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.     

Myocardial infarction and heart failure in the db/db diabetic mouse

Clinical studies have reported that the incidence and severity of myocardial infarction is significantly greater in diabetics compared with nondiabetics after correction for all other risk factors. The majority of studies investigating the pathophysiology of myocardial ischemia-reperfusion injury have focused on otherwise healthy animals. At present, there is a paucity of experimental investigations on the pathophysiology of heart failure in diabetic animals. We hypothesized that the severity of myocardial reperfusion injury and the development of congestive heart failure would be markedly enhanced in the db/db diabetic mouse. Accordingly, we studied the effects of varying durations of in vivo myocardial ischemia and reperfusion on the incidence of heart failure in db/db diabetic mice. Nondiabetic and db/db diabetic mice (10 wk of age) were subjected to 30, 45, or 60 min of left coronary artery occlusion and 28 days of reperfusion. Survival at 24 h of reperfusion was 100% in nondiabetic mice subjected to 30 min of myocardial ischemia and 88% in nondiabetic mice subjected to 45 min of myocardial ischemia. In contrast, survival was 53% in db/db diabetic mice subjected to 30 min of myocardial ischemia and 44% in db/db mice after 45 min of myocardial ischemia. Prolonged survival in nondiabetic mice was not significantly attenuated when compared during the 28-day follow-up period with all groups experiencing >90% survival. Prolonged survival was significantly decreased in db/db mice after both 30 and 45 min of myocardial ischemia compared with sham controls. Furthermore, we observed a significant degree or left ventricular dilatation, cardiac hypertrophy, and cardiac contractile dysfunction in db/db mice subjected to 45 min of myocardial ischemia and 28 days reperfusion. In nondiabetic mice subjected to 45 min of myocardial ischemia, we failed to observe any changes in left ventricular dimensions or fractional shortening. These studies provide a feasible experimental model system for the investigation of heart failure secondary to acute myocardial infarction in the db/db diabetic mouse.     

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.     

Increased cerebral matrix metalloprotease-9 activity is associated with compromised recovery in the diabetic db/db mouse following a stroke

Diabetes is a major risk factor of stroke and is associated with increased frequency of stroke and a poorer prognosis for recovery. In earlier studies we have utilized type 2 diabetic mouse models of stroke and demonstrated that diabetic db/db and ob/ob mice experience larger infarct volumes and impaired recovery associated with greater infiltration of macrophage following hypoxic-ischemic (H/I) insult than their heterozygous non-diabetic db/+ and ob/+ littermates. To obtain a better understanding of the pathogenesis of the impaired recovery, we have investigated the role of matrix metalloproteases and their endogenous inhibitors in the breakdown of the blood-brain barrier (BBB) following H/I. Diabetic db/db mice showed a significant and more rapid increase in matrix metalloprotease (MMP)-9 mRNA, protein and gelatinolytic activity compared with db/+, which resulted in an increased degradation of occludin and collagen IV and subsequently, an increased BBB permeability and greater infiltration of neutrophils into the infarct area. The expression of the MMPs, especially in the db/+ mice, is preceded by an elevated expression of their endogenous tissue inhibitors of metalloproteases (TIMPs) 1, 2, and 3, whereas in the db/db mice, a lower expression of the TIMPs is associated with greater MMP 3 and 9 expression. These results suggest that an imbalance in the MMPs/TIMPs cascade in the diabetic mouse, particularly MMP-9, results in a greater neutrophil invasion, a compromised BBB and consequently a greater insult.     

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.     

Exacerbated brain damage, edema and inflammation in type-2 diabetic mice subjected to focal ischemia

One of the limiting factors in stroke therapeutic development is the use of animal models that do not well represent the underlying medical conditions of patients. In humans, diabetes increases the risk of stroke incidence as well as post-stroke mortality. To understand the mechanisms that render diabetics to increased brain damage, we evaluated the effect of transient middle cerebral artery occlusion in adult db/db mice. The db/db mouse is a model of type-2 diabetes with four times higher blood sugar than its normoglycemic genetic control(db/+ mouse). Following transient middle cerebral artery occlusion, the db/db mice showed significantly higher mortality, bigger infarcts, increased cerebral edema, worsened neurological status compared to db/+ mice. The db/db mice also showed significantly higher post-ischemic inflammatory markers (ICAM1(+) capillaries, extravasated macrophages/neutrophils and exacerbated proinflammatory gene expression) compared to db/+ mice. In addition, the post-ischemic neuroprotective heat-shock chaperone gene expression was curtailed in the db/db compared to db/+ mice.     

Expressional changes of ganglioside GM3 during ovarian maturation and early embryonic development in db/db mice

Diabetes and obesity cause abnormal development of reproductive processes in a variety of species, but the mechanisms that underlie this effect have not been fully elucidated. This study examined the expressional changes of ganglioside GM3 during ovarian maturation, in vitro fertilization (IVF) and early embryonic development in diabetic/obese db/db mice. In high-performance thin-layer chromatography studies, GM3 expression was conspicuously low in the ovaries of db/db mice compared to non-diabetic db/+ mice. Signal detected by anti-GM3 monoclonal antibody was greatly reduced in the primary, secondary and graffian follicles of db/db mice compared to control mice. Results from IVF with ova and sperm from db/db mice showed that GM3 expression during early embryonic development was obviously decreased compared to db/+ mice. This study also elucidated the effects of high glucose (20 and 30 mm) on early embryonic development in ICR strain mice. High glucose caused a decrease in GM3 expression during early embryonic development. Taken together, the results of this study indicate decreased GM3 expression during ovarian maturation and embryonic development of db/db mice, suggesting that alteration of ganglioside expression induced by the diabetic condition may be implicated in the abnormal follicular embryonic development.     

IL-1beta-mediated innate immunity is amplified in the db/db mouse model of type 2 diabetes

Chronic inflammation appears to play a critical role in type 2 diabetes and its complications. Here we tested the hypothesis that this inflammatory dysregulation affects the IL-1beta system and has functional consequences in the brain. Diabetic, db/db, and nondiabetic, db/+, mice were administered i.p. LPS, a potent cytokine inducer, at a dose of 100 microg/kg/mouse. db/db mouse innate immune-associated sickness behavior was 14.8, 33, 44.7, and 34% greater than that of db/+ mice at 2, 4, 8, and 12 h, respectively. When a fixed dose of LPS was used (5 microg/mouse), db/db mouse sickness was again enhanced 18.4, 22.2, and 14.5% at 4, 8, and 12 h as compared with db/+ mice. In diabetic mice, peritoneal macrophages produced more IL-1beta in response to LPS, and peritoneal levels of IL-1beta induced by LPS were increased. Importantly, IL-1R antagonist and type 2 IL-1 receptor (IL-1R2) failed to up-regulate in response to LPS in db/db mice. Finally, both peripheral and central administration of IL-1beta, itself, induced sickness in db/db mice that mimicked the effects of peripheral LPS and was significantly greater than that seen in db/+ mice. Taken together, these results indicate that IL-1beta-mediated innate immunity is augmented in db/db mice both at the periphery and in the brain, and the mechanism is due to diabetes-associated loss of IL-1beta counterregulation.     

Circulating endothelial progenitor cells and cellular membrane microparticles in db/db diabetic mouse: possible implications in cerebral ischemic damage

For determining the implications of circulating endothelial progenitor cells (cEPCs) and cellular membrane microparticles (MPs) in diabetic stroke, levels of EPCs, EPC-MPs, and endothelium-derived MPs (EMPs) and their correlations with blood glucose concentration, cerebral microvascular density (cMVD), and ischemic damage were investigated in type 2 diabetic db/db and db/+ (wild-type control) mice. Therapeutic efficacy of EPC infusion (preincubated with MPs) was also explored. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery. Ischemic damage and cMVD were determined using histological analyses. The levels of cEPCs and MPs were determined using flow cytometric analyses. EPC generation and functions were evaluated by in vitro cell cultures. Results showed the following. 1) In db/db mice, the basal level of cEPCs was less and cMVDs were lower, but the levels of circulating EPC-MPs and EMPs were more; 2) MCAO induced a larger infarct volume and less of an increase in cEPCs in db/db mice; 3) the level of cEPCs correlated with blood glucose concentration (negatively), cMVD (positively), and ischemic damage (negatively), but the levels of EPC-MPs and EMPs correlated inversely with those parameters; 4) EPCs were reduced and dysfunctional in db/db mice, and preincubation with db/db MPs impaired EPC functions; and 5) infusion of EPCs preincubated with db/+ MPs increased the level of cEPCs and reduced ischemic damage, and these beneficial effects were reduced or lost in EPCs preincubated with db/db MPs. These data suggest that reduced cEPCs, impaired EPC generation/function, and increased production of MPs might be the mechanisms responsible for increased ischemic damage seen in db/db mice.     

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.     

Quercetin Attenuates Cell Apoptosis in Focal Cerebral Ischemia Rat Brain Via Activation of BDNF–TrkB–PI3K/Akt Signaling Pathway

Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF?CTrkB?CPI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF?CTrkB?CPI3K/Akt signaling pathway.     

Type 2 diabetes impairs insulin receptor substrate-2-mediated phosphatidylinositol 3-kinase activity in primary macrophages to induce a state of cytokine resistance to IL-4 in association with overexpression of suppressor of cytokine signaling-3

Chronic elevation of proinflammatory markers in type 2 diabetes (T2D) is well defined, but the role of anti-inflammatory cytokines in T2D is less clear. In this study, we report that normal IL-4-dependent elaboration of IL-1 receptor antagonist (IL-1RA) requires IRS-2-mediated PI3K activity in primary macrophages. We also show that macrophages isolated from obese/diabetic db/db mice have impaired IRS-2-mediated PI3K activity and constitutively overexpress suppressor of cytokine signaling (SOCS)-3, which impairs an important IL-4 anti-inflammatory function. Peritoneal proinflammatory cytokine levels were examined in diabese (db/db) mice, and IL-6 was found to be nearly 7-fold higher than in nondiabese (db/+) control mice. Resident peritoneal macrophages were isolated from db/db mice and were found to constitutively overexpress IL-6 and were unable to elaborate IL-1RA in response to IL-4-like db/+ mouse macrophages. Inhibition of PI3K with wortmannin or blockage of IRS-2/PI3K complex formation with a cell permeable IRS-2-derived tyrosine phosphopeptide inhibited IL-4-dependent IL-1RA production in db/+ macrophages. Examination of IL-4 signaling in db/db macrophages revealed that IL-4-dependent IRS-2/PI3K complex formation and IRS-2 tyrosine phosphorylation was reduced compared with db/+ macrophages. SOCS-3/IL-4 receptor complexes, however, were increased in db/db mouse macrophages compared with db/+ mice macrophages as was db/db mouse macrophage SOCS-3 expression. These results indicate that in the db/db mouse model of T2D, macrophage expression of SOCS-3 is increased, and impaired IL-4-dependent IRS-2/PI3K formation induces a state of IL-4 resistance that disrupts IL-4-dependent production of IL-1RA.     

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.     

The Akt/GSK-3β pathway mediates flurbiprofen-induced neuroprotection against focal cerebral ischemia/reperfusion injury in rats

Apoptosis is one of the major mechanisms of cell death during cerebral ischemia and reperfusion injury. Flurbiprofen has been shown to reduce cerebral ischemia/reperfusion injury in both focal and global cerebral ischemia models, but the mechanism remains unclear. This study aimed to investigate the potential association between the neuroprotective effect of flurbiprofen and the apoptosis inhibiting signaling pathways, in particularly the Akt/GSK-3β pathway. A focal cerebral ischemia rat model was subjected to middle cerebral artery occlusion (MCAO) for 120 min and then treated with flurbiprofen at the onset of reperfusion. The infarct volume and the neurological deficit scores were evaluated at 24 h after reperfusion. Cell apoptosis, apoptosis-related proteins and the levels of p-Akt and p-GSK-3β in ischemic penumbra were measured using TUNEL and western blot. The results showed that administration of flurbiprofen at the doses of 5 and 10 mg/kg significantly attenuated brain ischemia/reperfusion injury, as shown by a reduction in the infarct volume, neurological deficit scores and cell apoptosis. Moreover, flurbiprofen not only inhibited the expression of Bax protein and p-GSK-3β, but also increased the expression of Bcl-2 protein, the ratio of Bcl-2/Bax as well as the P-Akt level. Taken together, these results suggest that flurbiprofen protects the brain from ischemia/reperfusion injury by reducing apoptosis and this neuroprotective effect may be partly due to the activation of Akt/GSK-3β signaling pathway.     

Recent insights into diabetic renal injury from the db/db mouse model of type 2 diabetic nephropathy

The db/db mouse is the most widely used animal model of type 2 diabetic nephropathy. Recent studies have utilized genetic backcrossing with transgenic mouse strains to create novel db/db strains that either lack or overexpress specific genes. These novel strains [ICAM-1-/-, CCL2-/-, MKK3-/-, osteopontin-/-, plasminogen activator inhibitor-1 (PAI-1)-/-, endothelial nitric oxide synthase-/-, SOD-Tg, rCAT-Tg] have provided valuable insights into the molecular mechanisms which promote diabetic renal injury. In addition, surgical removal of one kidney has been shown to accelerate injury in the remaining kidney of diabetic db/db mice. A number of novel therapeutic agents have also been tested in db/db mice, including inhibitors of inflammation (chemokine receptor antagonists, anti-CCL2 RNA aptamer, anti-c-fms antibody); oxidative stress (oxykine, biliverdin); the renin-angiotensin-aldosterone system (aliskiren, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, eplerenone); advanced glycation end products (AGE; pyridoxamine, alagebrium, soluble AGE receptor); angiogenesis (NM-3, anti-CXCL12 RNA aptamer, soluble Flt-1); lipid accumulation (statins, farnesoid X receptor agonists, Omacor); intracellular signaling pathways (PKC-β or JNK inhibitors); and fibrosis [transforming growth factor (TGF)-β antibody, TGF-βR kinase inhibitor, soluble betaglycan, SMP-534, CTGF-antisense oligonucleotide, mutant PAI-1, pirfenidone], which have identified potential therapeutic targets for clinical translation. This review summarizes the advances in knowledge gained from studies in genetically modified db/db mice and treatment of db/db mice with novel therapeutic agents.     

Novel hypoglycemic effects of Ganoderma lucidum water-extract in obese/diabetic (+db/+db) mice

In this study, we evaluated the pharmacological effects of Ganoderma lucidum (G. lucidum) (water-extract) (0.003, 0.03 and 0.3 g/kg, 4-week oral gavage) consumption using the lean (+db/+m) and the obese/diabetic (+db/+db) mice. Different physiological parameters (plasma glucose and insulin levels, lipoproteins-cholesterol levels, phosphoenolpyruvate carboxykinase (PEPCK), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) and isolated aorta relaxation of both species were measured and compared. G. lucidum (0.03 and 0.3 g/kg) lowered the serum glucose level in +db/+db mice after the first week of treatment whereas a reduction was observed in +db/+m mice only fed with 0.3 g/kg of G. lucidum at the fourth week. A higher hepatic PEPCK gene expression was found in +db/+db mice. G. lucidum (0.03 and 0.3 g/kg) markedly reduced the PEPCK expression in +db/+db mice whereas the expression of PEPCK was attenuated in +db/+m mice (0.3 g/kg G. lucidum). HMG CoA reductase protein expression (in both hepatic and extra-hepatic organs) and the serum insulin level were not altered by G. lucidum. These data demonstrate that G. lucidum consumption can provide beneficial effects in treating type 2 diabetes mellitus (T2DM) by lowering the serum glucose levels through the suppression of the hepatic PEPCK gene expression.     

Klotho attenuates diabetic nephropathy in db/db mice and ameliorates high glucose-induced injury of human renal glomerular endothelial cells

Glomerular endothelial cell injury plays an important role in the development and progression of diabetic nephropathy (DN). The expression and function of klotho in glomerular endothelial cells remain unclear. Thus, this study aimed to investigate the expression and the functional role of klotho in DN progression in mice and in high glucose (HG)-induced cell injury of human renal glomerular endothelial cells (HRGECs) and the underlying mechanism. In this study, HRGECs were cultured with media containing HG to induce endothelial cell injury and db/db mice were used as DN model mice. Klotho was overexpressed or knocked down in HRECs to evaluate its role in HG-induced HRGECs injury. klotho-overexpressing adenovirus (rAAV-klotho) was injected into db/db mice via the tail vein to further validate the protective effect of klotho in DN. Decreased klotho expression was observed in DN patients, DN mice, and HG-exposed HRGECs. Furthermore, klotho overexpression significantly abolished the HG-induced HRGECs injury and activation of Wnt/β-catenin pathway and RAAS. In contrast, klotho knockdown exerted the opposite effects. Moreover, klotho attenuated diabetic nephropathy in db/db mice, which was also associated with inhibition of the Wnt/β-catenin pathway and RAAS. In conclusion, klotho attenuates DN in db/db mice and ameliorates HG-induced injury of HRGECs.     

Effects of rexinoids on glucose transport and insulin-mediated signaling in skeletal muscles of diabetic (db/db) mice

Rexinoids and thiazolidinediones (TZDs) are two classes of nuclear receptor ligands that induce insulin sensitization in diabetic rodents. TZDs are peroxisome proliferator-activated receptor gamma (PPARgamma) activators, whereas rexinoids are selective ligands for the retinoid X receptors (RXRs). Activation of both the insulin receptor substrates (IRSs)/Akt and the c-Cbl-associated protein (CAP)/c-Cbl pathways are important in regulating insulin-stimulated glucose transport. We have compared the effects of a rexinoid (LG268) and a TZD (rosiglitazone) on these two signal pathways in skeletal muscle of diabetic (db/db) mice. The results we have obtained show that treatment of db/db mice with either LG268 or rosiglitazone for 2 weeks results in a significant increase in insulin-stimulated glucose transport activity in skeletal muscle. Treatment with LG268 increases insulin-stimulated IRS-1 tyrosine phosphorylation and Akt phosphorylation in skeletal muscle without affecting the activity of the CAP/c-Cbl pathway. In contrast, rosiglitazone increases the levels of CAP expression and insulin-stimulated c-Cbl phosphorylation without affecting the IRS-1/Akt pathway. The effects of LG268 on the IRS-1/Akt pathway were associated with a decrease in the level of IRS-1 Ser(307) phosphorylation. Taken together, these data suggest that rexinoids improve insulin sensitivity via changes in skeletal muscle metabolism that are distinct from those induced by TZDs. Rexinoids represent a novel class of insulin sensitizers with potential applications in the treatment of insulin resistance.     

Alterations of the Ca2+ signaling pathway in pancreatic beta-cells isolated from db/db mice

Upon glucose elevation, pancreatic beta-cells secrete insulin in a Ca²⁺-dependent manner. In diabetic animal models, different aspects of the calcium signaling pathway in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca²⁺ ([Ca²⁺]_i) via Ca²⁺ influx, Ca²⁺ mobilization from endoplasmic reticulum (ER) calcium stores, and the removal of Ca²⁺ via multiple mechanisms in beta-cells from both diabetic db/db mice and nondiabetic C57BL/6J mice. We refined our previous quantitative model to describe the slow [Ca²⁺]_i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump, SERCA2 and SERCA3, were severely down-regulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduction in the Ca²⁺ concentration in the ER, a compensatory up-regulation of the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) and a reduction in depolarizationevoked Ca²⁺ influx. As a result, the patterns of glucosestimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.     

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF’s role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-κB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-κB activity and phosphorylation of the inhibitor of kappa B (IκBα) increased in ischemic brains, but IRF3, inhibitor of κB kinase complex-ε (IKKε), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-κB activity or p-IκBα induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-κB signaling and brain injury after acute cerebral I/R.