Oral Supplementation with Benzylamine Delays the Onset of Diabetes in Obese and Diabetic db-/- Mice.

Substrates of semicarbazide-sensitive amine oxidase (SSAO) exert insulin-like actions in adipocytes. One of them, benzylamine (Bza) exhibits antihyperglycemic properties in several rodent models of diabetes. To further study the antidiabetic potential of this naturally occurring amine, a model of severe type 2 diabetes, the obese db-/- mouse, was subjected to oral Bza administration. To this end, db-/- mice and their lean littermates were treated at 4 weeks of age by adding 0.5% Bza in drinking water for seven weeks. Body mass, fat content, blood glucose and urinary glucose output were followed while adipocyte insulin responsiveness and gene expression were checked at the end of supplementation, together with aorta nitrites. Bza supplementation delayed the appearance of hyperglycemia, abolished polydypsia and glycosuria in obese/diabetic mice without any detectable effect in lean control, except for a reduction in food intake observed in both genotypes. The improvement of glucose homeostasis was observed in db-/- mice at the expense of increased fat deposition, especially in the subcutaneous white adipose tissue (SCWAT), without sign of worsened inflammation or insulin responsiveness and with lowered circulating triglycerides and uric acid, while NO bioavailability was increased in aorta. The higher capacity of SSAO in oxidizing Bza in SCWAT, found in the obese mice, was unaltered by Bza supplementation and likely involved in the activation of glucose utilization by adipocytes. We propose that Bza oxidation in tissues, which produces hydrogen peroxide mainly in SCWAT, facilitates insulin-independent glucose utilization. Bza could be considered as a potential agent for dietary supplementation aiming at preventing diabetic complications.

Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin.

Apelin signaling plays an important role during embryo development and regulates angiogenesis, cardiovascular activity, and energy metabolism in adulthood. Overexpression and hyperactivity of this signaling pathway is observed in various pathologic states, such as cardiovascular diseases and cancer, which highlights the importance of inhibiting apelin receptor (APJ); therefore, we developed a cell-based screening assay that uses fluorescence microscopy to identify APJ antagonists. This approach led us to identify the U.S. Food and Drug Administration-approved compound protamine-already used clinically after cardiac surgery-as an agent to bind to heparin and thereby reverse its anticlotting activity. Protamine displays a 390-nM affinity for APJ and behaves as a full antagonist with regard to G protein and ß-arrestin-dependent intracellular signaling. Ex vivo and in vivo, protamine abolishes well-known apelin effects, such as angiogenesis, glucose tolerance, and vasodilatation. Remarkably, protamine antagonist activity is fully reversed by heparin treatment both in vitro and in vivo Thus, our results demonstrate a new pharmacologic property of protamine-blockade of APJ-that could explain some adverse effects observed in protamine-treated patients. Moreover, our data reveal that the established antiangiogenic activity of protamine would rely on APJ antagonism.-Le Gonidec, S., Chaves-Almagro, C., Bai, Y., Kang, H. J., Smith, A., Wanecq, E., Huang, X.-P., Prats, H., Knibiehler, B., Roth, B. L., Barak, L. S., Caron, M. G., Valet, P., Audigier, Y., Masri, B. Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin.

LEOPARD syndrome-associated SHP2 mutation confers leanness and protection from diet-induced obesity.

LEOPARD syndrome (multiple Lentigines, Electrocardiographic conduction abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth, sensorineural Deafness; LS), also called Noonan syndrome with multiple lentigines (NSML), is a rare autosomal dominant disorder associating various developmental defects, notably cardiopathies, dysmorphism, and short stature. It is mainly caused by mutations of the PTPN11 gene that catalytically inactivate the tyrosine phosphatase SHP2 (Src-homology 2 domain-containing phosphatase 2). Besides its pleiotropic roles during development, SHP2 plays key functions in energetic metabolism regulation. However, the metabolic outcomes of LS mutations have never been examined. Therefore, we performed an extensive metabolic exploration of an original LS mouse model, expressing the T468M mutation of SHP2, frequently borne by LS patients. Our results reveal that, besides expected symptoms, LS animals display a strong reduction of adiposity and resistance to diet-induced obesity, associated with overall better metabolic profile. We provide evidence that LS mutant expression impairs adipogenesis, triggers energy expenditure, and enhances insulin signaling, three features that can contribute to the lean phenotype of LS mice. Interestingly, chronic treatment of LS mice with low doses of MEK inhibitor, but not rapamycin, resulted in weight and adiposity gains. Importantly, preliminary data in a French cohort of LS patients suggests that most of them have lower-than-average body mass index, associated, for tested patients, with reduced adiposity. Altogether, these findings unravel previously unidentified characteristics for LS, which could represent a metabolic benefit for patients, but may also participate to the development or worsening of some traits of the disease. Beyond LS, they also highlight a protective role of SHP2 global LS-mimicking modulation toward the development of obesity and associated disorders.

Effects of dietary eicosapentaenoic acid (EPA) supplementation in high-fat fed mice on lipid metabolism and apelin/APJ system in skeletal muscle.

Various studies have shown that eicosapentaenoic acid (EPA) has beneficial effects on obesity and associated disorders. Apelin, the ligand of APJ receptor also exerts insulin-sensitizing effects especially by improving muscle metabolism. EPA has been shown to increase apelin production in adipose tissue but its effects in muscle have not been addressed. Thus, the effects of EPA supplementation (36 g/kg EPA) in high-fat diet (HFD) (45% fat, 20% protein, 35% carbohydrate) were studied in mice with focus on muscle lipid metabolism and apelin/APJ expression. Compared with HFD mice, HFD+EPA mice had significantly less weight gain, fat mass, lower blood glucose, insulinemia and hepatic steatosis after 10 weeks of diet. In addition, EPA prevented muscle metabolism alterations since intramuscular triglycerides were decreased and ß-oxidation increased. In soleus muscles of HFD+EPA mice, apelin and APJ expression were significantly increased compared to HFD mice. However, plasma apelin concentrations in HFD and HFD+EPA mice were similar. EPA-induced apelin expression was confirmed in differentiated C2C12 myocytes but in this model, apelin secretion was also increased in response to EPA treatment. In conclusion, EPA supplementation in HFD prevents obesity and metabolic alterations in mice, especially in skeletal muscle. Since EPA increases apelin/APJ expression in muscle, apelin may act in a paracrine/autocrine manner to contribute to these benefical effects.

Influence of secreted factors from human adipose tissue on glucose utilization and proinflammatory reaction

The objective of the present study was to characterize the nature of the autocrine/paracrine signal within human adipose tissue that may alter glucose metabolism and the inflammatory status in adipocytes. We prepared a conditioned medium from abdominal dermolipectomies in the absence (CM) or the presence (CMBSA) of bovine serum albumin (BSA), and we tested the influence of CM and CMBSA on glucose transport, maximal insulin response, and the expression of inflammation marker genes in differentiated human SGBS adipocytes. We found that CMBSA increased basal and reduced insulin-stimulated glucose incorporation along with a reduced mRNA level of the glucose transport GLUT4, and an increased expression of GLUT1. These effects were associated with a potent upregulation in the mRNA level of the proinflammatory cytokines IL-6 and MCP-1. These regulations were strongly attenuated in the absence of BSA during the preparation of CM, or after BSA depletion of CM, and were attributed to water-soluble molecules rather than lipids. Finally, fractionation of CMBSA by isoelectric focusing showed that part of its bioactivity could be reproduced with proteins with pHi ranging from 6.6 to 7.6. In conclusion, our results demonstrate that the production by human adipose tissue of autocrine/paracrine neutral proteins is able to increase the inflammatory status of the adipocytes and to deteriorate their glucose metabolism and maximal insulin response, and their release is greatly amplified by the presence of albumin (AU)

The intestinal glucose-apelin cycle controls carbohydrate absorption in mice.

BACKGROUND & AIMS: Glucose is absorbed into intestine cells via the sodium glucose transporter 1 (SGLT-1) and glucose transporter 2 (GLUT2); various peptides and hormones control this process. Apelin is a peptide that regulates glucose homeostasis and is produced by proximal digestive cells; we studied whether glucose modulates apelin secretion by enterocytes and the effects of apelin on intestinal glucose absorption. METHODS: We characterized glucose-related luminal apelin secretion in vivo and ex vivo by mass spectroscopy and immunologic techniques. The effects of apelin on (14)C-labeled glucose transport were determined in jejunal loops and in mice following apelin gavage. We determined levels of GLUT2 and SGLT-1 proteins and phosphorylation of AMPKα2 by immunoblotting. The net effect of apelin on intestinal glucose transepithelial transport was determined in mice. RESULTS: Glucose stimulated luminal secretion of the pyroglutaminated apelin-13 isoform ([Pyr-1]-apelin-13) in the small intestine of mice. Apelin increased specific glucose flux through the gastric epithelial barrier in jejunal loops and in vivo following oral glucose administration. Conversely, pharmacologic apelin blockade in the intestine reduced the increased glycemia that occurs following oral glucose administration. Apelin activity was associated with phosphorylation of AMPKα2 and a rapid increase of the GLUT2/SGLT-1 protein ratio in the brush border membrane. CONCLUSIONS: Glucose amplifies its own transport from the intestinal lumen to the bloodstream by increasing luminal apelin secretion. In the lumen, active apelin regulates carbohydrate flux through enterocytes by promoting AMPKα2 phosphorylation and modifying the ratio of SGLT-1:GLUT2. The glucose-apelin cycle might be pharmacologically handled to regulate glucose absorption and assess better control of glucose homeostasis.

Influence of secreted factors from human adipose tissue on glucose utilization and proinflammatory reaction.

The objective of the present study was to characterize the nature of the autocrine/paracrine signal within human adipose tissue that may alter glucose metabolism and the inflammatory status in adipocytes. We prepared a conditioned medium from abdominal dermolipectomies in the absence (CM) or the presence (CMBSA) of bovine serum albumin (BSA), and we tested the influence of CM and CMBSA on glucose transport, maximal insulin response, and the expression of inflammation marker genes in differentiated human SGBS adipocytes. We found that CMBSA increased basal and reduced insulin-stimulated glucose incorporation along with a reduced mRNA level of the glucose transport GLUT4, and an increased expression of GLUT1. These effects were associated with a potent upregulation in the mRNA level of the proinflammatory cytokines IL-6 and MCP-1. These regulations were strongly attenuated in the absence of BSA during the preparation of CM, or after BSA depletion of CM, and were attributed to water-soluble molecules rather than lipids. Finally, fractionation of CMBSA by isoelectric focusing showed that part of its bioactivity could be reproduced with proteins with pHi ranging from 6.6 to 7.6. In conclusion, our results demonstrate that the production by human adipose tissue of autocrine/paracrine neutral proteins is able to increase the inflammatory status of the adipocytes and to deteriorate their glucose metabolism and maximal insulin response, and their release is greatly amplified by the presence of albumin.

Apelin treatment increases complete Fatty Acid oxidation, mitochondrial oxidative capacity, and biogenesis in muscle of insulin-resistant mice.

Both acute and chronic apelin treatment have been shown to improve insulin sensitivity in mice. However, the effects of apelin on fatty acid oxidation (FAO) during obesity-related insulin resistance have not yet been addressed. Thus, the aim of the current study was to determine the impact of chronic treatment on lipid use, especially in skeletal muscles. High-fat diet (HFD)-induced obese and insulin-resistant mice treated by an apelin injection (0.1 µmol/kg/day i.p.) during 4 weeks had decreased fat mass, glycemia, and plasma levels of triglycerides and were protected from hyperinsulinemia compared with HFD PBS-treated mice. Indirect calorimetry experiments showed that apelin-treated mice had a better use of lipids. The complete FAO, the oxidative capacity, and mitochondrial biogenesis were increased in soleus of apelin-treated mice. The action of apelin was AMP-activated protein kinase (AMPK) dependent since all the effects studied were abrogated in HFD apelin-treated mice with muscle-specific inactive AMPK. Finally, the apelin-stimulated improvement of oxidative capacity led to decreased levels of acylcarnitines and enhanced insulin-stimulated glucose uptake in soleus. Thus, by promoting complete lipid use in muscle of insulin-resistant mice through mitochondrial biogenesis and tighter matching between FAO and the tricarboxylic acid cycle, apelin treatment could contribute to insulin sensitivity improvement.

Apelin prevents cardiac fibroblast activation and collagen production through inhibition of sphingosine kinase 1.

AIMS: Activation of cardiac fibroblasts and their differentiation into myofibroblasts is a key event in the progression of cardiac fibrosis that leads to end-stage heart failure. Apelin, an adipocyte-derived factor, exhibits a number of cardioprotective properties; however, whether apelin is involved in cardiac fibroblast activation and myofibroblast formation remains unknown. The aim of this study was to determine the effects of apelin in activated cardiac fibroblasts, the potential related mechanisms and impact on cardiac fibrotic remodelling process. METHODS AND RESULTS: In vitro experiments were performed in mouse cardiac fibroblasts obtained from normal and pressure-overload hearts. Pretreatment of naive cardiac fibroblasts with apelin (1-100 nM) inhibited Transforming growth factor-ß (TGF-ß)-mediated expression of the myofibroblast marker α-smooth muscle actin (α-SMA) and collagen production. Furthermore, apelin decreased the spontaneous collagen production in cardiac fibroblasts isolated from hearts after aortic banding. Knockdown strategy and pharmacological inhibition revealed that prevention of collagen accumulation by apelin was mediated by a reduction in sphingosine kinase 1 (SphK1) activity. In vivo studies using the aortic banding model indicated that pretreatment with apelin attenuated the development of myocardial fibrotic remodelling and inhibited cardiac SphK1 activity and α-SMA expression. Moreover, administration of apelin 2 weeks after aortic banding prevented cardiac remodelling by inhibiting myocyte hypertrophy, cardiac fibrosis, and ventricular dysfunction. CONCLUSION: Our data provide the first evidence that apelin inhibits TGF-ß-stimulated activation of cardiac fibroblasts through a SphK1-dependent mechanism. We also demonstrated that the administration of apelin during the phase of reactive fibrosis prevents structural remodelling of the myocardium and ventricular dysfunction. These findings may have important implications for designing future therapies for myocardial performance during fibrotic remodelling, affecting the clinical management of patients with progressive heart failure.

Isopropylnorsynephrine is a stronger lipolytic agent in human adipocytes than synephrine and other amines present in Citrus aurantium

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The weight loss observed in consumers of extracts of Citrus aurantium (bitter orange) has been tentatively attributed to the lipolytic and thermogenic effects of the alkaloids abundant in the unripe fruit. Synephrine, octopamine, tyramine, and other alkaloids have been repeatedly identified and quantified in Citrus members of the Rutaceae family or in their extracts incorporated in dietary supplements for weight management. However, there are only scarce reports on their lipolytic action. This study aimed at comparing the acute lipolytic activity of synephrine, octopamine, tyramine, andN-methyltyramine in rat and human adipocytes. Maximal respo (..) (AU)

Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA). ATX is secreted by adipose tissue and its expression is enhanced in obese/insulin-resistant individuals. Here, we analyzed the specific contribution of adipose-ATX to fat expansion associated with nutritional obesity and its consequences on plasma LPA levels. We established ATX(F/F)/aP2-Cre (FATX-KO) transgenic mice carrying a null ATX allele specifically in adipose tissue. FATX-KO mice and their control littermates were fed either a normal or a high-fat diet (HFD) (45% fat) for 13 weeks. FATX-KO mice showed a strong decrease (up to 90%) in ATX expression in white and brown adipose tissue, but not in other ATX-expressing organs. This was associated with a 38% reduction in plasma LPA levels. When fed an HFD, FATX-KO mice showed a higher fat mass and a higher adipocyte size than control mice although food intake was unchanged. This was associated with increased expression of peroxisome proliferator-activated receptor (PPAR)γ2 and of PPAR-sensitive genes (aP2, adiponectin, leptin, glut-1) in subcutaneous white adipose tissue, as well as in an increased tolerance to glucose. These results show that adipose-ATX is a negative regulator of fat mass expansion in response to an HFD and contributes to plasma LPA levels.

Isopropylnorsynephrine is a stronger lipolytic agent in human adipocytes than synephrine and other amines present in Citrus aurantium.

The weight loss observed in consumers of extracts of Citrus aurantium (bitter orange) has been tentatively attributed to the lipolytic and thermogenic effects of the alkaloids abundant in the unripe fruit. Synephrine, octopamine, tyramine, and other alkaloids have been repeatedly identified and quantified in Citrus members of the Rutaceae family or in their extracts incorporated in dietary supplements for weight management. However, there are only scarce reports on their lipolytic action. This study aimed at comparing the acute lipolytic activity of synephrine, octopamine, tyramine, and N-methyltyramine in rat and human adipocytes. Maximal response to the prototypical ß-adrenergic agonist isoprenaline was taken as reference in both species. In rat, octopamine was slightly more active than synephrine while tyramine and N-methyl tyramine did not stimulate-and even inhibited-lipolysis. In human adipocytes, none of these amines stimulated lipolysis when tested up to 10 µg/ml. At higher doses (≥100 µg/ml), tyramine and N-methyl tyramine induced only 20% of the maximal lipolysis and exhibited antilipolytic properties. Synephrine and octopamine were partially stimulatory at high doses. Since synephrine is more abundant than octopamine in C. aurantium, it should be the main responsible for the putative lipolytic action of the extracts claimed to mitigate obesity. Noteworthy, their common isopropyl derivative, isopropylnorsynephrine (also named isopropyloctopamine or betaphrine), was clearly lipolytic: active at 1 µg/ml and reproducing more than 60% of isoprenaline maximal effect in human adipocytes. This compound, not detected in C. aurantium, and which has few reported adverse effects to date, might be useful for in vivo triglyceride breakdown.

Apelin and APJ regulation in adipose tissue and skeletal muscle of type 2 diabetic mice and humans.

Apelin, an adipocyte-secreted factor upregulated by insulin, is increased in adipose tissue (AT) and plasma with obesity. Apelin was recently identified as a new player in the control of glucose homeostasis. However, the regulation of apelin and APJ (apelin receptor) expression in skeletal muscle in relation to insulin resistance or type 2 diabetes is not known. Thus we studied apelin and APJ expression in AT and muscle in different mice models of obesity and in type 2 diabetic patients. In insulin-resistant high-fat (HF)-fed mice, apelin and APJ expression were increased in AT compared with control. This was not the case in AT of highly insulin-resistant db/db mice. In skeletal muscle, apelin expression was similar in control and HF-fed mice and decreased in db/db mice. APJ expression was decreased in both HF-fed and db/db mice. Control subjects and type 2 diabetic patients were subjected to a hyperinsulinemic-euglycemic clamp, and tissues biopsies were obtained before and at the end of the clamp. There was no significant difference in basal apelin and APJ expression in AT and muscle between control and diabetic patients. However, apelin plasma levels were significantly increased in diabetic patients. During the clamp, hyperinsulinemia increased apelin and APJ expression in AT of control but not in diabetic subjects. In muscle, only APJ mRNA levels were increased in control but also in diabetic patients. Taken together, these data show that apelin and APJ expression in mice and humans is regulated in a tissue-dependent manner and according to the severity of insulin resistance.

Chronic benzylamine administration in the drinking water improves glucose tolerance, reduces body weight gain and circulating cholesterol in high-fat diet-fed mice.

Benzylamine is found in Moringa oleifera, a plant used to treat diabetes in traditional medicine. In mammals, benzylamine is metabolized by semicarbazide-sensitive amine oxidase (SSAO) to benzaldehyde and hydrogen peroxide. This latter product has insulin-mimicking action, and is involved in the effects of benzylamine on human adipocytes: stimulation of glucose transport and inhibition of lipolysis. This study examined whether chronic, oral administration of benzylamine could improve glucose tolerance and the circulating lipid profile without increasing oxidative stress in overweight and pre-diabetic mice. The benzylamine diffusion across the intestine was verified using everted gut sacs. Then, glucose handling and metabolic markers were measured in mice rendered insulin-resistant when fed a high-fat diet (HFD) and receiving or not benzylamine in their drinking water (3600micromol/(kgday)) for 17 weeks. HFD-benzylamine mice showed lower body weight gain, fasting blood glucose, total plasma cholesterol and hyperglycaemic response to glucose load when compared to HFD control. In adipocytes, insulin-induced activation of glucose transport and inhibition of lipolysis remained unchanged. In aorta, benzylamine treatment partially restored the nitrite levels that were reduced by HFD. In liver, lipid peroxidation markers were reduced. Resistin and uric acid, surrogate plasma markers of metabolic syndrome, were decreased. In spite of the putative deleterious nature of the hydrogen peroxide generated during amine oxidation, and in agreement with its in vitro insulin-like actions found on adipocytes, the SSAO-substrate benzylamine could be considered as a potential oral agent to treat metabolic syndrome.