Behavioral, metabolic, and lipidomic characterization of the 5xFADxTg30 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is associated with both extracellular amyloid-ß (Aß) plaques and intracellular tau-containing neurofibrillary tangles (NFT). We characterized the behavioral, metabolic and lipidomic phenotype of the 5xFADxTg30 mouse model which contains overexpression of both Aß and tau. Our results independently reproduce several phenotypic traits described previously for this model, while providing additional characterization. This model develops many aspects associated with AD including frailty, decreased survival, initiation of aspects of cognitive decline and alterations to specific lipid classes and molecular lipid species in the plasma and brain. Notably, some sex-specific differences exist in this model and motor impairment with aging in this model does compromise the utility of the model for some movement-based behavioral assessments of cognitive function. These findings provide a reference for individuals interested in using this model to understand the pathology associated with elevated Aß and tau or for testing potential therapeutics for the treatment of AD.

Yap regulates skeletal muscle fatty acid oxidation and adiposity in metabolic disease.

Obesity is a major risk factor underlying the development of metabolic disease and a growing public health concern globally. Strategies to promote skeletal muscle metabolism can be effective to limit the progression of metabolic disease. Here, we demonstrate that the levels of the Hippo pathway transcriptional co-activator YAP are decreased in muscle biopsies from obese, insulin-resistant humans and mice. Targeted disruption of Yap in adult skeletal muscle resulted in incomplete oxidation of fatty acids and lipotoxicity. Integrated 'omics analysis from isolated adult muscle nuclei revealed that Yap regulates a transcriptional profile associated with metabolic substrate utilisation. In line with these findings, increasing Yap abundance in the striated muscle of obese (db/db) mice enhanced energy expenditure and attenuated adiposity. Our results demonstrate a vital role for Yap as a mediator of skeletal muscle metabolism. Strategies to enhance Yap activity in skeletal muscle warrant consideration as part of comprehensive approaches to treat metabolic disease.

Evidence against a role for NLRP3-driven islet inflammation in db/db mice.

OBJECTIVES: Type 2 diabetes (T2D) is associated with chronic, low grade inflammation. Activation of the NLRP3 inflammasome and secretion of its target interleukin-1ß (IL-1ß) have been implicated in pancreatic ß cell failure in T2D. Specific targeting of the NLRP3 inflammasome to prevent pancreatic ß cell death could allow for selective T2D treatment without compromising all IL-1ß-associated immune responses. We hypothesized that treating a mouse model of T2D with MCC950, a compound that specifically inhibits NLRP3, would prevent pancreatic ß cell death, thereby preventing the onset of T2D. METHODS: Diabetic db/db mice were treated with MCC950 via drinking water for 8 weeks from 6 to 14 weeks of age, a period over which they developed pancreatic ß cell failure. We assessed metabolic parameters such as body composition, glucose tolerance, or insulin secretion over the course of the intervention. RESULTS: MCC950 was a potent inhibitor of NLRP3-induced IL-1ß in vitro and was detected at high levels in the plasma of treated db/db mice. Treatment of pre-diabetic db/db mice with MCC950, however, did not prevent pancreatic dysfunction and full onset of the T2D pathology. When examining the NLRP3 pathway in the pancreas of db/db mice, we could not detect an activation of this pathway nor increased levels of its target IL-1ß. CONCLUSIONS: NLRP3 driven-pancreatic IL-1ß inflammation does not play a key role in the pathogenesis of the db/db murine model of T2D.

Distinct patterns of tissue-specific lipid accumulation during the induction of insulin resistance in mice by high-fat feeding.

AIMS/HYPOTHESIS: While it is well known that diet-induced obesity causes insulin resistance, the precise mechanisms underpinning the initiation of insulin resistance are unclear. To determine factors that may cause insulin resistance, we have performed a detailed time-course study in mice fed a high-fat diet (HFD). METHODS: C57Bl/6 mice were fed chow or an HFD from 3 days to 16 weeks and glucose tolerance and tissue-specific insulin action were determined. Tissue lipid profiles were analysed by mass spectrometry and inflammatory markers were measured in adipose tissue, liver and skeletal muscle. RESULTS: Glucose intolerance developed within 3 days of the HFD and did not deteriorate further in the period to 12 weeks. Whole-body insulin resistance, measured by hyperinsulinaemic-euglycaemic clamp, was detected after 1 week of HFD and was due to hepatic insulin resistance. Adipose tissue was insulin resistant after 1 week, while skeletal muscle displayed insulin resistance at 3 weeks, coinciding with a defect in glucose disposal. Interestingly, no further deterioration in insulin sensitivity was observed in any tissue after this initial defect. Diacylglycerol content was increased in liver and muscle when insulin resistance first developed, while the onset of insulin resistance in adipose tissue was associated with increases in ceramide and sphingomyelin. Adipose tissue inflammation was only detected at 16 weeks of HFD and did not correlate with the induction of insulin resistance. CONCLUSIONS/INTERPRETATION: HFD-induced whole-body insulin resistance is initiated by impaired hepatic insulin action and exacerbated by skeletal muscle insulin resistance and is associated with the accumulation of specific bioactive lipid species.

Enhanced phosphoinositide 3-kinase(p110α) activity prevents diabetes-induced cardiomyopathy and superoxide generation in a mouse model of diabetes.

AIMS/HYPOTHESIS: Diabetic cardiomyopathy is characterised by diastolic dysfunction, oxidative stress, fibrosis, apoptosis and pathological cardiomyocyte hypertrophy. Phosphoinositide 3-kinase (PI3K)(p110α) is a cardioprotective kinase, but its role in the diabetic heart is unknown. The aim of this study was to assess whether PI3K(p110α) plays a critical role in the induction of diabetic cardiomyopathy, and whether increasing PI3K(p110α) activity in the heart can prevent the development of cardiac dysfunction in a setting of diabetes. METHODS: Type 1 diabetes was induced with streptozotocin in adult male cardiac-specific transgenic mice with increased PI3K(p110α) activity (constitutively active PI3K [p110α], caPI3K] or decreased PI3K(p110α) activity (dominant-negative PI3K [p110α], dnPI3K) and non-transgenic (Ntg) mice for 12 weeks. Cardiac function, histological and molecular analyses were performed. RESULTS: Diabetic Ntg mice displayed diastolic dysfunction and increased cardiomyocyte size, expression of atrial and B-type natriuretic peptides (Anp, Bnp), fibrosis and apoptosis, as well as increased superoxide generation and increased protein kinase C ß2 (PKCß2), p22 ( phox ) and apoptosis signal-regulating kinase 1 (Ask1) expression. Diabetic dnPI3K mice displayed an exaggerated cardiomyopathy phenotype compared with diabetic Ntg mice. In contrast, diabetic caPI3K mice were protected against diastolic dysfunction, pathological cardiomyocyte hypertrophy, fibrosis and apoptosis. Protection in diabetic caPI3K mice was associated with attenuation of left ventricular superoxide generation, attenuated Anp, Bnp, PKCß2, Ask1 and p22 ( phox ) expression, and elevated AKT. Further, in cardiomyocyte-like cells, increased PI3K(p110α) activity suppressed high glucose-induced superoxide generation and enhanced mitochondrial function. CONCLUSIONS/INTERPRETATION: These results demonstrate that reduced PI3K activity accelerates the development of diabetic cardiomyopathy, and that enhanced PI3K(p110α) activity can prevent adverse cardiac remodelling and dysfunction in a setting of diabetes.

Skeletal muscle-specific overproduction of constitutively activated c-Jun N-terminal kinase (JNK) induces insulin resistance in mice.

AIMS/HYPOTHESIS: Although skeletal muscle insulin resistance has been associated with activation of c-Jun N-terminal kinase (JNK), whether increased JNK activity causes insulin resistance in this organ is not clear. In this study we examined the metabolic consequences of isolated JNK phosphorylation in muscle tissue. METHODS: Plasmids containing genes encoding a wild-type JNK1 (WT-JNK) or a JNK1/JNKK2 fusion protein (rendering JNK constitutively active; CA-Jnk) were electroporated into one tibialis anterior (TA) muscle of C57Bl/6 mice, with the contralateral TA injected with an empty vector (CON) to serve as a within-animal control. RESULTS: Overproduction of WT-JNK resulted in a modest (~25%) increase in phosphorylation (Thr(183)/Tyr(185)) of JNK, but no differences were observed in Ser(307) phosphorylation of insulin receptor substrate 1 (IRS-1) or total IRS-1 protein, nor in insulin-stimulated glucose clearance into the TA muscle when comparing WT-JNK with CON. By contrast, overexpression of CA-Jnk, which markedly increased the phosphorylation of CA-JNK, also increased serine phosphorylation of IRS-1, markedly decreased total IRS-1 protein, and decreased insulin-stimulated phosphorylation of the insulin receptor (Tyr(1361)) and phosphorylation of Akt at (Ser(473) and Thr(308)) compared with CON. Moreover, overexpression of CA-Jnk decreased insulin-stimulated glucose clearance into the TA muscle compared with CON and these effects were observed without changes in intramuscular lipid species. CONCLUSIONS/INTERPRETATION: Constitutive activation of JNK in skeletal muscle impairs insulin signalling at the level of IRS-1 and Akt, a process which results in the disruption of normal glucose clearance into the muscle.

Interleukin-6-deficient mice develop hepatic inflammation and systemic insulin resistance.

AIMS/HYPOTHESIS: The role of IL-6 in the development of obesity and hepatic insulin resistance is unclear and still the subject of controversy. We aimed to determine whether global deletion of Il6 in mice (Il6 (-/-)) results in standard chow-induced and high-fat diet (HFD)-induced obesity, hepatosteatosis, inflammation and insulin resistance. METHODS: Male, 8-week-old Il6 (-/-) and littermate control mice were fed a standard chow or HFD for 12 weeks and phenotyped accordingly. RESULTS: Il6 (-/-) mice displayed obesity, hepatosteatosis, liver inflammation and insulin resistance when compared with control mice on a standard chow diet. When fed a HFD, the Il6 (-/-) and control mice had marked, equivalent gains in body weight, fat mass and ectopic lipid deposition in the liver relative to chow-fed animals. Despite this normalisation, the greater liver inflammation, damage and insulin resistance observed in chow-fed Il6 (-/-) mice relative to control persisted when both were fed the HFD. Microarray analysis from livers of mice fed a HFD revealed that genes associated with oxidative phosphorylation, the electron transport chain and tricarboxylic acid cycle were uniformly decreased in Il6 (-/-) relative to control mice. This coincided with reduced maximal activity of the mitochondrial enzyme ß-hydroxyacyl-CoA-dehydrogenase and decreased levels of mitochondrial respiratory chain proteins. CONCLUSIONS/INTERPRETATION: Our data suggest that IL-6 deficiency exacerbates HFD-induced hepatic insulin resistance and inflammation, a process that appears to be related to defects in mitochondrial metabolism.

Effects of the nitric oxide donor, sodium nitroprusside, on resting leg glucose uptake in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Nitric oxide (NO) has been implicated as an important signalling molecule in the contraction-mediated glucose uptake pathway and may represent a novel strategy for blood glucose control. The current study sought to determine whether acute infusion of the NO donor, sodium nitroprusside (SNP), increases leg glucose uptake at rest in patients with type 2 diabetes. METHODS: Fifteen male patients with type 2 diabetes (aged 54+/-4 years, mean+/-SD) were entered into a randomised, cross-over design study, examining the effect of a 30-min intra-femoral infusion of SNP on leg glucose uptake. Comparison was made with a 30-min infusion of verapamil, titrated to elicit similar leg blood flow responses to SNP. Leg blood flow was measured by thermodilution in the femoral vein, and leg glucose uptake was calculated as the product of leg blood flow and the femoral arterio-venous (A-V) glucose concentration gradient. RESULTS: The two drugs increased leg blood flow to a similar extent (p=0.50). Both leg A-V glucose concentration gradient (SNP 0.12+/-0.05, verapamil -0.06+/-0.04 mmol/l; mean+/- SEM, p=0.03) and leg glucose uptake (SNP 0.17+/-0.09, verapamil -0.09+/-0.06 mmol/min; p=0.03) were higher with the SNP treatment than with verapamil. These results occurred independently of any significant difference in plasma insulin concentration between drugs (p=0.56). CONCLUSIONS/INTERPRETATION: Acute infusion of SNP resulted in greater glucose uptake relative to verapamil. NO may therefore be an important mediator of peripheral glucose disposal and a potential therapeutic target in patients with type 2 diabetes.