Autophagy-mediated NCOR1 degradation is required for brown fat maturation and thermogenesis.

Brown adipose tissue (BAT) thermogenesis affects energy balance, and thereby it has the potential to induce weight loss and to prevent obesity. Here, we document a macroautophagic/autophagic-dependent mechanism of peroxisome proliferator-activated receptor gamma (PPARG) activity regulation that induces brown adipose differentiation and thermogenesis and that is mediated by TP53INP2. Disruption of TP53INP2-dependent autophagy reduced brown adipogenesis in cultured cells. In vivo specific-tp53inp2 ablation in brown precursor cells or in adult mice decreased the expression of thermogenic and mature adipocyte genes in BAT. As a result, TP53INP2-deficient mice had reduced UCP1 content in BAT and impaired maximal thermogenic capacity, leading to lipid accumulation and to positive energy balance. Mechanistically, TP53INP2 stimulates PPARG activity and adipogenesis in brown adipose cells by promoting the autophagic degradation of NCOR1, a PPARG co-repressor. Moreover, the modulation of TP53INP2 expression in BAT and in human brown adipocytes suggests that this protein increases PPARG activity during metabolic activation of brown fat. In all, we have identified a novel molecular explanation for the contribution of autophagy to BAT energy metabolism that could facilitate the design of therapeutic strategies against obesity and its metabolic complications.

Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles.

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

[Translational medicine and innovation in health: mechanisms and perspectives]. / Medicina traslacional e innovación en salud: mecanismos y perspectivas.

Many new discoveries in Life Sciences cannot be translated into products, services or new applications to improve human health. Translational medicine, defined as "from bench to bedside", refers to the transfer of results or new knowledge achieved in the laboratory into health innovation. We aim to review the state of art of translational medicine, its relationship with innovation processes and the different perspectives to consider. Finally, we contextualize the situation of Research and Development (R&D) in Chile and the main issues of the biotechnology market in the country.

Medicina traslacional e innovación en salud: mecanismos y perspectivas / Translational medicine and innovation in health: mechanisms and perspectives

Many new discoveries in Life Sciences cannot be translated into products, services or new applications to improve human health. Translational medicine, defined as "from bench to bedside", refers to the transfer of results or new knowledge achieved in the laboratory into health innovation. We aim to review the state of art of translational medicine, its relationship with innovation processes and the different perspectives to consider. Finally, we contextualize the situation of Research and Development (R&D) in Chile and the main issues of the biotechnology market in the country.

Insulin-mimetic compound hexaquis (benzylammonium) decavanadate is antilipolytic in human fat cells.

AIM: To assess in rodent and human adipocytes the antilipolytic capacity of hexaquis(benzylammonium) decavanadate (B6V10), previously shown to exert antidiabetic effects in rodent models, such as lowering free fatty acids (FFA) and glucose circulating levels. METHODS: Adipose tissue (AT) samples were obtained after informed consent from overweight women undergoing plastic surgery. Comparison of the effects of B6V10 and reference antilipolytic agents (insulin, benzylamine, vanadate) on the lipolytic activity was performed on adipocytes freshly isolated from rat, mouse and human AT. Glycerol release was measured using colorimetric assay as an index of lipolytic activity. The influence of B6V10 and reference agents on glucose transport into human fat cells was determined using the radiolabelled 2-deoxyglucose uptake assay. RESULTS: In all the species studied, B6V10 exhibited a dose-dependent inhibition of adipocyte lipolysis when triglyceride breakdown was moderately enhanced by ß-adrenergic receptor stimulation. B6V10 exerted on human adipocyte a maximal lipolysis inhibition of glycerol release that was stronger than that elicited by insulin. However, B6V10 did not inhibit basal and maximally stimulated lipolysis. When incubated at dose ≥ 10 µmol/L, B6V10 stimulated by twofold the glucose uptake in human fat cells, but - similarly to benzylamine - without reaching the maximal effect of insulin, while it reproduced one-half of the insulin-stimulation of lipogenesis in mouse fat cells. CONCLUSION: B6V10 exerts insulin-like actions in adipocytes, including lipolysis inhibition and glucose transport activation. B6V10 may be useful in limiting lipotoxicity related to obesity and insulin resistance.

Autophagy-regulating TP53INP2 mediates muscle wasting and is repressed in diabetes.

A precise balance between protein degradation and synthesis is essential to preserve skeletal muscle mass. Here, we found that TP53INP2, a homolog of the Drosophila melanogaster DOR protein that regulates autophagy in cellular models, has a direct impact on skeletal muscle mass in vivo. Using different transgenic mouse models, we demonstrated that muscle-specific overexpression of Tp53inp2 reduced muscle mass, while deletion of Tp53inp2 resulted in muscle hypertrophy. TP53INP2 activated basal autophagy in skeletal muscle and sustained p62-independent autophagic degradation of ubiquitinated proteins. Animals with muscle-specific overexpression of Tp53inp2 exhibited enhanced muscle wasting in streptozotocin-induced diabetes that was dependent on autophagy; however, TP53INP2 ablation mitigated experimental diabetes-associated muscle loss. The overexpression or absence of TP53INP2 did not affect muscle wasting in response to denervation, a condition in which autophagy is blocked, further indicating that TP53INP2 alters muscle mass by activating autophagy. Moreover, TP53INP2 expression was markedly repressed in muscle from patients with type 2 diabetes and in murine models of diabetes. Our results indicate that TP53INP2 negatively regulates skeletal muscle mass through activation of autophagy. Furthermore, we propose that TP53INP2 repression is part of an adaptive mechanism aimed at preserving muscle mass under conditions in which insulin action is deficient.

Oral insulin-mimetic compounds that act independently of insulin.

The hallmarks of insulin action are the stimulation and suppression of anabolic and catabolic responses, respectively. These responses are orchestrated by the insulin pathway and are initiated by the binding of insulin to the insulin receptor, which leads to activation of the receptor's intrinsic tyrosine kinase. Severe defects in the insulin pathway, such as in types A and B and advanced type 1 and 2 diabetes lead to severe insulin resistance, resulting in a partial or complete absence of response to exogenous insulin and other known classes of antidiabetes therapies. We have characterized a novel class of arylalkylamine vanadium salts that exert potent insulin-mimetic effects downstream of the insulin receptor in adipocytes. These compounds trigger insulin signaling, which is characterized by rapid activation of insulin receptor substrate-1, Akt, and glycogen synthase kinase-3 independent of insulin receptor phosphorylation. Administration of these compounds to animal models of diabetes lowered glycemia and normalized the plasma lipid profile. Arylalkylamine vanadium compounds also showed antidiabetic effects in severely diabetic rats with undetectable circulating insulin. These results demonstrate the feasibility of insulin-like regulation in the complete absence of insulin and downstream of the insulin receptor. This represents a novel therapeutic approach for diabetic patients with severe insulin resistance.

Short- and long-term insulin-like effects of monoamine oxidases and semicarbazide-sensitive amine oxidase substrates in cultured adipocytes.

Semicarbazide-sensitive amine oxidase (SSAO) is known to increase during in vitro adipogenesis and to be one of the most highly expressed membrane proteins of white adipocytes. Although less well documented, mitochondrial monoamine oxidases (MAOs) are also present in adipocytes and share with SSAO the capacity to generate hydrogen peroxide. This work therefore aimed to compare several biologic effects of MAO and SSAO substrates in 3T3-F442A adipocytes. In differentiated cells, tyramine oxidation was predominantly MAO dependent, whereas benzylamine oxidation was SSAO dependent. Both amines partially mimicked insulin actions, including stimulation of Akt phosphorylation and glucose uptake. In addition, tyramine and benzylamine impaired tumor necrosis factor alpha-dependent nitric oxide formation in a pargyline- and semicarbazide-sensitive manner, respectively. Various biogenic amines were tested in competition for tyramine or benzylamine oxidation and classified as MAO-preferring (methoxytyramine, tryptamine) or SSAO-preferring substrates (methylamine, octopamine). Short-term incubation with 1 mmol/L of all amines except histamine stimulated glucose uptake up to 20% to 50% of maximal insulin activation. One-week treatment with either MAO or SSAO substrates alone allowed postconfluent cells to differentiate into adipocytes, reproducing 60% of insulin-promoted lipid accumulation. All amines also exerted a slight improvement in the adipogenic action of insulin. Therefore, like SSAO, substrate activation of MAO can interact with adipocyte metabolism by mimicking diverse effects of insulin in addition to preventing tumor necrosis factor alpha-dependent responses.

Methylamine but not mafenide mimics insulin-like activity of the semicarbazide-sensitive amine oxidase-substrate benzylamine on glucose tolerance and on human adipocyte metabolism.

It has been reported that benzylamine reduces blood glucose in rabbits, stimulates hexose uptake, and inhibits lipolysis in mouse, rabbit, and human adipocytes. In the presence of vanadate, benzylamine is also able to improve glucose disposal in normoglycaemic and diabetic rats. Such insulin-mimicking properties are the consequence of hydrogen peroxide production during benzylamine oxidation by semicarbazide-sensitive amine oxidase (SSAO). The aim of the study was to determine whether other SSAO-substrates could share such potential antidiabetic properties. Thus, mafenide, a synthetic antimicrobial sulfonamide structurally related to benzylamine, and which has been recently reported to interact with SSAO, was tested in the above mentioned models, in parallel with methylamine, a proposed endogenous SSAO-substrate. All tested amines stimulated glucose uptake and inhibited lipolysis in rat and mouse fat cells. Methylamine and benzylamine, but not mafenide, reduced the hyperglycaemic response during a glucose tolerance test in rabbits while the three amines tested were devoid of insulin-releasing activity under both in vivo and in vitro conditions. In human adipocytes, mafenide did not stimulate glucose transport since it was not a high-affinity substrate for SSAO and generated less hydrogen peroxide than benzylamine or methylamine. Therefore, mafenide could not be considered as an antidiabetic drug despite being oxidized and exhibiting insulin-mimicking effects in rat and mouse adipocytes. By contrast, the endogenous substrate methylamine improved glucose utilization in all in vitro and in vivo models, leading to consider novel SSAO substrates as drugs with potential anti-hyperglycaemic properties.

The Charcot-Marie-Tooth type 2A gene product, Mfn2, up-regulates fuel oxidation through expression of OXPHOS system.

Mitofusin-2 (Mfn2) is a mitochondrial membrane protein that participates in mitochondrial fusion in mammalian cells and mutations in the Mfn2 gene cause Charcot-Marie-Tooth neuropathy type 2A. Here, we show that Mfn2 loss-of-function inhibits pyruvate, glucose and fatty acid oxidation and reduces mitochondrial membrane potential, whereas Mfn2 gain-of-function increases glucose oxidation and mitochondrial membrane potential. As to the mechanisms involved, we have found that Mfn2 loss-of-function represses nuclear-encoded subunits of OXPHOS complexes I, II, III and V, whereas Mfn2 overexpression induced the subunits of complexes I, IV and V. Obesity-induced Mfn2 deficiency in rat skeletal muscle was also associated with a decrease in the subunits of complexes I, II, III and V. In addition, the effect of Mfn2 overexpression on mitochondrial metabolism was mimicked by a truncated Mfn2 mutant that is inactive as a mitochondrial fusion protein. Our results indicate that Mfn2 triggers mitochondrial energization, at least in part, by regulating OXPHOS expression through signals that are independent of its role as a mitochondrial fusion protein.

Exploring the binding mode of semicarbazide-sensitive amine oxidase/VAP-1: identification of novel substrates with insulin-like activity.

We previously reported that substrates of semicarbazide-sensitive amine oxidase in combination with low concentrations of vanadate exert potent insulin-like effects. Here we performed homology modeling of the catalytic domain of mouse SSAO/VAP-1 and searched through chemical databases to identify novel SSAO substrates. The modeling of the catalytic domain revealed that aromatic residues Tyr384, Phe389, and Tyr394 define a pocket of stable size that may participate in the binding of apolar substrates. We identified a number of amines as substrates of human, rat, and mouse SSAO. The compounds PD0119035, 2,3-dimethoxy-benzylamine, and C-naphthalen-1-yl-methylamine showed high affinity as substrates of rat SSAO. C-Naphthalen-1-yl-methylamine was the only substrate that showed high affinity for human SSAO. C-Naphthalen-1-yl-methylamine and 4-aminomethyl-benzenesulfonamide showed the highest capacity to stimulate glucose transport in isolated rat adipocytes. The impact of these findings on the development of new treatments for diabetes is discussed.

Semicarbazide-sensitive amine oxidase activity exerts insulin-like effects on glucose metabolism and insulin-signaling pathways in adipose cells.

Semicarbazide-sensitive amine oxidase (SSAO) is very abundant at the plasma membrane in adipocytes. The combination of SSAO substrates and low concentrations of vanadate markedly stimulates glucose transport and GLUT4 glucose transporter recruitment to the cell surface in rat adipocytes by a mechanism that requires SSAO activity and hydrogen peroxide formation. Substrates of SSAO such as benzylamine or tyramine in combination with vanadate potently stimulate tyrosine phosphorylation of both insulin-receptor substrates 1 (IRS-1) and 3 (IRS-3) and phosphatidylinositol 3-kinase (PI 3-kinase) activity in adipose cells, which occurs in the presence of a weak stimulation of insulin-receptor kinase. Moreover, the acute administration of benzylamine and vanadate in vivo enhances glucose tolerance in non-diabetic and streptozotocin-induced diabetic rats and reduces hyperglycemia after chronic treatment in streptozotocin-diabetic rats. Based on these observations, we propose that SSAO activity and vanadate potently mimic insulin effects in adipose cells and exert an anti-diabetic action in an animal model of type 1 diabetes mellitus.

Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 activity exerts an antidiabetic action in Goto-Kakizaki rats.

In this study we have explored whether the bifunctional protein semicarbazide-sensitive amine oxidase (SSAO)/vascular adhesion protein-1 (VAP-1) represents a novel target for type 2 diabetes. To this end, Goto-Kakizaki (GK) diabetic rats were treated with the SSAO substrate benzylamine and with low ineffective doses of vanadate previously shown to have antidiabetic effects in streptozotocin-induced diabetic rats. The administration of benzylamine in combination with vanadate in type 2 diabetic rats acutely stimulated glucose tolerance, and the chronic treatment normalized hyperglycemia, stimulated glucose transport in adipocytes, and reversed muscle insulin resistance. Acute in vivo administration of benzylamine and vanadate stimulated skeletal muscle glucose transport, an effect that was also observed in incubated muscle preparations coincubated with adipose tissue explants or with human recombinant SSAO. Acute administration of benzylamine/vanadate also ameliorated insulin secretion in diabetic GK rats, and this effect was also observed in incubated pancreatic islets. In keeping with these observations, we also demonstrate that pancreatic islets express SSAO/VAP-1. As far as mechanisms of action, we have found that benzylamine/vanadate causes enhanced tyrosine phosphorylation of proteins and reduced protein tyrosine phosphatase activity in adipocytes. In addition, incubation of human recombinant SSAO, benzylamine, and vanadate generates peroxovanadium compounds in vitro. Based on these data, we propose that benzylamine/vanadate administration generates peroxovanadium locally in pancreatic islets, which stimulates insulin secretion and also produces peroxovanadium in adipose tissue, activating glucose metabolism in adipocytes and in neighboring muscle. This opens the possibility of using the SSAO/VAP-1 activity as a local generator of protein tyrosine phosphatase inhibitors in antidiabetic therapy.

Tyramine stimulates glucose uptake in insulin-sensitive tissues in vitro and in vivo via its oxidation by amine oxidases.

Tyramine and benzylamine have been described as stimulators of glucose transport in adipocytes. This effect is dependent on amine oxidation by monoamine oxidase (MAO) or semicarbazide-sensitive amine oxidase (SSAO) and on the subsequent hydrogen peroxide formation as already demonstrated by blockade with oxidase inhibitors or antioxidants and potentiation with vanadate. In this work, we extended these observations to skeletal muscle and cardiac myocytes using in vitro and in vivo approaches. Tissue distribution studies showed that substantial extrahepatic peripheral MAO activities exist in kidney and gut, but also in insulin-sensitive tissues: heart, adipose tissue, and skeletal muscles. SSAO activity is also widely distributed and present at a lower level than MAO, except in fat depots where both oxidases were equally involved in tyramine oxidation. When tested in vitro at millimolar doses, tyramine caused a large stimulation of glucose transport in rat adipocytes and in skeletal and cardiac muscles. In vivo administration of tyramine (4 mg/kg i.p.) lowered the hyperglycemic responses to a glucose challenge in control and in streptozotocin-treated rats. This positive effect on glucose disposal was obtained without vanadate and was abolished by SSAO and MAO inhibitors. Tyramine increased hexose uptake in vivo in insulin-sensitive tissues, whereas it induced only transient effects on plasma insulin or cardiovascular parameters. In conclusion, activation of the amine oxidases present in insulin-sensitive tissues induces insulin-like effects, readily detectable in vitro, and increasing peripheral glucose utilization in vivo.