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.     

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.     

Possible involvement of corticosterone in bone loss of genetically diabetic db/db mice.

The etiology of bone loss in non-insulin dependent diabetes mellitus is still unknown. We compared serum biochemical parameters and bone parameters of genetically diabetic db/db mice with those of their control non-diabetic +/+ mice. We found that serum corticosterone levels of the db/db mice were significantly elevated after 5 weeks while bone mineral density of femur metaphysis significantly decreased in the db/db mice after 12 weeks of age compared with age matched +/+ mice. To explore the causal relationship between the serum corticosterone levels and the bone loss, metyrapone (100 mg/kg, p.o., twice a day), a glucocorticoid synthesis inhibitor, was administered to these mice for 4 weeks after the age of 8 weeks. The compound significantly decreased serum corticosterone levels in both strains. Metyrapone prevented bone loss by increasing the bone mineral content of the metaphysis in the db/db mice. In addition, the treatment slightly improved the ratio of ash weight to dry weight in the db/db mice. These results suggest that increased serum corticosterone levels are concerned with the etiology of bone loss in non-insulin dependent diabetic db/db mice.     

Effects of oxytetracycline treatment on enzymes of hepatic glycogen metabolism in genetically diabetic (db/db) mice

The effects of daily oxytetracycline treatment on the activities of hepatic glycogen synthase, glycogen phosphorylase, plasma glucose, and insulin, and on liver glycogen, free fatty acid, and triglyceride levels were examined in 8- to 15-week-old genetically diabetic and lean mice. Oxytetracycline administration resulted in substantial reductions in the plasma glucose and immunoreactive-insulin levels in both diabetic and lean mice. The drug had no significant effect on the liver glycogen content in either phenotype, regardless of age, but it increased hepatic lipids and depressed body weights in lean animals. The most prominent effect of the drug was in markedly altering the activities of both glycogen synthase and phosphorylase in the liver of older diabetic mice. Oxytetracycline treatment produced a three-fold increase in the percentage of glycogen synthase I activity and reduced by one-third the percentage of glycogen phosphorylase a activity in 15-week-old diabetic mice. In age-matched lean mice treated with oxytetracycline, the percentage of glycogen synthase I activity increased significantly, but the percentage of phosphorylase a activity was unchanged. These data suggest that the drug may alter an aspect of hepatic glycogen metabolism which might lead to an inhibition of glycogenolysis and subsequent diminution of blood sugar levels in the diabetic. The present results show that, while oxytetracycline may be effective in reducing the severity of some of the diabetic symptoms associated with carbohydrate metabolism in this animal model of maturity-onset diabetes, the drug may have adverse effects on aspects of protein and lipid metabolism in these animals.     

Perfused hearts from Type 2 diabetic (db/db) mice show metabolic responsiveness to insulin

Diabetic (db/db) mice provide an animal model of Type 2 diabetes characterized by marked in vivo insulin resistance. The effect of insulin on myocardial metabolism has not been fully elucidated in this diabetic model. In the present study we tested the hypothesis that the metabolic response to insulin in db/db hearts will be diminished due to cardiac insulin resistance. Insulin-induced changes in glucose oxidation (GLUox) and fatty acid (FA) oxidation (FAox) were measured in isolated hearts from control and diabetic mice, perfused with both low as well as high concentration of glucose and FA: 10 mM glucose/0.5 mM palmitate and 28 mM glucose/1.1 mM palmitate. Both in the absence and presence of insulin, diabetic hearts showed decreased rates of GLUox and elevated rates of FAox. However, the insulin-induced increment in GLUox, as well as the insulin-induced decrement in FAox, was similar or even more pronounced in diabetic that in control hearts. During elevated FA and glucose supply, however, the effect of insulin was blunted in db/db hearts with respect to both FAox and GLUox. Finally, insulin-stimulated deoxyglucose uptake was markedly reduced in isolated cardiomyocytes from db/db mice, whereas glucose uptake in isolated perfused db/db hearts was clearly responsive to insulin. These results show that, despite reduced insulin-stimulated glucose uptake in isolated cardiomyocytes, isolated perfused db/db hearts are responsive to metabolic actions of insulin. These results should advocate the use of insulin therapy (glucose-insulin-potassium) in diabetic patients undergoing cardiac surgery or during reperfusion after an ischemic insult.     

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.     

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.     

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.     

Aberrations in the diurnal rhythms of plasma glucose, plasma insulin, liver glycogen, and hepatic glycogen synthase and phosphorylase activities in genetically diabetic (db/db) mice

The diurnal rhythms of plasma glucose, insulin, liver glycogen, and hepatic glycogen synthase and phosphorylase activities were determined in control and genetically diabetic (db/db) mice 8 weeks of age. The diabetic mice showed wide fluctuations in their plasma glucose levels, although being similar to controls near the end of the light period. Little variation was observed in their elevated plasma insulin levels. Liver glycogen levels in diabetic mice were not depleted to the low levels seen in controls during the last part of the light period but were maintained at significantly higher levels. However, maximum attained glycogen levels were similar in the two groups of mice. Alterations were also observed for the diurnal rhythms of glycogen synthase and phosphorylase activities, although again the daily maximums were similar in control and diabetic mice. These findings suggest that the reported changes of several of these metabolic parameters in the db/db mouse may be due to alterations in the diurnal pattern rather than to absolute changes.     

Glucose and insulin improve cardiac efficiency and postischemic functional recovery in perfused hearts from type 2 diabetic (db/db) mice

Hearts from type 2 diabetic (db/db) mice demonstrate altered substrate utilization with high rates of fatty acid oxidation, decreased functional recovery following ischemia, and reduced cardiac efficiency. Although db/db mice show overall insulin resistance in vivo, we recently reported that insulin induces a marked shift toward glucose oxidation in isolated perfused db/db hearts. We hypothesize that such a shift in metabolism should improve cardiac efficiency and consequently increase functional recovery following low-flow ischemia. Hearts from db/db and nondiabetic (db/+) mice were perfused with 0.7 mM palmitate plus either 5 mM glucose (G), 5 mM glucose and 300 microU/ml insulin (GI), or 33 mM glucose and 900 microU/ml insulin (HGHI). Substrate oxidation and postischemic recovery were only moderately affected by GI and HGHI in db/+ hearts. In contrast, GI and particularly HGHI markedly increased glucose oxidation and improved postischemic functional recovery in db/db hearts. Cardiac efficiency was significantly improved in db/db, but not in db/+ hearts, in the presence of HGHI. In conclusion, insulin and glucose normalize cardiac metabolism, restore efficiency, and improve postischemic recovery in type 2 diabetic mouse hearts. These findings may in part explain the beneficial effect of glucose-insulin-potassium therapy in diabetic patients with cardiac complications.     

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.

     

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.     

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.     

Association of monoamine oxidase and malate dehydrogenase with liver peroxisomes of genetically obese (ob/ob and db/db) mice.

1. Liver post-nuclear supernatants (PNS) from genetically obese (ob/ob and db/db), lean (+/?), and albino mice were fractionated by dual centrifugation in B-XIV zonal rotors and subcellular fractions were analysed by marker-enzyme estimations and by electron microscopy. 2. Rate-dependent banding of PNS yielded a peroxisome-enriched region (PER) well-separated from mitochondria. 3. Density-dependent banding of PER in ob/ob and db/db mice only, yielded purified peroxisomes which were associated with malate dehydrogenase (cytosolic) and monoamine oxidase. 4. Markers for the mitochondrial matrix, intermembrane space and inner membrane compartments were absent from the peroxisomes. 5. The experimental results are interpreted as indicating that peroxisomes of genetically obese mice are either altered so that protein import is imprecise or so that their attachment to mitochondria is more extensive.     

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.