Jamie Soto

GLUT1 reductions exacerbate Alzheimer's disease vasculo-neuronal dysfunction and degeneration

The glucose transporter GLUT1 at the blood-brain barrier (BBB) mediates glucose transport into the brain. Alzheimers disease is characterized by early reductions in glucose transport associated with diminished GLUT1 expression at the BBB. Whether GLUT1 reduction influences disease pathogenesis remains, however, elusive. Here we show that GLUT1 deficiency in mice overexpressing amyloid β-peptide (Aβ) precursor protein leads to early cerebral microvascular degeneration, blood flow reductions and dysregulation and BBB breakdown, and to accelerated amyloid β-peptide (Aβ) pathology, reduced Aβ clearance, diminished neuronal activity, behavioral deficits, and progressive neuronal loss and neurodegeneration that develop after initial cerebrovascular degenerative changes. We also show that GLUT1 deficiency in endothelium, but not in astrocytes, initiates the vascular phenotype as shown by BBB breakdown. Thus, reduced BBB GLUT1 expression worsens Alzheimers disease cerebrovascular degeneration, neuropathology and cognitive function, suggesting that GLUT1 may represent a therapeutic target for Alzheimers disease vasculo-neuronal dysfunction and degeneration.

Insulin-like growth factor I receptor signaling is required for exercise-induced cardiac hypertrophy.

The receptors for IGF-I (IGF-IR) and insulin (IR) have been implicated in physiological cardiac growth, but it is unknown whether IGF-IR or IR signaling are critically required. We generated mice with cardiomyocyte-specific knockout of IGF-IR (CIGF1RKO) and compared them with cardiomyocyte-specific insulin receptor knockout (CIRKO) mice in response to 5 wk exercise swim training. Cardiac development was normal in CIGF1RKO mice, but the hypertrophic response to exercise was prevented. In contrast, despite reduced baseline heart size, the hypertrophic response of CIRKO hearts to exercise was preserved. Exercise increased IGF-IR content in control and CIRKO hearts. Akt phosphorylation increased in exercise-trained control and CIRKO hearts and, surprisingly, in CIGF1RKO hearts as well. In exercise-trained control and CIRKO mice, expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and glycogen content were both increased but were unchanged in trained CIGF1RKO mice. Activation of AMP-activated protein kinase (AMPK) and its downstream target eukaryotic elongation factor-2 was increased in exercise-trained CIGF1RKO but not in CIRKO or control hearts. In cultured neonatal rat cardiomyocytes, activation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) prevented IGF-I/insulin-induced cardiomyocyte hypertrophy. These studies identify an essential role for IGF-IR in mediating physiological cardiomyocyte hypertrophy. IGF-IR deficiency promotes energetic stress in response to exercise, thereby activating AMPK, which leads to phosphorylation of eukaryotic elongation factor-2. These signaling events antagonize Akt signaling, which although necessary for mediating physiological cardiac hypertrophy, is insufficient to promote cardiac hypertrophy in the absence of myocardial IGF-I signaling.

Tissue-Specific Remodeling of the Mitochondrial Proteome in Type 1 Diabetic Akita Mice

OBJECTIVE To elucidate the molecular basis for mitochondrial dysfunction, which has been implicated in the pathogenesis of diabetes complications. RESEARCH DESIGN AND METHODS Mitochondrial matrix and membrane fractions were generated from liver, brain, heart, and kidney of wild-type and type 1 diabetic Akita mice. Comparative proteomics was performed using label-free proteome expression analysis. Mitochondrial state 3 respirations and ATP synthesis were measured, and mitochondrial morphology was evaluated by electron microscopy. Expression of genes that regulate mitochondrial biogenesis, substrate utilization, and oxidative phosphorylation (OXPHOS) were determined. RESULTS In diabetic mice, fatty acid oxidation (FAO) proteins were less abundant in liver mitochondria, whereas FAO protein content was induced in mitochondria from all other tissues. Kidney mitochondria showed coordinate induction of tricarboxylic acid (TCA) cycle enzymes, whereas TCA cycle proteins were repressed in cardiac mitochondria. Levels of OXPHOS subunits were coordinately increased in liver mitochondria, whereas mitochondria of other tissues were unaffected. Mitochondrial respiration, ATP synthesis, and morphology were unaffected in liver and kidney mitochondria. In contrast, state 3 respirations, ATP synthesis, and mitochondrial cristae density were decreased in cardiac mitochondria and were accompanied by coordinate repression of OXPHOS and peroxisome proliferator–activated receptor (PPAR)-γ coactivator (PGC)-1α transcripts. CONCLUSIONS Type 1 diabetes causes tissue-specific remodeling of the mitochondrial proteome. Preservation of mitochondrial function in kidney, brain, and liver, versus mitochondrial dysfunction in the heart, supports a central role for mitochondrial dysfunction in diabetic cardiomyopathy.

Nuclear receptor SHP, a death receptor that targets mitochondria, induces apoptosis and inhibits tumor growth

ABSTRACT Small heterodimer partner (SHP) is an epigenetically regulated nuclear transcriptional repressor that suppresses the development of liver cancer by inhibiting cellular growth. Here we report a novel cytoplasmic function of SHP through its regulation of mitochondrial activity. SHP is a pivotal cell death receptor that targets mitochondria, where it binds with Bcl-2, disrupts Bcl-2/Bid interaction, and induces cytochrome c release. The apoptosis inducer AHPN {retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid} acts by regulating SHP gene expression and promotes the translocation of SHP from the nucleus to the mitochondria. Induction of apoptosis by SHP activation inhibits peritoneal pancreatic tumor growth. Our findings provide for the first time a mechanism by which SHP regulates cell survival, namely, by controlling mitochondrial function via modulating the activity of Bcl-2 through AHPN-mediated or AHPN-independent action. Thus, SHP regulates a mechanism by which apoptotic signals can mediate local control of mitochondrial function and apoptosis, which in turn may limit tumorigenesis.

Mitochondrial Fusion and Function in Charcot-Marie-Tooth Type 2A Patient Fibroblasts with Mitofusin 2 Mutations

Charcot-Marie-Tooth Type 2A is a dominantly inherited peripheral neuropathy characterized by axonal degeneration of sensory and motor nerves. The disease is caused by mutations in the mitochondrial fusion gene MFN2. Mfn2 is an integral outer mitochondrial membrane protein composed of a large GTPase domain and two heptad repeat (HR) domains that face the cytoplasm. Mitochondrial membrane fusion and division are balanced processes that are necessary to maintain tubular mitochondrial morphology, respiratory function, and uniform distribution of the organelle throughout the cell. We have utilized primary fibroblasts from CMT2A patients to survey mitochondrial phenotypes associated with heterozygous MFN2 alleles expressed at physiological levels. Our results indicate that, in fibroblasts, mitofusin expression, mitochondrial morphology, ultrastructure, mtDNA content, and respiratory capacity are not affected by the presence of mutant Mfn2 protein. Consistent with a lack of mitochondrial dysfunction, we also show that mitochondrial fusion occurs efficiently in CMT2A patient-derived fibroblasts. Our observations are in agreement with the neuronal specificity of the disease and are consistent with a recent finding that mitochondrial fusion can be maintained in cells that express mutant Mfn2 protein due to complementation by a second mitofusin, Mfn1. We discuss our results and those of others in terms of a comprehensive model for the mechanism(s) by which mutations in MFN2 may lead to CMT2A disease.

Iron Overload and Diabetes Risk: A Shift From Glucose to Fatty Acid Oxidation and Increased Hepatic Glucose Production in a Mouse Model of Hereditary Hemochromatosis

OBJECTIVE Excess tissue iron levels are a risk factor for diabetes, but the mechanisms underlying the association are incompletely understood. We previously published that mice and humans with a form of hereditary iron overload, hemochromatosis, exhibit loss of β-cell mass. This effect by itself is not sufficient, however, to fully explain the diabetes risk phenotype associated with all forms of iron overload. RESEARCH DESIGN AND METHODS We therefore examined glucose and fatty acid metabolism and hepatic glucose production in vivo and in vitro in a mouse model of hemochromatosis in which the gene most often mutated in the human disease, HFE, has been deleted (Hfe−/−). RESULTS Although Hfe−/− mice exhibit increased glucose uptake in skeletal muscle, glucose oxidation is decreased and the ratio of fatty acid to glucose oxidation is increased. On a high-fat diet, the Hfe−/− mice exhibit increased fatty acid oxidation and are hypermetabolic. The decreased glucose oxidation in skeletal muscle is due to decreased pyruvate dehydrogenase (PDH) enzyme activity related, in turn, to increased expression of PDH kinase 4 (pdk4). Increased substrate recycling to liver contributes to elevated hepatic glucose production in the Hfe−/− mice. CONCLUSIONS Increased hepatic glucose production and metabolic inflexibility, both of which are characteristics of type 2 diabetes, may contribute to the risk of diabetes with excessive tissue iron.

Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart

Significance Mitochondrial Ca2+ is a fundamental signal that allows for adaptation to physiological stress but a liability during ischemia-reperfusion injury in heart. On one hand, mitochondrial Ca2+ entry coordinates energy supply and demand in myocardium by increasing the activity of matrix dehydrogenases to augment ATP production by oxidative phosphorylation. On the other hand, inhibiting mitochondrial Ca2+ overload is promulgated as a therapeutic approach to preserve myocardial tissue following ischemia-reperfusion injury. We developed a new mouse model of myocardial-targeted transgenic dominant-negative mitochondrial Ca2+ uniporter (MCU) expression to test consequences of chronic loss of MCU-mediated Ca2+ entry in heart. Here we show that MCU inhibition has unanticipated consequences on extramitochondrial pathways affecting oxygen utilization, cytoplasmic Ca2+ homeostasis, physiologic responses to stress, and pathologic responses to ischemia-reperfusion injury. Myocardial mitochondrial Ca2+ entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca2+ are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca2+ uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca2+ entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU–permeabilized myocardial fibers or isolated mitochondria in low Ca2+ were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca2+ homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca2+] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca2+ homeostasis. Mitochondrial Ca2+ overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca2+ homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.

Central Leptin Signaling Is Required to Normalize Myocardial Fatty Acid Oxidation Rates in Caloric-Restricted ob/ob Mice

OBJECTIVE ob/ob and db/db mice manifest myocardial hypertrophy, insulin resistance, altered substrate utilization, mitochondrial dysfunction, and lipid accumulation. This study was designed to determine the contribution of central and peripheral leptin signaling to myocardial metabolism and function in ob/ob and db/db mice in the absence of diabetes and morbid obesity. RESEARCH DESIGN AND METHODS Male ob/ob mice (aged 4 weeks) were caloric restricted by pairfeeding to a leptin-treated ob/ob group. In addition to determining glucose tolerance and circulating lipid concentrations, myocardial substrate metabolism and mitochondrial function were determined in saponin-permeabilized cardiac fibers. Second, experiments were performed to determine whether leptin treatment by intraperitoneal injection or intracerebroventricular infusion could normalize myocardial palmitate oxidation in caloric-restricted ob/ob mouse hearts. RESULTS Despite normalizing body weight and glucose tolerance, fat mass and circulating lipid levels remained increased in caloric-restricted ob/ob animals. Palmitate oxidation remained elevated in caloric-restricted ob/ob hearts and was normalized by intraperitoneal or intracerebroventricular leptin. Intraperitoneal and intracerebroventricular treatment also normalized circulating free fatty acid levels, myocardial fatty acid oxidation gene expression, and myocardial insulin sensitivity. CONCLUSIONS These data suggest that impaired hypothalamic leptin signaling is sufficient to increase myocardial fatty acid oxidation by increasing delivery of free fatty acid substrates and peroxisome proliferator–activated receptor-α ligands to the heart.

Insulin Signaling Regulates Mitochondrial Function in Pancreatic β-Cells

Insulin/IGF-I signaling regulates the metabolism of most mammalian tissues including pancreatic islets. To dissect the mechanisms linking insulin signaling with mitochondrial function, we first identified a mitochondria-tethering complex in β-cells that included glucokinase (GK), and the pro-apoptotic protein, BADS. Mitochondria isolated from β-cells derived from β-cell specific insulin receptor knockout (βIRKO) mice exhibited reduced BADS, GK and protein kinase A in the complex, and attenuated function. Similar alterations were evident in islets from patients with type 2 diabetes. Decreased mitochondrial GK activity in βIRKOs could be explained, in part, by reduced expression and altered phosphorylation of BADS. The elevated phosphorylation of p70S6K and JNK1 was likely due to compensatory increase in IGF-1 receptor expression. Re-expression of insulin receptors in βIRKO cells partially restored the stoichiometry of the complex and mitochondrial function. These data indicate that insulin signaling regulates mitochondrial function and have implications for β-cell dysfunction in type 2 diabetes.

Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity.

Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs.