Tisochrysis lutea F&M-M36 Mitigates Risk Factors of Metabolic Syndrome and Promotes Visceral Fat Browning through ß3-Adrenergic Receptor/UCP1 Signaling.

Pre-metabolic syndrome (pre-MetS) may represent the best transition phase to start treatments aimed at reducing cardiometabolic risk factors of MetS. In this study, we investigated the effects of the marine microalga Tisochrysis lutea F&M-M36 (T. lutea) on cardiometabolic components of pre-MetS and its underlying mechanisms. Rats were fed a standard (5% fat) or a high-fat diet (20% fat) supplemented or not with 5% of T. lutea or fenofibrate (100 mg/Kg) for 3 months. Like fenofibrate, T. lutea decreased blood triglycerides (p < 0.01) and glucose levels (p < 0.01), increased fecal lipid excretion (p < 0.05) and adiponectin (p < 0.001) without affecting weight gain. Unlike fenofibrate, T. lutea did not increase liver weight and steatosis, reduced renal fat (p < 0.05), diastolic (p < 0.05) and mean arterial pressure (p < 0.05). In visceral adipose tissue (VAT), T. lutea, but not fenofibrate, increased the ß3-adrenergic receptor (ß3ADR) (p < 0.05) and Uncoupling protein 1 (UCP-1) (p < 0.001) while both induced glucagon-like peptide-1 receptor (GLP1R) protein expression (p < 0.001) and decreased interleukin (IL)-6 and IL-1ß gene expression (p < 0.05). Pathway analysis on VAT whole-gene expression profiles showed that T. lutea up-regulated energy-metabolism-related genes and down-regulated inflammatory and autophagy pathways. The multitarget activity of T. lutea suggests that this microalga could be useful in mitigating risk factors of MetS.

Redox Properties of 3-Iodothyronamine (T1AM) and 3-Iodothyroacetic Acid (TA1).

3-iodothyronamine (T1AM) and 3-iodothyroacetic acid (TA1) are thyroid-hormone-related compounds endowed with pharmacological activity through mechanisms that remain elusive. Some evidence suggests that they may have redox features. We assessed the chemical activity of T1AM and TA1 at pro-oxidant conditions. Further, in the cell model consisting of brown adipocytes (BAs) differentiated for 6 days in the absence (M cells) or in the presence of 20 nM T1AM (M + T1AM cells), characterized by pro-oxidant metabolism, or TA1 (M + TA1 cells), we investigated the expression/activity levels of pro- and anti-oxidant proteins, including UCP-1, sirtuin-1 (SIRT1), mitochondrial monoamine (MAO-A and MAO-B), semicarbazide-sensitive amine oxidase (SSAO), and reactive oxygen species (ROS)-dependent lipoperoxidation. T1AM and TA1 showed in-vitro antioxidant and superoxide scavenging properties, while only TA1 acted as a hydroxyl radical scavenger. M + T1AM cells showed higher lipoperoxidation levels and reduced SIRT1 expression and activity, similar MAO-A, but higher MAO-B activity in terms of M cells. Instead, the M + TA1 cells exhibited increased levels of SIRT1 protein and activity and significantly lower UCP-1, MAO-A, MAO-B, and SSAO in comparison with the M cells, and did not show signs of lipoperoxidation. Our results suggest that SIRT1 is the mediator of T1AM and TA1 pro-or anti-oxidant effects as a result of ROS intracellular levels, including the hydroxyl radical. Here, we provide evidence indicating that T1AM and TA1 administration impacts on the redox status of a biological system, a feature that indicates the novel mechanism of action of these two thyroid-hormone-related compounds.

Angiotensin-II Drives Human Satellite Cells Toward Hypertrophy and Myofibroblast Trans-Differentiation by Two Independent Pathways.

Skeletal muscle regeneration is ensured by satellite cells (SC), which upon activation undergo self-renewal and myogenesis. The correct sequence of healing events may be offset by inflammatory and/or fibrotic factors able to promote fibrosis and consequent muscle wasting. Angiotensin-II (Ang) is an effector peptide of the renin angiotensin system (RAS), of which the direct role in human SCs (hSCs) is still controversial. Based on the hypertrophic and fibrogenic effects of Ang via transient receptor potential canonical (TRPC) channels in cardiac and renal tissues, we hypothesized a similar axis in hSCs. Toward this aim, we demonstrated that hSCs respond to acute Ang stimulation, dose-dependently enhancing p-mTOR, p-AKT, p-ERK1/2 and p-P38. Additionally, sub-acute Ang conditioning increased cell size and promoted trans-differentiation into myofibroblasts. To provide a mechanistic hypothesis on TRPC channel involvement in the processes, we proved that TRPC channels mediate a basal calcium entry into hSCs that is stimulated by acute Ang and strongly amplified by sub-chronic Ang conditioning. Altogether, these findings demonstrate that Ang induces a fate shift of hSCs into myofibroblasts and provide a basis to support a benefit of RAS and TRPC channel blockade to oppose muscle fibrosis.

Single Construct Suppression and Replacement Gene Therapy for the Treatment of All CALM1-, CALM2-, and CALM3-Mediated Arrhythmia Disorders.

BACKGROUND: CaM (calmodulin)-mediated long-QT syndrome is a genetic arrhythmia disorder (calmodulinopathies) characterized by a high prevalence of life-threatening ventricular arrhythmias occurring early in life. Three distinct genes (CALM1, CALM2, and CALM3) encode for the identical CaM protein. Conventional pharmacotherapies fail to adequately protect against potentially lethal cardiac events in patients with calmodulinopathy. METHODS: Five custom-designed CALM1-, CALM2-, and CALM3-targeting short hairpin RNAs (shRNAs) were tested for knockdown (KD) efficiency using TSA201 cells and reverse transcription-quantitative polymerase chain reaction. A dual-component suppression and replacement (SupRep) CALM gene therapy (CALM-SupRep) was created by cloning into a single construct CALM1-, CALM2-, and CALM3-specific shRNAs that produce KD (suppression) of each respective gene and a shRNA-immune CALM1 cDNA (replacement). CALM1-F142L, CALM2-D130G, and CALM3-D130G induced pluripotent stem cell-derived CMs were generated from patients with CaM-mediated long-QT syndrome. A voltage-sensing dye was used to measure action potential duration at 90% repolarization (APD90). RESULTS: Following shRNA KD efficiency testing, a candidate shRNA was identified for CALM1 (86% KD), CALM2 (71% KD), and CALM3 (94% KD). The APD90 was significantly prolonged in CALM2-D130G (647±9 ms) compared with CALM2-WT (359±12 ms; P<0.0001). Transfection with CALM-SupRep shortened the average APD90 of CALM2-D130G to 457±19 ms (66% attenuation; P<0.0001). Additionally, transfection with CALM-SupRep shortened the APD90 of CALM1-F142L (665±9 to 410±15 ms; P<0.0001) and CALM3-D130G (978±81 to 446±6 ms; P<0.001). CONCLUSIONS: We provide the first proof-of-principle suppression-replacement gene therapy for CaM-mediated long-QT syndrome. The CALM-SupRep gene therapy shortened the pathologically prolonged APD90 in CALM1-, CALM2-, and CALM3-variant CaM-mediated long-QT syndrome induced pluripotent stem cell-derived CM lines. The single CALM-SupRep construct may be able to treat all calmodulinopathies, regardless of which of the 3 CaM-encoding genes are affected.

The 3-iodothyronamine (T1AM) and the 3-iodothyroacetic acid (TA1) indicate a novel connection with the histamine system for neuroprotection.

The 3-iodothyronamine (T1AM) and 3-iodothryoacetic acid (TA1), are endogenous occurring compounds structurally related with thyroid hormones (THs, the pro-hormone T4 and the active hormone T3) initially proposed as possible mediators of the rapid effects of T3. However, after years from their identification, the physio-pathological meaning of T1AM and TA1 tissue levels remains an unsolved issue while pharmacological evidence indicates both compounds promote in rodents central and peripheral effects with mechanisms which remain mostly elusive. Pharmacodynamics of T1AM includes the recognition of G-coupled receptors, ion channels but also biotransformation into an active metabolite, i.e. the TA1. Furthermore, long term T1AM treatment associates with post-translational modifications of cell proteins. Such array of signaling may represent an added value, rather than a limit, equipping T1AM to play different functions depending on local expression of targets and enzymes involved in its biotransformation. Up to date, no information regarding TA1 mechanistic is available. We here review some of the main findings describing effects of T1AM (and TA1) which suggest these compounds interplay with the histaminergic system. These data reveal T1AM and TA1 are part of a network of signals involved in neuronal plasticity including neuroprotection and suggest T1AM and TA1 as lead compounds for a novel class of atypical psychoactive drugs.

3-Iodothyronamine Affects Thermogenic Substrates' Mobilization in Brown Adipocytes.

We investigated the effect of 3-iodothyronamine (T1AM) on thermogenic substrates in brown adipocytes (BAs). BAs isolated from the stromal fraction of rat brown adipose tissue were exposed to an adipogenic medium containing insulin in the absence (M) or in the presence of 20 nM T1AM (M+T1AM) for 6 days. At the end of the treatment, the expression of p-PKA/PKA, p-AKT/AKT, p-AMPK/AMPK, p-CREB/CREB, p-P38/P38, type 1 and 3 beta adrenergic receptors (ß1-ß3AR), GLUT4, type 2 deiodinase (DIO2), and uncoupling protein 1 (UCP-1) were evaluated. The effects of cell conditioning with T1AM on fatty acid mobilization (basal and adrenergic-mediated), glucose uptake (basal and insulin-mediated), and ATP cell content were also analyzed in both cell populations. When compared to cells not exposed, M+T1AM cells showed increased p-PKA/PKA, p-AKT/AKT, p-CREB/CREB, p-P38/P38, and p-AMPK/AMPK, downregulation of DIO2 and ß1AR, and upregulation of glycosylated ß3AR, GLUT4, and adiponectin. At basal conditions, glycerol release was higher for M+T1AM cells than M cells, without any significant differences in basal glucose uptake. Notably, in M+T1AM cells, adrenergic agonists failed to activate PKA and lipolysis and to increase ATP level, but the glucose uptake in response to insulin exposure was more pronounced than in M cells. In conclusion, our results suggest that BAs conditioning with T1AM promote a catabolic condition promising to fight obesity and insulin resistance.

Thyroid Hormone, Thyroid Hormone Metabolites and Mast Cells: A Less Explored Issue.

Mast cells are primary players in immune and inflammatory diseases. In the brain, mast cells are located at the brain side of the blood brain barrier (BBB) exerting a crucial role in protecting the brain from xenobiotic invasion. Furthermore, recent advances in neuroscience indicate mast cells may play an important role in glial cell-neuron communication through the release of mediators, including histamine. Interestingly, brain mast cells contain not only 50% of the brain histamine but also hormones, proteases and lipids or amine mediators; and cell degranulation may be triggered by different stimuli activating membrane bound receptors including the four types of histaminergic receptors. Among hormones, mast cells can store thyroid hormone (T3) and express membrane-bound thyroid stimulating hormone receptors (TSHRs), thus suggesting from one side that thyroid function may affect mast cells function, from the other that mast cell degranulation may impact on thyroid function. In this respect, the research on hormones in mast cells is scarce. Recent pharmacological evidence indicates the existence of a non-genomic portion of the thyroid secretion including thyroid hormone metabolites. Among which the 3,5 diiodothyronine (3,5-T2), 3-iodothyroanamine (T1AM) and 3-iodothyroacetic acid (TA1) are the most studied. All these compounds are endogenously occurring and found to be increased in inflammatory-based diseases involving mast cells. T1AM and TA1 induce, as T3, neuroprotective effects and itch but also hyperalgesia in rodents with a mechanism largely unknown but mediated by the release of histamine. Due to the rapid onset of their effectiveness they may trigger histamine release from a cell where it is "ready-to-be released," i.e., mast cells. Following a very thin path which passes through old experimental and clinical evidence, at the light of novel acquisitions on endogenous T3 metabolites, we aim to stimulate the attention on the possibility that mast cell histamine may be the connector of a novel (neuro) endocrine pathway linking the thyroid with mast cells.

N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide (EPPTB) prevents 3-iodothyronamine (T1AM)-induced neuroprotection against kainic acid toxicity.

Thyroid hormone and thyroid hormone metabolites, including 3-iodothyronamine (T1AM) and 3-iodothyroacetic acid (TA1), activate AKT signaling in hippocampal neurons affording protection from excitotoxic damage. We aim to explore whether the mechanism of T1AM neuroprotection against kainic acid (KA)-induced excitotoxicity included the activation of the trace amine associated receptor isoform 1 (TAAR1), one of T1AM targets. Rat organotypic hippocampal slices were exposed to vehicle (Veh) or to 5 µM kA for 24 h in the absence or presence of 0.1, 1 and 10 µM T1AM or to 0.1, 1 and 10 µM T1AM and 1 µM N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide (EPPTB), the only available TAAR1 antagonist, or to 1 µM T1AM in the absence or in the presence of 10 µM LY294002, an inhibitor of phosphoinositide 3-kinases (PI3Ks). Cell death was evaluated by measuring propidium iodide (PI) levels of fluorescence 24 h after treatment. In parallel, the expression levels of p-AKT and p-PKA were evaluated by Western blot analysis of slice lysates. The activity of mitochondrial monoamine oxidases (MAO) was assayed fluorimetrically. 24 h exposure of slices to T1AM resulted in the activation of AKT and PKA. KA exposure induced cell death in the CA3 region and significantly reduced p-AKT and p-PKA levels. The presence of 1 and 10 µM T1AM significantly protected neurons from death and conserved both kinase levels with the essential role of AKT in neuroprotection. Furthermore, EPPTB prevented T1AM-induced neuroprotection, activation of PKA and AKT. Of note, in the presence of EPPTB T1AM degradation by MAO was reduced. Our results indicate that the neuroprotection offered by T1AM depends, as for TA1, on AKT activation but do not allow to conclusively indicate TAAR1 as the target implicated.

Brain Histamine Modulates the Antidepressant-Like Effect of the 3-Iodothyroacetic Acid (TA1).

3-iodothyroacetic acid (TA1), an end metabolite of thyroid hormone, has been shown to produce behavioral effects in mice that are dependent on brain histamine. We now aim to verify whether pharmacologically administered TA1 has brain bioavailability and is able to induce histamine-dependent antidepressant-like behaviors. TA1 brain, liver and plasma levels were measured by LC/MS-MS in male CD1 mice, sacrificed 15 min after receiving a high TA1 dose (330 µgkg-1). The hypothalamic mTOR/AKT/GSK-ß cascade activation was evaluated in mice treated with 0.4, 1.32, 4 µgkg-1 TA1 by Western-blot. Mast cells were visualized by immuno-histochemistry in brain slices obtained from mice treated with 4 µgkg-1 TA1. Histamine release triggered by TA1 (20-1000 nM) was also evaluated in mouse peritoneal mast cells. After receiving TA1 (1.32, 4 or 11 µgkg-1; i.p.) CD1 male mice were subjected to the forced swim (FST) and the tail suspension tests (TST). Spontaneous locomotor and exploratory activities, motor incoordination, and anxiolytic or anxiogenic effects, were evaluated. Parallel behavioral tests were also carried out in mice that, prior to receiving TA1, were pre-treated with pyrilamine (10 mgkg-1; PYR) or zolantidine (5 mgkg-1; ZOL), histamine type 1 and type 2 receptor antagonists, respectively, or with p-chloro-phenylalanine (100 mgkg-1; PCPA), an inhibitor of serotonin synthesis. TA1 given i.p. to mice rapidly distributes in the brain, activates the hypothalamic mTOR/AKT and GSK-3ß cascade and triggers mast cells degranulation. Furthermore, TA1 induces antidepressant effects and stimulates locomotion with a mechanism that appears to depend on the histaminergic system. TA1 antidepressant effect depends on brain histamine, thus highlighting a relationship between the immune system, brain inflammation and the thyroid.