A 7-day high-fat, high-calorie diet induces fibre-specific increases in intramuscular triglyceride and perilipin protein expression in human skeletal muscle.

KEY POINTS: We have recently shown that a high-fat, high-calorie (HFHC) diet decreases whole body glucose clearance without impairing skeletal muscle insulin signalling, in healthy lean individuals. These diets are also known to increase skeletal muscle IMTG stores, but the effect on lipid metabolites leading to skeletal muscle insulin resistance has not been investigated. This study measured the effect of 7 days' HFHC diet on (1) skeletal muscle concentration of lipid metabolites, and (2) potential changes in the perilipin (PLIN) content of the lipid droplets storing intramuscular triglyceride (IMTG). The HFHC diet increased PLIN3 protein expression and redistributed PLIN2 to lipid droplet stores in type I fibres. The HFHC diet increased IMTG content in type I fibres, while lipid metabolite concentrations remained the same. The data suggest that the increases in IMTG stores assists in reducing the accumulation of lipid metabolites known to contribute to skeletal muscle insulin resistance. ABSTRACT: A high-fat, high-calorie (HFHC) diet reduces whole body glucose clearance without impairing skeletal muscle insulin signalling in healthy lean individuals. HFHC diets also increase skeletal muscle lipid stores. However, unlike certain lipid metabolites, intramuscular triglyceride (IMTG) stored within lipid droplets (LDs) does not directly contribute to skeletal muscle insulin resistance. Increased expression of perilipin (PLIN) proteins and colocalisation to LDs has been shown to assist in IMTG storage. We aimed to test the hypothesis that 7 days on a HFHC diet increases IMTG content while minimising accumulation of lipid metabolites known to disrupt skeletal muscle insulin signalling in sedentary and obese individuals. We also aimed to identify changes in expression and subcellular distribution of proteins involved in IMTG storage. Muscle biopsies were obtained from the m. vastus lateralis of 13 (11 males, 2 females) healthy lean individuals (age: 23 ± 2.5 years; body mass index: 24.5 ± 2.4 kg m-2 ), following an overnight fast, before and after consuming a high-fat (64% energy), high-calorie (+47% kcal) diet for 7 days. After the HFHC diet, IMTG content increased in type I fibres only (+101%; P < 0.001), whereas there was no change in the concentration of either total diacylglycerol (P = 0.123) or total ceramides (P = 0.150). Of the PLINs investigated, only PLIN3 content increased (+50%; P < 0.01) solely in type I fibres. LDs labelled with PLIN2 increased (+80%; P < 0.01), also in type I fibres only. We propose that these adaptations of LDs support IMTG storage and minimise accumulation of lipid metabolites to protect skeletal muscle insulin signalling following 7 days' HFHC diet.

Differential metabolic effects of oral butyrate treatment in lean versus metabolic syndrome subjects.

BACKGROUND: Gut microbiota-derived short-chain fatty acids (SCFAs) have been associated with beneficial metabolic effects. However, the direct effect of oral butyrate on metabolic parameters in humans has never been studied. In this first in men pilot study, we thus treated both lean and metabolic syndrome male subjects with oral sodium butyrate and investigated the effect on metabolism. METHODS: Healthy lean males (n = 9) and metabolic syndrome males (n = 10) were treated with oral 4 g of sodium butyrate daily for 4 weeks. Before and after treatment, insulin sensitivity was determined by a two-step hyperinsulinemic euglycemic clamp using [6,6-2H2]-glucose. Brown adipose tissue (BAT) uptake of glucose was visualized using 18F-FDG PET-CT. Fecal SCFA and bile acid concentrations as well as microbiota composition were determined before and after treatment. RESULTS: Oral butyrate had no effect on plasma and fecal butyrate levels after treatment, but did alter other SCFAs in both plasma and feces. Moreover, only in healthy lean subjects a significant improvement was observed in both peripheral (median Rd: from 71 to 82 µmol/kg min, p < 0.05) and hepatic insulin sensitivity (EGP suppression from 75 to 82% p < 0.05). Although BAT activity was significantly higher at baseline in lean (SUVmax: 12.4 ± 1.8) compared with metabolic syndrome subjects (SUVmax: 0.3 ± 0.8, p < 0.01), no significant effect following butyrate treatment on BAT was observed in either group (SUVmax lean to 13.3 ± 2.4 versus metabolic syndrome subjects to 1.2 ± 4.1). CONCLUSIONS: Oral butyrate treatment beneficially affects glucose metabolism in lean but not metabolic syndrome subjects, presumably due to an altered SCFA handling in insulin-resistant subjects. Although preliminary, these first in men findings argue against oral butyrate supplementation as treatment for glucose regulation in human subjects with type 2 diabetes mellitus.

Ceramides are associated with inflammatory processes in human mediastinal adipose tissue.

BACKGROUND AND AIM: Ceramides are poorly characterized in human adipose tissue. The aim of this study was to investigate concentrations of different ceramide species in human subcutaneous and visceral adipose tissue depots and to determine associations between ceramides and global gene expression profiles. METHODS AND RESULTS: Concentrations of six ceramide species were determined in plasma and in subcutaneous and mediastinal adipose tissue from 10 overweight subjects (BMI 29.4 ± 4.9 kg/m(2)). In the adipose tissue biopsies gene expression arrays were performed and relationships between ceramides and gene expression analyzed. Immunostaining of the two adipose tissue depots was performed in an independent group of 10 patients. Mediastinal adipose tissue contained significantly higher concentrations (p < 0.05) of all six ceramide species than the subcutaneous depot. Of the six ceramides in plasma, concentrations of only two (Cer d18:1/18:0 and Cer d18:1/22:0) correlated significantly (p < 0.05) with the corresponding species in mediastinal adipose tissue, but there were no significant correlations between ceramides in plasma and subcutaneous adipose tissue. Multivariate analysis identified significant correlations between the total ceramide concentration and global gene expression within mediastinal, but not subcutaneous adipose tissue, according to cross-validation. Gene ontology analysis of genes related to ceramides in the mediastinal depot revealed that genes positively correlated with ceramides were associated mainly with immune and inflammatory categories, while genes negatively correlated with ceramides were associated mainly with lipid and carbohydrate metabolism. CONCLUSIONS: Ceramides in human mediastinal adipose tissue may be involved in inflammation and lipid and carbohydrate metabolism.

Clinical dyslipidaemia is associated with changes in the lipid composition and inflammatory properties of apolipoprotein-B-containing lipoproteins from women with type 2 diabetes.

AIMS/HYPOTHESIS: The aim of this study was to use lipidomics to determine if the lipid composition of apolipoprotein-B-containing lipoproteins is modified by dyslipidaemia in type 2 diabetes and if any of the identified changes potentially have biological relevance in the pathophysiology of type 2 diabetes. METHODS: VLDL and LDL from normolipidaemic and dyslipidaemic type 2 diabetic women and controls were isolated and quantified with HPLC and mass spectrometry. A detailed molecular characterisation of VLDL triacylglycerols (TAG) was also performed using the novel ozone-induced dissociation method, which allowed us to distinguish vaccenic acid (C18:1 n-7) from oleic acid (C18:1 n-9) in specific TAG species. RESULTS: Lipid class composition was very similar in VLDL and LDL from normolipidaemic type 2 diabetic and control participants. By contrast, dyslipidaemia was associated with significant changes in both lipid classes (e.g. increased diacylglycerols) and lipid species (e.g. increased C16:1 and C20:3 in phosphatidylcholine and cholesteryl ester and increased C16:0 [palmitic acid] and vaccenic acid in TAG). Levels of palmitic acid in VLDL and LDL TAG correlated with insulin resistance, and VLDL TAG enriched in palmitic acid promoted increased secretion of proinflammatory mediators from human smooth muscle cells. CONCLUSIONS: We showed that dyslipidaemia is associated with major changes in both lipid class and lipid species composition in VLDL and LDL from women with type 2 diabetes. In addition, we identified specific molecular lipid species that both correlate with clinical variables and are proinflammatory. Our study thus shows the potential of advanced lipidomic methods to further understand the pathophysiology of type 2 diabetes.

LDL-associated apolipoprotein J and lysozyme are associated with atherogenic properties of LDL found in type 2 diabetes and the metabolic syndrome.

OBJECTIVES: Exchangeable low-density lipoprotein (LDL)-associated proteins can affect the atherogenic properties of LDL. Our aim was to analyse the protein composition of LDL from individuals with or without type 2 diabetes and the metabolic syndrome (T2DM) in relation to other LDL particle characteristics, to assess whether certain proteins associate more with certain subclasses of LDL typical for T2DM, such as small, apoCIII-rich LDL. DESIGN: Low-density lipoprotein from two cohorts of 61-year-old men (n = 19 and 64) with or without T2DM was isolated using size-exclusion chromatography or deuterium oxide-based ultracentrifugation. LDL-associated proteins were identified using mass spectrometry and quantified using two-dimensional gel electrophoresis or enzyme-linked immunosorbent assay. Differently expressed LDL-associated proteins apolipoprotein (apo)J and lysozyme were also measured in serum from a third cohort of women (n = 71) with or without T2DM. Lysozyme binding to advanced glycation end product (AGE)-LDL was examined in vitro. RESULTS: ApoJ and lysozyme were increased in LDL particles with increased apoCIII content and decreased cholesterol content. When isolated with size-exclusion chromatography, LDL from individuals with T2DM contained more apoJ and lysozyme and less apoA1 than LDL from control individuals. LDL content of apoJ correlated with a smaller LDL particle size. Serum levels of lysozyme, but not apoJ, were increased in individuals with T2DM. In vitro, lysozyme associated more with AGE-LDL than with unmodified LDL. CONCLUSIONS: Our results indicate that apoJ and lysozyme are increased in LDL with characteristics of small dense LDL in T2DM. Small dense LDL is easily glycated, and the increased affinity of lysozyme for AGE-LDL provides a possible partial explanation for an increase lysozyme in LDL from those with type 2 diabetes.

Retracted: 147 reduced syntaxin-5 in skeletal muscle of patients with type 2 diabetes. A link between lipid storage and insulin resistance.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This abstract has been retracted at the request of Jan Borén, co-author, because of conscious fabrication, corruption or suppression of basic material and conscious preparation and presentation of falsified results in the abstract by one of the authors.

TGF-beta1 perturbs vascular development and inhibits epithelial differentiation in fetal lung in vivo.

Members of the transforming growth factor beta (TGF-beta) family of polypeptides have been implicated in morphogenesis and differentiation in numerous tissues, including the lung. In order to further define effects of TGF-beta signaling in lung morphogenesis, a constitutively active form of TGF-beta1 was expressed in respiratory epithelial cells of the fetal mouse lung in vivo. Expression of TGF-beta1 arrested lung morphogenesis in the pseudoglandular stage of development, inhibiting synthesis of differentiation-dependent proteins, SP-B, SP-C, and CCSP, and maintaining embryonic patterns of staining for thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor-3beta (HNF-3beta). The pulmonary mesenchyme was thickened and vascular density was increased by TGF-beta1. TGF-beta1 decreased expression of vascular endothelial growth factor-A (VEGF-A) mRNA and protein, and the abundance of Flk-1 mRNA in the lung mesenchyme. Distribution of platelet-endothelial cell adhesion molecule (PECAM)-1, a marker of pulmonary blood vessels, was altered, and ultrastructural studies demonstrated that TGF-beta1 inhibited vascular development in the fetal lung. TGF-beta1 perturbed both epithelial cell differentiation and formation of the pulmonary vasculature, supporting the concept that precise control of signaling via the TGF-beta receptor pathway is critical for normal lung morphogenesis.

FGF-10 disrupts lung morphogenesis and causes pulmonary adenomas in vivo.

Transgenic mice in which fibroblast growth factor (FGF)-10 was expressed in the lungs of fetal and postnatal mice were generated with a doxycycline-inducible system controlled by surfactant protein (SP) C or Clara cell secretory protein (CCSP) promoter elements. Expression of FGF-10 mRNA in the fetal lung caused adenomatous malformations, perturbed branching morphogenesis, and caused respiratory failure at birth. When expressed after birth, FGF-10 caused multifocal pulmonary tumors. FGF-10-induced tumors were highly differentiated papillary and lepidic pulmonary adenomas. Epithelial cells lining the tumors stained intensely for thyroid transcription factor (TTF)-1 and SP-C but not CCSP, indicating that FGF-10 enhanced differentiation of cells to a peripheral alveolar type II cell phenotype. Withdrawal from doxycycline caused rapid regression of the tumors associated with rapid loss of the differentiation markers TTF-1, SP-B, and proSP-C. FGF-10 disrupted lung morphogenesis and induced multifocal pulmonary tumors in vivo and caused reversible type II cell differentiation of the respiratory epithelium.

Lamellar body formation in normal and surfactant protein B-deficient fetal mice.

Surfactant protein B (SP-B) -/- mice die of lethal respiratory distress syndrome shortly after birth. Alveolar type II epithelial cells in SP-B-deficient mice are characterized by a complete absence of lamellar bodies, the intracellular storage form of pulmonary surfactant, and the presence of inclusions containing numerous small vesicles and electron-dense masses. The present study was undertaken to characterize the formation of these inclusions during fetal lung development and clarify their relationship to lamellar bodies. In wild-type and SP-B +/- mice, small lamellar bodies with loosely organized lamellae and distinct limiting membranes were first detected on day 16 to 16.5 of gestation. SP-B -/- mice were readily identified on day 16 by the absence of immature lamellar bodies, the appearance of vesicular inclusions similar to those previously described in late gestation SP-B -/- mice, and the accumulation of misprocessed SP-C protein. Vesicular inclusions were rarely detected in SP-B +/- mice and were never detected in wild-type littermates. Classical multivesicular bodies were observed fusing with lamellar bodies in wild-type mice, and with the vesicular inclusions in SP-B -/- mice that occasionally contained a few membrane lamellae. On day 18, the airways of SP-B -/- mice lacked tubular myelin and were filled with vesicles and electron-dense masses, suggesting that the contents of the vesicular inclusions were secreted. Taken together, these observations suggest that vesicular inclusions in SP-B -/- mice are disorganized lamellar bodies in which the absence of SP-B leads to failure to package surfactant phospholipids into concentric lamellae.

Utility of surfactant protein B precursor and thyroid transcription factor 1 in differentiating adenocarcinoma of the lung from malignant mesothelioma.

Differentiation of malignant mesothelioma from adenocarcinoma, particularly from a lung primary, remains a difficult diagnostic problem. Surfactant protein B precursor (pro-SP-B) and thyroid transcription factor 1 (ITF-1) are expressed selectively in the normal respiratory epithelium and in adenocarcinomas of the lung. In this study, we evaluated the utility of pro-SP-B and ITF-1 in distinguishing pulmonary adenocarcinomas and malignant mesotheliomas. Immunoreactivity for pro-SP-B and TTF-1 was examined in paraffin sections of 370 primary lung carcinomas (208 adenocarcinomas, 101 squamous cell carcinomas, and 61 large cell carcinomas) and 95 malignant mesotheliomas, using a pro-SP-B antiserum and a monoclonal TTF-1 antibody with a biotin-streptavidin detection system. Immunostaining for pro-SP-B was detected in 57% of adenocarcinomas, and 20% of large cell carcinomas. Immunoreactivity for TTF-1 was shown in 76% of adenocarcinomas and 26% of large cell carcinomas. Malignant mesotheliomas and squamous cell carcinomas did not stain with either antibody. The expression of pro-SP-B and TTF-1 in adenocarcinomas of the lung but not in malignant mesotheliomas shows that pro-SP-B and TTF-1 staining is useful in differentiating these neoplasms.

Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo.

Surfactant protein D (SP-D) is a 43-kDa member of the collectin family of collagenous lectin domain-containing proteins that is expressed in epithelial cells of the lung. The SP-D gene was targeted by homologous recombination in embryonic stem cells that were used to produce SP-D (+/-) and SP-D (-/-) mice. Both SP-D (-/-) and SP-D (+/-) mice survived normally in the perinatal and postnatal periods. Whereas no abnormalities were observed in SP-D (+/-) mice, alveolar and tissue phosphatidylcholine pool sizes were markedly increased in SP-D (-/-) mice. Increased numbers of large foamy alveolar macrophages and enlarged alveoli were also observed in SP-D (-/-) mice. Phospholipid composition was unaltered in SP-D (-/-) mice, but surfactant morphology was abnormal, consisting of dense phospholipid membranous arrays with decreased tubular myelin. The pulmonary lipoidosis in the SP-D (-/-) mice was not associated with accumulation of surfactant proteins B or C, or their mRNAs, distinguishing the disorder from alveolar proteinosis syndromes. Surfactant protein A mRNA was reduced and, SP-A protein appeared to be reduced in SP-D (-/-) compared with wild type mice. Targeting of the mouse SP-D gene caused accumulation of surfactant lipid and altered phospholipid structures, demonstrating a previously unsuspected role for SP-D in surfactant lipid homeostasis in vivo.

Temporal-spatial distribution of hepatocyte nuclear factor-3beta in developing human lung and other foregut derivatives.

We assessed the temporal-spatial distribution of hepatocyte nuclear factor-3beta (HNF-3beta) in developing human lung and other foregut derivatives. Tissue from 31 fetuses (10-40 weeks) and 24 infants with hyaline membrane disease (HMD) or bronchopulmonary dysplasia (BPD) (2 days to 7 months) was studied. HNF-3beta was detected in nuclei of epithelial cells of trachea and of conducting and terminal airways at 10 weeks. Thereafter, epithelial nuclei were immunolabeled more widely in peripheral than proximal airways. HNF-3beta was confined to bronchiolo-alveolar portals and Type II cells in nonfetal lung. In infants with BPD, HNF-3beta was expressed abundantly in regenerating epithelial cells at the periphery of lung lobules. HNF-3beta was also detected in fetal esophagus, pancreas, duodenum, stomach, and gallbladder, suggesting that it is a marker for progenitor cells in foregut derivatives. The pattern of expression of HNF-3beta in the lung was similar to that of thyroid transcription factor-1 (TTF-1) at all ages. The temporal-spatial patterns of HNF-3beta and TTF-1 in the developing and regenerating lung are consistent with their proposed role in epithelial cell differentiation, regeneration, and surfactant protein gene expression. (J Histochem Cytochem 46:955-962, 1998)

Immunogold EM localization of neurochemicals in human pulmonary neuroendocrine cells.

Antibodies against the pulmonary neuroendocrine cell peptides gastrin-releasing peptide (bombesin), calcitonin and calcitonin gene-related peptide (CGRP) have been labeled with colloidal gold spheres for immunocytochemical localization in human fetal and newborn lung tissue. In general, the presence and amount of immunolabeling increased with increasing gestational age, with only calcitonin appearing late in fetal life. The largest percentage of neuroepithelial body (NEB) cells labeled and the largest number of labeled dense core vesicles (DCV) were in infants with chronic lung disease (bronchopulmonary dysplasia). Serial ribbons allowed identification of more than one peptide in a single NEB cell. The use of two antibodies labeled with colloidal gold spheres of different sizes allowed the identification of two peptides in the same DCV. Quantification of relative amounts of labeled peptides was not possible, as the peptide labeling with the larger size gold sphere was consistently underestimated. Colocalization to the same DCV has been shown in humans for bombesin and calcitonin, calcitonin and CGRP, bombesin and CGRP and, by others for cholecystokinin (CCK) and serotonin. Colocalization of two or more peptides or an amine to a single DCV within the same cell implies simultaneous discharge by exocytosis. The action of the two (or more) substances might be in concert, perhaps with one acting in a paracrine fashion, and the second in an autocrine fashion. In this case, the second peptide or amine might have a regulatory function in the parent cell, influencing DCV storage or rate of release.

Expression of surfactant protein B precursor and surfactant protein B mRNA in adenocarcinoma of the lung.

Surfactant protein B (SP-B) is a 79-amino acid, hydrophobic protein that plays important roles in surfactant function and homeostasis. SP-B is produced in the respiratory epithelium by proteolytic processing of a glycosylated precursor (pro-SP-B) of relative molecular mass 42,000 to 46,000. To develop diagnostic markers for pulmonary adenocarcinomas, we examined the incidence and distribution of pro-SP-B and SP-B mRNA in paraffin sections of 35 non-small cell lung carcinomas (15 adenocarcinomas, 15 squamous cell carcinomas, and 5 large cell carcinomas), using immunohistochemical techniques and in situ hybridization. Fifteen nonpulmonary adenocarcinomas were used as controls. Pro-SP-B and SP-B mRNA were detected in 60% and 53% of pulmonary adenocarcinomas, respectively. Expression was seen in adenocarcinomas with acinar, papillary, bronchioloalveolar, and solid growth patterns. Squamous cell and large cell carcinomas of the lung and nonpulmonary adenocarcinomas did not contain pro-SP-B immunoreactivity or SP-B mRNA. The specificity of SP-B gene expression in adenocarcinomas of the lung supports the usefulness of pro-SP-B and SP-B mRNA in the study and diagnosis of these neoplasms.

Ontogeny of Clara cell-specific protein and its mRNA: their association with neuroepithelial bodies in human fetal lung and in bronchopulmonary dysplasia.

Clara cell-specific 10-KD protein (CCSP) is an abundant product of nonciliated bronchiolar epithelial (Clara) cells in the lung. We have determined the temporal-spatial distribution of CCSP and its mRNA in developing human lung and in neonatal lung disease, using immunohistochemistry and in situ hybridization. CCSP immunoreactivity was found in nonciliated bronchiolar epithelial cells from 12 weeks of gestation onward. Tracheal and bronchial epithelia showed positive immunoreactivity at each gestational week after 15 weeks and 14 weeks, respectively. CCSP mRNA was seen in the bronchial and bronchiolar epithelia from 16 weeks onward and was detected in the trachea from 19 through 23 weeks of gestation. CCSP immunoreactivity and mRNA were present in nonciliated single cells of bronchial and bronchiolar epithelia in fetuses and in infants with and without lung disease. CCSP- and CCSP mRNA-containing epithelial cells also formed dusters around neuroepithelial bodies (NEBs), especially at airway branch points, suggesting that NEBs and Clara cells might interact during development and during pulmonary regeneration. Because of evidence of overlapping of some but not all cells expressing CCSP, SP-A, and pro-SP-B during lung development, a common cell lineage is proposed, with subsequent divergence of phenotypes.

Expression of thyroid transcription factor-1(TTF-1) in fetal and neonatal human lung.

We assessed the immunohistochemical localization of thyroid transcription factor-1 (TTF-1) in the lungs of 24 human fetuses (11-23 weeks), three infants without pulmonary pathology (36-42 weeks), and 24 infants (2 days-6.5 months) with hyaline membrane disease (HMD) or bronchopulmonary dysplasia (BPD). TTF-1 was detected in fetal lung epithelial cell nuclei by 11 weeks' gestation. Budding tips of terminal airways had prominently labeled nuclei. By 17 weeks, labeling was present in scattered nonciliated columnar and cuboidal cells. Throughout gestation, TTF-1 nuclear staining was prominent in airways abutting pleural, peribronchial, or perivascular connective tissue, being less prominent in centers of lobules. By 23 weeks, many cells in cuboidal but not columnar cell-lined airways had labeled nuclei. At term, TTF-1 was detected primarily in Type II epithelial cells. In HMD with alveolar hemorrhage, edema, or airway collapse, little or no TTF-1 was present except in open terminal airways. In BDP lungs, TTF-1 was absent in areas of alveolar collapse or infection, being present in regenerating open airways. The temporal-spatial distribution of TTF-1, in general, follows patterns of distribution of surfactant protein-B in developing and pathological lungs, consistent with its role in the regulation of epithelial cell gene expression in the lung.