👤 M Wabitsch

Melanocortin 4 receptor (MC4R) deficiency is the most common monogenic cause of obesity, yet remains underdiagnosed. Patients with monogenic obesity often undergo a frustrating diagnostic and therapeutic odyssey of years of ineffective lifestyle interventions before a causal diagnosis is made. We report a four-generation family where genetic testing in a child identified a likely pathogenic MC4R variant also carried by three ancestors. The studied family included a 7-year-old index patient, her mother, grandmother, and great-grandmother with a history of early-onset obesity. Panel sequencing of monogenic obesity genes was performed in the index patient whereas in the relatives targeted analysis of the familial MC4R variant was performed by Sanger sequencing. The index patient developed severe obesity by age 2 years, with hyperphagia, tall stature, and dyslipidemia. Despite lifestyle interventions, her body mass index (BMI) continued to increase. At the age of 7 years, genetic panel testing identified a rare monoallelic variant in the MC4R gene c.913C > T; p.Arg305Trp, previously shown to impair receptor function. Treatment with liraglutide (3.0 mg/day) was initiated at age 8 years, resulting in marked reduction in BMI during the first year of treatment. Subsequent genetic testing of family members identified the same variant in her mother, grandmother, and great-grandmother, all of whom had a history of early-onset obesity and related comorbidities, consistent with segregation of the variant within the family. This case underscores the importance of early genetic testing in severe childhood obesity to avoid ineffective treatments and enable targeted therapies (e.g., GLP-1 analogues). Diagnosing (likely) pathogenic MC4R variants can also identify at-risk relatives, providing psychological and clinical benefits across generations. Show less

Obesity causes dyslipidemia and is a major risk factor for cardiovascular disease. However, the mechanisms coupling weight gain and lipid metabolism are poorly understood. Brain melanocortin 4 receptors (MC4Rs) regulate body weight and lipid metabolism in mice, but the relevance of these findings to humans is unclear. Here we investigated lipid levels in men and women with obesity due to MC4R deficiency. Among 7,719 people from the Genetics of Obesity Study cohort, we identified 316 probands and 144 adult family members with loss-of-function (LoF) MC4R mutations. Adults with MC4R deficiency had lower levels of total and low-density lipoprotein (LDL)-cholesterol and triglycerides than 336,728 controls from the UK Biobank, after adjusting for adiposity. Carriers of LoF MC4R variants within the UK Biobank had lower lipid levels and a lower risk of cardiovascular disease, after accounting for body weight, compared to noncarriers. After a high-fat meal, the postprandial rise in triglyceride-rich lipoproteins and metabolomic markers of fatty acid oxidation were reduced in people with MC4R deficiency compared to controls, changes that favor triglyceride storage in adipose tissue. We concluded that central MC4Rs regulate lipid metabolism and cardiovascular disease risk in humans, highlighting potential therapeutic approaches for cardiovascular risk reduction. Show less

A total of 150 clinicians and researchers representing 19 countries came together in person and online to participate in the highly anticipated 2nd International Meeting on Pathway-Related Obesity: Vision & Evidence (IMPROVE), held on 13-15 December 2023 in Paris, France. Building on the success of the inaugural event in 2022, this gathering served as a pivotal platform for attendees to delve into the latest scientific and clinical developments in hyperphagia and early-onset obesity caused by rare melanocortin-4 receptor (MC4R) pathway disease. The central objective of the meeting was to explore the complexities of MC4R pathway-related diseases and generate opportunities for collaborative dialogue among delegates for the advancement of this field. The event unfolded across three distinct sessions, with a dedicated focus on monogenic MC4R pathway disease, Bardet-Biedl syndrome (BBS) and hypothalamic obesity, together with a discussion on the future of the field. Additionally, the agenda featured three insightful workshops designed to facilitate in-depth discussions. One workshop focused on the genetics of monogenic MC4R pathway diseases, another scrutinised the genetics of BBS and the final workshop examined patient management through the exploration of clinical cases. As we reflect on the wealth of information disseminated and the collaborative spirit that permeated the meeting, it becomes clear that IMPROVE 2023 was not merely an assembly of professionals; it was a forum where the future of research in rare MC4R pathway diseases and patient care took centre stage. Here, we encapsulate the key insights, discussions, and initiatives that emerged from this important meeting. Show less

Characteristics of hyperphagia include heightened and prolonged hunger, longer time to satiation, shorter duration of satiety, severe preoccupation with food (i.e., hyperphagic drive), abnormal food-seeking behaviors, and distress or functional impairment when food is unavailable. Patients with melanocortin-4 receptor (MC4R) pathway diseases including those caused by variants in one of multiple key genes of the pathway often present with hyperphagia that results in early-onset, severe obesity because this pathway plays a critical role in regulation of hunger/satiation and energy balance. Patients with syndromic obesity (e.g., Bardet-Biedl syndrome) may also have hyperphagia as a result of neurodevelopmental disruptions in the MC4R pathway. Genetic testing is suggested in patients with early-onset, severe obesity and clinical features of genetic obesity (e.g., hyperphagia, neurodevelopmental differences, dysmorphic features); however, only a small percentage of individuals who meet these criteria undergo testing, potentially owing to limited availability, overlapping symptoms with other obesity types, and infrequent use of genetic testing during diagnosis. Diagnosing hyperphagia may be challenging, as no guidelines have been established for individuals with MC4R pathway diseases. Identifying these individuals is crucial to addressing the challenges of hyperphagia and associated obesity, which often limit quality of life and place overwhelming burdens on patients and families. Show less

Monogenic defects in the leptin-melanocortin pathway are associated with hyperphagia and severe, early-onset obesity. Early childhood growth patterns in height, weight, and BMI, might serve as phenotypic markers for specific genetic disorders; however, reliable data are scarce. This study aimed to evaluate the natural history of height, weight, and BMI in early childhood in a large European group of individuals with monogenic obesity. This multicentre observational study analysed height, weight, and BMI from birth to age 5 years in individuals diagnosed with biallelic (likely) pathogenic LEP, LEPR, POMC, PCSK1, or MC4R variants or monoallelic (likely) pathogenic MC4R variants from six European centres (Berlin and Ulm, Germany; Cambridge, UK; Madrid, Spain; Paris, France; Rotterdam, Netherlands). All patient data up to May 31, 2022 were included in this analysis. All individuals had at least two height or weight measurements between birth and age 5 years. Early childhood growth trajectories were compared with those of control children with obesity without a known genetic cause, following a negative next-generation sequencing panel. Diagnostic performance of BMI as a predictor test for monogenic obesity was also evaluated. We included 147 individuals with monogenic obesity. From the age of 6 months onwards, children with biallelic variants (n=88, 55% female vs 45% male) had substantially higher BMIs than those with monoallelic MC4R variants (n=59, 53% female vs 47% male) and control children (n=113, 59% female vs 41% male). Children with biallelic LEP, LEPR, and MC4R variants showed a steep BMI increase during the first year of life, followed by a plateau until age 5 years, whereas those with biallelic POMC variants did not plateau. Accelerated linear growth was only observed in children with biallelic MC4R variants starting from age 1 year. The optimal BMI cut-off for distinguishing individuals with biallelic variants from control individuals was identified at age 2 years, with a test positivity cutoff of 24·0 kg/m This study identified characteristic early childhood BMI trajectories for different forms of monogenic obesity. From age 6 months onwards, individuals with biallelic variants can be distinguished from those with monoallelic variants and common obesity. A BMI ≥24 kg/m Federal Ministry of Education and Research as part of the German Center for Child and Adolescent Health, German Research Foundation, Spanish Ministry of Health, The Wellcome Trust, Botnar Fondation, Leducq Foundation, National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre, and NIHR Senior Investigator Award. Show less

Hyperphagia is a condition associated with rare obesity-related diseases, presenting as a pathologic, insatiable hunger accompanied by abnormal food-seeking behaviors. In October 2023, a group of researchers and clinicians with expert knowledge on hyperphagia convened at the annual ObesityWeek meeting to discuss the need for a unified definition of hyperphagia and key items necessary to improve the identification, assessment, and treatment of hyperphagia in patients with melanocortin 4 receptor (MC4R) pathway-associated diseases. The definition of hyperphagia proposed by this group is a pathologic, insatiable hunger accompanied by abnormal food-seeking behaviors. Suggested methods to accurately identify patients with hyperphagia include increased physician and parent/caregiver education and standardized efficient screening procedures for use in the clinic. The etiology of hyperphagia as related to abnormal MC4R signaling was also reviewed and proposed as a central cause of the condition across several underlying diseases. Given this potential unified underlying pathology, the expert group recommends that patients with hyperphagia undergo genetic testing and that treatment include comprehensive weight-management strategies incorporating lifestyle and pharmacotherapies targeted at addressing hyperphagia. Show less

Smith Magenis Syndrome (SMS) is a rare genetic disorder caused by RAI1 haploinsufficiency. Obesity in people with SMS is believed partially due to dysfunction of the proximal melanocortin 4 receptor (MC4R) pathway. We therefore studied effects of treatment with the MC4R agonist setmelanotide on obesity and hunger, as well as metabolic, cardiac and safety, in individuals with SMS. People with SMS received once-daily setmelanotide injections, with the dose titrated bi-weekly to a maximum of 3 mg over ∼1 month; and a full-dose treatment duration of 3mo. The primary outcome was percent change in body weight. Secondary outcomes included hunger, waist circumference, body composition, and safety. 12 individuals, ages 11-39 y, enrolled and 10 completed the full-dose treatment phase. Mean percent change in body weight at end-treatment was - 0.28 % [(95 % CI, -2.1 % to 1.5 %; n = 12; P = 0.66]. Participants experienced a significant decrease in total cholesterol associated with a significant decrease in HDL-cholesterol and a trend for lower LDL-cholesterol. Self-reported hunger was reduced at end-treatment (p = 0.011). All participants reported adverse events (AEs), most commonly injection-site reactions and skin hyperpigmentation. No AEs led to withdrawal or death. In this trial, setmelanotide did not significantly reduce body weight in participants with SMS. Participants reported significant differences in hunger, but such self-reports are difficult to interpret without a placebo-treated group. The changes in lipid profiles require further investigation. Results of this study do not suggest that dysfunction of the proximal MC4R pathway is the main etiology for obesity in people with SMS. Show less

Nearly 90 clinicians and researchers from around the world attended the first IMPROVE 2022 International Meeting on Pathway-Related Obesity. Delegates attended in person or online from across Europe, Argentina and Israel to hear the latest scientific and clinical developments in hyperphagia and severe, early-onset obesity, and set out a vision of excellence for the future for improving the diagnosis, treatment, and care of patients with melanocortin-4 receptor (MC4R) pathway-related obesity. The meeting co-chair Peter Kühnen, Charité Universitätsmedizin Berlin, Germany, indicated that change was needed with the rapidly increasing prevalence of obesity and the associated complications to improve the understanding of the underlying mechanisms and acknowledge that monogenic forms of obesity can play an important role, providing insights that can be applied to a wider group of patients with obesity. World-leading experts presented the latest research and led discussions on the underlying science of obesity, diagnosis (including clinical and genetic approaches such as the role of defective MC4R signalling), and emerging clinical data and research with targeted pharmacological approaches. The aim of the meeting was to agree on the questions that needed to be addressed in future research and to ensure that optimised diagnostic work-up was used with new genetic testing tools becoming available. This should aid the planning of new evidence-based treatment strategies for the future, as explained by co-chair Martin Wabitsch, Ulm University Medical Center, Germany. Show less

Abdominal obesity increases the risk for non-alcoholic fatty liver disease (NAFLD), now known as metabolic dysfunction-associated steatotic liver disease (MASLD). To elucidate the directional cell-type level biological mechanisms underlying the association between abdominal obesity and MASLD, we integrated adipose and liver single nucleus RNA-sequencing and bulk cis-expression quantitative trait locus (eQTL) data with the UK Biobank genome-wide association study (GWAS) data using colocalization. Then we used colocalized cis-eQTL variants as instrumental variables in Mendelian randomization (MR) analyses, followed by functional validation experiments on the target genes of the cis-eQTL variants. We identified 17 colocalized abdominal obesity GWAS variants, regulating 17 adipose cell-type marker genes. Incorporating these 17 variants into MR discovers a putative tissue-of-origin, cell-type-aware causal effect of abdominal obesity on MASLD consistently with multiple MR methods without significant evidence for pleiotropy or heterogeneity. Single cell data confirm the adipocyte-enriched mean expression of the 17 genes. Our cellular experiments across human adipogenesis identify risk variant -specific epigenetic and transcriptional mechanisms. Knocking down two of the 17 genes, PPP2R5A and SH3PXD2B, shows a marked decrease in adipocyte lipidation and significantly alters adipocyte function and adipogenesis regulator genes, including DGAT2, LPL, ADIPOQ, PPARG, and SREBF1. Furthermore, the 17 genes capture a characteristic MASLD expression signature in subcutaneous adipose tissue. Overall, we discover a significant cell-type level effect of abdominal obesity on MASLD and trace its biological effect to adipogenesis. NIH grants R01HG010505, R01DK132775, and R01HL170604; the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant No. 802825), Academy of Finland (Grants Nos. 333021), the Finnish Foundation for Cardiovascular Research the Sigrid Jusélius Foundation and the Jane and Aatos Erkko Foundation; American Association for the Study of Liver Diseases (AASLD) Advanced Transplant Hepatology award and NIH/NIDDK (P30DK41301) Pilot and Feasibility award; NIH/NIEHS F32 award (F32ES034668); Finnish Diabetes Research Foundation, Kuopio University Hospital Project grant (EVO/VTR grants 2005-2021), the Academy of Finland grant (Contract no. 138006); Academy of Finland (Grant Nos 335443, 314383, 272376 and 266286), Sigrid Jusélius Foundation, Finnish Medical Foundation, Finnish Diabetes Research Foundation, Novo Nordisk Foundation (#NNF20OC0060547, NNF17OC0027232, NNF10OC1013354) and Government Research Funds to Helsinki University Hospital; Orion Research Foundation, Maud Kuistila Foundation, Finish Medical Foundation, and University of Helsinki. Show less

Obesity is a major health problem because it is associated with increased risk of cardiovascular disease, diabetes, hypertension, and some cancers. Strategies to prevent or reduce obesity focus mainly on the possible effects of natural compounds that can induce a phenotype of browning adipocytes capable of releasing energy in the form of heat. Allicin, a bioactive component of garlic with numerous pharmacological functions, is known to stimulate energy metabolism. In the present study, the effects of allicin on human Simpson-Golabi-Behmel Syndrome (SGBS) cells were investigated by quantifying the dynamics of lipid droplets (LDs) and mitochondria, as well as transcriptomic changes after six days of differentiation. Allicin significantly promoted the reduction in the surface area and size of LDs, leading to the formation of multilocular adipocytes, which was confirmed by the upregulation of genes related to lipolysis. The increase in the number and decrease in the mean aspect ratio of mitochondria in allicin-treated cells indicate a shift in mitochondrial dynamics toward fission. The structural results are confirmed by transcriptomic analysis showing a significant arrangement of gene expression associated with beige adipocytes, in particular increased expression of T-box transcription factor 1 (TBX1), uncoupling protein 1 (UCP1), PPARG coactivator 1 alpha (PPARGC1A), peroxisome proliferator-activated receptor alpha (PPARA), and OXPHOS-related genes. The most promising targets are nuclear genes such as retinoid X receptor alpha (RXRA), retinoid X receptor gamma (RXRG), nuclear receptor subfamily 1 group H member 3 (NR1H3), nuclear receptor subfamily 1 group H member 4 (NR1H4), PPARA, and oestrogen receptor 1 (ESR1). Transcriptomic data and the network pharmacology-based approach revealed that genes and potential targets of allicin are involved in ligand-activated transcription factor activity, intracellular receptor signalling, regulation of cold-induced thermogenesis, and positive regulation of lipid metabolism. The present study highlights the potential role of allicin in triggering browning in human SGBS cells by affecting the LD dynamics, mitochondrial morphology, and expression of brown marker genes. Understanding the potential targets through which allicin promotes this effect may reveal the underlying signalling pathways and support these findings. Show less

Hyperphagia is a key symptom in patients with monogenic obesity, but the assessment is challenging. We aimed to investigate the applicability of Dykens' Hyperphagia Questionnaire in patients with monogenic and syndromic obesity to assess the quality and severity of hyperphagia, and to compare our results with those reported in the literature. Patients with biallelic leptin receptor variants (LEPR, n = 8), heterozygous melanocortin-4 receptor variants (MC4R, n = 7) and 16p11.2 deletions, leading to a deletion of the Src homology 2B adaptor protein gene (n = 5) were included in the study. Hyperphagia was assessed by the parent-based, 13-item hyperphagia questionnaire from Dykens et al. (2007). A literature research was performed to identify published hyperphagia scores assessed by Dykens' Hyperphagia Questionnaire. The total hyperphagia scores were similar in patients with biallelic LEPR and monoallelic MC4R variants (32.0 ± 9.3 vs. 31.4 ± 5.4), but significantly lower in patients with 16p11.2 deletions (21.4 ± 5.5, p < 0.05). Compared to patients with syndromic obesity (27.6 ± 9.0) from the literature, patients with LEPR and MC4R variants had higher total hyperphagia scores. Total hyperphagia scores in patients with 16p11.2 deletions were lower than for patients with other syndromic obesity forms (21.4 ± 5.5 vs. 24.6 ± 8.1), but similar to those for individuals with obesity without a genetic cause (22.9 ± 7.2). Dykens' Hyperphagia Questionnaire seems to be a useful tool to assess hyperphagic behaviour in patients with monogenic and syndromic obesity. Show less

Rare homozygous or biallelic variants in To characterize the historical weight trajectory in these patients. This analysis included data from 2 pivotal single-arm, open-label, Phase 3 trials (NCT02896192, NCT03287960). These were multicenter trials. Patients had obesity due to POMC/PCSK1 or LEPR deficiency. During the trial, patients were treated with setmelanotide. Historical data on measured weight and height were obtained during screening. A total of 17 patients (POMC, n = 8; PCSK1, n = 1; LEPR, n = 8) with historical weight and height data were included in this analysis. Before setmelanotide treatment, patients with obesity due to POMC/PCSK1 or LEPR deficiency were above the 95th percentile for weight throughout childhood, demonstrated continuous weight gain, and did not show long-term weight loss upon interventions (eg, diet, surgery, exercise). Setmelanotide treatment attenuated weight and body mass index trajectories over the observation period of 1 year. In patients with POMC, PCSK1, or LEPR deficiency, traditional interventions for weight loss had limited impact on the trajectory of severe early-onset obesity. However, setmelanotide treatment attenuated weight and body mass index trajectories and led to weight loss associated with health benefits in most individuals. Show less

The melanocortin 4 receptor (MC4R), a component of the leptin-melanocortin pathway, plays a part in bodyweight regulation. Severe early-onset obesity can be caused by biallelic variants in genes that affect the MC4R pathway. We report the results from trials of the MC4R agonist setmelanotide in individuals with severe obesity due to either pro-opiomelanocortin (POMC) deficiency obesity or leptin receptor (LEPR) deficiency obesity. These single-arm, open-label, multicentre, phase 3 trials were done in ten hospitals across Canada, the USA, Belgium, France, Germany, the Netherlands, and the UK. Participants aged 6 years or older with POMC or LEPR deficiency obesity received open-label setmelanotide for 12 weeks. Participants with at least 5 kg weight loss (or ≥5% if weighing <100 kg at baseline) entered an 8-week placebo-controlled withdrawal sequence (including 4 weeks each of blinded setmelanotide and placebo treatment) followed by 32 additional weeks of open-label treatment. The primary endpoint, which was assessed in participants who received at least one dose of study medication and had a baseline assessment (full analysis set), was the proportion of participants with at least 10% weight loss compared with baseline at approximately 1 year. A key secondary endpoint was mean percentage change in the most hunger score of the 11-point Likert-type scale at approximately 1 year on the therapeutic dose, which was assessed in a subset of participants aged 12 years or older in the full analysis set who demonstrated at least 5 kg weight loss (or ≥5% in paediatric participants if baseline bodyweight was <100 kg) over the 12-week open-label treatment phase and subsequently proceeded into the placebo-controlled withdrawal sequence, regardless of later disposition. These studies are registered with ClinicalTrials.gov, NCT02896192 and NCT03287960. Between Feb 14, 2017, and Sept 7, 2018, ten participants were enrolled in the POMC trial and 11 participants were enrolled in the LEPR trial, and included in the full analysis and safety sets. Eight (80%) participants in the POMC trial and five (45%) participants in the LEPR trial achieved at least 10% weight loss at approximately 1 year. The mean percentage change in the most hunger score was -27·1% (n=7; 90% CI -40·6 to -15·0; p=0·0005) in the POMC trial and -43·7% (n=7; -54·8 to -29·1; p<0·0001) in the LEPR trial. The most common adverse events were injection site reaction and hyperpigmentation, which were reported in all ten participants in the POMC trial; nausea was reported in five participants and vomiting in three participants. In the LEPR trial, the most commonly reported treatment-related adverse events were injection site reaction in all 11 participants, skin disorders in five participants, and nausea in four participants. No serious treatment-related adverse events occurred in both trials. Our results support setmelanotide for the treatment of obesity and hyperphagia caused by POMC or LEPR deficiency. Rhythm Pharmaceuticals. Show less

The clinical phenotype of patients with monogenic obesity due to mutations in the leptin receptor (LEPR) or melanocortin 4 receptor (MC4R) gene is characterized by impaired satiety and hyperphagia, leading to extreme, sometimes life-threatening weight gain. In a case series, we analysed the effect of an off-label methylphenidate (MPH) use for 1 year as an individual treatment approach on eating behaviour (Child Eating Behaviour Questionnaire [CEBQ]), appetite (visual analogue scales) and body mass index (BMI) trajectories in five patients with severe obesity due to mutations in the LEPR (n = 3) or MC4R (n = 2) gene. After 1 year use of MPH (20 mg/day divided in two to three doses), BMI (Δ BMI The observed decrease in BMI trajectories with MPH use for one year is clinically meaningful in this group of patients, since the natural course would have been associated with a pronounced increase in BMI, leading to comorbidities and complications over time. Show less

Conjugated linoleic acid (CLA) reduces adiposity in vivo. However, mechanisms mediating these changes are unclear. Therefore, we treated cultures of human adipocytes with trans-10, cis-12 (10,12) CLA, cis-9, trans-11 (9,11) CLA or other trans fatty acids (FA), and measured indices of lipid metabolism. The lipid-lowering effects of 10,12 CLA were unique, as other trans FA did not reduce TG content to the same extent. Using low levels of [(14)C]-CLA isomers, it was shown that both isomers were readily incorporated into acylglycerols and phospholipids, albeit at lower levels than [(14)C]-oleic or [(14)C]-linoleic acids. When using [(14)C]-acetic acid and [(14)C]-pyruvic acid as substrates, 30 μM 10,12 CLA, but not 9,11 CLA, decreased de novo synthesis of triglyceride, free FA, diacylglycerol, cholesterol esters, cardiolipin, phospholipids and ceramides within 3-24 h. Treatment with 30 μM 10,12 CLA, but not 9,11 CLA, decreased total cellular lipids within 3 days and the ratio of monounsaturated FA (MUFA) to saturated FA, and increased C18:0 acyl-CoA levels within 24 h. Consistent with these data, stearoyl-CoA desaturase (SCD)-1 mRNA and protein levels were down-regulated by 10,12 CLA within 7-12 h, respectively. The mRNA levels of liver X receptor (LXR)α and sterol regulatory element binding protein (SREBP)-1c, transcription factors that regulate SCD-1, were decreased by 10,12 CLA within 5 h. These data suggest that the isomer-specific decrease in de novo lipid synthesis by 10,12 CLA is due, in part, to the rapid repression of lipogenic transcription factors that regulate MUFA synthesis, suggesting an anti-obesity mechanism unique to this trans FA. Show less

Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation. Show less

To establish that human adipocytes express functional glucose-dependent insulinotropic peptide (GIP) receptors and in particular the regulation of GIP receptor (GIPR) expression in the context of the dynamic process of adipocyte differentiation. A combination of semiquantitative real-time PCR and measurement of GIP-stimulated cAMP accumulation was used to establish the expression and functional coupling of GIPRs during in vitro differentiation of human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes. Semiquantitative real-time PCR revealed that GIPR expression was substantially increased by day 4 of differentiation, reaching a maximum around 6-8 days (approximately 200-fold increase above undifferentiated cells, n=2). We also analysed the expression of the adipocyte fatty acid binding protein (FABP4) to relate GIPR expression to a molecular differentiation marker of adipogenesis. FABP4 expression was barely detectable in undifferentiated cells. However, following exposure to adipogenic medium, FABP4 expression gradually increased, with a maximal expression level around 10 days (approximately 1,600,000-fold increase above undifferentiated cells, n=2). Thus, the increases in GIPR mRNA during adipogenesis occur earlier than FABP4, suggesting that it might represent a gene expressed early in terminal differentiation and thus plays a role in fat droplet formation. A unit of 1 microM GIP failed to raise intracellular cAMP levels above basal levels in undifferentiated cells (n=3). In stark contrast, the 9-day differentiated cells produced a robust concentration-dependent increase in cAMP accumulation following stimulation with GIP, with an EC(50) value of 2.3 nM (n=3). The maximal response represented a 9-34-fold increase in cAMP accumulation above basal levels. This study demonstrates that GIPRs are expressed by human adipocytes, both GIPR mRNA and functional receptor expression being present in differentiated adipocytes but not in preadipocytes. Further investigation into the functional effects of GIP on differentiated SGBS cells could help towards understanding exactly how GIP regulates fat accumulation in human adipocytes. Show less