Hyperleptinemia as a contributing factor for the impairment of glucose intolerance in obesity.

Obesity has emerged as a major risk factor for insulin resistance leading to the development of type 2 diabetes (T2D). The condition is characterized by high circulating levels of the adipose-derived hormone leptin and a state of chronic low-grade inflammation. Pro-inflammatory signaling in the hypothalamus is associated with a decrease of central leptin- and insulin action leading to impaired systemic glucose tolerance. Intriguingly, leptin not only regulates body weight and glucose homeostasis but also acts as a pro-inflammatory cytokine. Here we demonstrate that increasing leptin levels (62,5 µg/kg/d, PEGylated leptin) in mice fed a high-fat diet (HFD) exacerbated body weight gain and aggravated hypothalamic micro- as well as astrogliosis. In contrast, administration of a predetermined dose of a long-acting leptin antagonist (100 µg/kg/d, PESLAN) chosen to block excessive leptin signaling during diet-induced obesity (DIO) showed the opposite effect and significantly improved glucose tolerance as well as decreased the total number of microglia and astrocytes in the hypothalamus of mice fed HFD. These results suggest that high levels of leptin, such as in obesity, worsen HFD-induced micro-and astrogliosis, whereas the partial reduction of hyperleptinemia in DIO mice may have beneficial metabolic effects and improves hypothalamic gliosis.

Defining the Transcriptional Targets of Leptin Reveals a Role for Atf3 in Leptin Action.

Leptin acts via its receptor (LepRb) to modulate gene expression in hypothalamic LepRb-expressing neurons, thereby controlling energy balance and glucose homeostasis. Despite the importance of the control of gene expression in hypothalamic LepRb neurons for leptin action, the transcriptional targets of LepRb signaling have remained undefined because LepRb cells contribute a small fraction to the aggregate transcriptome of the brain regions in which they reside. We thus employed translating ribosome affinity purification followed by RNA sequencing to isolate and analyze mRNA from the hypothalamic LepRb neurons of wild-type or leptin-deficient (Lepob/ob) mice treated with vehicle or exogenous leptin. Although the expression of most of the genes encoding the neuropeptides commonly considered to represent the main targets of leptin action were altered only following chronic leptin deprivation, our analysis revealed other transcripts that were coordinately regulated by leptin under multiple treatment conditions. Among these, acute leptin treatment increased expression of the transcription factor Atf3 in LepRb neurons. Furthermore, ablation of Atf3 from LepRb neurons (Atf3LepRbKO mice) decreased leptin efficacy and promoted positive energy balance in mice. Thus, this analysis revealed the gene targets of leptin action, including Atf3, which represents a cellular mediator of leptin action.

Essential Role for Hypothalamic Calcitonin Receptor‒Expressing Neurons in the Control of Food Intake by Leptin.

The adipocyte-derived hormone leptin acts via its receptor (LepRb) on central nervous system neurons to communicate the repletion of long-term energy stores, to decrease food intake, and to promote energy expenditure. We generated mice that express Cre recombinase from the calcitonin receptor (Calcr) locus (Calcrcre mice) to study Calcr-expressing LepRb (LepRbCalcr) neurons, which reside predominantly in the arcuate nucleus (ARC). Calcrcre-mediated ablation of LepRb in LepRbCalcrknockout (KO) mice caused hyperphagic obesity. Because LepRb-mediated transcriptional control plays a crucial role in leptin action, we used translating ribosome affinity purification followed by RNA sequencing to define the transcriptome of hypothalamic Calcr neurons, along with its alteration in LepRbCalcrKO mice. We found that ARC LepRbCalcr cells include neuropeptide Y (NPY)/agouti-related peptide (AgRP)/γ-aminobutyric acid (GABA) ("NAG") cells as well as non-NAG cells that are distinct from pro-opiomelanocortin cells. Furthermore, although LepRbCalcrKO mice exhibited dysregulated expression of several genes involved in energy balance, neither the expression of Agrp and Npy nor the activity of NAG cells was altered in vivo. Thus, although direct leptin action via LepRbCalcr cells plays an important role in leptin action, our data also suggest that leptin indirectly, as well as directly, regulates these cells.

Association of metreleptin treatment and dietary intervention with neurological outcomes in Celia's encephalopathy.

Celia's encephalopathy (progressive encephalopathy with/without lipodystrophy, PELD) is a recessive neurodegenerative disease that is fatal in childhood. It is caused by a c.985C>T variant in the BSCL2/seipin gene that results in an aberrant seipin protein. We evaluated neurological development before and during treatment with human recombinant leptin (metreleptin) plus a dietary intervention rich in polyunsaturated fatty acids (PUFA) in the only living patient. A 7 years and 10 months old girl affected by PELD was treated at age 3 years with metreleptin, adding at age 6 omega-3 fatty acid supplementation. Her mental age was evaluated using the Battelle Developmental Inventory Screening Test (BDI), and brain PET/MRI was performed before treatment and at age 5, 6.5, and 7.5 years. At age 7.5 years, the girl remains alive and leads a normal life for her mental age of 30 months, which increased by 4 months over the last 18 months according to BDI. PET images showed improved glucose uptake in the thalami, cerebellum, and brainstem. This patient showed a clear slowdown in neurological regression during leptin replacement plus a high PUFA diet. The aberrant BSCL2 transcript was overexpressed in SH-SY5Y cells and was treated with docosahexaenoic acid (200 µM) plus leptin (0.001 mg/ml) for 24 h. The relative expression of aberrant BSCL2 transcript was measured by qPCR. In vitro studies showed significant reduction (32%) in aberrant transcript expression. This therapeutic approach should be further studied in this devastating disease.

The Effects of Leptin Replacement on Neural Plasticity.

Leptin, an adipokine synthesized and secreted mainly by the adipose tissue, has multiple effects on the regulation of food intake, energy expenditure, and metabolism. Its recently-approved analogue, metreleptin, has been evaluated in clinical trials for the treatment of patients with leptin deficiency due to mutations in the leptin gene, lipodystrophy syndromes, and hypothalamic amenorrhea. In such patients, leptin replacement therapy has led to changes in brain structure and function in intra- and extrahypothalamic areas, including the hippocampus. Furthermore, in one of those patients, improvements in neurocognitive development have been observed. In addition to this evidence linking leptin to neural plasticity and function, observational studies evaluating leptin-sufficient humans have also demonstrated direct correlation between blood leptin levels and brain volume and inverse associations between circulating leptin and risk for the development of dementia. This review summarizes the evidence in the literature on the role of leptin in neural plasticity (in leptin-deficient and in leptin-sufficient individuals) and its effects on synaptic activity, glutamate receptor trafficking, neuronal morphology, neuronal development and survival, and microglial function.

Long-Acting PASylated Leptin Ameliorates Obesity by Promoting Satiety and Preventing Hypometabolism in Leptin-Deficient Lep(ob/ob) Mice.

Body weight loss of Lep(ob/ob) mice in response to leptin is larger than expected from the reduction in energy intake alone, suggesting a thermogenic action of unknown magnitude. We exploited the superior pharmacological properties of a novel long-acting leptin prepared via PASylation to study the contribution of its anorexigenic and thermogenic effects. PASylation, the genetic fusion of leptin with a conformationally disordered polypeptide comprising 600 Pro/Ala/Ser (PAS) residues, provides a superior way to increase the hydrodynamic volume of the fusion protein, thus retarding kidney filtration and extending plasma half-life. Here a single PAS(600)-leptin injection (300 pmol/g) resulted in a maximal weight reduction of 21% 6 days after application. The negative energy balance of 300 kJ/(4 d) was driven by a decrease in energy intake, whereas energy expenditure remained stable. Mice that were food restricted to the same extent showed an energy deficit of only 220 kJ/(4 d) owing to recurring torpor bouts. Therefore, the anorexigenic effect of PAS(600)-leptin contributes 75% to weight loss, whereas the thermogenic action accounts for 25% by preventing hypometabolism. In a second experiment, just four injections of PAS(600)-leptin (100 pmol/g) administered in 5- to 6-day intervals rectified the Lep(ob/ob) phenotype. In total, 16 nmol of PAS(600)-leptin per mouse triggered a weight loss of 43% within 20 days and normalized hypothermia and glucose homeostasis as well as hepatic steatosis. The beneficial properties of PAS(600)-leptin are substantiated by a comparison with previous studies in which approximately 400 nmol (∼25-fold) unmodified leptin was mandatory to achieve similar improvements.

Leptin therapy alters appetite and neural responses to food stimuli in brain areas of leptin-sensitive subjects without altering brain structure.

CONTEXT: Leptin is a key regulator of energy intake and expenditure. Individuals with congenital leptin deficiency demonstrate structural and functional brain changes when given leptin. However, whether acquired leptin deficiency may operate similarly is unclear. OBJECTIVE: We set out to determine whether the brains of individuals with acquired leptin deficiency may react to leptin in a similar manner. DESIGN: We used functional magnetic resonance imaging before and after short- and long-term metreleptin treatment in three leptin-sensitive patients with acquired hypoleptinemia. Nine healthy women were scanned as normoleptinemic controls. SETTING: The setting was an academic medical center. PATIENTS OR OTHER PARTICIPANTS: The participants were 3 hypoleptinemic women and nine normoleptinemic, matched women. INTERVENTIONS: We used metreleptin, recombinant leptin, therapy for 24 weeks in hypoleptinemic women only. MAIN OUTCOME MEASURE: We measured neural changes in response to viewing food as compared to nonfood images. We hypothesized that metreleptin treatment would increase brain activity in areas related to cognitive control and inhibition and would decrease brain activity in areas related to reward processing, as compared to the normoleptinemic counterparts. RESULTS: Unlike patients with congenital leptin deficiency, hypoleptinemic patients demonstrated no structural brain differences from healthy controls and/or structural changes in response to treatment. Short-term metreleptin treatment in leptin-sensitive hypoleptinemic subjects enhances areas involved in detecting the salience and rewarding value of food during fasting, whereas long-term treatment decreases attention to food and the rewarding value of food after feeding. Furthermore, hypothalamic activity is modulated by metreleptin treatment, and leptin decreases functional connectivity of the hypothalamus to key feeding-related areas in these hypoleptinemic subjects. CONCLUSIONS: Leptin replacement in acutely hypoleptinemic women did not alter brain structure but did alter functional cortical activity to food cues in key feeding and reward-related areas.

Early leptin blockade predisposes fat-fed rats to overweight and modifies hypothalamic microRNAs.

Perinatal leptin impairment has long-term consequences on energy homeostasis leading to body weight gain. The underlying mechanisms are still not clearly established. We aimed to analyze the long-term effects of early leptin blockade. In this study, newborn rats received daily injection of a pegylated rat leptin antagonist (pRLA) or saline from day 2 (d2) to d13 and then body weight gain, insulin/leptin sensitivity, and expression profile of microRNAs (miRNAs) at the hypothalamic level were determined at d28, d90, or d153 (following 1 month of high-fat diet (HFD) challenge). We show that pRLA treatment predisposes rats to overweight and promotes leptin/insulin resistance in both hypothalamus and liver at adulthood. pRLA treatment also modifies the hypothalamic miRNA expression profile at d28 leading to the upregulation of 34 miRNAs and the downregulation of four miRNAs. For quantitative RT-PCR confirmation, we show the upregulation of rno-miR-10a at d28 and rno-miR-200a, rno-miR-409-5p, and rno-miR-125a-3p following HFD challenge. Finally, pRLA treatment modifies the expression of genes involved in energy homeostasis control such as UCPs and AdipoRs. In pRLA rat muscle, Ucp2/3 and Adipor1/r2 are upregulated at d90. In liver, pRLA treatment upregulates Adipor1/r2 following HFD challenge. These genes are known to be involved in insulin resistance and type 2 diabetes. In conclusion, we demonstrate that the impairment of leptin action in early life promotes insulin/leptin resistance and modifies the hypothalamic miRNA expression pattern in adulthood, and finally, this study highlights the potential link between hypothalamic miRNA expression pattern and insulin/leptin responsiveness.

Novel approaches to the treatment of obesity and type 2 diabetes mellitus: bioactive leptin-related synthetic peptide analogs.

Leptin, the protein product of the ob gene, is primarily an adipocyte-secreted hormone, whose functional significance is rapidly expanding. Although early research efforts were focused on defining leptin's role in reversing obesity in rodents, there is now substantial evidence indicating that its influence extends to a number of hypothalamic-pituitaryendocrine axes, including adrenal, gonadal, growth hormone, pancreatic islets, and thyroid. The pleiotropic nature of leptin has been confirmed by demonstration of a role for leptin in hematopoiesis, angiogenesis, immune function, osteogenesis, reproduction, and wound healing. Unfortunately, the results of the majority of clinical trials with recombinant human leptin indicated that its effectiveness in restoring energy balance and correcting obesity-related endocrinopathies in genetically obese rodent models extended only to the management of those rare forms of human obesity caused by mutation in the ob gene. Failure of leptin in the clinic, and withdrawal of phentermine from Europe, and fenfluramine and sibutrimine from clinical use in the United States, have stimulated new approaches in the development of anti-obesity and anti-diabetes pharmacophores. These efforts are focused on utilizing leptin-related synthetic peptides as leptin receptor antagonists or leptin-related synthetic peptide analogs or mimetics. This review summarizes patents on leptin-related peptide analogs, antagonists and mimetics.

Basal, endogenous leptin is metabolically active in newborn rat pups.

OBJECTIVE: The adipocyte-derived hormone leptin regulates food intake and body weight via the activation of JAK-STAT pathway in mammalian adult hypothalamic neurons. To investigate whether endogenous leptin is metabolically active in newborn rat pups, the JAK-STAT leptin signaling pathway was analyzed following leptin antagonist challenge. METHODS: One day old male control pups were injected with either (i) saline, (ii) leptin (10 µg/g, s.c; n=4), (iii) pegylated leptin antagonist (PEG-MLA, 20 µg/g, s.c, n=4), or (iv) leptin plus PEG-MLA. Hypothalamus was dissected from individual pups at 30, 45, and 60 min. Protein expression of ObR, STAT3, pSTAT3, and SOCS3 was analyzed by Western blot. RESULTS: Leptin, but not PEG-MLA, produced a significant increase in hypothalamic pSTAT3 relative to saline treatment. Systemically administered PEG-MLA effectively blocks leptin signal induction of hypothalamic JAK-STAT signaling. The presence of PEG-MLA in combination with leptin attenuated the leptin-induced increase in pSTAT3. CONCLUSIONS: Thus, basal leptin levels are metabolically active in the newborn rats. These results brings new insights in considering the importance of endogenous leptin at birth, especially in low birth weight offspring who may be predisposed to altered neurogenesis and later obesity, and provide potential therapeutic strategies for programmed or diet-induced obesity.

Tat-modified leptin is more accessible to hypothalamus through brain-blood barrier with a significant inhibition of body-weight gain in high-fat-diet fed mice.

Obesity in human was found mainly due to the poor transportation of leptin through brain-blood barrier (BBB), called as leptin resistance. To produce a leptin capable of penetrating BBB, we have added Tat-PTD(9) to the C terminal of leptin to construct a fusion protein. The fusion Tat-leptin and native leptin genes were synthesized by single-step insertion of a polymerase chain reaction and expressed in Escherichia coli BL21 (Rosseta). The expressing products were purified and renatured by Ni-NTA affinity chromatography, and identified by the molecular size in SDS-PAGE gel and by its immunoreactivity to specific antibody with Western-blotting assay. To bio-functionally evaluate the fusion protein, Balb/c mice fed with high-fat diet (HFD) were given Tat-leptin, leptin or saline for 19 days. The immunohistochemical staining showed the increases in positive stains for the leptin in the region of hypothalamus of the HFD mice with either Tat-leptin or leptin as compared to saline group, but the staining intensity and frequency in the group with Tat-leptin were stronger and higher than those in the group with leptin. Furthermore, the most efficiency in preventing the body-weight gain caused by HFD was found in Tat-leptin group among these three groups. These results suggest that Tat-modified leptin may become a great potential candidate for the prevention or therapy of obese patients.

Leptin responsiveness restored by amylin agonism in diet-induced obesity: evidence from nonclinical and clinical studies.

Body weight is regulated by complex neurohormonal interactions between endocrine signals of long-term adiposity (e.g., leptin, a hypothalamic signal) and short-term satiety (e.g., amylin, a hindbrain signal). We report that concurrent peripheral administration of amylin and leptin elicits synergistic, fat-specific weight loss in leptin-resistant, diet-induced obese rats. Weight loss synergy was specific to amylin treatment, compared with other anorexigenic peptides, and dissociable from amylin's effect on food intake. The addition of leptin after amylin pretreatment elicited further weight loss, compared with either monotherapy condition. In a 24-week randomized, double-blind, clinical proof-of-concept study in overweight/obese subjects, coadministration of recombinant human leptin and the amylin analog pramlintide elicited 12.7% mean weight loss, significantly more than was observed with either treatment alone (P < 0.01). In obese rats, amylin pretreatment partially restored hypothalamic leptin signaling (pSTAT3 immunoreactivity) within the ventromedial, but not the arcuate nucleus and up-regulated basal and leptin-stimulated signaling in the hindbrain area postrema. These findings provide both nonclinical and clinical evidence that amylin agonism restored leptin responsiveness in diet-induced obesity, suggesting that integrated neurohormonal approaches to obesity pharmacotherapy may facilitate greater weight loss by harnessing naturally occurring synergies.

Leptin antagonist reverses hypertension caused by leptin overexpression, but fails to normalize obesity-related hypertension.

OBJECTIVE: The present study employed a rat leptin antagonist to evaluate the role of elevated leptin in obesity-associated hypertension. METHODS: First, leptin was overexpressed in the hypothalamus of lean rats for 155 days through the administration of a recombinant adeno-associated viral-mediated central vector-encoding leptin. Then a leptin antagonist was infused intracerebroventricularly for 14 days. In a second experiment, rats were fed with a high-fat diet or chow for 5 months, then the leptin antagonist was infused intracerebroventricularly for 14 days. RESULTS: Hypothalamic overexpression of leptin elevated blood pressure by 18 mmHg, but 14-day central infusion of the leptin antagonist reversed leptin-induced hypertension. High-fat feeding increased blood pressure (by approximately 8-9 mmHg) and tyrosine hydroxylase activity (by 76%) in superior cervical ganglia compared with chow feeding. Leptin antagonist infusion accelerated weight gain, food intake, and adiposity in high-fat-fed rats compared with chow-fed rats, and tyrosine hydroxylase activity was also reversed in the superior cervical ganglia. Elevated mean arterial pressure was not affected, although there was a small decrease in heart rate in both chow and high-fat-fed groups. CONCLUSION: Central overexpression of leptin leads to hypertension that can be reversed by a leptin antagonist. In contrast, this leptin antagonist does not reverse the high-fat feeding-induced elevation of blood pressure, even though there is apparent blockade of other leptin-mediated metabolic and sympatho-excitatory responses.