Neira Sáinz

Acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes

Objectives:The orexigenic hormone ghrelin circulates mainly in two forms, acylated and desacyl ghrelin. We evaluated the impact of obesity and obesity-associated type 2 diabetes (T2D) on ghrelin forms and the potential role of acylated and desacyl ghrelin in the control of adipogenesis in humans.Methods:Plasma concentrations of the different ghrelin forms were measured in 80 subjects. The expression of the ghrelin receptor (growth hormone secretagogue receptor, GHS-R) was analyzed in omental adipose tissue using western blot and immunohistochemistry, and the effect of acylated ghrelin and desacyl ghrelin (0.1–1000 pmol l−1) on adipogenesis was determined in vitro in omental adipocytes.Results:Circulating concentrations of acylated ghrelin were increased, whereas desacyl ghrelin levels were decreased, in obesity and obesity-associated T2D. Body mass index, waist circumference, insulin and HOMA (homeostasis model assessment) index were positively correlated with acylated ghrelin levels. Obese individuals showed a lower protein expression of GHS-R in omental adipose tissue. In differentiating omental adipocytes, incubation with both acylated and desacyl ghrelin significantly increased PPARγ (peroxisome proliferator-activated receptor γ) and SREBP1 (sterol-regulatory element binding protein-1) mRNA levels, as well as several fat storage-related proteins, including acetyl-CoA carboxylase, fatty acid synthase, lipoprotein lipase and perilipin. Consequently, both the ghrelin forms stimulated intracytoplasmatic lipid accumulation.Conclusions:Both acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes. Given the lipogenic effect of acylated ghrelin on visceral adipocytes, the herein-reported elevation of its circulating concentrations in obese individuals may play a role in excessive fat accumulation in obesity.

BAT: a new target for human obesity?

Two types of adipose tissue can be distinguished histologically and functionally: white (WAT) and brown adipose tissue (BAT). Whereas BAT is specialized in the production of heat, WAT stores excess energy as triacylglycerols. BAT is present throughout life in rodents, whereas in humans it was thought to involute rapidly postnatally, having essentially disappeared within the first years after birth. However, positron emission tomography has provided evidence that adults retain metabolically active BAT depots that can be induced in response to cold and sympathetic nervous system activation. These findings together with the recent identification of specific molecular determinants (PRDM16 and BMP7) activating brown adipogenesis highlights BAT as a potential relevant target for pharmacological and gene expression manipulation to combat human obesity.

Leptin resistance and diet-induced obesity: central and peripheral actions of leptin

Obesity is a chronic disease that represents one of the most serious global health burdens associated to an excess of body fat resulting from an imbalance between energy intake and expenditure, which is regulated by environmental and genetic interactions. The adipose-derived hormone leptin acts via a specific receptor in the brain to regulate energy balance and body weight, although this protein can also elicit a myriad of actions in peripheral tissues. Obese individuals, rather than be leptin deficient, have in most cases, high levels of circulating leptin. The failure of these high levels to control body weight suggests the presence of a resistance process to the hormone that could be partly responsible of disturbances on body weight regulation. Furthermore, leptin resistance can impair physiological peripheral functions of leptin such as lipid and carbohydrate metabolism and nutrient intestinal utilization. The present document summarizes those findings regarding leptin resistance development and the role of this hormone in the development and maintenance of an obese state. Thus, we focused on the effect of the impaired leptin action on adipose tissue, liver, skeletal muscle and intestinal function and the accompanying relationships with diet-induced obesity. The involvement of some inflammatory mediators implicated in the development of obesity and their roles in leptin resistance development are also discussed.

Leptin Administration Favors Muscle Mass Accretion by Decreasing FoxO3a and Increasing PGC-1α in ob/ob Mice

Absence of leptin has been associated with reduced skeletal muscle mass in leptin-deficient ob/ob mice. The aim of our study was to examine the effect of leptin on the catabolic and anabolic pathways regulating muscle mass. Gastrocnemius, extensor digitorum longus and soleus muscle mass as well as fiber size were significantly lower in ob/ob mice compared to wild type littermates, being significantly increased by leptin administration (P<0.001). This effect was associated with an inactivation of the muscle atrophy-related transcription factor forkhead box class O3 (FoxO3a) (P<0.05), and with a decrease in the protein expression levels of the E3 ubiquitin-ligases muscle atrophy F-box (MAFbx) (P<0.05) and muscle RING finger 1 (MuRF1) (P<0.05). Moreover, leptin increased (P<0.01) protein expression levels of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a regulator of muscle fiber type, and decreased (P<0.05) myostatin protein, a negative regulator of muscle growth. Leptin administration also activated (P<0.01) the regulators of cell cycle progression proliferating cell nuclear antigen (PCNA) and cyclin D1, and increased (P<0.01) myofibrillar protein troponin T. The present study provides evidence that leptin treatment may increase muscle mass of ob/ob mice by inhibiting myofibrillar protein degradation as well as enhancing muscle cell proliferation.

Association of plasma acylated ghrelin with blood pressure and left ventricular mass in patients with metabolic syndrome.

Objective The gut-derived hormone, ghrelin, improves cardiac function in healthy individuals and patients with chronic heart failure. The aim of this study was to investigate whether the major isoforms of the hormone, acylated and desacyl ghrelin, are related to inappropriate left ventricular mass in patients with the metabolic syndrome (MetS). Methods and results Plasma concentrations of ghrelin forms were measured in 180 white participants (65 normal weight, 60 obese without MetS and 55 obese with MetS; 56% men). MetS was defined according to Adult Treatment Panel III criteria. The presence of left ventricular hypertrophy (LVH) was diagnosed by sex-specific left ventricular mass/height2.7 cut-off values (>49.2 g/m2.7 for men and >46.7 g/m2.7 for women). Circulating concentrations of acylated ghrelin were increased in obesity and MetS, whereas desacyl ghrelin levels were decreased. Compared with participants in the lowest tertiles, the age-adjusted and sex-adjusted odds of having MetS were lower in the highest category of desacyl ghrelin (odds ratio 0.1, 95% confidence interval 0.1–0.4, P < 0.001). The prevalence of LVH was increased in the highest tertile of acylated ghrelin (odds ratio 3.4, 95% confidence interval 1.7–5.6, P < 0.05). Plasma acylated ghrelin was increased (P < 0.05) in patients with MetS exhibiting LVH compared with those with appropriate left ventricular mass, whereas plasma desacyl ghrelin was not changed (P = 0.490). Conclusion Acylated ghrelin was positively associated with SBP and left ventricular mass indices, even after correction for BMI. These results suggest that the increased acylated ghrelin concentrations may represent a compensatory mechanism to overcome the development of hypertension and LVH in patients with MetS.

Leptin administration activates irisin-induced myogenesis via nitric oxide-dependent mechanisms, but reduces its effect on subcutaneous fat browning in mice

Background/objetives:Obese leptin-deficient ob/ob mice exhibit high adiposity and reduced muscle mass with leptin replacement promoting weight loss and inducing muscle accretion through PGC-1α-dependent mechanisms. Our aim was to analyze in vivo and in vitro the effect of leptin on FNDC5, a novel PGC-1α-dependent myokine that is synthesized and cleaved to form irisin that induces white adipose browning.Methods/results:Twelve-week-old male wild-type and ob/ob mice were divided in three groups as follows: control, leptin-treated (1 mg kg−1 day−1) and pair-fed. Leptin administration was associated with increased gastrocnemius weight and cell surface area, higher Pgc1a and Fndc5 transcript levels and a slight increase in circulating irisin. Leptin upregulated Fndc5 expression through nitric oxide (NO)-dependent mechanisms in murine C2C12 myocytes and stimulated both basal and irisin-stimulated myogenesis, as evidenced by increased myocyte cell proliferation, higher myogenin and myonectin transcript levels together with lower mRNA expression of myostatin and dystrophin and the muscle atrophy-related factors MuRF1 and MAFbx. Interestingly, leptin downregulated Fndc5 expression in a NO-independent manner in murine differentiated subcutaneous adipocytes. Furthermore, leptin prevented the irisin-induced upregulation of both brown (Ucp1 and Cidec) and beige (Tmem26) adipocyte-specific genes and the increase in uncoupling protein-1-positive cells.Conclusions:Taken together, our results provide evidence for a regulatory role of leptin on FNDC5/irisin, favoring muscle accretion but reducing fat browning.

The inhibitory effect of leptin on angiotensin II-induced vasoconstriction is blunted in spontaneously hypertensive rats.

Objective Leptin attenuates the angiotensin II-induced increase of cytosolic calcium ([Ca2+]i) and vasoconstriction in the aorta of normotensive Wistar rats. To determine whether these effects may be altered in hypertension, we assessed the effect of leptin on angiotensin II-induced vascular response in the aorta of 10-week-old spontaneously hypertensive rats (SHR). Methods Contractile responses to angiotensin II (100 nmol/l) in the presence of different concentrations of leptin (0.1, 1, 10, 100 nmol/l) were evaluated in isolated aortic rings by the organ bath system. [Ca2+]i was measured in vascular smooth muscle cells (VSMCs) using Fura-2 fluorescence. The expression of the short (OB-Ra) and long (OB-Rb) isoforms of the leptin receptor in VSMCs was evaluated by real-time reverse transcriptase-polymerase chain reaction and western-blot analysis. Results Circulating leptin concentrations were increased in SHR. Serum metabolic parameters, including glucose, insulin, total cholesterol and triglyceride levels, were also elevated in SHR. Leptin did not modify the angiotensin II-induced vasoconstriction in SHR either in intact or endothelium-denuded aortic rings. In addition, leptin was not able either to diminish the angiotensin II-induced the peak rise of [Ca2+]i or to accelerate the recovery rate to basal calcium levels in VSMCs from SHR. However, OB-Ra and OB-Rb mRNA and protein expression were increased in SHR VSMCs. Conclusions The lack of effect of leptin on angiotensin II-induced contraction in the aorta of SHR is due to an impaired handling of [Ca2+]i in VSMCs. Hyperleptinemia and overexpression of OB-R in VSMCs could be compensatory mechanisms against VSMC leptin resistance in genetically hypertensive rats.

Osteopontin Deletion Prevents the Development of Obesity and Hepatic Steatosis via Impaired Adipose Tissue Matrix Remodeling and Reduced Inflammation and Fibrosis in Adipose Tissue and Liver in Mice

Osteopontin (OPN) is a multifunctional extracellular matrix (ECM) protein involved in multiple physiological processes. OPN expression is dramatically increased in visceral adipose tissue in obesity and the lack of OPN protects against the development of insulin resistance and inflammation in mice. We sought to unravel the potential mechanisms involved in the beneficial effects of the absence of OPN. We analyzed the effect of the lack of OPN in the development of obesity and hepatic steatosis induced by a high-fat diet (HFD) using OPN-KO mice. OPN expression was upregulated in epididymal white adipose tissue (EWAT) and liver in wild type (WT) mice with HFD. OPN-KO mice had higher insulin sensitivity, lower body weight and fat mass with reduced adipose tissue ECM remodeling and reduced adipocyte size than WT mice under a HFD. Reduced MMP2 and MMP9 activity was involved in the decreased ECM remodeling. Crown-like structure number in EWAT as well as F4/80-positive cells and Emr1 expression in EWAT and liver increased with HFD, while OPN-deficiency blunted the increase. Moreover, our data show for the first time that OPN-KO under a HFD mice display reduced fibrosis in adipose tissue and liver, as well as reduced oxidative stress in adipose tissue. Gene expression of collagens Col1a1, Col6a1 and Col6a3 in EWAT and liver, as well as the profibrotic cytokine Tgfb1 in EWAT were increased with HFD, while OPN-deficiency prevented this increase. OPN deficiency prevented hepatic steatosis via reduction in the expression of molecules involved in the onset of fat accumulation such as Pparg, Srebf1, Fasn, Mogat1, Dgat2 and Cidec. Furthermore, OPN-KO mice exhibited higher body temperature and improved BAT function. The present data reveal novel mechanisms of OPN in the development of obesity, pointing out the inhibition of OPN as a promising target for the treatment of obesity and fatty liver.

Deletion of Inducible Nitric-Oxide Synthase in Leptin- Deficient Mice Improves Brown Adipose Tissue Function

Background Leptin and nitric oxide (NO) on their own participate in the control of non-shivering thermogenesis. However, the functional interplay between both factors in this process has not been explored so far. Therefore, the aim of the present study was to analyze the impact of the absence of the inducible NO synthase (iNOS) gene in the regulation of energy balance in ob/ob mice. Methods and Findings Double knockout (DBKO) mice simultaneously lacking the ob and iNOS genes were generated, and the expression of molecules involved in the control of brown fat cell function was analyzed by real-time PCR, western-blot and immunohistochemistry. Twelve week-old DBKO mice exhibited reduced body weight (p<0.05), decreased amounts of total fat pads (p<0.05), lower food efficiency rates (p<0.05) and higher rectal temperature (p<0.05) than ob/ob mice. Ablation of iNOS also improved the carbohydrate and lipid metabolism of ob/ob mice. DBKO showed a marked reduction in the size of brown adipocytes compared to ob/ob mutants. In this sense, in comparison to ob/ob mice, DBKO rodents showed an increase in the expression of PR domain containing 16 (Prdm16), a transcriptional regulator of brown adipogenesis. Moreover, iNOS deletion enhanced the expression of mitochondria-related proteins, such as peroxisome proliferator-activated receptor γ coactivator-1 α (Pgc-1α), sirtuin-1 (Sirt-1) and sirtuin-3 (Sirt-3). Accordingly, mitochondrial uncoupling proteins 1 and 3 (Ucp-1 and Ucp-3) were upregulated in brown adipose tissue (BAT) of DBKO mice as compared to ob/ob rodents. Conclusion Ablation of iNOS improved the energy balance of ob/ob mice by decreasing food efficiency through an increase in thermogenesis. These effects may be mediated, in part, through the recovery of the BAT phenotype and brown fat cell function improvement.

Leptin Administration Downregulates the Increased Expression Levels of Genes Related to Oxidative Stress and Inflammation in the Skeletal Muscle of ob/ob Mice

Obese leptin-deficient ob/ob mice exhibit a low-grade chronic inflammation together with a low muscle mass. Our aim was to analyze the changes in muscle expression levels of genes related to oxidative stress and inflammatory responses in leptin deficiency and to identify the effect of in vivo leptin administration. Ob/ob mice were divided in three groups as follows: control ob/ob, leptin-treated ob/ob (1 mg/kg/d) and leptin pair-fed ob/ob mice. Gastrocnemius weight was lower in control ob/ob than in wild type mice (P < .01) exhibiting an increase after leptin treatment compared to control and pair-fed (P < .01) ob/ob animals. Thiobarbituric acid reactive substances, markers of oxidative stress, were higher in serum (P < .01) and gastrocnemius (P = .05) of control ob/ob than in wild type mice and were significantly decreased (P < .01) by leptin treatment. Leptin deficiency altered the expression of 1,546 genes, while leptin treatment modified the regulation of 1,127 genes with 86 of them being involved in oxidative stress, immune defense and inflammatory response. Leptin administration decreased the high expression of Crybb1, Hspb3, Hspb7, Mt4, Cat, Rbm9, Serpinc1 and Serpinb1a observed in control ob/ob mice, indicating that it improves inflammation and muscle loss.