Letizia Palomba

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.

Obesity-driven synaptic remodeling affects endocannabinoid control of orexinergic neurons.

Significance Endocannabinoids act retrogradely at presynaptic sites to activate cannabinoid receptor type 1 (CB1) receptors, thereby inhibiting neurotransmitter release and fine-tuning synaptic transmission. In murine models of obesity with leptin deficiency, we report that orexin-A neurons undergo a shift from predominant control by CB1-expressing excitatory to CB1-expressing inhibitory inputs. In addition, endocannabinoid biosynthesis is increased in these neurons. CB1 activation by endocannabinoids reduces the inhibition of orexinergic neurons in obese mice, thereby enhancing orexin-A release in target brain areas and contributing to hyperphagia and increased body weight, as well as to alterations of hormone levels typical of obesity. Acute or chronic alterations in energy status alter the balance between excitatory and inhibitory synaptic transmission and associated synaptic plasticity to allow for the adaptation of energy metabolism to new homeostatic requirements. The impact of such changes on endocannabinoid and cannabinoid receptor type 1 (CB1)-mediated modulation of synaptic transmission and strength is not known, despite the fact that this signaling system is an important target for the development of new drugs against obesity. We investigated whether CB1-expressing excitatory vs. inhibitory inputs to orexin-A–containing neurons in the lateral hypothalamus are altered in obesity and how this modifies endocannabinoid control of these neurons. In lean mice, these inputs are mostly excitatory. By confocal and ultrastructural microscopic analyses, we observed that in leptin-knockout (ob/ob) obese mice, and in mice with diet-induced obesity, orexinergic neurons receive predominantly inhibitory CB1-expressing inputs and overexpress the biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol, which retrogradely inhibits synaptic transmission at CB1-expressing axon terminals. Patch-clamp recordings also showed increased CB1-sensitive inhibitory innervation of orexinergic neurons in ob/ob mice. These alterations are reversed by leptin administration, partly through activation of the mammalian target of rapamycin pathway in neuropeptide-Y-ergic neurons of the arcuate nucleus, and are accompanied by CB1-mediated enhancement of orexinergic innervation of target brain areas. We propose that enhanced inhibitory control of orexin-A neurons, and their CB1-mediated disinhibition, are a consequence of leptin signaling impairment in the arcuate nucleus. We also provide initial evidence of the participation of this phenomenon in hyperphagia and hormonal dysregulation in obesity.

Apoptosis and necrosis following exposure of U937 cells to increasing concentrations of hydrogen peroxide: the effect of the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide.

A 3-hr exposure of U937 cells to hydrogen peroxide (H2O2) followed by a 6-hr posttreatment incubation in fresh culture medium promotes apoptosis or necrosis, depending on the oxidant concentration. Addition of 3-aminobenzamide (3AB) during the recovery phase prevented necrosis and caused apoptosis. 3AB did not, however, affect the apoptotic response of cells treated with apogenic concentrations of H2O2. Cells exposed for 3 hr to 1.5 mM H2O2, while showing some signs of suffering, maintained a normal nuclear organization and good organelle morphology. At the biochemical level, the oxidant promoted the formation of Mb-sized DNA fragments and rapidly depleted both the adenine nucleotide and non-protein sulphydryl pools, which did not recover during posttreatment incubation in the absence or presence of 3AB. These results allow a novel interpretation of the concentration-dependent switch from apoptosis to necrosis. We propose that H2O2 activates the apoptotic response at the early times of peroxide exposure and that this process can be completed, or inhibited, during the posttreatment incubation phase. Inhibition of apoptosis leads to necrosis and can be prevented by 3AB via a mechanism independent of inhibition of poly(ADP-ribose)polymerase. As a corollary, the necrotic response promoted by high concentrations of H2O2 in U937 cells appears to be the result of specific inhibition of the late steps of apoptosis.

Prevention of necrosis and activation of apoptosis in oxidatively injured human myeloid leukemia U937 cells

A 3 h exposure to 1 mM H2O2 followed by 6 h post‐challenge growth in peroxide‐free medium induces necrosis in U937 cells. Addition of the poly(ADP‐ribose)polymerase inhibitor 3‐aminobenzamide during recovery prevents necrosis and triggers apoptosis, as shown by the appearance of apoptotic bodies, extensive blebbing and formation of multimeric DNA fragments as well as 50 kb double stranded DNA fragments. Thus, the same initial damage can be a triggering event for both apoptotic and necrotic cell death. Furthermore, necrosis does not appear to be a passive response to overwhelming damage.

Beta-amyloid inhibits NOS activity by subtracting NADPH availability.

The amyloid peptides Aβ1–42 and Aβ25–35 strongly inhibited the activity of constitutive neuronal and endothelial nitric oxide synthases (i.e., NOS‐I and NOS‐III, respectively) in cell‐free assays. The molecular mechanism of NOS inhibition by Aβ fragments was studied in detail with Aβ25–35. The inhibitory ability was mostly NADPH‐dependent and specific for the soluble form of Aβ25–35. Optical, fluorescence, and NMR spectroscopy showed that the soluble, but not aggregated, Aβ25–35 interacted with NADPH, thus suggesting that a direct recruitment of NADPH may result in diminished availability of the redox cofactor for NOS functioning. To assess the physiological relevance of our findings, rat neuronal‐like PC12 and glioma C6 cell lines were used as cellular models. After Aβ25–35 internalization into cells was verified, the activity of constitutive NOS was measured using the DAF‐2DA detection system and found to be severely impaired upon Aβ25–35 uptake. Consistent with previous results on the molecular cross‐talk between NOS isoforms, repression of constitutive NOS by Aβ25–35 resulted in enhanced expression of inducible NOS (NOS‐II) mRNA in C6 cells. Our results represent the first evidence that amyloid fragments impair constitutive NOS activity in cell‐free and cellular systems, providing a possible molecular mechanism for the onset and/or maintenance of Alzheimers disease.

Peroxynitrite-mediated release of arachidonic acid from PC12 cells.

A short term exposure of PC12 cells to a concentration of tert‐butylhydroperoxide (tB‐OOH) causing peroxynitrite‐dependent DNA damage and cytotoxiticity promoted a release of arachidonic acid (AA) that was sensitive to phospholipase A2 (PLA2) inhibitors and insensitive to phospholipase C or diacylglycerol lipase inhibitors. The extent of AA release was also mitigated by nitric oxide synthase (NOS) inhibitors and peroxynitrite scavengers. Low levels (10 μM) of authentic peroxynitrite restored the release of AA mediated by tB‐OOH in NOS‐inhibited cells whereas concentrations of peroxynitrite of 20 μM, or higher, effectively stimulated a PLA2 inhibitor‐sensitive release of AA also in the absence of additional treatments. These results are consistent with the possibility that endogenous as well as exogenous peroxynitrite promotes activation of PLA2.

The Raf/MEK inhibitor PD98059 enhances ERK1/2 phosphorylation mediated by peroxynitrite via enforced mitochondrial formation of reactive oxygen species

Exposure of PC12 cells to 100 μM peroxynitrite promotes phosphorylation of extracellular signal‐regulated kinases 1 and 2 (ERK1/2) sensitive to PD98059 or U0126. At higher concentrations, however, ERK1/2 phosphorylation was prevented by U0126 and increased by PD98059 via a U0126‐sensitive mechanism. PD98059, unlike U0126, enhanced the peroxynitrite‐dependent formation of reactive oxygen species (ROS). These results, along with others obtained using respiratory chain inhibitors and respiration‐deficient cells, lead to the conclusion that PD98059, while effectively inhibiting the peroxynitrite‐induced Raf/MEK signaling leading to ERK1/2 phosphorylation, promotes an enforced mitochondrial formation of ROS inducing ERK1/2 phosphorylation via a Raf‐1‐independent/MEK‐dependent mechanism.

Orexin-A represses satiety-inducing POMC neurons and contributes to obesity via stimulation of endocannabinoid signaling

Significance Both evolutionarily and functionally, wakefulness requires, and is accompanied by, food search and intake for survival. From the molecular perspective, the neuropeptide orexin-A (OX-A) promotes wakefulness, α–melanocyte-stimulating hormone (α-MSH) promotes satiety, and the endocannabinoid 2-arachidonoylglycerol (2-AG) promotes appetite. In the cerebrospinal fluid of obese mice and in the plasma of human obese subjects, we found an inverse correlation between OX-A and α-MSH levels, which led us to uncover the role of OX-A in promoting hyperphagia by enhancing 2-AG levels and subsequently activating CB1 receptor-mediated down-regulation of POMC synthesis and α-MSH release. Pharmacological inhibition of OX-A receptor type 1 counteracted the impairment of α-MSH signaling and the associated hyperphagia, obesity, and steatosis, thus providing a potential therapy for these pathological conditions. In the hypothalamic arcuate nucleus (ARC), proopiomelanocortin (POMC) neurons and the POMC-derived peptide α–melanocyte-stimulating hormone (α-MSH) promote satiety. POMC neurons receive orexin-A (OX-A)-expressing inputs and express both OX-A receptor type 1 (OX-1R) and cannabinoid receptor type 1 (CB1R) on the plasma membrane. OX-A is crucial for the control of wakefulness and energy homeostasis and promotes, in OX-1R–expressing cells, the biosynthesis of the endogenous counterpart of marijuanas psychotropic and appetite-inducing component Δ9-tetrahydrocannabinol, i.e., the endocannabinoid 2-arachidonoylglycerol (2-AG), which acts at CB1R. We report that OX-A/OX-1R signaling at POMC neurons promotes 2-AG biosynthesis, hyperphagia, and weight gain by blunting α-MSH production via CB1R-induced and extracellular-signal-regulated kinase 1/2 activation- and STAT3 inhibition-mediated suppression of Pomc gene transcription. Because the systemic pharmacological blockade of OX-1R by SB334867 caused anorectic effects by reducing food intake and body weight, our results unravel a previously unsuspected role for OX-A in endocannabinoid-mediated promotion of appetite by combining OX-induced alertness with food seeking. Notably, increased OX-A trafficking was found in the fibers projecting to the ARC of obese mice (ob/ob and high-fat diet fed) concurrently with elevation of OX-A release in the cerebrospinal fluid and blood of mice. Furthermore, a negative correlation between OX-A and α-MSH serum levels was found in obese mice as well as in human obese subjects (body mass index > 40), in combination with elevation of alanine aminotransferase and γ-glutamyl transferase, two markers of fatty liver disease. These alterations were counteracted by antagonism of OX-1R, thus providing the basis for a therapeutic treatment of these diseases.

tert-Butylhydroperoxide induces peroxynitrite-dependent mitochondrial permeability transition leading PC12 cells to necrosis

A short‐term exposure of PC12 cells to tert‐butylhydroperoxide, followed by recovery in fresh culture medium, causes cell death and the extent of this response progressively increases during the 120 min of post‐treatment incubation. Morphological and biochemical analyses of these cells revealed that the mode of cell death was necrosis. Cell killing induced by the hydroperoxide seems to be in part mediated by peroxynitrite because the lethal response was markedly and similarly reduced by the nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine methylester and by scavengers of nitric oxide or peroxynitrite. This peroxynitrite‐dependent mechanism of cytotoxicity was blunted by antioxidants and inhibitors of mitochondrial permeability transition and the onset of cell death was preceded by mitochondrial depolarization and loss of cellular ATP. We conclude that tert‐butylhydroperoxide promotes peroxynitrite‐dependent PC12 cell necrosis causally linked to peroxidation of membrane lipids and mitochondrial permeability transition. J. Neurosci. Res. 65:387–395, 2001.

Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons

Background: In hypothalamic neurons, leptin induces ROS production via PPAR-γ inhibition. Results: CB1 agonism prevents leptin-induced ROS accumulation by reversing PPAR-γ and catalase inhibition. Inhibition of endocannabinoid inactivation also counteracts leptin effects. Conclusion: CB1 inhibits effects of leptin that underlie part of its anorexic actions. Significance: During conditions of increased endocannabinoid tone CB1 might reduce leptin activity in the hypothalamus. The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB1 receptor activity. Here we investigated the possibility of a negative regulation by CB1 receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2′-chloroethylamide (ACEA) in a CB1-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL−/− mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB1 activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.