Leptin Receptor Activation Research Articles

Novel Leptin-Based Therapeutic Strategies to Limit Synaptic Dysfunction in Alzheimer's Disease.

Accumulation of hyper-phosphorylated tau and amyloid beta (Aβ) are key pathological hallmarks of Alzheimer's disease (AD). Increasing evidence indicates that in the early pre-clinical stages of AD, phosphorylation and build-up of tau drives impairments in hippocampal excitatory synaptic function, which ultimately leads to cognitive deficits. Consequently, limiting tau-related synaptic abnormalities may have beneficial effects in AD. There is now significant evidence that the hippocampus is an important brain target for the endocrine hormone leptin and that leptin has pro-cognitive properties, as activation of synaptic leptin receptors markedly influences higher cognitive processes including learning and memory. Clinical studies have identified a link between the circulating leptin levels and the risk of AD, such that AD risk is elevated when leptin levels fall outwith the physiological range. This has fuelled interest in targeting the leptin system therapeutically. Accumulating evidence supports this possibility, as numerous studies have shown that leptin has protective effects in a variety of models of AD. Recent findings have demonstrated that leptin has beneficial effects in the preclinical stages of AD, as leptin prevents the early synaptic impairments driven by tau protein and amyloid β. Here we review recent findings that implicate the leptin system as a potential novel therapeutic target in AD.

Long-duration leptin transgene expression in dorsal vagal complex does not alter bone parameters in female Sprague Dawley rats

The hypothalamus and dorsal vagal complex (DVC) are both important for integration of signals that regulate energy balance. Increased leptin transgene expression in either the hypothalamus or DVC of female rats was shown to decrease white adipose tissue and circulating levels of leptin and adiponectin. However, in contrast to hypothalamus, leptin transgene expression in the DVC had no effect on food intake, circulating insulin, ghrelin and glucose, nor on thermogenic energy expenditure. These findings imply different roles for hypothalamus and DVC in leptin signaling. Leptin signaling is required for normal bone accrual and turnover. Leptin transgene expression in the hypothalamus normalized the skeletal phenotype of leptin-deficient ob/ob mice but had no long-duration (≥10 weeks) effects on the skeleton of leptin-replete rats. The goal of this investigation was to determine the long-duration effects of leptin transgene expression in the DVC on the skeleton of leptin-replete rats. To accomplish this goal, we analyzed bone from three-month-old female rats that were microinjected with recombinant adeno-associated virus encoding either rat leptin (rAAV-Leptin, n = 6) or green fluorescent protein (rAAV-GFP, control, n = 5) gene. Representative bones from the appendicular (femur) and axial (3rd lumbar vertebra) skeleton were evaluated following 10 weeks of treatment. Selectively increasing leptin transgene expression in the DVC had no effect on femur cortical or cancellous bone microarchitecture. Additionally, increasing leptin transgene expression had no effect on vertebral osteoblast-lined or osteoclast-lined bone perimeter or marrow adiposity. Taken together, the findings suggest that activation of leptin receptors in the DVC has minimal specific effects on the skeleton of leptin-replete female rats.

Leptin excites basolateral amygdala principal neurons and reduces food intake by LepRb-JAK2-PI3K-dependent depression of GIRK channels.

Leptin is an adipocyte-derived hormone that modulates food intake, energy balance, neuroendocrine status, thermogenesis, and cognition. Whereas a high density of leptin receptors has been detected in the basolateral amygdala (BLA) neurons, the physiological functions of leptin in the BLA have not been determined yet. We found that application of leptin excited BLA principal neurons by activation of the long form leptin receptor, LepRb. The LepRb-elicited excitation of BLA neurons was mediated by depression of the G protein-activated inwardly rectifying potassium (GIRK) channels. Janus Kinase 2 (JAK2) and phosphoinositide 3-kinase (PI3K) were required for leptin-induced excitation of BLA neurons and depression of GIRK channels. Microinjection of leptin into the BLA reduced food intake via activation of LepRb, JAK2, and PI3K. Our results may provide a cellular and molecular mechanism to explain the physiological roles of leptin in vivo.

Structural insights into the mechanism of leptin receptor activation

Leptin is an adipocyte-derived protein hormone that promotes satiety and energy homeostasis by activating the leptin receptor (LepR)–STAT3 signaling axis in a subset of hypothalamic neurons. Leptin signaling is dysregulated in obesity, however, where appetite remains elevated despite high levels of circulating leptin. To gain insight into the mechanism of leptin receptor activation, here we determine the structure of a stabilized leptin-bound LepR signaling complex using single particle cryo-EM. The structure reveals an asymmetric architecture in which a single leptin induces LepR dimerization via two distinct receptor-binding sites. Analysis of the leptin–LepR binding interfaces reveals the molecular basis for human obesity-associated mutations. Structure-based design of leptin variants that destabilize the asymmetric LepR dimer yield both partial and biased agonists that partially suppress STAT3 activation in the presence of wild-type leptin and decouple activation of STAT3 from LepR negative regulators. Together, these results reveal the structural basis for LepR activation and provide insights into the differential plasticity of signaling pathways downstream of LepR.

Role of the adipocyte immune brain axis in Parkinson's disease: friend or foe?

Role of the adipocyte immune brain axis in Parkinson's disease: friend or foe?

Leptin Promotes Striatal Dopamine Release via Cholinergic Interneurons and Regionally Distinct Signaling Pathways.

Dopamine (DA) is a critical regulator of striatal network activity and is essential for motor activation and reward-associated behaviors. Previous work has shown that DA is influenced by the reward value of food, as well as by hormonal factors that reguate food intake and energy expenditure. Changes in striatal DA signaling also have been linked to aberrant eating patterns. Here we test the effect of leptin, an adipocyte-derived hormone involved in feeding and energy homeostasis regulation, on striatal DA release and uptake. Immunohistochemical evaluation identified leptin receptor (LepR) expression throughout mouse striatum, including on striatal cholinergic interneurons (ChIs) and their extensive processes. Using fast-scan cyclic voltammetry (FSCV), we found that leptin causes a concentration-dependent increase in evoked extra-cellular DA concentration ([DA]o) in dorsal striatum (dStr) and nucleus accumbens (NAc) core and shell in male mouse striatal slices, and also an increase in the rate of DA uptake. Further, we found that leptin increases ChI excitability, and that the enhancing effect of leptin on evoked [DA]o is lost when nicotinic acetylcholine (ACh) receptors are antagonized or when examined in striatal slices from mice lacking ACh synthesis. Evaluation of signaling pathways underlying leptin's action revealed a requirement for intracellular Ca2+, and the involvement of different downstream pathways in dStr and NAc core versus NAc shell. These results provide the first evidence for dynamic regulation of DA release and uptake by leptin within brain motor and reward pathways, and highlight the involvement of ChIs in this process.SIGNIFICANCE STATEMENT Given the importance of striatal dopamine (DA) in reward, motivation, motor behavior and food intake, identifying the actions of metabolic hormones on DA release in striatal subregions should provide new insight into factors that influence DA-dependent motivated behaviors. We find that one of these hormones, leptin, boosts striatal DA release through a process involving striatal cholinergic interneurons (ChIs) and nicotinic acetylcholine (ACh) receptors. Moreover, we find that the intracellular cascades downstream from leptin receptor (LepR) activation that lead to enhanced DA release differ among striatal subregions. Thus, we not only show that leptin regulates DA release, but also identify characteristics of this process that could be harnessed to alter pathologic eating behaviors.

Leptin receptor-expressing cells in the ventromedial nucleus of the hypothalamus contribute to enhanced CCK-induced satiety following central leptin injection.

Others have shown that leptin and cholecystokinin (CCK) act synergistically to suppress food intake. Experiments described here tested whether leptin in the ventromedial hypothalamus (VMH) contributes to the synergy with peripheral CCK in male Sprague Dawley rats. A subthreshold injection of 50-ng leptin into the VMH 1 h before a peripheral injection of 1 µg/kg CCK did not change the response to CCK in rats offered chow or low-fat purified diet, but did exaggerate the reduction in intake of high-fat diet 30 min and 1 h after injection in rats that had been food deprived for 8 h. By contrast, deletion of leptin receptor-expressing cells in the VMH using leptin-conjugated saporin (Lep-Sap) abolished the response to peripheral CCK in chow-fed rats. Lateral ventricle injection of 2-µg leptin combined with peripheral CCK exaggerated the inhibition of chow intake for up to 6 h in control rats treated with Blank-saporin, but not in Lep-Sap rats. Blank-Saporin rats offered low- or high-fat purified diet also demonstrated a dose-response inhibition of intake that reached significance with 1 µg/kg of CCK for both diets. CCK did not inhibit intake of Lep-Sap rats in either low- or high-fat-fed rats. Thus, although basal activation of VMH leptin receptors makes a significant contribution to the synergy with CCK, increased leptin activity in the VMH does not exaggerate the response to CCK in intact rats offered low-fat diets, but does enhance the response in those offered high-fat diet.NEW & NOTEWORTHY Leptin is a feedback signal in the control of energy balance, whereas cholecystokinin (CCK) is a short-term satiety signal that inhibits meal size. The two hormones synergize to promote satiety. We tested whether leptin receptors in the ventromedial nucleus of the hypothalamus (VMH) contribute to the synergy. The results suggest that there is a requirement for a baseline level of activation of leptin receptors in the VMH in order for CCK to promote satiety.

Diabetogenic and Obesogenic Effects of Cadmium in Db/Db Mice and Rats at a Clinically Relevant Level of Exposure.

Studies show an association between cadmium (Cd) exposure and prediabetes or type II diabetes mellitus. We have previously reported that Cd causes decreased levels of serum leptin in rats following 12 weeks of daily Cd dosing (0.6 mg/kg/b.w./day). Since leptin plays an important role in metabolism, we examined the effects of Cd on rats and db/db mice, which are deficient in leptin receptor activity. We gave rats and mice daily subcutaneous injections of saline (control) or CdCl2 at a dose of 0.6 mg/kg of Cd for 2 weeks, followed by 2 weeks of no dosing. At the end of the 4-week study, exposure to Cd resulted in a more rapid increase in blood glucose levels following an oral glucose tolerance test in db/db vs. lean mice. During the two weeks of no Cd dosing, individual rat bodyweight gain was greater (p ≤ 0.05) in Cd-treated animals. At this time point, the combined epididymal and retroperitoneal fat pad weight was significantly greater (p ≤ 0.05) in the Cd-treated lean mice compared to saline-treated controls. Although this pilot study had relatively low N values (4 per treatment group for mice and 6 for rats) the results show that clinically relevant levels of Cd exposure resulted in diabetogenic as well as obesogenic effects.

Atomistic mechanism of leptin and leptin-receptor association

The leptin-leptin receptor complex is at the very core of energy homeostasis and immune system regulation, among many other functions. In this work, we built homology models of leptin and the leptin binding domain (LBD) of the receptor from humans and mice. Docking analyses were used to obtain the coordinates of the native leptin-LBD complexes and a mixed heterodimer formed by human leptin and mouse LBD. Molecular dynamics (MD) simulations were performed using all models (monomers and heterodimers) as initial coordinates and the GROMACS program. The overall structural and dynamical behaviors are similar for the three complexes. Upon MD simulations, several new interactions appear. In particular, hydrophobic interactions, with more than 90% persistence, seem to be the most relevant for the stability of the dimers, as well as the pair formed by Asp85Lep and Arg468LBD. This in silico analysis provides structural and dynamical information, at the atomistic level, about the mechanism of leptin-LBD complex formation and leptin receptor activation. This knowledge might be used in the rational drug design of therapeutics to modulate leptin signaling. Communicated by Ramaswamy H. Sarma

Interactions of Lipids, Lipoproteins, and Apolipoproteins with the Blood-Brain Barrier.

Lipids and lipoproteins are a diverse group of substances and their interactions with the blood-brain barrier (BBB) is similarly diverse. Some lipoproteins such as high density lipoprotein (HDL), apolipoprotein (apo) A-I, apoJ, some free fatty acids, and triglycerides cross the BBB whereas others such as apoE do not. Some forms of cholesterol can cross the BBB and others do not. Lipids can have effects on BBB preservation and function: HDL may protect the BBB during multiple sclerosis, cholesterol can disrupt the BBB, and triglycerides inhibit the transport of leptin across the BBB and the activation of the hypothalamic leptin receptor. ApoE is associated with many effects on the BBB, with the specific isoform apoE4 having detrimental effects. In summary, the diverse ways in which lipids, lipoproteins, and apolipoproteins interact with the BBB is important in both health and disease.

Central Leptin Receptor Blockade Restored Renal Glucose Handling in a Model of Estrogen Deficiency

Hormonal changes and estrogen deficiency accompanying menopause have several effects on women such as weight gain in the form of adipose tissue. An increase in adiposity associates with an increased production of leptin, an adipocyte derived hormone, that acts mainly in the central nervous system. Leptin regulates energy balance and plays a primary role in the regulation of glucose homeostasis. In this study, we aim to determine leptin's effect on renal glucose handling in an estrogen deficient adult female rat model (ovariectomized; OVX). We observed significant weight gain, increase in glomerular filtration rate (GFR, increase in creatinine clearance from 1.3 ml/min to 2.3 ml/min) and protein level of an early biomarker of kidney injury, the neutrophil gelatinase-associated lipocalin (NGAL, 90% increase in both glomeruli and tubules, measured by immunohistochemistry) in OVX rats when compared to their sham-operated controls. The mechanism proposed for the increase in GFR and renal damage is lipotoxicity due to intrarenal lipid accumulation associated with increased perirenal adipose tissue. Indeed, a significant accumulation of renal lipid, assessed using oil red O staining, was detected in the renal tubules of the OVX rats when compared with their controls. All these effects were partially reversed by treatment with a leptin receptor antagonist (LAN-6, 3µg/day), delivered for four weeks into the lateral ventricle by an osmotic pump. The sodium-glucose cotransporter-2 (SGLT2), the main mediator of renal glucose handling, is co-expressed in renal proximal tubules (PTs) with Na/H exchanger-3 (NHE3), a key player of salt regulation. Using immunohistochemistry, we measured significant reduction of renal SGLT2 protein expression level in OVX rats when compared with their controls. NHE3 protein expression level was also decreased in the same PTs. The decrease in SGLT2 and NHE3 protein expression measured in OVX rats and glycosuria detected in the urine of OVX rats was significantly reduced in rats treated with LAN-6. Serum glucose levels did not change significantly in the conditions under study. These findings suggest that in the absence of estrogen: 1. infiltration of renal lipid leads to renal injury, 2. kidney injury combines with an increase in GFR, 3. protein expression levels of SGLT2 and NHE3 are decreased and renal glycosuria develops. Finally, blocking activation of central leptin receptors in part restores kidney function. This may shed light in the understanding of leptin's regulation of glucose homeostasis and lead to potential benefits against kidney dysfunction caused by intrarenal lipid accumulation that develops during estrogen deficiency.

Perivascular adipose tissue (PVAT)‐derived leptin improves aortic endothelial function via attenuating endothelial glycolysis in a mouse model of lipodystrophy

Endothelial dysfunction, characterized by impaired endothelial-dependent relaxation (EDR), is a hallmark and first step in the chain of events leading to cardiovascular disorders. Several vascular pathologies have been associated with aberrant increases in endothelial glycolysis and 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) expression/activity, however, whether endothelial glycolysis contributes to EDR regulation remains unknown. Additionally, endothelial dysfunction is present in conditions associated with severe alterations in body fat content including obesity (excess) and lipodystrophy (absence of adipose tissue). However, the exact link between endothelial function and adipose tissue, particularly the one surrounding blood vessels (perivascular adipose tissue, PVAT) remains ill-defined. As leptin, the adipokine highly involved in the regulation of cardiovascular function, has also been shown to modulate cellular bioenergetics in immune and cancer cells, we hypothesized that leptin derived from PVAT controls EDR via endothelial leptin receptor activation and attenuation of PFKFB3-mediated glycolysis. To determine the role of PFKFB3 in endothelial function, we overexpressed PFKFB3 in mouse endothelial cells via adenovirus transduction (Ad-PFKFB3), and reported impaired aorta EDR. Additionally, using seahorse analyzer and glycolysis stress test we found that human aortic endothelial cells (HAEC) transduced with Ad-PFKFB3 showed a significant increase in basal glycolysis (p<0.05) which was blunted by exposure to leptin (10µg/ml, 2h). To determine the role of PVAT-derived leptin in EDR, we used a mouse model of lipodystrophy, with total deficiency in adipose tissue and leptin (Bscl2-/-) characterized by vascular and metabolic dysfunctions as evidenced by impaired EDR and glucose intolerance. We also found that aortas from Bscl2-/- mice exhibited a marked increase in basal and maximum glycolysis measured using seahorse analyzer and glycolysis stress test. This is in line with the PCR data showing increased expression of PFKFB3 mRNA compared to WT mice. Interestingly, inhibition of PFKFB3 using 3 PO or PFK158 (20uM) restored EDR in Bscl2-/- mice. Additionally, we transplanted 25 mg PVAT from WT mice to the abdominal aorta of Bscl2-/-. After 4 weeks, PVAT significantly reduced aortic expression of PFKFB3 and corrected systemic glycemia as well as EDR in Bscl2-/- vs sham (P<0.05). However, leptin receptor blockade (Allo-Aca, 0.05 mg/kg/day) blunted PVAT-mediated restoration of EDR in PVAT-transplanted Bscl2-/- mice. In conclusion, these data demonstrate that PVAT releases a vasodilatory factor, potentially, leptin that acts via activation of endothelial leptin receptor to improve endothelial function via a mechanism that involves regulation of PFKFB3-mediated endothelial glycolysis.

Estrogen Regulates the Trafficking of Leptin Receptors

During menopause, when endogenous estrogen levels are depleted, a rise in hypertension is observed in 75% of post-menopausal women, suggesting there is a cardioprotective effect from estrogen. In previous studies, estrogen has been shown to effect hypothalamic paraventricular nuclei by its action to lessen the impact of a glutamate induced increase in blood pressure. These data, along with others, suggest that the cardioprotective effect of estrogen is regionally specific in the central nervous system. However, the mechanism(s) behind estrogen's attenuating effect on blood pressure have not been fully elucidated. We propose that estrogen regulates blood pressure in part, by inhibiting the actions of leptin. Leptin is an adipocyte-derived peptide hormone. Activation of its signaling pathway within the central nervous system can alter blood pressure. Studies with injections of leptin in rodent's dorsomedial hypothalamus showed an increase in blood pressure, while antagonism of leptin receptors correlated with diminished blood pressure. Both results suggest that adjustment of leptin signaling in the dorsomedial hypothalamus is sufficient to alter blood pressure. In this study, we hypothesize that estrogen can block leptin receptor activation and downstream signaling events by increasing the expression of SOCS3 (suppressor of cytokine signaling 3), a negative modulator of leptin signaling. We tested our hypothesis by utilizing an in vitro system (i.e. GT1-7 mouse hypothalamic neuronal cells) and an in vivo system (i.e. female C57B6 mice) that were treated with estradiol (100nM). Our data demonstrated that administering estrogen decreased the expression of leptin receptors in the hypothalamus and we proposed this occurred due to the increase in SOCS3 expression we observed when estradiol was administered. Taken together, these data suggest that estrogen can attenuate leptin's physiological response on blood pressure by increasing an inhibitor (i.e. SOCS3) of leptin's signaling.

Fasting reduces the number of TRH immunoreactive neurons in the hypothalamic paraventricular nucleus of male rats, but not in mice

During fasting or weight loss, the fall in leptin levels leads to suppression of thyrotropin-releasing hormone (TRH) expression in the paraventricular nucleus of the hypothalamus (PVH) and, consequently, inhibition of the hypothalamic-pituitary-thyroid (HPT) axis. However, differently than rats, just few PVHTRH neurons express the leptin receptor in mice. In the present study, male adult rats and mice were submitted to 48 -h fasting to evaluate the consequences on proTRH peptide expression at the PVH level. Additionally, the proTRH peptide expression was also assessed in the brains of leptin-deficient (Lepob/ob) mice. We observed that approximately 50 % of PVHTRH neurons of leptin-injected rats exhibited phosphorylation of the signal transducer and activator of transcription 3 (pSTAT3), a marker of leptin receptor activation. In contrast, very few PVHTRH neurons of leptin-injected mice exhibited pSTAT3. Rats submitted to 48 -h fasting showed a significant reduction in the number of PVHTRH immunoreactive neurons, as compared to fed rats. On the other hand, no changes in the number of PVHTRH immunoreactive neurons were observed between fasted and fed mice. Next, the number of TRH immunoreactive cells was determined in the PVH, dorsomedial nucleus of the hypothalamus and nucleus raphe pallidus of Lepob/ob and wild-type mice and no significant differences were observed, despite reduced plasma T4 levels in Lepob/ob mice. Taken together, these findings provide additional evidence of the important species-specific differences in the mechanisms used by fasting and/or leptin to regulate the HPT axis.

The Neuronal Actions of Leptin and the Implications for Treating Alzheimer's Disease.

It is widely accepted that the endocrine hormone leptin controls food intake and energy homeostasis via activation of leptin receptors expressed on hypothalamic arcuate neurons. The hippocampal formation also displays raised levels of leptin receptor expression and accumulating evidence indicates that leptin has a significant impact on hippocampal synaptic function. Thus, cellular and behavioural studies support a cognitive enhancing role for leptin as excitatory synaptic transmission, synaptic plasticity and glutamate receptor trafficking at hippocampal Schaffer collateral (SC)-CA1 synapses are regulated by leptin, and treatment with leptin enhances performance in hippocampus-dependent memory tasks. Recent studies indicate that hippocampal temporoammonic (TA)-CA1 synapses are also a key target for leptin. The ability of leptin to regulate TA-CA1 synapses has important functional consequences as TA-CA1 synapses are implicated in spatial and episodic memory processes. Moreover, degeneration is initiated in the TA pathway at very early stages of Alzheimer’s disease, and recent clinical evidence has revealed links between plasma leptin levels and the incidence of Alzheimer’s disease (AD). Additionally, accumulating evidence indicates that leptin has neuroprotective actions in various AD models, whereas dysfunctions in the leptin system accelerate AD pathogenesis. Here, we review the data implicating the leptin system as a potential novel target for AD, and the evidence that boosting the hippocampal actions of leptin may be beneficial.

Leptin regulation of hippocampal synaptic function in health and disease.

It is widely accepted that the metabolic hormone leptin regulates food intake and body weight via activation of hypothalamic leptin receptors. However, as leptin receptors are also highly expressed in other brain regions, such as the hippocampus, alterations in leptin responsiveness also impacts on key functions of the hippocampus, like learning and memory. Within the hippocampus, high levels of leptin receptors are expressed at excitatory synapses, and in accordance with a synaptic localization, leptin potently regulates synaptic transmission at both Schaffer collateral (SC) and temporoammonic (TA) inputs to CA1 pyramidal neurons. Increasing evidence from cellular and behavioral studies examining leptin action at CA1 synapses support the notion that leptin is a potential cognitive enhancer. However, the capacity of leptin to regulate synaptic efficacy at SC-CA1 and TA-CA1 synapses declines in an age-dependent manner. Moreover, clinical evidence that supports a link between circulating leptin levels and the risk of the age-related neurodegenerative disorder, Alzheimer's disease (AD) is accumulating. Consequently, it has been proposed that the leptin system is a potential therapeutic target in AD, and that boosting the hippocampal actions of leptin may be beneficial in the treatment of AD. Here we review recent progress in our understanding of the neuronal and hippocampal synaptic functions that are regulated by leptin and how alterations in the leptin system influence age-related CNS-related disorders like AD.

The relationship between leptin, leptin receptor, and FGFR1 in primary human breast tumors.

e13578 Background: General/overall obesity and visceral/central obesity are associated with increased breast cancer risk and poorer cancer outcomes; however, the precise etiology of these observations is not fully known. Our previous research suggests that adipose tissue-derived fibroblast growth factor 2 (FGF2) promotes malignant transformation of mammary epithelial cells through the activation of its primary receptor, fibroblast growth factor receptor 1 (FGFR1). Both FGF2 and leptin have been shown to be elevated in the serum in obesity (body mass index (BMI) &gt; 25kg/m2). It is well established that elevated sera leptin promotes breast cancer through multiple mechanisms. Therefore, we hypothesized that tumors that express elevated FGFR1 and leptin receptor (LepR) may have a poorer prognosis in obese patients. Methods: Databases that house gene-sequenced breast cancer tumors and normal breast tissue were analyzed to discern relationships between gene copy number and mRNA levels of leptin, leptin receptor, FGFR1 and janus kinase 2 (Jak2), as Jak2 was shown to be a common downstream mediator of both FGFR1 and leptin receptor activation by Cytoscape network analysis. Furthermore, we tested the efficacy of Jak2 inhibitors for preventing mammary epithelial cell malignant transformation using our well-established models of adipose tissue-stimulated growth in soft agar. The anchorage-independent growth in soft agar is a surrogate marker for malignant transformation. Results: A much stronger significant relationship exists between FGFR1 mRNA and leptin mRNA in primary breast cancer (p = 3.17*10−9) compared with normal breast epithelium (p = 0.0013). In primary breast tumors, gene copy number of FGFR1 positively correlates with leptin copy number (p = 8.956*10−8, r = 0.19). Moreover, two separate Jak2 inhibitors attenuated both leptin+FGF2-stimulated and mouse adipose tissue-stimulated MCF-10A cell transformation. Conclusions: Taken together, these results suggest that elevated sera FGF2 and leptin in obese patients may promote breast tumorigenesis in tumors that express elevated FGFR1 and LepR and that Jak2 inhibitors may be a novel therapeutic option for adiposity-driven breast cancers. Future work will examine the relationship between FGFR1/leptin receptor expression and survival and how these relationships are influenced by BMI status.

Lipid Nephrotoxicity and Increased Risk of Nephrolithiasis in Model of Estrogen Deficiency

Menopause is a period during which women tend to gain body fat. Estrogen deficiency is proposed to impact body fat composition and energy balance in these women by influencing the levels of leptin, an adipocyte‐derived hormone that acts primarily on the central nervous system. Specifically, lipid nephrotoxicity increases the risk for kidney disease and nephrolithiasis due to renal lipid accumulation and overly acidic urinary pH. Mechanisms underlying these kidney disturbances and extent of their effects when women become estrogen deficient (i.e. post‐menopausal) are not well‐defined. Utilizing an estrogen deficient (i.e. ovariectomized) rodent model, we hypothesize that central leptin receptor activation regulates renal function by augmenting the accumulation of renal lipids and urinary pH acidification. We measured the glomerular filtration rate (GFR) by creatinine clearance and protein level of the neutrophil gelatinase‐associated lipocalin (NGAL), an early biomarker of kidney injury, by immunohistochemistry. An increase in creatinine clearance (from 1.3 ml/min to 2.3 ml/min) and protein levels of NGAL (over 90% increase in both glomeruli and tubules) was measured in ovariectomized adult female rats when compared with their sham‐operated controls. Treatment with leptin receptor antagonist (LAN‐6, 3μg/day), delivered for four weeks into the lateral ventricle by an osmotic pump, significantly attenuated these effects. Furthermore, we determined that perirenal fat accumulation was markedly higher in the same estrogen deficiency model when compared with the sham‐operated control. Perirenal fat accumulation has been associated with lipid accumulation in renal tissues and excess of intrarenal lipid is increasingly recognized to contribute to kidney injury through lipotoxicity. A significant accumulation of renal lipid, tested using oil red O staining, was detected in the renal tubules of the ovariectomized animals when compared with their controls. We also found significant acidification of urinary pH and decreased protein expression of a key player in urinary pH regulation, Na/H exchanger‐3 (NHE3), in ovariectomized animals when compared with their controls. Renal lipid accumulation, urinary pH acidification and decrease in NHE3 expression were all partially reversed by blocking activation of central leptin receptors. We propose that: 1. An increase in GFR combined with kidney injury presupposes kidney dysfunction, 2. infiltration of renal lipid leads to renal injury, through lipotoxicity, 3. urinary pH acidification and reduction of NHE3 expression increases the risk of nephrolithiasis. Taken together, these findings suggest that leptin plays a key role in the development of menopausal‐associated kidney disease.

The complicated clinical course in a case of atypical lipodystrophy after development of neutralizing antibody to metreleptin: treatment with setmelanotide.

SummaryA patient with atypical partial lipodystrophy who had a transient initial response to metreleptin experienced acute worsening of her metabolic state when neutralizing antibodies against metreleptin appeared. Because her metabolic status continued to deteriorate, a therapeutic trial with melanocortin-4 receptor agonist setmelanotide, that is believed to function downstream from leptin receptor in the leptin signaling system, was undertaken in an effort to improve her metabolic status for the first time in a patient with lipodystrophy. To achieve this, a compassionate use (investigational new drug application; IND) was initiated (NCT03262610). Glucose control, body fat by dual-energy X-ray absorptiometry and MRI, and liver fat by proton density fat fraction were monitored. Daily hunger scores were assessed by patient filled questionnaires. Although there was a slight decrease in hunger scales and visceral fat, stimulating melanocortin-4 receptor by setmelanotide did not result in any other metabolic benefit such as improvement of hypertriglyceridemia or diabetes control as desired. Targeting melanocortin-4 receptor to regulate energy metabolism in this setting was not sufficient to obtain a significant metabolic benefit. However, complex features of our case make it difficult to generalize these observations to all cases of lipodystrophy. It is still possible that melanocortin-4 receptor agonistic action may offer some therapeutic benefits in leptin-deficient patients.Learning points:A patient with atypical lipodystrophy with an initial benefit with metreleptin therapy developed neutralizing antibodies to metreleptin (Nab-leptin), which led to substantial worsening in metabolic control. The neutralizing activity in her serum persisted for longer than 3 years.Whether the worsening in her metabolic state was truly caused by the development of Nab-leptin cannot be fully ascertained, but there was a temporal relationship. The experience noted in our patient at least raises the possibility for concern for substantial metabolic worsening upon emergence and persistence of Nab-leptin. Further studies of cases where Nab-leptin is detected and better assay systems to detect and characterize Nab-leptin are needed.The use of setmelanotide, a selective MC4R agonist targeting specific neurons downstream from the leptin receptor activation, was not effective in restoring metabolic control in this complex patient with presumed diminished leptin action due to Nab-leptin.Although stimulating the MC4R pathway was not sufficient to obtain a significant metabolic benefit in lowering triglycerides and helping with her insulin resistance as was noted with metreleptin earlier, there was a mild reduction in reported food intake and appetite.Complex features of our case make it difficult to generalize our observation to all leptin-deficient patients. It is possible that some leptin-deficient patients (especially those who need primarily control of food intake) may still theoretically benefit from MC4R agonistic action, and further studies in carefully selected patients may help to tease out the differential pathways of metabolic regulation by the complex network of leptin signaling system.

Leptin receptor-expressing neurons in ventromedial nucleus of the hypothalamus contribute to weight loss caused by fourth ventricle leptin infusions.

Leptin administration into the hindbrain, and specifically the nucleus of the solitary tract, increases phosphorylated signal transducer and activator of transcription 3 (pSTAT3), a marker of leptin receptor activation, in hypothalamic nuclei known to express leptin receptors. The ventromedial nucleus of the hypothalamus (VMH) shows the greatest response, with a threefold increase in pSTAT3. This experiment tested the importance of VMH leptin receptor-expressing neurons in mediating weight loss caused by fourth ventricle (4V) leptin infusion. Male Sprague-Dawley rats received bilateral VMH 75-nL injections of 260 ng/μL of leptin-conjugated saporin (Lep-Sap) or blank-saporin (Blk-Sap). After 23 days they were fitted with 4V infusion cannulas and 1 wk later adapted to housing in a calorimeter before they were infused with 0.9 μg leptin/day for 14 days. There was no effect of VMH Lep-Sap on weight gain or glucose clearance before leptin infusion. Leptin inhibited food intake and respiratory exchange ratio in Blk-Sap but not Lep-Sap rats. Leptin had no effect on energy expenditure or brown adipose tissue temperature of either group. Inguinal and epididymal fat were significantly reduced in leptin-treated Blk-Sap rats, but the response was greatly attenuated in Lep-Sap rats. VMH pSTAT3 was increased in leptin-treated Blk-Sap but not Lep-Sap rats. These results support the concept that leptin-induced weight loss results from an integrated response across different brain areas. They also support previous reports that VMH leptin receptors do not play a significant role in maintaining energy balance in basal conditions but limit weight gain during positive energy balance.