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.     

Elevated Expression of Carboxy-Terminal Modulator Protein (CTMP) Aggravates Brain Ischemic Injury in Diabetic db/db Mice

Deregulation of Akt signaling is important in the brain injuries caused by cerebral ischemia in diabetic animals, and the underlying mechanism is not fully understood. We investigated the role of carboxy-terminal modulator protein (CTMP), an endogenous Akt inhibitor, in brain injury following focal cerebral ischemia in type 2 diabetic db/db mice and their control littermates non-diabetic db/+ mice. db/db mice showed a significant elevation in the expression of CTMP compared to db/+ mice under normal physiological conditions. After ischemia, db/db mice exhibit higher levels of CTMP expression, decreased Akt kinase activity, adverse neurological deficits and cerebral infarction than db/+ mice. To further certain the effectiveness of Akt signaling to the final outcome of cerebral ischemia, the animals were treated with LY294002, an inhibitor of the Akt pathway, which aggravated the ischemic injury in db/+ mice but not in db/db mice. RNA interference-mediated depletion of CTMP were finally applied in db/db mice, which restored Akt activity, improved neurological scores and reduced infarct volume. These results suggest that elevation of CTMP in diabetic mice suppresses Akt activity and ultimately negatively affects the outcome of ischemia. Inhibitors specifically targeting CTMP may be beneficial in the treatment of cerebral ischemia in patients with diabetes.     

Estradiol restores diabetes-induced reductions in sex steroid receptor expression and distribution in the vagina of db/db mouse model

Sex steroid hormones and receptors play an important role in maintaining vaginal physiology. Disruptions in steroid receptor signaling adversely impact vaginal function. Limited studies are available investigating the effects of diabetic complications on steroid receptor expression and distribution in the vagina. The goals of this study were to investigate type 2 diabetes-induced changes in expression, localization and distribution of estrogen (ER), progesterone (PR) and androgen receptors (AR) in the vagina and to determine if estradiol treatment ameliorates these changes. Eight-week-old female diabetic (db/db) mice (strain BKS.Cg-m+/+ Lepr^db/J) were divided into two subgroups: untreated diabetic and diabetic animals treated with pellets containing estradiol. Control normoglycemic littermates were subcutaneously implanted with pellets devoid of estradiol. At 16 weeks of age, animals were sacrificed, vaginal tissues excised and analyzed by Western blot and immunohistochemical methods. Diabetes produced marked reductions in protein expression of ER, PR, and AR. Diabetes also resulted in marked differences in the distribution, staining intensity and proportion of immunoreactive cells containing these steroid receptors in the epithelium, lamina propria and muscularis. Treatment of diabetic animals with estradiol restored receptor protein expression and distribution similar to those levels observed in control animals. This study demonstrates that type 2 diabetes markedly reduces steroid receptor protein expression and distribution in the vagina. Estradiol treatment of diabetic animals ameliorates these diabetes-induced changes.     

The effect of diabetes mellitus on aortic prostanoid synthesis and serum cholesterol levels in the rat fed a high cholesterol diet

The effect of diabetes mellitus on serum cholesterol and aortic microsomal prostanoid synthesis was studied in cholesterol fed male Lewis rats. Normal, diabetic and diabetic rats treated with pancreatic islets were divided into three diet subgroups, control diet, control +2% cholesterol for 8 weeks and control +2% cholesterol diet for 16 weeks. Serum glucose levels were elevated three-fold in the diabetic group compared to normal. Treatment with islets restored serum glucose to normal levels in diabetic rats. The 2% cholesterol diet did not significantly alter serum glucose levels in any of the groups. Body weights in the diabetic group were significantly lower than normal or diabetic rats treated with islets. Feeding 2% cholesterol for 16 weeks significantly increased weight in normal and islet treated diabetic rats but not in the diabetic group. Aortic microsomal prostanoid synthesis was similar in all experimental groups with 6-keto-PGF1 alpha (PGI2 metabolite) being the major product synthesized in all groups. Aortic microsomal prostanoid levels were not altered by the 2% cholesterol diet. Serum cholesterol levels increased 14-fold in the diabetic group which returned to the normal level in the diabetic animals treated with islets. These data show that diabetes does not alter aortic microsomal prostanoid levels in the rat. However, diabetes significantly increased serum cholesterol levels which were reversed by islet transplantation.     

Reversal of diabetes by allogenic islet transplantation without immunosuppression

Streptozotocin-induced diabetes is temporarily reversed following the allo-transplantation of BALB/c (H-2d) islet tissue to normal CBA (H-2k) recipients, but by 2-4 week post-transplantation these animals return to their initial diabetic condition. Organ culture of allogeneic islet tissue in 95% O2 and 5% CO2 for 7 days prior to transplantation reduces the immunogenicity of the tissue, and cultured allografts give prolonged (greater than 110 days) reversal of diabetes in normal allogeneic recipients. The non-fasting blood sugar level remains in or very close to the normal range, urine glucose output is one to two orders of magnitude less than that of diabetic control animals and allografted animals regain their pre-morbid body weight within 60 days of transplantation. Surgical removal of the allograft results in a rapid return of the animal to the initial diabetic condition.     

Characterisation of Age-Dependent Beta Cell Dynamics in the Male db/db Mice

Aim

To characterise changes in pancreatic beta cell mass during the development of diabetes in untreated male C57BLKS/J db/db mice.

Methods

Blood samples were collected from a total of 72 untreated male db/db mice aged 5, 6, 8, 10, 12, 14, 18, 24 and 34 weeks, for measurement of terminal blood glucose, HbA_1c, plasma insulin, and C-peptide. Pancreata were removed for quantification of beta cell mass, islet numbers as well as proliferation and apoptosis by immunohistochemistry and stereology.

Results

Total pancreatic beta cell mass increased significantly from 2.1 ± 0.3 mg in mice aged 5 weeks to a peak value of 4.84 ± 0.26 mg (P < 0.05) in 12-week-old mice, then gradually decreased to 3.27 ± 0.44 mg in mice aged 34 weeks. Analysis of islets in the 5-, 10-, and 24-week age groups showed increased beta cell proliferation in the 10-week-old animals whereas a low proliferation is seen in older animals. The expansion in beta cell mass was driven by an increase in mean islet mass as the total number of islets was unchanged in the three groups.

Conclusions/Interpretation

The age-dependent beta cell dynamics in male db/db mice has been described from 5-34 weeks of age and at the same time alterations in insulin/glucose homeostasis were assessed. High beta cell proliferation and increased beta cell mass occur in young animals followed by a gradual decline characterised by a low beta cell proliferation in older animals. The expansion of beta cell mass was caused by an increase in mean islet mass and not islet number.     

Insulin secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control.

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Effects of Combining Linagliptin Treatment with BI-38335, A Novel SGLT2 Inhibitor, on Pancreatic Islet Function and Inflammation in db/db Mice

Dipeptidyl peptidase-4 (DPP-4) inhibitors enhance incretin actions and beta-cell function. Concurrently, sodium-glucose co-transporter 2 (SGLT2) inhibitors block renal glucose reabsorption promoting excretion. In this study, we investigated the effects of linagliptin (a DPP-4 inhibitor) and BI-38335 (an SGLT2 inhibitor), individually and in combination, on glucose homeostasis, islet function, and pancreatic islet morphology in db/db mice. Diabetic and non-diabetic mice received linagliptin (3 mg/kg), BI-38335 (1 mg/kg), the two drugs in combination or control once daily for 8 weeks. Blood glucose homeostasis and insulin sensitivity were assessed. Pancreatic islet function and morphology as well as inflammatory factors and toll like receptor 2 (TLR2) pathways involved in islet inflammation were investigated. Active treatments markedly reduced blood glucose and glycated hemoglobin A1c (HbA1c) levels, with the combined treatment showing the greater effects. Insulin resistance was improved in the BI-38335 and combination groups with the enhancement of insulin sensitivity and significant increase of serum adiponectin levels. The combined treatment exhibited greater effects on enhanced islet glucose-stimulated insulin secretion and improved glucose tolerance. Moreover, the combination restored the islet beta-/alpha-cell ratio, reduced beta-cell apoptosis, decreased expression of islet immune cell markers, and suppressed factors related to the TLR2 pathway. In addition, all active treatments reduced serum lipid profiles, though the combination produced more robust effects. Collectively, our data show that combined treatment with BI-38335 and linagliptin work, at least in part, synergistically to benefit islet cell function/architecture and insulin resistance, thus improving glycemic control.     

Glucokinase in B-cell-depleted islets of Langerhans

Glucose phosphorylation was studied in B-cell-enriched or in B-cell-depleted pancreatic islets from normal or streptozotocin-diabetic rats, respectively, using quantitative histochemical procedures. The data indicate that B-cell-enriched preparations from normal animals and whole islets from normals, diabetics, and insulin-treated diabetic animals have comparable glucokinase activities. Average maximum velocities were (mmol/kg dry tissue/hr) 134.1 +/- 7.3 for whole islets and 125.6 +/- 10.7 for the B-cell-enriched preparations from normal rats, 143.1 +/- 13.6 for B-cell-depleted islets from diabetic rats, and 124.4 +/- 10.7 for B-cell-depleted islets from insulin-treated diabetic animals. The Kmax for glucose of the enzyme in islets from untreated diabetic rats was 16 mM, comparable to the Kmax found for glucokinase from normal rat islets. Mannoheptulose, previously shown to be a competitive inhibitor of glucokinase from liver and normal islets, also inhibited glucokinase in B-cell-depleted islets from diabetic rats. The data indicate that glucokinase is not selectively located in the B-cell, as was previously assumed, but is also found in A- and/or D-cells of diabetic rats. This observation raises significant questions about the functional role of islet glucokinase under control and diabetic conditions.     

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.     

Endocrine control of cytosolic factors stimulating adenylate cyclase in rat lung

Endocrine control of cytoplasmic factors modulating adenylate cyclase activity in rat lung membranes was investigated. Hypophysectomy, adrenalectomy and thyroidectomy showed an adverse effect on the body and organ weights. Lung protein, glycogen and DNA contents were decreased in the endocrine ablated animals which were restored to the normal values on hormone treatment. Phosphodiesterase and phosphorylase activities were increased and decreased in adrenalectomized and thyroidectomized animals, respectively. The activities of these enzymes were restored to normal values on hormone treatment. Adrenalectomy and thyroidectomy affected ATPases differently. Basal adenylate cyclase activity in rat lung membranes was not affected by adrenalectomy and hormone treatment. However, the total enzyme activity was increased by both dexamethasone (DEX) and thyroxine (T4) treatments. The activation of the particulate adenylate cyclase by the cytoplasmic factors was markedly decreased in the lung from hypophysectomized, adrenalectomized and thyroidectomized rats. This decrease in the cytoplasmic activation of adenylate cyclase was restored to or above the control values on hormone treatment. Alteration in the activation of enzyme by cytoplasmic factors did not appear to be due to the change in the responsiveness of the enzyme. Glucocorticoids appeared to have a specific effect on the cytoplasmic factors modulating the enzyme.     

Capillary area in early low-dose streptozocin-treated mice.

It has been reported that vasoconstriction of intra-islet capillaries plays an important role in the initiation of the insulitis seen in the islets of Langerhans of diabetic animals. Nevertheless, only a few studies have concentrated on islet vessels. This led us to perform an experiment with the aim to compare the islet capillary area of normal untreated and multiple low-dose streptozocin (LDS) (40 mg/kg b.wt. i.p./5 days)-treated mice. In order to identify endothelial cells a method devised by Gomori, based on the fact that these cells present alkaline phosphatases on their surface, was used. Results revealed that in LDS-treated animals the capillary area per islet is significantly reduced when compared to the vascular area of controls (p less than 0.05). This could be due to a vasoconstriction phenomenon that occurs in the islet capillaries after the streptozocin administration and before the appearance of any inflammation. Our findings could demonstrate that vasoconstriction events are involved in initiation of the diabetic disease.     

Low doses of L-tryptophan are lethal in rats with adrenal insufficiency

Adrenalectomy decreased the LD50 value for L-tryptophan from greater than 1000 mg/kg in normal rats to 11.6 mg/kg. The LD50 in adrenalectomized rats was restored to normal by corticosterone replacement therapy. Administration of metyrapone, which blocks the synthesis of adrenal steroids, to normal rats decreased plasma corticosterone levels by approximately 50% and decreased the LD50 from greater than 1000 mg/kg to 24.9 mg/kg. Neurochemical analysis revealed a large increase in tissue tryptamine levels following administration of L-tryptophan in rats with adrenal insufficiency. Therefore, it appears that death due to L-tryptophan in animals with adrenal insufficiency is due to formation of excess tryptamine and, consequently, an elevation in blood pressure and other cardiovascular dysfunctions.     

Capillary area in early low-dose streptozocin-treated mice

Summary It has been reported that vasoconstriction of intra-islet capillaries plays an important role in the initiation of the insulitis seen in the islets of Langerhans of diabetic animals. Nevertheless, only a few studies have concentrated on islet vessels. This led us to perform an experiment with the aim to compare the islet capillary area of normal untreated and mulitple low-dose streptozocin (LDS) (40 mg/kg b.wt. i.p./5 days)-treated mice. In order to identify endothelial cells a method devised by Gomori, based on the fact that these cells present alkaline phosphatases on their surface, was used. Results revealed that in LDS-treated animals the capillary area per islet is significantly reduced when compared to the vascular area of controls (p<0.05). This could be due to a vasoconstriction phenomenon that occurs in the islet capillaries after the streptozocin administration and before the appearance of any inflammation. Our findings could demonstrate that vasoconstriction events are involved in initiation of the diabetic disease.     

Diabetes-induced rat hyposensitivity to compound 48/80

Rat mortality and contractile responses of isolated tracheas to compound 48/80 from rats made diabetic 4 days before by a single intravenous injection of alloxan and from diabetic rats that had been treated with insulin 6 h before were compared with control animals. Diabetic animals and tracheal segments from diabetic rats were significantly less responsive to compound 48/80 than control and insulin-treated diabetic animals. On the other hand, diabetic animals have a lower quantity of peritoneal mast cells than control rats, and insulin restored the normal quantity of cells in diabetic animals. These data indicate that diabetes elicits an hyposensitivity to compound 48/80, possibly related to a diabetes-induced decrease in the mast cell count.     

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.     

Regeneration of beta cells in islets of Langerhans of pancreas of alloxan diabetic rats by acetone extract of Momordica charantia (Linn.) (bitter gourd) fruits

Acetone extract of whole fruit powder of M. charantia (bitter gourd) in doses 25, 50 and 75 mg/100 g body weight lowered the blood glucose from 13.30 to 50% after 8 to 30 days treatment in alloxan diabetic albino rats, confirming antihyperglycemic effect of this plant in diabetic animals and humans. Histological observations with acetone extract showed different phases of recovery of beta cells of the islets of Langerhans of pancreas, which in the untreated diabetic rats were less in number and showed varied degree of atrophy. The most important finding of the present study was observation of the presence of small scattered islets among the acinar tissue in some experimental animals, which may reflect neoformation of islets from pre-existing islet cells. The liver of alloxan diabetic rats showed hydropic degeneration, fatty change and necrosis at some places but liver of extract treated animals was normal. Glycogen localization in liver of diabetic rats was faint but after 30 days treatment with different doses of extract, normal to heavy glycogen localization was observed.