Background. 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. Methods. Male C57Bl6/J mice were fed a control or WD (32%/57%/11% calories from fat/carbohydrates/protein) for 12 wks, with subgroups restrained for 30 min/day over the final 3 wks. Metabolism, behavior, tolerance of perfused hearts to ischemia/reperfusion (I/R), and cardiac 'death proteins' were assessed. Results. The WD resulted in insignificant trends to 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 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 non-additive, whereas anhedonia (reduced sucrose consumption) only arose with their combination. Neuroinflammation markers (hippocampal TNF-a, Il-1b) were unchanged. Myocardial I/R tolerance was unaltered with stress or WD alone, while combination worsened dysfunction and oncosis (LDH efflux). Apoptosis (nucleosome accumulation) and death protein expression (BAK, BAX, BCL-2, RIP-1, TNF-α, cleaved caspase-3 and PARP) were unchanged. Conclusion. 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).

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.

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 β (GSK3β); 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-GSK3β, 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.

Caveolins regulate myocardial substrate handling, survival signaling, and stress resistance; however, control of expression is incompletely defined. We test how metabolic features of type 2 diabetes (T2D), and modulation of cell signaling, influence caveolins in H9c2 cardiomyoblasts. Cells were exposed to glucose (25 vs. 5 mM), insulin (100 nM), or palmitate (0.1 mM), individually or combined, and the effects of adenylate cyclase (AC) activation (50 μM forskolin), focal adhesion kinase (FAK) or protein kinase C β2 (PKCβ2) inhibition (1 μM FAK inhibitor 14 or CGP-53353, respectively) or the polyunsaturated fatty acid (PUFA) α-linolenic acid (ALA; 10 μM) were tested. Simulated T2D (elevated glucose + insulin + palmitate) depressed caveolin-1 and -3 without modifying caveolin-2. Caveolin-3 repression was primarily palmitate dependent, whereas high glucose (HG) and insulin independently increased caveolin-3 (while reducing expression when combined). Differential control was evident: baseline caveolin-3 was suppressed by FAK/PKCβ2 and insensitive to AC activities, with baseline caveolin-1 and -2 suppressed by AC and insensitive to FAK/PKCβ2. Forskolin and ALA selectively preserved caveolin-3 in T2D cells, whereas PKCβ2 and FAK inhibition increased caveolin-3 under all conditions. Despite preservation of caveolin-3, ALA did not modify nucleosome content (apoptosis marker) or transcription of proinflammatory mediators in T2D cells. In summary, caveolin-1 and -3 are strongly repressed with simulated T2D, with caveolin-3 particularly sensitive to palmitate; intrinsic PKCβ2 and FAK activities depress caveolin-3 in healthy and stressed cells; ALA and AC activation and PKCβ2 inhibition preserve caveolin-3 under T2D conditions; and caveolin-3 changes with T2D and ALA appear unrelated to inflammatory signaling or extent of apoptosis.

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