Early life stress exacerbates the obesogenic and anxiogenic effects of a Western diet without worsening cardiac ischaemic tolerance in male mice.

Early life stress (ELS) and a Western diet (WD) promote mood and cardiovascular disorders, however, how these risks interact in disease pathogenesis is unclear. We assessed effects of ELS with or without a subsequent WD on behaviour, cardiometabolic risk factors, and cardiac function/ischaemic tolerance in male mice. Fifty-six new-born male C57BL/6J mice were randomly allocated to a control group (CON) undisturbed before weaning, or to maternal separation (3h/day) and early (postnatal day 17) weaning (MSEW). Mice consumed standard rodent chow (CON, n = 14; MSEW, n = 15) or WD chow (WD, n = 19; MSEW + WD, n = 19) from week 8 to 24. Fasted blood was sampled and open field test and elevated plus maze (EPM) tests undertaken at 7, 15, and 23 weeks of age, with hearts excised at 24 weeks for Langendorff perfusion (evaluating pre- and post-ischaemic function). MSEW alone transiently increased open field activity at 7 weeks; body weight and serum triglycerides at 4 and 7 weeks, respectively; and final blood glucose levels and insulin resistance at 23 weeks. WD increased insulin resistance and body weight gain, the latter potentiated by MSEW. MSEW + WD was anxiogenic, reducing EPM open arm activity vs. WD alone. Although MSEW had modest metabolic effects and did not influence cardiac function or ischaemic tolerance in lean mice, it exacerbated weight gain and anxiogenesis, and improved ischaemic tolerance in WD fed animals. MSEW-induced increases in body weight (obesity) in WD fed animals in the absence of changes in insulin resistance may have protected the hearts of these mice.

Circulating TMAO, the gut microbiome and cardiometabolic disease risk: an exploration in key precursor disorders.

BACKGROUND: Elevations in the gut metabolite trimethylamine-N-oxide (TMAO) have been linked to cardiovascular and metabolic diseases. Whether elevated TMAO levels reflect early mechanistic involvement or a sequela of evolving disease awaits elucidation. The purpose of this study was to further explore these potential associations. METHODS: We investigated relationships between circulating levels of TMAO and its pre-cursor substrates, dietary factors, gut microbiome profiles and disease risk in individuals with a Healthy BMI (18.5 < BMI < 25, n = 41) or key precursor states for cardiometabolic disease: Overweight (25 < BMI < 30 kg/m2, n = 33), Obese (BMI > 30, n = 27) and Metabolic Syndrome (MetS; ≥ 3 ATPIII report criteria, n = 39). RESULTS: Unexpectedly, plasma [TMAO] did not vary substantially between groups (means of 3-4 µM; p > 0.05), although carnitine was elevated in participants with MetS. Gut microbial diversity and Firmicutes were also significantly reduced in the MetS group (p < 0.05). Exploratory analysis across diverse parameters reveals significant correlations between circulating [TMAO] and seafood intake (p = 0.007), gut microbial diversity (p = 0.017-0.048), and plasma [trimethylamine] (TMA; p = 0.001). No associations were evident with anthropometric parameters or cardiometabolic disease risk. Most variance in [TMAO] within and between groups remained unexplained. CONCLUSIONS: Data indicate that circulating [TMAO] may be significantly linked to seafood intake, levels of TMA substrate and gut microbial diversity across healthy and early disease phenotypes. However, mean concentrations remain < 5 µM, with little evidence of links between TMAO and cardiometabolic disease risk. These observations suggest circulating TMAO may not participate mechanistically in cardiometabolic disease development, with later elevations likely a detrimental sequela of extant disease.

Behavioural disruption in diabetic mice: Neurobiological correlates and influences of dietary α-linolenic acid.

AIM: Evolving type 2 diabetes (T2D) may influence locomotion and affective state, promoting metabolic dysfunction. We examined behaviour and neurobiology in a model of T2D, testing for benefits with dietary n-3 polyunsaturated fatty acid (PUFA). METHODS: Male C57Bl/6 mice received vehicle or 75 mg/kg streptozotocin (STZ) and 21 wks of control or Western diets (43 % fat, 40 % carbohydrate, 17 % protein). Sub-sets received dietary α-linolenic acid (ALA; 10 % of fat intake) for 6 wks. Behaviour was examined via open field and sucrose preference tests, and hippocampal and frontal cortex (FC) leptin and dopamine levels and inflammatory signalling assessed. KEY FINDINGS: T2D mice exhibited weight gain (+15 %), hyperglycemia (+35 %), hyperinsulinemia (+60 %) and insulin-resistance (+80 % higher HOMA-IR), together with anxiety-like behaviour (without anhedonia) that appeared independent of body weight and glycemic status. Cortical leptin declined whereas receptor mRNA increased. Supplementation with ALA did not influence metabolic state, while enhancing locomotion and reducing anxiety-like behaviours in healthy but not T2D mice. Hippocampal dopamine was selectively increased by ALA in T2D mice, with a trend to reduced circulating leptin in both groups. Across all groups, anxiety-like behaviour was associated with declining cortical and hippocampal leptin levels and increasing receptor mRNA, while declining dopamine levels were accompanied by decreased dopamine/serotonin receptor transcripts. SIGNIFICANCE: Chronic T2D induced anxiogenesis in mice appears to be independent of metabolic homeostasis but linked to central leptin-resistance, together with disturbed dopamine and serotonin signalling. Despite anxiolytic effects of ALA in healthy mice, no metabolic or behavioural benefits were evident in T2D.

Stress-induced body weight loss and improvements in cardiometabolic risk factors do not translate to improved myocardial ischemic tolerance in western diet-fed mice.

Although both diet-induced obesity and psychological stress are recognized as significant independent contributors to cardiometabolic and behavioral disorders, our understanding of how these two disorders interact and influence cardiometabolic risk and myocardial ischemic tolerance is limited. The aim of this study was to assess the combined effects of an obesogenic diet and psychological stress on cardiometabolic risk factors (body weight, dyslipidemia, insulin sensitivity) and postischemic cardiovascular outcomes. C57Bl/6J mice (n = 48) were subject to a combination of 22 weeks of western diet (WD) feeding and chronic restraint stress (CRS) for the last 4 weeks. Metabolic and behavioral changes were assessed using glucose tolerance tests and open field tests (OFTs), respectively. After 22 weeks, cardiac function and ischemic tolerance were assessed in Langendorff perfused hearts. WD feeding increased body weight and worsened blood lipids and insulin sensitivity. WD-fed mice also exhibited reduced exploratory behavior within the OFT. CRS reduced body weight and increased locomotion in both dietary groups and had differential effects on fasting glucose metabolism in the two dietary groups while not impacting non-fasting insulin. Although the WD only marginally reduced reperfusion left ventricular developed pressure recovery, CRS worsened reperfusion diastolic dysfunction in both dietary groups. Interestingly, despite WD+CRS animals exhibiting improved cardiometabolic parameters compared to the WD group, these changes did not translate to marked improvements to postischemic cardiac outcomes. In conclusion, in this study, combined WD feeding and CRS did not act synergistically to worsen cardiometabolic risk factors but instead improved them. Despite these cardiometabolic improvements, WD+CRS increased reperfusion end diastolic pressure which may be indicative of worsened ischemia/reperfusion injury.

Effect of chronic restraint stress and western-diet feeding on colonic regulatory gene expression in mice.

BACKGROUND: Diet-induced obesity (DIO) and psychological stress are significant independent regulators of gastrointestinal physiology; however, our understanding of how these two disorders influence the host-microbe interface is still poorly characterized. The aim of this study was to assess the combined influences of diet-induced obesity and psychological stress on microbiome composition and colonic gene expression. METHODS: C57BL/6J mice (n = 48) were subject to a combination of 22 weeks of Western diet (WD) feeding and a chronic restraint stressor (CRS) for the last 4 weeks of feeding. At the end of the combined intervention, microbiome composition was determined from cecal contents, and colonic tissue gene expression was assessed by multiplex analysis using NanoString nCounter System and real-time qPCR. RESULTS: WD feeding induced a DIO phenotype with increased body weight, worsened metabolic markers, and alterations to microbiome composition. CRS reduced body weight in both dietary groups while having differential effects on glucose metabolism. CRS improved the Firmicutes/Bacteroidetes ratio in WD-fed animals while expanding the Proteobacteria phyla. Significantly lower expression of colonic Tlr4 (p = 0.008), Ocln (p = 0.004), and Cldn3 (p = 0.004) were noted in WD-fed animals compared to controls with no synergistic effects observed when combined with CRS. No changes to colonic expression of downstream inflammatory mediators were observed. Interestingly, higher levels of expression of Cldn2 (p = 0.04) and bile acid receptor Nr1h4 (p = 0.02) were seen in mice exposed to CRS. CONCLUSION: Differential but not synergistic effects of WD and CRS were noted at the host-microbe interface suggesting multifactorial responses that require further investigation.

A YOLO based software for automated detection and analysis of rodent behaviour in the open field arena.

Rodent models are important in mechanistic studies of the physiological and pathophysiological determinants of behaviour. The Open Field Test (OFT) is one of the most commonly utilised tests to assess rodent behaviour in a novel open environment. The key variables assessed in an OFT are general locomotor activity and exploratory behaviours and can be assessed manually or by automated systems. Although several automated systems exist, they are often expensive, difficult to use, or limited in the type of video that can be analysed. Here we describe a machine-learning algorithm - dubbed Cosevare - that uses a trained YOLOv3 DNN to identify and track movement of mice in the open-field arena. We validated Cosevare's capacity to accurately track locomotive and exploratory behaviour in 10 videos, comparing outputs generated by Cosevare with analysis by 5 manual scorers. Behavioural differences between control mice and those with diet-induced obesity (DIO) were also documented. We found the YOLOv3 based tracker to be accurate at identifying and tracking the mice within the open-field arena and in instances with variable backgrounds. Additionally, kinematic and spatial-based analysis demonstrated highly consistent scoring of locomotion, centre square duration (CSD) and entries (CSE) between Cosevare and manual scorers. Automated analysis was also able to distinguish behavioural differences between healthy control and DIO mice. The study found that a YOLOv3 based tracker is able to easily track mouse behaviour in the open field arena and supports machine learning as a potential future alternative for the assessment of animal behaviour in a wide range of species in differing environments and behavioural tests.

Trimethylamine N-oxide: heart of the microbiota-CVD nexus?

We critically review potential involvement of trimethylamine N-oxide (TMAO) as a link between diet, the gut microbiota and CVD. Generated primarily from dietary choline and carnitine by gut bacteria and hepatic flavin-containing mono-oxygenase (FMO) activity, TMAO could promote cardiometabolic disease when chronically elevated. However, control of circulating TMAO is poorly understood, and diet, age, body mass, sex hormones, renal clearance, FMO3 expression and genetic background may explain as little as 25 % of TMAO variance. The basis of elevations with obesity, diabetes, atherosclerosis or CHD is similarly ill-defined, although gut microbiota profiles/remodelling appear critical. Elevated TMAO could promote CVD via inflammation, oxidative stress, scavenger receptor up-regulation, reverse cholesterol transport (RCT) inhibition, and cardiovascular dysfunction. However, concentrations influencing inflammation, scavenger receptors and RCT (≥100 µm) are only achieved in advanced heart failure or chronic kidney disease (CKD), and greatly exceed pathogenicity of <1-5 µm levels implied in some TMAO-CVD associations. There is also evidence that CVD risk is insensitive to TMAO variance beyond these levels in omnivores and vegetarians, and that major TMAO sources are cardioprotective. Assessing available evidence suggests that modest elevations in TMAO (≤10 µm) are a non-pathogenic consequence of diverse risk factors (ageing, obesity, dyslipidaemia, insulin resistance/diabetes, renal dysfunction), indirectly reflecting CVD risk without participating mechanistically. Nonetheless, TMAO may surpass a pathogenic threshold as a consequence of CVD/CKD, secondarily promoting disease progression. TMAO might thus reflect early CVD risk while providing a prognostic biomarker or secondary target in established disease, although mechanistic contributions to CVD await confirmation.

Dietary α-Linolenic Acid Counters Cardioprotective Dysfunction in Diabetic Mice: Unconventional PUFA Protection.

Whether dietary omega-3 (n-3) polyunsaturated fatty acid (PUFA) confers cardiac benefit in cardiometabolic disorders is unclear. We test whether dietary -linolenic acid (ALA) enhances myocardial resistance to ischemia-reperfusion (I-R) and responses to ischemic preconditioning (IPC) in type 2 diabetes (T2D); and involvement of conventional PUFA-dependent mechanisms (caveolins/cavins, kinase signaling, mitochondrial function, and inflammation). Eight-week male C57Bl/6 mice received streptozotocin (75 mg/kg) and 21 weeks high-fat/high-carbohydrate feeding. Half received ALA over six weeks. Responses to I-R/IPC were assessed in perfused hearts. Localization and expression of caveolins/cavins, protein kinase B (AKT), and glycogen synthase kinase-3 ß (GSK3ß); mitochondrial function; and inflammatory mediators were assessed. ALA reduced circulating leptin, without affecting body weight, glycemic dysfunction, or cholesterol. While I-R tolerance was unaltered, paradoxical injury with IPC was reversed to cardioprotection with ALA. However, post-ischemic apoptosis (nucleosome content) appeared unchanged. Benefit was not associated with shifts in localization or expression of caveolins/cavins, p-AKT, p-GSK3ß, or mitochondrial function. Despite mixed inflammatory mediator changes, tumor necrosis factor-a (TNF-a) was markedly reduced. Data collectively reveal a novel impact of ALA on cardioprotective dysfunction in T2D mice, unrelated to caveolins/cavins, mitochondrial, or stress kinase modulation. Although evidence suggests inflammatory involvement, the basis of this "un-conventional" protection remains to be identified.

Synergistic effects of low-level stress and a Western diet on metabolic homeostasis, mood, and myocardial ischemic tolerance.

How low-level psychological stress and overnutrition interact in influencing cardiometabolic disease is unclear. Mechanistic overlaps suggest potential synergies; however, findings are contradictory. We test whether low-level stress and Western diet (WD) feeding synergistically influence homeostasis, mood, and myocardial ischemic tolerance. Male C57BL6/J mice were fed a control diet or WD (32%/57%/11% calories from fat/carbohydrates/protein) for 12 wk, with subgroups restrained for 30 min/day over the final 3 wk. Metabolism, behavior, tolerance of perfused hearts to ischemia-reperfusion (I/R), and cardiac "death proteins" were assessed. The WD resulted in insignificant trends toward increased body weight (+5%), glucose (+40%), insulin (+40%), triglycerides (+15%), and cholesterol (+20%) and reduced leptin (-20%) while significantly reducing insulin sensitivity [100% rise in homeostasis model assessment of insulin resistance (HOMA-IR), P < 0.05]. Restraint did not independently influence metabolism while increasing HOMA-IR a further 50% (and resulting in significant elevations in insulin and glucose to 60-90% above control) in WD mice (P < 0.05), despite blunting weight gain in control and WD mice. Anxiogenesis with restraint or WD was nonadditive, whereas anhedonia (reduced sucrose consumption) only arose with their combination. Neuroinflammation markers (hippocampal TNF-α, Il-1b) were unchanged. Myocardial I/R tolerance was unaltered with stress or WD alone, whereas the combination worsened dysfunction and oncosis [lactate dehydrogenase (LDH) efflux]. Apoptosis (nucleosome accumulation) and death protein expression (BAK, BAX, BCL-2, RIP-1, TNF-α, cleaved caspase-3, and PARP) were unchanged. We conclude that mild, anxiogenic yet cardio-metabolically "benign" stress interacts synergistically with a WD to disrupt homeostasis, promote anhedonia (independently of neuroinflammation), and impair myocardial ischemic tolerance (independently of apoptosis and death protein levels).

Chronic type 2 but not type 1 diabetes impairs myocardial ischaemic tolerance and preconditioning in C57Bl/6 mice.

NEW FINDINGS: • What is the central question of this study? What is the impact of chronic adult-onset diabetes on cardiac ischaemic outcomes and preconditioning? • What is the main finding and its importance? Chronic adult-onset type 2 but not type 1 diabetes significantly impairs myocardial ischaemic tolerance and ischaemic preconditioning. Preconditioning may be detrimental in type 2 diabetes, exaggerating nitrosative stress and apoptotic protein expression. ABSTRACT: Effects of diabetes on myocardial responses to ischaemia-reperfusion (I-R) and cardioprotective stimuli remain contentious, potentially reflecting influences of disease duration and time of onset. Chronic adult-onset type 1 diabetes (T1D) and type 2 diabetes (T2D) were modelled non-genetically in male C57Bl/6 mice via 5 × 50 mg kg-1 daily streptozotocin (STZ) injections + 12 weeks' standard chow or 1 × 75 mg kg-1 STZ injection + 12 weeks' obesogenic diet (32% calories as fat, 57% carbohydrate, 11% protein), respectively. Systemic outcomes were assessed and myocardial responses to I-R ± ischaemic preconditioning (IPC; 3 × 5 min I-R) determined in Langendorff perfused hearts. Uncontrolled T1D was characterised by pronounced hyperglycaemia (25 mm fasting glucose), glucose intolerance and ∼10% body weight loss, whereas T2D mice exhibited moderate hyperglycaemia (15 mm), hyperinsulinaemia, glucose intolerance and 17% weight gain. Circulating ghrelin, resistin and noradrenaline were unchanged with T1D, while leptin increased and noradrenaline declined in T2D mice. Ischaemic tolerance and IPC were preserved in T1D hearts. In contrast, T2D worsened post-ischaemic function (∼40% greater diastolic and contractile dysfunction) and cell death (100% higher troponin efflux), and abolished IPC protection. Whereas IPC reduced post-ischaemic nitrotyrosine and pro-apoptotic Bak and Bax levels in non-diabetic hearts, these effects were reduced in T1D and IPC augmented Bax and nitrosylation in T2D hearts. The data demonstrate chronic T1D does not inhibit myocardial I-R tolerance or IPC, whereas metabolic and endocrine disruption in T2D is associated with ischaemic intolerance and inhibition of IPC. Indeed, normally protective IPC may exaggerate damage mechanisms in T2D hearts.

The brain-adipocyte-gut network: Linking obesity and depression subtypes.

Major depressive disorder (MDD) and obesity are dominant and inter-related health burdens. Obesity is a risk factor for MDD, and there is evidence MDD increases risk of obesity. However, description of a bidirectional relationship between obesity and MDD is misleading, as closer examination reveals distinct unidirectional relationships in MDD subtypes. MDD is frequently associated with weight loss, although obesity promotes MDD. In contrast, MDD with atypical features (MDD-AF) is characterised by subsequent weight gain and obesity. The bases of these distinct associations remain to be detailed, with conflicting findings clouding interpretation. These associations can be viewed within a systems biology framework-the psycho-immune neuroendocrine (PINE) network shared between MDD and metabolic disorders. Shared PINE subsystem perturbations may underlie increased MDD in overweight and obese people (obesity-associated depression), while obesity in MDD-AF (depression-associated obesity) involves more complex interactions between behavioural and biomolecular changes. In the former, the chronic PINE dysfunction triggering MDD is augmented by obesity-dependent dysregulation in shared networks, including inflammatory, leptin-ghrelin, neuroendocrine, and gut microbiome systems, influenced by chronic image-associated psychological stress (particularly in younger or female patients). In MDD-AF, behavioural dysregulation, including hypersensitivity to interpersonal rejection, fundamentally underpins energy imbalance (involving hyperphagia, lethargy, hypersomnia), with evolving obesity exaggerating these drivers via positive feedback (and potentially augmenting PINE disruption). In both settings, sex and age are important determinants of outcome, associated with differences in emotional versus cognitive dysregulation. A systems biology approach is recommended for further research into the pathophysiological networks underlying MDD and linking depression and obesity.

Bilirubin acts as a multipotent guardian of cardiovascular integrity: more than just a radical idea.

Bilirubin, a potentially toxic catabolite of heme and indicator of hepatobiliary insufficiency, exhibits potent cardiac and vascular protective properties. Individuals with Gilbert's syndrome (GS) may experience hyperbilirubinemia in response to stressors including reduced hepatic bilirubin excretion/increased red blood cell breakdown, with individuals usually informed by their clinician that their condition is of little consequence. However, GS appears to protect from all-cause mortality, with progressively elevated total bilirubin associated with protection from ischemic heart and chronic obstructive pulmonary diseases. Bilirubin may protect against these diseases and associated mortality by reducing circulating cholesterol, oxidative lipid/protein modifications, and blood pressure. In addition, bilirubin inhibits platelet activation and protects the heart from ischemia-reperfusion injury. These effects attenuate multiple stages of the atherosclerotic process in addition to protecting the heart during resultant ischemic stress, likely underpinning the profound reduction in cardiovascular mortality in hyperbilirubinemic GS. This review outlines our current knowledge of and uses for bilirubin in clinical medicine and summarizes recent progress in revealing the physiological importance of this poorly understood molecule. We believe that this review will be of significant interest to clinicians, medical researchers, and individuals who have GS.

Pravastatin improves risk factors but not ischaemic tolerance in obese rats.

Statins are effective in management of dyslipidaemia, and a cornerstone of CVD prevention strategies. However, the impacts of their pleiotropic effects on other cardiovascular risk factors and myocardial responses to infarction are not well characterised. We hypothesised that pravastatin treatment in obesity improves lipid profiles, insulin-resistance and myocardial resistance to ischaemia/reperfusion (I/R) injury. Wistar rats were fed a control (C) chow or high carbohydrate and fat diet (HCFD) for 16 weeks with vehicle or pravastatin (prava 7.5 mg/kg/day) treatment for 8 weeks. At 16 weeks HOMAs were performed, blood samples collected and hearts excised for Langendorff perfusions/biochemical analyses. Anti-oxidant activity and proteins regulating mitochondrial fission/fusion and apoptosis were assessed. The HCFD increased body weight (736±15 vs. 655±12 g for C; P<0.001), serum triglycerides (2.91±0.52 vs. 1.64±0.26 mmol/L for C; P<0.001) and insulin-resistance (HOMA- 6.9±0.8 vs. 4.2±0.5 for C; P<0.05) while prava prevented diet induced changes and paradoxically increased lipid peroxidation. The HCFD increased infarct size (34.1±3.1% vs. 18.8±3.0% of AAR for C; P<0.05), which was unchanged by prava in C and HCFD animals. The HCFD decreased cardiac TxR activity and mitochondrial MFN-1 and increased mitochondrial DRP-1 (reducing MFN-1:DRP-1 ratio) and Bax expression, with the latter changes prevented by prava. While unaltered by diet, cytosolic levels of Bax and caspase-3 were reduced by prava in C and HCFD hearts (without changes in cleaved caspase-3). We conclude that obesity, hyper-triglyceridemia and impaired glycemic control in HCFD rats are countered by prava. Despite improved risk factors, prava did not reduce myocardial infarct size, potentially reflecting its complex pleiotropic impacts on cardiac GPX activity and MFN-1, DRP-1, caspase-3 and Bcl-2 proteins.

Myocyte membrane and microdomain modifications in diabetes: determinants of ischemic tolerance and cardioprotection.

Cardiovascular disease, predominantly ischemic heart disease (IHD), is the leading cause of death in diabetes mellitus (DM). In addition to eliciting cardiomyopathy, DM induces a 'wicked triumvirate': (i) increasing the risk and incidence of IHD and myocardial ischemia; (ii) decreasing myocardial tolerance to ischemia-reperfusion (I-R) injury; and (iii) inhibiting or eliminating responses to cardioprotective stimuli. Changes in ischemic tolerance and cardioprotective signaling may contribute to substantially higher mortality and morbidity following ischemic insult in DM patients. Among the diverse mechanisms implicated in diabetic impairment of ischemic tolerance and cardioprotection, changes in sarcolemmal makeup may play an overarching role and are considered in detail in the current review. Observations predominantly in animal models reveal DM-dependent changes in membrane lipid composition (cholesterol and triglyceride accumulation, fatty acid saturation vs. reduced desaturation, phospholipid remodeling) that contribute to modulation of caveolar domains, gap junctions and T-tubules. These modifications influence sarcolemmal biophysical properties, receptor and phospholipid signaling, ion channel and transporter functions, contributing to contractile and electrophysiological dysfunction, cardiomyopathy, ischemic intolerance and suppression of protective signaling. A better understanding of these sarcolemmal abnormalities in types I and II DM (T1DM, T2DM) can inform approaches to limiting cardiomyopathy, associated IHD and their consequences. Key knowledge gaps include details of sarcolemmal changes in models of T2DM, temporal patterns of lipid, microdomain and T-tubule changes during disease development, and the precise impacts of these diverse sarcolemmal modifications. Importantly, exercise, dietary, pharmacological and gene approaches have potential for improving sarcolemmal makeup, and thus myocyte function and stress-resistance in this ubiquitous metabolic disorder.

Dependence of Cardiac Systolic Function on Elevated Fatty Acid Availability in Obese, Insulin-Resistant Rats.

BACKGROUND: Clinical data advocating an adverse effect of obesity on left ventricular (LV) systolic function independent of comorbidities is controversial. We hypothesized that in obesity with prediabetic insulin resistance, circulating fatty acids (FAs) become a valuable fuel source in the maintenance of normal systolic function. METHODS: Male Wistar rats were fed a high caloric diet for 32 weeks to induce obesity. Myocardial LV systolic function was assessed using echocardiography and isolated heart preparations. RESULTS: Aortic output was reduced in obese rat hearts over a range of filling pressures (for example: 15 cmH2O, obese: 32.6 ± 1.2 ml/min vs control: 46.2 ± 0.9 ml/min, P < .05) when perfused with glucose alone. Similarly, the slope of the LV end-systolic pressure-volume relationship decreased, and there was a right shift in the LV end-systolic stress-strain relationship as determined in Langendorff perfused, isovolumic rat heart preparations in the presence of isoproterenol (10(-8)M) (LV systolic stress-strain relationship and a reduced load-independent intrinsic systolic myocardial function, obese: 791 ± 62 g/cm(2) vs control: 1186 ± 74 g/cm(2), P < .01). The addition of insulin to the perfusion buffer improved aortic output, whereas the addition of FAs completely normalized aortic output. LV function was maintained in obese animals in vivo during an inotropic challenge. CONCLUSIONS: Elevated circulating FA levels may be important to maintain myocardial systolic function in the initial stages of obesity and insulin resistance.

Pre- or post-ischemic bilirubin ditaurate treatment reduces oxidative tissue damage and improves cardiac function.

BACKGROUND: Unconjugated bilirubin (UCB), an endogenous antioxidant, may protect the heart against ischemia-reperfusion (I-R) injury. However, the 'cardioprotective' potential of bilirubin therapy remains unclear. We tested whether pre- or post-ischemic treatment of ex vivo perfused hearts with bilirubin ditaurate (BRT) improves post-ischemic functional outcomes and myocardial oxidative damage. METHODS: Isolated Langendorff perfused hearts (male, Wistar rats) were treated with 50 µM BRT for 30 min before (Pre) or after (Post) 30 min of zero-flow ischemia. Functional outcomes were monitored, with myocardial damage estimated from creatine kinase efflux, infarct size, and left ventricular lipid/protein oxidation assessed by measuring malondialdehyde and protein carbonyls. Ischemia induced contractile dysfunction and cellular injury, with both BRT treatments improving I-R outcomes. RESULTS: Final post-ischemic recoveries for left ventricular diastolic/developed pressures were significantly enhanced in treated groups: end-diastolic pressure (Control, 78±14, Pre, 51±15*, Post, 51±13 mm Hg*); left ventricular developed pressure, (LVDP; Control 44±15, Pre, 71±19*, Post, 84±13 mm Hg*). Myocardial injury/infarction (MI) was also significantly reduced with BRT treatment: post-ischemic creatine kinase efflux (Control, 1.24±0.41, Pre, 0.86±0.31*, Post, 0.51±0.29 U/g/mL*; infarct size, Control, 67±17, Pre, 39±15*, Post, 22±11%*). These changes were accompanied by significantly reduced malondialdehyde and protein carbonyl content in Pre and Post treated hearts (*P<0.05 vs. Control). CONCLUSIONS: These data collectively reveal significant cardioprotection upon BRT treatment, with post-treatment being particularly effective. Significant reductions in infarct size and lipid and protein oxidation indicate a mechanism related to protection from oxidative damage and indicate the potential utility of this molecule as a post-MI treatment.

Trenbolone Improves Cardiometabolic Risk Factors and Myocardial Tolerance to Ischemia-Reperfusion in Male Rats With Testosterone-Deficient Metabolic Syndrome.

The increasing prevalence of obesity adds another dimension to the pathophysiology of testosterone (TEST) deficiency (TD) and potentially impairs the therapeutic efficacy of classical TEST replacement therapy. We investigated the therapeutic effects of selective androgen receptor modulation with trenbolone (TREN) in a model of TD with the metabolic syndrome (MetS). Male Wistar rats (n=50) were fed either a control standard rat chow (CTRL) or a high-fat/high-sucrose (HF/HS) diet. After 8 weeks of feeding, rats underwent sham surgery or an orchiectomy (ORX). Alzet miniosmotic pumps containing either vehicle, 2-mg/kg·d TEST or 2-mg/kg·d TREN were implanted in HF/HS+ORX rats. Body composition, fat distribution, lipid profile, and insulin sensitivity were assessed. Infarct size was quantified to assess myocardial damage after in vivo ischaemia reperfusion, before cardiac and prostate histology was performed. The HF/HS+ORX animals had increased sc and visceral adiposity; circulating triglycerides, cholesterol, and insulin; and myocardial damage, with low circulating TEST compared with CTRLs. Both TEST and TREN protected HF/HS+ORX animals against sc fat accumulation, hypercholesterolaemia, and myocardial damage. However, only TREN protected against visceral fat accumulation, hypertriglyceridaemia, and hyperinsulinaemia and reduced myocardial damage relative to CTRLs. TEST caused widespread cardiac fibrosis and prostate hyperplasia, which were less pronounced with TREN. We propose that TEST replacement therapy may have contraindications for males with TD and obesity-related MetS. TREN treatment may be more effective in restoring androgen status and reducing cardiovascular risk in males with TD and MetS.

A refined high carbohydrate diet is associated with changes in the serotonin pathway and visceral obesity.

Consumption of palatable foods high in refined carbohydrate has been implicated as a contributing factor to the epidemic levels of obesity. Such foods may disrupt appetite regulation in the hypothalamus through alterations in hunger and satiety signalling. This investigation examined whether a palatable high refined carbohydrate (HRC) diet with the potential to induce obesity was linked to modulation of serotonin and dopamine signalling within the hypothalamus of rats. Male Wistar rats were allowed ad libitum access to either a palatable refined carbohydrate enriched (HRC) diet or standard chow (SC). Visceral fat percentage was used as a measure of the animals' weight gain during the trial. Real-time PCR was applied to determine any variation in levels of expression of the serotonin (Slc6A4 or Sert) and dopamine transporter (Slc6A3 or Dat) genes. After 29 weeks, the HRC group showed a significant increase in visceral fat percentage accompanied by increased expression of Sert. Higher levels of circulating triglycerides were also seen. This investigation determined that a refined high carbohydrate diet is associated with visceral obesity, increased circulating lipids in the blood and distorted serotonergic signalling, which possibly alters satiety and hunger signals.

Impact of Diet-Induced Obesity and Testosterone Deficiency on the Cardiovascular System: A Novel Rodent Model Representative of Males with Testosterone-Deficient Metabolic Syndrome (TDMetS).

INTRODUCTION: Current models of obesity utilise normogonadic animals and neglect the strong relationships between obesity-associated metabolic syndrome (MetS) and male testosterone deficiency (TD). The joint presentation of these conditions has complex implications for the cardiovascular system that are not well understood. We have characterised and investigated three models in male rats: one of diet-induced obesity with the MetS; a second using orchiectomised rats mimicking TD; and a third combining MetS with TD which we propose is representative of males with testosterone deficiency and the metabolic syndrome (TDMetS). METHODS: Male Wistar rats (n = 24) were randomly assigned to two groups and provided ad libitum access to normal rat chow (CTRL) or a high fat/high sugar/low protein "obesogenic" diet (OGD) for 28 weeks (n = 12/group). These groups were further sub-divided into sham-operated or orchiectomised (ORX) animals to mimic hypogonadism, with and without diet-induced obesity (n = 6/group). Serum lipids, glucose, insulin and sex hormone concentrations were determined. Body composition, cardiovascular structure and function; and myocardial tolerance to ischemia-reperfusion were assessed. RESULTS: OGD-fed animals had 72% greater fat mass; 2.4-fold greater serum cholesterol; 2.3-fold greater serum triglycerides and 3-fold greater fasting glucose (indicative of diabetes mellitus) compared to CTRLs (all p<0.05). The ORX animals had reduced serum testosterone and left ventricle mass (p<0.05). In addition to the combined differences observed in each of the isolated models, the OGD, ORX and OGD+ORX models each had greater CK-MB levels following in vivo cardiac ischemia-reperfusion insult compared to CTRLs (p<0.05). CONCLUSION: Our findings provide evidence to support that the MetS and TD independently impair myocardial tolerance to ischemia-reperfusion. The combined OGD+ORX phenotype described in this study is a novel animal model with associated cardiovascular risk factors and complex myocardial pathology which may be representative of male patients presenting with TDMetS.

Improvements in body composition, cardiometabolic risk factors and insulin sensitivity with trenbolone in normogonadic rats.

Trenbolone (TREN) is used for anabolic growth-promotion in over 20 million cattle annually and continues to be misused for aesthetic purposes in humans. The current study investigated TREN's effects on body composition and cardiometabolic risk factors; and its tissue-selective effects on the cardiovascular system, liver and prostate. Male rats (n=12) were implanted with osmotic infusion pumps delivering either cyclodextrin vehicle (CTRL) or 2mg/kg/day TREN for 6 weeks. Dual-energy X-ray Absorptiometry assessment of body composition; organ wet weights and serum lipid profiles; and insulin sensitivity were assessed. Cardiac ultrasound examinations were performed before in vivo studies assessed myocardial susceptibility to ischemia-reperfusion (I/R) injury. Circulating sex hormones and liver enzyme activities; and prostate and liver histology were examined. In 6 weeks, fat mass increased by 34±7% in CTRLs (p<0.01). Fat mass decreased by 37±6% and lean mass increased by 11±4% with TREN (p<0.05). Serum triglycerides, HDL and LDL were reduced by 62%, 57% and 78% (p<0.05) respectively in TREN rats. Histological examination of the prostates from TREN-treated rats indicated benign hyperplasia associated with an increased prostate mass (149% compared to CTRLs, p<0.01). No evidence of adverse cardiac or hepatic effects was observed. In conclusion, improvements in body composition, lipid profile and insulin sensitivity (key risk factors for cardiometabolic disease) were achieved with six-week TREN treatment without evidence of adverse cardiovascular or hepatic effects that are commonly associated with traditional anabolic steroid misuse. Sex hormone suppression and benign prostate hyperplasia were confirmed as adverse effects of the treatment.