Alessandra Valerio

Branched-Chain Amino Acid Supplementation Promotes Survival and Supports Cardiac and Skeletal Muscle Mitochondrial Biogenesis in Middle-Aged Mice

Recent evidence points to a strong relationship between increased mitochondrial biogenesis and increased survival in eukaryotes. Branched-chain amino acids (BCAAs) have been shown to extend chronological life span in yeast. However, the role of these amino acids in mitochondrial biogenesis and longevity in mammals is unknown. Here, we show that a BCAA-enriched mixture (BCAAem) increased the average life span of mice. BCAAem supplementation increased mitochondrial biogenesis and sirtuin 1 expression in primary cardiac and skeletal myocytes and in cardiac and skeletal muscle, but not in adipose tissue and liver of middle-aged mice, and this was accompanied by enhanced physical endurance. Moreover, the reactive oxygen species (ROS) defense system genes were upregulated, and ROS production was reduced by BCAAem supplementation. All of the BCAAem-mediated effects were strongly attenuated in endothelial nitric oxide synthase null mutant mice. These data reveal an important antiaging role of BCAAs mediated by mitochondrial biogenesis in mammals.

TNF-α downregulates eNOS expression and mitochondrial biogenesis in fat and muscle of obese rodents

Obesity is associated with chronic low-grade inflammation. Thus, at metabolically relevant sites, including adipose tissue and muscle, there is abnormal production of proinflammatory cytokines such as TNF-alpha. Here we demonstrate that eNOS expression was reduced, with a concomitant reduction of mitochondrial biogenesis and function, in white and brown adipose tissue and in the soleus muscle of 3 different animal models of obesity. The genetic deletion of TNF receptor 1 in obese mice restored eNOS expression and mitochondrial biogenesis in fat and muscle; this was associated with less body weight gain than in obese wild-type controls. Furthermore, TNF-alpha downregulated eNOS expression and mitochondrial biogenesis in cultured white and brown adipocytes and muscle satellite cells of mice. The NO donors DETA-NO and SNAP prevented the reduction of mitochondrial biogenesis observed with TNF-alpha. Our findings demonstrate that TNF-alpha impairs mitochondrial biogenesis and function in different tissues of obese rodents by downregulating eNOS expression and suggest a novel pathophysiological process that sustains obesity.

CB(1) signaling in forebrain and sympathetic neurons is a key determinant of endocannabinoid actions on energy balance.

The endocannabinoid system (ECS) plays a critical role in obesity development. The pharmacological blockade of cannabinoid receptor type 1 (CB(1)) has been shown to reduce body weight and to alleviate obesity-related metabolic disorders. An unsolved question is at which anatomical level CB(1) modulates energy balance and the mechanisms involved in its action. Here, we demonstrate that CB(1) receptors expressed in forebrain and sympathetic neurons play a key role in the pathophysiological development of diet-induced obesity. Conditional mutant mice lacking CB(1) expression in neurons known to control energy balance, but not in nonneuronal peripheral organs, displayed a lean phenotype and resistance to diet-induced obesity. This phenotype results from an increase in lipid oxidation and thermogenesis as a consequence of an enhanced sympathetic tone and a decrease in energy absorption. In conclusion, CB(1) signaling in the forebrain and sympathetic neurons is a key determinant of the ECS control of energy balance.

Cannabinoid Type 1 Receptor Blockade Promotes Mitochondrial Biogenesis Through Endothelial Nitric Oxide Synthase Expression in White Adipocytes

OBJECTIVE—Cannabinoid type 1 (CB1) receptor blockade decreases body weight and adiposity in obese subjects; however, the underlying mechanism is not yet fully understood. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) induces mitochondrial biogenesis and function in adipocytes. This study was undertaken to test whether CB1 receptor blockade increases the espression of eNOS and mitochondrial biogenesis in white adipocytes. RESEARCH DESIGN AND METHODS—We examined the effects on eNOS and mitochondrial biogenesis of selective pharmacological blockade of CB1 receptors by SR141716 (rimonabant) in mouse primary white adipocytes. We also examined eNOS expression and mitochondrial biogenesis in white adipose tissue (WAT) and isolated mature white adipocytes of CB1 receptor–deficient (CB1−/−) and chronically SR141716-treated mice on either a standard or high-fat diet. RESULTS—SR141716 treatment increased eNOS expression in cultured white adipocytes. Moreover, SR141716 increased mitochondrial DNA amount, mRNA levels of genes involved in mitochondrial biogenesis, and mitochondrial mass and function through eNOS induction, as demonstrated by reversal of SR141716 effects by small interfering RNA–mediated decrease in eNOS. While high-fat diet–fed wild-type mice showed reduced eNOS expression and mitochondrial biogenesis in WAT and isolated mature white adipocytes, genetic CB1 receptor deletion or chronic treatment with SR141716 restored these parameters to the levels observed in wild-type mice on the standard diet, an effect linked to the prevention of adiposity and body weight increase. CONCLUSIONS—CB1 receptor blockade increases mitochondrial biogenesis in white adipocytes by inducing the expression of eNOS. This is linked to the prevention of high-fat diet–induced fat accumulation, without concomitant changes in food intake.

IDENTIFICATION AND CHARACTERIZATION OF A KAPPA B/REL BINDING SITE IN THE REGULATORY REGION OF THE AMYLOID PRECURSOR PROTEIN GENE

Several observations support the hypothesis that pathogenetic mechanisms of β amyloid formation in Alzheimers disease may involve alterations in amyloid precursor protein (APP) gene expression. In this regard, molecular dissection of the APP gene transcriptional regulation is of primary importance. We report evidence that members of the family of transcription factors NFκB/Rel can specifically recognize two identical sequences located in the 5′-regulatory region of APP. These sequences, which we refer to as APPκB sites, interact preferentially with p50-containing members of the family. In particular, p50 homodimers and p50/p65 and p50/c-Rel heterodimers act as transcriptional activators at the APPκB site. Finally, the nuclear complex specifically binding to the APPκB sites proves to be an integral part of neurons and lymphocytes.

Metabotropic glutamate receptor mrna expression in rat spinal cord

IT has recently become clear that, analogous to the situation observed in the brain, glutamatergic neurotransmission in the spinal cord relies upon the activation of ionotropic and metabotropic glutamate receptors (mGluRs). Although electrophysiological studies have emphasized the relevance of mGluR activation in the modulation of spinal neuron responses to glutamate, a detailed study of the molecular forms of receptors belonging to the mGluR family in the spinal cord is still lacking. Using a reverse transcriptase-polymerase chain reaction technique with primers specifically recognizing each cloned mGluR subtype, we found that rat adult spinal cord specifically expresses high levels of mRNA encoding mGluR1a but not mGluR1b. The expression of mRNAs for mGluRs 5a and 5b was also found at levels roughly comparable to that observed in mGluR5-positive brain areas. mGluR2, mGluR3, mGluR4 and mGluR7 mRNAs appeared to be expressed at lower levels, while mGluR6 and mGluR8 mRNAs were not detectable.

Cannabinoid Receptor Stimulation Impairs Mitochondrial Biogenesis in Mouse White Adipose Tissue, Muscle, and Liver: The Role of eNOS, p38 MAPK, and AMPK Pathways

OBJECTIVE Cannabinoid type 1 (CB1) receptor is involved in whole-body and cellular energy metabolism. We asked whether CB1 receptor stimulation was able to decrease mitochondrial biogenesis in different metabolically active tissues of obese high-fat diet (HFD)-fed mice. RESEARCH DESIGN AND METHODS The effects of selective CB1 agonist arachidonyl-2-chloroethanolamide (ACEA) and endocannabinoids anandamide and 2-arachidonoylglycerol on endothelial nitric oxide synthase (eNOS) expression were examined, as were mitochondrial DNA amount and mitochondrial biogenesis parameters in cultured mouse and human white adipocytes. These parameters were also investigated in white adipose tissue (WAT), muscle, and liver of mice chronically treated with ACEA. Moreover, p38 mitogen-activated protein kinase (MAPK) phosphorylation was investigated in WAT and isolated mature adipocytes from eNOS−/− and wild-type mice. eNOS, p38 MAPK, adenosine monophosphate–activated protein kinase (AMPK), and mitochondrial biogenesis were investigated in WAT, muscle, and liver of HFD mice chronically treated with ACEA. RESULTS ACEA decreased mitochondrial biogenesis and eNOS expression, activated p38 MAPK, and reduced AMPK phosphorylation in white adipocytes. The ACEA effects on mitochondria were antagonized by nitric oxide donors and by p38 MAPK silencing. White adipocytes from eNOS−/− mice displayed higher p38 MAPK phosphorylation than wild-type animals under basal conditions, and ACEA was ineffective in cells lacking eNOS. Moreover, mitochondrial biogenesis was downregulated, while p38 MAPK phosphorylation was increased and AMPK phosphorylation was decreased in WAT, muscle, and liver of ACEA-treated mice on a HFD. CONCLUSIONS CB1 receptor stimulation decreases mitochondrial biogenesis in white adipocytes, through eNOS downregulation and p38 MAPK activation, and impairs mitochondrial function in metabolically active tissues of dietary obese mice.

MGLUR5 METABOTROPIC GLUTAMATE RECEPTOR DISTRIBUTION IN RAT AND HUMAN SPINAL CORD : A DEVELOPMENTAL STUDY

By combining biochemical, molecular and immunohistochemical approaches, we have investigated the presence of metabotropic glutamate receptors (mGluRs) belonging to the subtype 5 in rat and human spinal cords and the developmental changes in their expression. A polyclonal antibody raised against the carboxy-terminal portion of mGluR5 was used to study the distribution of the receptor in rat foetal (Et15), neonatal (P8) and adult spinal cords and dorsal root ganglia (DRG). mGluR5 appeared to be predominantly expressed in regions containing the primary sensory afferents. Immunoblotting with anti-mGluR5 antibody revealed lower receptor protein levels in rat adult spinal cord when compared with P8 rat spinal cord. Reverse transcriptase-polymerase chain reaction showed both mGluR5a and mGluR5b mRNAs expression in rat spinal cord. The mGluR5a variant was found more abundant in young animals than in adults. The pattern of mGluR5 immunostaining was also studied in foetal (6-8, 10, 12 and 22 weeks of gestation) and adult human spinal cord. At all stages of human development, a strong mGluR5 immunoreactivity was observed in the dorsal roots and in the dorsal and dorsolateral funiculi with maximum levels of staining at week 12 of gestation. Foetal DRG neurons were heterogeneously labeled. mGluR5 was also diffusely detectable in the mantle layer. In adult human spinal cords, immunoreactivity was confined to laminae I and II of the dorsal horns. These results demonstrate for the first time the presence of mGluR5 in human spinal cord. The distribution of this receptor suggests a role in the development of somatosensory pathways and in the control of nociceptive neurotransmission.

Leptin increases axonal growth cone size in developing mouse cortical neurons by convergent signals inactivating glycogen synthase kinase-3β

We examined the effects of the adipose hormone leptin on the development of mouse cortical neurons. Treatment of neonatal and adult mice with intraperitoneal leptin (5 mg/kg) induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in pyriform and entorhinal cortex neurons. Stimulation of cultured embryonic cortical neurons with leptin evoked Janus kinase 2 and ERK1/2 phosphorylation and activated the downstream effector 90-kDa ribosomal protein S6 kinase. Moreover, leptin elicited the phosphorylation of the phosphatidylinositol 3-kinase effector Akt and evoked Ser-9 phosphorylation of glycogen synthase kinase-3β (GSK3β), an event inactivating this kinase. Leptin-mediated GSK3β phosphorylation was prevented by the MEK/ERK inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, or the protein kinase C inhibitor GF109203X. Exposure of cortical neurons to leptin also induced Ser-41 phosphorylation of the neuronal growth-associated protein GAP-43, an effect prevented by LY294002 and GF109203X but not by PD98059. Ser-41-GAP-43 phosphorylation is usually high in expanding axonal growth cones. Neurons exposed to 100 ng/ml leptin for 72 h displayed reduced rate of growth cone collapse, a shift of growth cone size distribution toward higher values, and a 4-fold increase in mean growth cone surface area compared with control cultures. The leptin-induced growth cone spreading was hampered in cortical neurons from Leprdb/db mice lacking functional leptin receptors; it was associated with localized Ser-9-GSK3β phosphorylation and mimicked by the GSK3β inhibitor SB216763. At concentrations preventing GSK3β phosphorylation, PD98059, LY294002, or GF109203X reversed the leptin-induced growth cone surface enlargement. We concluded that the leptin-mediated regulation of growth cone morphogenesis in cortical neurons relies on upstream regulators of GSK3β activity.

Prevention of neuron and oligodendrocyte degeneration by interleukin-6 (IL-6) and IL-6 receptor/IL-6 fusion protein in organotypic hippocampal slices

We investigated the effects of IL-6 and a chimeric derivative of IL-6 and soluble IL-6 receptor (IL6RIL6 chimera) on excitotoxic injury in rat organotypic hippocampal slices. Brief application of N-methyl-d-aspartate (NMDA) induced astrocyte reactivity, neuron cell death, and oligodendrocyte degeneration, the latter caused by secondary activation of AMPA/kainate receptors. Both these cytokines rescued neurons and oligodendrocytes, albeit the chimeric compound was much more potent and efficient than IL-6. No change was produced on reactive astrocytosis. The cytokines preserved myelin basic protein (MBP) production in slices exposed to excitotoxic insult, and when applied singularly for a week, they also enhanced both MBP and proteolipid protein expression. These effects occurred through activating the signal transducer gp130 and were associated with stimulation of transcription factors STAT1 and STAT3. Our results suggest that IL-6 and IL6RIL6 may prove to be valuable in treating neurodegenerative and demyelinating diseases.