BAT), epididymal white adipose tissue and the liver of genetically diabetic (db/db) mice and their normal siblings. Lipogenesis was measured in vivo with 3H2O, from weaning (21 days of age) until 20 weeks of age. Hyperinsulinaemia was evident in db/db mice at all ages. Low rates of lipogenesis were observed at weaning in tissues of both groups of mice, but the rate rose rapidly in the first few days post-weaning. In normal mice, peak lipogenesis was obtained in each tissue at 4-5 weeks of age, and there were no major changes (on a whole-tissue basis) thereafter. A different developmental pattern was apparent in db/db mice. The rate of lipogenesis in BAT rose sharply after weaning, reaching a peak at 26 days of age (several times higher than normal mice), and then falling rapidly such that by 45 days of age it was lower than in normal mice; at age 20 weeks lipogenesis in BAT of the diabetic animals was negligible. In white adipose tissue of the db/db mutants lipogenesis (per tissue) reached a maximum at 5 weeks of age, and fell substantially between 10 and 20 weeks of age. Hepatic lipogenesis in the db/db mice rose progressively from weaning until 8 weeks of age, and then decreased. Except at weaning, hepatic lipogenesis (per tissue) was much greater in db/db mice than in normal mice, and the liver was a more important site of lipogenesis in diabetic mice than in normals, accounting for up to 60% of the whole-body total. In contrast, BAT accounted for a considerably smaller proportion of whole-body lipogenesis in db/db mice than in normal mice. It is concluded that there are major developmental differences in lipogenesis between tissues of db/db mice, and between diabetic and normal animals. The data suggest that there is an early and preferential development of insulin resistance in BAT of the db/db mutant.     

Impairment of cell-mediated immunity in mutation diabetic mice (db/db).

Mutation diabetes in the mouse occurs in the C57BL/Ks strain. All homozygous animals (db/db) develop obesity, elevated blood sugar levels and increased or normal blood insulin concentration. The defects in cellular immunity in db/db mice and their littermate controls were examined both in vivo and in vitro. Significant suppression of delayed footpad swelling and first and second set skin allograft rejection time were observed. In addition, DNA synthesis in spleen cells after nonspecific mitogen stimulation was markedly inhibited. Diabetic animals with a mean blood sugar of 512 +/- 101 mg/100 ml did not respond to exogenous insulin therapy by lowering their blood sugar levels or reversing the defect in DNA synthesis. Adding insulin to spleen cell culture in vitro had no demonstrable effect on their response to mitogens. Thus, mutation diabetic mice with their known defect in the peripheral utilization of insulin have markedly suppressed cell-mediated immune mechanisms.     

Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice

Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-(3)H]glucose, [U-(14)C]glucose, and [9,10-(3)H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts.     

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.     

Curcumin restores mitochondrial functions and decreases lipid peroxidation in liver and kidneys of diabetic db/db mice

Background

Nitrosative and oxidative stress play a key role in obesity and diabetes-related mitochondrial dysfunction. The objective was to investigate the effect of curcumin treatment on state 3 and 4 oxygen consumption, nitric oxide (NO) synthesis, ATPase activity and lipid oxidation in mitochondria isolated from liver and kidneys of diabetic db/db mice.

Results

Hyperglycaemia increased oxygen consumption and decreased NO synthesis in liver mitochondria isolated from diabetic mice relative to the control mice. In kidney mitochondria, hyperglycaemia increased state 3 oxygen consumption and thiobarbituric acid-reactive substances (TBARS) levels in diabetic mice relative to control mice. Interestingly, treating db/db mice with curcumin improved or restored these parameters to normal levels; also curcumin increased liver mitochondrial ATPase activity in db/db mice relative to untreated db/db mice.

Conclusions

These findings suggest that hyperglycaemia modifies oxygen consumption rate, NO synthesis and increases TBARS levels in mitochondria from the liver and kidneys of diabetic mice, whereas curcumin may have a protective role against these alterations.     

Prevention of diabetic nephropathy by treatment with astaxanthin in diabetic db/db mice

Oxidative stress is implicated as an important mechanism by which diabetes causes nephropathy. Astaxanthin, which is found as a common pigment in algae, fish, and birds, is a carotenoid with significant potential for antioxidative activity. In this study, we examined whether chronic administration of astaxanthin could prevent the progression of diabetic nephropathy induced by oxidative stress in mice. We used female db/db mice, a rodent model of type 2 diabetes, and their non-diabetic db/m littermates. The mice were divided into three groups as follows: non-diabetic db/m, diabetic db/db, and diabetic db/db treated with astaxanthin. Blood glucose level, body weight, urinary albumin, and urinary 8-hydroxydeoxyguanosine (8-OHdG) were measured during the experiments. Histological and 8-OHdG immunohistochemical studies were performed for 12 weeks from the beginning of treatment. After 12 weeks of treatment, the astaxanthin-treated group showed a lower level of blood glucose compared with the non-treated db/db group; however, both groups had a significantly high level compared with the db/m mice. The relative mesangial area calculated by the mesangial area/total glomerular area ratio was significantly ameliorated in the astaxanthin-treated group compared with the non-treated db/db group. The increases in urinary albumin and 8-OHdG at 12 weeks of treatment were significantly inhibited by chronic treatment with astaxanthin. The 8-OHdG immunoreactive cells in glomeruli of non-treated db/db mice were more numerous than in the astaxanthin-treated db/db mice. In this study, treatment with astaxanthin ameliorated the progression and acceleration of diabetic nephropathy in the rodent model of type 2 diabetes. The results suggested that the antioxidative activity of astaxanthin reduced the oxidative stress on the kidneys and prevented renal cell damage. In conclusion, administration of astaxanthin might be a novel approach for the prevention of diabetes nephropathy.     

Anti-diabetic properties of genistein-chromium (III) complex in db/db diabetic mice and its sub-acute toxicity evaluation in normal mice

BackgroundIn this study, chromium (III) complex was synthesized from genistein (GEN) which had good hypoglycemic activity and inorganic chromium (III) element, and its hypoglycemic activity and sub-acute toxicity were studied.MethodsThe genistein-chromium (III) complex was synthesized by chelating chromium with genistein in ethanol and its structure was determined by LC–MS, atomic absorption spectroscopy, UV–vis spectroscopy, infrared spectroscopy, elemental and thermodynamic analysis. The anti-diabetic activity of the complex was assessed in db/db mice and C57 mice by daily oral gavage for 4 weeks. The sub-acute toxicity test was carried out on KM mice with this complex.ResultsThe molecular structure of this complex was inferred as a complex [CrGEN₃] formed by three ligands and one chromium element. The complex could significantly improve the body weight of db/db mice, fasting blood glucose, random blood glucose, organ index, glycogen levels and the performance of OGTT (Oral Glucose Tolerance Test) and ITT (Insulin Tolerance Test) in db/db mice (p < 0.05). The morphology of liver, kidney, pancreas and skeletal muscle also had obviously improvement and repairment. Effects on serum indices and antioxidant enzymes activities of db/db mice showed that the serum profiles and antioxidant ability of complex group had significant improvement compared with the diabetic control group (p < 0.05 or p < 0.01), and some indices even returned to normal levels. In addition, this complex did not produce any hazardous symptoms or deaths in sub-acute toxicity test. High dose of [CrGEN₃] had no significant influence on serum indices and antioxidant capacity in normal mice, and the organ tissues maintained organized and integrity in the sub-acute toxicity study.ConclusionThe study of the genistein-chromium (III) complex showed that the complex had good hypoglycemic activity in vivo, and did not have the potential toxicity. These results would provide an important reference for the development of functional hypoglycemic foods or pharmaceuticals.     

Echocardiographic assessment of cardiac function in diabetic db/db and transgenic db/db-hGLUT4 mice

Control db/+ and diabetic db/db mice at 6 and 12 wk of age were subjected to echocardiography to determine whether contractile function was reduced in vivo and restored in transgenic db/db-human glucose transporter 4 (hGLUT4) mice (12 wk old) in which cardiac metabolism has been normalized. Systolic function was unchanged in 6-wk-old db/db mice, but fractional shortening and velocity of circumferential fiber shortening were reduced in 12-wk-old db/db mice (43.8 +/- 2.1% and 8.3 +/- 0.5 circs/s, respectively) relative to db/+ control mice (59.5 +/- 2.3% and 11.8 +/- 0.4 circs/s, respectively). Doppler flow measurements were unchanged in 6-wk-old db/db mice. The ratio of E and A transmitral flows was reduced from 3.56 +/- 0.29 in db/+ mice to 2.40 +/- 0.20 in 12-wk-old db/db mice, indicating diastolic dysfunction. Thus a diabetic cardiomyopathy with systolic and diastolic dysfunction was evident in 12-wk-old diabetic db/db mice. Cardiac function was normalized in transgenic db/db-hGLUT4 mice, indicating that altered cardiac metabolism can produce contractile dysfunction in diabetic db/db hearts.