Obesity has become a major public health problem. Given the current increase in life expectancy, the prevalence of obesity also raises steadily among older age groups. The increase in life expectancy is often accompanied with additional years of susceptibility to chronic ill health associated with obesity in the elderly. Both obesity and ageing are conditions leading to serious health problems and increased risk for disease and death. Ageing is associated with an increase in abdominal obesity, a major contributor to insulin resistance and the metabolic syndrome. Obesity in the elderly is thus a serious concern and comprehension of the key mechanisms of ageing and age-related diseases has become a necessary matter. Here, we aimed to identify similarities underlying mechanisms related to both obesity and ageing. We bring together evidence that age-related changes in body fat distribution and metabolism might be key factors of a vicious cycle that can accelerate the ageing process and onset of age-related diseases.

Susceptibility to obesity changes during the course of life. We utilized the C57BL/6J (B6) and 129S mouse as a genetic model for variation in diet‐induced obesity to define the adiposity phenotypes from birth to maturity at 8 weeks‐of‐age. From birth to 8 weeks‐of‐age, both male and female 129S mice had significantly higher fat mass and adiposity index than B6 mice, although they were not obese. After 8 weeks‐of‐age, B6 had greater adiposity/obesity than 129S mice in response to a high fat (HF). We sought to determine the mechanism activating the fat accumulation in B6 mice at 8‐weeks‐of‐age. We used microarray analysis of gene expression during development of inguinal fat to show that molecular networks of lipogenesis were maximally expressed at 8 weeks‐of‐age. In addition, the DNA methylation analysis of the Sfrp5 promoter and binding of acetylated histones to Sfrp5 and Acly promoter regions showed that major differences in the expression of genes of lipogenesis and chromatin structure occur during development. Differences in lipogenesis networks could account for the strain‐dependent differences in adiposity up to 8 weeks‐of‐age; however, changes in the expression of genes in these networks were not associated with the susceptibility to DIO in B6 male mice beyond 8 weeks‐of‐age.

Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Defects in the function of hypothalamic neurons cause a spectrum of human diseases, including obesity, infertility, growth defects, sleep disorders, social disorders, and stress disorders. These diseases have been studied in animal models such as mice, but the rarity and relative inaccessibility of mouse hypothalamic neurons and species-specific differences between mice and humans highlight the need for human cellular models of hypothalamic diseases. We and others have developed methods to differentiate human pluripotent stem cells (hPSCs) into hypothalamic neurons and related cell types, such as astrocytes. This protocol builds on published studies by providing detailed step-by-step instructions for neuronal differentiation, quality control, long-term neuronal maintenance, and the functional interrogation of hypothalamic cells by calcium imaging. Together, these protocols should enable any group with appropriate facilities to generate and study human hypothalamic cells. © 2017 by John Wiley & Sons, Inc.

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Brain-derived neurotrophic factor (BDNF) signals through its high affinity receptor Tropomyosin receptor kinase-B (TrkB) to regulate neuronal development, synapse formation and plasticity. In rodents, genetic disruption of Bdnf and TrkB leads to weight gain and a spectrum of neurobehavioural phenotypes. Here, we functionally characterised a de novo missense variant in BDNF and seven rare variants in TrkB identified in a large cohort of people with severe, childhood-onset obesity. In cells, the E183K BDNF variant resulted in impaired processing and secretion of the mature peptide. Multiple variants in the kinase domain and one variant in the extracellular domain of TrkB led to a loss of function through multiple signalling pathways, impaired neurite outgrowth and dominantly inhibited glutamatergic synaptogenesis in hippocampal neurons. BDNF/TrkB variant carriers exhibited learning difficulties, impaired memory, hyperactivity, stereotyped and sometimes, maladaptive behaviours. In conclusion, human loss of function BDNF/TrkB variants that impair hippocampal synaptogenesis may contribute to a spectrum of neurobehavioural disorders. The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) is widely expressed in the mammalian brain and signals via the Tropomyosin receptor kinase B (TrkB) to regulate neuronal differentiation and survival, synapse formation and activity-dependent changes in synapse structure and function. Bdnf and TrkB null mice are embryonically lethal 1,2. Bdnf haplo-insufficient mice and mice in which Bdnf is deleted in the postnatal brain, survive and exhibit hyperactivity, impaired pain sensation, increased food intake and weight gain 3. In humans, deletions encompassing the BDNF gene on chromosome 11p.12.3 and very rare loss of function coding variants in TrkB have been reported in individuals with speech and language delay, hyperphagia and severe obesity 4-6. BDNF is synthesised as a precursor protein, pre-pro-BDNF, which is converted into pro-BDNF by removal of the signal peptide and packaged into vesicles before being transported distally to dendrites or axons 7. Only once the protein is destined for secretion, is pro-BDNF converted to mature BDNF through proteolytic cleavage by furin and other proprotein convertases in the trans-Golgi network or secretory vesicles, releasing mature BDNF from the pro-domain 8. Processing of pro-BDNF and secretion are thought to occur almost simultaneously 9. The regulated equilibrium between pro-BDNF and mature BDNF appears to be physiologically relevant as a hippocampus-specific deletion of the serine protease tissue plasminogen activator (tPA), which is involved in the cleavage of pro-BDNF to BDNF extracellularly, increases depression and anxiety-like behaviour in adult mice 10 .

ObjectiveThe lack of pro-opiomelanocortin (POMC)-derived melanocortin peptides results in hypoadrenalism and severe obesity in both humans and rodents that is treatable with synthetic melanocortins. However, there are significant differences in POMC processing between humans and rodents, and little is known about the relative physiological importance of POMC products in the human brain. The aim of this study was to determine which POMC-derived peptides are present in the human brain, to establish their relative concentrations, and to test if their production is dynamically regulated.MethodsWe analysed both fresh post-mortem human hypothalamic tissue and hypothalamic neurons derived from human pluripotent stem cells (hPSCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the sequence and quantify the production of hypothalamic neuropeptides, including those derived from POMC.ResultsIn both in vitro and in vivo hypothalamic cells, LC-MS/MS revealed the sequence of hundreds of neuropeptides as a resource for the field. Although the existence of β-melanocyte stimulating hormone (MSH) is controversial, we found that both this peptide and desacetyl α-MSH (d-α-MSH) were produced in considerable excess of acetylated α-MSH. In hPSC-derived hypothalamic neurons, these POMC derivatives were appropriately trafficked, secreted, and their production was significantly (P < 0.0001) increased in response to the hormone leptin.ConclusionsOur findings challenge the assumed pre-eminence of α-MSH and suggest that in humans, d-α-MSH and β-MSH are likely to be the predominant physiological products acting on melanocortin receptors.

Human obesity can result from the aberrant production or processing of proopiomelanocortin (POMC) in hypothalamic neurons, but it is unclear which human POMC-derived peptides are most relevant to body weight regulation. To address this question, we analysed both hypothalamic neurons derived from human pluripotent stem cells (hPSCs) and primary human hypothalamic tissue using quantitative liquid chromatography tandem mass spectroscopy (LC-MS/MS). In both in vitro-and in vivo-derived samples, we found that POMC was processed into b-melanocyte stimulating hormone (b-MSH), whose existence in the human brain has been controversial. b-MSH and desacetyl a-MSH (d-a-MSH) were produced at roughly equimolar concentrations and in vast excess to acetylated a-MSH (5-to 200-fold), suggesting that the importance of both d-a-MSH and b-MSH to human obesity has been underestimated. Since body weight is sensitive to changes in MSH concentration, we asked whether hPSC-derived hypothalamic neurons could provide mechanistic insights into the processing and secretion of MSH peptides. We found that cultured human hypothalamic neurons appropriately trafficked POMC and its derivatives, and robustly (P<0.0001) secreted them when depolarised. Furthermore, the adipocyte-derived hormone leptin significantly (P<0.01) promoted their production of both d-a-MSH and b-MSH. These results establish hPSC-derived hypothalamic neurons as a model system for studying human-specific aspects of POMC processing that might be therapeutically harnessed to treat obesity.

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