Preparation of Superactive Prolactin Receptor Antagonists.

Most breast cancer deaths are caused by malignant estrogen receptor-positive breast tumors that later recur as metastatic disease. Prolactin (PRL) has been documented as a factor promoting breast cancer development and metastasis. We therefore developed superactive prolactin receptor (PRLR) antagonists aimed at blocking PRL action. We purified 12 novel mutants to homogeneity as monomers, and the most potent antagonist was over 95-fold more active than the previously reported weak antagonist, the mutant Del 1-9 human PRL G129R. This enhanced antagonistic activity resulted mostly from prolonged interaction with the extracellular domain (ECD) of PRLR. All mutants were properly refolded, as indicated by interaction with human PRLR-ECD and by circular dichroism analysis. We then prepared monopegylated variants of the most active mutants to extend their biological half-life in vivo.

A Leptin Receptor Antagonist Attenuates Adipose Tissue Browning and Muscle Wasting in Infantile Nephropathic Cystinosis-Associated Cachexia.

Mice lacking the functional cystinosin gene (Ctns-/-), a model of infantile nephropathic cystinosis (INC), exhibit the cachexia phenotype with adipose tissue browning and muscle wasting. Elevated leptin signaling is an important cause of chronic kidney disease-associated cachexia. The pegylated leptin receptor antagonist (PLA) binds to but does not activate the leptin receptor. We tested the efficacy of this PLA in Ctns-/- mice. We treated 12-month-old Ctns-/- mice and control mice with PLA (7 mg/kg/day, IP) or saline as a vehicle for 28 days. PLA normalized food intake and weight gain, increased fat and lean mass, decreased metabolic rate and improved muscle function. It also attenuated perturbations of energy homeostasis in adipose tissue and muscle in Ctns-/- mice. PLA attenuated adipose tissue browning in Ctns-/- mice. PLA increased gastrocnemius weight and fiber size as well as attenuated muscle fat infiltration in Ctns-/- mice. This was accompanied by correcting the increased expression of muscle wasting signaling while promoting the decreased expression of myogenesis in gastrocnemius of Ctns-/- mice. PLA attenuated aberrant expressed muscle genes that have been associated with muscle atrophy, increased energy expenditure and lipolysis in Ctns-/- mice. Leptin antagonism may represent a viable therapeutic strategy for adipose tissue browning and muscle wasting in INC.

COVID-19 Severity in Obesity: Leptin and Inflammatory Cytokine Interplay in the Link Between High Morbidity and Mortality.

Obesity is one of the foremost risk factors in coronavirus infection resulting in severe illness and mortality as the pandemic progresses. Obesity is a well-known predisposed chronic inflammatory condition. The dynamics of obesity and its impacts on immunity may change the disease severity of pneumonia, especially in acute respiratory distress syndrome, a primary cause of death from SARS-CoV-2 infection. The adipocytes of adipose tissue secret leptin in proportion to individuals' body fat mass. An increase in circulating plasma leptin is a typical characteristic of obesity and correlates with a leptin-resistant state. Leptin is considered a pleiotropic molecule regulating appetite and immunity. In immunity, leptin functions as a cytokine and coordinates the host's innate and adaptive responses by promoting the Th1 type of immune response. Leptin induced the proliferation and functions of antigen-presenting cells, monocytes, and T helper cells, subsequently influencing the pro-inflammatory cytokine secretion by these cells, such as TNF-α, IL-2, or IL-6. Leptin scarcity or resistance is linked with dysregulation of cytokine secretion leading to autoimmune disorders, inflammatory responses, and increased susceptibility towards infectious diseases. Therefore, leptin activity by leptin long-lasting super active antagonist's dysregulation in patients with obesity might contribute to high mortality rates in these patients during SARS-CoV-2 infection. This review systematically discusses the interplay mechanism between leptin and inflammatory cytokines and their contribution to the fatal outcomes in COVID-19 patients with obesity.

Technical note: Effects of pegylation and route of administration on leptin kinetics in newborn lambs1.

Chronic energy insufficiency resulting from inadequate feed intake or increased nutrient demand reduces plasma leptin in ruminants. Treatment of energy-deficient ruminants with exogenous leptin has identified some physiological consequences of reduced plasma leptin, but their full complement remains unknown. Additional leptin-dependent responses could be identified by using strategies that interfere with leptin signaling such as administration of leptin mutants that act as competitive antagonists. The effectiveness of these antagonists depends on their fold excess over endogenous leptin, and this condition can be achieved under in vivo conditions by extending the half-life (t1/2) of the antagonist by addition of a polyethylene glycol (PEG) molecule (pegylation). Use of this approach in ruminants, however, is limited by the lack of information on the t1/2 of native and pegylated leptin and on the most effective route of administration. To answer these questions, newborn lambs (n = 3) were injected with an intravenous (i.v.) bolus of 150 µg of human leptin followed by blood sampling over the next 12 h. Analysis of the semilog plasma leptin concentration over time yielded a t1/2 of 43 ± 4.9 min; an i.v. bolus of 276 µg of bovine leptin yielded a comparable t1/2 (P > 0.05). Next, newborn lambs (n = 4) received a single dose of 229 µg/kg of metabolic body weight (BW0.75) of pegylated super human leptin antagonist (PEG-SHLA) via the i.v. or subcutaneous (s.c.) route. Plasma PEG-SHLA concentration reached a peak of 1,528 ± 78 ng/mL after 1 min and a nadir of 71 ± 9 ng/mL after 24 h with the i.v. route versus a peak of 423 ± 43 ng/mL after 300 min and a nadir of 146 ± 22 ng/mL after 24 h for the s.c. route; the t1/2 of PEG-SHLA was 394 ± 29 min for the i.v. route and 433 ± 58 min for the s.c. route. Finally, plasma concentration of PEG-SHLA was modeled when given either i.v. or s.c. at a dose of 229 µg/kg BW0.75 every 12 h. Once a steady state was reached, peak and lowest concentrations PEG-SHLA over the 12-h windows were 2,269 and 403 ng/mL for the i.v. route and 814 and 555 ng/mL for the s.c. route. Weighted PEG-SHLA concentrations over the 12-h period were 1,455 and 713 ng/mL for the i.v. and s.c. route, translating into 364- and 178-fold excess over endogenous plasma leptin. These data confirm the effectiveness of pegylation in extending the t1/2 of leptin antagonists in newborn lambs and in increasing their circulation in fold excess over endogenous leptin.

Pegylated Human Leptin D23L Mutant-Preparation and Biological Activity In Vitro and In Vivo in Male ob/ob Mice.

Recombinant monomeric human leptin (hLEP) and its D23L mutant were prepared in Escherichia coli and pegylated at their N-terminus using 20-kDa methoxy pegylated (PEG)-propionylaldehyde. As determined by both SDS-PAGE and size-exclusion chromatography, the pegylated proteins consisted of >90% monopegylated and <10% double-pegylated species. Circular dichroism spectra showed that their secondary structure, characteristic of all four α-helix bundle cytokines, was not affected by either the D23L mutation or pegylation. Because of the D23L mutation, affinity for hLEP receptor increased 25- and 40-fold for the pegylated and nonpegylated mutant, respectively. However, whereas the proliferation-promoting activity in vitro of nonmutated and mutated nonpegylated hLEP was identical, that of the respective pegylated mutant was approximately sixfold higher compared with the pegylated nonmutated hLEP. This difference was also seen in vivo. Both pegylated hLEPs at all doses significantly decreased body weight and food consumption, as compared with the vehicle-treated control. Once-daily administration of pegylated hLEP D23L at doses of 0.1, 0.3, and 1 mg/kg for 14 consecutive days in ob/ob mice resulted in significantly decreased body weight and food consumption as compared with respective pegylated hLEP-treated animals, with the biggest difference observed at 0.1 mg/kg. Repeated administration of either pegylated hLEP D23L or pegylated hLEP significantly decreased blood glucose levels compared with the control before glucose challenge and after oral glucose tolerance test, but with no difference between the two treatments. The pegylated hLEP D23L mutant seems to be a more potent reagent suitable for in vivo studies than the pegylated nonmutated hLEP.

Preparation of biologically active monomeric recombinant zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) recombinant growth hormones.

Fish growth hormones (GHs) play an important role in regulating growth, metabolism, reproduction, osmoregulation, and immunity and have thus garnered attention for their application in aquaculture. Zebrafish GH (zGH) cDNA or rainbow trout GH (rtGH) cDNA was cloned into the pMon3401 vector, expressed in MON105-competent Escherichia coli and purified to homogeneity. Their biological activity was evidenced by their ability to interact with ovine GH receptor extracellular domain and stimulate GH receptor-mediated proliferation in FDC-P1-3B9 cells stably transfected with rabbit GH receptor. The relative affinity of zGH and rtGH, estimated by IC50, was about 38-fold and 512-fold lower, respectively, than ovine GH. This is likely the reason for the low biological activity in cells with rabbit GH receptor, ~ 36-fold lower for zGH and ~ 107-fold lower for rtGH than for human GH. This was not due to improper refolding, as evidenced by circular dichroism analysis. Predicting the activity of fish GHs is problematic as there is no one single optimal in vitro bioassay; heterologous assays may be ambiguous, and only homologous assays are suitable for measuring activity.

New reagents for poultry research: preparation, purification, and in vitro evaluation of non-PEGylated and mono-PEGylated chicken prolactin.

Recombinant chicken prolactin (chPRL), expressed in Escherichia coli and purified as a monomer, was successfully PEGylated and purified to homogeneity as a mono-PEGylated protein (PEG-chPRL). Its biological activity was estimated by its ability to interact with human prolactin receptor extracellular domain (hPRLR-ECD) and stimulate PRLR-mediated proliferation in Nb2-11C cells. PEG-chPRL activity in a cell bioassay was 10-fold lower than that of non-PEGylated chPRL, but only 2-fold lower in a binding assay to hPRLR-ECD. The CD spectra of non-PEGylated and PEGylated chPRL were almost identical and similar to that of hPRL, indicating proper refolding. Although the PEGylation of chPRL resulted in lower activity in vitro, PEG-chPRL was absorbed more slowly than chPRL, remained in the circulation 16 h longer. Furthermore the effects of PEG-chPRL injections in chickens on subsequent corticosteroid levels in blood were significantly profound compared to chPRL. These favorable PEGylation-induced pharmacokinetic alterations should improve efficacy of PEG-chPRL in in vivo experiments, as dosing frequency can be reduced due to its prolonged persistence in the circulation, and thus reduce the frequency of dosing. Furthermore, hydrophobic interaction chromatography was successfully adopted to isolate PEG-chPRL as a better alternative for separation of PEGylated PRL, and is likely to be successfully applicable to other proteins.

Hyperglycemia drives intestinal barrier dysfunction and risk for enteric infection.

Obesity, diabetes, and related manifestations are associated with an enhanced, but poorly understood, risk for mucosal infection and systemic inflammation. Here, we show in mouse models of obesity and diabetes that hyperglycemia drives intestinal barrier permeability, through GLUT2-dependent transcriptional reprogramming of intestinal epithelial cells and alteration of tight and adherence junction integrity. Consequently, hyperglycemia-mediated barrier disruption leads to systemic influx of microbial products and enhanced dissemination of enteric infection. Treatment of hyperglycemia, intestinal epithelial-specific GLUT2 deletion, or inhibition of glucose metabolism restores barrier function and bacterial containment. In humans, systemic influx of intestinal microbiome products correlates with individualized glycemic control, indicated by glycated hemoglobin levels. Together, our results mechanistically link hyperglycemia and intestinal barrier function with systemic infectious and inflammatory consequences of obesity and diabetes.

Preparation of Potent Leptin Receptor Antagonists and Their Therapeutic Use in Mouse Models of Uremic Cachexia and Kidney Fibrosis.

Leptin antagonists (L39A/D40A/F4lA mutants) of mouse, human, rat and ovine leptins were developed in our laboratory by rational mutagenesis, expressed in Escherichia coli, refolded and purified to homogeneity. Pegylation of these antagonists resulted in long-acting reagents suitable for in-vivo studies. Further selection of high-affinity leptin antagonists was achieved by random mutagenesis of the whole open reading frame followed by yeast- surface display; an additional mutation (D23L) increased their affinity toward leptin receptor 60-fold. This superactive pegylated mouse leptin antagonist (PLA) exhibited a strong orexigenic effect, leading, in 10-14 days, to a 40% increase in body weight resulting mainly from obesity; this was reversed once PLA treatment was ceased. Cachexia is common in patients with Chronic Kidney Disease (CKD). Our studies suggested that leptin mediates cachexia by decreasing food intake while increasing energy consumption in CKD mice. We showed that PLA ameliorates CKD-associated cachexia in mice. Leptin may also contribute to the development of muscle and renal fibrosis in CKD, serious complications associated with increased morbidity and mortality. Transforming growth factor (TGF)-ß signaling may be the most potent mediator of fibrogenesis in multiple organs, and leptin is a co-activator of TGF-ß. Muscle fibrosis was evident in our CKD mice and PLA treatment significantly reduced the mRNA levels of TGF- ß1 and its downstream targets in their muscle and renal tissues. PLA may offer a novel therapeutic strategy for CKD-associated cachexia, muscle and renal fibrosis to improve CKD patients' survival and quality of life.

Novel reagents for human prolactin research: large-scale preparation and characterization of prolactin receptor extracellular domain, non-pegylated and pegylated prolactin and prolactin receptor antagonist.

To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for large-scale preparation of untagged human PRL (hPRL), a hPRL antagonist (del 1-9-G129R hPRL) that acts as a pure antagonist of hPRL in binding to hPRL receptor extracellular domain (hPRLR-ECD), and hPRLR-ECD are demonstrated. The interaction of del 1-9-G129R hPRL with hPRLR-ECD was demonstrated by competitive non-radioactive binding assay using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, by formation of stable 1:1 complexes with hPRLR-ECD under non-denaturing conditions using size-exclusion chromatography, and by surface plasmon resonance methodology. In all three types of experiments, the interaction of del 1-9-G129R hPRL was equal to that of unmodified hPRL. Del 1-9-G129R hPRL inhibited the hPRL-induced proliferation of Baf/LP cells stably expressing hPRLR. Overall, the biological properties of del 1-9-G129R hPRL prepared by the protocol described herein were similar to those of the antagonist prepared using the protocol reported in the original study; however, the newly described protocol improved yields by >6-fold. To provide long-lasting hPRL as a new reagent needed for in vivo experiments, we prepared its mono-pegylated analogue and found that pegylation lowers its biological activity in a homologous in vitro assay. As its future use will require the development of a PRL antagonist with highly elevated affinity, del 1-9-G129R hPRL was expressed on the surface of yeast cells. It retained its binding capacity for hPRLR-ECD, and this methodology was shown to be suitable for future development of high-affinity hPRL antagonists using a library of randomly mutated open reading frame of del 1-9-G129R hPRL and selecting high-affinity mutants by yeast surface display methodology.

Comparison of in vitro bioactivity of chicken prolactin and mammalian lactogenic hormones.

Recombinant chicken prolactin, expressed in Escherichia coli as an unfolded protein, was successfully refolded and purified to homogeneity as a monomeric protein. Its biological activity was evidenced by its ability to interact with rabbit prolactin receptor extracellular domain and stimulate prolactin receptor-mediated proliferation in three cell types possessing mammalian prolactin receptors. Chicken prolactin activity in those assays was 20-100-fold lower than that of mammalian lactogenic hormones, likely due to lower affinity for mammalian prolactin receptors and not to improper refolding, because in two homologous bioassays, chicken prolactin activity was equal to or higher than that of ovine prolactin and the CD spectra of chicken and human prolactin were almost identical. Our results using seven mammalian lactogenic hormones from five species in three bioassays revealed the major role of species specificity in testing biological activity in vitro. Heterologous bioassays may be misleading and homologous assays are strongly recommended for predicting the activity of species-specific lactogenic hormones in vivo.

Persistent microbiome alterations modulate the rate of post-dieting weight regain.

In tackling the obesity pandemic, considerable efforts are devoted to the development of effective weight reduction strategies, yet many dieting individuals fail to maintain a long-term weight reduction, and instead undergo excessive weight regain cycles. The mechanisms driving recurrent post-dieting obesity remain largely elusive. Here we identify an intestinal microbiome signature that persists after successful dieting of obese mice and contributes to faster weight regain and metabolic aberrations upon re-exposure to obesity-promoting conditions. Faecal transfer experiments show that the accelerated weight regain phenotype can be transmitted to germ-free mice. We develop a machine-learning algorithm that enables personalized microbiome-based prediction of the extent of post-dieting weight regain. Additionally, we find that the microbiome contributes to diminished post-dieting flavonoid levels and reduced energy expenditure, and demonstrate that flavonoid-based 'post-biotic' intervention ameliorates excessive secondary weight gain. Together, our data highlight a possible microbiome contribution to accelerated post-dieting weight regain, and suggest that microbiome-targeting approaches may help to diagnose and treat this common disorder.

Local Application of Leptin Antagonist Attenuates Angiotensin II-Induced Ascending Aortic Aneurysm and Cardiac Remodeling.

BACKGROUND: Ascending thoracic aortic aneurysm (ATAA) is driven by angiotensin II (AngII) and contributes to the development of left ventricular (LV) remodeling through aortoventricular coupling. We previously showed that locally available leptin augments AngII-induced abdominal aortic aneurysms in apolipoprotein E-deficient mice. We hypothesized that locally synthesized leptin mediates AngII-induced ATAA. METHODS AND RESULTS: Following demonstration of leptin synthesis in samples of human ATAA associated with different etiologies, we modeled in situ leptin expression in apolipoprotein E-deficient mice by applying exogenous leptin on the surface of the ascending aorta. This treatment resulted in local aortic stiffening and dilation, LV hypertrophy, and thickening of aortic/mitral valve leaflets. Similar results were obtained in an AngII-infusion ATAA mouse model. To test the dependence of AngII-induced aortic and LV remodeling on leptin activity, a leptin antagonist was applied to the ascending aorta in AngII-infused mice. Locally applied single low-dose leptin antagonist moderated AngII-induced ascending aortic dilation and protected mice from ATAA rupture. Furthermore, LV hypertrophy was attenuated and thickening of aortic valve leaflets was moderated. Last, analysis of human aortic valve stenosis leaflets revealed de novo leptin synthesis, whereas exogenous leptin stimulated proliferation and promoted mineralization of human valve interstitial cells in culture. CONCLUSIONS: AngII-induced ATAA is mediated by locally synthesized leptin. Aortoventricular hemodynamic coupling drives LV hypertrophy and promotes early aortic valve lesions, possibly mediated by valvular in situ leptin synthesis. Clinical implementation of local leptin antagonist therapy may attenuate AngII-induced ATAA and moderate related LV hypertrophy and pre-aortic valve stenosis lesions. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov/. Unique identifier: NCT00449306.

Central Resistin/TLR4 Impairs Adiponectin Signaling, Contributing to Insulin and FGF21 Resistance.

Adiponectin, an insulin-sensitizing hormone, and resistin, known to promote insulin resistance, constitute a potential link between obesity and type 2 diabetes. In addition, fibroblast growth factor (FGF)21 has effects similar to those of adiponectin in regulating glucose and lipid metabolism and insulin sensitivity. However, the interplay between adiponectin, FGF21, and resistin signaling pathways during the onset of insulin resistance is unknown. Here, we investigated whether central resistin promotes insulin resistance through the impairment of adiponectin and FGF21 signaling. We show that chronic intracerebroventricular resistin infusion downregulated both hypothalamic and hepatic APPL1, a key protein in adiponectin signaling, associated with decreased Akt-APPL1 interaction and an increased Akt association with its endogenous inhibitor tribbles homolog 3. Resistin treatment also decreased plasma adiponectin levels and reduced both hypothalamic and peripheral expression of adiponectin receptors. Additionally, we report that intracerebroventricular resistin increased plasma FGF21 levels and downregulated its receptor components in the hypothalamus and peripheral tissues, promoting FGF21 resistance. Interestingly, we also show that resistin effects were abolished in TLR4 knockout mice and in neuronal cells expressing TLR4 siRNAs. Our study reveals a novel mechanism of insulin resistance onset orchestrated by a central resistin-TLR4 pathway that impairs adiponectin signaling and promotes FGF21 resistance.

Identification of functional leptin receptors expressed in ventricular mitochondria.

Leptin is a 16 kDa pro-satiety peptide produced primarily not only by white adipocytes but also by numerous other tissues including the heart. Circulating leptin exerts its effect through specific receptors, although its principle actions are dependent on the activation of the long form of the leptin receptor, termed OBRb. As leptin is also produced within the cardiomyocyte, we hypothesized that the peptide can also exert effects by targeting intracellular sites. Accordingly, we determined whether cardiac mitochondria express functional leptin receptors. The presence of mitochondrial OBRb was identified through Western blotting of isolated mitochondria, immunofluorescence as well as immunogold labeling with electron microscopy. Although leptin had no direct effect on mitochondrial integrity, it profoundly enhanced the ability of calcium to induce mitochondrial swelling, an effect partially reversed by an OBR antagonist. 24 h exposure to leptin (50 ng/ml) was without effect on mitochondria in cultured neonatal rat ventricular myocytes in contrast to leptin tagged with a 10 amino acid membrane translocation sequence which significantly induced mitochondrial permeability transition pore opening, whereas both leptins produced a hypertrophic response. Our results therefore show that mitochondria express functional OBR which may be of importance toward understanding the role of intracellularly derived leptin in cardiac physiology and pathology.

Diet-induced obese mice retain endogenous leptin action.

Obesity is characterized by hyperleptinemia and decreased response to exogenous leptin. This has been widely attributed to the development of leptin resistance, a state of impaired leptin signaling proposed to contribute to the development and persistence of obesity. To directly determine endogenous leptin activity in obesity, we treated lean and obese mice with a leptin receptor antagonist. The antagonist increased feeding and body weight (BW) in lean mice, but not in obese models of leptin, leptin receptor, or melanocortin-4 receptor deficiency. In contrast, the antagonist increased feeding and BW comparably in lean and diet-induced obese (DIO) mice, an increase associated with decreased hypothalamic expression of Socs3, a primary target of leptin. These findings demonstrate that hyperleptinemic DIO mice retain leptin suppression of feeding comparable to lean mice and counter the view that resistance to endogenous leptin contributes to the persistence of DIO in mice.

Blockage of the Neonatal Leptin Surge Affects the Gene Expression of Growth Factors, Glial Proteins, and Neuropeptides Involved in the Control of Metabolism and Reproduction in Peripubertal Male and Female Rats.

Leptin (Lep) is important in the development of neuroendocrine circuits involved in metabolic control. Because both Lep and metabolism influence pubertal development, we hypothesized that early changes in Lep signaling could also modulate hypothalamic (HT) systems involved in reproduction. We previously demonstrated that a single injection of a Lep antagonist (Antag) on postnatal day (PND)9, coincident with the neonatal Lep peak, induced sexually dimorphic modifications in trophic factors and markers of cell turnover and neuronal maturation in the HT on PND13. Here, our aim was to investigate whether the alterations induced by Lep antagonism persist into puberty. Accordingly, male and female rats were treated with a pegylated super Lep Antag from PND5 to PND9 and killed just before the normal appearance of external signs of puberty (PND33 in females and PND43 in males). There was no effect on body weight, but in males food intake increased, subcutaneous adipose tissue decreased and HT neuropeptide Y and Agouti-related peptide mRNA levels were reduced, with no effect in females. In both sexes, the Antag increased HT mRNA levels of the kisspeptin receptor, G protein-coupled recepter 54 (Gpr54). Expression of the Lep receptor, trophic factors, and glial markers were differently affected in the HT of peripubertal males and females. Lep production in adipose tissue was decreased in Antag-treated rats of both sexes, with production of other cytokines being differentially regulated between sexes. In conclusion, in addition to the long-term effects on metabolism, changes in neonatal Lep levels modifies factors involved in reproduction that could possibly affect sexual maturation.

Hippocampal GSK3ß as a Molecular Link Between Obesity and Depression.

Obesity is considered as a risk factor for mood disorders including depression. Nevertheless, the mechanisms underlying this association are not clearly understood. To address this issue, we investigated the impact of high-fat (HF)-diet-induced obesity on depressive-like behavior and on serotonin (5-HT)-dependent Akt/glycogen synthase kinase 3ß (GSK3ß) signaling in the dentate gyrus (DG) of the hippocampus, which has been associated with mood regulation. We first showed that a HF diet induced significant overweight and hyperglycemia as well as a depressive-like behavior in adult Wistar rats. By using an ex vivo approach on brain slices, we demonstrated that 5-HT activates the Akt/GSK3ß cascade in the DG of control chow (C) diet-fed animals and that a 16-week HF diet feeding abolishes this activation, concurrently with a desensitization of leptin and insulin signaling in the same region. Furthermore, depressive-like behavior inversely correlated with 5-HT-induced phosphorylation of GSK3ß in the subgranular neurons of the DG. Interestingly, a substitution of HF with C diet for 6 weeks induced a total loss of depressive symptoms, whereas body weight and glycemia remained significantly higher compared to control rats. In addition, food restoration led to a recovery of the Akt/GSK3ß signaling pathway activation in the DG. In parallel, we observed a negative correlation between body weight and cell proliferation in the subgranular zone of the DG. To conclude, we provide evidence for a desensitization of 5-HT-induced Akt/GSK3ß signaling and an impaired cell proliferation in the DG by HF diet, suggesting novel molecular mechanisms linking obesity to depression.

Exploiting the therapeutic potential of leptin signaling in cachexia.

PURPOSE OF REVIEW: The anorexia-cachexia syndrome is a complication of many chronic conditions including cancer, chronic obstructive pulmonary disease, congestive heart failure, and chronic kidney disease (CKD). Leptin levels are significantly elevated in CKD patients and are associated with markers of poor nutritional status as well as mortality and morbidity. This review will focus on the mechanism and exploit the therapeutic potential of leptin signaling in CKD-associated cachexia. RECENT FINDINGS: Studies in db/db mice show that the lack of leptin receptor is protective against CKD-induced cachexia. Blockade of leptin's downstream mediators, such as melanocortin-4 receptor, attenuated CKD-associated cachexia. Pegylation of leptin antagonists resulted in a potent and effective long-acting reagents suitable for in-vivo studies or therapies. Pegylated leptin antagonist treatment ameliorates CKD-associated cachexia in mice. SUMMARY: Leptin antagonism may represent a viable therapeutic strategy for cachexia in CKD.

UCP1 is present in porcine adipose tissue and is responsive to postnatal leptin.

Intrauterine growth restriction (IUGR) may be accompanied by inadequate thermoregulation, especially in piglets that are not considered to possess any brown adipose tissue (BAT) and are thus entirely dependent on shivering thermogenesis in order to maintain body temperature after birth. Leptin can stimulate heat production by promoting non-shivering thermogenesis in BAT, but whether this response occurs in piglets is unknown. Newborn female piglets that were characterised as showing IUGR (mean birth weight of approximately 0.98 kg) were therefore administered injections of either saline or leptin once a day for the first 5 days of neonatal life. The dose of leptin was 0.5 mg/kg, which is sufficient to increase plasma leptin by approximately tenfold and on the day of birth induced a rapid increase in body temperature to values similar to those of normal-sized 'control' piglets (mean birth weight of ∼1.47 kg). Perirenal adipose tissue was then sampled from all offspring at 21 days of age and the presence of the BAT-specific uncoupling protein 1 (UCP1) was determined by immunohistochemistry and immunoblotting. UCP1 was clearly detectable in all samples analysed and its abundance was significantly reduced in the IUGR piglets that had received saline compared with controls, but was raised to the same amount as in controls in those IUGR females given leptin. There were no differences in gene expression between primary markers of brown and white adipose tissues between groups. In conclusion, piglets possess BAT that when stimulated exogenously by leptin can promote increased body temperature.