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Context: Bone marrow (BM) in adult long bones is rich in adipose tissue, but the functions of BM adipocytes are largely unknown. We set out to elucidate the metabolic and molecular characteristics of BM adipose tissue (BMAT) in humans., Objective: Our aim was to determine if BMAT is an insulin-sensitive tissue, and whether the insulin sensitivity is altered in obesity or type 2 diabetes (T2DM)., Design: This was a cross-sectional and longitudinal study., Setting: The study was conducted in a clinical research center., Patients or Other Participants: Bone marrow adipose tissue glucose uptake (GU) was assessed in 23 morbidly obese subjects (9 with T2DM) and 9 healthy controls with normal body weight. In addition, GU was assessed in another 11 controls during cold exposure. Bone marrow adipose tissue samples for molecular analyses were collected from non-DM patients undergoing knee arthroplasty., Intervention(s): Obese subjects were assessed before and 6 months after bariatric surgery and controls at 1 time point., Main Outcome Measure: We used positron emission tomography imaging with 2-[18F]fluoro-2-deoxy-D-glucose tracer to characterize GU in femoral and vertebral BMAT. Bone marrow adipose tissue molecular profile was assessed using quantitative RT-PCR., Results: Insulin enhances GU in human BMAT. Femoral BMAT insulin sensitivity was impaired in obese patients with T2DM compared to controls, but it improved after bariatric surgery. Furthermore, gene expression analysis revealed that BMAT was distinct from brown and white adipose tissue., Conclusions: Bone marrow adipose tissue is a metabolically active, insulin-sensitive and molecularly distinct fat depot that may play a role in whole body energy metabolism., (© Endocrine Society 2020.)

Adaptation to the environment and extraction of energy are essential for survival. Some species have found niches and specialized in using a particular source of energy, whereas others-including humans and several other mammals-have developed a high degree of flexibility 1 . A lot is known about the general metabolic fates of different substrates but we still lack a detailed mechanistic understanding of how cells adapt in their use of basic nutrients 2 . Here we show that the closely related fasting/starvation-induced forkhead transcription factors FOXK1 and FOXK2 induce aerobic glycolysis by upregulating the enzymatic machinery required for this (for example, hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase), while at the same time suppressing further oxidation of pyruvate in the mitochondria by increasing the activity of pyruvate dehydrogenase kinases 1 and 4. Together with suppression of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 this leads to increased phosphorylation of the E1α regulatory subunit of the pyruvate dehydrogenase complex, which in turn inhibits further oxidation of pyruvate in the mitochondria-instead, pyruvate is reduced to lactate. Suppression of FOXK1 and FOXK2 induce the opposite phenotype. Both in vitro and in vivo experiments, including studies of primary human cells, show how FOXK1 and/or FOXK2 are likely to act as important regulators that reprogram cellular metabolism to induce aerobic glycolysis.

Adapting to the cold extrauterine environment after birth is a great challenge for the newborn. Due to their high surface area-to-volume ratio, infants tend to lose more heat to the environment as compared to adults. In addition, human newborns lack sufficiently developed skeletal muscle mass to maintain body temperature through shivering thermogenesis, an important source of heat in cold-exposed adults. Evolution has provided humans and other placental mammals with brown adipose tissue (BAT), a tissue that converts chemically stored energy, in the form of fatty acids and glucose, into heat through non-shivering thermogenesis. The thermogenic activity of this tissue is significant for the human infant's ability to maintain a sufficiently high core body temperature. Although BAT has been studied in human infants for more than a century, the literature covering different aspects of the tissue is rather limited. The aim of this review is to summarize the literature and describe what is actually known about the tissue and its importance for early human life.

Recruitment and activation of thermogenic adipocytes have received increasing attention as a strategy to improve systemic metabolic control. The analysis of brown and brite adipocytes is complicated by the complexity of adipose tissue biopsies. Here, we provide an in-depth analysis of pure brown, brite, and white adipocyte transcriptomes. By combining mouse and human transcriptome data, we identify a gene signature that can classify brown and white adipocytes in mice and men. Using a machine-learning-based cell deconvolution approach, we develop an algorithm proficient in calculating the brown adipocyte content in complex human and mouse biopsies. Applying this algorithm, we can show in a human weight loss study that brown adipose tissue (BAT) content is associated with energy expenditure and the propensity to lose weight. This online available tool can be used for in-depth characterization of complex adipose tissue samples and may support the development of therapeutic strategies to increase energy expenditure in humans., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Peroxisome proliferator-activated receptors (PPARs) have been suggested as the master regulators of adipose tissue formation. However, their role in regulating brown fat functionality has not been resolved. To address this question, we generated mice with inducible brown fat-specific deletions of PPARα, β/δ, and γ, respectively. We found that both PPARα and β/δδ are dispensable for brown fat function. In contrast, we could show that ablation of PPARγ in vitro and in vivo led to a reduced thermogenic capacity accompanied by a loss of inducibility by β-adrenergic signaling, as well as a shift from oxidative fatty acid metabolism to glucose utilization. We identified glycerol kinase (Gyk) as a partial mediator of PPARγ function and could show that Gyk expression correlates with brown fat thermogenic capacity in human brown fat biopsies. Thus, Gyk might constitute the link between PPARγ-mediated regulation of brown fat function and activation by β-adrenergic signaling., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: To study if repeated cold-exposure increases metabolic rate and/or brown adipose tissue (BAT) volume in humans when compared with avoiding to freeze., Design: Randomized, open, parallel-group trial., Methods: Healthy non-selected participants were randomized to achieve cold-exposure 1hour/day, or to avoid any sense of feeling cold, for 6weeks. Metabolic rate (MR) was measured by indirect calorimetry before and after acute cold-exposure with cold vests and ingestion of cold water. The BAT volumes in the supraclavicular region were measured with magnetic resonance imaging (MRI)., Results: Twenty-eight participants were recruited, 12 were allocated to controls and 16 to cold-exposure. Two participants in the cold group dropped out and one was excluded. Both the non-stimulated and the cold-stimulated MR were lowered within the group randomized to avoid cold (MR at room temperature from 1841±199 kCal/24h to 1795±213 kCal/24h, p=0.047 cold-activated MR from 1900±150 kCal/24h to 1793±215 kCal/24h, p=0.028). There was a trend towards increased MR at room temperature following the intervention in the cold-group (p=0.052). The difference between MR changes by the interventions between groups was statistically significant (p=0.008 at room temperature, p=0.032 after cold-activation). In an on-treatment analysis after exclusion of two participants that reported ≥8days without cold-exposure, supraclavicular BAT volume had increased in the cold-exposure group (from 0.0175±0.015l to 0.0216±0.014l, p=0.049)., Conclusions: We found evidence for plasticity in metabolic rate by avoiding to freeze compared with cold-exposure in a randomized setting in non-selected humans., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Brown adipose tissue (BAT) dissipates nutritional energy as heat via the uncoupling protein-1 (UCP1) and BAT activity correlates with leanness in human adults. Here we profile G protein-coupled receptors (GPCRs) in brown adipocytes to identify druggable regulators of BAT. Twenty-one per cent of the GPCRs link to the Gq family, and inhibition of Gq signalling enhances differentiation of human and murine brown adipocytes. In contrast, activation of Gq signalling abrogates brown adipogenesis. We further identify the endothelin/Ednra pathway as an autocrine activator of Gq signalling in brown adipocytes. Expression of a constitutively active Gq protein in mice reduces UCP1 expression in BAT, whole-body energy expenditure and the number of brown-like/beige cells in white adipose tissue (WAT). Furthermore, expression of Gq in human WAT inversely correlates with UCP1 expression. Thus, our data indicate that Gq signalling regulates brown/beige adipocytes and inhibition of Gq signalling may be a novel therapeutic approach to combat obesity.

Background: To evaluate the possibility of quantifying brown adipose tissue (BAT) volume and fat concentration with a high resolution, long echo time, dual-echo Dixon imaging protocol., Methods: A 0.42 mm isotropic resolution water-fat separated MRI protocol was implemented by using the second opposite-phase echo and third in-phase echo. Fat images were calibrated with regard to the intensity of nearby white adipose tissue (WAT) to form relative fat content (RFC) images. To evaluate the ability to measure BAT volume and RFC contrast dynamics, rats were divided into two groups that were kept at 4° or 22°C for 5 days. The rats were then scanned in a 70 cm bore 3.0 Tesla MRI scanner and a human dual energy CT. Interscapular, paraaortal, and perirenal BAT (i/pa/pr-BAT) depots as well as WAT and muscle were segmented in the MRI and CT images. Biopsies were collected from the identified BAT depots., Results: The biopsies confirmed that the three depots identified with the RFC images consisted of BAT. There was a significant linear correlation (P < 0.001) between the measured RFC and the Hounsfield units from DECT. Significantly lower iBAT RFC (P = 0.0064) and significantly larger iBAT and prBAT volumes (P = 0.0017) were observed in the cold stimulated rats., Conclusion: The calibrated Dixon images with RFC scaling can depict BAT and be used to measure differences in volume, and fat concentration, induced by cold stimulation. The high correlation between RFC and HU suggests that the fat concentration is the main RFC image contrast mechanism., (© 2015 Wiley Periodicals, Inc.)

Brown adipose tissue (BAT) is capable of transforming chemically stored energy, in the form of triglycerides, into heat. Recent studies have shown that metabolically active BAT is present in a large proportion of adult humans, where its activity correlates with a favorable metabolic status. Hence, the tissue is now regarded as an interesting target for therapies against obesity and associated diseases such as type 2 diabetes, the hypothesis being that an induction of BAT would be beneficial for these disease states. Apart from the association between BAT activity and a healthier metabolic status, later studies have also shown a positive correlation between BAT volume and both bone cross-sectional area and bone mineral density, suggesting that BAT might stimulate bone anabolism. The aim of this review is to give the reader a brief overview of the BAT research field and to summarize and discuss recent findings regarding BAT being a potential player in bone metabolism.

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells-so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Brown adipose tissue (BAT) has attracted scientific interest as an antidiabetic tissue owing to its ability to dissipate energy as heat. Despite a plethora of data concerning the role of BAT in glucose metabolism in rodents, the role of BAT (if any) in glucose metabolism in humans remains unclear. To investigate whether BAT activation alters whole-body glucose homeostasis and insulin sensitivity in humans, we studied seven BAT-positive (BAT(+)) men and five BAT-negative (BAT(-)) men under thermoneutral conditions and after prolonged (5-8 h) cold exposure (CE). The two groups were similar in age, BMI, and adiposity. CE significantly increased resting energy expenditure, whole-body glucose disposal, plasma glucose oxidation, and insulin sensitivity in the BAT(+) group only. These results demonstrate a physiologically significant role of BAT in whole-body energy expenditure, glucose homeostasis, and insulin sensitivity in humans, and support the notion that BAT may function as an antidiabetic tissue in humans., (© 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Obesity and related diseases are a major cause of human morbidity and mortality and constitute a substantial economic burden for society. Effective treatment regimens are scarce, and new therapeutic targets are needed. Brown adipose tissue, an energy-expending tissue that produces heat, represents a potential therapeutic target. Its presence is associated with low body mass index, low total adipose tissue content and a lower risk of type 2 diabetes mellitus. Knowledge about the development and function of thermogenic adipocytes in brown adipose tissue has increased substantially in the last decade. Important transcriptional regulators have been identified, and hormones able to modulate the thermogenic capacity of the tissue have been recognized. Intriguingly, it is now clear that humans, like rodents, possess two types of thermogenic adipocytes: the classical brown adipocytes found in the interscapular brown adipose organ and the so-called beige adipocytes primarily found in subcutaneous white adipose tissue after adrenergic stimulation. The presence of two distinct types of energy-expending adipocytes in humans is conceptually important because these cells might be stimulated and recruited by different signals, raising the possibility that they might be separate potential targets for therapeutic intervention. In this review, we will discuss important features of the energy-expending brown adipose tissue and highlight those that may serve as potential targets for pharmacological intervention aimed at expanding the tissue and/or enhancing its function to counteract obesity., (© 2014 The Association for the Publication of the Journal of Internal Medicine.)

During the last years the existence of metabolically active brown adipose tissue in adult humans has been widely accepted by the research community. Its unique ability to dissipate chemical energy stored in triglycerides as heat makes it an attractive target for new drugs against obesity and its related diseases. Hence the tissue is now subject to intense research, the hypothesis being that an expansion and/or activation of the tissue is associated with a healthy metabolic phenotype. Animal studies provide evidence for the existence of at least two types of brown adipocytes. Apart from the classical brown adipocyte that is found primarily in the interscapular region where it constitutes a thermogenic organ, a second type of brown adipocyte, the so-called beige adipocyte, can appear within white adipose tissue depots. The fact that the two cell types develop from different precursors suggests that they might be recruited and stimulated by different cues and therefore represent two distinct targets for therapeutic intervention. The aim of this commentary is to discuss recent work addressing the question whether also humans possess two types of brown adipocytes and to highlight some issues when looking for molecular markers for such cells.

Context: Brown adipose tissue (BAT) is a metabolically highly active organ with increased thermogenic activity in rodents exposed to cold temperature. Recently its presence in the cervical adipose tissue of human adults and its association with a favorable metabolic phenotype have been reported., Objective: The objective of the study was to determine the prevalence of retroperitoneal BAT in human adults., Design: This was an observational cohort study., Setting: The study was conducted at a tertiary referral hospital., Patients: Fifty-seven patients who underwent surgery for benign adrenal tumors were included in this study., Main Outcome Measures: Prevalence of retroperitoneal BAT adjacent to the removed adrenal tumor as determined by uncoupling protein 1 (UCP1) protein and mRNA expression was measured., Results: Using protein and mRNA expression analysis, we detected UCP1 protein in 26 of 57 patients (45.6%) as well as high mRNA expression of genes characteristic for brown adipocytes, independent of the adrenal tumor type. The presence of brown adipocytes within the retroperitoneal fat was associated with a significantly lower outdoor temperature during the month prior to surgery. Importantly, UCP1 expression on both mRNA and protein level was inversely correlated to outdoor temperature, whereas body mass index, sex, age, and diabetes status were not., Conclusions: These findings suggest that human retroperitoneal adipose tissue can acquire a BAT phenotype, thereby adapting to environmental challenges. These adaptive processes might provide a valuable therapeutic target in the treatment of obesity and insulin resistance.

The previously observed supraclavicular depot of brown adipose tissue (BAT) in adult humans was commonly believed to be the equivalent of the interscapular thermogenic organ of small mammals. This view was recently disputed on the basis of the demonstration that this depot consists of beige (also called brite) brown adipocytes, a newly identified type of brown adipocyte that is distinct from the classical brown adipocytes that make up the interscapular thermogenic organs of other mammals. A combination of high-resolution imaging techniques and histological and biochemical analyses showed evidence for an anatomically distinguishable interscapular BAT (iBAT) depot in human infants that consists of classical brown adipocytes, a cell type that has so far not been shown to exist in humans. On the basis of these findings, we conclude that infants, similarly to rodents, have the bona fide iBAT thermogenic organ consisting of classical brown adipocytes that is essential for the survival of small mammals in a cold environment.

We investigated the metabolism of human brown adipose tissue (BAT) in healthy subjects by determining its cold-induced and insulin-stimulated glucose uptake and blood flow (perfusion) using positron emission tomography (PET) combined with computed tomography (CT). Second, we assessed gene expression in human BAT and white adipose tissue (WAT). Glucose uptake was induced 12-fold in BAT by cold, accompanied by doubling of perfusion. We found a positive association between whole-body energy expenditure and BAT perfusion. Insulin enhanced glucose uptake 5-fold in BAT independently of its perfusion, while the effect on WAT was weaker. The gene expression level of insulin-sensitive glucose transporter GLUT4 was also higher in BAT as compared to WAT. In conclusion, BAT appears to be differently activated by insulin and cold; in response to insulin, BAT displays high glucose uptake without increased perfusion, but when activated by cold, it dissipates energy in a perfusion-dependent manner., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: Combination antiretroviral therapy (cART) is associated with lipodystrophy, i.e., loss of subcutaneous adipose tissue in the abdomen, limbs, and face and its accumulation intra-abdominally. No fat is lost dorsocervically and it can even accumulate in this region (buffalo hump). It is unknown how preserved dorsocervical fat differs from abdominal subcutaneous fat in HIV-1-infected cART-treated patients with (cART+LD+) and without (cART+LD-) lipodystrophy., Research Design and Methods: We used histology, microarray, PCR, and magnetic resonance imaging to compare dorsocervical and abdominal subcutaneous adipose tissue in cART+LD+ (n=21) and cART+LD- (n=11)., Results: Albeit dorsocervical adipose tissue in cART+LD+ seems spared from lipoatrophy, its mitochondrial DNA (mtDNA; copies/cell) content was significantly lower (by 62%) than that of the corresponding tissue in cART+LD-. Expression of CD68 mRNA, a marker of macrophages, and numerous inflammatory genes in microarray were significantly lower in dorsocervical versus abdominal subcutaneous adipose tissue. Genes with the greatest difference in expression between the two depots were those involved in regulation of transcription and regionalization (homeobox genes), irrespective of lipodystrophy status. There was negligible mRNA expression of uncoupling protein 1, a gene characteristic of brown adipose tissue, in either depot., Conclusions: Because mtDNA is depleted even in the nonatrophic dorsocervical adipose tissue, it is unlikely that the cause of lipoatrophy is loss of mtDNA. Dorsocervical adipose tissue is less inflamed than lipoatrophic adipose tissue. It does not resemble brown adipose tissue. The greatest difference in gene expression between dorsocervical and abdominal subcutaneous adipose tissue is in expression of homeobox genes., (© 2011 by the American Diabetes Association.)

Objective: Previous findings demonstrate that enhanced expression of the forkhead transcription factor Foxc2 in adipose tissue leads to a lean and insulin-sensitive phenotype. These findings prompted us to further investigate the role of Foxc2 in the regulation of genes of fundamental importance for metabolism and mitochondrial function., Research Design and Methods: The effects of Foxc2 on expression of genes involved in mitochondriogenesis and mitochondrial function were assessed by quantitative real-time PCR. The potential of a direct transcriptional regulation of regulated genes was tested in promoter assays, and mitochondrial morphology was investigated by electron microscopy. Mitochondrial function was tested by measuring oxygen consumption and extracellular acidification rates as well as palmitate oxidation., Results: Enhanced expression of FOXC2 in adipocytes or in cells with no endogenous Foxc2 expression induces mitochondriogenesis and an elongated mitochondrial morphology. Together with increased aerobic metabolic capacity, increased palmitate oxidation, and upregulation of genes encoding respiratory complexes and of brown fat-related genes, Foxc2 also specifically induces mitochondrial fusion genes in adipocytes. Among tested forkhead genes, Foxc2 is unique in its ability to trans-activate the nuclear-encoded mitochondrial transcription factor A (mtTFA/Tfam) gene--a master regulator of mitochondrial biogenesis. In human adipose tissue the expression levels of mtTFA/Tfam and of fusion genes also correlate with that of Foxc2., Conclusions: We previously showed that a high-calorie diet and insulin induce Foxc2 in adipocytes; the current findings identify a previously unknown role for Foxc2 as an important metabo-regulator of mitochondrial morphology and metabolism.

New targets for pharmacological interventions are of great importance to combat the epidemic of obesity. Brown adipose tissue could potentially represent one such target. Unlike white adipose tissue, brown adipose tissue has the ability to dissipate energy by producing heat rather than storing it as triglycerides. In small mammals, the presence of active brown adipose tissue is pivotal for the maintenance of body temperature and possibly to protect against the detrimental effects of surplus energy intake. Animal studies have shown that expansion and/or activation of brown adipose tissue counteracts diet-induced weight gain and related disorders such as type 2 diabetes mellitus. Several independent studies have now confirmed the presence of functional brown adipose tissue in adult humans, for whom this tissue is probably metabolically beneficial given its association with both low BMI and low total adipose tissue content. Over the past few years, knowledge of the transcriptional control and development of brown adipose tissue has increased substantially. Thus, several possible targets that may be useful for the expansion and/or activation of this tissue by pharmacological means have been identified. Whether or not brown adipose tissue will be useful in the battle against obesity remains to be seen. However, this possibility is certainly well worth exploring.

In this study, we explore the effects of several FOX and mutant FOX transcription factors on adipocyte determination, differentiation, and metabolism. In addition to Foxc2 and Foxo1, we report that Foxf2, Foxp1, and Foxa1 are other members of the Fox family that show regulated expression during adipogenesis. Although enforced expression of FOXC2 inhibits adipogenesis, Foxf2 slightly enhances the rate of differentiation. Constitutively active FOXC2-VP16 inhibits adipogenesis through multiple mechanisms. FOXC2-VP16 impairs the transient induction of C/EBPbeta during adipogenesis and induces expression of the transcriptional repressor Hey1 as well as the activator of Wnt/beta-catenin signaling, Wnt10b. The constitutive transcriptional repressor, FOXC2-Eng, enhances adipogenesis of preadipocytes and multipotent mesenchymal precursors and determines NIH-3T3 and C2C12 cells to the adipocyte lineage. Although PPARgamma ligand or C/EBPalpha are not necessary for stimulation of adipogenesis by FOXC2-Eng, at least low levels of PPARgamma protein are absolutely required. Finally, expression of FOXC2-Eng in adipocytes increases insulin-stimulated glucose uptake, further expanding the profound and pleiotropic effects of FOX transcription factors on adipocyte biology.

Using positron-emission tomography (PET), we found that cold-induced glucose uptake was increased by a factor of 15 in paracervical and supraclavicular adipose tissue in five healthy subjects. We obtained biopsy specimens of this tissue from the first three consecutive subjects and documented messenger RNA (mRNA) and protein levels of the brown-adipocyte marker, uncoupling protein 1 (UCP1). Together with morphologic assessment, which showed numerous multilocular, intracellular lipid droplets, and with the results of biochemical analysis, these findings document the presence of substantial amounts of metabolically active brown adipose tissue in healthy adult humans., (2009 Massachusetts Medical Society)

MUC1 is a mucin glycoprotein containing multiple tandem repeats of 20 amino acids, with five serines and threonines that can be O-glycosylated. Here, we investigated the O-glycosylation site occupancy in MUC1 glycoproteins produced in two mutant CHO cell lines, Lec3.2.8.1 and ldlD. We found that the average site occupancy was higher in MUC1 from Lec3.2.8.1 than from ldlD and that the occupancy increased with the number of tandem repeats in the protein and also depended on the culture conditions used for production. Moreover, we describe the successful use of electron-capture dissociation (ECD) fragmentation, coupled to online liquid chromatography mass spectrometry, to determine the glycosylation of individual sites in recombinant MUC1 proteins with 16 tandem repeats. We analyzed MUC1 tandem repeat peptides with 1-5 GalNAc residues by ECD fragmentation and found that the first site to be glycosylated was either Ser-5 or Thr-6, with the addition of a second GalNAc at Thr-14. For peptides with three GalNAc residues, several different variants of glycopeptides were found, indicating a heterogeneous order of glycosylation at this stage. In contrast, only one variant was found for peptides with four GalNAc residues, where Thr-19 in the PDTR motif was left unglycosylated, indicating that this site is glycosylated last. The results gave novel insight into the order of GalNAc substitution in MUC1 in vivo.

Mucins are large glycoproteins protecting mucosal surfaces throughout the body. Their expressions are tissue-specific, but in disease states such as cystic fibrosis, inflammation and cancer, this specificity can be disturbed. MUC5AC is normally expressed in the mucous cells of the epithelia lining the stomach and the trachea, where it constitutes a major component of the gastric and respiratory mucus. A number of mAbs have been raised against the gastric M1 antigen, an early marker for colonic carcinogenesis. Several of these mAbs recognize epitopes present on MUC5AC, suggesting that MUC5AC is the antigen. However, some of the mAbs raised against the gastric M1 antigen are widely used as antibodies against MUC5AC, despite the fact that their specificity for MUC5AC has not been clearly shown. In this study, we have tested the reactivity of the latter antibodies against a recombinantly expressed C-terminal cysteine-rich part of human MUC5AC. We demonstrate for the first time that the widely used mAb 45M1, as well as 2-12M1 and 166M1, are true antibodies against MUC5AC, with epitopes located in the C-terminal cysteine-rich part of the mucin.

MUC5AC is the main gel-forming mucin expressed by goblet cells of the airways and stomach where it protects the underlying epithelia. We expressed the C-terminal cysteine-rich part of the human MUC5AC mucin in CHO-K1 cells (Chinese-hamster ovary K1 cells) where it formed disulfide-linked dimers in the ER (endoplasmic reticulum). After reducing the disulfide bonds of these dimers, not only the expected monomers were found, but also two smaller fragments, indicating that the protein was partially cleaved. The site of cleavage was located at an Asp-Pro bond situated in a GDPH (Gly-Asp-Pro-His) sequence found in the vWD4 (von Willebrand D4) domain. This sequence is also found in the human MUC2 mucin, previously shown to be cleaved at the same site by a slow, non-enzymatic process triggered by a pH below 6 [Lidell, Johansson and Hansson (2003) J. Biol. Chem. 278, 13944-13951]. In contrast with this, the cleavage of MUC5AC started already in the neutral ER. However, it continued and was slightly accelerated at a pH below 6.5, a pH found in the later parts of the secretory pathway. The cleavage generated a reactive group in the new C-terminus that could link the protein to a primary amine. No cleavage of MUC5AC has so far been reported. By using an antibody reacting with the C-terminal cleavage fragment, we could verify that the cleavage occurs in wild-type MUC5AC produced by HT-29 cells. The cleavage of MUC5AC and the generation of the reactive new C-terminus could contribute to the adherent and viscous mucus found at chronic lung diseases such as asthma and cystic fibrosis, characterized by mucus hypersecretion and lowered pH of the airways.

In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N- and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel.

Background: In a hitherto unconfirmed report, orosomucoid was reported to ameliorate the nephrotic syndrome induced by puromycin aminonucleoside (PAN) in rats., Methods: We wanted to test this hypothesis and extend the analysis of the effects on the glomerular barrier. Glomerular filtration rate (GFR), and fractional clearance for albumin (theta(albumin)) and for neutral Ficolls were estimated in cooled isolated perfused kidneys. Modern transport equations were used to estimate glomerular size selectivity and charge selectivity. Also, podocyte morphology was studied. Four groups of rats (4 x n = 8) were administered PAN intraperitoneally and treated daily for 5 days with orosomucoid in two different doses (groups A and B), albumin (group C) or saline (group D). Two additional groups of rats (2 x n = 8) were used as controls and these rats received either saline (group E) or orosomucoid (group F) but no PAN., Results: Treatment with orosomucoid restored podocyte morphology and renal function from the damaging effects of PAN in a dose-dependent manner. GFR was significantly reduced by PAN (groups C and D) when compared with controls (groups E and F). This effect was partly (group A) or completely (group B) reversed by orosomucoid. The theta(albumin) was 0.002+/-0.001 (mean+/-SEM) in controls (group E) and was unaffected by orosomucoid per se (group F). PAN increased theta(albumin) to 0.020+/-0.001 in group C and to 0.021+/-0.001 in group D, while it was significantly less in group A, 0.014+/-0.001, P<0.05. The heterogeneous charged fibre model analysis revealed that PAN reduced the relative volume of negatively charged fibres, phi, from 7.1+/-0.08% (group E) to 48% of this value in groups C and D (P<0.001); phi was 4.5+/-0.04% in group A, 5.3+/-0.44% in group B, and 6.1+/-0.11% in group F., Conclusion: High doses of orosomucoid completely normalized the glomerular barrier in six out of eight animals with puromycin-induced nephrotic syndrome. Thus, orosomucoid has a promising therapeutic potential for certain kidney disorders.

The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin kappa-chain signal sequence to direct the protein to the secretory pathway. Pulse-chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.

During purification of a recombinant MUC2 C terminus expressed in CHO-K1 cells, the protein was partly cleaved when buffers with a pH of 6.0 were used. When buffers with higher pH values were used, less cleavage was found. Disulfide bonds held the two fragments generated together as these were only observed after reduction. Edman sequencing of the C-terminal 110-kDa fragment revealed that the cleavage had occurred at an Asp-Pro bond, a site described previously to generate the so-called "link peptide" after disulfide bond reduction. In vitro studies on the conditions for cleavage showed that it occurred in a time-dependent manner at a pH below 6.0. Furthermore, the reaction was not enzyme-mediated as it occurred in pure preparations of the MUC2 C terminus and was not inhibited by protease inhibitors. When expressed in the mucin producing cell line LS 174T, the C terminus was cleaved to a higher extent compared with the CHO-K1 cells. Neutralizing the secretory pathway with either NH(4)Cl or bafilomycin A1 inhibited this cleavage. Altogether, our results suggest that the cleavage is an autocatalytic reaction that occurs in the acidic environment of the late secretory pathway. Furthermore, the cleavage produced a new, reactive C terminus that has the potential to attach the mucin to itself or other molecules. Because a pH below 6 can be reached in the late secretory pathway and on mucosal surfaces, the cleavage and possible cross-linking are likely to be of biological importance.

The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of approximately 240 kDa. Upon reduction, peptides of approximately 85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.