KEY POINTS: The heat of activation of cardiac muscle reflects the metabolic cost of restoring ionic homeostasis following a contraction. The accuracy of its measurement depends critically on the abolition of crossbridge cycling. We abolished crossbridge activity in isolated rat ventricular trabeculae by use of blebbistatin, an agent that selectively inhibits myosin II ATPase. We found cardiac activation heat to be muscle length independent and to account for 15-20% of total heat production at body temperature. We conclude that it can be accurately estimated at minimal muscle length. ABSTRACT: Activation heat arises from two sources during the contraction of striated muscle. It reflects the metabolic expenditure associated with Ca2+ pumping by the sarcoplasmic reticular Ca2+ -ATPase and Ca2+ translocation by the Na+ /Ca2+ exchanger coupled to the Na+ ,K+ -ATPase. In cardiac preparations, investigators are constrained in estimating its magnitude by reducing muscle length to the point where macroscopic twitch force vanishes. But this experimental protocol has been criticised since, at zero force, the observed heat may be contaminated by residual crossbridge cycling activity. To eliminate this concern, the putative thermal contribution from crossbridge cycling activity must be abolished, at least at minimal muscle length. We achieved this using blebbistatin, a selective inhibitor of myosin II ATPase. Using a microcalorimeter, we measured the force production and heat output, as functions of muscle length, of isolated rat trabeculae from both ventricles contracting isometrically at 5 Hz and at 37°C. In the presence of blebbistatin (15 μmol l-1 ), active force was zero but heat output remained constant, at all muscle lengths. Activation heat measured in the presence of blebbistatin was not different from that estimated from the intercept of the heat-stress relation in its absence. We thus reached two conclusions. First, activation heat is independent of muscle length. Second, residual crossbridge heat is negligible at zero active force; hence, the intercept of the cardiac heat-force relation provides an estimate of activation heat uncontaminated by crossbridge cycling. Both results resolve long-standing disputes in the literature.

KEY POINTS: Uteroplacental insufficiency compromises maternal mammary development, milk production and pup organ development; this is ameliorated by cross-fostering, which improves pup growth and organ development and prevents adult diseases in growth-restricted (Restricted) offspring by enhancing postnatal nutrition. Leptin is transported to the fetus from the mother by the placenta; we report reduced plasma leptin concentrations in Restricted fetuses associated with sex-specific alterations in placental leptin transporter expression. Pup plasma leptin concentrations were also reduced during suckling, which may suggest reduced milk leptin transport or leptin reabsorption. Mothers suckled by Restricted pups had impaired mammary development and changes in milk fatty acid composition with no alterations in milk leptin; cross-fostering restored pup plasma leptin concentrations, which may be correlated to improved milk composition and intake. Increased plasma leptin and altered milk fatty acid composition in Restricted pups suckling mothers with normal lactation may improve postnatal growth and prevent adult diseases. ABSTRACT: Uteroplacental insufficiency reduces birth weight and adversely affects fetal organ development, increasing adult disease risk. Cross-fostering improves postnatal nutrition and restores these deficits. Mothers with growth-restricted pups have compromised milk production and composition; however, the impact cross-fostering has on milk production and composition is unknown. Plasma leptin concentrations peak during the completion of organogenesis, which occurs postnatally in rats. Leptin is transferred to the fetus via the placenta and to the pup via the lactating mammary gland. This study investigated the effect of uteroplacental insufficiency on pup plasma leptin concentrations and placental leptin transporters. We additionally examined whether cross-fostering improves mammary development, milk composition and pup plasma leptin concentrations. Fetal growth restriction was induced by bilateral uterine vessel ligation surgery on gestation day 18 in Wistar Kyoto rats (termed uteroplacental insufficiency surgery mothers). Growth-restricted (Restricted) fetuses had reduced plasma leptin concentrations, persisting throughout lactation, and sex-specific alterations in placental leptin transporters. Mothers suckled by Restricted pups had impaired mammary development, altered milk fatty acid composition and increased plasma leptin concentrations, despite no changes in milk leptin. Milk intake was reduced in Restricted pups suckling uteroplacental insufficiency surgery mothers compared to Restricted pups suckling sham-operated mothers. Cross-fostering Restricted pups onto a sham-operated mother improved postnatal growth and restored plasma leptin concentrations compared to Restricted pups suckling uteroplacental insufficiency surgery mothers. Uteroplacental insufficiency alters leptin homeostasis. This is ameliorated with cross-fostering and enhanced milk fatty acid composition and consumption, which may protect the pups from developing adverse health conditions in adulthood.

Babies born small are at an increased risk of developing myriad adult diseases. While growth restriction increases disease risk in all individuals, often a second hit is required to unmask 'programmed' impairments in physiology. Programmed disease outcomes are demonstrated more commonly in male offspring compared with females, with these sex-specific outcomes partly attributed to different placenta-regulated growth strategies of the male and female fetus. Pregnancy is known to be a major risk factor for unmasking a number of conditions and can be considered a 'second hit' for women who were born small. As such, female offspring often develop impairments of physiology for the first time during pregnancy that present as pregnancy complications. Numerous maternal stressors can further increase the risk of developing a maternal complication during pregnancy. Importantly, these maternal complications can have long-term consequences for both the mother after pregnancy and the developing fetus. Conditions such as preeclampsia, gestational diabetes and hypertension as well as thyroid, liver and kidney diseases are all conditions that can complicate pregnancy and have long-term consequences for maternal and offspring health. Babies born to mothers who develop these conditions are often at a greater risk of developing disease in adulthood. This has implications as a mechanism for transmission of disease across generations. In this review, we discuss the evidence surrounding long-term intergenerational implications of being born small and/or experiencing stress during pregnancy on programming outcomes.

This study seeks to determine whether knockdown of basal forebrain p75 neurotrophin receptor (p75(NTR) ) expression elicits increased hippocampal choline acetyltransferase (ChAT) activity in mature animals. Antisense (AS) oligonucleotides (oligos) targeting p75(NTR) were infused into the medial septal area of mature rats continuously for 4 weeks. In all rats, the cannula outlet was placed equidistant between the left and the right sides of the vertical diagonal band of Broca. We tested phosphorothioate (PS), morpholino (Mo), and gapmer (mixed PS/RNA) oligos. Gapmer AS infusions of 7.5 and 22 μg/day decreased septal p75(NTR) mRNA by 34% and 48%, respectively. The same infusions increased hippocampal ChAT activity by 41% and 55%. Increased hippocampal ChAT activity correlated strongly with septal p75(NTR) downregulation in individual rats. Infusions of PS and Mo AS oligos did not downregulate p75(NTR) mRNA or stimulate ChAT activity. These results demonstrate that p75(NTR) can dynamically regulate hippocampal ChAT activity in the mature CNS. They also reveal the different efficacies of three diverse AS oligo chemistries when infused intracerebrally. Among the three types, gapmer oligos worked best.

OBJECTIVE: To develop a frailty index (FI) and explore the relationship of frailty to subsequent adverse outcomes on the effectiveness and safety of more intensive control of both blood glucose and blood pressure (BP), among participants with type 2 diabetes in the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial. RESEARCH DESIGN AND METHODS: Cox proportional hazard models were used to estimate the effectiveness and safety of intensive glucose control and BP intervention according to frailty (defined as FI >0.21) status. The primary outcomes were macro- and microvascular events. The secondary outcomes were all-cause mortality, cardiovascular mortality, severe hypoglycemia, and discontinuation of BP treatment due to hypotension/dizziness. RESULTS: There were 11,140 participants (mean age, 65.8 years; 42.5% women, 25.7% frail). Frailty was an independent predictor of all primary outcomes and secondary outcomes. The effect of intensive glucose treatment on primary outcomes showed some evidence of attenuation in the frail: hazard ratios for combined major macro- and microvascular events 1.03 (95% CI 0.90-1.19) in the frail versus 0.84 (95% CI 0.74-0.94) in the nonfrail (P = 0.02). A similar trend was observed with BP intervention. Severe hypoglycemia rates (per 1,000 person-years) were higher in the frail: 8.39 (6.15-10.63) vs. 4.80 (3.84-5.76) in nonfrail (P < 0.001). There was no significant difference in discontinuation of BP treatment between frailty groups. CONCLUSIONS: It was possible to retrospectively estimate frailty in a trial population, and this FI identified those at higher risk of poor outcomes. Participants with frailty had some attenuation of benefit from intensive glucose-lowering and BP-lowering treatments.

AIMS/HYPOTHESIS: Type 2 diabetes increases the risk of cardiovascular and renal complications, but early risk prediction could lead to timely intervention and better outcomes. Genetic information can be used to enable early detection of risk. METHODS: We developed a multi-polygenic risk score (multiPRS) that combines ten weighted PRSs (10 wPRS) composed of 598 SNPs associated with main risk factors and outcomes of type 2 diabetes, derived from summary statistics data of genome-wide association studies. The 10 wPRS, first principal component of ethnicity, sex, age at onset and diabetes duration were included into one logistic regression model to predict micro- and macrovascular outcomes in 4098 participants in the ADVANCE study and 17,604 individuals with type 2 diabetes in the UK Biobank study. RESULTS: The model showed a similar predictive performance for cardiovascular and renal complications in different cohorts. It identified the top 30% of ADVANCE participants with a mean of 3.1-fold increased risk of major micro- and macrovascular events (p = 6.3 × 10-21 and p = 9.6 × 10-31, respectively) and a 4.4-fold (p = 6.8 × 10-33) higher risk of cardiovascular death. While in ADVANCE overall, combined intensive blood pressure and glucose control decreased cardiovascular death by 24%, the model identified a high-risk group in whom it decreased the mortality rate by 47%, and a low-risk group in whom it had no discernible effect. High-risk individuals had the greatest absolute risk reduction with a number needed to treat of 12 to prevent one cardiovascular death over 5 years. CONCLUSIONS/INTERPRETATION: This novel multiPRS model stratified individuals with type 2 diabetes according to risk of complications and helped to target earlier those who would receive greater benefit from intensive therapy.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry-based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and in situ muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross-species phosphosite responses, as well as unique model-specific regulation. We identified > 22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK-mediated phosphorylation of STIM1 negatively regulates store-operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross-species resource of exercise-regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

AIMS: To evaluate 1,5-anhydroglucitol (1,5-AG) according to clinical outcomes and assess the effects of glucose- and blood pressure-lowering interventions on change in 1,5-AG levels in people with type 2 diabetes. METHODS: We measured 1,5-AG in 6826 stored samples at baseline and in a random subsample of 684 participants at the 1-year follow-up visit in the ADVANCE trial. We examined baseline 1,5-AG [< 39.7, 39.7-66.2, ≥ 66.2 μmol/L (<6, 6-10, ≥10 μg/mL)] and microvascular and macrovascular events and mortality using Cox regression models during 5 years of follow-up. Using an intention-to-treat approach, we examined 1-year change in 1,5-AG (mean and percent) in response to the glucose- and blood pressure-lowering interventions in the subsample. RESULTS: Low 1,5-AG level [<39.7 μmol/L vs ≥ 66.2 μmol/L (<6 μg/mL vs ≥10 μg/mL)] was associated with microvascular events (hazard ratio 1.28, 95% confidence interval 1.03-1.60) after adjustment for risk factors and baseline glycated haemoglobin (HbA1c); however, the associations for macrovascular events and mortality were not independent of HbA1c. The glucose-lowering intervention was associated with a significant 1-year increase in 1,5-AG (vs standard control) of 6.69 μmol/L (SE 2.52) [1.01 μg/mL (SE 0.38)], corresponding to an 8.26% (SE 0.10%) increase from baseline. We also observed an increase in 1,5-AG of similar magnitude in response to the blood pressure intervention independent of the glucose-lowering effect. CONCLUSIONS: Our results suggest that 1,5-AG is a marker of risk in adults with type 2 diabetes, but only for microvascular events independently of HbA1c. We found that 1,5-AG was improved (increased) in response to an intensive glucose-lowering intervention, although the independent effect of the blood pressure-lowering intervention on 1,5-AG suggests potential non-glycaemic influences.

KEY POINTS: Linoleic acid consumption is increasing in Western populations. We investigated whether elevated linoleic acid in pregnancy was deleterious to mothers or offspring. Maternal and fetal body and organ weights were not affected by elevated linoleic acid consumption. Maternal lipids and leptin were altered following elevated linoleic acid consumption. Male offspring numbers were reduced following elevated linoleic acid consumption. ABSTRACT: Dietary intakes of linoleic acid (LA) have increased dramatically in Western populations, including in women of reproductive age. Pro-inflammatory effects of LA may have detrimental effects on maternal and offspring outcomes. We aimed to investigate whether consumption of a maternal diet with elevated LA altered maternal inflammatory or metabolic markers during pregnancy, fetal growth and/or the sex ratio of the offspring. Female Wistar Kyoto rats consumed a diet high in LA (HLA) (6.21% of energy) or a diet low in LA (LLA) (1.44% of energy) for 10 weeks prior to mating and during pregnancy. Pregnant rats were killed at embryonic day 20 (E20). There were no differences in maternal or fetal body weights or organ weights in the HLA group compared to the LLA group. There was no difference in maternal circulating cytokine concentrations between dietary groups. In the maternal liver, IL-1α concentrations were significantly lower, and TNF-α and IL-7 significantly higher in the HLA group. Total plasma cholesterol, LDL-cholesterol, HDL cholesterol and the total:HDL cholesterol ratio were lower in dams fed the HLA diet. mRNA expression of sterol regulatory element binding transcription factor 1 (SREBF-1) and leptin in maternal adipose tissue was lower in the HLA group, as were circulating leptin concentrations. The proportion of male fetuses was lower and circulating prostaglandin E metabolite concentrations were increased in the HLA group. In conclusion, consumption of a maternal diet high in linoleic acid alters cholesterol metabolism and prostaglandin E metabolite concentrations, which may contribute to the reduced proportion of male offspring.

KEY POINTS: Fetal growth is dependent on effective placental nutrient transportation, which is regulated by mammalian target of rapamycin (mTOR) complex 1 modulation of nutrient transporter expression. These transporters are dysregulated in pregnancies affected by uteroplacental insufficiency and maternal obesity. Nutrient transporters and mTOR were altered in placentae of mothers born growth restricted compared to normal birth weight dams, with maternal diet- and fetal sex-specific responses. Exercise initiated during pregnancy downregulated mTOR protein expression, despite an increase in mTOR activation in male associated placentae, and reduced nutrient transporter gene abundance, which was also dependent on maternal diet and fetal sex. Limited changes were characterized with exercise initiated before and continued throughout pregnancy in nutrient transporter and mTOR expression. Maternal exercise during pregnancy differentially regulated mTOR and nutrient transporters in a diet- and sex-specific manner, which likely aimed to improve late gestational placental growth and neonatal survival. ABSTRACT: Adequate transplacental nutrient delivery is essential for fetoplacental development. Intrauterine growth restriction and maternal obesity independently alter placental nutrient transporter expression. Although exercise is beneficial for maternal health, limited studies have characterized how the timing of exercise initiation influences placental nutrient transport. Therefore, this study investigated the impact of maternal exercise on placental mechanistic target of rapamycin (mTOR) and nutrient transporter expression in growth restricted mothers and whether these outcomes were dependent on maternal diet or fetal sex. Uteroplacental insufficiency or sham surgery was induced on embryonic day (E) 18 in Wistar-Kyoto rats. F1 offspring were fed a chow or high-fat diet from weaning and at 16 weeks were randomly allocated to an exercise protocol: sedentary, exercised prior to and during pregnancy, or exercised during pregnancy only. Females were mated with normal males (20 weeks) and F2 placentae collected at E20. Exercise during pregnancy only, reduced mTOR protein expression in all groups and increased mTOR activation in male associated placentae. Exercise during pregnancy only, decreased the expression of amino acid transporters in a diet- and sex-specific manner. Maternal growth restriction altered mTOR and system A amino acid transporter expression in a sex- and diet-specific manner. These data highlight that maternal exercise initiated during pregnancy alters placental mTOR expression, which may directly regulate amino acid transporter expression, to a greater extent than exercise initiated prior to and continued during pregnancy, in a diet- and fetal sex-dependent manner. These findings highlight that the timing of exercise initiation is important for optimal placental function.

AIMS: Patients with type 2 diabetes have a high risk of cardiovascular disease (CVD). Central obesity has been particularly associated with this risk relationship. We aimed to evaluate waist to height ratio (WHtR) as a predictor of risk in such patients. METHODS: WHtR was evaluated as a predictor of the risk of CVD and mortality amongst 11 125 participants with type 2 diabetes in the ADVANCE and ADVANCE-ON studies, and was compared with body mass index (BMI), waist circumference and waist hip ratio (WHR). Primary outcome was a composite of death from CVD, non-fatal myocardial infarction or non-fatal stroke. Secondary outcomes were myocardial infarction, stroke, cardiovascular death and death from any cause. Cox models were used, with bootstrapping to compare associations between anthropometric measures for the primary outcome. RESULTS: Median follow-up time was 9.0 years. There was a positive association between WHtR and adverse outcomes. The hazard ratio (HR) (confidence interval), per SD higher WHtR, was 1.16 (1.11-1.22) for the primary endpoint, with no heterogeneity by sex or region, but a stronger effect in individuals aged 66 years or older. The other 3 anthropometric measurements showed similar associations, although there was evidence that WHtR marginally outperformed BMI and WHR. Based on commonly used BMI cut-points, the equivalent WHtR cut-points were estimated to be 0.55 and 0.6, with no evidence of a difference across subgroups. CONCLUSIONS: In patients with diabetes, WHtR is a useful indicator of future adverse risk, with similar effects in different population subgroups.

AIM: To estimate the associations between risk factors and cognitive decline (CD)/dementia, and the sex differences in these risk factors in individuals with type 2 diabetes, while accounting for the competing risk of death. MATERIALS AND METHODS: The Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial of 11,140 individuals with type 2 diabetes was used to estimate the odds of CD/dementia using multinomial logistic regression. RESULTS: During a median 5-year follow-up, 1827 participants (43.2% women) had CD/dementia (1718 with CD only; 21 with dementia only; 88 with CD and dementia), and 929 (31.0% women) died without CD/dementia. Women had lower odds of CD/dementia than men (odds ratio [OR] [95% confidence interval], 0.88 [0.77, 1.00]); older age, higher total cholesterol, HbA1c, waist circumference, waist-to-height ratio, moderately increased albumin-creatinine ratio, stroke/transient ischaemic attack and retinal disease were each associated with greater odds of CD/dementia; higher years at education completion, baseline cognitive function, taller stature and current alcohol use were inversely associated. Higher waist circumference (women-to-men ratio of ORs [ROR], 1.05 [1.00, 1.10] per 5 cm) and presence of anxiety/depression (ROR, 1.28 [1.01, 1.63]) were associated with greater ORs for CD/dementia in women than men. CONCLUSIONS: Several risk factors were associated with CD/dementia. Higher waist circumference and mental health symptoms were more strongly associated with CD/dementia in women than men. Further studies should examine the mechanisms that underlie these sex differences.

Increased heart size is a major risk factor for heart failure and premature mortality. Although abnormal heart growth subsequent to hypertension often accompanies disturbances in mechano-energetics and cardiac efficiency, it remains uncertain whether hypertrophy is their primary driver. In this study, we aimed to investigate the direct association between cardiac hypertrophy and cardiac mechano-energetics using isolated left-ventricular trabeculae from a rat model of primary cardiac hypertrophy and its control. We evaluated energy expenditure (heat output) and mechanical performance (force length work production) simultaneously at a range of preloads and afterloads in a microcalorimeter, we determined energy expenditure related to cross-bridge cycling and Ca2+ cycling (activation heat), and we quantified energy efficiency. Rats with cardiac hypertrophy exhibited increased cardiomyocyte length and width. Their trabeculae showed mechanical impairment, evidenced by lower force production, extent and kinetics of shortening, and work output. Lower force was associated with lower energy expenditure related to Ca2+ cycling and to cross-bridge cycling. However, despite these changes, both mechanical and cross-bridge energy efficiency were unchanged. Our results show that cardiac hypertrophy is associated with impaired contractile performance and with preservation of energy efficiency. These findings provide direction for future investigations targeting metabolic and Ca2+ disturbances underlying cardiac mechanical and energetic impairment in primary cardiac hypertrophy.

AIM: To examine possible sex differences in the excess risk of myocardial infarction (MI) consequent to a range of conventional risk factors in a large-scale international cohort of patients with diabetes, and to quantify these potential differences both on the relative and absolute scales. MATERIALS AND METHODS: Eleven thousand and sixty-five participants (42% women) with type 2 diabetes in the ADVANCE trial and its post-trial follow-up study, ADVANCE-ON, were included. Cox regression models were used to estimate hazard ratios (HRs) for associations between risk factors and MI (fatal and non-fatal) by sex, and the women-to-men ratio of HRs (RHR). RESULTS: Over a median of 9.6 years of follow-up, 719 patients experienced MI. Smoking status, smoking intensity, higher systolic blood pressure (SBP), HbA1c, total and LDL cholesterol, duration of diabetes, triglycerides, body mass index (BMI) and lower HDL cholesterol were associated with an increased risk of MI in both sexes. Furthermore, some variables were associated with a greater relative risk of MI in women than men: RHRs were 1.75 (95% CI: 1.05-2.91) for current smoking, 1.53 (1.00-2.32) for former smoking, 1.18 (1.02-1.37) for SBP, and 1.13 (95% CI, 1.003-1.26) for duration of diabetes. Although incidence rates of MI were higher in men (9.3 per 1000 person-years) compared with women (5.8 per 1000 person-years), rate differences associated with risk factors were greater in women than men, except for HDL cholesterol and BMI. CONCLUSIONS: In patients with type 2 diabetes, smoking, higher SBP and longer duration of diabetes had a greater relative and absolute effect in women than men, highlighting the importance of routine sex-specific approaches and early interventions in women with diabetes.

Maternal nutrition plays a critical role in fetal development and can influence adult onset of disease. Linoleic acid (LA) and alpha-linolenic acid (ALA) are major omega-6 (n-6) and n-3 polyunsaturated fatty acids (PUFA), respectively, that are essential in our diet. LA and ALA are critical for the development of the fetal neurological and immune systems. However, in recent years, the consumption of n-6 PUFA has increased gradually worldwide, and elevated n-6 PUFA consumption may be harmful to human health. Consumption of diets with high levels of n-6 PUFA before or during pregnancy may have detrimental effects on fetal development and may influence overall health of offspring in adulthood. This review discusses the role of n-6 PUFA in fetal programming, the importance of a balance between n-6 and n-3 PUFAs in the maternal diet, and the need of further animal models and human studies that critically evaluate both n-6 and n-3 PUFA contents in diets.

The endocannabinoid system (ECS), modulated by metabolites of linoleic acid (LA), is important in regulating cardiovascular function. In pregnancy, LA is vital for foetal development. We investigated the effects of elevated LA in H9c2 cardiomyoblasts in vitro and of a high linoleic acid (HLA, 6.21%) or low linoleic acid (LLA, 1.44%) diet during pregnancy in maternal and offspring hearts. H9c2 cell viability was reduced following LA exposure at concentrations between 300 and 1000 µM. HLA diet decreased cannabinoid receptor type 2 (CB2) mRNA expression in foetal hearts from both sexes. However, HLA diet increased CB2 expression in maternal hearts. The mRNA expression of fatty acid amide hydrolase (FAAH) in foetal hearts was higher in females than in males irrespective of diet and N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) mRNA expression showed an interaction between diet and sex. Data indicate that a high LA diet alters cell viability and CB2 expression, potentially influencing cardiac function during pregnancy and development of the offspring's heart.

Dietary intakes of linoleic acid (LA) have increased, including in women of reproductive age. Changes in maternal gut microbiome have been implicated in the metabolic adaptions that occur during pregnancy. We aimed to investigate whether consumption of a diet with elevated LA altered fecal microbiome diversity before and during pregnancy. Female Wistar-Kyoto rats consumed a high-LA diet (HLA: 6.21% of energy) or a low-LA diet (LLA: 1.44% of energy) for 10 wk before mating and during pregnancy. DNA was isolated from fecal samples before pregnancy [embryonic day 0 (E0)], or during pregnancy at E10 and E20. The microbiome composition was assessed with 16S rRNA sequencing. At E0, the beta-diversity of LLA and HLA groups differed with HLA rats having significantly lower abundance of the genera Akkermansia, Peptococcus, Sutterella, and Xo2d06 but higher abundance of Butyricimonas and Coprococcus. Over gestation, in LLA but not HLA rats, there was a reduction in alpha-diversity and an increase in beta-diversity. In the LLA group, the abundance of Akkermansia, Blautia, rc4.4, and Streptococcus decreased over gestation, whereas Coprococcus increased. In the HLA group; only the abundance of Butyricimonas decreased. At E20, there were no differences in alpha- and beta-diversity, and the abundance of Roseburia was significantly increased in the HLA group. In conclusion, consumption of a HLA diet alters gut microbiota composition, as does pregnancy in rats consuming a LLA diet. In pregnancy, consumption of a HLA diet does not alter gut microbiota composition.

Gliomas are the most common central nervous system malignancies and present with significant morbidity and mortality. Treatment modalities are currently limited to surgical resection, chemotherapy and radiotherapy. Increases in survival rate over the previous decades are negligible, further pinpointing an unmet clinical need in this field. There is a continual struggle with the development of effective glioma diagnostics and therapeutics, largely due to a multitude of factors, including the presence of the blood-brain barrier and significant intertumoural and intratumoural heterogeneity. Importantly, there is a lack of reliable biomarkers for glioma, particularly in aiding tumour subtyping and measuring response to therapy. There is a need for biomarkers that would both overcome the complexity of the disease and allow for a minimally invasive means of detection and analysis. This is a comprehensive review evaluating the potential of current cellular, proteomic and molecular biomarker candidates for glioma. Significant hurdles faced in glioma diagnostics and therapy are also discussed here.

OBJECTIVES: Preferential damage to fast, glycolytic myofibers is common in many muscle-wasting diseases, including Duchenne muscular dystrophy (DMD). Promoting an oxidative phenotype could protect muscles from damage and ameliorate the dystrophic pathology with therapeutic relevance, but developing efficacious strategies requires understanding currently unknown biological roles for dystrophin and utrophin in dystrophic muscle adaptation and plasticity. METHODS: Combining whole transcriptome RNA sequencing and mitochondrial proteomics with assessments of metabolic and contractile function, we investigated the roles of dystrophin and utrophin in fast-to-slow muscle remodeling with low-frequency electrical stimulation (LFS, 10 Hz, 12 h/d, 7 d/wk, 28 d) in mdx (dystrophin null) and dko (dystrophin/utrophin null) mice, two established preclinical models of DMD. RESULTS: Novel biological roles in adaptation were demonstrated by impaired transcriptional activation of estrogen-related receptor alpha-responsive genes supporting oxidative phosphorylation in dystrophic muscles. Further, utrophin expression in dystrophic muscles was required for LFS-induced remodeling of mitochondrial respiratory chain complexes, enhanced fiber respiration, and conferred protection from eccentric contraction-mediated damage. CONCLUSIONS: These findings reveal novel roles for dystrophin and utrophin during LFS-induced metabolic remodeling of dystrophic muscle and highlight the therapeutic potential of LFS to ameliorate the dystrophic pathology and protect from contraction-induced injury with important implications for DMD and related muscle disorders.

Multiple myeloma is an incurable malignancy which despite progressive improvements in overall survival over the last decade remains characterised by recurrent relapse with progressively shorter duration of response and treatment-free intervals with each subsequent treatment. Efforts to unravel the complex and heterogeneous genomic alterations, the marked dysregulation of the immune system and the multifarious interplay between malignant plasma cells and those of the tumour microenvironment have not only led to improved understanding of myelomagenesis and disease progression but have facilitated the rapid development of novel therapeutics including immunotherapies and small molecules bringing us a step closer to therapies that no doubt will extend survival. Novel therapeutic combinations both in the upfront and relapsed setting as well as novel methods to assess response and guide management are rapidly transforming the management of myeloma.

The male germ line is capable of transmitting a legacy of stress exposure to the next generation of offspring. This transgenerational process manifests by altering offspring affective behaviours, cognition and metabolism. Paternal early life trauma causes hippocampal serotonergic dysregulation in male offspring. We previously showed a transgenerational modification to male offspring anxiety-like behaviours by treatment of adult male breeders with corticosterone (CORT) prior to mating. In the present study, we used offspring from our paternal CORT model and characterised offspring serotonergic function by examining their responses to the 5HT1AR agonist, 8-OH-DPAT, and the selective serotonin reuptake inhibitor, sertraline. We also examined whether post-weaning environmental enrichment, a paradigm well-known to modulate serotonergic signalling in the brain, had the capacity to normalise the anxiety phenotype of male offspring. Finally, we assessed gene expression levels of 5HT1AR and serotonin transporter in the offspring hippocampus to determine whether deficits in gene transcription contributed to the male-only anxiety phenotype. We report that male and female offspring of CORT-treated fathers are hypersensitive to sertraline but have normal hypothermic responses to 8-OH-DPAT. No deficits in htr1a and sert were found in association with paternal CORT treatment, and environmental enrichment did not rescue the anxiety phenotype of male offspring on the elevated-plus maze. These findings indicate that varying forms of paternal stress exert different effects on offspring brain serotonergic function.

In an emerging bioelectronics era, there is a clinical need for physiological devices incorporating biofeedback that permits natural and demand-dependent control in real time. Here, we describe a novel device termed a central pattern generator (CPG) that uses cutting edge analog circuitry producing temporally controlled, electrical stimulus outputs based on the real time integration of physiological feedback. Motivated by the fact that respiratory sinus arrhythmia (RSA), which is the cyclical changes in heart rate every breath, is an essential component of heart rate variability (HRV) (an indicator of cardiac health), we have explored the versatility and efficiency of the CPG for producing respiratory modulation of heart rate in anesthetized, spontaneously breathing rats. Diaphragmatic electromyographic activity was used as the input to the device and its output connected to either the right cervical vagus nerve or the right atrium for pacing heart rate. We found that the CPG could induce respiratory related heart rate modulation that closely mimicked RSA. Whether connected to the vagus nerve or right atrium, the versatility of the device was demonstrated by permitting: (i) heart rate modulation in any phase of the respiratory cycle, (ii) control of the magnitude of heart rate modulation, and (iii) instant adaptation to changes in respiratory frequency. Vagal nerve pacing was only possible following transection of the nerve limiting its effective use chronically. Pacing via the right atrium permitted better flexibility and control of heart rate above its intrinsic level. This investigation now lays the foundation for future studies using this biofeedback technology permitting closer analysis of both the function and dysfunction of RSA.

Narrow arterioles in the retina have been shown to predict hypertension as well as other vascular diseases, likely through an increase in the peripheral resistance of the microcirculatory flow. In this study, we performed a genome-wide association study in 18,722 unrelated individuals of European ancestry from the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium and the Blue Mountain Eye Study, to identify genetic determinants associated with variations in retinal arteriolar caliber. Retinal vascular calibers were measured on digitized retinal photographs using a standardized protocol. One variant (rs2194025 on chromosome 5q14 near the myocyte enhancer factor 2C MEF2C gene) was associated with retinal arteriolar caliber in the meta-analysis of the discovery cohorts at genome-wide significance of P-value <5×10(-8). This variant was replicated in an additional 3,939 individuals of European ancestry from the Australian Twins Study and Multi-Ethnic Study of Atherosclerosis (rs2194025, P-value = 2.11×10(-12) in combined meta-analysis of discovery and replication cohorts). In independent studies of modest sample sizes, no significant association was found between this variant and clinical outcomes including coronary artery disease, stroke, myocardial infarction or hypertension. In conclusion, we found one novel loci which underlie genetic variation in microvasculature which may be relevant to vascular disease. The relevance of these findings to clinical outcomes remains to be determined.

Glycogen is a highly branched glucose polymer functioning as a glucose buffer in animals. Multiple-detector size exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis were used to examine the structure of undegraded native liver glycogen (both whole and enzymatically debranched) as a function of molecular size, isolated from the livers of healthy and db/db mice (the latter a type 2 diabetic model). Both the fully branched and debranched levels of glycogen structure showed fundamental differences between glycogen from healthy and db/db mice. Healthy glycogen had a greater population of large particles, with more α particles (tightly linked assemblages of smaller β particles) than glycogen from db/db mice. These structural differences suggest a new understanding of type 2 diabetes.

Cholera toxin (CT) is well established to produce diarrhea by producing hyperactivity of the enteric neural circuits that regulate water and electrolyte secretion. Its effects on intestinal motor patterns are less well understood. We examined the effects of luminal CT on motor activity of guinea-pig jejunum in vitro. Segments of jejunum were cannulated at either end and mounted horizontally. Their contractile activity was video-imaged and the recordings were used to construct spatiotemporal maps of contractile activity with CT (1.25 or 12.5 μg/ml) in the lumen. Both concentrations of CT induced propulsive motor activity in jejunal segments. The effect of 1.25 μg/ml CT was markedly enhanced by co-incubation with granisetron (5-HT(3) antagonist, 1 μM), which prevents the hypersecretion induced by CT. The increased propulsive activity was not accompanied by increased segmentation and occurred very early after exposure to CT in the presence of granisetron. Luminal CT also reduced the pressure threshold for saline distension evoked propulsive reflexes, an effect resistant to granisetron. In contrast, CT prevented the induction of segmenting contractions by luminal decanoic acid, so its effects on propulsive and segmenting contractile activity are distinctly different. Thus, in addition to producing hypersecretion, CT excites propulsive motor activity with an entirely different time course and pharmacology, but inhibits nutrient-induced segmentation. This suggests that CT excites more than one enteric neural circuit and that propulsive and segmenting motor patterns are differentially regulated.

Endocannabinoids and their G-protein coupled receptors (GPCR) are a current research focus in the area of obesity due to the system's role in food intake and glucose and lipid metabolism. Importantly, overweight and obese individuals often have higher circulating levels of the arachidonic acid-derived endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) and an altered pattern of receptor expression. Consequently, this leads to an increase in orexigenic stimuli, changes in fatty acid synthesis, insulin sensitivity, and glucose utilisation, with preferential energy storage in adipose tissue. As endocannabinoids are products of dietary fats, modification of dietary intake may modulate their levels, with eicosapentaenoic and docosahexaenoic acid based endocannabinoids being able to displace arachidonic acid from cell membranes, reducing AEA and 2-AG production. Similarly, oleoyl ethanolamide, a product of oleic acid, induces satiety, decreases circulating fatty acid concentrations, increases the capacity for β -oxidation, and is capable of inhibiting the action of AEA and 2-AG in adipose tissue. Thus, understanding how dietary fats alter endocannabinoid system activity is a pertinent area of research due to public health messages promoting a shift towards plant-derived fats, which are rich sources of AEA and 2-AG precursor fatty acids, possibly encouraging excessive energy intake and weight gain.

Depression is a commonly reported co-morbidity during rehabilitation from alcohol use disorders and its presence is associated with an increased likelihood of relapse. Interventions which impede the development of depression could be of potential benefit if incorporated into treatment programs. We previously demonstrated an ameliorative effect of physical exercise on depressive behaviors in a mouse model of alcohol abstinence. Here, we show that environmental enrichment (cognitive and social stimulation) has a similar beneficial effect. The hypothalamic-pituitary-adrenal (HPA) axis is a key physiological system regulating stress responses and its dysregulation has been separably implicated in the pathophysiology of depression and addiction disorders. We performed a series of dexamethasone challenges and found that mice undergoing 2 weeks of alcohol abstinence had significantly greater corticosterone and ACTH levels following a DEX-CRH challenge compared to water controls. Environmental enrichment during alcohol abstinence corrected the abnormal DEX-CRH corticosterone response despite a further elevation of ACTH levels. Examination of gene expression revealed abstinence-associated alterations in glucocorticoid receptor (Gr), corticotrophin releasing hormone (Crh) and pro-opiomelanocortin (Pomc1) mRNA levels which were differentially modulated by environmental enrichment. Overall, our study demonstrates a benefit of environmental enrichment on alcohol abstinence-associated depressive behaviors and HPA axis dysregulation.

Depression is the most common psychiatric disorder in Huntington's disease (HD) patients. In the general population, women are more prone to develop depression and such susceptibility might be related to serotonergic dysregulation. There is yet to be a study of sexual dimorphism in the development and presentation of depression in HD patients. We investigated whether 8-week-old male and female R6/1 transgenic HD mice display depressive-like endophenotypes associated with serotonergic impairments. We also studied the behavioral effects of acute treatment with sertraline. We found that only female HD mice exhibited a decreased preference for saccharin as well as impaired emotionality-related behaviors when assessed on the novelty-suppressed feeding test (NSFT) and the forced-swimming test (FST). The exaggerated immobility time displayed by female HD in the FST was reduced by acute administration of sertraline. We also report an increased response to the 5-HT(1A) receptor agonist 8-OH-DPAT in inducing hypothermia and a decreased 5-HT(2A) receptor function in HD animals. While tissue levels of serotonin were reduced in both male and female HD mice, we found that serotonin concentration and hydroxylase-2 (TPH2) mRNA levels were higher in the hippocampus of males compared to female animals. Finally, the antidepressant-like effects of sertraline in the FST were blunted in male HD animals. This study reveals sex-specific depressive-related behaviors during an early stage of HD prior to any cognitive and motor deficits. Our data suggest a crucial role for disrupted serotonin signaling in mediating the sexually dimorphic depression-like phenotype in HD mice.

Dysregulated lipid metabolism and inflammation are linked to the development of insulin resistance in obesity, and the intracellular accumulation of the sphingolipid ceramide has been implicated in these processes. Here, we explored the role of circulating ceramide on the pathogenesis of insulin resistance. Ceramide transported in LDL is elevated in the plasma of obese patients with type 2 diabetes and correlated with insulin resistance but not with the degree of obesity. Treating cultured myotubes with LDL containing ceramide promoted ceramide accrual in cells and was accompanied by reduced insulin-stimulated glucose uptake, Akt phosphorylation, and GLUT4 translocation compared with LDL deficient in ceramide. LDL-ceramide induced a proinflammatory response in cultured macrophages via toll-like receptor-dependent and -independent mechanisms. Finally, infusing LDL-ceramide into lean mice reduced insulin-stimulated glucose uptake, and this was due to impaired insulin action specifically in skeletal muscle. These newly identified roles of LDL-ceramide suggest that strategies aimed at reducing hepatic ceramide production or reducing ceramide packaging into lipoproteins may improve skeletal muscle insulin action.

c-Myc transformed human Burkitt's lymphoma (BL) cells are highly sensitive to TGF-β-induced apoptosis. Previously we demonstrated that TGF-β-mediated cell death in BL cells is regulated via the mitochondrial intrinsic apoptosis pathway, which is dependent on the activation of BAX and/or BAK. TGF-β directly induces transcription of the BH3-only protein BIK and represses expression of the pro-survival factor BCL-X(L) but has no effect on the direct BAX/BAK "activators" BIM or BID (tBID). Here we show that TGF-β induces the BH3-only activator PUMA to aid induction of the intrinsic cell death pathway. TGF-β also induced PUMA in normal germinal center CD77-positive centroblasts isolated from human tonsil tissue. PUMA was a direct TGF-β target gene in B-cells, and we identify a putative Smad-binding region within the human PUMA promoter that recruits Smad3 and Smad4 in cells in response to TGF-β signaling. Constitutive activity of the isolated Smad-binding region in luciferase reporter assays was dependent on Smad consensus sequences and was partially dependent on endogenous TGF-β signaling and Smad4. Knockdown of PUMA in BL cells using lentiviral shRNA resulted in slower kinetics of the TGF-β-mediated apoptotic response. Analysis of Eμ-Myc cell lines demonstrated that c-myc-driven murine lymphomas are also sensitive to TGF-β-mediated apoptosis. Moreover, Puma(-/-) Eμ-Myc lines demonstrated significantly delayed kinetics of the apoptotic response when compared with wild type lymphomas. TGF-β therefore induces a polygenic response in Myc-driven lymphomas involving transcription of PUMA, which is necessary for the rapid induction of cell death.

Neuroadrenergic function in type 2 diabetic (T2D) patients without neuropathy is poorly characterized. We therefore compared sympathetic nervous system activity at rest and during an oral glucose tolerance test in obese metabolic syndrome (MetS) subjects classified as glucose intolerant (impaired glucose tolerance [IGT]; n = 17) or treatment-naive T2D (n = 17). Untreated subjects, matched for age (mean 59 ± 1 year), sex, BMI (32.4 ± 0.6 kg/m(2)), and family history of diabetes were studied. We measured resting muscle sympathetic nerve activity (MSNA) by microneurography, whole-body norepinephrine kinetics by isotope dilution, insulin sensitivity by euglycemic-hyperinsulinemic clamp (steady-state glucose utilization adjusted for fat-free mass and steady-state insulin concentration [M/I]), and MetS components. T2D subjects had higher resting MSNA burst incidence (67 ± 4 versus 55 ± 3 bursts per 100 heartbeats; P = 0.05) and arterial norepinephrine levels (264 ± 33 versus 167 ± 16 pg/mL; P = 0.02), lower plasma norepinephrine clearance (by 17%; P = 0.03), and reduced neuronal reuptake compared with IGT subjects (by 46%; P = 0.04). Moreover, norepinephrine spillover responses to glucose ingestion were blunted in T2D subjects. The M/I value independently predicted whole-body norepinephrine spillover (r = -0.47; P = 0.008), whereas fasting insulin level related to neuronal norepinephrine reuptake (r = -0.35, P = 0.047). These findings demonstrate that progression to T2D is associated with increased central sympathetic drive, blunted sympathetic responsiveness, and altered norepinephrine disposition.

BACKGROUND AND PURPOSE: Locally-active growth factors have been implicated in the pathogenesis of many diseases in which organ fibrosis is a characteristic feature. In the setting of chronic kidney disease (CKD), two such pro-fibrotic factors, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) have emerged as lead potential targets for intervention. Given the incomplete organ protection afforded by blocking the actions of TGF-β or PDGF individually, we sought to determine whether an agent that inhibited the actions of both may have broader effects in ameliorating the key structural and functional abnormalities of CKD. EXPERIMENTAL APPROACH: Accordingly, we studied the effects of a recently described, small molecule anti-fibrotic drug, 3-methoxy-4-propargyloxycinnamoyl anthranilate (FT011, Fibrotech Therapeutics, Australia), which should have these effects. KEY RESULTS: In the in vitro setting, FT011 inhibited both TGF-β1 and PDGF-BB induced collagen production as well as PDGF-BB-mediated mesangial proliferation. Consistent with these in vitro actions, when studied in a robust model of non-diabetic kidney disease, the 5/6 nephrectomised rat, FT011 attenuated the decline in GFR, proteinuria and glomerulosclerosis (p<0.05 for all). Similarly, in the streptozotocin-diabetic Ren-2 rat, a model of advanced diabetic nephropathy, FT011 reduced albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. CONCLUSIONS AND IMPLICATIONS: Together these studies suggest that broadly antagonising growth factor actions, including those of TGF-β1 and PDGF-BB, has the potential to protect the kidney from progressive injury in both the diabetic and non-diabetic settings.

BACKGROUND: The nature of synaptic transmission at functionally distinct synapses in intestinal reflex pathways has not been fully identified. In this study, we investigated whether transmission between interneurons in the descending inhibitory pathway is mediated by a purine acting at P2Y receptors to produce slow excitatory synaptic potentials (EPSPs). METHODOLOGY/PRINCIPAL FINDINGS: Myenteric neurons from guinea-pig ileum in vitro were impaled with intracellular microelectrodes. Responses to distension 15 mm oral to the recording site, in a separately perfused stimulation chamber and to electrical stimulation of local nerve trunks were recorded. A subset of neurons, previously identified as nitric oxide synthase immunoreactive descending interneurons, responded to both stimuli with slow EPSPs that were reversibly abolished by a high concentration of PPADS (30 μM, P2 receptor antagonist). When added to the central chamber of a three chambered organ bath, PPADS concentration-dependently depressed transmission through that chamber of descending inhibitory reflexes, measured as inhibitory junction potentials in the circular muscle of the anal chamber. Reflexes evoked by distension in the central chamber were unaffected. A similar depression of transmission was seen when the specific P2Y(1) receptor antagonist MRS 2179 (10 μM) was in the central chamber. Blocking either nicotinic receptors (hexamethonium 200 μM) or 5-HT(3) receptors (granisetron 1 μM) together with P2 receptors had no greater effect than blocking P2 receptors alone. CONCLUSIONS/SIGNIFICANCE: Slow EPSPs mediated by P2Y(1) receptors, play a primary role in transmission between descending interneurons of the inhibitory reflexes in the guinea-pig ileum. This is the first demonstration for a primary role of excitatory metabotropic receptors in physiological transmission at a functionally identified synapse.

Two major processes govern T cell proliferation and survival: interleukin-7-mediated homeostasis and antigen-induced selection. How cells transit between the two states is unknown. Here we show that T cell receptor ligation actively inhibits homeostatic survival signals while initiating a new, dominant survival programme. This switch is mediated by a change in the expression of pro- and anti-apoptosis proteins through the downregulation of Bcl-2 and the induction of Bim, A1 and Bcl-xL. Calcineurin inhibitors prevent the initiation of the new survival programme, while permitting the dominant repression of Bcl-2. Thus, in the presence of these drugs the response to antigen receptor ligation is cell death. Our results identify a molecular switch that can serve as an attractive target for inducing antigen-specific tolerance in treating autoimmune disease patients and transplant recipients.

BACKGROUND: High dietary fructose has structural and metabolic cardiac impact, but the potential for fructose to exert direct myocardial action is uncertain. Cardiomyocyte functional responsiveness to fructose, and capacity to transport fructose has not been previously demonstrated. OBJECTIVE: The aim of the present study was to seek evidence of fructose-induced modulation of cardiomyocyte excitation-contraction coupling in an acute, in vitro setting. METHODS AND RESULTS: The functional effects of fructose on isolated adult rat cardiomyocyte contractility and Ca²⁺ handling were evaluated under physiological conditions (37°C, 2 mM Ca²⁺, HEPES buffer, 4 Hz stimulation) using video edge detection and microfluorimetry (Fura2) methods. Compared with control glucose (11 mM) superfusate, 2-deoxyglucose (2 DG, 11 mM) substitution prolonged both the contraction and relaxation phases of the twitch (by 16 and 36% respectively, p<0.05) and this effect was completely abrogated with fructose supplementation (11 mM). Similarly, fructose prevented the Ca²⁺ transient delay induced by exposure to 2 DG (time to peak Ca²⁺ transient: 2 DG: 29.0±2.1 ms vs. glucose: 23.6±1.1 ms vs. fructose +2 DG: 23.7±1.0 ms; p<0.05). The presence of the fructose transporter, GLUT5 (Slc2a5) was demonstrated in ventricular cardiomyocytes using real time RT-PCR and this was confirmed by conventional RT-PCR. CONCLUSION: This is the first demonstration of an acute influence of fructose on cardiomyocyte excitation-contraction coupling. The findings indicate cardiomyocyte capacity to transport and functionally utilize exogenously supplied fructose. This study provides the impetus for future research directed towards characterizing myocardial fructose metabolism and understanding how long term high fructose intake may contribute to modulating cardiac function.

Obesity is associated with chronic low-grade inflammation that contributes to defects in energy metabolism and insulin resistance. Suppressor of cytokine signaling (SOCS)-3 expression is increased in skeletal muscle of obese humans. SOCS3 inhibits leptin signaling in the hypothalamus and insulin signal transduction in adipose tissue and the liver. Skeletal muscle is an important tissue for controlling energy expenditure and whole-body insulin sensitivity; however, the physiological importance of SOCS3 in this tissue has not been examined. Therefore, we generated mice that had SOCS3 specifically deleted in skeletal muscle (SOCS MKO). The SOCS3 MKO mice had normal muscle development, body mass, adiposity, appetite, and energy expenditure compared with wild-type (WT) littermates. Despite similar degrees of obesity when fed a high-fat diet, SOCS3 MKO mice were protected against the development of hyperinsulinemia and insulin resistance because of enhanced skeletal muscle insulin receptor substrate 1 (IRS1) and Akt phosphorylation that resulted in increased skeletal muscle glucose uptake. These data indicate that skeletal muscle SOCS3 does not play a critical role in regulating muscle development or energy expenditure, but it is an important contributing factor for inhibiting insulin sensitivity in obesity. Therapies aimed at inhibiting SOCS3 in skeletal muscle may be effective in reversing obesity-related glucose intolerance and insulin resistance.

Huntington's disease (HD) has long been regarded as a disease of the central nervous system, partly due to typical disease symptoms that include loss of motor control, cognitive deficits and neuropsychiatric disturbances. However, the huntingtin gene is ubiquitously expressed throughout the body. We had previously reported a female-specific depression-related behavioural phenotype in the R6/1 transgenic mouse model of HD. One hypothesis suggests that pathology of the hypothalamic-pituitary-adrenal (HPA) axis, the key physiological stress-response system that links central and peripheral organs, is a cause of depression. There is evidence of HPA axis pathology in HD, but whether it contributes to the female R6/1 behavioural phenotype is unclear. We have examined HPA axis response of R6/1 mice following acute stress and found evidence of a female-specific dysregulation of the HPA axis in R6/1 mice, which we further isolated to a hyper-response of adrenal cortical cells to stimulation by adrenocorticotrophin hormone. Interestingly, the adrenal pathophysiology was not detected in mice that had been housed in environmentally enriching conditions, an effect of enrichment that was also reproduced in vitro. This constitutes the first evidence that environmental enrichment can in fact exert a lasting influence on peripheral organ function. Cognitive stimulation may therefore not only have benefits for mental function, but also for overall physiological wellbeing.

Ischemic heart disease is a leading cause of death, and there is considerable imperative to identify effective therapeutic interventions. Cardiomyocyte Ca(2+) overload is a major cause of ischemia and reperfusion injury, initiating a cascade of events culminating in cardiomyocyte death, myocardial dysfunction, and occurrence of lethal arrhythmias. Responsive to fluctuations in intracellular Ca(2+), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has emerged as an enticing therapeutic target in the management of ischemic heart injury. CaMKII is activated early in ischemia and to a greater extent in the first few minutes of reperfusion, at a time when reperfusion arrhythmias are particularly prominent. CaMKII phosphorylates and upregulates many of the key proteins involved in intracellular Na(+) and Ca(2+) loading in ischemia and reperfusion. Experimentally, selective inhibition of CaMKII activity reduces cardiomyocyte death and arrhythmic incidence post-ischemia. New evidence is emerging that CaMKII actions in ischemia and reperfusion involve specific splice variant targeted actions, selective and localized post-translational modifications, and organelle-directed substrate interactions. A more complete mechanistic understanding of CaMKII mode of action in ischemia and reperfusion is required to optimize intervention opportunities. This review summarizes the current experimentally derived understanding of CaMKII participation in mediating the pathophysiology of the heart in ischemia and in reperfusion, and highlights priority future research directions.

The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.

OBJECTIVE: Skeletal muscle insulin resistance is associated with lipid accumulation, but whether insulin resistance is due to reduced or enhanced flux of long-chain fatty acids into the mitochondria is both controversial and unclear. We hypothesized that skeletal muscle-specific overexpression of the muscle isoform of carnitine palmitoyltransferase 1 (CPT1), the enzyme that controls the entry of long-chain fatty acyl CoA into mitochondria, would enhance rates of fatty acid oxidation and improve insulin action in muscle in high-fat diet insulin-resistant rats. RESEARCH DESIGN AND METHODS: Rats were fed a standard (chow) or high-fat diet for 4 weeks. After 3 weeks, in vivo electrotransfer was used to overexpress the muscle isoform of CPT1 in the distal hindlimb muscles (tibialis anterior and extensor digitorum longus [EDL]). Skeletal muscle insulin action was examined in vivo during a hyperinsulinemic-euglycemic clamp. RESULTS: In vivo electrotransfer produced a physiologically relevant increase of approximately 20% in enzyme activity; and although the high-fat diet produced insulin resistance in the sham-treated muscle, insulin action was improved in the CPT1-overexpressing muscle. This improvement was associated with a reduction in triacylglycerol content, the membrane-to-cytosolic ratio of diacylglycerol, and protein kinase C theta activity. Importantly, overexpression of CPT1 did not affect markers of mitochondrial capacity or function, nor did it alter skeletal muscle acylcarnitine profiles irrespective of diet. CONCLUSIONS: Our data provide clear evidence that a physiological increase in the capacity of long-chain fatty acyl CoA entry into mitochondria is sufficient to ameliorate lipid-induced insulin resistance in muscle.

While high doses of estrogen, in combination with androgens, can initiate prostate cancer (PCa) via activation of the estrogen receptor α (ERα), the role of ERα in PCa cells within established tumors is largely unknown. Here we show that expression of ERα is increased in high grade human PCa. Similarly, ERα is elevated in mouse models of aggressive PCa driven by MYC overexpression or deletion of PTEN. Within the prostate of PTEN-deficient mice, there is a progressive pattern of ERα expression: low in benign glands, moderate in tumors within the dorsal, lateral and ventral lobes, and high in tumors within the anterior prostate. This expression significantly correlates with the proliferation marker Ki67. Furthermore, in vitro knockdown of ERα in cells derived from PTEN-deficient tumors causes a significant and sustained decrease in proliferation. Depletion of ERα also reduces the activity of the PI3K and MAPK pathways, both downstream targets of non-genomic ERα action. Finally, ERα knockdown reduces the levels of the MYC protein and lowers the sensitivity of cellular proliferation to glucose withdrawal, which correlates with decreased expression of the glucose transporter GLUT1. Collectively, these results demonstrate that ERα orchestrates proliferation and metabolism to promote the neoplastic growth of PCa cells.

Objective. It has been suggested that atypical antipsychotics confer their effects via brain-derived neurotrophic factor (BDNF). We investigated the effect of quetiapine on serum levels of BDNF and vascular endothelial growth factor (VEGF) in drug-naive first-episode psychosis subjects. Methods. Fifteen patients drawn from a larger study received quetiapine treatment for twelve weeks. Baseline levels of serum BDNF and VEGF were compared to age- and sex-matched healthy controls and to levels following treatment. Linear regression analyses were performed to determine the relationship of BDNF and VEGF levels with outcome measures at baseline and week 12. Results. The mean serum BDNF level was significantly higher at week 12 compared to baseline and correlated with reductions in Brief Psychiatric Rating Scale (BPRS) and general psychopathology scores. Changes in serum VEGF levels also correlated significantly with a reduction in BPRS scores, a significant improvement in PANNS positive symptoms scores, and displayed a positive relationship with changes in BDNF levels. Conclusions. Our findings suggest that BDNF and VEGF are potential biomarkers for gauging improvement of psychotic symptoms. This suggests a novel neurotrophic-based mechanism of the drug effects of quetiapine on psychosis. This is the first report of VEGF perturbation in psychosis.

Induction of heat shock protein (HSP)72 protects against obesity-induced insulin resistance, but the underlying mechanisms are unknown. Here, we show that HSP72 plays a pivotal role in increasing skeletal muscle mitochondrial number and oxidative metabolism. Mice overexpressing HSP72 in skeletal muscle (HSP72Tg) and control wild-type (WT) mice were fed either a chow or high-fat diet (HFD). Despite a similar energy intake when HSP72Tg mice were compared with WT mice, the HFD increased body weight, intramuscular lipid accumulation (triacylglycerol and diacylglycerol but not ceramide), and severe glucose intolerance in WT mice alone. Whole-body VO2, fatty acid oxidation, and endurance running capacity were markedly increased in HSP72Tg mice. Moreover, HSP72Tg mice exhibited an increase in mitochondrial number. In addition, the HSP72 coinducer BGP-15, currently in human clinical trials for type 2 diabetes, also increased mitochondrial number and insulin sensitivity in a rat model of type 2 diabetes. Together, these data identify a novel role for activation of HSP72 in skeletal muscle. Thus, the increased oxidative metabolism associated with activation of HSP72 has potential clinical implications not only for type 2 diabetes but also for other disorders where mitochondrial function is compromised.

BACKGROUND: Current therapy for relieving bronchoconstriction may be ineffective in severe asthma, particularly in the small airways. The aim of this study was to further characterise responses to the recently identified novel bronchodilators rosiglitazone (RGZ) and chloroquine (CQ) under conditions where β-adrenoceptor agonist efficacy was limited or impaired in mouse small airways within lung slices. METHODS: Relaxation to RGZ and CQ was assessed following submaximal methacholine (MCh) pre-contraction, in slices treated overnight with either RGZ, CQ or albuterol (ALB) (to induce β-adrenoceptor desensitization), and in slices treated with caffeine/ryanodine in which contraction is associated with increases in Ca2+ sensitivity in the absence of contractile agonist-induced Ca2+ oscillations. Furthermore, the effects of RGZ, CQ, ALB and isoproterenol (ISO) on the initiation and development of methacholine-induced contraction were also compared. RESULTS: RGZ and CQ, but not ALB or ISO, elicited complete relaxation with increasing MCh pre-contraction and maintained their potency and efficacy following β-adrenoceptor desensitization. RGZ, CQ and ALB maintained efficacy following overnight incubation with RGZ or CQ. Relaxation responses to all dilators were generally maintained but delayed after caffeine/ryanodine. Pre-treatment with RGZ, but not CQ, ALB or ISO, reduced MCh potency. CONCLUSIONS: This study demonstrates the superior effectiveness of RGZ in comparison to CQ and β-adrenoceptor agonists as a dilator of mouse small airways. Further investigation of the mechanisms underlying the relatively greater efficacy of RGZ under these conditions are warranted and should be extended to include studies in human asthmatic airways.

The cannabinoid receptor 2 (CB2) is well known for its immune modulatory role. However, recent localisation of CB2 receptors in metabolically active tissue suggests that the CB2 receptor plays a significant role in energy homeostasis. This study was designed to investigate the impact of chronic CB2 receptor stimulation on food intake, body weight and mood. Lean male C57BL/6 mice were injected i.p. with the selective CB2 receptor agonist, JWH-015 (0.0, 1.0, 5.0 and 10.0 mg kg-1) to establish dose response parameters. Mice made obese following exposure to a diet consisting of 19.4 MJ/kg (4641 Kcal/kg) of energy (19.0% protein, 21.0% total fat, 4.7% crude fiber, and 4.7% AD fiber were given either vehicle or 10 mg/kg JWH-015. Impact on mood, food intake, body weight, plasma metabolites, expression of key metabolic proteins in the brown adipose tissue (BAT) and white adipose tissue (WAT), and markers of inflammation were measured. High dose (10 mg/kg) JWH-015 reduced food intake after 1, 2, 4, and 24 h in lean mice. When given to diet induced obese (DIO) mice, a 10 mg/kg dose of JWH-015 significantly reduced body weight compared to vehicle. This dose led to a shift in markers of lipid metabolism and inflammation in WAT consistent with lipolysis and improved immune response. Furthermore, JWH-015 (10 mg/kg) produced a transient reduction in food intake and significant reduction in fat mass and adipocyte cell size. Importantly, JWH-015 produced an anxiolytic response in the elevated plus maze while having no effect on immobility time in the forced swim test. It should be noted that though the 10 mg/kg dose produced positive effects on the obese state, the possibility that these effects are mediated via non-CB2 receptor mechanisms cannot be ruled out. These results demonstrate a role for CB2 receptors in modulating energy homeostasis and obesity associated metabolic pathologies in the absence of any adverse impact on mood.

BACKGROUND: Ischemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain's ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke. RESULTS: Ischemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; P<0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia. CONCLUSION: Our study has identified that glycogen breakdown is impaired during ischemic stroke, the molecular basis of which includes reduced glycogen debranching enzyme expression level together with reduced glycogen phosphorylase and PKA activity.

Huntington's disease (HD) is an autosomal dominant disorder caused by a tandem repeat expansion encoding an expanded tract of glutamines in the huntingtin protein. HD is progressive and manifests as psychiatric symptoms (including depression), cognitive deficits (culminating in dementia), and motor abnormalities (including chorea). Having reached the twentieth anniversary of the discovery of the "genetic stutter" which causes HD, we still lack sophisticated insight into why so many HD patients exhibit affective disorders such as depression at very early stages, prior to overt appearance of motor deficits. In this review, we will focus on depression as the major psychiatric manifestation of HD, discuss potential mechanisms of pathogenesis identified from animal models, and compare depression in HD patients with that of the wider gene-negative population. The discovery of depressive-like behaviors as well as cellular and molecular correlates of depression in transgenic HD mice has added strong support to the hypothesis that the HD mutation adds significantly to the genetic load for depression. A key question is whether HD-associated depression differs from that in the general population. Whilst preclinical studies, clinical data, and treatment responses suggest striking similarities, there are also some apparent differences. We discuss various molecular and cellular mechanisms which may contribute to depression in HD, and whether they may generalize to other depressive disorders. The autosomal dominant nature of HD and the existence of models with excellent construct validity provide a unique opportunity to understand the pathogenesis of depression and associated gene-environment interactions. Thus, understanding the pathogenesis of depression in HD may not only facilitate tailored therapeutic approaches for HD sufferers, but may also translate to the clinical depression which devastates the lives of so many people.

BACKGROUND: To secure pH homeostasis, the central respiratory network must permanently adapt its rhythmic motor drive to environment and behaviour. In neonates, it is commonly admitted that the retrotrapezoid/parafacial respiratory group of neurons of the ventral medulla plays the primary role in the respiratory response to acidosis, although the serotonergic system may also contribute to this response. METHODOLOGY/PRINCIPAL FINDINGS: Using en bloc medullary preparations from neonatal mice, we have shown for the first time that the respiratory response to acidosis is abolished after pre-treatment with the serotonin-transporter blocker fluoxetine (25-50 µM, 20 min), a commonly used antidepressant. Using mRNA in situ hybridization and immunohistology, we have also shown the expression of the serotonin transporter mRNA and serotonin-containing neurons in the vicinity of the RTN/pFRG of neonatal mice. CONCLUSIONS: These results reveal that the serotonergic system plays a pivotal role in pH homeostasis. Although obtained in vitro in neonatal mice, they suggest that drugs targeting the serotonergic system should be used with caution in infants, pregnant women and breastfeeding mothers.

Diabetic retinopathy features inflammation as well as injury to glial cells and the microvasculature, which are influenced by hypertension and overactivity of the renin-angiotensin system. FT011 is an anti-inflammatory and anti-fibrotic agent that has been reported to attenuate organ damage in diabetic rats with cardiomyopathy and nephropathy. However, the potential therapeutic utility of FT011 for diabetic retinopathy has not been evaluated. We hypothesized that FT011 would attenuate retinopathy in diabetic Ren-2 rats, which exhibit hypertension due to an overactive extra-renal renin-angiotensin system. Diabetic rats were studied for 8 and 32 weeks and received intravitreal injections of FT011 (50 μM) or vehicle (0.9% NaCl). Comparisons were to age-matched controls. In the 8-week study, retinal inflammation was examined by quantitating vascular leukocyte adherence, microglial/macrophage density and the expression of inflammatory mediators. Macroglial Müller cells, which exhibit a pro-inflammatory and pro-angiogenic phenotype in diabetes, were evaluated in the 8-week study as well as in culture following exposure to hyperglycaemia and FT011 (10, 30, 100 μM) for 72 hours. In the 32-week study, severe retinal vasculopathy was examined by quantitating acellular capillaries and extracellular matrix proteins. In diabetic rats, FT011 reduced retinal leukostasis, microglial density and mRNA levels of intercellular adhesion molecule-1 (ICAM-1). In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach.

The microtubule associated protein tau causes primary and secondary tauopathies by unknown molecular mechanisms. Post-translational O-GlcNAc-ylation of brain proteins was demonstrated here to be beneficial for Tau.P301L mice by pharmacological inhibition of O-GlcNAc-ase. Chronic treatment of ageing Tau.P301L mice mitigated their loss in body-weight and improved their motor deficits, while the survival was 3-fold higher at the pre-fixed study endpoint at age 9.5 months. Moreover, O-GlcNAc-ase inhibition significantly improved the breathing parameters of Tau.P301L mice, which underpinned pharmacologically the close correlation of mortality and upper-airway defects. O-GlcNAc-ylation of brain proteins increased rapidly and stably by systemic inhibition of O-GlcNAc-ase. Conversely, biochemical evidence for protein Tau.P301L to become O-GlcNAc-ylated was not obtained, nor was its phosphorylation consistently or markedly affected. We conclude that increasing O-GlcNAc-ylation of brain proteins improved the clinical condition and prolonged the survival of ageing Tau.P301L mice, but not by direct biochemical action on protein tau. The pharmacological effect is proposed to be located downstream in the pathological cascade initiated by protein Tau.P301L, opening novel venues for our understanding, and eventually treating the neurodegeneration mediated by protein tau.

There is increasing evidence of prodromal manifestation of neuropsychiatric symptoms in a variety of neurodegenerative diseases such as Parkinson's disease (PD) and Huntington's disease (HD). These affective symptoms may be observed many years before the core diagnostic symptoms of the neurological condition. It is becoming more apparent that depression is a significant modifying factor of the trajectory of disease progression and even treatment outcomes. It is therefore crucial that we understand the potential pathophysiologies related to the primary condition, which could contribute to the development of depression. The hypothalamic-pituitary-adrenal (HPA)-axis is a key neuroendocrine signaling system involved in physiological homeostasis and stress response. Disturbances of this system lead to severe hormonal imbalances, and the majority of such patients also present with behavioral deficits and/or mood disorders. Dysregulation of the HPA-axis is also strongly implicated in the pathology of major depressive disorder. Consistent with this, antidepressant drugs, such as the selective serotonin reuptake inhibitors have been shown to alter HPA-axis activity. In this review, we will summarize the current state of knowledge regarding HPA-axis pathology in Alzheimer's, PD and HD, differentiating between prodromal and later stages of disease progression when evidence is available. Both clinical and preclinical evidence will be examined, but we highlight animal model studies as being particularly useful for uncovering novel mechanisms of pathology related to co-morbid mood disorders. Finally, we purpose utilizing the preclinical evidence to better inform prospective, intervention studies.

BACKGROUND: When tested in a randomized controlled trial (RCT) of 31,411 patients, the nurse-led 6-PACK falls prevention program did not reduce falls. Poor implementation fidelity (i.e., program not implemented as intended) may explain this result. Despite repeated calls for the examination of implementation fidelity as an essential component of evaluating interventions designed to improve the delivery of care, it has been neglected in prior falls prevention studies. This study examined implementation fidelity of the 6-PACK program during a large multi-site RCT. METHODS: Based on the 6-PACK implementation framework and intervention description, implementation fidelity was examined by quantifying adherence to program components and organizational support. Adherence indicators were: 1) falls-risk tool completion; and for patients classified as high-risk, provision of 2) a 'Falls alert' sign; and 3) at least one additional 6-PACK intervention. Organizational support indicators were: 1) provision of resources (executive sponsorship, site clinical leaders and equipment); 2) implementation activities (modification of patient care plans; training; implementation tailoring; audits, reminders and feedback; and provision of data); and 3) program acceptability. Data were collected from daily bedside observation, medical records, resource utilization diaries and nurse surveys. RESULTS: All seven intervention components were delivered on the 12 intervention wards. Program adherence data were collected from 103,398 observations and medical record audits. The falls-risk tool was completed each day for 75% of patients. Of the 38% of patients classified as high-risk, 79% had a 'Falls alert' sign and 63% were provided with at least one additional 6-PACK intervention, as recommended. All hospitals provided the recommended resources and undertook the nine outlined program implementation activities. Most of the nurses surveyed considered program components important for falls prevention. CONCLUSIONS: While implementation fidelity was variable across wards, overall it was found to be acceptable during the RCT. Implementation failure is unlikely to be a key factor for the observed lack of program effectiveness in the 6-PACK trial. TRIAL REGISTRATION: The 6-PACK cluster RCT is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12611000332921 (29 March 2011).

There is growing evidence that the preconceptual lifestyle and other environmental exposures of a father can significantly alter the physiological and behavioral phenotypes of their children. We and others have shown that paternal preconception stress, regardless of whether the stress was experienced during early-life or adulthood, results in offspring with altered anxiety and depression-related behaviors, attributed to hypothalamic-pituitary-adrenal axis dysregulation. The transgenerational response to paternal preconceptual stress is believed to be mediated by sperm-borne small noncoding RNAs, specifically microRNAs. As physical activity confers physical and mental health benefits for the individual, we used a model of voluntary wheel-running and investigated the transgenerational response to paternal exercise. We found that male offspring of runners had suppressed reinstatement of juvenile fear memory, and reduced anxiety in the light-dark apparatus during adulthood. No changes in these affective behaviors were observed in female offspring. We were surprised to find that running had a limited impact on sperm-borne microRNAs. The levels of three unique microRNAs (miR-19b, miR-455 and miR-133a) were found to be altered in the sperm of runners. In addition, we discovered that the levels of two species of tRNA-derived RNAs (tDRs)-tRNA-Gly and tRNA-Pro-were also altered by running. Taken together, we believe this is the first evidence that paternal exercise is associated with an anxiolytic behavioral phenotype of male offspring and altered levels of small noncoding RNAs in sperm. These small noncoding RNAs are known to have an impact on post-transcriptional gene regulation and can thus change the developmental trajectory of offspring brains and associated affective behaviors.

The enteric nervous system (ENS) situated within the gastrointestinal tract comprises an intricate network of neurons and glia which together regulate intestinal function. The exact neuro-glial circuitry and the signaling molecules involved are yet to be fully elucidated. Vasoactive intestinal peptide (VIP) is one of the main neurotransmitters in the gut, and is important for regulating intestinal secretion and motility. However, the role of VIP and its VPAC receptors within the enteric circuitry is not well understood. We investigated this in the submucosal plexus of mouse jejunum using calcium (Ca2+)-imaging. Local VIP application induced Ca2+-transients primarily in neurons and these were inhibited by VPAC1- and VPAC2-antagonists (PG 99-269 and PG 99-465 respectively). These VIP-evoked neural Ca2+-transients were also inhibited by tetrodotoxin (TTX), indicating that they were secondary to action potential generation. Surprisingly, VIP induced Ca2+-transients in glia in the presence of the VPAC2 antagonist. Further, selective VPAC1 receptor activation with the agonist ([K15, R16, L27]VIP(1-7)/GRF(8-27)) predominantly evoked glial responses. However, VPAC1-immunoreactivity did not colocalize with the glial marker glial fibrillary acidic protein (GFAP). Rather, VPAC1 expression was found on cholinergic submucosal neurons and nerve fibers. This suggests that glial responses observed were secondary to neuronal activation. Trains of electrical stimuli were applied to fiber tracts to induce endogenous VIP release. Delayed glial responses were evoked when the VPAC2 antagonist was present. These findings support the presence of an intrinsic VIP/VPAC-initiated neuron-to-glia signaling pathway. VPAC1 agonist-evoked glial responses were inhibited by purinergic antagonists (PPADS and MRS2179), thus demonstrating the involvement of P2Y1 receptors. Collectively, we showed that neurally-released VIP can activate neurons expressing VPAC1 and/or VPAC2 receptors to modulate purine-release onto glia. Selective VPAC1 activation evokes a glial response, whereas VPAC2 receptors may act to inhibit this response. Thus, we identified a component of an enteric neuron-glia circuit that is fine-tuned by endogenous VIP acting through VPAC1- and VPAC2-mediated pathways.

OBJECTIVE: Pigment epithelium-derived factor (PEDF) is an adipocyte-secreted factor involved in the development of insulin resistance in obesity. Previous studies have identified PEDF as a regulator of triacylglycerol metabolism in the liver that may act through adipose triglyceride lipase (ATGL). We used ATGL(-/-) mice to determine the role of PEDF in regulating lipid and glucose metabolism. RESEARCH DESIGN AND METHODS: Recombinant PEDF was administered to ATGL(-/-) and wild-type mice, and whole-body energy metabolism was studied by indirect calorimetry. Adipose tissue lipolysis and skeletal muscle fatty acid metabolism was determined in isolated tissue preparations. Muscle lipids were assessed by electrospray ionization-tandem mass spectrometry. Whole-body insulin sensitivity and skeletal muscle glucose uptake were assessed. RESULTS: PEDF impaired the capacity to adjust substrate selection, resulting in a delayed diurnal decline in the respiratory exchange ratio, and suppressed daily fatty acid oxidation. PEDF enhanced adipocyte lipolysis and triacylglycerol lipase activity in skeletal muscle. Muscle fatty acid uptake and storage were unaffected, whereas fatty acid oxidation was impaired. These changes in lipid metabolism were abrogated in ATGL(-/-) mice and were not attributable to hypothalamic actions. ATGL(-/-) mice were also refractory to PEDF-mediated insulin resistance, but this was not related to changes in lipid species in skeletal muscle. CONCLUSIONS: The results are the first direct demonstration that 1) PEDF influences systemic fatty acid metabolism by promoting lipolysis in an ATGL-dependent manner and reducing fatty acid oxidation and 2) ATGL is required for the negative effects of PEDF on insulin action.

BACKGROUND: The functional polymorphism that explains the established association of the androgen receptor (AR) with androgenetic alopecia (AGA) remains unidentified, but Copy Number Variation (CNV) might be relevant. CNV involves changes in copy number of large segments of DNA, leading to the altered dosage of gene regulators or genes themselves. Two recent reports indicate regions of CNV in and around AR, and these have not been studied in relation to AGA. The aim of this preliminary case-control study was to determine if AR CNV is associated with AGA, with the hypothesis that CNV is the functional AR variant contributing to this condition. METHODOLOGY/PRINCIPAL FINDINGS: Multiplex Ligation-dependent Probe Amplification was used to screen for CNV in five AR exons and a conserved, non-coding region upstream of AR in 85 men carefully selected as cases and controls for maximal phenotypic contrast. There was no evidence of CNV in AR in any of the cases or controls, and thus no evidence of significant association between AGA and AR CNV. CONCLUSIONS/SIGNIFICANCE: The results suggest this form of genomic variation at the AR locus is unlikely to predispose to AGA.

There are established epidemiological links between obesity and the severity of prostate cancer. We directly tested this relationship by assessing tumorigenicity of patient-derived xenografts (PDXs) of moderate-grade localized prostate cancer in lean and obese severe combined immunodeficiency (SCID) mice. Mice were rendered obese and insulin resistant by high-fat feeding for 6 weeks prior to transplantation, and PDXs were assessed 10 weeks thereafter. Histological analysis of PDX grafts showed no differences in tumor pathology, prostate-specific antigen, androgen receptor and homeobox protein Nkx-3.1 expression, or proliferation index in lean versus obese mice. Whilst systemic obesity per se did not promote prostate tumorigenicity, we next asked whether the peri-prostatic adipose tissue (PPAT), which covers the prostate anteriorly, plays a role in prostate tumorigenesis. In vitro studies in a cellularized co-culture model of stromal and epithelial cells demonstrated that factors secreted from human PPAT are pro-tumorigenic. Accordingly, we recapitulated the prostate-PPAT spatial relationship by co-grafting human PPAT with prostate cancer in PDX grafts. PDX tissues were harvested 10 weeks after grafting, and histological analysis revealed no evidence of enhanced tumorigenesis with PPAT compared to prostate cancer grafts alone. Altogether, these data demonstrate that prostate cancer tumorigenicity is not accelerated in the setting of diet-induced obesity or in the presence of human PPAT, prompting the need for further work to define the at-risk populations of obesity-driven tumorigenesis and the biological factors linking obesity, adipose tissue and prostate cancer pathogenesis.

EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions. We recently reported that Efnb3 gene deletion results in hypertension in female but not male mice. These data suggest that EFNB3 regulates blood pressure in a sex- and sex hormone-dependent way. In the present study, we conducted a human genetic study to assess the association of EFNB3 single nucleotide polymorphisms with human hypertension risks, using 3,448 patients with type 2 diabetes from the ADVANCE study (Action in Diabetes and Vascular Disease: Peterax and Diamicron MR Controlled Evaluation). We have observed significant association between 2 SNPs in the 3' untranslated region or within the adjacent region just 3' of the EFNB3 gene with hypertension, corroborating our findings from the mouse model. Thus, our investigation has shown that EFNB3 is a hypertension risk gene in certain individuals.

BACKGROUND: The prevalence of diabetic nephropathy varies according to ethnicity. Environmental as well as genetic factors contribute to the heterogeneity in the presentation of diabetic nephropathy. Our objective was to evaluate this heterogeneity within the Caucasian population. METHODS: The geo-ethnic origin of the 3409 genotyped Caucasian type 2 diabetes (T2D) patients of Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation was determined using principal component analysis. Genome-wide association studies analyses of age of onset of T2D were performed for geo-ethnic groups separately and combined. RESULTS: The first principal component separated the Caucasian study participants into Slavic and Celtic ethnic origins. Age of onset of diabetes was significantly lower in Slavic patients (P = 7.3 × 10), whereas the prevalence of hypertension (P = 4.9 × 10) and albuminuria (5.1 × 10) were significantly higher. Age of onset of T2D and albuminuria appear to have an important genetic component as the values of these traits were also different between Slavic and Celtic individuals living in the same countries. Common and geo-ethnic-specific loci were found to be associated to age of onset of diabetes. Among the latter, the PROX1/PROX1-AS1 genes (rs340841) had the highest impact. Single-nucleotide polymorphism rs340841 CC genotype was associated with a 4.4 year earlier onset of T2D in Slavic patients living or not in countries with predominant Slavic populations. CONCLUSION: These results reveal the presence of distinct genetic architectures between Caucasian ethnic groups that likely have clinical relevance, among them PROX1 gene is a strong candidate of early onset of diabetes with variations depending on ethnicity.

Sugar-sweetened beverages (SSBs) are consumed globally, and have been associated with adverse health outcomes, including weight gain, high blood pressure, type 2 diabetes (T2D), and cardiovascular disease (CVD). There is global variation in beverage formulation in terms of glucose and fructose concentration, which may pose unique health risks linked to glycemic control for Australian consumers. However, previous systematic reviews have overlooked Australian-based literature. A systematic review was performed to synthesise evidence for the associations between consumption of SSBs and intense-sweetened beverages with clinical cardiometabolic risk factors in the Australian population. Articles were sourced from Global Health, Health Source: Nursing/Academic Edition, Medline, and Culmative Index to Nursing and Allied Health Literature. To be eligible for review, studies had to report on the consumption of sugar-sweetened (including fruit juice and fruit drinks) and/or intense-sweetened beverages, and at least one clinical cardiometabolic risk factor. Eighteen studies were included in this review. Research has mostly focused on the relationship between SSB consumption and adiposity-related outcomes. No studies have examined indices of glycaemic control (glucose/insulin), and the evidence for the health impact of intense-sweetened drinks is limited. In addition, studies have primarily been of cross-sectional design, and have examined children and adolescents, as opposed to adult populations. In the Australian population, there is modest but consistent evidence that SSB consumption has adverse associations with weight, but there is insufficient data to assess relationships with cardiometabolic outcomes.

[This corrects the article on p. 170 in vol. 5, PMID: 24822049.].

BACKGROUND: The regulation of microRNAs (miRNAs) at different stages of the progression of type 2 diabetes mellitus (T2DM) and their role in glucose homeostasis was investigated. METHODS: Microarrays were used to assess miRNA expression in skeletal muscle biopsies taken from healthy individuals and patients with pre-diabetes or T2DM, and insulin resistant offspring of rat dams fed a high fat diet during pregnancy. RESULTS: Twenty-three miRNAs were differentially expressed in patients with T2DM, and 7 in the insulin resistant rat offspring compared to their controls. Among these, only one miRNA was similarly regulated: miR-194 expression was significantly reduced by 25 to 50% in both the rat model and in human with pre-diabetes and established diabetes. Knockdown of miR-194 in L6 skeletal muscle cells induced an increase in basal and insulin-stimulated glucose uptake and glycogen synthesis. This occurred in conjunction with an increased glycolysis, indicated by elevated lactate production. Moreover, oxidative capacity was also increased as we found an enhanced glucose oxidation in presence of the mitochondrial uncoupler FCCP. When miR-194 was down-regulated in vitro, western blot analysis showed an increased phosphorylation of AKT and GSK3β in response to insulin, and an increase in expression of proteins controlling mitochondrial oxidative phosphorylation. CONCLUSIONS: Type 2 diabetes mellitus is associated with regulation of several miRNAs in skeletal muscle. Interestingly, miR-194 was a unique miRNA that appeared regulated across different stages of the disease progression, from the early stages of insulin resistance to the development of T2DM. We have shown miR-194 is involved in multiple aspects of skeletal muscle glucose metabolism from uptake, through to glycolysis, glycogenesis and glucose oxidation, potentially via mechanisms involving AKT, GSK3 and oxidative phosphorylation. MiR-194 could be down-regulated in patients with early features of diabetes as an adaptive response to facilitate tissue glucose uptake and metabolism in the face of insulin resistance.

In the thymus, hematopoietic progenitors commit to the T cell lineage and undergo sequential differentiation to generate diverse T cell subsets, including major histocompatibility complex (MHC)-restricted αβ T cell receptor (TCR) T cells and non-MHC-restricted γδ TCR T cells. The factors controlling precursor commitment and their subsequent maturation and specification into αβ TCR versus γδ TCR T cells remain unclear. Here, we show that the tyrosine phosphatase PTPN2 attenuates STAT5 (signal transducer and activator of transcription 5) signaling to regulate T cell lineage commitment and SRC family kinase LCK and STAT5 signaling to regulate αβ TCR versus γδ TCR T cell development. Our findings identify PTPN2 as an important regulator of critical checkpoints that dictate the commitment of multipotent precursors to the T cell lineage and their subsequent maturation into αβ TCR or γδ TCR T cells.

The impact of statin treatment on the abnormal plasma lipidome of mixed dyslipidemic patients with metabolic syndrome (MetS), a group at increased risk of developing diabetes, was evaluated. Insulin-resistant hypertriglyceridemic hypertensive obese males (n = 12) displaying MetS were treated with pitavastatin (4 mg/day) for 180 days; healthy normolipidemic age-matched nonobese males (n = 12) acted as controls. Statin treatment substantially normalized triglyceride (-41%), remnant cholesterol (-55%), and LDL-cholesterol (-39%), with minor effect on HDL-cholesterol (+4%). Lipidomic analysis, normalized to nonHDL-cholesterol in order to probe statin-induced differences in molecular composition independently of reduction in plasma cholesterol, revealed increment in 132 of 138 lipid species that were subnormal at baseline and significantly shifted toward the control group on statin treatment. Increment in alkyl- and alkenylphospholipids (plasmalogens) was prominent, and consistent with significant statin-induced increase in plasma polyunsaturated fatty acid levels. Comparison of the statin-mediated lipidomic changes in MetS with the abnormal plasma lipidomic profile characteristic of prediabetes and T2D in the Australian Diabetes, Obesity, and Lifestyle Study and San Antonio Family Heart Study cohorts by hypergeometric analysis revealed a significant shift toward the lipid profile of controls, indicative of a marked trend toward a normolipidemic phenotype. Pitavastatin attenuated the abnormal plasma lipidome of MetS patients typical of prediabetes and T2D.

Mitochondria are morphologically dynamic organelles constantly undergoing processes of fission and fusion that maintain integrity and bioenergetics of the organelle: these processes are vital for cell survival. Disruption in the balance of mitochondrial fusion and fission is thought to play a role in several pathological conditions including ischemic heart disease. Proteins involved in regulating the processes of mitochondrial fusion and fission are therefore potential targets for pharmacological therapies. Mdivi-1 is a small molecule inhibitor of the mitochondrial fission protein Drp1. Inhibiting mitochondrial fission with Mdivi-1 has proven cytoprotective benefits in several cell types involved in a wide array of cardiovascular injury models. On the other hand, Mdivi-1 can also exert antiproliferative and cytotoxic effects, particularly in hyperproliferative cells. In this review, we discuss these divergent effects of Mdivi-1 on cell survival, as well as the potential and limitations of Mdivi-1 as a therapeutic agent.

Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways, however, it remains unclear as to what extent these pathways can be exploited, either individually or in combination, in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro, but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways, we developed a high-density microbioreactor array (HDMA)--a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt, Hedgehog, IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation, inducing cell cycle activity marked by Ki67, and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration.

BACKGROUND: The inability of the adult mammalian heart to regenerate following injury represents a major barrier in cardiovascular medicine. In contrast, the neonatal mammalian heart retains a transient capacity for regeneration, which is lost shortly after birth. Defining the molecular mechanisms that govern regenerative capacity in the neonatal period remains a central goal in cardiac biology. Here, we assemble a transcriptomic framework of multiple cardiac cell populations during postnatal development and following injury, which enables comparative analyses of the regenerative (neonatal) versus nonregenerative (adult) state for the first time. METHODS: Cardiomyocytes, fibroblasts, leukocytes, and endothelial cells from infarcted and noninfarcted neonatal (P1) and adult (P56) mouse hearts were isolated by enzymatic dissociation and fluorescence-activated cell sorting at day 3 following surgery. RNA sequencing was performed on these cell populations to generate the transcriptome of the major cardiac cell populations during cardiac development, repair, and regeneration. To complement our transcriptomic data, we also surveyed the epigenetic landscape of cardiomyocytes during postnatal maturation by performing deep sequencing of accessible chromatin regions by using the Assay for Transposase-Accessible Chromatin from purified mouse cardiomyocyte nuclei (P1, P14, and P56). RESULTS: Profiling of cardiomyocyte and nonmyocyte transcriptional programs uncovered several injury-responsive genes across regenerative and nonregenerative time points. However, the majority of transcriptional changes in all cardiac cell types resulted from developmental maturation from neonatal stages to adulthood rather than activation of a distinct regeneration-specific gene program. Furthermore, adult leukocytes and fibroblasts were characterized by the expression of a proliferative gene expression network following infarction, which mirrored the neonatal state. In contrast, cardiomyocytes failed to reactivate the neonatal proliferative network following infarction, which was associated with loss of chromatin accessibility around cell cycle genes during postnatal maturation. CONCLUSIONS: This work provides a comprehensive framework and transcriptional resource of multiple cardiac cell populations during cardiac development, repair, and regeneration. Our findings define a regulatory program underpinning the neonatal regenerative state and identify alterations in the chromatin landscape that could limit reinduction of the regenerative program in adult cardiomyocytes.

BACKGROUND: Microvascular disease is associated with a high risk of macrovascular events in patients with type 2 diabetes, but the impact of macrovascular disease on the risk of microvascular events remains unknown. We sought to evaluate the respective effects of prior microvascular and macrovascular disease on the risk of major outcomes, including microvascular events, in these patients. METHODS: Participants in the Action in Diabetes and Vascular Disease: PreterAx and DiamicroN Modified-Release Controlled Evaluation (ADVANCE) trial (n = 11,140) and the ADVANCE-ON post-trial study (n = 8494) were categorized into 4 groups at baseline: dual absence of microvascular or macrovascular disease (n = 6789), presence of microvascular disease alone (n = 761), macrovascular disease alone (n = 3196), and both (n = 394). Outcomes were all-cause mortality, major macrovascular events (MACE), and major clinical microvascular events. RESULTS: All-cause mortality, MACE, and major clinical microvascular events occurred in 2265 (20%), 2166 (19%), and 807 (7%) participants respectively, during a median follow-up of 9.9 (inter-quartile interval 5.6-10.9) years. The adjusted hazard ratios [95% CI] of death, MACE, and major clinical microvascular events were each greater in patients with baseline microvascular disease (1.43 [1.20-1.71], 1.64 [1.37-1.97], and 4.74 [3.86-5.82], respectively), macrovascular disease (1.43 [1.30-1.57], 2.04 [1.86-2.25], and 1.26 [1.06-1.51]) or both (2.01 [1.65-2.45], 2.92 [2.40-3.55], and 6.30 [4.93-8.06]) compared with those without these conditions. No interaction was observed between baseline microvascular and macrovascular disease for these events. The addition of microvascular disease (change in c-statistic [95% CI] 0.005 [0.002-0.008], p = 0.02) or macrovascular disease (0.005 [0.002-0.007], p < 0.0001) considered separately or together (0.011 [0.007-0.014], p < 0.0001) improved the discrimination and the classification (integrated discrimination improvement (IDI): 0.013 [0.010-0.016], p < 0.001; net reclassification improvement (NRI): 0.021 [0.011-0.032], p < 0.001) of the risk of all-cause mortality. Microvascular disease improved discrimination (0.009 [0.003-0.014]) and classification (IDI: 0.008 [0.006-0.010]; NRI: 0.011 [0.001-0.020]) of MACE. Baseline macrovascular disease modestly enhanced IDI (0.002 [0.001-0.002]) and NRI (0.041 [0.002-0.087]), but not discrimination, of major clinical microvascular events. CONCLUSIONS: Microvascular and macrovascular disease are independently associated with the 10-year risk of death, MACE, and major clinical microvascular events in patients with type 2 diabetes. The coexistence of these conditions was associated with the highest risks.

Background and Aims: Cholera toxin (CT)-induced hypersecretion requires activation of secretomotor pathways in the enteric nervous system (ENS). AH neurons, which have been identified as a population of intrinsic sensory neurons (ISNs), are a source of excitatory input to the secretomotor pathways. We therefore examined effects of CT in the intestinal lumen on myenteric and submucosal AH neurons. Methods: Isolated segments of guinea pig jejunum were incubated for 90 min with saline plus CT (12.5 μg/ml) or CT + neurotransmitter antagonist, or CT + tetrodotoxin (TTX) in their lumen. After washing CT away, submucosal or myenteric plexus preparations were dissected keeping circumferentially adjacent mucosa intact. Submucosal AH neurons were impaled adjacent to intact mucosa and myenteric AH neurons were impaled adjacent to, more than 5 mm from, and in the absence of intact mucosa. Neuronal excitability was monitored by injecting 500 ms current pulses through the recording electrode. Results: After CT pre-treatment, excitability of myenteric AH neurons adjacent to intact mucosa (n = 29) was greater than that of control neurons (n = 24), but submucosal AH neurons (n = 33, control n = 27) were unaffected. CT also induced excitability increases in myenteric AH neurons impaled distant from the mucosa (n = 6) or in its absence (n = 5). Coincubation with tetrodotoxin or SR142801 (NK3 receptor antagonist), but not SR140333 (NK1 antagonist) or granisetron (5-HT3 receptor antagonist) prevented the increased excitability induced by CT. Increased excitability was associated with a reduction in the characteristic AHP and an increase in the ADP of these neurons, but not a change in the hyperpolarization-activated inward current, Ih . Conclusions: CT increases excitability of myenteric, but not submucosal, AH neurons. This is neurally mediated and depends on NK3, but not 5-HT3 receptors. Therefore, CT may act to amplify the secretomotor response to CT via an increase in the activity of the afferent limb of the enteric reflex circuitry.

Global assessments of burden of disease suggests there are sex differences in risk factors for chronic disease, including overweight/obesity, dietary patterns and habitual physical activity. Given that prevention efforts aim to target such factors to reduce disease risk, the age at which sex differences may occur is of particular interest. Early life to young adulthood is the optimal time for intervention, with lifestyle habits typically forming during this period. This study aimed to identify the sex differences in risk factors for chronic disease during childhood (5-9 years), adolescence (10-17 years) and emerging adulthood (18-25 years) in a large population-representative Australian sample. Among children in this study (n = 739), no sex-related differences were observed. Among adolescents (n = 1304), females were more likely than males to meet daily fruit and vegetable recommendations (12.9% vs. 7.5%; OR = 1.84, 95% CI = 1.16, 2.93, p < 0.05). Among emerging adults (n = 909), females were less likely to be overweight/obese (30.1% vs. 39.8%; OR = 0.65, 95% CI = 0.44, 0.95, p < 0.05) and more likely to meet physical activity recommendations (52.1% vs. 42.3%; OR = 1.44, 95% CI = 1.01, 2.06, p < 0.05). These findings suggest that sex differences for risk factors of chronic disease occur during adolescence and emerging adulthood, although the differences are not consistent across age periods. From adolescence onwards, it appears that females exhibit lower risk factors than males and a life span approach to risk factor monitoring is warranted.