Partial inhibition of adipose tissue lipolysis does not increase fat mass but improves glucose metabolism and insulin sensitivity through modulation of fatty acid turnover and induction of fat cell de novo lipogenesis.

When energy is needed, white adipose tissue (WAT) provides fatty acids (FAs) for use in peripheral tissues via stimulation of fat cell lipolysis. FAs have been postulated to play a critical role in the development of obesity-induced insulin resistance, a major risk factor for diabetes and cardiovascular disease. However, whether and how chronic inhibition of fat mobilization from WAT modulates insulin sensitivity remains elusive. Hormone-sensitive lipase (HSL) participates in the breakdown of WAT triacylglycerol into FAs. HSL haploinsufficiency and treatment with a HSL inhibitor resulted in improvement of insulin tolerance without impact on body weight, fat mass, and WAT inflammation in high-fat-diet–fed mice. In vivo palmitate turnover analysis revealed that blunted lipolytic capacity is associated with diminution in FA uptake and storage in peripheral tissues of obese HSL haploinsufficient mice. The reduction in FA turnover was accompanied by an improvement of glucose metabolism with a shift in respiratory quotient, increase of glucose uptake in WAT and skeletal muscle, and enhancement of de novo lipogenesis and insulin signalling in liver. In human adipocytes, HSL gene silencing led to improved insulin-stimulated glucose uptake, resulting in increased de novo lipogenesis and activation of cognate gene expression. In clinical studies, WAT lipolytic rate was positively and negatively correlated with indexes of insulin resistance and WAT de novo lipogenesis gene expression, respectively. In obese individuals, chronic inhibition of lipolysis resulted in induction of WAT de novo lipogenesis gene expression. Thus, reduction in WAT lipolysis reshapes FA fluxes without increase of fat mass and improves glucose metabolism through cell-autonomous induction of fat cell de novo lipogenesis, which contributes to improved insulin sensitivity.

Author Summary

In periods of energy demand, mobilization of fat stores in mammals (i.e., adipose tissue lipolysis) is essential to provide energy in the form of fatty acids. In excess, however, fatty acids induce resistance to the action of insulin, which serves to regulate glucose metabolism in skeletal muscle and liver. Insulin resistance (or low insulin sensitivity) is believed to be a cornerstone of the complications of obesity such as type 2 diabetes and cardiovascular diseases. In this study, our clinical observation of natural variation in fat cell lipolysis in individuals reveals that a high lipolytic rate is associated with low insulin sensitivity. Furthermore, partial genetic and pharmacologic inhibition of hormone-sensitive lipase, one of the enzymes involved in the breakdown of white adipose tissue lipids, results in improvement of insulin sensitivity in mice without gain in body weight and fat mass. We undertake a series of mechanistic studies in mice and in human fat cells to show that blunted lipolytic capacity increases the synthesis of new fatty acids from glucose in fat cells, a pathway that has recently been shown by others to be a major determinant of whole body insulin sensitivity. In conclusion, partial inhibition of adipose tissue lipolysis is a plausible strategy in the treatment of obesity-related insulin resistance.

The phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2, or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells of a patient with an unclassified syndrome of congenital, progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated p.His1047Leu mutation in PIK3CA, which encodes the p110α catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in 10 further patients with overlapping syndromes identified either p.His1047Leu or a second cancer-associated mutation, p.His1047Arg, in 9 cases. Affected dermal fibroblasts showed enhanced basal and EGF-stimulated phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation and concomitant activation of downstream signaling. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.

The strongest BMI–associated GWAS locus in humans is the FTO gene. Rodent studies demonstrate a role for FTO in energy homeostasis and body composition. The phenotypes observed in loss of expression studies are complex with perinatal lethality, stunted growth from weaning, and significant alterations in body composition. Thus understanding how and where Fto regulates food intake, energy expenditure, and body composition is a challenge. To address this we generated a series of mice with distinct temporal and spatial loss of Fto expression. Global germline loss of Fto resulted in high perinatal lethality and a reduction in body length, fat mass, and lean mass. When ratio corrected for lean mass, mice had a significant increase in energy expenditure, but more appropriate multiple linear regression normalisation showed no difference in energy expenditure. Global deletion of Fto after the in utero and perinatal period, at 6 weeks of age, removed the high lethality of germline loss. However, there was a reduction in weight by 9 weeks, primarily as loss of lean mass. Over the subsequent 10 weeks, weight converged, driven by an increase in fat mass. There was a switch to a lower RER with no overall change in food intake or energy expenditure. To test if the phenotype can be explained by loss of Fto in the mediobasal hypothalamus, we sterotactically injected adeno-associated viral vectors encoding Cre recombinase to cause regional deletion. We observed a small reduction in food intake and weight gain with no effect on energy expenditure or body composition. Thus, although hypothalamic Fto can impact feeding, the effect of loss of Fto on body composition is brought about by its actions at sites elsewhere. Our data suggest that Fto may have a critical role in the control of lean mass, independent of its effect on food intake.

Author Summary

The fat mass and obesity (FTO) gene has one of the strongest links with body mass index (BMI) in the human population. One in six people have the “risk” alteration and weigh 3 kg more than those with the unaltered gene, but it is not understood how this gene influences BMI and obesity. We set out to understand how and where in the body FTO affects food intake, energy expenditure, and body composition using a mouse model that can be manipulated to lack FTO at particular times and/or places. Removing FTO everywhere from conception had a dramatic effect on body composition and resulted in stunted growth and some lethality. Removing FTO everywhere but only in adult animals resulted in better viability and normal growth but, surprisingly, reduced lean mass and increased fat mass with a change in the type of metabolic fuel being used. Finally, we removed FTO from the hypothalamus of adult animals, an important brain region involved in energy metabolism. These animals showed a mild reduction in food intake and weight gain. Our experiments show that FTO has an important role in body composition and that other brain areas outside of the hypothalamus are also important in determining its effects.

Src homology 2 B adapter protein 1 (SH2B1) modulates signaling by a variety of ligands that bind to receptor tyrosine kinases or JAK-associated cytokine receptors, including leptin, insulin, growth hormone (GH), and nerve growth factor (NGF). Targeted deletion of Sh2b1 in mice results in increased food intake, obesity, and insulin resistance, with an intermediate phenotype seen in heterozygous null mice on a high-fat diet. We identified SH2B1 loss-of-function mutations in a large cohort of patients with severe early-onset obesity. Mutation carriers exhibited hyperphagia, childhood-onset obesity, disproportionate insulin resistance, and reduced final height as adults. Unexpectedly, mutation carriers exhibited a spectrum of behavioral abnormalities that were not reported in controls, including social isolation and aggression. We conclude that SH2B1 plays a critical role in the control of human food intake and body weight and is implicated in maladaptive human behavior.

Summary

Work on obesity is evolving, and obesity is a consequence of our evolutionary history. In the space of 50 years, we have become an obese species. The reasons why can be addressed at a number of different levels. These include separating between whether the primary cause lies on the food intake or energy expenditure side of the energy balance equation, and determining how genetic and environmental effects contribute to weight variation between individuals. Opinion on whether increased food intake or decreased energy expenditure drives the obesity epidemic is still divided, but recent evidence favours the idea that food intake, rather than altered expenditure, is most important. There is more of a consensus that genetics explains most (probably around 65%) of weight variation between individuals. Recent advances in genome-wide association studies have identified many polymorphisms that are linked to obesity, yet much of the genetic variance remains unexplained. Finding the causes of this unexplained variation will be an impetus of genetic and epigenetic research on obesity over the next decade. Many environmental factors – including gut microbiota, stress and endocrine disruptors – have been linked to the risk of developing obesity. A better understanding of gene-by-environment interactions will also be key to understanding obesity in the years to come.

Background

Niaspan® (extended-release niacin) is a nicotinic acid formulation used to treat dyslipidemia in obese subjects. Niaspan binds to the GPR109A receptor in adipose tissue and stimulates adiponectin secretion, which should improve insulin sensitivity. However, Niaspan therapy often causes insulin resistance. The purpose of this study was to evaluate whether Niaspan-induced changes in plasma adiponectin concentration are associated with a blunting of Niaspan's adverse effect on insulin action in obese subjects with non-alcoholic fatty liver disease (NAFLD).

Methods

A hyperinsulinemic-euglycemic clamp procedure was used to assess muscle insulin sensitivity before and after 16 weeks of Niaspan therapy in 9 obese subjects with NAFLD [age 43 ± 5 years; BMI 35.1 ± 1.3 (means ± SEM)].

Results

Niaspan therapy did not affect body weight (99.1 ± 4.2 vs. 100 ± 4.4 kg) or percent body fat (37.8 ± 2.5 vs. 37.0 ± 2.5%). However, Niaspan therapy caused a 22% reduction in insulin-mediated glucose disposal (p < 0.05). The deterioration in glucose disposal was inversely correlated with the Niaspan-induced increase in plasma adiponectin concentration (r = 0.67, p = 0.05).

Conclusions

These results demonstrate that Niaspan causes skeletal muscle insulin resistance, independent of changes in body weight or body fat, and the Niaspan-induced increase in plasma adiponectin concentration might partially ameliorate Niaspan's adverse effect on insulin action in obese subjects with NAFLD.

OBJECTIVE

Obesity-associated insulin resistance is characterized by a state of chronic, low-grade inflammation that is associated with the accumulation of M1 proinflammatory macrophages in adipose tissue. Although different evidence explains the mechanisms linking the expansion of adipose tissue and adipose tissue macrophage (ATM) polarization, in the current study we investigated the concept of lipid-induced toxicity as the pathogenic link that could explain the trigger of this response.

RESEARCH DESIGN AND METHODS

We addressed this question using isolated ATMs and adipocytes from genetic and diet-induced murine models of obesity. Through transcriptomic and lipidomic analysis, we created a model integrating transcript and lipid species networks simultaneously occurring in adipocytes and ATMs and their reversibility by thiazolidinedione treatment.

RESULTS

We show that polarization of ATMs is associated with lipid accumulation and the consequent formation of foam cell–like cells in adipose tissue. Our study reveals that early stages of adipose tissue expansion are characterized by M2-polarized ATMs and that progressive lipid accumulation within ATMs heralds the M1 polarization, a macrophage phenotype associated with severe obesity and insulin resistance. Furthermore, rosiglitazone treatment, which promotes redistribution of lipids toward adipocytes and extends the M2 ATM polarization state, prevents the lipid alterations associated with M1 ATM polarization.

CONCLUSIONS

Our data indicate that the M1 ATM polarization in obesity might be a macrophage-specific manifestation of a more general lipotoxic pathogenic mechanism. This indicates that strategies to optimize fat deposition and repartitioning toward adipocytes might improve insulin sensitivity by preventing ATM lipotoxicity and M1 polarization.

OBJECTIVE

Genetic defects in human pericentrin (PCNT), encoding the centrosomal protein pericentrin, cause a form of osteodysplastic primordial dwarfism that is sometimes reported to be associated with diabetes. We thus set out to determine the prevalence of diabetes and insulin resistance among patients with PCNT defects and examined the effects of pericentrin depletion on insulin action using 3T3-L1 adipocytes as a model system.

RESEARCH DESIGN AND METHODS

A cross-sectional metabolic assessment of 21 patients with PCNT mutations was undertaken. Pericentrin expression in human tissues was profiled using quantitative real-time PCR. The effect of pericentrin knockdown on insulin action and adipogenesis in 3T3-L1 adipocytes was determined using Oil red O staining, gene-expression analysis, immunoblotting, and glucose uptake assays. Pericentrin expression and localization also was determined in skeletal muscle.

RESULTS

Of 21 patients with genetic defects in PCNT, 18 had insulin resistance, which was severe in the majority of subjects. Ten subjects had confirmed diabetes (mean age of onset 15 years [range 5–28]), and 13 had metabolic dyslipidemia. All patients without insulin resistance were younger than 4 years old. Knockdown of pericentrin in adipocytes had no effect on proximal insulin signaling but produced a twofold impairment in insulin-stimulated glucose uptake, approximately commensurate with an associated defect in cell proliferation and adipogenesis. Pericentrin was highly expressed in human skeletal muscle, where it showed a perinuclear distribution.

CONCLUSIONS

Severe insulin resistance and premature diabetes are common features of PCNT deficiency but are not congenital. Partial failure of adipocyte differentiation may contribute to this, but pericentrin deficiency does not impair proximal insulin action in adipocytes.

Objective:

Lipid accumulation in skeletal muscle and the liver is strongly implicated in the development of insulin resistance and type 2 diabetes, but the mechanisms underpinning fat accrual in these sites remain incompletely understood. Accumulating evidence of muscle mitochondrial dysfunction in insulin-resistant states has fuelled the notion that primary defects in mitochondrial fat oxidation may be a contributory mechanism. The purpose of our study was to determine whether patients with congenital lipodystrophy, a disorder primarily affecting white adipose tissue, manifest impaired mitochondrial oxidative phosphorylation in skeletal muscle.

Research Design and Methods:

Mitochondrial oxidative phosphorylation was assessed in quadriceps muscle using 31P-magnetic resonance spectroscopy measurements of phosphocreatine recovery kinetics after a standardized exercise bout in nondiabetic patients with congenital lipodystrophy and in age-, gender-, body mass index-, and fitness-matched controls.

Results:

The phosphocreatine recovery rate constant (k) was significantly lower in patients with congenital lipodystrophy than in healthy controls (P < 0.001). This substantial (∼35%) defect in mitochondrial oxidative phosphorylation was not associated with significant changes in basal or sleeping metabolic rates.

Conclusions:

Muscle mitochondrial oxidative phosphorylation is impaired in patients with congenital lipodystrophy, a paradigmatic example of primary adipose tissue dysfunction. This finding suggests that changes in mitochondrial oxidative phosphorylation in skeletal muscle could, at least in some circumstances, be a secondary consequence of adipose tissue failure. These data corroborate accumulating evidence that mitochondrial dysfunction can be a consequence of insulin-resistant states rather than a primary defect. Nevertheless, impaired mitochondrial fat oxidation is likely to accelerate ectopic fat accumulation and worsen insulin resistance.

The close correspondence between energy intake and expenditure over prolonged time periods, coupled with an apparent protection of the level of body adiposity in the face of perturbations of energy balance, has led to the idea that body fatness is regulated via mechanisms that control intake and energy expenditure. Two models have dominated the discussion of how this regulation might take place. The set point model is rooted in physiology, genetics and molecular biology, and suggests that there is an active feedback mechanism linking adipose tissue (stored energy) to intake and expenditure via a set point, presumably encoded in the brain. This model is consistent with many of the biological aspects of energy balance, but struggles to explain the many significant environmental and social influences on obesity, food intake and physical activity. More importantly, the set point model does not effectively explain the ‘obesity epidemic’ – the large increase in body weight and adiposity of a large proportion of individuals in many countries since the 1980s. An alternative model, called the settling point model, is based on the idea that there is passive feedback between the size of the body stores and aspects of expenditure. This model accommodates many of the social and environmental characteristics of energy balance, but struggles to explain some of the biological and genetic aspects. The shortcomings of these two models reflect their failure to address the gene-by-environment interactions that dominate the regulation of body weight. We discuss two additional models – the general intake model and the dual intervention point model – that address this issue and might offer better ways to understand how body fatness is controlled.

Perilipin (PLIN1) is a constitutive adipocyte lipid droplet coat protein. N-terminal amphipathic helices and central hydrophobic stretches are thought to anchor it on the lipid droplet, where it appears to function as a scaffold protein regulating lipase activity. We recently identified two different C-terminal PLIN1 frame shift mutations (Leu-404fs and Val-398fs) in patients with a novel subtype of partial lipodystrophy, hypertriglyceridemia, severe insulin resistance, and type 2 diabetes (Gandotra, S., Le Dour, C., Bottomley, W., Cervera, P., Giral, P., Reznik, Y., Charpentier, G., Auclair, M., Delépine, M., Barroso, I., Semple, R. K., Lathrop, M., Lascols, O., Capeau, J., O'Rahilly, S., Magré, J., Savage, D. B., and Vigouroux, C. (2011) N. Engl. J. Med. 364, 740–748.) When overexpressed in preadipocytes, both mutants fail to inhibit basal lipolysis. Here we used bimolecular fluorescence complementation assays to show that the mutants fail to bind ABHD5, permitting its constitutive coactivation of ATGL, resulting in increased basal lipolysis. siRNA-mediated knockdown of either ABHD5 or ATGL expression in the stably transfected cells expressing mutant PLIN1 reduced basal lipolysis. These insights from naturally occurring human variants suggest that the C terminus sequesters ABHD5 and thus inhibits basal ATGL activity. The data also suggest that pharmacological inhibition of ATGL could have therapeutic potential in patients with this rare but metabolically serious disorder.

Obesity is a highly heritable and genetically heterogeneous disorder1. Here we investigated the contribution of copy number variation to obesity in 300 Caucasian patients with severe early-onset obesity, 143 of whom also had developmental delay. Large (>500 kilobases), rare (<1%) deletions were significantly enriched in patients compared to 7,366 controls (P < 0.001). We identified several rare copy number variants that were recurrent in patients but absent or at much lower prevalence in controls. We identified five patients with overlapping deletions on chromosome 16p11.2 that were found in 2 out of 7,366 controls (P < 5 × 10−5). In three patients the deletion co-segregated with severe obesity. Two patients harboured a larger de novo 16p11.2 deletion, extending through a 593-kilobase region previously associated with autism2-4 and mental retardation5; both of these patients had mild developmental delay in addition to severe obesity. In an independent sample of 1,062 patients with severe obesity alone, the smaller 16p11.2 deletion was found in an additional two patients. All 16p11.2 deletions encompass several genes but include SH2B1, which is known to be involved in leptin and insulin signalling6. Deletion carriers exhibited hyperphagia and severe insulin resistance disproportionate for the degree of obesity. We show that copy number variation contributes significantly to the genetic architecture of human obesity.

Mitochondrial dysfunction is associated with insulin resistance and type 2 diabetes. It has thus been suggested that primary and/or genetic abnormalities in mitochondrial function may lead to accumulation of toxic lipid species in muscle and elsewhere, impairing insulin action on glucose metabolism. Alternatively, however, defects in insulin signaling may be primary events that result in mitochondrial dysfunction, or there may be a bidirectional relationship between these phenomena. To investigate this, we examined mitochondrial function in patients with genetic defects in insulin receptor (INSR) signaling. We found that phosphocreatine recovery after exercise, a measure of skeletal muscle mitochondrial function in vivo, was significantly slowed in patients with INSR mutations compared with that in healthy age-, fitness-, and BMI-matched controls. These findings suggest that defective insulin signaling may promote mitochondrial dysfunction. Furthermore, consistent with previous studies of mouse models of mitochondrial dysfunction, basal and sleeping metabolic rates were both significantly increased in genetically insulin-resistant patients, perhaps because mitochondrial dysfunction necessitates increased nutrient oxidation in order to maintain cellular energy levels.

Data from mice lacking all endogenous melanocortin peptides suggest Agouti-related-peptide acts in vivo as a melanocortin antagonist rather than an inverse agonist.

The hypothalamic melanocortin system is unique among neuropeptide systems controlling energy homeostasis, in that both anorexigenic proopiomelanocortin (POMC)-derived and orexigenic Agouti related-peptide (AgRP)-derived ligands act at the same receptors, namely melanocortin 3 and 4 receptors (MC3/4R). AgRP clearly acts as a competitive antagonist at MC3R and MC4R but may also have an inverse agonist action at these receptors. The physiological relevance of this remains uncertain. We generated a mouse lacking both POMC and AgRP [double knockout (DKO) mouse]. Phenotyping was performed in the absence and presence of glucocorticoids, and the response to central peptide administration was studied. The phenotype of DKO mice is indistinguishable from that of mice lacking Pomc alone, with both exhibiting highly similar degrees of hyperphagia and increased body length, fat, and lean mass compared with wild-type controls. After a 24-h fast, there was no difference in the refeeding response between Pomc−/− and DKO mice. Similarly, corticosterone supplementation caused an equivalent increase in food intake and body weight in both genotypes. Although the central administration of [Nle4, d-Phe7]-α-MSH to DKO mice caused a decrease in food intake and an increase in brown adipose tissue Ucp1 expression, both of which could be antagonized with the coadministration of AgRP, there was no effect of AgRP alone. These data suggest AgRP acts predominantly as a melanocortin antagonist. If AgRP has significant melanocortin-independent actions, these are of insufficient magnitude in vivo to impact any of the detailed phenotypes we have measured under a wide variety of conditions.

Lipoprotein receptor LRP1 play critical roles in lipid metabolism, and this study reveals a novel role for LRP1 in controlling food intake and obesity in the central nervous system of the adult mouse.

Obesity is a growing epidemic characterized by excess fat storage in adipocytes. Although lipoprotein receptors play important roles in lipid uptake, their role in controlling food intake and obesity is not known. Here we show that the lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis. Conditional deletion of the Lrp1 gene in the brain resulted in an obese phenotype characterized by increased food intake, decreased energy consumption, and decreased leptin signaling. LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. We further showed that deletion of the Lrp1 gene specifically in the hypothalamus by Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results demonstrate that the lipoprotein receptor LRP1, which is critical in lipid metabolism, also regulates food intake and energy homeostasis in the adult central nervous system.

Author Summary

The World Health Organization estimates that at least 1 in 10 adults worldwide are obese, and in some western countries, a far greater percentage (25% or more) is affected. Obesity is a serious concern because it increases the risk of cardiovascular disease, type 2 diabetes, and some cancers, among other health problems. Despite recent advances in understanding the disease mechanism, effective treatments are still lacking. Lipoprotein receptors play critical roles in lipid metabolism, but their potential roles in controlling food intake and obesity in the central nervous system have not been examined. Here we show that deletion of LRP1, a member of the LDL (low density lipoprotein) receptor family, in the adult mouse brain results in obese phenotype characterized by increased food intake, decreased energy consumption and decreased leptin signaling. We further show that deletion of the Lrp1 gene specifically in the hypothalamus (a region of the brain) by using Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results present a novel function of LRP1: the direct regulation of leptin signaling and energy balance in the adult central nervous system. Hence, LRP1 represents a very promising new therapeutic target for the design of innovative and more effective therapies for obesity.

OBJECTIVE

Single nucleotide polymorphisms (SNPs) in intron 1 of fat mass– and obesity-associated gene (FTO) are strongly associated with human adiposity, whereas Fto−/− mice are lean and Fto+/− mice are resistant to diet-induced obesity. We aimed to determine whether FTO mutations are disproportionately represented in lean or obese humans and to use these mutations to understand structure-function relationships within FTO.

RESEARCH DESIGN AND METHODS

We sequenced all coding exons of FTO in 1,433 severely obese and 1,433 lean individuals. We studied the enzymatic activity of selected nonsynonymous variants.

RESULTS

We identified 33 heterozygous nonsynonymous variants in lean (2.3%) and 35 in obese (2.4%) individuals, with 8 mutations unique to the obese and 11 unique to the lean. Two novel mutations replace absolutely conserved residues: R322Q in the catalytic domain and R96H in the predicted substrate recognition lid. R322Q was unable to catalyze the conversion of 2-oxoglutarate to succinate in the presence or absence of 3-methylthymidine. R96H retained some basal activity, which was not enhanced by 3-methylthymidine. However, both were found in lean and obese individuals.

CONCLUSIONS

Heterozygous, loss-of-function mutations in FTO exist but are found in both lean and obese subjects. Although intron 1 SNPs are unequivocally associated with obesity in multiple populations and murine studies strongly suggest that FTO has a role in energy balance, it appears that loss of one functional copy of FTO in humans is compatible with being either lean or obese. Functional analyses of FTO mutations have given novel insights into structure-function relationships in this enzyme.

Genetic diagnosis of inherited metabolic disease is conventionally achieved through syndrome recognition and targeted gene sequencing, but many patients receive no specific diagnosis. Next-generation sequencing allied to capture of expressed sequences from genomic DNA now offers a powerful new diagnostic approach. Barriers to routine diagnostic use include cost, and the complexity of interpreting results arising from simultaneous identification of large numbers of variants. We applied exome-wide sequencing to an individual, 16-year-old daughter of consanguineous parents with a novel syndrome of short stature, severe insulin resistance, ptosis, and microcephaly. Pulldown of expressed sequences from genomic DNA followed by massively parallel sequencing was undertaken. Single nucleotide variants were called using SAMtools prior to filtering based on sequence quality and existence in control genomes and exomes. Of 485 genetic variants predicted to alter protein sequence and absent from control data, 24 were homozygous in the patient. One mutation – the p.Arg732X mutation in the WRN gene – has previously been reported in Werner's syndrome (WS). On re-evaluation of the patient several early features of WS were detected including loss of fat from the extremities and frontal hair thinning. Lymphoblastoid cells from the proband exhibited a defective decatenation checkpoint, consistent with loss of WRN activity. We have thus diagnosed WS some 15 years earlier than average, permitting aggressive prophylactic therapy and screening for WS complications, illustrating the potential of exome-wide sequencing to achieve early diagnosis and change management of rare autosomal recessive disease, even in individual patients of consanguineous parentage with apparently novel syndromes.

Sepsis describes a complex clinical syndrome that results from an infection, setting off a cascade of systemic inflammatory responses that can lead to multiple organ failure and death. Leptin is a 16 kDa adipokine that, among its multiple known effects, is involved in regulating immune function. Here we demonstrate that leptin deficiency in ob/ob mice leads to higher mortality and more severe organ damage in a standard model of sepsis in mice (cecal ligation and puncture, CLP). Moreover, systemic leptin replacement improved the immune response to CLP. Based on the molecular mechanisms of leptin regulation of energy metabolism and reproductive function, we hypothesized that leptin acts in the central nervous system (CNS) to efficiently coordinate peripheral immune defense in sepsis. We now report that leptin signaling in the brain increases survival during sepsis in leptin-deficient as well as in wild-type mice and that endogenous CNS leptin action is required for an adequate systemic immune response. These findings reveal the existence of a relevant neuroendocrine control of systemic immune defense, and suggest a possible therapeutic potential for leptin analogues in infectious disease.

Genetic studies in patients with severe early-onset obesity have provided insights into the molecular and physiological pathways that regulate body weight in humans. We report a 19-year-old male with hyperphagia and severe obesity, mild learning difficulties and hypogonadism, in whom diagnostic tests for Prader–Willi syndrome (PWS) had been negative. We carried out detailed clinical and metabolic phenotyping of this patient and investigated the genetic basis of this obesity syndrome using Agilent 185 k array comparative genomic hybridization (aCGH) and Affymetrix 6.0 genotyping arrays. The identified deletion was validated using multiplex ligation-dependent probe amplification and long-range PCR, followed by breakpoint sequencing which enabled precise localization of the deletion. We identified a ∼187 kb microdeletion at chromosome 15q11–13 that encompasses non-coding small nucleolar RNAs (including HBII-85 snoRNAs) which were not expressed in peripheral lymphocytes from the patient. Characterization of the clinical phenotype revealed increased ad libitum food intake, normal basal metabolic rate when adjusted for fat-free mass, partial hypogonadotropic hypogonadism and growth failure. We have identified a novel deletion on chromosome 15q11–13 in an individual with hyperphagia, obesity, hypogonadism and other features associated with PWS, which is normally caused by deficiency of several paternally expressed imprinted transcripts within chromosome 15q11–13, a region that includes multiple protein-coding genes as well as several non-coding snoRNAs. These findings provide direct evidence for the role of a particular family of non-coding RNAs, the HBII-85 snoRNA cluster, in human energy homeostasis, growth and reproduction.

The transcription factor SREBP1c (sterol-regulatory-element-binding protein 1c) is highly expressed in adipose tissue and plays a central role in several aspects of adipocyte development including the induction of PPARγ (peroxisome-proliferator-activated receptor γ), the generation of an endogenous PPARγ ligand and the expression of several genes critical for lipid biosynthesis. Despite its significance, the regulation of SREBP1c expression during adipogenesis is not well characterized. We have noted that in several models of adipogenesis, SREBP1c expression closely mimics that of known C/EBPβ (CCAAT/enhancer-binding protein β) targets. Inhibition of C/EBP activity during adipogenesis by expressing either the dominant-negative C/EBPβ LIP (liver-enriched inhibitory protein) isoform, the co-repressor ETO (eight-twenty one/MTG8) or using siRNAs (small interfering RNAs) targeting either C/EBPβ or C/EBPδ significantly impaired early SREBP1c induction. Furthermore, ChIP (chromatin immunoprecipitation) assays identified specific sequences in the SREBP1c promoter to which C/EBPβ and C/EBPδ bind in intact cells, demonstrating that these factors may directly regulate SREBP1c expression. Using cells in which C/EBPα expression is inhibited using shRNA (short hairpin RNA) and ChIP assays we show that C/EBPα replaces C/EBPβ and C/EBPδ as a regulator of SREBP1c expression in maturing adipocytes. These results provide novel insight into the induction of SREBP1c expression during adipogenesis. Moreover, the findings of the present study identify an important additional mechanism via which the C/EBP transcription factors may control a network of gene expression regulating adipogenesis, lipogenesis and insulin sensitivity.

Lipodystrophic syndromes are characterized by adipose tissue deficiency. Although rare, they are of considerable interest as, like obesity, they typically lead to ectopic lipid accumulation, dyslipidaemia and insulin resistant diabetes. Here we describe a female patient with partial lipodystrophy (affecting limb, femorogluteal and subcutaneous abdominal fat), white adipocytes with multiloculated lipid droplets and insulin-resistant diabetes who was found to be homozygous for a premature truncation mutation in the lipid droplet protein CIDEC (E186X). The truncation disrupts the highly conserved CIDE-C domain and the mutant protein is mistargeted and fails to increase lipid droplet size in transfected cells. In mice, Cidec deficiency also reduces fat mass and induces the formation of white adipocytes with multilocular lipid droplets, but in contrast to our patient, Cidec null mice are protected against diet-induced obesity and insulin resistance. As well as describing a novel autosomal recessive form of familial partial lipodystrophy these observations suggest that CIDEC is required for unilocular lipid droplet formation and optimal energy storage in human fat.

Sequence variants in the first intron of FTO are strongly associated with human obesity and human carriers of the risk alleles show evidence for increased appetite and food intake. Mice globally lacking Fto display a complex phenotype characterised by both increased energy expenditure and increased food intake. The site of action of FTO on energy balance is unclear. Fasting reduces levels of Fto mRNA in the arcuate nucleus (ARC) of the hypothalamus, a site where Fto expression is particularly high. In this study, we have extended this nutritional link by demonstrating that consumption of a high fat diet (45%) results in a 2.5 fold increase in Arc Fto expression. We have further explored the role of hypothalamic Fto in the control of food intake by using stereotactic injections coupled with AAV technology to bi-directionally modulate Fto expression. An over expression of Fto protein by 2.5-fold in the ARC results in a 14% decrease in average daily food intake in the first week. In contrast, knocking down Arc Fto expression by 40% increases food intake by 16%. mRNA levels of Agrp, Pomc and Npy, ARC-expressed genes classically associated with the control of food intake, were not affected by the manipulation of Fto expression. However, over expression of Fto resulted in a 4-fold increase in the mRNA levels of Stat3, a signalling molecule critical for leptin receptor signalling, suggesting a possible candidate for the mediation of Fto's actions. These data provide further support for the notion that FTO itself can influence key components of energy balance, and is therefore a strong candidate for the mediation of the robust association between FTO intronic variants and adiposity. Importantly, this provide the first indication that selective alteration of FTO levels in the hypothalamus can influence food intake, a finding consistent with the reported effects of FTO alleles on appetite and food intake in man.

Background

Expansion of a CAG repeat in the coding region of exon 1 in the ATXN2 gene located in human chromosome 12q24.1 causes the neurodegenerative disease spinocerebellar ataxia type 2 (SCA2). In contrast to other polyglutamine (polyQ) disorders, the SCA2 repeat is not highly polymorphic in central European (CEU) controls with Q22 representing 90% of alleles, and Q23 contributing between 5–7% of alleles. Recently, the ATXN2 CAG repeat has been identified as a target of adaptive selection in the CEU population. Mouse lines deficient for atxn2 develop marked hyperphagia and obesity raising the possibility that loss-of-function mutations in the ATXN2 gene may be related to energy balance in humans. Some linkage studies of obesity related phenotypes such as antipsychotic induced weight gain have reported significant lod scores on chromosome 12q24. We tested the hypothesis that rare loss-of-function ATXN2 variants cause obesity analogous to rare mutations in the leptin, leptin receptor and MC4R genes.

Methodology/Principal Findings

We sequenced the coding region of ATXN2 including intron-exon boundaries in 92 severely obese children with a body mass index (BMI) >3.2 standard deviations above age- and gender-adjusted means. We confirmed five previously identified single nucleotide polymorphisms (SNPs) and three new SNPs resulting in two synonymous substitutions and one intronic polymorphism. Alleles encoding >Q22 were overrepresented in our sample of obese children and contributed 15% of alleles in children identified by their parents as white. SNP rs695872 closely flanking the CAG repeat showed a greatly increased frequency of C/C homozygotes and G/C heterozygotes compared with reported frequencies in the CEU population.

Conclusions/Significance

Although we did not identify variants leading to novel amino acid substitutions, nonsense or frameshift mutations, this study warrants further examination of variation in the ATXN2 gene in obesity and related phenotypes in a larger case-control study with emphasis on rs695872 and CAG repeat structure.

The adipocyte-derived protein adiponectin is highly heritable and inversely associated with risk of type 2 diabetes mellitus (T2D) and coronary heart disease (CHD). We meta-analyzed 3 genome-wide association studies for circulating adiponectin levels (n = 8,531) and sought validation of the lead single nucleotide polymorphisms (SNPs) in 5 additional cohorts (n = 6,202). Five SNPs were genome-wide significant in their relationship with adiponectin (P≤5×10−8). We then tested whether these 5 SNPs were associated with risk of T2D and CHD using a Bonferroni-corrected threshold of P≤0.011 to declare statistical significance for these disease associations. SNPs at the adiponectin-encoding ADIPOQ locus demonstrated the strongest associations with adiponectin levels (P-combined = 9.2×10−19 for lead SNP, rs266717, n = 14,733). A novel variant in the ARL15 (ADP-ribosylation factor-like 15) gene was associated with lower circulating levels of adiponectin (rs4311394-G, P-combined = 2.9×10−8, n = 14,733). This same risk allele at ARL15 was also associated with a higher risk of CHD (odds ratio [OR] = 1.12, P = 8.5×10−6, n = 22,421) more nominally, an increased risk of T2D (OR = 1.11, P = 3.2×10−3, n = 10,128), and several metabolic traits. Expression studies in humans indicated that ARL15 is well-expressed in skeletal muscle. These findings identify a novel protein, ARL15, which influences circulating adiponectin levels and may impact upon CHD risk.

Author Summary

Through a meta-analysis of genome-wide association studies of 14,733 individuals, we identified common base-pair variants in the genome which influence circulating adiponectin levels. Since adiponectin is an adipocyte-derived circulating protein which has been inversely associated with risk of obesity-related diseases such as type 2 diabetes (T2D) and coronary heart disease (CHD), we next sought to understand if the identified variants influencing adiponectin levels also influence risk of T2D, CHD, and several metabolic traits. In addition to confirming that variation at the ADIPOQ locus influences adiponectin levels, our analyses point to a variant in the ARL15 (ADP-ribosylation factor-like 15) locus which decreases adiponectin levels and increases risk of CHD and T2D. Further, this same variant was associated with increased fasting insulin levels and glycated hemoglobin. While the function of ARL15 is not known, we provide insight into the tissue specificity of ARL15 expression. These results thus provide novel insights into the physiology of the adiponectin pathway and obesity-related diseases.