Association Between Type 2 Diabetes and Changes in Myocardial Structure, Contractile Function, Energetics, and Blood Flow Before and After Aortic Valve Replacement in Patients With Severe Aortic Stenosis.

BACKGROUND: Type 2 diabetes (T2D) is associated with an increased risk of left ventricular dysfunction after aortic valve replacement (AVR) in patients with severe aortic stenosis (AS). Persistent impairments in myocardial energetics and myocardial blood flow (MBF) may underpin this observation. Using phosphorus magnetic resonance spectroscopy and cardiovascular magnetic resonance, this study tested the hypothesis that patients with severe AS and T2D (AS-T2D) would have impaired myocardial energetics as reflected by the phosphocreatine to ATP ratio (PCr/ATP) and vasodilator stress MBF compared with patients with AS without T2D (AS-noT2D), and that these differences would persist after AVR. METHODS: Ninety-five patients with severe AS without coronary artery disease awaiting AVR (30 AS-T2D and 65 AS-noT2D) were recruited (mean, 71 years of age [95% CI, 69, 73]; 34 [37%] women). Thirty demographically matched healthy volunteers (HVs) and 30 patients with T2D without AS (T2D controls) were controls. One month before and 6 months after AVR, cardiac PCr/ATP, adenosine stress MBF, global longitudinal strain, NT-proBNP (N-terminal pro-B-type natriuretic peptide), and 6-minute walk distance were assessed in patients with AS. T2D controls underwent identical assessments at baseline and 6-month follow-up. HVs were assessed once and did not undergo 6-minute walk testing. RESULTS: Compared with HVs, patients with AS (AS-T2D and AS-noT2D combined) showed impairment in PCr/ATP (mean [95% CI]; HVs, 2.15 [1.89, 2.34]; AS, 1.66 [1.56, 1.75]; P<0.0001) and vasodilator stress MBF (HVs, 2.11 mL min g [1.89, 2.34]; AS, 1.54 mL min g [1.41, 1.66]; P<0.0001) before AVR. Before AVR, within the AS group, patients with AS-T2D had worse PCr/ATP (AS-noT2D, 1.74 [1.62, 1.86]; AS-T2D, 1.44 [1.32, 1.56]; P=0.002) and vasodilator stress MBF (AS-noT2D, 1.67 mL min g [1.5, 1.84]; AS-T2D, 1.25 mL min g [1.22, 1.38]; P=0.001) compared with patients with AS-noT2D. Before AVR, patients with AS-T2D also had worse PCr/ATP (AS-T2D, 1.44 [1.30, 1.60]; T2D controls, 1.66 [1.56, 1.75]; P=0.04) and vasodilator stress MBF (AS-T2D, 1.25 mL min g [1.10, 1.41]; T2D controls, 1.54 mL min g [1.41, 1.66]; P=0.001) compared with T2D controls at baseline. After AVR, PCr/ATP normalized in patients with AS-noT2D, whereas patients with AS-T2D showed no improvements (AS-noT2D, 2.11 [1.79, 2.43]; AS-T2D, 1.30 [1.07, 1.53]; P=0.0006). Vasodilator stress MBF improved in both AS groups after AVR, but this remained lower in patients with AS-T2D (AS-noT2D, 1.80 mL min g [1.59, 2.0]; AS-T2D, 1.48 mL min g [1.29, 1.66]; P=0.03). There were no longer differences in PCr/ATP (AS-T2D, 1.44 [1.30, 1.60]; T2D controls, 1.51 [1.34, 1.53]; P=0.12) or vasodilator stress MBF (AS-T2D, 1.48 mL min g [1.29, 1.66]; T2D controls, 1.60 mL min g [1.34, 1.86]; P=0.82) between patients with AS-T2D after AVR and T2D controls at follow-up. Whereas global longitudinal strain, 6-minute walk distance, and NT-proBNP all improved after AVR in patients with AS-noT2D, no improvement in these assessments was observed in patients with AS-T2D. CONCLUSIONS: Among patients with severe AS, those with T2D demonstrate persistent abnormalities in myocardial PCr/ATP, vasodilator stress MBF, and cardiac contractile function after AVR; AVR effectively normalizes myocardial PCr/ATP, vasodilator stress MBF, and cardiac contractile function in patients without T2D.

Endothelial IGF-1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity.

Changes in composition of the intestinal microbiota are linked to the development of obesity and can lead to endothelial cell (EC) dysfunction. It is unknown whether EC can directly influence the microbiota. Insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) are critical for coupling nutritional status and cellular growth; IGF-1R is expressed in multiple cell types including EC. The role of ECIGF-1R in the response to nutritional obesity is unexplored. To examine this, we use gene-modified mice with EC-specific overexpression of human IGF-1R (hIGFREO) and their wild-type littermates. After high-fat feeding, hIGFREO weigh less, have reduced adiposity and have improved glucose tolerance. hIGFREO show an altered gene expression and altered microbial diversity in the gut, including a relative increase in the beneficial genus Akkermansia. The depletion of gut microbiota with broad-spectrum antibiotics induces a loss of the favourable metabolic differences seen in hIGFREO mice. We show that IGF-1R facilitates crosstalk between the EC and the gut wall; this crosstalk protects against diet-induced obesity, as a result of an altered gut microbiota.

Temporal analysis of skeletal muscle remodeling post hindlimb ischemia reveals intricate autophagy regulation.

Hind limb ischemia (HLI) is the most severe form of peripheral arterial disease, associated with a substantial reduction of limb blood flow that impairs skeletal muscle homeostasis to promote functional disability. The molecular regulators of HLI-induced muscle perturbations remain poorly defined. This study investigated whether changes in the molecular catabolic-autophagy signaling network were linked to temporal remodeling of skeletal muscle in HLI. HLI was induced in mice via hindlimb ischemia (femoral artery ligation) and confirmed by Doppler echocardiography. Experiments were terminated at time points defined as early- (7 days; n = 5) or late- (28 days; n = 5) stage HLI. Ischemic and nonischemic (contralateral) limb muscles were compared. Ischemic versus nonischemic muscles demonstrated overt remodeling at early-HLI but normalized at late-HLI. Early-onset fiber atrophy was associated with excessive autophagy signaling in ischemic muscle; protein expression increased for Beclin-1, LC3, and p62 (P < 0.05) but proteasome-dependent markers were reduced (P < 0.05). Mitophagy signaling increased in early-stage HLI that aligned with an early and sustained loss of mitochondrial content (P < 0.05). Upstream autophagy regulators, Sestrins, showed divergent responses during early-stage HLI (Sestrin2 increased while Sestrin1 decreased; P < 0.05) in parallel to increased AMP-activated protein kinase (AMPK) phosphorylation (P < 0.05) and lower antioxidant enzyme expression. No changes were found in markers for mechanistic target of rapamycin complex 1 signaling. These data indicate that early activation of the sestrin-AMPK signaling axis may regulate autophagy to stimulate rapid and overt muscle atrophy in HLI, which is normalized within weeks and accompanied by recovery of muscle mass. A complex interplay between Sestrins to regulate autophagy signaling during early-to-late muscle remodeling in HLI is likely.

Sexual dimorphism in adipose tissue mitochondrial function and metabolic flexibility in obesity.

OBJECTIVE: The prevalence of obesity is growing globally. Adiposity increases the risk for metabolic syndrome, type 2 diabetes and cardiovascular disease. Adipose tissue distribution influences systemic metabolism and impacts metabolic disease risk. The link between sexual dimorphisms of adiposity and metabolism is poorly defined. We hypothesise that depot-specific adipose tissue mitochondrial function contributes to the sexual dimorphism of metabolic flexibility in obesity. METHODS: Male and female mice fed high fat diet (HFD) or standard diet (STD) from 8-18 weeks of age underwent whole animal calorimetry and high-resolution mitochondrial respirometry analysis on adipose tissue depots. To determine translatability we used RT-qPCR to examine key brown adipocyte-associated gene expression: peroxisome proliferator-activated receptor co-activator 1α, Uncoupling protein 1 and cell death inducing DFFA like effector a in brown adipose tissue (BAT) and subcutaneous adipose tissue (sWAT) of 18-week-old mice and sWAT from human volunteers. RESULTS: Male mice exhibited greater weight gain compared to female mice when challenged with HFD. Relative to increased body mass, the adipose to body weight ratio for BAT and sWAT depots was increased in HFD-fed males compared to female HFD-fed mice. Oxygen consumption, energy expenditure, respiratory exchange ratio and food consumption did not differ between males and females fed HFD. BAT mitochondria from obese females showed increased Complex I & II respiration and maximal respiration compared to lean females whereas obese males did not exhibit adaptive mitochondrial BAT respiration. Sexual dimorphism in BAT-associated gene expression in sWAT was also associated with Body Mass Index in humans. CONCLUSIONS: We show that sexual dimorphism of weight gain is reflected in mitochondrial respiration analysis. Female mice have increased metabolic flexibility to adapt to changes in energy intake by regulating energy expenditure through increased complex II and maximal mitochondrial respiration within BAT when HFD challenged and increased proton leak in sWAT mitochondria.

Elevated ambient temperature accelerates aspects of torpor phenology in an obligate hibernator.

The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) is assumed to be an obligate hibernator - commencing and terminating hibernation on a circannual rhythm, regardless of environmental conditions - but, until now, this assumption had never been fully tested. We housed three groups of captive-born ground squirrels from Aug. 2017 to Aug. 2018 under constant photoperiod (12 h L:12 h D) at 5, 16 or 25 °C, and monitored hibernation using body temperature loggers. At 5 and 16 °C all animals hibernated from autumn to spring with no differences in date of first/last torpor or duration of interbout euthermic periods (IBE), but torpor bout duration was 25% shorter at 16 °C. One of 4 animals housed at 25 °C did not hibernate. For the other three 25 °C animals, the first torpor date did not differ from the other groups, but the last torpor bout (5 Feb.) occurred almost 8 weeks earlier. These animals aroused from torpor more frequently and IBE lasted significantly longer, so the total time spent torpid was less than 50% of the other groups. Unlike the 5 or 16 °C animals, 25 °C animals re-entered torpor in late spring 2018. Taken together these data suggest that this species is an obligate hibernator, but that high ambient temperatures can accelerate the endogenous circannual hibernation rhythm.

Environmental temperature effects on adipose tissue growth in a hibernator.

Obligate hibernators express circannual patterns of body mass and hibernation, which persist under constant laboratory conditions. Brown adipose tissue (BAT) is important for thermogenesis during arousals from hibernation, whereas white adipose tissue (WAT) serves as energy storage and thermal insulation. The goal of this study was to investigate the effects of environmental temperature on BAT and WAT. We hypothesized that changes to environmental temperature would not influence the pattern of mass gain or BAT and WAT volume in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus). To test this, we housed animals at thermoneutral 25°C (warm-housed) or 5°C (cold-housed), with the same photoperiod (12 h light:12 h dark) over an entire year. Throughout the year we measured the volume and water:fat ratio of WAT and BAT using magnetic resonance imaging (MRI). We found no evidence of torpor in the warm-housed animals, indicating that this species might not be an obligate hibernator, as previously assumed. Regardless of ambient temperature, BAT volume increased prior to winter, then decreased in late winter with no change in water:fat ratio. By contrast, both body mass and WAT volume of cold-housed animals declined throughout the winter and recovered after hibernation, but thermoneutral housing produced no circannual pattern in body mass, even though WAT volume declined in late winter. Cold exposure appears to be a primary regulator for WAT but BAT may exhibit an endogenous circannual rhythm in terms of depot volume.

Identification of a lipid-rich depot in the orbital cavity of the thirteen-lined ground squirrel.

We discovered a previously undescribed orbital lipid depot in the thirteen-lined ground squirrel during the first ever magnetic resonance image (MRI) of this common experimental model of mammalian hibernation. In animals housed at constant ambient temperatures (5°C or 25°C, 12 h:12 h light:dark photoperiod), the volume of this depot increased in the autumn and decreased in the spring, suggesting an endogenous circannual pattern. Water-fat MRI revealed that throughout the year this depot is composed of ∼40% lipid, similar to brown adipose tissue (BAT). During arousal from torpor, thermal images showed higher surface temperatures near this depot before the rest of the head warmed, suggesting a thermoregulatory function. This depot, however, does not contain uncoupling protein 1, a BAT biomarker, or uncoupling protein 3. Histology shows blood vessels in close proximity to each other, suggesting it may serve as a vascular rete, perhaps to preferentially warm the eye and brain during arousals.

Thermoneutral temperature reduces liver volume but increases fat content in a mammalian hibernator.

Hibernators survive challenging winters by entering torpor, which lowers body temperature (Tb) to ∼5 °C for 12-14 days, followed by spontaneous arousals where Tb increases to ∼37 °C for 10-12 h before entering another torpor bout. This Tb cycle is accompanied by significant fluctuations in metabolic rate. Little is known about the role of the liver in lipid metabolism during hibernation. In this study we measured the effect of ambient temperature on liver volume and lipid content in 13-lined ground squirrels (Ictidomys tridecemlineatus). We housed animals at thermoneutral (25 °C) or cold (5 °C) ambient temperatures, with the same photoperiod (12 h light:12 h dark) for an entire year. We determined volume and water-fat ratio of the liver using magnetic resonance imaging (MRI). Ambient temperature significantly affected both liver volume and fat content. From October to August squirrels housed at 25 °C had 25% smaller livers compared to the squirrels housed at 5 °C, but their average lipid content (13.3%) was 37% higher. Because the squirrels housed at 25 °C appeared to continue feeding throughout the winter but did not enter extended torpor, more carbohydrates may have been diverted to lipid stores. By contrast, animals housed at 5 °C did not appear to feed, and carbohydrates would likely be preferentially stored in the liver as glycogen to supply glucose for brain metabolism. These results suggest that the fat burden caused by hibernators preparing for winter can lead to symptoms of metabolic syndrome, but that these symptoms are reversible in the spring.

An improved method for detecting torpor entrance and arousal in a mammalian hibernator using heart rate data.

We used electrocardiogram (ECG) telemeters to measure the heart rate of hibernating Ictidomys tridecemlineatus (thirteen-lined ground squirrel). An increase in heart rate from 2.2 to 5 beats min-1 accurately identified arousal from torpor before any change in body temperature was detected. Variability in raw heart rate data was significantly reduced by a forward-backward Butterworth low-pass filter, allowing for discrete differential analysis. A decrease in filtered heart rate to 70% of maximum values in interbout euthermia (from approximately 312 to 235 beats min-1) accurately detected entrance into torpor bouts. At this point, body temperature had fallen from 36.1°C to only 34.7°C, much higher than the 30°C typically used to identify entrance. Using these heart rate criteria allowed advanced detection of entrance and arousal (detected 51.9 and 76 min earlier, respectively), compared with traditional body temperature criteria. This method will improve our ability to detect biochemical and molecular markers underlying these transition periods, during which many physiological changes occur.

MiR-214-3p Regulates Piezo1, Lysyl Oxidases and Mitochondrial Function in Human Cardiac Fibroblasts.

Cardiac fibroblasts are pivotal regulators of cardiac homeostasis and are essential in the repair of the heart after myocardial infarction (MI), but their function can also become dysregulated, leading to adverse cardiac remodelling involving both fibrosis and hypertrophy. MicroRNAs (miRNAs) are noncoding RNAs that target mRNAs to prevent their translation, with specific miRNAs showing differential expression and regulation in cardiovascular disease. Here, we show that miR-214-3p is enriched in the fibroblast fraction of the murine heart, and its levels are increased with cardiac remodelling associated with heart failure, or in the acute phase after experimental MI. Tandem mass tagging proteomics and in-silico network analyses were used to explore protein targets regulated by miR-214-3p in cultured human cardiac fibroblasts from multiple donors. Overexpression of miR-214-3p by miRNA mimics resulted in decreased expression and activity of the Piezo1 mechanosensitive cation channel, increased expression of the entire lysyl oxidase (LOX) family of collagen cross-linking enzymes, and decreased expression of an array of mitochondrial proteins, including mitofusin-2 (MFN2), resulting in mitochondrial dysfunction, as measured by citrate synthase and Seahorse mitochondrial respiration assays. Collectively, our data suggest that miR-214-3p is an important regulator of cardiac fibroblast phenotypes and functions key to cardiac remodelling, and that this miRNA represents a potential therapeutic target in cardiovascular disease.

Measurement of Fatty Acid Oxidation by High-Resolution Respirometry: Special Considerations for Analysis of Skeletal and Cardiac Muscle and Adipose Tissue.

High-resolution respirometry is a state-of-the-art approach for the quantitation of mitochondrial function. Isolated mitochondria, cultured cells, or tissues/fibers are suspended in oxygenated respiration medium within a closed chamber and substrates or inhibitors added in a stepwise manner. The dissolved oxygen concentration decreases as aerobic metabolism in the specimen proceeds, recorded by an oxygen sensor within the chamber to give a quantifiable measure of oxygen consumption by the sample. Measuring oxygen consumption using a variety of respiratory substrates or respiratory complex-targeted inhibitors enables multiple respiratory pathways to be interrogated to determine the functional capacity of the mitochondria in real time. Using a substrate-uncoupler-inhibitor titration (SUIT) protocol, we have developed a method which makes use of differing chain length fatty acids to derive a measure of fatty acid-stimulated respiration through ß-oxidation in a variety of tissue types including skeletal and cardiac muscles and brown and white adipose tissues. This report provides technical details of the protocol, and the adaptations employed, to generate robust analysis of mitochondrial fatty acid ß-oxidation.

Metabokines in the regulation of systemic energy metabolism.

Metabolism consists of life-sustaining chemical reactions involving metabolites. Historically, metabolites were defined as the intermediates or end products of metabolism and considered to be passive participants changed by metabolic processes. However, recent research has redefined how we view metabolism. There is emerging evidence of metabolites which function to mediate cellular signalling and interorgan crosstalk, regulating local metabolism and systemic physiology. These bioactive metabolite signals have been termed metabokines. Metabokines regulate diverse energy metabolism pathways across multiple tissues, including fatty acid ß-oxidation, mitochondrial oxidative phosphorylation, lipolysis, glycolysis and gluconeogenesis. There is increasing impetus to uncover novel metabokine signalling axes to better understand how these may be perturbed in metabolic diseases and determine their utility as therapeutic targets.

Prospective Longitudinal Characterization of the Relationship between Diabetes and Cardiac Structural and Functional Changes.

OBJECTIVES: In a cohort of type 2 diabetic (T2D) patients who underwent baseline cardiac magnetic resonance (CMR) and biomarker testing, during a median follow-up of 6 years, we aimed to determine longitudinal changes in the phenotypic expression of heart disease in diabetes, report clinical outcomes, and compare baseline clinical characteristics and CMR findings of patients who experienced major adverse cardiovascular events (MACE) to those remaining MACE free. BACKGROUND: T2D increases the risk of heart failure (HF) and cardiovascular mortality. The long-term impact of T2D on cardiac phenotype in the absence of cardiovascular disease and other clinical events is unknown. METHODS: Patients with T2D (n = 100) with no history of cardiovascular disease or hypertension were recruited at baseline. Biventricular volumes, function, and myocardial extracellular volume fraction (ECV) were assessed by CMR, and blood biomarkers were taken. Follow-up CMR was repeated in those without interim clinical events after 6 years. RESULTS: Follow-up was successful in 83 participants. Of those, 29 experienced cardiovascular/clinical events (36%). Of the remaining 59, 32 patients who experienced no events received follow-up CMR. In this cohort, despite no significant changes in blood pressure, weight, or glycated hemoglobin, significant reductions in biventricular end-diastolic volumes and ejection fractions occurred over time. The mean ECV was unchanged. Baseline plasma high-sensitivity cardiac troponin T (hs-cTnT) was significantly associated with a change in left ventricular (LV) ejection fraction. Patients who experienced MACE had higher LV mass and greater LV concentricity than those who remained event free. CONCLUSIONS: T2D results in reductions in biventricular size and systolic function over time even in the absence of cardiovascular/clinical events.

Long-chain ceramides are cell non-autonomous signals linking lipotoxicity to endoplasmic reticulum stress in skeletal muscle.

The endoplasmic reticulum (ER) regulates cellular protein and lipid biosynthesis. ER dysfunction leads to protein misfolding and the unfolded protein response (UPR), which limits protein synthesis to prevent cytotoxicity. Chronic ER stress in skeletal muscle is a unifying mechanism linking lipotoxicity to metabolic disease. Unidentified signals from cells undergoing ER stress propagate paracrine and systemic UPR activation. Here, we induce ER stress and lipotoxicity in myotubes. We observe ER stress-inducing lipid cell non-autonomous signal(s). Lipidomics identifies that palmitate-induced cell stress induces long-chain ceramide 40:1 and 42:1 secretion. Ceramide synthesis through the ceramide synthase 2 de novo pathway is regulated by UPR kinase Perk. Inactivation of CerS2 in mice reduces systemic and muscle ceramide signals and muscle UPR activation. The ceramides are packaged into extracellular vesicles, secreted and induce UPR activation in naïve myotubes through dihydroceramide accumulation. This study furthers our understanding of ER stress by identifying UPR-inducing cell non-autonomous signals.

Brown and beige adipose tissue regulate systemic metabolism through a metabolite interorgan signaling axis.

Brown and beige adipose tissue are emerging as distinct endocrine organs. These tissues are functionally associated with skeletal muscle, adipose tissue metabolism and systemic energy expenditure, suggesting an interorgan signaling network. Using metabolomics, we identify 3-methyl-2-oxovaleric acid, 5-oxoproline, and ß-hydroxyisobutyric acid as small molecule metabokines synthesized in browning adipocytes and secreted via monocarboxylate transporters. 3-methyl-2-oxovaleric acid, 5-oxoproline and ß-hydroxyisobutyric acid induce a brown adipocyte-specific phenotype in white adipocytes and mitochondrial oxidative energy metabolism in skeletal myocytes both in vitro and in vivo. 3-methyl-2-oxovaleric acid and 5-oxoproline signal through cAMP-PKA-p38 MAPK and ß-hydroxyisobutyric acid via mTOR. In humans, plasma and adipose tissue 3-methyl-2-oxovaleric acid, 5-oxoproline and ß-hydroxyisobutyric acid concentrations correlate with markers of adipose browning and inversely associate with body mass index. These metabolites reduce adiposity, increase energy expenditure and improve glucose and insulin homeostasis in mouse models of obesity and diabetes. Our findings identify beige adipose-brown adipose-muscle physiological metabokine crosstalk.

Water-fat MRI in a hibernator reveals seasonal growth of white and brown adipose tissue without cold exposure.

Obligate hibernators, such as ground squirrels, display circannual patterns which persist even under constant laboratory conditions, suggesting that they are regulated by endogenous rhythms. Brown adipose tissue (BAT) is important for thermogenesis during periodic arousals from hibernation when core body temperature rises spontaneously from 5 to 37 °C. In most small eutherians BAT growth requires several weeks of cold exposure. We hypothesized that in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), a hibernator, BAT growth is regulated, in part, by an endogenous rhythm and we predicted that this growth would precede the hibernation season without cold exposure. We tested this prediction using repeated water-fat magnetic resonance imaging over a year, including the hibernation season. Thoracic BAT depots increased in volume from spring through autumn even though animals were housed at ~22 °C. Subsequent cold exposure (5 °C) enlarged the thoracic BAT further. The fat fraction of this tissue fell significantly during the period of peak growth, indicating relative increases in non-triglyceride components, perhaps mitochondria or vasculature. We also found that the proportion of the body consisting of white adipose tissue (WAT) increased steadily from spring through autumn, and fell throughout hibernation, mirroring changes in body mass. Unlike BAT, WAT fat fractions remained constant (near 90%) throughout the year. Future studies will evaluate the significance of photoperiod and cold exposure on the growth of these tissues. We also found tissue with a fat fraction characteristic of BAT in the head near the eyes, a potentially novel discovery that requires further confirmation.