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.

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.

PKR is not obligatory for high-fat diet-induced obesity and its associated metabolic and inflammatory complications.

Protein kinase R (PKR) has previously been suggested to mediate many of the deleterious consequences of a high-fat diet (HFD). However, previous studies have observed substantial phenotypic variability when examining the metabolic consequences of PKR deletion. Accordingly, herein, we have re-examined the role of PKR in the development of obesity and its associated metabolic complications in vivo as well as its putative lipid-sensing role in vitro. Here we show that the deletion of PKR does not affect HFD-induced obesity, hepatic steatosis or glucose metabolism, and only modestly affects adipose tissue inflammation. Treatment with the saturated fatty acid palmitate in vitro induced comparable levels of inflammation in WT and PKR KO macrophages, demonstrating that PKR is not necessary for the sensing of pro-inflammatory lipids. These results challenge the proposed role for PKR in obesity, its associated metabolic complications and its role in lipid-induced inflammation.

Targeting gp130 to prevent inflammation and promote insulin action.

Obesity and type 2 diabetes are now the most prevalent metabolic diseases in the Western world and the development of new strategies to treat these metabolic diseases is most warranted. Obesity results in a state of chronic low-grade inflammation in metabolically active tissues such as the liver, adipose tissue, brain and skeletal muscle. Work in our laboratory has focussed on the role of the cytokine interleukin-6 (IL)-6 and other IL-6-like cytokines that signal through the gp130 receptor complex. We have focussed on the role of blocking IL-6 trans-signalling to prevent inflammation on the one hand, and activating membrane-bound signalling to promote insulin sensitivity on the other hand. Since the cloning of the IL-6 gene nearly 30 years ago, a pattern has emerged associating IL-6 with a number of diseases associated with inflammation including rheumatoid arthritis (RA), Crohn's disease and several cancers. Accordingly, tocilizumab, an IL-6 receptor-inhibiting monoclonal antibody, is now useful for the treatment of RA. However, this may not be the most optimal strategy to block inflammation associated with IL-6 and may result in unwanted side effects that, paradoxically, could actually promote metabolic disease.

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.

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.

Deficiency of haematopoietic-cell-derived IL-10 does not exacerbate high-fat-diet-induced inflammation or insulin resistance in mice.

AIMS/HYPOTHESIS: Recent work has identified the important roles of M1 pro-inflammatory and M2 anti-inflammatory macrophages in the regulation of insulin sensitivity. Specifically, increased numbers of M2 macrophages and a decrease in M1 macrophages within the adipose tissue are associated with a state of enhanced insulin sensitivity. IL-10 is an anti-inflammatory cytokine and is a critical effector molecule of M2 macrophages. METHODS: In the present study, we examined the contribution of haematopoietic-cell-derived IL-10 to the development of obesity-induced inflammation and insulin resistance. We hypothesised that haematopoietic-cell-restricted deletion of IL-10 would exacerbate obesity-induced inflammation and insulin resistance. Lethally irradiated wild-type recipient mice receiving bone marrow from either wild-type or Il10-knockout mice were placed on either a chow or a high-fat diet for a period of 12 weeks and assessed for alterations in body composition, tissue inflammation and glucose and insulin tolerance. RESULTS: Contrary to our hypothesis, neither inflammation, as measured by the activation of pro-inflammatory stress kinases and gene expression of several pro-inflammatory cytokines in the adipose tissue and liver, nor diet-induced obesity and insulin resistance were exacerbated by the deletion of haematopoietic-cell-derived IL-10. Interestingly, however, Il10 mRNA expression and IL-10 protein production in liver and/or adipose tissue were markedly elevated in Il10-knockout bone-marrow-transplanted mice relative to wild-type bone marrow-transplanted mice. CONCLUSIONS/INTERPRETATION: These data show that deletion of IL-10 from the haematopoietic system does not potentiate high-fat diet-induced inflammation or insulin resistance.

Effect of prolonged exercise and carbohydrate ingestion on type 1 and type 2 T lymphocyte distribution and intracellular cytokine production in humans.

The present study was undertaken to examine the role of the exercise-induced stress hormone response on the regulation of type 1 and type 2 T lymphocyte intracellular cytokine production. Subjects performed 2.5 h of cycling exercise at 65% maximal O2 uptake while ingesting a 6.4% carbohydrate (CHO) solution, 12.8% CHO solution, or a placebo. Peripheral whole blood samples were stimulated and stained for T lymphocyte surface antigens (CD4 and CD8). Cells were then permeabilized, stained for intracellular cytokines, and analyzed using flow cytometry. Exercise resulted in a decrease (P < 0.05) in the number and percentage of IFN-gamma positive CD4+ and CD8+ T lymphocytes. These stimulated cells produced less IFN-gamma immediately postexercise (P < 0.05) and 2-h postexercise (P < 0.05) compared with preexercise. However, CHO ingestion, which attenuated the exercise-induced stress hormone response compared with placebo (P < 0.05), prevented both the decrease in the number and percentage of IFN-gamma-positive CD4+ and CD8+ T lymphocytes and the suppression of IFN-gamma production from stimulated CD4+ and CD8+ T lymphocytes. There was no effect of exercise on the number of, or cytokine production from, IL-4-positive CD4+ or CD8+ T lymphocytes. These data provide support for the role of exercise-induced elevations in stress hormones in the regulation of type 1 T lymphocyte cytokine production and distribution.

Exercise induces the release of heat shock protein 72 from the human brain in vivo.

The present study tested the hypothesis that in response to physical stress the human brain has the capacity to release heat shock protein 72 (Hsp72) in vivo. Therefore, 6 humans (males) cycled for 180 minutes at 60% of their maximal oxygen uptake, and the cerebral Hsp72 response was determined on the basis of the internal jugular venous to arterial difference and global cerebral blood flow. At rest, there was a net balance of Hsp72 across the brain, but after 180 minutes of exercise, we were able to detect the release of Hsp72 from the brain (335 +/- 182 ng/min). However, large individual differences were observed as 3 of the 6 subjects had a marked increase in the release of Hsp72, whereas exercise had little effect on the cerebral Hsp72 balance in the remaining 3 subjects. Given that cerebral blood flow was unchanged during exercise compared with values obtained at rest, it is unlikely that the cerebral Hsp72 release relates to necrosis of specific cells within the brain. These data demonstrate that the human brain is able to release Hsp72 in vivo in response to a physical stressor such as exercise. Further study is required to determine the biological significance of these observations.

Leukocyte heat shock protein expression before and after intensified training.

The purpose of the current research was to test the hypothesis that exercise induced leukocyte heat shock protein (HSP) expression is increased during periods of intensified exercise training. Seven male endurance cyclists carried out tests of maximal oxygen consumption and endurance capacity. These standard exercise tests were carried out prior to and following 6 days of prescribed intensified training. Sampled leukocytes were examined for Hsp27 and Hsp70 expression using a Fluorescence Activated Cell Scanner (EPICS XL, Coulter). During a period of overreaching, as signified by a drop in time to fatigue following the intensified training period (p = 0.02), the number of extracellular Hsp27 positive granulocytes increased in response to the VO(2)max test. Acute, intracellular HSP responses were observed in both baseline and overreached conditions. The present study showed that a period of intensified training caused adaptations in the acute heat shock protein exercise response, reflected by a greater increase of cell surface HSP positive leukocytes following heavy training.

Prior aspirin use in unstable angina predisposes to higher risk: the aspirin paradox.

BACKGROUND: Aspirin is the most widely prescribed agent used in prevention of coronary thrombosis. Thus, many patients presenting with acute coronary syndromes have a history of aspirin usage. METHODS: To assess response to medical therapy in patients taking aspirin prior to admission with an acute coronary syndrome, we reviewed outcomes data from the Efficacy and Safety of Subcutaneous Enoxaparin in Non-Q-wave Coronary Events (ESSENCE) and Platelet Receptor Inhibition in Ischemic Syndrome Management in Patients Limited by Unstable Signs and Symptoms (PRISM-PLUS) studies. RESULTS: Patients with acute coronary syndromes who had taken aspirin prior to enrollment were less likely to have non-Q-wave myocardial infarction on admission (ESSENCE: 16.0% vs. 29.2%, p<0.001; PRISM-PLUS: 34.2% vs. 57.7%, p<0.001). However, prior aspirin users were more likely to be failed by standard medical therapy with unfractionated heparin than non-prior aspirin users (ESSENCE: 21.5% vs. 16.5%, p=0.017; PRISM-PLUS: 23.5% vs. 12.1%, p<0.001). Prior aspirin users received greater benefit from both enoxaparin (21.5% vs. 16.8%, p=0.009) and tirofiban with unfractionated heparin (23.5% vs. 16.0%, p=0.007) than from unfractionated heparin alone. Non-prior aspirin users presented with higher rates of non-Q-wave myocardial infarction. CONCLUSIONS: Prior aspirin users admitted with acute coronary syndromes may have a more benign presentation, but are more likely to be failed by medical therapy with unfractionated heparin and should be considered as a high-risk group. Enoxaparin or the combination of tirofiban and unfractionated heparin are both more effective than unfractionated heparin in this group.

Nutritional strategies to minimise exercise-induced immunosuppression in athletes.

Strenuous prolonged exertion and heavy training are associated with depressed immune function. Furthermore, improper nutrition can compound the negative influence of heavy exertion on immunocompetence. Dietary deficiencies of protein and specific micronutrients have long been associated with immune dysfunction. An adequate intake of iron, zinc, and vitamins A, E, B6 and B12 is particularly important but excess intakes can also impair immune function. Immune system impairment has also been associated with excess intake of fat. To maintain immune function, athletes should eat a well balanced diet sufficient to meet their energy requirements. An athlete exercising in a carbohydrate-depleted state experiences larger increases in circulating stress hormones and a greater perturbation of several immune function indices. Conversely, consuming carbohydrate during exercise attenuates rises in stress hormones such as cortisol and appears to limit the degree of exercise-induced immunosuppression, at least for non-fatiguing bouts of exercise. Strong evidence that high doses of antioxidant vitamins, glutamine supplementation or echinacea extracts can prevent exercise-induced immunosuppression is lacking.