A Low-Frequency APOB p.(Pro955Ser) Variant Contributes to the Severity of/Variability in Familial Hypercholesterolemia.

CONTEXT: Heterozygous familial hypercholesterolemia (HeFH) is caused by a rare pathogenic variant in the LDLR, APOB, and PCSK9 genes. However, the causative variants in these genes have not been identified in approximately 40% of HeFH patients. OBJECTIVE: Our aim was to identify novel (or additional) genes/variants that contribute to HeFH. METHODS: Whole-exome sequencing was performed for 215 family members from 122 families with HeFH without pathogenic variants in the LDLR or PCSK9 genes. RESULTS: We could not find novel causative familial hypercholesterolemia (FH) genes/variants by family analysis. Next, we examined all APOB variants. Twenty-four nonsynonymous APOB variants were identified. The allele frequencies of the c.2863C > T:p.(Pro955Ser) variant in the HeFH probands and the general Japanese population were 0.15 and 0.034, respectively [odds ratio 4.9 (95% CI 3.4-7.1); P = 6.9 × 10-13]. The patients harboring the c.2863C > T:p.(Pro955Ser) variant accounted for 9.8% (n = 63) of unrelated patients with HeFH (n = 645). The penetrance of the c.2863C > T:p.(Pro955Ser) variant was low in the pedigree-based genetic analysis. In an in vitro assay, low-density lipoprotein (LDL) uptake from patients with the homozygous c.2863C > T:p.(Pro955Ser) variant was 44% of the LDL uptake from control subjects, and it was similar to that of the LDL uptake from patients with the known pathogenic heterozygous p.(Arg3527Gln) variant. CONCLUSIONS: The low-frequency APOB c.2863C > T:p.(Pro955Ser) variant is not an FH-causative variant, but it has a moderate effect size in HeFH. These findings suggest that the combination of the APOB c.2863C > T:p.(Pro955Ser) variant and age, environmental factors, or other genetic factors contributes to the severity of or variability in the HeFH phenotype.

The impact of gene variants on the thickness and softness of the Achilles tendon in familial hypercholesterolemia.

BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is characterized by high low-density lipoprotein (LDL)-cholesterol, xanthoma of the Achilles tendon (AT), and premature coronary artery disease (CAD). Ultrasonography can assess AT thickness and softness, making it useful for evaluating AT in FH diagnosis. We aimed to clarify whether FH-causative LDLR or PCSK9 variants affect AT thickness or softness. METHODS: In total, 248 FH and 60 non-familial hypercholesterolemia (non-FH) patients (total: 308) aged ≥30 years were enrolled. Patients with FH were classified according to genotype. AT thickness and elasticity index (EI) as softness were measured by ultrasonography. RESULTS: In FH patients with LDLR variants, AT was significantly thicker and softer than it was in FH patients with PCSK9 variants, FH patients without LDLR or PCSK9 variants, and patients with non-FH. The proportion of patients harboring LDLR variants significantly increased with AT thickness (p = 2.0 × 10-31) and softness (p = 1.4 × 10-18). Among those with AT thickness>8.5 mm and EI < 3.9, patients with LDLR variants accounted for 89% and 83%, respectively. The odds of AT thickening, AT softening and CAD increased 13.3-fold, 4.9-fold, and 2.1-fold, adjusted for the LDL-C year score by the presence of LDLR variants compared with those of patients without LDLR or PCSK9 variants. PCSK9 variants did not influence AT thickening or softening or CAD prevalence. CONCLUSIONS: LDLR variants affected AT thickness and softness independent of the LDL-C year score, but PCSK9 variants did not. Evaluating AT is important for identifying FH patients with LDLR variants at high risk for CAD.

Identification of a novel large duplication (exon2_6dup): copy number variation in the LDLR gene in a large family with familial hypercholesterolemia by whole-genome sequencing.

BACKGROUND: We found a large family with familial hypercholesterolemia (FH) that included 7 siblings who all developed myocardial infarction. OBJECTIVE: The aim of this study was to identify the pathogenic gene underlying FH in the family. METHODS: Whole-genome sequencing (WGS) was performed in 12 affected and 10 unaffected individuals in the family. RESULTS: WGS identified a novel large duplication: copy number variation (CNV) in the LDLR gene, exon2_6dup (c.68-499_940+252dup), that was present in the 12 affected family members but not in any of the 10 unaffected family members. The exact extent and genomic breakpoint sequence of the duplication caused by nonallelic homologous recombination between Alu sequences were identified based on bioinformatic analysis of WGS data for the LDLR gene. CONCLUSIONS: A novel c.68-499_940+252dup variant in the LDLR gene was identified based on bioinformatic analysis of WGS data. WGS is a powerful tool that can be used to precisely identify CNVs in addition to small-scale variations in FH-related genes.

Association between Achilles Tendon Softness and Atherosclerotic Cardiovascular Disease in Patients with Familial Hypercholesterolemia.

AIMS: Achilles tendon (AT) xanthomas are a specific physical finding of familial hypercholesterolemia (FH) and AT thickness has been used for its diagnosis and evaluation of its severity. Recently, we reported that the AT of FH patients was softer than that of non-FH patients and the combined use of a cut-off value for AT softness with that for AT thickness improved diagnostic accuracy. However, an association between AT softness and severity of atherosclerosis has not been reported. Accordingly, the present study aimed to investigate whether AT softness was associated with carotid atherosclerosis and presence of atherosclerotic cardiovascular disease (ASCVD) in FH. METHODS: The AT of 176 genetically diagnosed FH patients and 98 non-FH patients was examined to measure AT thickness and the elasticity index (EI) as an indicator for assessing AT softness using ultrasonography. RESULTS: Increased age was associated with AT softness, and overweight was negatively related to AT softness. There were significant inverse correlations between EI and maximum and mean intima-media thickness (IMT) within the common carotid artery only among FH patients. In multiple linear regression analysis, although the relationship between EI and mean IMT was attenuated, the association between EI and maximum IMT remained robust. In logistic regression analysis adjusted for age, sex and traditional cardiovascular risk factors (smoking history, presence of hypertension, presence of diabetes mellitus, overweight, LDL-cholesterol, HDL-cholesterol, and Log triglycerides), EI was associated with presence of ASCVD (Odds ratio per 1-SD increase, 0.37; 95% CI, 0.15 - 0.86; P=0.0252). CONCLUSION: The degree of lipid deposition in the AT of FH patients could be assessed by its thickness as well as its softness. AT softness is not only useful in diagnosing FH but is also associated with the severity of carotid atherosclerosis and presence of ASCVD. In addition, these findings suggest that AT softness would be helpful in risk assessment for FH patients.

Overeating Risk in Overweight Young Women Is Divided into Two Types According to Appetite and Eating Behavior.

Background: The problem of obesity in young women leads to future chronic diseases, effects on reproductive health, and next-generation obesity. Thus, it is necessary to provide effective support for these women's behavioral change. The purpose of this study was to evaluate dietary-related indicators to clarify the appetite and eating behavior problems among young women. Methods: Healthy women 18-39 years of age were enrolled. Interoceptive awareness (IA) was quantified using a heartbeat perception task score. Eating behavior was examined in three ways: Three-Factor Eating Questionnaire (TFEQ), visual analog scales of subjective appetite sensations, and a food consumption test. Results: In all, 15 participants who were overweight and 50 with normal weight were analyzed. The overweight women were clustered into two groups according to the heartbeat perception task score: a low-score group (women with overweight who have low IA [OW-LOW]) and high-score group (women with overweight who have high IA [OW-HIGH]). The OW-LOW group had significantly smaller intermeal changes in hunger score compared with women with normal weight. The disinhibition score on the TFEQ for the OW-HIGH group was significantly higher than the normal-weight women, and the prospective consumption score in the fasting condition was significantly higher in women with normal weight and a high heartbeat perception task score. Conclusions: Overweight young women were characterized into two groups with different appetite and eating behavior, which is connected to the risk of overeating. An appetite characteristic is associated with a high risk of obesity among the normal-weight population. Individualized interventions tailored to the IA levels may help in improving and preventing obesity.

Impact of LDLR and PCSK9 pathogenic variants in Japanese heterozygous familial hypercholesterolemia patients.

BACKGROUND AND AIMS: More than 4970 variants in the low-density lipoprotein receptor (LDLR) gene and 350 variants in the proprotein convertase subtilisin/kexin 9 (PCSK9) gene have been reported in familial hypercholesterolemia (FH) patients. However, the effects of these variants on FH pathophysiology have not been fully clarified. We aimed to update the LDLR and PCSK9 variants in Japanese heterozygous FH (HeFH) patients and annotate their clinical significance for the genetic diagnosis of HeFH. METHODS: A genetic analysis of the LDLR and PCSK9 genes was performed in 801 clinically diagnosed HeFH patients. The association of the pathogenic variants with the clinical FH phenotype was examined. RESULTS: Pathogenic variants in the LDLR and PCSK9 genes were found in 46% (n = 296) and 7.8% (n = 51) of unrelated FH patients (n = 650), respectively. The prevalence of Achilles tendon thickness was low (44%) in patients harbouring PCSK9 pathogenic variants. Furthermore, 17% of unrelated FH patients harboured one of five frequent LDLR pathogenic variants: c.1845+2T > C, c.1012T > A: p.(Cys338Ser), c.1297G > C: p.(Asp433His), c.1702C > G: p.(Leu568Val), and c.2431A > T: p.(Lys811*). Patients harbouring the c.1845+2T > C and c.1702C > G: p.(Leu568Val) variants had significantly lower serum LDL-cholesterol levels and higher serum HDL-cholesterol levels, respectively, compared with those harbouring the other LDLR pathogenic variants. The proportion of LDLR pathogenic variants was higher in patients with a younger age of coronary artery disease (CAD) onset and significantly decreased as the age of CAD onset increased. CONCLUSIONS: This study annotated the clinical significance and characteristics of LDLR and PCSK9 pathogenic variants in Japanese HeFH patients.

Familial Hypercholesterolemia and Lipoprotein Apheresis.

Lipoprotein apheresis has been developed as the treatment for refractory familial hypercholesterolemia (FH) to remove low-density lipoprotein (LDL), which is the main pathogenic factor. Currently, three procedures are available in Japan, including the plasma exchange, double-membrane filtration, and selective LDL adsorption. Selective LDL adsorption, which was developed in Japan, has been one of the most common treatment methods in the world. Lipoprotein apheresis enabled the prevention of atherosclerosis progression even in homozygous FH (HoFH) patients. However, in our observational study, HoFH patients who started lipoprotein apheresis in adulthood had a poorer prognosis than those who started in childhood. Therefore, HoFH patients need to start lipoprotein apheresis as early as possible. Although the indication for lipoprotein apheresis in heterozygous FH (HeFH) patients has been decreasing with the advent of strong statins, our observational study showed that HeFH patients who discontinued lipoprotein apheresis had a poorer prognosis than patients who continued apheresis therapy. These results suggest that it is beneficial for very-high-risk HeFH patients to be treated by lipoprotein apheresis even if their LDL cholesterol is controlled well by lipid-lowering agents. Since launching a new class of lipid-lowering agents, proprotein convertase subtilisin/kexin type 9 (PCSK9) antibody and microsome triglyceride transfer protein inhibitors, the indication for lipoprotein apheresis in FH has been changing. However, despite the development of these drugs, lipoprotein apheresis is still an option with a high therapeutic effect for FH patients with severe atherosclerotic cardiovascular disease.

Non-obese visceral adiposity is associated with the risk of atherosclerosis in Japanese patients with rheumatoid arthritis: a cross-sectional study.

Rheumatoid arthritis (RA) patients often have altered body composition including reduced muscle mass and increased fat mass. Some RA patients are likely to increase visceral fat without obesity [Body Mass Index (BMI) ≥ 25]. The objective of the study was to determine the association between obesity and/or visceral adiposity and the risk for atherosclerosis in Japanese RA patients. Obesity was evaluated using the BMI, with visceral adiposity evaluated using the visceral fat area (VFA) and the visceral/subcutaneous fat ratio (V/S ratio), quantified using the dual bioelectrical impedance method. Atherosclerosis was evaluated based on the intima-media thickness (IMT) and Plaque score (PS) of the carotid artery, measured using ultrasonography. Multivariate analysis was performed to determine the factors associated with IMT and PS. IMT and PS were compared among groups of patients sub-classified according to BMI and VFA levels. The V/S ratio was higher in RA patients than healthy controls, after adjustment for age, BMI, and waist circumference. On multivariate analysis, the V/S ratio, but not the BMI, was independently associated with the IMT and PS. Among the sub-classifications for BMI and VFA, non-obese patients with a high visceral adiposity (18.5 ≤ BMI < 25 kg/m2 and VFA ≥ 100 cm2) had the highest IMT (mean IMT, 0.93 ± 0.29 mm; maximum IMT, 1.44 ± 0.71 mm) and PS (1.43 ± 0.61), compared to all other BMI and VFA subgroups. RA patients have increased visceral adiposity, which is associated with a high prevalence of atherosclerotic of plaques. Non-obese RA patients who have visceral adiposity have a specifically higher risk for atherosclerosis.

Analysis of mitochondrial function in human induced pluripotent stem cells from patients with mitochondrial diabetes due to the A3243G mutation.

We previously established human induced pluripotent stem (iPS) cells in two diabetic patients from different families with the mitochondrial A3243G mutation and isolated isogenic iPS cell clones with either undetectable or high levels of the mutation in both patients. In the present study, we analyzed the mitochondrial functions of two mutation-undetectable and two mutation-high clones in each patient through four methods to assess complex I activity, mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production. In the first patient, complex I activity, mitochondrial respiration, and mitochondrial ATP production were decreased in the mutation-high clones compared with the mutation-undetectable clones, and mitochondrial membrane potential was decreased in a mutation-high clone compared with a mutation-undetectable clone. In the second patient, complex I activity was decreased in one mutation-high clone compared with the other clones. The other parameters showed no differences in any clones. In addition, the complex I activity and mitochondrial respiration of the mutation-undetectable clones from both patients were located in the range of those of iPS cells from healthy subjects. The present study suggests that the mitochondrial function of the mutation-undetectable iPS cell clones obtained from two patients with the A3243G mutation is comparable to the control iPS cells.

Achilles Tendon Ultrasonography for Diagnosis of Familial Hypercholesterolemia Among Japanese Subjects.

BACKGROUND: Difficulty in detecting and measuring Achilles tendon (AT) xanthomas may be responsible for underdiagnosis of familial hypercholesterolemia (FH). We aimed to determine a cutoff value for AT thickness (AT-T) using ultrasonography to diagnose FH, and to investigate the relationship between AT-T and atherosclerosis.Methods and Results:Ultrasonographic AT-T and carotid intima-media thickness (IMT) were evaluated in 130 genetically diagnosed FH patients and 155 non-FH patients. The outline and internal properties of the AT could be clearly determined using ultrasonography, and a good correlation in AT-T was observed between ultrasonography and the conventional method of X-ray radiography (r=0.924, P<0.001). Cutoff values for the diagnosis of FH derived from receiver-operating curves were 5.8 mm (sensitivity 71%, specificity 78%) in men, and 5.5 mm (sensitivity 80%, specificity 81%) in women. Importantly, increased AT-T was positively associated with carotid IMT only in the FH group. Additionally, increased AT-T was associated with the presence of coronary artery disease in a logistic regression analysis adjusted for traditional cardiovascular risk factors. CONCLUSIONS: This is the first study to determine a cutoff value for AT-T based on ultrasonography for the diagnosis of FH in Japanese subjects. Clearer detection and easier measurement of AT-T using ultrasonography would encourage clinicians to diagnose FH more actively, and could solve the problem of underdiagnosis of FH.

A Large Difference in Dose Timing of Basal Insulin Introduces Risk of Hypoglycemia and Overweight: A Cross-Sectional Study.

INTRODUCTION: Basal insulin should be injected at the same time each day, but people with diabetes sometimes mistime their injections. It is not known whether irregular daily dose timing affects diabetes-related factors. We report here our evaluation of the effects of deviations from a regular dosing schedule on glycemic control and hypoglycemia on patients treated with long-acting insulin (insulin glargine U100). We also consider the effects of ultra-long-acting insulin (insulin degludec) in this context. METHODS: Nineteen individuals with type 1 diabetes and 58 with type 2 diabetes were enrolled. Demographic data on all participants were retrieved from their medical records. Variation in dose timing was determined as the difference between the time of the earliest mistimed dose and the time of the latest mistimed dose, for each participant, over a 2-week period. All participants completed the Summary of Diabetes Self-Care Activities questionnaire, Problem Areas in Diabetes scale and 5-Item World Health Organization Well-being Index. Glargine U100 was switched to degludec in those individuals with type 2 diabetes who achieved inadequate glycemic control or suffered from frequent hypoglycemic episodes or who required two injections per day, and changes in hemoglobin A1c level and frequency of hypoglycemic episodes during the 12-week period were compared. RESULTS: A greater difference in dose timing was related to a higher frequency of hypoglycemic episodes and overweight in persons with type 2 diabetes. Smoking, drinking and living alone were independently associated with a greater difference in dose timing. Insulin degludec decreased the frequency of hypoglycemia and improved glycemic control in participants whose dose mistiming was >120 min. CONCLUSION: Fixed dose timing should be employed for basal insulin, as a larger difference in dose timing worsens diabetes-related factors. Insulin degludec improved glycemic control and lowered the hypoglycemia rate in persons with more irregular dose timing.

Impaired adipogenic capacity in induced pluripotent stem cells from lipodystrophic patients with BSCL2 mutations.

OBJECTIVE: Congenital generalized lipodystrophy (CGL) is an autosomal recessive disorder characterized by marked scarcity of adipose tissue, extreme insulin resistance, hypertriglyceridemia, hepatic steatosis and early-onset diabetes. Mutation of the BSCL2/SEIPIN gene causes the most severe form of CGL. The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with CGL harboring BSCL2/SEIPIN mutations. METHODS: Skin biopsies were obtained from two Japanese patients with CGL harboring different nonsense mutations (E189X and R275X) in BSCL2/SEIPIN. The fibroblasts thus obtained were infected with retroviruses encoding OCT4, SOX2, c-MYC, and KLF4. The generated iPS cells were evaluated for pluripotency by examining the expression of pluripotency markers (alkaline phosphatase, SSEA-4, TRA-1-60, and NANOG) and their ability to differentiate to three germ layers in vitro by forming embryoid bodies, and to form teratomas in vivo. Adipogenic capacity of differentiated BSCL2-iPS cells was determined by oil red O and adipose differentiation-related protein (ADRP) staining. Rescue experiments were also performed using stable expression of wild-type BSCL2. A coimmunoprecipitation assay was conducted to investigate the interaction of SEIPIN with ADRP. RESULTS: iPS cells were generated from fibroblasts of the two patients with CGL. Each of the patient-derived iPS (BSCL2-iPS) clones showed all of the hallmarks of pluripotency and could differentiate into derivatives of all three germ layers in vitro by forming embryoid bodies, and form teratomas after injection into mouse testes. BSCL2-iPS cells maintained the mutations in BSCL2 and lacked intact BSCL2. Upon adipogenic differentiation, BSCL2-iPS cells exhibited marked reduction of lipid droplet formation concomitant with diffuse cytoplasmic distribution of ADRP, compared with iPS cells from healthy individuals. Forced expression of BSCL2 not only rescued the lipid accumulation defects, but also restored cytoplasmic punctate localization of ADRP in BSCL2-iPS cells. Coimmunoprecipitation indicated SEIPIN interacted with ADRP. CONCLUSION: BSCL2-iPS cells that recapitulate the lipodystrophic phenotypes in vitro could provide valuable models with which to study the physiology of lipid accumulation and the pathology of human lipodystrophy. We found that BSCL2 defines the localization of ADRP, which has a role in lipid accumulation and adipogenic differentiation.

Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus.

Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus-derived cell lines, termed adult mouse hypothalamus (AMH) cells, by developing transgenic mice in which SV40 Tag was overexpressed in chromogranin A-positive cells in a tamoxifen-dependent manner. In order to obtain leptin-responsive clones, we selected clones based on the phosphorylation levels of STAT3 induced by leptin. The selected clones were fairly responsive to leptin in terms of STAT3, ERK, and Akt phosphorylation and induction of c-Fos mRNA induction. Pretreatment with leptin, insulin, and palmitate attenuated the c-Fos mRNA response to leptin, suggesting that certain aspects of leptin resistance might be reconstituted in this cellular model. These cell lines are useful tools for understanding the molecular nature of the signal disturbance in the leptin-resistant state and for identifying potential target molecules for drugs that relieve leptin resistance, although they have drawbacks including de-differentiated nature and lack of long-time stability.

Comprehensive Profiling of GPCR Expression in Ghrelin-Producing Cells.

To determine the comprehensive G protein-coupled receptor (GPCR) expression profile in ghrelin-producing cells and to elucidate the role of GPCR-mediated signaling in the regulation of ghrelin secretion, we determined GPCR expression profiles by RNA sequencing in the ghrelin-producing cell line MGN3-1 and analyzed the effects of ligands for highly expressed receptors on intracellular signaling and ghrelin secretion. Expression of selected GPCRs was confirmed in fluorescence-activated cell-sorted fluorescently tagged ghrelin-producing cells from ghrelin-promoter CreERT2/Rosa-CAG-LSL-ZsGreen1 mice. Expression levels of GPCRs previously suggested to regulate ghrelin secretion including adrenergic-ß1 receptor, GPR81, oxytocin receptor, GPR120, and somatostatin receptor 2 were high in MGN3-1 cells. Consistent with previous reports, isoproterenol and oxytocin stimulated the Gs and Gq pathways, respectively, whereas lactate, palmitate, and somatostatin stimulated the Gi pathway, confirming the reliability of current assays. Among other highly expressed GPCRs, prostaglandin E receptor 4 agonist prostaglandin E2 significantly stimulated the Gs pathway and ghrelin secretion. Muscarine, the canonical agonist of cholinergic receptor muscarinic 4, stimulated both the Gq and Gi pathways. Although muscarine treatment alone did not affect ghrelin secretion, it did suppress forskolin-induced ghrelin secretion, suggesting that the cholinergic pathway may play a role in counterbalancing the stimulation of ghrelin by Gs (eg, by adrenaline). In addition, GPR142 ligand tryptophan stimulated ghrelin secretion. In conclusion, we determined the comprehensive expression profile of GPCRs in ghrelin-producing cells and identified two novel ghrelin regulators, prostaglandin E2 and tryptophan. These results will lead to a greater understanding of the physiology of ghrelin and facilitate the development of ghrelin-modulating drugs.

Leptin restores the insulinotropic effect of exenatide in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and high-fat diet.

Leptin may reduce pancreatic lipid deposition, which increases with progression of obesity and can impair ß-cell function. The insulinotropic effect of glucagon-like peptide-1 (GLP-1) and the efficacy of GLP-1 receptor agonist are reduced associated with impaired ß-cell function. In this study, we examined whether leptin could restore the efficacy of exenatide, a GLP-1 receptor agonist, in type 2 diabetes with increased adiposity. We chronically administered leptin (500 µg·kg⁻¹·day⁻¹) and/or exenatide (20 µg·kg⁻¹·day⁻¹) for 2 wk in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and high-fat diet (STZ/HFD mice). The STZ/HFD mice exhibited hyperglycemia, overweight, increased pancreatic triglyceride level, and reduced glucose-stimulated insulin secretion (GSIS); moreover, the insulinotropic effect of exenatide was reduced. However, leptin significantly reduced pancreatic triglyceride level, and adding leptin to exenatide (LEP/EX) remarkably enhanced GSIS. These results suggested that the leptin treatment restored the insulinotropic effect of exenatide in the mice. In addition, LEP/EX reduced food intake, body weight, and triglyceride levels in the skeletal muscle and liver, and corrected hyperglycemia to a greater extent than either monotherapy. The pair-feeding experiment indicated that the marked reduction of pancreatic triglyceride level and enhancement of GSIS by LEP/EX occurred via mechanisms other than calorie restriction. These results suggest that leptin treatment may restore the insulinotropic effect of exenatide associated with the reduction of pancreatic lipid deposition in type 2 diabetes with increased adiposity. Combination therapy with leptin and exenatide could be an effective treatment for patients with type 2 diabetes with increased adiposity.

Cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis through dysregulated adipocytokine production.

Like many other human cancers, the development of malignant mesothelioma is closely associated with a chronic inflammatory condition. Both macrophages and mesothelial cells play crucial roles in the inflammatory response caused by asbestos exposure. Here, we show that adipocytes can also contribute to asbestos-induced inflammation through dysregulated adipocytokine production. 3T3-L1 preadipocytes were differentiated into mature adipocytes prior to use. These cells took up asbestos fibers (chrysotile, crocidolite and amosite) but were more resistant to asbestos-induced injury than macrophages and mesothelial cells. Expression microarray analysis followed by reverse transcription-PCR revealed that adipocytes respond directly to asbestos exposure with an increased production of proinflammatory adipocytokines [e.g. monocyte chemoattractant protein-1 (MCP-1)], whereas the production of anti-inflammatory adipocytokines (e.g. adiponectin) is suppressed. This was confirmed in epididymal fat pad of mice after intraperitoneal injection of asbestos fibers. Such dysregulated adipocytokine production favors the establishment of a proinflammatory environment. Furthermore, MCP-1 marginally promoted the growth of MeT-5A mesothelial cells and significantly enhanced the wound healing of Y-MESO-8A and Y-MESO-8D human mesothelioma cells. Our results suggest that increased levels of adipocytokines, such as MCP-1, can potentially contribute to the promotion of mesothelial carcinogenesis through the enhanced recruitment of inflammatory cells as well as a direct growth and migration stimulatory effect on mesothelial and mesothelioma cells. Taken together, our findings support a potential cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis.

In vitro characterization and engraftment of adipocytes derived from human induced pluripotent stem cells and embryonic stem cells.

Human induced pluripotent stem (iPS) and embryonic stem (ES) cells can differentiate into a variety of cell types. We reported on adipogenic potential of human iPS and ES cells in vitro. In the present study, we investigate the survival and maintenance of adipocytes differentiated in vitro from human iPS and ES cells after transplantation. Following adipogenic induction in vitro, the differentiated cells exhibited functional properties of adipocytes such as lipid storage, lipolysis, and insulin responsiveness. Subsequently, Matrigel containing the differentiated human iPS and ES cells was transplanted into the subcutaneous tissue of nude mice. After 1-4 weeks, the cells with adipocyte-like features were observed in transplanted Matrigel by histological analysis. The human origin of the cells, their lipid accumulation, and gene expression of adipocyte markers in transplanted cells were then confirmed, suggesting the presence of adipocytes in transplanted Matrigel. When the relative areas of these cells were calculated by dividing the adipocyte areas by the total Matrigel areas, we found that they peaked at 2 weeks after transplantation, and that the adipocytes persisted at 4 weeks. The present study demonstrates that human iPS and ES cells can differentiate into adipocytes with functional properties and that adipocytes derived from human iPS and ES cells can survive and maintain the differentiated properties of adipocytes for at least 4 weeks after transplantation. Adipocytes derived from human iPS and ES cells thus have the potential to open new avenues for stem cell-based research into metabolic diseases and future therapeutic applications.

Cell transplantation therapy for diabetes mellitus: endocrine pancreas and adipocyte.

Experimental transplantation of endocrine tissues has led to significant advances in our understanding of endocrinology and metabolism. Endocrine cell transplantation therapy is expected to be applied to the treatment of metabolic endocriopathies. Restoration of functional pancreatic beta-cell mass or of functional adipose mass are reasonable treatment approaches for patients with diabetes or lipodystrophy, respectively. Human induced pluripotent stem (iPS) cell research is having a great impact on life sciences. Doctors Takahashi and Yamanaka discovered that the forced expression of a set of genes can convert mouse and human somatic cells into a pluripotent state [1, 2]. These iPS cells can differentiate into a variety of cell types. Therefore, iPS cells from patients may be a potential cell source for autologous cell replacement therapy. This review briefly summarizes the current knowledge about transplantation therapy for diabetes mellitus, the development of the endocrine pancreas and adipocytes, and endocrine-metabolic disease-specific iPS cells.

GPR119 expression in normal human tissues and islet cell tumors: evidence for its islet-gastrointestinal distribution, expression in pancreatic beta and alpha cells, and involvement in islet function.

OBJECTIVE: GPR119 is reportedly involved in regulating glucose metabolism and food intake in rodents, but little is known about its expression and functional significance in humans. To begin to assess the potential clinical importance of GPR119, the distribution of GPR119 gene expression in humans was examined. MATERIALS/METHODS: Expression of GPR119 mRNA in fresh samples of normal human pancreas (n=19) and pancreatic islets (n=3) and in insulinomas (n=2) and glucagonomas (n=2), all collected at surgery, was compared with the mRNA expression of various receptors highly expressed and operative in human pancreatic islets. RESULTS: GPR119 mRNA was most abundant in the pancreas, followed by the duodenum, stomach, jejunum, ileum and colon. Pancreatic levels of GPR119 mRNA were similar to those of GPR40 mRNA and were higher than those of GLP1R and SUR1 mRNA, which are strongly expressed in human pancreatic islets. Moreover, levels of GPR119 mRNA in pancreatic islets were more than 10 times higher than in adjacent pancreatic tissue, as were levels of GPR40 mRNA. GPR119 mRNA was also abundant in two cases of insulinoma and two cases of glucagonoma, but was undetectable in a pancreatic acinar cell tumor. Similar results were obtained with mouse pancreatic islets, MIN6 insulinoma cells and alpha-TC glucagonoma cells. CONCLUSIONS: The results provide evidence of an islet-gastrointestinal distribution of GPR119, its expression in pancreatic beta and alpha cells, and its possible involvement in islet function. They also provide the basis for a better understanding of the potential clinical importance of GPR119.