Mitochondrial respiration is lower in the intrauterine growth-restricted fetal sheep heart.

Fetuses affected by intrauterine growth restriction have an increased risk of developing heart disease and failure in adulthood. Compared with controls, late gestation intrauterine growth-restricted (IUGR) fetal sheep have fewer binucleated cardiomyocytes, reflecting a more immature heart, which may reduce mitochondrial capacity to oxidize substrates. We hypothesized that the late gestation IUGR fetal heart has a lower capacity for mitochondrial oxidative phosphorylation. Left (LV) and right (RV) ventricles from IUGR and control (CON) fetal sheep at 90% gestation were harvested. Mitochondrial respiration (states 1-3, LeakOmy, and maximal respiration) in response to carbohydrates and lipids, citrate synthase (CS) activity, protein expression levels of mitochondrial oxidative phosphorylation complexes (CI-CV), and mRNA expression levels of mitochondrial biosynthesis regulators were measured. The carbohydrate and lipid state 3 respiration rates were lower in IUGR than CON, and CS activity was lower in IUGR LV than CON LV. However, relative CII and CV protein levels were higher in IUGR than CON; CV expression level was higher in IUGR than CON. Genes involved in lipid metabolism had lower expression in IUGR than CON. In addition, the LV and RV demonstrated distinct differences in oxygen flux and gene expression levels, which were independent from CON and IUGR status. Low mitochondrial respiration and CS activity in the IUGR heart compared with CON are consistent with delayed cardiomyocyte maturation, and CII and CV protein expression levels may be upregulated to support ATP production. These insights will provide a better understanding of fetal heart development in an adverse in utero environment. KEY POINTS: Growth-restricted fetuses have a higher risk of developing and dying from cardiovascular diseases in adulthood. Mitochondria are the main supplier of energy for the heart. As the heart matures, the substrate preference of the mitochondria switches from carbohydrates to lipids. We used a sheep model of intrauterine growth restriction to study the capacity of the mitochondria in the heart to produce energy using either carbohydrate or lipid substrates by measuring how much oxygen was consumed. Our data show that the mitochondria respiration levels in the growth-restricted fetal heart were lower than in the normally growing fetuses, and the expression levels of genes involved in lipid metabolism were also lower. Differences between the right and left ventricles that are independent of the fetal growth restriction condition were identified. These results indicate an impaired metabolic maturation of the growth-restricted fetal heart associated with a decreased capacity to oxidize lipids postnatally.

Single-leg exercise training augments in vivo skeletal muscle oxidative flux and vascular content and function in adults with type 2 diabetes.

KEY POINTS: People with type 2 diabetes (T2D) have impaired skeletal muscle oxidative flux due to limited oxygen delivery. In the current study, this impairment in oxidative flux in people with T2D was abrogated with a single-leg exercise training protocol. Additionally, single-leg exercise training increased skeletal muscle CD31 content, calf blood flow and state 4 mitochondrial respiration in all participants. ABSTRACT: Cardiorespiratory fitness is impaired in type 2 diabetes (T2D), conferring significant cardiovascular risk in this population; interventions are needed. Previously, we reported that a T2D-associated decrement in skeletal muscle oxidative flux is ameliorated with acute use of supplemental oxygen, suggesting that skeletal muscle oxygenation is rate-limiting to in vivo mitochondrial oxidative flux during exercise in T2D. We hypothesized that single-leg exercise training (SLET) would improve the T2D-specific impairment in in vivo mitochondrial oxidative flux during exercise. Adults with (n = 19) and without T2D (n = 22) with similar body mass indexes and levels of physical activity participated in two weeks of SLET. Following SLET, in vivo oxidative flux measured by 31 P-MRS increased in participants with T2D, but not people without T2D, measured by the increase in initial phosphocreatine synthesis (P = 0.0455 for the group × exercise interaction) and maximum rate of oxidative ATP synthesis (P = 0.0286 for the interaction). Additionally, oxidative phosphorylation increased in all participants with SLET (P = 0.0209). After SLET, there was no effect of supplemental oxygen on any of the in vivo oxidative flux measurements in either group (P > 0.02), consistent with resolution of the T2D-associated oxygen limitation previously observed at baseline in subjects with T2D. State 4 mitochondrial respiration also improved in muscle fibres ex vivo. Skeletal muscle vasculature content and calf blood flow increased in all participants with SLET (P < 0.0040); oxygen extraction in the calf increased only in T2D (P = 0.0461). SLET resolves the T2D-associated impairment of skeletal muscle in vivo mitochondrial oxidative flux potentially through improved effective blood flow/oxygen delivery.

Lower citrate synthase activity, mitochondrial complex expression, and fewer oxidative myofibers characterize skeletal muscle from growth-restricted fetal sheep.

Skeletal muscle from the late gestation sheep fetus with intrauterine growth restriction (IUGR) has evidence of reduced oxidative metabolism. Using a sheep model of placental insufficiency and IUGR, we tested the hypothesis that by late gestation, IUGR fetal skeletal muscle has reduced capacity for oxidative phosphorylation because of intrinsic deficits in mitochondrial respiration. We measured mitochondrial respiration in permeabilized muscle fibers from biceps femoris (BF) and soleus (SOL) from control and IUGR fetal sheep. Using muscles including BF, SOL, tibialis anterior (TA), and flexor digitorum superficialis (FDS), we measured citrate synthase (CS) activity, mitochondrial complex subunit abundance, fiber type distribution, and gene expression of regulators of mitochondrial biosynthesis. Ex vivo mitochondrial respiration was similar in control and IUGR muscle. However, CS activity was lower in IUGR BF and TA, indicating lower mitochondrial content, and protein expression of individual mitochondrial complex subunits was lower in IUGR TA and BF in a muscle-specific pattern. IUGR TA, BF, and FDS also had lower expression of type I oxidative fibers. Fiber-type shifts that support glycolytic instead of oxidative metabolism may be advantageous for the IUGR fetus in a hypoxic and nutrient-deficient environment, whereas these adaptions may be maladaptive in postnatal life.

(-)-Epicatechin Reverses Glucose Intolerance in Rats Housed at Thermoneutrality.

Diabetes is a life-threatening and debilitating disease with pathological hallmarks, including glucose intolerance and insulin resistance. Plant compounds are a source of novel and effective therapeutics, and the flavonoid (-)-epicatechin, common to popular foods worldwide, has been shown to improve carbohydrate metabolism in both clinical studies and preclinical models. We hypothesized that (-)-epicatechin would alleviate thermoneutral housing-induced glucose intolerance. Male rats were housed at either thermoneutral (30 °C) or room temperature (24 °C) for 16 weeks and gavaged with either 1 mg/kg body weight or vehicle for the last 15 days before sacrifice. Rats housed at thermoneutrality had a significantly elevated serum glucose area under the curve (p < 0.05) and reduced glucose-mediated insulin secretion. In contrast, rats at thermoneutrality treated with (-)-epicatechin had improved glucose tolerance and increased insulin secretion (p < 0.05). Insulin tolerance tests revealed no differences in insulin sensitivity in any of the four groups. Pancreatic immunohistochemistry staining showed significantly greater islet insulin positive cells in animals housed at thermoneutrality. In conclusion, (-)-epicatechin improved carbohydrate tolerance via increased insulin secretion in response to glucose challenge without a change in insulin sensitivity.

Saxagliptin restores vascular mitochondrial exercise response in the Goto-Kakizaki rat.

Cardiovascular disease risk and all-cause mortality are largely predicted by physical fitness. Exercise stimulates vascular mitochondrial biogenesis through endothelial nitric oxide synthase (eNOS), sirtuins, and PPARγ coactivator 1α (PGC-1α), a response absent in diabetes and hypertension. We hypothesized that an agent regulating eNOS in the context of diabetes could reconstitute exercise-mediated signaling to mitochondrial biogenesis. Glucagon-like peptide 1 (GLP-1) stimulates eNOS and blood flow; we used saxagliptin, an inhibitor of GLP-1 degradation, to test whether vascular mitochondrial adaptation to exercise in diabetes could be restored. Goto-Kakizaki (GK) rats, a nonobese, type 2 diabetes model, and Wistar controls were exposed to an 8-day exercise intervention with or without saxagliptin (10 mg·kg·d). We evaluated the impact of exercise and saxagliptin on mitochondrial proteins and signaling pathways in aorta. Mitochondrial protein expression increased with exercise in the Wistar aorta and decreased or remained unchanged in the GK animals. GK rats treated with saxagliptin plus exercise showed increased expression of mitochondrial complexes, cytochrome c, eNOS, nNOS, PGC-1α, and UCP3 proteins. Notably, a 3-week saxagliptin plus exercise intervention significantly increased running time in the GK rats. These data suggest that saxagliptin restores vascular mitochondrial adaptation to exercise in a diabetic rodent model and may augment the impact of exercise on the vasculature.

Targeting mitochondria to restore failed adaptation to exercise in diabetes.

Our translational research group focuses on addressing the problem of exercise defects in diabetes with basic research efforts in cell and rodent models and clinical research efforts in subjects with diabetes mellitus. CREB (cAMP-response-element-binding protein) regulates cellular differentiation of neurons, ß-cells, adipocytes and smooth muscle cells; it is also a potent survival factor and an upstream regulator of mitochondrial biogenesis. In diabetes and cardiovascular disease, CREB protein content is decreased in the vascular media, and its regulation in aberrant in ß-cells, neurons and cardiomyocytes. Loss of CREB content and function leads to decreased vascular target tissue resilience when exposed to stressors such as metabolic, oxidative or sheer stress. This basic research programme set the stage for our central hypothesis that diabetes-mediated CREB dysfunction predisposes the diabetes disease progression and cardiovascular complications. Our clinical research programme revealed that diabetes mellitus leads to defects in functional exercise capacity. Our group has determined that the defects in exercise correlate with insulin resistance, endothelial dysfunction, decreased cardiac perfusion and diastolic dysfunction, slowed muscle perfusion kinetics, decreased muscle perfusion and slowed oxidative phosphorylation. Combined basic and clinical research has defined the relationship between exercise and vascular function with particular emphasis on how the signalling to CREB and eNOS [endothelial NOS (nitric oxide synthase)] regulates tissue perfusion, mitochondrial dynamics, vascular function and exercise capacity. The present review summarizes our current working hypothesis that restoration of eNOS/NOS dysfunction will restore cellular homoeostasis and permit an optimal tissue response to an exercise training intervention.

Perivascular adipose tissue remodeling impairs vasoreactivity in thermoneutral-housed rats.

Objective: Vascular pathology, characterized by impaired vasoreactivity and mitochondrial respiration, differs between the sexes. Housing rats under thermoneutral (TN) conditions causes vascular dysfunction and perturbed metabolism. We hypothesized that perivascular adipose tissue (PVAT), a vasoregulatory adipose depot with brown adipose tissue (BAT) phenotype, remodels to a white adipose (WAT) phenotype in rats housed at TN, driving diminished vasoreactivity in a sex-dependent manner. Methods: Male and female Wistar rats were housed at either room temperature (RT) or TN. Endpoints included changes in PVAT morphology, vasoreactivity in vessels with intact PVAT or transferred to PVAT of the oppositely-housed animal, vessel stiffness, vessel mitochondrial respiration and cellular signaling. Results: Remodeling of PVAT was observed in rats housed at TN; animals in this environment showed PVAT whitening and displayed diminished aortae vasodilation (p<0.05), different between the sexes. Juxtaposing PVAT from RT rats onto aortae from TN rats in females corrected vasodilation (p<0.05); this did not occur in males. In aortae of all animals housed at TN, mitochondrial respiration was significantly diminished in lipid substrate experiments (p<0.05), and there was significantly less expression of peNOS (p<0.001). Conclusions: These data are consistent with TN-induced remodeling of PVAT, notably associated with sex-specific blunting of vasoreactivity, diminished mitochondrial respiration, and altered cellular signaling.

Muscle mitochondrial function is impaired in adults with type 1 diabetes.

AIMS: Type 1 diabetes has been associated with mitochondrial dysfunction. However, the mechanism of this dysfunction in adults remains unclear. METHODS: A secondary analysis was conducted using data from several clinical trials measuring in-vivo and ex-vivo mitochondrial function in adults with type 1 diabetes (n = 34, age 38.8 ± 14.6 years) and similarly aged controls (n = 59, age 44.6 ± 13.9 years). In-vivo mitochondrial function was assessed before, during, and after isometric exercise with 31phosphorous magnetic resonance spectroscopy. High resolution respirometry of vastus lateralis muscle tissue was used to assess ex-vivo measures. RESULTS: In-vivo data showed higher rates of anaerobic glycolysis (p = 0.013), and a lower maximal mitochondrial oxidative capacity (p = 0.012) and mitochondrial efficiency (p = 0.024) in adults with type 1 diabetes. After adjustment for age and percent body fat maximal mitochondrial capacity (p = 0.014) continued to be lower and anaerobic glycolysis higher (p = 0.040) in adults with type 1 diabetes. Ex-vivo data did not demonstrate significant differences between the two groups. CONCLUSIONS: The in-vivo analysis demonstrates that adults with type 1 diabetes have mitochondrial dysfunction. This builds on previous research showing in-vivo mitochondrial dysfunction in youths with type 1 diabetes and suggests that defects in substrate or oxygen delivery may play a role in in-vivo dysfunction.

Nitric oxide regulates vascular adaptive mitochondrial dynamics.

Cardiovascular disease risk factors, such as diabetes, hypertension, dyslipidemia, obesity, and physical inactivity, are all correlated with impaired endothelial nitric oxide synthase (eNOS) function and decreased nitric oxide (NO) production. NO-mediated regulation of mitochondrial biogenesis has been established in many tissues, yet the role of eNOS in vascular mitochondrial biogenesis and dynamics is unclear. We hypothesized that genetic eNOS deletion and 3-day nitric oxide synthase (NOS) inhibition in rodents would result in impaired mitochondrial biogenesis and defunct fission/fusion and autophagy profiles within the aorta. We observed a significant, eNOS expression-dependent decrease in mitochondrial electron transport chain (ETC) protein subunits from complexes I, II, III, and V in eNOS heterozygotes and eNOS null mice compared with age-matched controls. In response to NOS inhibition with NG-nitro-L-arginine methyl ester (L-NAME) treatment in Sprague Dawley rats, significant decreases were observed in ETC protein subunits from complexes I, III, and IV as well as voltage-dependent anion channel 1. Decreased protein content of upstream regulators of mitochondrial biogenesis, cAMP response element-binding protein and peroxisome proliferator-activated receptor-γ coactivator-1α, were observed in response to 3-day L-NAME treatment. Both genetic eNOS deletion and NOS inhibition resulted in decreased manganese superoxide dismutase protein. L-NAME treatment resulted in significant changes to mitochondrial dynamic protein profiles with decreased fusion, increased fission, and minimally perturbed autophagy. In addition, L-NAME treatment blocked mitochondrial adaptation to an exercise intervention in the aorta. These results suggest that eNOS/NO play a role in basal and adaptive mitochondrial biogenesis in the vasculature and regulation of mitochondrial turnover.

Sex Differences in the Skeletal Muscle Response to a High Fat, High Sucrose Diet in Rats.

Men are diagnosed with type 2 diabetes at lower body mass indexes than women; the role of skeletal muscle in this sex difference is poorly understood. Type 2 diabetes impacts skeletal muscle, particularly in females who demonstrate a lower oxidative capacity compared to males. To address mechanistic differences underlying this sex disparity, we investigated skeletal muscle mitochondrial respiration in female and male rats in response to chronic high-fat, high-sugar (HFHS) diet consumption. Four-week-old Wistar Rats were fed a standard chow or HFHS diet for 14 weeks to identify sex-specific adaptations in mitochondrial respirometry and characteristics, transcriptional patterns, and protein profiles. Fat mass was greater with the HFHS diet in both sexes when controlled for body mass (p < 0.0001). Blood glucose and insulin resistance were greater in males (p = 0.01) and HFHS-fed rats (p < 0.001). HFHS-fed males had higher mitochondrial respiration compared with females (p < 0.01 sex/diet interaction). No evidence of a difference by sex or diet was found for mitochondrial synthesis, dynamics, or quality to support the mitochondrial respiration sex/diet interaction. However, transcriptomic analyses indicate sex differences in nutrient handling. Sex-specific differences occurred in PI3K/AKT signaling, PPARα/RXRα, and triacylglycerol degradation. These findings may provide insight into the clinical sex differences in body mass index threshold for diabetes development and tissue-specific progression of insulin resistance.

(-)-Epicatechin Improves Vasoreactivity and Mitochondrial Respiration in Thermoneutral-Housed Wistar Rat Vasculature.

Cardiovascular disease (CVD) is a global health concern. Vascular dysfunction is an aspect of CVD, and novel treatments targeting vascular physiology are necessary. In the endothelium, eNOS regulates vasodilation and mitochondrial function; both are disrupted in CVD. (−)-Epicatechin, a botanical compound known for its vasodilatory, eNOS, and mitochondrial-stimulating properties, is a potential therapy in those with CVD. We hypothesized that (−)-epicatechin would support eNOS activity and mitochondrial respiration, leading to improved vasoreactivity in a thermoneutral-derived rat model of vascular dysfunction. We housed Wistar rats at room temperature or in thermoneutral conditions for a total of 16 week and treated them with 1mg/kg body weight (−)-epicatechin for 15 day. Vasoreactivity, eNOS activity, and mitochondrial respiration were measured, in addition to the protein expression of upstream cellular signaling molecules including AMPK and CaMKII. We observed a significant improvement of vasodilation in those housed in thermoneutrality and treated with (−)-epicatechin (p < 0.05), as well as dampened mitochondrial respiration (p < 0.05). AMPK and CaMKIIα and ß expression were lessened with (−)-epicatechin treatment in those housed at thermoneutrality (p < 0.05). The opposite was observed with animals housed at room temperature supplemented with (−)-epicatechin. These data illustrate a context-dependent vascular response to (−)-epicatechin, a candidate for CVD therapeutic development.

CREB downregulation in vascular disease: a common response to cardiovascular risk.

OBJECTIVE: To examine the impact of low-density lipoprotein (LDL), an established mediator of atherosclerosis, on the transcription factor cAMP-response element-binding protein (CREB), which is a regulator of vascular smooth muscle cell (VSMC) quiescence. METHODS AND RESULTS: VSMC CREB content is diminished in rodent models of diabetes and pulmonary hypertension. We examined aortic CREB content in rodent models of aging, hypertension, and insulin resistance, and we determined nuclear CREB protein in the medial VSMC of high-fat-fed LDL receptor-null mice. There was significant loss of CREB protein in all models. In vitro, primary culture rat aortic VSMC exposed to LDL and oxidized LDL exhibited a rapid, transient increase in CREB phosphorylation and transient phosphorylation/activation of Akt, ERK, JNK, ans p38 MAPK. Exposure to oxidized LDL, but not to LDL, for 24 to 48 hours decreased CREB protein in a dose-dependent fashion and led to nuclear exclusion of CREB. Pharmacological reactive oxygen species scavengers and inhibition of ERK activation blocked oxidized LDL-mediated CREB downregulation. CONCLUSIONS: These data support a model wherein loss of VSMC CREB protein, which renders these cells more susceptible to activation and apoptosis, is a common pathological response to vascular injury and potentially contributes to plaque progression.

Breast Cancer Endocrine Therapy Promotes Weight Gain With Distinct Adipose Tissue Effects in Lean and Obese Female Mice.

Breast cancer survivors treated with tamoxifen and aromatase inhibitors report weight gain and have an elevated risk of type 2 diabetes, especially if they have obesity. These patient experiences are inconsistent with, preclinical studies using high doses of tamoxifen which reported acute weight loss. We investigated the impact of breast cancer endocrine therapies in a preclinical model of obesity and in a small group of breast adipose tissue samples from women taking tamoxifen to understand the clinical findings. Mature female mice were housed at thermoneutrality and fed either a low-fat/low-sucrose (LFLS) or a high-fat/high-sucrose (HFHS) diet. Consistent with the high expression of Esr1 observed in mesenchymal stem cells from adipose tissue, endocrine therapy was associated with adipose accumulation and more preadipocytes compared with estrogen-treated control mice but resulted in fewer adipocyte progenitors only in the context of HFHS. Analysis of subcutaneous adipose stromal cells revealed diet- and treatment-dependent effects of endocrine therapies on various cell types and genes, illustrating the complexity of adipose tissue estrogen receptor signaling. Breast cancer therapies supported adipocyte hypertrophy and associated with hepatic steatosis, hyperinsulinemia, and glucose intolerance, particularly in obese females. Current tamoxifen use associated with larger breast adipocyte diameter only in women with obesity. Our translational studies suggest that endocrine therapies may disrupt adipocyte progenitors and support adipocyte hypertrophy, potentially leading to ectopic lipid deposition that may be linked to a greater type 2 diabetes risk. Monitoring glucose tolerance and potential interventions that target insulin action should be considered for some women receiving life-saving endocrine therapies for breast cancer.

(-)-Epicatechin Modulates Mitochondrial Redox in Vascular Cell Models of Oxidative Stress.

Diabetes mellitus affects 451 million people worldwide, and people with diabetes are 3-5 times more likely to develop cardiovascular disease. In vascular tissue, mitochondrial function is important for vasoreactivity. Diabetes-mediated generation of excess reactive oxygen species (ROS) may contribute to vascular dysfunction via damage to mitochondria and regulation of endothelial nitric oxide synthase (eNOS). We have identified (-)-epicatechin (EPICAT), a plant compound and known vasodilator, as a potential therapy. We hypothesized that mitochondrial ROS in cells treated with antimycin A (AA, a compound targeting mitochondrial complex III) or high glucose (HG, global perturbation) could be normalized by EPICAT, and correlate with improved mitochondrial dynamics and cellular signaling. Human umbilical vein endothelial cells (HUVEC) were treated with HG, AA, and/or 0.1 or 1.0 µM of EPICAT. Mitochondrial and cellular superoxide, mitochondrial respiration, and cellular signaling upstream of mitochondrial function were assessed. EPICAT at 1.0 µM significantly attenuated mitochondrial superoxide in HG-treated cells. At 0.1 µM, EPICAT nonsignificantly increased mitochondrial respiration, agreeing with previous reports. EPICAT significantly increased complex I expression in AA-treated cells, and 1.0 µM EPICAT significantly decreased mitochondrial complex V expression in HG-treated cells. No significant effects were seen on either AMPK or eNOS expression. Our study suggests that EPICAT is useful in mitigating moderate ROS concentrations from a global perturbation and may modulate mitochondrial complex activity. Our data illustrate that EPICAT acts in the cell in a dose-dependent manner, demonstrating hormesis.

Glucagon-like peptide-1 receptor antagonism impairs basal exercise capacity and vascular adaptation to aerobic exercise training in rats.

Cardiorespiratory fitness (CRF) inversely predicts cardiovascular (CV) mortality and CRF is impaired in people with type 2 diabetes (T2D). Aerobic exercise training (ET) improves CRF and is associated with decreased risk of premature death in healthy and diseased populations. Understanding the mechanisms contributing to ET adaptation may identify targets for reducing CV mortality of relevance to people with T2D. The antihyperglycemic hormone glucagon-like peptide-1 (GLP-1) influences many of the same pathways as exercise and may contribute to CV adaptation to ET. We hypothesized that GLP-1 is necessary for adaptation to ET. Twelve-week-old male Wistar rats were randomized (n = 8-12/group) to receive PBS or GLP-1 receptor antagonist (exendin 9-39 (Ex(9-39)) via osmotic pump for 4 weeks ± ET. CRF was greater with ET (P < 0.01). Ex(9-39) treatment blunted CRF in both sedentary and ET rats (P < 0.001). Ex(9-39) attenuated acetylcholine-mediated vasodilation, while this response was maintained with Ex(9-39)+ET (P = 0.04). Aortic stiffness was greater with Ex(9-39) (P = 0.057) and was made worse when Ex(9-39) was combined with ET (P = 0.004). Ex vivo aortic vasoconstriction with potassium and phenylephrine was lower with Ex(9-39) (P < 0.0001). Carotid strain improved with PBS + ET but did not change in the Ex(9-39) rats with ET (P < 0.0001). Left ventricular mitochondrial respiration was elevated with Ex(9-39) (P < 0.02). GLP-1 receptor antagonism impairs CRF with and without ET, attenuates the vascular adaptation to ET, and elevates cardiac mitochondrial respiration. These data suggest that GLP-1 is integral to the adaptive vascular response to ET.

Raw and processed microscope images of fixed cells at baseline and following various experimental perturbations.

The data included in this article comprise raw and processed images of fixed cells at baseline and subjected to various experimental perturbations. This dataset includes images of HUVEC cells fixed and subsequently incubated at either 37 °C or room temperature, primary rat vascular smooth muscle cells exposed to 25 mM glucose, and SH-SY5Y neurons exposed to hydrogen peroxide. Raw images appear exactly as they were captured on the microscope, while processed images show the binarization provided by software used for measurements of mitochondrial morphology. For in-depth discussion of the experiments and computational methods pertaining to this data, please refer to the corresponding research article titled "Fully automated software for quantitative measurements of mitochondrial morphology" (McClatchey et al., in press) [1].

Fully automated software for quantitative measurements of mitochondrial morphology.

Mitochondria undergo dynamic changes in morphology in order to adapt to changes in nutrient and oxygen availability, communicate with the nucleus, and modulate intracellular calcium dynamics. Many recent papers have been published assessing mitochondrial morphology endpoints. Although these studies have yielded valuable insights, contemporary assessment of mitochondrial morphology is typically subjective and qualitative, precluding direct comparison of outcomes between different studies and likely missing many subtle effects. In this paper, we describe a novel software technique for measuring the average length, average width, spatial density, and intracellular localization of mitochondria from a fluorescent microscope image. This method was applied to distinguish baseline characteristics of Human Umbilical Vein Endothelial Cells (HUVECs), primary Goto-Kakizaki rat aortic smooth muscle cells (GK SMCs), primary Wistar rat aortic smooth muscle cells (Wistar SMCs), and SH-SY5Ys (human neuroblastoma cell line). Consistent with direct observation, our algorithms found SH-SY5Ys to have the greatest mitochondrial density, while HUVECs were found to have the longest mitochondria. Mitochondrial morphology responses to temperature, nutrient, and oxidative stressors were characterized to test algorithm performance. Large morphology changes recorded by the software agreed with direct observation, and subtle but consistent morphology changes were found that would not otherwise have been detected. Endpoints were consistent between experimental repetitions (R=0.93 for length, R=0.93 for width, R=0.89 for spatial density, and R=0.74 for localization), and maintained reasonable agreement even when compared to images taken with compromised microscope resolution or in an alternate imaging plane. These results indicate that the automated software described herein allows quantitative and objective characterization of mitochondrial morphology from fluorescent microscope images.

Differential Mitochondrial Adaptation in Primary Vascular Smooth Muscle Cells from a Diabetic Rat Model.

Diabetes affects more than 330 million people worldwide and causes elevated cardiovascular disease risk. Mitochondria are critical for vascular function, generate cellular reactive oxygen species (ROS), and are perturbed by diabetes, representing a novel target for therapeutics. We hypothesized that adaptive mitochondrial plasticity in response to nutrient stress would be impaired in diabetes cellular physiology via a nitric oxide synthase- (NOS-) mediated decrease in mitochondrial function. Primary smooth muscle cells (SMCs) from aorta of the nonobese, insulin resistant rat diabetes model Goto-Kakizaki (GK) and the Wistar control rat were exposed to high glucose (25 mM). At baseline, significantly greater nitric oxide evolution, ROS production, and respiratory control ratio (RCR) were observed in GK SMCs. Upon exposure to high glucose, expression of phosphorylated eNOS, uncoupled respiration, and expression of mitochondrial complexes I, II, III, and V were significantly decreased in GK SMCs (p < 0.05). Mitochondrial superoxide increased with high glucose in Wistar SMCs (p < 0.05) with no change in the GK beyond elevated baseline concentrations. Baseline comparisons show persistent metabolic perturbations in a diabetes phenotype. Overall, nutrient stress in GK SMCs caused a persistent decline in eNOS and mitochondrial function and disrupted mitochondrial plasticity, illustrating eNOS and mitochondria as potential therapeutic targets.

Impaired response to exercise intervention in the vasculature in metabolic syndrome.

Physical activity decreases risk for diabetes and cardiovascular disease morbidity and mortality; however, the specific impact of exercise on the diabetic vasculature is unexamined. We hypothesized that an acute, moderate exercise intervention in diabetic and hypertensive rats would induce mitochondrial biogenesis and mitochondrial antioxidant defence to improve vascular resilience. SHHF/Mcc-fa(cp) lean (hypertensive) and obese (hypertensive, insulin resistant), as well as Sprague Dawley (SD) control rats were run on a treadmill for 8 days. In aortic lysates from SD rats, we observed a significant increase in subunit proteins from oxidative phosphorylation (OxPhos) complexes I-III, with no changes in the lean or obese SHHF rats. Exercise also increased the expression of mitochondrial antioxidant defence uncoupling protein 3 (UCP3) (p < 0.05) in SHHF lean rats, whereas no changes were observed in the SD or SHHF obese rats with exercise. We evaluated upstream signalling pathways for mitochondrial biogenesis, and only peroxisome proliferators-activated receptor gamma coactivator 1α (PGC-1α) significantly decreased in SHHF lean rats (p < 0.05) with exercise. In these experiments, we demonstrate absent mitochondrial induction with exercise exposure in models of chronic vascular disease. These findings suggest that chronic vascular stress results in decreased sensitivity of vasculature to the adaptive mitochondrial responses normally induced by exercise.

Skeletal muscle-specific deletion of lipoprotein lipase enhances insulin signaling in skeletal muscle but causes insulin resistance in liver and other tissues.

OBJECTIVE: Skeletal muscle-specific LPL knockout mouse (SMLPL(-/-)) were created to study the systemic impact of reduced lipoprotein lipid delivery in skeletal muscle on insulin sensitivity, body weight, and composition. RESEARCH DESIGN AND METHODS: Tissue-specific insulin sensitivity was assessed using a hyperinsulinemic-euglycemic clamp and 2-deoxyglucose uptake. Gene expression and insulin-signaling molecules were compared in skeletal muscle and liver of SMLPL(-/-) and control mice. RESULTS: Nine-week-old SMLPL(-/-) mice showed no differences in body weight, fat mass, or whole-body insulin sensitivity, but older SMLPL(-/-) mice had greater weight gain and whole-body insulin resistance. High-fat diet feeding accelerated the development of obesity. In young SMLPL(-/-) mice, insulin-stimulated glucose uptake was increased 58% in the skeletal muscle, but was reduced in white adipose tissue (WAT) and heart. Insulin action was also diminished in liver: 40% suppression of hepatic glucose production in SMLPL(-/-) vs. 90% in control mice. Skeletal muscle triglyceride was 38% lower, and insulin-stimulated phosphorylated Akt (Ser473) was twofold greater in SMLPL(-/-) mice without changes in IRS-1 tyrosine phosphorylation and phosphatidylinositol 3-kinase activity. Hepatic triglyceride and liver X receptor, carbohydrate response element-binding protein, and PEPCK mRNAs were unaffected in SMLPL(-/-) mice, but peroxisome proliferator-activated receptor (PPAR)-gamma coactivator-1alpha and interleukin-1beta mRNAs were higher, and stearoyl-coenzyme A desaturase-1 and PPARgamma mRNAs were reduced. CONCLUSIONS: LPL deletion in skeletal muscle reduces lipid storage and increases insulin signaling in skeletal muscle without changes in body composition. Moreover, lack of LPL in skeletal muscle results in insulin resistance in other key metabolic tissues and ultimately leads to obesity and systemic insulin resistance.