Edoardo Dalla Nora

The Human Lipodystrophy Gene BSCL2/Seipin May Be Essential for Normal Adipocyte Differentiation

OBJECTIVE—Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) is a recessive disorder featuring near complete absence of adipose tissue. Remarkably, although the causative gene, BSCL2, has been known for several years, its molecular function and its role in adipose tissue development have not been elucidated. Therefore, we examined whether BSCL2 is involved in the regulation of adipocyte differentiation and the mechanism whereby pathogenic mutations in BSCL2 cause lipodystrophy. RESEARCH DESIGN AND METHODS—Following the characterization of BSCL2 expression in developing adipocytes, C3H10T1/2 mesenchymal stem cells were generated in which BSCL2 expression was knocked down using short hairpin RNA (shRNA). These cells were used to investigate whether BSCL2 is required for adipogenesis. BSCL2 constructs harboring pathogenic mutations known to cause lipodystrophy were also generated and characterized. RESULTS—BSCL2 expression was strongly induced during adipocyte differentiation, and the induction of BSCL2 expression was essential for adipogenesis to occur. The initial induction of key adipogenic transcription factors, including peroxisome proliferator–activated receptor (PPAR)γ and CAAT/enhancer-binding protein-α, was preserved in cells lacking BSCL2. However, the expression of these critical factors was not sustained, suggesting that the activity of PPARγ was impaired. Moreover, expression of key genes mediating triglyceride synthesis, including AGPAT2, lipin 1, and DGAT2, was persistently reduced and lipid accumulation was inhibited. Analysis of pathogenic missense mutants of BSCL2 revealed that the amino acid substitution A212P causes aberrant targeting of BSCL2 within the cell, suggesting that subcellular localization of BSCL2 may be critical to its function. CONCLUSIONS—This study demonstrates that BSCL2 is an essential, cell-autonomous regulator of adipogenesis.

Leptin Deficiency Unmasks the Deleterious Effects of Impaired Peroxisome Proliferator–Activated Receptor γ Function (P465L PPARγ) in Mice

Peroxisome proliferator–activated receptor (PPAR)γ is a key transcription factor facilitating fat deposition in adipose tissue through its proadipogenic and lipogenic actions. Human patients with dominant-negative mutations in PPARγ display lipodystrophy and extreme insulin resistance. For this reason it was completely unexpected that mice harboring an equivalent mutation (P465L) in PPARγ developed normal amounts of adipose tissue and were insulin sensitive. This finding raised important doubts about the interspecies translatability of PPARγ-related findings, bringing into question the relevance of other PPARγ murine models. Here, we demonstrate that when expressed on a hyperphagic ob/ob background, the P465L PPARγ mutant grossly exacerbates the insulin resistance and metabolic disturbances associated with leptin deficiency, yet reduces whole-body adiposity and adipocyte size. In mouse, coexistence of the P465L PPARγ mutation and the leptin-deficient state creates a mismatch between insufficient adipose tissue expandability and excessive energy availability, unmasking the deleterious effects of PPARγ mutations on carbohydrate metabolism and replicating the characteristic clinical symptoms observed in human patients with dominant-negative PPARγ mutations. Thus, adipose tissue expandability is identified as an important factor for the development of insulin resistance in the context of positive energy balance.

Dact1, a Nutritionally Regulated Preadipocyte Gene, Controls Adipogenesis by Coordinating the Wnt/β-Catenin Signaling Network

OBJECTIVE—Wnt signaling inhibits adipogenesis, but its regulation, physiological relevance, and molecular effectors are poorly understood. Here, we identify the Wnt modulator Dapper1/Frodo1 (Dact1) as a new preadipocyte gene involved in the regulation of murine and human adipogenesis. RESEARCH DESIGN AND METHODS—Changes in Dact1 expression were investigated in three in vitro models of adipogenesis. In vitro gain- and loss-of-function studies were used to investigate the mechanism of Dact1 action during adipogenesis. The in vivo regulation of Dact1 and Wnt/β-catenin signaling were investigated in murine models of altered nutritional status, of pharmacological stimulation of in vivo adipogenesis, and during the development of dietary and genetic obesity. RESULTS—Dact1 is a preadipocyte gene that decreases during adipogenesis. However, Dact1 knockdown impairs adipogenesis through activation of the Wnt/β-catenin signaling pathway, and this is reversed by treatment with the secreted Wnt antagonist, secreted Frizzled-related protein 1 (Sfrp1). In contrast, constitutive Dact1 overexpression promotes adipogenesis and confers resistance to Wnt ligand-induced antiadipogenesis through increased expression of endogenous Sfrps and reduced expression of Wnts. In vivo, in white adipose tissue, Dact1 and Wnt/β-catenin signaling also exhibit coordinated expression profiles in response to altered nutritional status, in response to pharmacological stimulation of in vivo adipogenesis, and during the development of dietary and genetic obesity. CONCLUSIONS—Dact1 regulates adipogenesis through coordinated effects on gene expression that selectively alter intracellular and paracrine/autocrine components of the Wnt/β-catenin signaling pathway. These novel insights into the molecular mechanisms controlling adipose tissue plasticity provide a functional network with therapeutic potential against diseases, such as obesity and associated metabolic disorders.

SEQUENTIAL REGULATION OF DGAT2 EXPRESSION BY C/EBPβ AND C/EBPα DURING ADIPOGENESIS

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triacylglycerol (TG) synthesis. Despite the existence of an alternative acyltransferase (DGAT1), mice lacking DGAT2 have a severe deficiency of TG in adipose tissue, indicating a nonredundant role for this enzyme in adipocyte TG synthesis. We have studied the regulation of DGAT2 expression during adipogenesis. In both isolated murine preadipocytes and 3T3-L1 cells the temporal pattern of DGAT2 expression closely mimicked that of genes whose expression is regulated by CAAT/enhancer-binding protein β (C/EBPβ). Inhibition of C/EBPβ expression in differentiating preadipocytes reduced DGAT2 expression, and electrophoretic mobility shift assay and chromatin immunoprecipitation experiments identified a promoter element in the DGAT2 gene that is likely to mediate this effect. The importance of C/EBPβ in adipocyte expression of DGAT2 was confirmed by the finding of reduced DGAT2 expression in the adipose tissue of C/EBPβ-null animals. However, DGAT2 expression is maintained at high levels during the later stages of adipogenesis, when C/EBPβ levels decline. We show that, at these later stages of differentiation, C/EBPα is capable of substituting for C/EBPβ at the same promoter element. These observations provide novel insight into the transcriptional regulation of DGAT2 expression. Moreover, they further refine the complex and serial roles of the C/EBP family of transcription factors in inducing and maintaining the metabolic properties of mature adipocytes.

PPARγ Pro12Ala and ACE ID polymorphisms are associated with BMI and fat distribution, but not metabolic syndrome

BackgroundMetabolic Syndrome (MetS) results from the combined effect of environmental and genetic factors. We investigated the possible association of peroxisome proliferator-activated receptor-γ2 (PPARγ2) Pro12Ala and Angiotensin Converting Enzyme (ACE) I/D polymorphisms with MetS and interaction between these genetic variants.MethodsThree hundred sixty four unrelated Caucasian subjects were enrolled. Waist circumference, blood pressure, and body mass index (BMI) were recorded. Body composition was estimated by impedance analysis; MetS was diagnosed by the NCEP-ATPIII criteria. A fasting blood sample was obtained for glucose, insulin, lipid profile determination, and DNA isolation for genotyping.ResultsThe prevalence of MetS did not differ across PPARγ2 or ACE polymorphisms. Carriers of PPARγ2 Ala allele had higher BMI and fat-mass but lower systolic blood pressure compared with Pro/Pro homozygotes. A significant PPARγ2 gene-gender interaction was observed in the modulation of BMI, fat mass, and blood pressure, with significant associations found in women only. A PPARγ2-ACE risk genotype combination for BMI and fat mass was found, with ACE DD/PPARγ2 Ala subjects having a higher BMI (p = 0.002) and Fat Mass (p = 0.002). Pro12Ala was independently associated with waist circumference independent of BMI and gender.ConclusionsCarriers of PPARγ2 Ala allele had higher BMI and fat-mass but not a worse metabolic profile, possibly because of a more favorable adipose tissue distribution. A gene interaction exists between Pro12Ala and ACE I/D on BMI and fat mass. Further studies are needed to assess the contribution of Pro12Ala polymorphism in adiposity distribution.

Plasma 24S-hydroxycholesterol levels in elderly subjects with late onset Alzheimer's disease or vascular dementia: a case-control study.

BackgroundIn central nervous system cholesterol cannot be degraded but is secreted into circulation predominantly in the form of its polar metabolite 24(S)-hydroxycholesterol (24S-OH-Chol). Some studies suggested an association between 24S-OH-Chol metabolism and different neurological diseases including dementia. A possible decrease in 24S-OH-Chol plasma levels has been reported late onset Alzheimers disease (LOAD) and vascular dementia (VD), but results of previous studies are partially contradictory.MethodsBy high-speed liquid chromatography/tandem mass spectrometry we evaluated the plasma levels of 24S-OH-Chol in a sample of 160 older individuals: 60 patients with LOAD, 35 patients with VD, 25 subjects affected by cognitive impairment no-dementia (CIND), and 40 (144 for genetics study) cognitively normal Controls. We also investigated the possible association between PPARgamma Pro12Ala polymorphism and dementia or 24S-OH-Chol levels.ResultsCompared with Controls, plasma 24S-OH-Chol levels were higher in LOAD and lower in VD; a slight not-significant increase in CIND was observed (ANOVA p: 0.001). A positive correlation between 24S-OH-Chol/TC ratio and plasma C reactive protein (CRP) levels was found in the whole sample, independent of possible confounders (multiple regression p: 0.04; r2: 0.10). This correlation was strong in LOAD (r: 0.39), still present in CIND (r: 0.20), but was absent in VD patients (r: 0.08). The PPARgamma Pro12Ala polymorphism was not associated with the diagnosis of LOAD, VD, or CIND; no correlation emerged between the Ala allele and 24S-OH-Chol plasma levels.ConclusionsOur results suggest that plasma 24S-OH-Chol levels might be increased in the first stages of LOAD, and this phenomenon might be related with systemic inflammation. The finding of lower 24S-OH-Chol concentrations in VD might be related with a more advanced stage of VD compared with LOAD in our sample, and/or to different pathogenetic mechanisms and evolution of these two forms of dementia.

Plasma triglycerides predict ten-years all-cause mortality in outpatients with type 2 diabetes mellitus: a longitudinal observational study

BackgroundCardiovascular disease (CVD) is the leading cause of death in type 2 diabetes mellitus (T2DM). American Diabetes Association standards of care set a series of targets recommended for the CVD prevention: blood pressure, LDL and HDL cholesterol (LDL-C and HDL-C), triglycerides and HbA1c goals. The aim of this study was to evaluate cardiovascular risk factors in a T2DM outpatient population in order to estimate their specific clinical value in predicting long-term overall mortality.MethodsOur study population was co mposed of 1917 T2DM outpatients attending the hospital-based Diabetes Clinic of Ferrara for a mean follow-up period of 10 years; recorded information included personal, clinical and biochemical data, and pharmacological treatment.ResultsA Cox proportional hazard analysis was performed, pointing out as age (HR:1.08; IC95%: 1.06-1.11), sex (males: HR:1.97; IC95%: 1.26-3.07), mean triglycerides levels during follow-up (III vs I tertile: HR:1.87; IC95%: 1.12-3.12) and lipid-lowering treatment (HR:0.56; IC95%: 0.35-0.90) were significantly associated with all-cause mortality, independent of confounding factors such as mean values of LDL-C, HDL-C, HbA1c, blood pressure, BMI, fasting glucose, and antihypertensive and antidiabetic treatment.ConclusionsThis finding suggests that more attention should be given to the management of cardiovascular risk in type 2 diabetic patients with high triglycerides levels.

Gene expression regional differences in human subcutaneous adipose tissue

BackgroundAccumulation of visceral adipose tissue (VAT) is clearly associated with an increased risk of obesity-related diseases and all-cause mortality, whereas gluteal subcutaneous fat accumulation (g-SAT) is associated with a lower risk. The relative contribution, in term of cardiovascular risk, of abdominal subcutaneous adipose tissue (a-SAT) is still controversial with studies showing both a detrimental effect and a protective role.Animal and in vitro studies demonstrated that adipocytes from visceral and subcutaneous depots have distinct morphological, metabolic and functional characteristics. These regional differences have a key role in the pathogenesis of obesity-related diseases. There is recent evidence that differentiation between upper-body and lower-body adipose tissues might be under control of site-specific sets of developmental genes, such as Homebox (HOX) genes, a group of related genes that control the body plan of an embryo along the anterior-posterior axis. However, the possible heterogeneity between different subcutaneous regions has not been extensively investigated.Here we studied global mRNA expression in g-SAT and a-SAT with a microarray approach. RNA was isolated from g-SAT and a-SAT biopsy, from eight healthy subjects, and hybridized on RNA microarray chips in order to detect regional differences in gene expression.ResultsA total of 131 genes are significantly and differently (>1.5 fold change, p < 0.05) expressed in a-SAT and g-SAT. Expression profiling reveals significant differences in expression of several HOX genes. Interestingly, two molecular signature of visceral adipocyte lineage, homebox genes HOXA5 and NR2F1, are up-regulated in a-SAT versus g-SAT by a 2.5 fold change.ConclusionsOur study shows that g-SAT and a-SAT have distinct expression profiles. The finding of a different expression of HOX genes, fundamental during the embryo development, suggests an early regional differentiation of subcutaneous adipose depots. Moreover, the higher expression of HOXA5 and NR2F1, two molecular signatures of visceral adipocytes, in a-SAT suggests that this subcutaneous adipose depot could be more similar to VAT than g-SAT.Our data suggest that we should look at SAT as composed of distinct depots with possibly different impact in obesity associated metabolic complications.

Age-related differences in plasma BDNF levels after prolonged bed rest

Brain-derived neurotrophic factor (BDNF) is a member of the family of neurotrophins and has been implicated in brain resistance to insults. Murine studies have demonstrated increased hippocampal concentration after acute immobilization and decreased concentration after chronic immobilization. In humans, chronic stress and sedentary lifestyle result in decreased plasma BDNF levels, but there no data exist regarding acute immobilization. The aim of our study was to evaluate age-related responses [comparing 7 younger subjects (age 23 ± 3 yr) and 8 older subjects (age 60 ± 4 yr)] of plasma BDNF before (baseline data collection, BDC) and after 14 days (BR14) of horizontal bed rest (BR). At BDC, BDNF levels were not different between the two groups (P = 0.101), although at BR14, BDNF levels were higher in older subjects (62.02 ± 18.31) than in younger subjects (34.36 ± 15.24 pg/ml) (P = 0.002). A general linear model for repeated measures showed a significant effect of BR on BDNF (P = 0.002). The BDC BDNF levels correlated with fat-free mass in both populations (ALL) (R = 0.628, P = 0.012), (older, R = 0.753, P = 0.031; younger, R = 0.772, P = 0.042), and with total cholesterol in ALL (R = 0.647, P = 0.009) and older study subjects (R = 0.805, P = 0.016). At BR14, BDNF correlated with total cholesterol (R = 0.579, P = 0.024) and age (R = 0.647, P = 0.009) in ALL. With an increase in age, the brain could become naturally less resistant to acute stressors, including the detrimental effects of prolonged bed rest, and thus the increase in BDNF in the older study group might reflect a protective overshooting of the brain to counteract the negative effects in such conditions.

Computerized cognitive training and brain derived neurotrophic factor during bed rest: Mechanisms to protect individual during acute stress

Acute stress, as bed rest, was shown to increase plasma level of the neurotrophin brain-derived neurotrophic factor (BDNF) in older, but not in young adults. This increase might represent a protective mechanism towards acute insults in aging subjects. Since computerized cognitive training (CCT) is known to protect brain, herein we evaluated the effect of CCT during bed rest on BDNF, muscle mass, neuromuscular function and metabolic parameters. The subjects that underwent CCT did not show an increase of BDNF after bed rest, and showed an anti-insular modification pattern in metabolism. Neuromuscular function parameters, already shown to beneficiate from CCT, negatively correlated with BDNF in research participants undergoing CCT, while positively correlated in the control group. In conclusion, BDNF increase can be interpreted as a standardized protective mechanism taking place whenever an insult occurs; it gives low, but consistent preservation of neuromuscular function. CCT, acting as an external protective mechanism, seems to modify this standardized response, avoiding BDNF increase or possibly modifying its time course. Our results suggest the possibility of differential neuroprotective mechanisms among ill and healthy individuals, and the importance of timing in determining the effects of protective mechanisms.