Impairment of substrate-mediated mitochondrial respiration in cardiac cells by chloroquine.

Chloroquine (CQ) has a long clinical history as an anti-malarial agent and also being used for the treatment of other infections and autoimmune diseases. Recently, this lysosomotropic agent and its derivatives are also been tested as adjuncts alongside conventional anti-cancer treatments in combinatorial therapies. However, their reported cardiotoxicity tends to raise concern over their indiscriminate use. Even though the influence of CQ and its derivatives on cardiac mitochondria is extensively studied in disease models, their impact on cardiac mitochondrial respiration under physiological conditions remains inconclusive. In this study, we aimed to evaluate the impact of CQ on cardiac mitochondrial respiration using both in-vitro and in-vivo model systems. Using high-resolution respirometry in isolated cardiac mitochondria from male C57BL/6 mice treated with intraperitoneal injection of 10 mg/kg/day of CQ for 14 days, CQ was found to impair substrate-mediated mitochondrial respiration in cardiac tissue. In an in-vitro model of H9C2 cardiomyoblasts, incubation with 50 µM of CQ for 24 h disrupted mitochondrial membrane potential, produced mitochondrial fragmentation, decreased mitochondrial respiration and induced superoxide generation. Altogether, our study results indicate that CQ has a deleterious impact on cardiac mitochondrial bioenergetics which in turn suggests that CQ treatment could be an added burden, especially in patients affected with diseases with underlying cardiac complications. As CQ is an inhibitor of the lysosomal pathway, the observed effect could be an outcome of the accumulation of dysfunctional mitochondria due to autophagy inhibition.

The Effect of Glucagon on Protein Catabolism During Insulin Deficiency: Exchange of Amino Acids Across Skeletal Muscle and the Splanchnic Bed.

Transient insulin deprivation with concurrent hyperglucagonemia is a catabolic state that can occur in type 1 diabetes. To evaluate glucagon's catabolic effect in the setting of its glucogenic effect, we measured the regional exchanges of amino acid metabolites (amino-metabolites) across muscle and splanchnic beds in 16 healthy humans during either somatostatin followed by glucagon or saline infusion alone. Despite a twofold or greater increase in the regional exchange of amino-metabolites by glucagon, whole-body kinetics and concentrations of amino acids (AA) remained stable. Glucagon increased the splanchnic uptake of not only gluconeogenic but also essential (EAA) AA while increasing their release from the muscle bed. Regional tracer-based kinetics and 3-methylhistidine release indicate that EAA release from muscle is likely caused by reduced protein synthesis rather than increased protein degradation. Furthermore, many metabolites known to affect insulin action and metabolism were altered by hyperglucagonemia including increase in branched-chain AA and keto acids of leucine and isoleucine in arterial plasma. Further, an increase in arterial concentrations of α-aminoadipic acid arising from increased conversion from lysine in the splanchnic bed was noted. These results demonstrate that hyperglucagonemia during hypoinsulinemia increases net muscle protein catabolism and substantially increases the exchange of amino metabolites across splanchnic and muscle beds.

Diminished substrate-mediated cardiac mitochondrial respiration and elevated autophagy in adult male offspring of gestational diabetic rats.

OBJECTIVE: Heart diseases are common in offspring of gestational diabetic mother (ODM). Defects in mitochondrial metabolism and autophagy may be one of the basic reasons behind the abnormal structural and functional behaviour of offspring's heart. So the main objective of the present study was to explore the cardiac mitochondrial respiration and autophagy in male and female offspring of diabetic pregnancy at two different developmental stages of life. METHODS: High-resolution respirometry was used to measure substrate-mediated mitochondrial respiration in isolated mitochondria from ventricular tissues of offspring of streptozotocin-induced diabetic mother rats. To find the expression of proteins involved in autophagic process and oxidative stress, western blotting and densitometric analysis were done. RESULTS: Mitochondrial complex I and complex II respiration was found to be decreased in adult male offspring while it was unaltered or less affected in weaning male and female offspring. Elevated autophagy was shown by adult male, while there was no change observed in adult female. Also absence of elevated expression of oxidative stress markers was observed in all groups. CONCUSION: The present study reports altered cardiac mitochondrial respiration and autophagy in male offspring of diabetic mothers than the control ones. The study also analysed the expression of various candidates of cardiac autophagic process in male and female offspring of diabetic pregnancy at two time points of development.

Diminished substrate-mediated cardiac mitochondrial respiration and elevated autophagy in adult male offspring of gestational diabetic rats.

Heart diseases are common in the offspring of diabetic mother (ODM). Defects in mitochondrial metabolism and autophagy may, in part, be responsible for the adverse structural and functional alterations in the heart. The principal objective of this study was to investigate cardiac mitochondrial respiration and autophagy in male and female offspring of diabetic pregnancy at two different developmental stages of life, weaning and adult. Male and female offspring of rats with streptozotocin-induced gestational diabetes were used for the study and compared with offspring of control (non-diabetic) mother (OCM) rats. High-resolution respirometry was used to measure substrate-mediated respiration in mitochondria isolated from ventricular tissues of ODM and OCM. Expression of proteins associated with autophagy and oxidative stress was examined by western blot analysis. Mitochondrial complex I and complex II respiration was significantly reduced in adult male ODM while it was unaltered or less affected in weaning male, adult and weaning female ODM. Elevated autophagy was observed in adult male but not in adult female ODM. Expression of oxidative stress markers was observed to be similar in all the groups. Altered cardiac mitochondrial respiration and autophagy were observed in adult male ODM compared to OCM, while the male and female offspring at weaning stage were less affected. The results of the study show that maternal hyperglycemia affects mitochondrial respiration and autophagy in the ODM heart, which may potentially be responsible for the cardiovascular complications observed in adult life.

Impaired substrate-mediated cardiac mitochondrial complex I respiration with unaltered regulation of fatty acid metabolism and oxidative stress status in type 2 diabetic Asian Indians.

BACKGROUND: The cardiovascular complications associated with type 2 diabetes mellitus could be attributed to changes in myocardial mitochondrial metabolism. Though it is a known fact that permeabilized cardiac muscle fibers and isolated mitochondria are metabolically compromised in the Caucasian population, studies of Asian Indian myocardial mitochondrial function are lacking. Thus, the objective of the present study is to analyze if there is altered cardiac mitochondrial substrate utilization in diabetic Asian Indians. METHODS: Mitochondrial substrate utilization was measured using high-resolution respirometry in isolated mitochondria prepared from right atrial appendage tissues of diabetic and nondiabetic subjects undergoing coronary artery bypass graft surgery. Western blotting and densitometric analysis were also done to compare the levels of proteins involved in fatty acid metabolism and regulation. RESULTS: The mitochondrial oxygen consumption rate for fatty acid substrate was shown to be decreased in diabetic subjects compared to nondiabetic subjects along with an unvaried mitochondrial DNA copy number and uniform levels of electron transport chain complex proteins and proteins involved in fatty acid metabolism and regulation. Decreased glutamate but unchanged pyruvate-mediated state 3 respiration were also observed in diabetic subjects. CONCLUSION: The current study reports deranged cardiac mitochondrial fatty acid-mediated complex I respiration in type 2 diabetic Asian Indians with comparable levels of regulators of fatty acid oxidation to that of nondiabetic myocardium. Altered glutamate-mediated mitochondrial respiration also points toward possible alterations in mitochondrial complex I activity. When compared with previous reports on other ethnic populations, the current study suggests that Asian Indian population too have altered cardiac mitochondrial substrate utilization.

TFAM Enhances Fat Oxidation and Attenuates High-Fat Diet-Induced Insulin Resistance in Skeletal Muscle.

Diet-induced insulin resistance (IR) adversely affects human health and life span. We show that muscle-specific overexpression of human mitochondrial transcription factor A (TFAM) attenuates high-fat diet (HFD)-induced fat gain and IR in mice in conjunction with increased energy expenditure and reduced oxidative stress. These TFAM effects on muscle are shown to be exerted by molecular changes that are beyond its direct effect on mitochondrial DNA replication and transcription. TFAM augmented the muscle tricarboxylic acid cycle and citrate synthase facilitating energy expenditure. TFAM enhanced muscle glucose uptake despite increased fatty acid (FA) oxidation in concert with higher ß-oxidation capacity to reduce the accumulation of IR-related carnitines and ceramides. TFAM also increased pAMPK expression, explaining enhanced PGC1α and PPARß, and reversing HFD-induced GLUT4 and pAKT reductions. TFAM-induced mild uncoupling is shown to protect mitochondrial membrane potential against FA-induced uncontrolled depolarization. These coordinated changes conferred protection to TFAM mice against HFD-induced obesity and IR while reducing oxidative stress with potential translational opportunities.

Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes.

Despite the strong association between diabetes and dementia, it remains to be fully elucidated how insulin deficiency adversely affects brain functions. We show that insulin deficiency in streptozotocin-induced diabetic mice decreased mitochondrial ATP production and/or citrate synthase and cytochrome oxidase activities in the cerebrum, hypothalamus, and hippocampus. Concomitant decrease in mitochondrial fusion proteins and increased fission proteins in these brain regions likely contributed to altered mitochondrial function. Although insulin deficiency did not cause any detectable increase in reactive oxygen species (ROS) emission, inhibition of monocarboxylate transporters increased ROS emission and further reduced ATP production, indicating the causative roles of elevated ketones and lactate in counteracting oxidative stress and as a fuel source for ATP production during insulin deficiency. Moreover, in healthy mice, intranasal insulin administration increased mitochondrial ATP production, demonstrating a direct regulatory role of insulin on brain mitochondrial function. Proteomics analysis of the cerebrum showed that although insulin deficiency led to oxidative post-translational modification of several proteins that cause tau phosphorylation and neurofibrillary degeneration, insulin administration enhanced neuronal development and neurotransmission pathways. Together these results render support for the critical role of insulin to maintain brain mitochondrial homeostasis and provide mechanistic insight into the potential therapeutic benefits of intranasal insulin.-Ruegsegger, G. N., Manjunatha, S., Summer, P., Gopala, S., Zabeilski, P., Dasari, S., Vanderboom, P. M., Lanza, I. R., Klaus, K. A., Nair, K. S. Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes.

Functional and proteomic alterations of plasma high density lipoproteins in type 1 diabetes mellitus.

OBJECTIVE: Higher HDL-cholesterol (HDL-C) is linked to lower cardiovascular risk but individuals with type 1 diabetes mellitus (T1DM) with normal or high HDL-C have higher cardiovascular events compared to age matched non-diabetic controls (ND). We determined whether altered HDL functions despite having normal HDL-C concentration may explain increased cardiovascular risk in T1DM individuals. We also determined whether irreversible posttranslational modifications (PTMs) of HDL bound proteins occur in T1DM individuals with altered HDL functions. METHODS: T1DM with poor glycemic control (T1D-PC, HbA1c≥8.5%, n=15) and T1DM with good glycemic control (T1D-GC, HbA1c≤6.6%, n=15) were compared with equal numbers of NDs, ND-PC and ND-GC respectively, matched for age, sex and body mass index (BMI). We measured cholesterol efflux capacity (CEC) of HDL in the serum using J774 macrophages, antioxidant function of HDL as the ability to reverse the oxidative damage of LDL and PON1 activity using commercially available kit. For proteomic analysis, HDL was isolated by density gradient ultracentrifugation and was analyzed by mass spectrometry and shotgun proteomics method. RESULTS: Plasma HDL-C concentrations in both T1DM groups were similar to their ND. However, CEC (%) of T1D-PC (16.9±0.8) and T1D-GC (17.1±1) were lower than their respective ND (17.9±1, p=0.01 and 18.2±1.4, p=0.02). HDL antioxidative function also was lower (p<0.05). The abundance of oxidative PTMs of apolipoproteins involved in CEC and antioxidative functions of HDL were higher in T1D-PC (ApoA4, p=0.041) and T1D-GC (ApoA4, p=0.025 and ApoE, p=0.041) in comparison with ND. Both T1D-PC and T1D-GC groups had higher abundance of amadori modification of ApoD (p=0.002 and p=0.041 respectively) and deamidation modification of ApoA4 was higher in T1D-PC (p=0.025). CONCLUSIONS: Compromised functions of HDL particles in T1DM individuals, irrespective of glycemic control, could be explained by higher abundance of irreversible PTMs of HDL proteins. These results lend mechanistic support to the hypothesis that HDL quality rather than quantity determines HDL function in T1DM and suggest that measurements of concentrations of HbA1c and HDL-C are not sufficient as biomarkers of effective treatment to lower cardiovascular risk in T1DM individuals.

Indian medical students' perspectives on problem-based learning experiences in the undergraduate curriculum: One size does not fit all.

PURPOSE: Problem-based learning (PBL) is a well-established method for facilitating the learning of basic science concepts in the context of clinical cases. Relevant evidence is lacking regarding PBL's effectiveness and acceptability as perceived by students accustomed to classical traditional teaching in India. Hence, this study gathered students' opinions on PBL versus Traditional teaching methods to generate a foundation for institutional policymaking and ultimately, changes in the policy of regulatory bodies. METHODS: A total of 773 first year medical students admitted from 2007-2010 in Kasturba Medical College Manipal, Manipal University, India were asked to respond to a 15-item questionnaire evaluating their preferences for PBL or traditional methods such as lectures after undergoing a systematically conducted PBL session in physiology. Their responses were analyzed with an unpaired t-test. Their comments were also collected. RESULTS: PBL scored significantly higher for most items in the questionnaire for "learning efficiency" and "student-teacher relationship". The students' comments highlighted the importance of a trained tutor/facilitator to enhance the learning process. CONCLUSION: Our students are willing to adapt to the PBL method, although they recognize certain benefits of traditional pedagogy. For learning efficiency and the student-teacher relationship, the students feel that neither method holds an advantage. We recommend that the future medical curriculum in India be a hybrid form of PBL and traditional methods with specific training on the unfamiliar PBL approach for both faculty and students.