The impact of obesity and uncontrolled asthma during pregnancy on metabolic and inflammatory pathways.

OBJECTIVE: Asthma and obesity are both inflammatory complications of pregnancy and when combined contribute to an increased risk of uncontrolled asthma during pregnancy and poor perinatal outcomes. Our previous work has identified the presence of maternal asthma is associated with a proinflammatory milieu in the placenta and reduced fetal growth. The current study was designed to determine the relationships between immunomodulatory metabolic pathways and inflammation and establish whether these pathways are associated with uncontrolled asthma in obese pregnant women. METHODS: Fifty-three obese (BMI >30) pregnant women were recruited prospectively. Participants were classified as having no asthma, controlled asthma, and uncontrolled asthma based on a doctor diagnosis and assessment using the Asthma Control Questionnaire (ACQ). Circulating plasma concentrations of metabolic hormones leptin, adiponectin, insulin, glucose, and extracellular vesicle (EVs) associated cytokines were measured at 18- and 36-weeks gestation. RESULTS: Concentrations of metabolic and inflammatory markers among obese participants with or without asthma were not significantly different throughout gestation. However total adiponectin concentrations increased as gestation progressed in obese, non-asthmatic women but did not increase in women with asthma. Plasma adiponectin and leptin levels in women with uncontrolled asthma were positively correlated with EV inflammatory markers including GM-CSF, IL-6, TNFα and IFNγ protein. CONCLUSIONS: This study demonstrated that most metabolic markers remain unchanged with the presence and severity of asthma in obese pregnant women. However, differences in the associations between metabolic and inflammatory pathways were observed in women with asthma and may be one of the mechanisms contributing to uncontrolled asthma in obese pregnant women.

Ectodermal-Neural Cortex 1 Isoforms Have Contrasting Effects on MC3T3-E1 Osteoblast Mineralization and Gene Expression.

The importance of Wnt pathway signaling in development of bone has been well established. Here we investigated the role of a known Wnt target, ENC1 (ectodermal-neural cortex 1; NRP/B), in osteoblast differentiation. Enc1 expression was detected in mouse osteoblasts, chondrocytes, and osteocytes by in situ hybridization, and osteoblastic expression was verified in differentiating primary cultures and MC3T3-E1 pre-osteoblast cells, with 57 kDa and 67 kDa ENC1 protein isoforms detected throughout differentiation. Induced knockdown of both ENC1 isoforms reduced alkaline phosphatase staining and virtually abolished MC3T3-E1 mineralization. At culture confluence, Alpl (alkaline phosphatase liver/bone/kidney) expression was markedly reduced compared with control cells, and there was significant and coordinated alteration of other genes involved in cellular phosphate biochemistry. In contrast, with 67 kDa-selective knockdown mineralized nodule formation was enhanced and there was a two-fold increase in Alpl expression at confluence. There was enhanced expression of Wnt/ß-catenin target genes with knockdown of both isoforms at this time-point and a five-fold increase in Frzb (Frizzled related protein) with 67 kDa-selective knockdown at mineralization, indicating possible ENC1 interactions with Wnt signaling in osteoblasts. These results are the first to demonstrate a role for ENC1 in the control of osteoblast differentiation. Additionally, the contrasting mineralization phenotypes and transcriptional patterns seen with coordinate knockdown of both ENC1 isoforms vs selective knockdown of 67 kDa ENC1 suggest opposing roles for the isoforms in regulation of osteoblastic differentiation, through effects on Alpl expression and phosphate cellular biochemistry. This study is the first to report differential roles for the ENC1 isoforms in any cell lineage. J. Cell. Biochem. 118: 2141-2150, 2017. © 2016 Wiley Periodicals, Inc.

Identification of carboxypeptidase X (CPX)-1 as a positive regulator of adipogenesis.

Adipose tissue expansion occurs through a combination of hypertrophy of existing adipocytes and generation of new adipocytes via the process of hyperplasia, which involves the proliferation and subsequent differentiation of preadipocytes. Deficiencies in hyperplasia contribute to adipose tissue dysfunction and the association of obesity with chronic cardiometabolic diseases. Thus, increased understanding of hyperplastic pathways may be expected to afford novel therapeutic strategies. We have reported that fibroblast growth factor (FGF)-1 promotes proliferation and differentiation of human preadipocytes and recently demonstrated that bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a central, proximal effector. Herein, we describe the identification and characterization of carboxypeptidase X (CPX)-1, a secreted collagen-binding glycoprotein, as a novel downstream effector in human primary and Simpson-Golabi-Behmel syndrome preadipocytes. CPX-1 expression increased after treatment of preadipocytes with FGF-1, BAMBI knockdown, or induction of differentiation. CPX-1 knockdown compromised preadipocyte differentiation coincident with reduced collagen expression. Furthermore, preadipocytes differentiated on matrix derived from CPX-1 knockdown cells exhibited reduced Glut4 expression and insulin-stimulated glucose uptake. Finally, CPX-1 expression was increased in adipose tissue from obese mice and humans. Collectively, these findings establish CPX-1 as a positive regulator of adipogenesis situated downstream of FGF-1/BAMBI that may contribute to hyperplastic adipose tissue expansion via affecting extracellular matrix remodeling.-Kim, Y.-H., Barclay, J. L., He, J., Luo, X., O'Neill, H. M., Keshvari, S., Webster, J. A., Ng, C., Hutley, L. J., Prins, J. B., Whitehead, J. P. Identification of carboxypeptidase X (CPX)-1 as a positive regulator of adipogenesis.

Fibroblast growth factor-1 (FGF-1) promotes adipogenesis by downregulation of carboxypeptidase A4 (CPA4) - a negative regulator of adipogenesis implicated in the modulation of local and systemic insulin sensitivity.

Fibroblast growth factor-1 (FGF-1) promotes differentiation of human preadipocytes into mature adipocytes via modulation of a BMP and Activin Membrane-Bound Inhibitor (BAMBI)/Peroxisome proliferator-activated receptor (PPARγ)-dependent network. Here, we combined transcriptomic and functional investigations to identify novel downstream effectors aligned with complementary analyses of gene expression in human adipose tissue to explore relationships with insulin sensitivity. RNA-Seq and qRT-PCR analysis revealed significant down-regulation of carboxypeptidase A4 (CPA4) following FGF-1 treatment or induction of differentiation of human preadipocytes in a BAMBI/PPARγ-independent manner. siRNA-mediated knockdown of CPA4 resulted in enhanced differentiation of human preadipocytes. Furthermore, expression of CPA4 in subcutaneous adipose tissue correlated negatively with indices of local and systemic (liver and muscle) insulin sensitivity. These results identify CPA4 as a negative regulator of adipogenesis that is down-regulated by FGF-1 and a putative deleterious modulator of local and systemic insulin sensitivity. Further investigations are required to define the molecular mechanism(s) involved and potential therapeutic opportunities.

Nutrient and immune sensing are obligate pathways in metabolism, immunity, and disease.

The growth and survival of multicellular organisms depend upon their abilities to acquire and metabolize nutrients, efficiently store and harness energy, and sense and fight infection. Systems for sensing and using nutrients have consequently coevolved alongside systems for sensing and responding to danger signals, including pathogens, and share many of the same cell signaling proteins and networks. Diets rich in carbohydrates and fats can overload these systems, leading to obesity, metabolic dysfunction, impaired immunity, and cardiovascular disease. Excessive nutrient intake promotes adiposity, typically altering adipocyte function and immune cell distribution, both of which trigger metabolic dysfunction. Here, we discuss novel mechanistic links between metabolism and immunity that underlie metabolic dysfunction in obesity. We aim to stimulate debate about how the endocrine and immune systems are connected through autocrine, paracrine, and neuroendocrine signaling in sophisticated networks that are only now beginning to be resolved. Understanding the expression and action of signaling proteins, together with modulating their receptors or pattern recognition using agonists or antagonists, will enable rational intervention in immunometabolism that may lead to novel treatments for obesity and metabolic dysfunction.

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein.

Carboxypeptidase X-1 (CPX-1) is an atypical member of the carboxypeptidase (CP) family of proteins involved in a variety of physiological and pathological processes. However, unlike most other family members CPX-1 lacks catalytic activity making its biological function unclear. CPX-1 contains a 160 amino acid discoidin domain (DSD) that serves as a binding domain in other proteins prompting us to investigate a putative functional role for this domain in CPX-1. Sequence alignment confirmed the overarching homology between the DSD of CPX-1 and other DSDs whilst more detailed analysis revealed conservation of the residues known to form the collagen-binding trench within the DSD of the discoidin domain receptors (DDRs) 1 and 2. Biochemical characterisation of transiently expressed human CPX-1 revealed that CPX-1 was secreted in an N-glycosylation-dependent manner as treatment with the N-glycosylation inhibitor tunicamycin inhibited secretion concomitant with a reduction in CPX-1 mobility on Western blot. Using a collagen pull-down assay we found that secreted CPX-1 bound collagen and this appeared independent of N-glycosylation as treatment with PNGaseF did not affect binding. Further analysis under non-reducing and reducing (+DTT) conditions revealed that CPX-1 was secreted in both monomeric and dimeric forms and only the former bound collagen. Finally, mutation of a key residue situated within the putative collagen-binding trench within the DSD of CPX-1 (R192A) significantly reduced secretion and collagen-binding by 40% and 60%, respectively. Collectively these results demonstrate that CPX-1 is a secreted collagen-binding glycoprotein and provide a foundation for future studies investigating the function of CPX-1.

Resveratrol does not benefit patients with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD), characterized by accumulation of hepatic triglycerides (steatosis), is associated with abdominal obesity, insulin resistance, and inflammation. Although weight loss via calorie restriction reduces features of NAFLD, there is no pharmacologic therapy. Resveratrol is a polyphenol that prevents high-energy diet-induced steatosis and insulin resistance in animals by up-regulating pathways that regulate energy metabolism. We performed a placebo-controlled trial to assess the effects of resveratrol in patients with NAFLD. METHODS: Overweight or obese men diagnosed with NAFLD were recruited from hepatology outpatient clinics in Brisbane, Australia from 2011 through 2012. They were randomly assigned to groups given 3000 mg resveratrol (n = 10) or placebo (n = 10) daily for 8 weeks. Outcomes included insulin resistance (assessed by the euglycemic-hyperinsulinemic clamp), hepatic steatosis, and abdominal fat distribution (assessed by magnetic resonance spectroscopy and imaging). Plasma markers of inflammation, as well as metabolic, hepatic, and antioxidant function, were measured; transcription of target genes was measured in peripheral blood mononuclear cells. Resveratrol pharmacokinetics and safety were assessed. RESULTS: Eight-week administration of resveratrol did not reduce insulin resistance, steatosis, or abdominal fat distribution when compared with baseline. No change was observed in plasma lipids or antioxidant activity. Levels of alanine and aspartate aminotransferases increased significantly among patients in the resveratrol group until week 6 when compared with the placebo group. Resveratrol did not significantly alter transcription of NQO1, PTP1B, IL6, or HO1 in peripheral blood mononuclear cells. Resveratrol was well-tolerated. CONCLUSIONS: Eight weeks administration of resveratrol did not significantly improve any features of NAFLD, compared with placebo, but it increased hepatic stress, based on observed increases in levels of liver enzymes. Further studies are needed to determine whether agents that are purported to mimic calorie restriction, such as resveratrol, are safe and effective for complications of obesity. Clinical trials registration no: ACTRN12612001135808.

A randomized trial of sodium-restriction on kidney function, fluid volume and adipokines in CKD patients.

BACKGROUND: Dietary sodium restriction is a key management strategy in chronic kidney disease (CKD). Recent evidence has demonstrated short-term reduction in blood pressure (BP) and proteinuria with sodium restriction, however the effect on other cardiovascular-related risk factors requires investigation in CKD. METHODS: The LowSALT CKD study involved 20 hypertensive Stage III-IV CKD patients counselled by a dietitian to consume a low-sodium diet (<100 mmol/day). The study was a randomised crossover trial comparing 2 weeks of high-sodium (additional 120 mmol sodium tablets) and low-sodium intake (placebo). Measurements were taken after each crossover arm including BP (peripheral and central), adipokines (inflammation markers and adiponectin), volume markers (extracellular-to-intracellular [E/I] fluid ratio; N-terminal pro-brain natriuretic peptide [NT-proBNP]), kidney function (estimated Glomerular Filtration Rate [eGFR]) and proteinuria (urine protein-creatinine ratio [PCR] and albumin-creatinine ratio [ACR]). Outcomes were compared using paired t-test for each cross-over arm. RESULTS: BP-lowering benefits of a low-sodium intake (peripheral BP (mean ± SD) 148/82 ± 21/12 mmHg) from high-sodium (159/87 ± 15/10 mmHg) intake were reflected in central BP and a reduction in eGFR, PCR, ACR, NTproBNP and E/I ratio. There was no change in inflammatory markers, total or high molecular weight adiponectin. CONCLUSIONS: Short-term benefits of sodium restriction on BP were reflected in significant change in kidney function and fluid volume parameters. Larger, long-term adequately powered trials in CKD are necessary to confirm these results. TRIAL REGISTRATION: Universal Trial Number U1111-1125-2149 registered on 13/10/2011; Australian New Zealand Clinical Trials Registry Number ACTRN12611001097932 registered on 21/10/2011.

Characterisation of the adiponectin receptors: the non-conserved N-terminal region of AdipoR2 prevents its expression at the cell-surface.

Adiponectin is a beneficial adipokine with insulin-sensitizing, anti-inflammatory and anti-atherogenic effects. These effects are mediated by two poorly characterised, closely related, atypical seven-transmembrane receptors. In the current report we have used C-terminal, epitope-tagged AdipoR1 and AdipoR2 constructs to monitor cell-surface expression by indirect immunofluorescence microscopy and quantitative plate-based analysis. We demonstrate that only AdipoR1 is constitutively expressed on the cell-surface. Further investigations, involving characterisation of a number of chimeric and truncated constructs, show the non-conserved region of AdipoR2 (residues 1-81) restricts its cell-surface expression. Introduction or deletion of this region, into AdipoR1 or AdipoR2, resulted in inhibition or promotion of cell-surface expression, respectively. We also confirmed that AdipoR1 and AdipoR2 can form heterodimers when co-expressed and that co-expression leads to the cell-surface expression of AdipoR2. Collectively these studies demonstrate that the non-conserved region of AdipoR2 restricts its cell-surface expression and raise the possibility that the majority of cell-surface AdipoR2 may be present in the form of heterodimers.

The effect of 25-hydroxyvitamin D on insulin sensitivity in obesity: is it mediated via adiponectin?

There has been substantial recent interest in using vitamin D to improve insulin sensitivity and preventing/delaying diabetes in those at risk. There is little consensus on the physiological mechanisms and whether the association is direct or indirect through enhanced production of insulin-sensitising chemicals, including adiponectin. We examined cross-sectional associations between serum 25-hydroxyvitamin D (25(OH)D) and insulin sensitivity (Matsuda index), parathyroid hormone (PTH), waist circumference, body mass index (BMI), triglycerides (TG), total and high molecular weight (HMW) adiponectin, HMW : total adiponectin ratio (HMW : total adiponectin), and total cholesterol : HDL cholesterol ratio (TC:HDL cholesterol) in 137 Caucasian adults of mean age 43.3 ± 8.3 years and BMI 38.8 ± 6.9 kg/m(2). Total adiponectin (standardised ß = 0.446; p < 0.001), waist circumference (standardised ß = -0.216; p < 0.05), BMI (standardised ß = -0.212; p < 0.05), and age (standardised ß = -0.298; p < 0.001) were independently associated with insulin sensitivity. Serum 25(OH)D (standardised ß = 0.114; p = 0.164) was not associated with insulin sensitivity, total or HMW adiponectin, HMW : total adiponectin, or lipids. Our results provide the novel finding that 25(OH)D is not associated with HMW adiponectin or HMW : total adiponectin in nondiabetic, obese adults and support the lack of association between 25(OH)D and lipids noted by others in similar groups of patients.

An observational study evaluating the efficacy of microfragmented adipose tissue in the treatment of osteoarthritis.

Aim: Osteoarthritis (OA) prevalence is increased in ageing and obese populations. This prospective single-arm cohort study aimed to investigate the efficacy of autologous microfragmented adipose tissue treatment of severe knee or shoulder OA. Materials & methods: Participants received an intra-articular microfragmented adipose tissue injection to the affected joint(s). Multiple patient reported outcome measures (PROMS) were recorded from 0 to 52 weeks for 63 consecutive joints. Results: Compared with baseline, there were significant improvements in all PROMS from 2 to 12 weeks and maintained at 52 weeks. Regression analysis revealed an inverse correlation with BMI and change in PROMS for knee joints. Conclusion: Our observed findings suggest this approach represents a safe, effective treatment for moderate-to-severe knee and shoulder OA, although efficacy may be reduced with increasing obesity.

Swelling and pain in the joints is common and found more often in older and overweight people. Osteoarthritis causes swelling and pain in joints because of a loss of tough, flexible tissue called cartilage. This study looks to see if injection of fat tissue into knee or shoulder joints can improve symptoms. The fat tissue used was called microfragmented adipose tissue (MFAT). This uses a technique to break down the fat tissue before injection. These cells were from the patient's own body. All patients had an injection of MFAT into their painful joints. In total, 59 patients took part. Reports were directly collected from the patient of how well they were doing. This was done before and after the injection at weeks 2, 6, 12, 24 and 52. There were three different types of report collected for knee joints and three for shoulder joints. Scores were then compared from these reports to see if there was a difference. An improvement was found in all three of the combined reports for both knees and shoulders. This stayed until 52 weeks. BMI is a measure of body weight in relation to height. Patients with a higher BMI were found to have had a smaller improvement in their scores. This study shows MFAT injections are safe and effective in treating painful joints.

Vitamin C protects against hypoxia, inflammation, and ER stress in primary human preadipocytes and adipocytes.

Dysregulation of adipose tissue involves increased cellular hypoxia, ER stress, and inflammation and altered adipokine production, contributing to the aetiology of obesity-related diseases including type 2 diabetes and cardiovascular disease. This study aimed to investigate the effects of Vitamin C supplementation on these processes in primary human preadipocytes and adipocytes. Treatment of preadipocytes and adipocytes with the proinflammatory cytokine TNFα and palmitic acid (PA), to mimic the obesogenic milieu, significantly increased markers of hypoxia, ER stress and inflammation and reduced secretion of high molecular weight (HMW) adiponectin. Importantly, Vitamin C abolished TNFα+PA induced hypoxia and significantly reduced the increases in ER stress and inflammation in both cell types. Vitamin C also significantly increased the secretion of HMW adiponectin from adipocytes. These findings indicate that Vitamin C can reduce obesity-associated cellular stress and thus provide a rationale for future investigations.

Adiponectin profiles are affected by chronic and acute changes in carbohydrate intake in healthy cats.

Adiponectin is a key adipokine that regulates carbohydrate and lipid metabolism. It circulates in stable low (LMW) and high molecular weight (HMW) forms. The aims of this study were to characterize baseline adiponectin profiles (total, LMW and HMW multimers) in healthy cats and to assess the effects of varying dietary carbohydrate content on adiponectin profiles. Cats were maintained on a diet with moderate carbohydrate content (37% metabolisable energy [ME]) for 4 weeks and then randomly allocated to either a low carbohydrate (19% ME) or high carbohydrate (52% ME) diet for 4 weeks. Fasting and postprandial plasma adiponectin profiles were measured by ELISA and sucrose gradient/Western blot. After consuming the moderate carbohydrate diet for 4 weeks, fasting total, HMW and LMW plasma adiponectin concentrations were 5.0±0.6, 2.5±0.5 and 2.6±0.2 µg/mL, respectively. After changing to the low carbohydrate diet, fasting total adiponectin was unchanged but HMW adiponectin increased and LMW adiponectin decreased. No significant postprandial changes were observed. Cats consuming the high carbohydrate diet had increased fasting total and LMW adiponectin with no change in HMW adiponectin. In the postprandial state total adiponectin was reduced and there was a trend towards a decrease in HMW (p=0.086) but not LMW multimers. These data indicate that feline adiponectin multimer profiles are similar to those reported in other species and demonstrate that changes in plasma adiponectin occur in response to chronic and acute carbohydrate intake and these reflect differential changes in adiponectin multimers.

ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling.

The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2beta, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.

Inhibition of inosine monophosphate dehydrogenase reduces adipogenesis and diet-induced obesity.

We previously described a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in lipid accumulation. Here we present data which demonstrate that IMPDH activity is required for differentiation of preadipocytes into mature, lipid-laden adipocytes and maintenance of adipose tissue mass. In 3T3-L1 preadipocytes inhibition of IMPDH with mycophenolic acid (MPA) reduced intracellular GTP levels by 60% (p<0.05) and blocked adipogenesis (p<0.05). Co-treatment with guanosine, a substrate in the salvage pathway of nucleotide biosynthesis, restored GTP levels and adipogenesis demonstrating the specificity of these effects. Treatment of diet-induced obese mice with mycophenolate mofetil (MMF), the prodrug of MPA, for 28 days did not affect food intake or lean body mass but reduced body fat content (by 36%, p=0.002) and adipocyte size (p=0.03) and number. These data suggest that inhibition of IMPDH may represent a novel strategy to reduce adipose tissue mass.

Reduced phosphorylation of AS160 contributes to glucocorticoid-mediated inhibition of glucose uptake in human and murine adipocytes.

Excess glucocorticoids induce insulin resistance and reduce glucose uptake although the underlying mechanisms are unclear. Here we demonstrate that Dex (1 microM for 24h) inhibits basal and insulin (1 nM) stimulated glucose uptake in human and murine adipocytes by 50% with a concomitant reduction in the levels of GLUT1/4 at the plasma membrane but no change in total GLUT1/4 levels. Expression and phosphorylation of proximal insulin signalling molecules (IRS1, PI3K, AKT) was unaffected by Dex as was phosphorylation of mTOR and FOXO1. In contrast, phosphorylation of AKT substrate 160kDa (AS160) at T642, which is essential for 14-3-3 recruitment and GLUT4 translocation, was reduced by 50% in basal and insulin-stimulated cells and this was mirrored by decreased 14-3-3 association. Co-treatment with the glucocorticoid receptor antagonist RU486 (10 microM) abrogated the Dex effect on AS160-T642 phosphorylation and restored glucose uptake by 80%. These data suggest Dex inhibits glucose uptake in adipocytes, at least in part, by reducing AS160 phosphorylation and interaction with 14-3-3.

Characterisation of inosine monophosphate dehydrogenase expression during retinal development: differences between variants and isoforms.

In mammals there are two ubiquitous, catalytically indistinguishable isoforms of inosine monophosphate dehydrogenase and mutations in the type I isoform, but not type II, cause retina-specific disorders. We have characterised the spatio-temporal expression of these proteins during development of the rat retina and performed functional investigations of the recently described retinal type I variants. Inosine monophosphate dehydrogenase was present in all immature cells throughout the retina during embryonic and neonatal development. Following eye opening and cell differentiation its distribution was restricted to the photoreceptors and bipolar cells, becoming prominent in Müller cells with aging. Type II was present in early, developing retinae whilst type I was undetectable. An isoform switch occurred around P10, after which the type I variants, type Ialpha and type Igamma, were the major forms. Functional investigations indicate type Igamma has greater catalytic activity compared with other variants and isoforms. Finally, all forms of type I show an increased propensity to form intracellular macrostructures compared to type II and these structures appear to be regulated in response to changing intracellular GTP levels. Collectively these data demonstrate that (i) type I does not play a role in early retinal development, (ii) type Igamma has greater activity and (iii) there are differences between type I and type II isoforms. These observations are consistent with the aetiology of retinitis pigmentosa and raise the possibility that programmed expression of specific inosine monophosphate dehydrogenase proteins may have arisen to meet the requirements of the cellular environment.

Characterization of the transcriptional and functional effects of fibroblast growth factor-1 on human preadipocyte differentiation.

We recently established that fibroblast growth factor (FGF)-1 promotes adipogenesis of primary human preadipocytes (phPA). In the current report, we have characterized the adipogenic effects of FGF-1 in phPA and also in a human PA strain derived from an individual with Simpson-Golabi-Behmel syndrome (SGBS PA), which exhibit an intrinsic capacity to differentiate with high efficiency. In further studies, we compared these models with the well-characterized murine 3T3-L1 preadipocyte cell line (3T3-L1 PA). FGF-1 up-regulated the adipogenic program in phPA, with increased expression of peroxisome proliferator-activated receptor-gamma in confluent PA prior to induction of differentiation and increased expression of adipocyte markers during differentiation. Moreover, phPA differentiated in the presence of FGF-1 were more insulin responsive and secreted increased levels of adiponectin. FGF-1 treatment of SGBS PA further enhanced differentiation. For the most part, the adipogenic program in phPA paralleled that observed in 3T3-L1 PA; however, we found no evidence of mitotic clonal expansion in the phPA. Finally, we investigated a role for extracellular regulated kinase 1/2 (ERK1/2) in adipogenesis of phPA. FGF-1 induced robust phosphorylation of ERK1/2 in early differentiation and inhibition of ERK1/2 activity significantly reduced phPA differentiation. These data suggest that FGF-1 treated phPA represent a valuable in vitro model for the study of adipogenesis and insulin action and indicate that ERK1/2 activation is necessary for human adipogenesis in the absence of mitotic clonal expansion.

Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: evidence for regulation of multimerization by alterations in posttranslational modifications.

Adiponectin is a secreted, multimeric protein with insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Serum adiponectin consists of trimer, hexamer, and larger high-molecular-weight (HMW) multimers, and these HMW multimers appear to be the more bioactive forms. Multimer composition of adiponectin appears to be regulated; however, the molecular mechanisms involved are unknown. We hypothesize that regulation of adiponectin multimerization and secretion occurs via changes in posttranslational modifications (PTMs). Although a structural role for intertrimer disulfide bonds in the formation of hexamers and HMW multimers is established, the role of other PTMs is unknown. PTMs identified in murine and bovine adiponectin include hydroxylation of multiple conserved proline and lysine residues and glycosylation of hydroxylysines. By mass spectrometry, we confirmed the presence of these PTMs in human adiponectin and identified three additional hydroxylations on Pro71, Pro76, and Pro95. We also investigated the role of the five modified lysines in multimer formation and secretion of recombinant human adiponectin expressed in mammalian cell lines. Mutation of modified lysines in the collagenous domain prevented formation of HMW multimers, whereas a pharmacological inhibitor of prolyl- and lysyl-hydroxylases, 2,2'-dipyridyl, inhibited formation of hexamers and HMW multimers. Bacterially expressed human adiponectin displayed a complete lack of differentially modified isoforms and failed to form bona fide trimers and larger multimers. Finally, glucose-induced increases in HMW multimer production from human adipose explants correlated with changes in the two-dimensional electrophoresis profile of adiponectin isoforms. Collectively, these data suggest that adiponectin multimer composition is affected by changes in PTM in response to physiological factors.

Muscle-specific overexpression of AdipoR1 or AdipoR2 gives rise to common and discrete local effects whilst AdipoR2 promotes additional systemic effects.

Hypoadiponectinemia and adiponectin resistance are implicated in the aetiology of obesity-related cardiometabolic disorders, hence represent a potential therapeutic axis. Here we characterised the effects of in vivo electrotransfer-mediated overexpression of the adiponectin receptors, AdipoR1 or AdipoR2, into tibialis anterior muscle (TAM) of lean or obese mice. In lean mice, TAM-specific overexpression of AdipoR1 (TAMR1) or AdipoR2 (TAMR2) increased phosphorylation of AMPK, AKT and ERK and expression of the insulin responsive glucose transporter glut4. In contrast, only TAMR2 increased pparα and a target gene acox1. These effects were decreased in obese mice despite no reduction in circulating adiponectin levels. TAMR2 also increased expression of adipoQ in TAM of lean and obese mice. Furthermore, in obese mice TAMR2 promoted systemic effects including; decreased weight gain; reduced epididymal fat mass and inflammation; increased epididymal adipoQ expression; increased circulating adiponectin. Collectively, these results demonstrate that AdipoR1 and AdipoR2 exhibit overlapping and distinct effects in skeletal muscle consistent with enhanced adiponectin sensitivity but these appear insufficient to ameliorate established obesity-induced adiponectin resistance. We also identify systemic effects upon TAMR2 in obese mice and postulate these are mediated by altered myokine production. Further studies are warranted to investigate this possibility which may reveal novel therapeutic approaches.