Fetuin B in white adipose tissue induces inflammation and is associated with peripheral insulin resistance in mice and humans.

OBJECTIVE: Fetuin B is a steatosis-responsive hepatokine that causes glucose intolerance in mice, but the underlying mechanisms remain incompletely described. This study aimed to elucidate the mechanisms of action of fetuin B by investigating its putative effects on white adipose tissue metabolism. METHODS: First, fetuin B gene and protein expression was measured in multiple organs in mice and in cultured adipocytes. Next, the authors performed a hyperinsulinemic-euglycemic clamp in mice and in humans to examine the link between white adipose tissue fetuin B content and indices of insulin sensitivity. Finally, the effect of fetuin B on inflammation was investigated in cultured adipocytes by quantitative polymerase chain reaction and full RNA sequencing. RESULTS: This study demonstrated in adipocytes and mice that fetuin B was produced and secreted by the liver and taken up by adipocytes and adipose tissue. There was a strong negative correlation between white adipose tissue fetuin B content and peripheral insulin sensitivity in mice and in humans. RNA sequencing and polymerase chain reaction analysis revealed that fetuin B induced an inflammatory response in adipocytes. CONCLUSIONS: Fetuin B content in white adipose tissue strongly associated with peripheral insulin resistance in mice and humans. Furthermore, fetuin B induced a proinflammatory response in adipocytes, which might drive peripheral insulin resistance.

Fecal carriage of vanB antibiotic resistance gene affects adipose tissue function under vancomycin use.

Detrimental consequences of antibiotic treatment may include long-lasting disruption of the gut microbiota. Previous studies found no negative effects of antibiotics on metabolic health, although individualized responses were observed. Here, we aimed to investigate the subject-specific response to vancomycin use in tissue-specific insulin sensitivity by stratifying individuals based on the presence of antibiotic resistance genes (ARGs) or opportunistic pathogens (OPs) in the baseline fecal microbiota. Quantitative Polymerase Chain Reaction (qPCR) was used to detect ARGs and OPs in DNA isolated from fecal samples of 56 males with overweight/obesity (Body Mass Index: 25-35 kg/m2) and impaired glucose metabolism (fasting plasma glucose ≥5.6 mmol/L and/or 2-hour glucose 7.8-11.1 mmol/L). A two-step hyperinsulinemic-euglycemic clamp was performed to determine tissue-specific insulin sensitivity. Abdominal subcutaneous adipose tissue (AT) gene expression was assessed using Affymetrix microarray. Gut microbial composition was determined using the Human Intestinal Tract Chip (HITChip) microarray. At baseline, the vancomycin resistance gene vanB was present in 60% of our population. In individuals that were vanB-negative at baseline, AT insulin sensitivity (insulin-mediated suppression of plasma free fatty acids) improved during vancomycin use, while it decreased among vanB-positive individuals (% change post versus baseline: 14.1 ± 5.6 vs. -6.7 ± 7.5% (p = .042)). The vancomycin-induced increase in AT insulin sensitivity was accompanied by downregulation of inflammatory pathways and enrichment of extracellular matrix remodeling pathways in AT. In the vanB-positive group, well-known vanB-carrying bacteria, Enterococcus and Streptococcus, expanded in the gut microbiome. In conclusion, microbiome composition and adipose tissue biology were differentially affected by vancomycin treatment based on fecal vanB carriage.

Weight Gain in Children during the COVID-19 Pandemic and the Protective Effect of Lifestyle Intervention in Children with Obesity.

INTRODUCTION: School closures due to the COVID-19 pandemic affect children's daily structure, mealtimes, physical activity, and sleeping habits, possibly exacerbating weight gain, particularly in vulnerable children with overweight and obesity. This study aimed to evaluate both perceived and objectively measured weight gain in children in the Netherlands during the COVID-19 pandemic and the effect of prior lifestyle intervention. METHODS: A total of 150 children of the Children, Obesity and Lifestyle during COVID-19 (COLC) study (cohort A) reported perceptions of weight change during the COVID-19 pandemic. Anthropometric data of 65 children with overweight and obesity were collected at the expertise Centre for Overweight Adolescent and Children's Healthcare in the same period (COACH; cohort B). RESULTS: In cohort A, 43% of children with overweight and obesity perceived weight gain during the pandemic, compared to 15% of lean children. In cohort B, the BMI z-score increased significantly (+0.065 SD) within 5 months. Participation in a lifestyle intervention for >1 year and having parents with Dutch background was associated with less weight gain, specifically in children with obesity. DISCUSSION/CONCLUSION: In particular, children with overweight and obesity seem to be at risk for accelerated weight gain during the COVID-19 pandemic. Prior long-term participation in a lifestyle intervention protects against this weight gain, which emphasizes the importance of strong support for vulnerable populations during health crises and pleads for wide implementation of lifestyle interventions for children.

Maternal cold exposure induces distinct transcriptome changes in the placenta and fetal brown adipose tissue in mice.

BACKGROUND: Brown adipose tissue (BAT) is specialized to dissipate energy in the form of heat. BAT-mediated heat production in rodents and humans is critical for effective temperature adaptation of newborns to the extrauterine environment immediately after birth. However, very little is known about whether and how fetal BAT development is modulated in-utero in response to changes in maternal thermal environment during pregnancy. Using BL6 mice, we evaluated the impact of different maternal environmental temperatures (28 °C and 18 °C) on the transcriptome of the placenta and fetal BAT to test if maternal cold exposure influences fetal BAT development via placental remodeling. RESULTS: Maternal weight gain during pregnancy, the average number of fetuses per pregnancy, and placental weight did not differ between the groups at 28 °C and 18 °C. However, the average fetal weight at E18.5 was 6% lower in the 18 °C-group compared to the 28 °C-group. In fetal BATs, cold exposure during pregnancy induced increased expression of genes involved in de novo lipogenesis and lipid metabolism while decreasing the expression of genes associated with muscle cell differentiation, thus suggesting that maternal cold exposure may promote fetal brown adipogenesis by suppressing the myogenic lineage in bidirectional progenitors. In placental tissues, maternal cold exposure was associated with upregulation of genes involved in complement activation and downregulation of genes related to muscle contraction and actin-myosin filament sliding. These changes may coordinate placental adaptation to maternal cold exposure, potentially by protecting against cold stress-induced inflammatory damage and modulating the vascular and extravascular contractile system in the placenta. CONCLUSIONS: These findings provide evidence that environmental cold temperature sensed by the mother can modulate the transcriptome of placental and fetal BAT tissues. The ramifications of the observed gene expression changes warrant future investigation.

Physical activity behaviour and screen time in Dutch children during the COVID-19 pandemic: Pre-, during- and post-school closures.

BACKGROUND: Measures during the COVID-19 pandemic, including the closure of schools and sports facilities, may have lasting impact on the physical activity (PA) of children that persists for a long time. OBJECTIVE: To investigate the effect of COVID-19 measures on screen time and PA in Dutch children pre-, during- and post-school closures. METHODS: In cohort A (n = 102, 10.5 ± 3.6 years, 42.4% boys), data on PA and screen time during the lockdown were collected using a questionnaire. In cohort B (n = 131, 10.2 ± 0.9 years, 43.5% boys), data on PA and screen time were collected using a questionnaire and accelerometry 1 year before and after school closure. RESULTS: In cohort A, 62% reported less total PA. Self-reported screen time on week days increased 34 ± 105 min/d during the lockdown. In cohort B, sedentary time as measured by accelerometry, increased by 45 ± 67 min/d and only 20% reached PA levels of 60 min/d compared to 64% in May 2019. Self-reported screen time increased by 59 ± 112 min/d and 62 ± 130 min/d during week and weekend days, respectively. CONCLUSIONS: Children were less physically active, and screen time was higher during and after the school closures due to the COVID-19 lockdown. This is alarming as an active lifestyle in children is crucial in preventing chronic diseases such as obesity.

Complement in Reproductive White Adipose Tissue Characterizes the Obese Preeclamptic-Like BPH/5 Mouse Prior to and During Pregnancy.

Preeclampsia (PE) is a serious hypertensive disorder of pregnancy characterized by abnormal placental development with an unknown etiology. To better understand which women will develop PE, a number of maternal risk factors have been identified, including obesity. Visceral white adipose tissue (WAT) contains inflammatory mediators that may contribute to PE. To explore this, we utilized the blood pressure high (BPH)/5 mouse model of superimposed PE that spontaneously recapitulates the maternal PE syndrome. We hypothesized that BPH/5 visceral WAT adjacent to the female reproductive tract (reproductive WAT) is a source of complement factors that contribute to the inflammatory milieu and angiogenic imbalance at the maternal-fetal interface in this model and in preeclamptic women. To test our hypothesis, we calorie-restricted BPH/5 females for two weeks prior to pregnancy and the first seven days of pregnancy, which attenuated complement component 3 (C3) but not complement factor B, nor complement factor D, (adipsin) in the reproductive WAT or the implantation site in BPH/5. Furthermore, calorie restriction during pregnancy restored vascular endothelial and placental growth factor mRNA levels in the BPH/5 implantation site. These data show maternal reproductive WAT may be a source of increased C3 during pregnancy, which is increased at the maternal-fetal interface in preeclamptic BPH/5 mice. It also suggests that calorie restriction could regulate inflammatory mediators thought to contribute to placental dysfunction in PE. Future studies are necessary to examine the effect of calorie restriction on C3 throughout pregnancy and the role of maternal obesity in PE.

Individual and cohort-specific gut microbiota patterns associated with tissue-specific insulin sensitivity in overweight and obese males.

A growing body of evidence suggests that the human gut microbiota plays a role in the development of obesity and related metabolic diseases. However, there is little consensus between studies, which could be due to biological as well as technical variation. In addition, little human data are available to investigate whether tissue-specific insulin sensitivity is related to specific microbial patterns. We examined this relation in two independent cohorts of overweight and obese pre-diabetic men, using phylogenetic microarray data and hepatic, peripheral and adipose tissue insulin sensitivity that were determined by a two-step hyperinsulinemic-euglycemic clamp with [6,6-2H2]-glucose tracer infusion. Despite a prominent subject-specific microbiota, we found significant associations of microbial taxa with tissue-specific insulin sensitivity using regression analysis. Using random forests we found moderate associations with other measures of glucose homeostasis in only one of the cohorts (fasting glucose concentrations AUC = 0.66 and HbA1c AUC = 0.65). However, all findings were cohort-specific due to pronounced variation in microbiota between cohorts, suggesting the existence of alternative states for dysbiosis in metabolic syndrome patients. Our findings suggest individual or group related dynamics, instead of universal microbiota signals, related to the host when the overweight or obese state has already developed and argue that care should be taken with extrapolating significant correlations from single cohorts, into generalized biological relevance.

Towards Prepared mums (TOP-mums) for a healthy start, a lifestyle intervention for women with overweight and a child wish: study protocol for a randomised controlled trial in the Netherlands.

INTRODUCTION: Periconception obesity is associated with a higher risk for adverse perinatal outcomes such as gestational diabetes mellitus, preeclampsia, large for gestational age, operative delivery and preterm birth. Lifestyle interventions during pregnancy have resulted in insufficient effects on reducing these perinatal complications. A few reasons for this disappointing effect can be suggested: (1) the time period during pregnancy for improvement of developmental circumstances is too short; (2) the periconception period in which complications originate is not included; and (3) lifestyle interventions may not have been sufficiently multidisciplinary and customised. A preconception lifestyle intervention might be more effective to reduce perinatal complications. Therefore, the aim of the Towards Prepared mums study is to evaluate the effect of a lifestyle intervention starting prior to conception on lifestyle behaviour change. METHODS AND ANALYSIS: This protocol outlines a non-blinded, randomised controlled trial. One hundred and twelve women (18-40 years of age) with overweight or obesity (body mass index≥25.0 kg/m2) who plan to conceive within 1 year will be randomised to either the intervention or care as usual group. The intervention group will receive a multidisciplinary, customised lifestyle intervention stimulating physical activity, a healthy diet and smoking cessation, if applicable. The lifestyle intervention and monitoring will take place until 12 months postpartum. The primary outcome is difference in weight in kg from baseline to 6 weeks postpartum. Secondary outcomes are gestational weight gain, postpartum weight retention, smoking cessation, dietary and physical activity habits. Furthermore, exploratory outcomes include body composition, cardiometabolic alterations, time to pregnancy, need for assisted reproductive technologies, perinatal complications of mother and child, and lung function of the child. Vaginal and oral swabs, samples of faeces, breast milk, placenta and cord blood will be stored for evaluation of microbial flora, epigenetic markers and breast milk composition. Furthermore, a cost-effectiveness analysis will take place. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Medical Ethical Committee of Maastricht University Medical Centre+ (NL52452.068.15/METC152026). Knowledge derived from this study will be made available by publications in international peer-reviewed scientific journals and will be presented at (inter)national scientific conferences. A dissemination plan for regional and national implementation of the intervention is developed. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT02703753.

Circulating but not faecal short-chain fatty acids are related to insulin sensitivity, lipolysis and GLP-1 concentrations in humans.

Microbial-derived short-chain fatty acids (SCFA) acetate, propionate and butyrate may provide a link between gut microbiota and whole-body insulin sensitivity (IS). In this cross-sectional study (160 participants, 64% male, BMI: 19.2-41.0 kg/m2, normal or impaired glucose metabolism), associations between SCFA (faecal and fasting circulating) and circulating metabolites, substrate oxidation and IS were investigated. In a subgroup (n = 93), IS was determined using a hyperinsulinemic-euglycemic clamp. Data were analyzed using multiple linear regression analysis adjusted for sex, age and BMI. Fasting circulating acetate, propionate and butyrate concentrations were positively associated with fasting GLP-1 concentrations. Additionally, circulating SCFA were negatively related to whole-body lipolysis (glycerol), triacylglycerols and free fatty acids levels (standardized (std) ß adjusted (adj) -0.190, P = 0.023; std ß adj -0.202, P = 0.010; std ß adj -0.306, P = 0.001, respectively). Circulating acetate and propionate were, respectively, negatively and positively correlated with IS (M-value: std ß adj -0.294, P < 0.001; std ß adj 0.161, P = 0.033, respectively). We show that circulating rather than faecal SCFA were associated with GLP-1 concentrations, whole-body lipolysis and peripheral IS in humans. Therefore, circulating SCFA are more directly linked to metabolic health, which indicates the need to measure circulating SCFA in human prebiotic/probiotic intervention studies as a biomarker/mediator of effects on host metabolism.

Dyslipidemia and the role of adipose tissue in early pregnancy in the BPH/5 mouse model for preeclampsia.

The hypertensive pregnancy disorder preeclampsia (PE) is a leading cause of fetal and maternal morbidity/mortality. Obesity increases the risk to develop PE, presumably via the release of inflammatory mediators from the adipose tissue, but the exact etiology remains largely unknown. Using obese PE-like blood pressure high subline 5 (BPH/5) and lean gestational age-matched C57Bl6 mice, we aimed to obtain insight into differential reproductive white adipose tissue (rWAT) gene expression, circulating lipids and inflammation at the maternal-fetal interface during early pregnancy. In addition, we investigated the effect of 7 days 25% calorie restriction (CR) in early pregnancy on gene expression in rWAT and implantation sites. Compared with C57Bl6, female BPH/5 are dyslipidemic before pregnancy and show an amplification of rWAT mass, circulating cholesterol, free fatty acids, and triacylglycerol levels throughout pregnancy. RNA sequencing showed that pregnant BPH/5 mice have elevated gene enrichment in pathways related to inflammation and cholesterol biosynthesis at embryonic day (e) 7.5. Expression of cholesterol-related HMGCS1, MVD, Cyp51a1, and DHCR was validated by quantitative reverse-transcription-polymerase chain reaction. CR during the first 7 days of pregnancy restored the relative mRNA expression of these genes to a level comparable to C57Bl6 pregnant females and reduced the expression of circulating leptin and proinflammatory prostaglandin synthase 2 in both rWAT and implantation sites in BPH/5 mice at e7.5. Our data suggest a possible role for rWAT in the dyslipidemic state and inflammatory uterine milieu that might underlie the pathogenesis of PE. Future studies should further address the physiological functioning of the adipose tissue in relation to PE-related pregnancy outcomes.

Short-Term Microbiota Manipulation and Forearm Substrate Metabolism in Obese Men: A Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: To investigate the impact of gut microbiota manipulation on fasting and postprandial skeletal muscle metabolism in humans. METHODS: 40 obese, insulin-resistant males were randomized to amoxicillin (broad-spectrum antibiotic), vancomycin (narrow-spectrum antibiotic), or placebo (7 days, 1,500 mg/day). Before and after treatment, forearm blood flow and metabolite fluxes across forearm muscle were measured under fasting and postprandial (high-fat mixed-meal) conditions. RESULTS: Vancomycin decreased bacterial diversity, reduced the abundance of Gram-positive Firmicutes, and increased the abundance of Gram-negative Proteobacteria, whereas amoxicillin did not affect microbial composition. Neither vancomycin nor amoxicillin treatment affected fasting and postprandial plasma glucose, free fatty acid (FFA), triacylglycerol (TAG), glycerol, lactate, and insulin concentrations or forearm blood flow. Fasting and postprandial net forearm muscle glucose uptake and the release of lactate were not significantly altered by antibiotic treatment as compared to placebo. Finally, antibiotic treatment did not change fasting and postprandial glycerol, FFA, and TAG fluxes across forearm muscle. CONCLUSION: The present study demonstrates that short-term antibiotic treatment has no effects on fasting and postprandial forearm substrate metabolism and blood flow in obese men with impaired glucose metabolism. These data suggest that short-term strategies targeting the gut microbiota to improve metabolic health may not be effective in obese humans.

Effects of gut microbiota manipulation on ex vivo lipolysis in human abdominal subcutaneous adipocytes.

The intestinal microbiota may contribute to the development of obesity by affecting host lipid metabolism and insulin sensitivity. To investigate the effects of microbiota manipulation on ex vivo basal and ß-adrenergically-stimulated lipolysis in human adipocytes, 36 obese men were randomized to amoxicillin (broad-spectrum antibiotic), vancomycin (narrow-spectrum antibiotic) or placebo treatment (7 d, 1500 mg/d). Before and after treatment, ex vivo adipose tissue lipolysis was assessed under basal conditions and during stimulation with the non-selective ß-agonist isoprenaline using freshly isolated mature adipocytes. Gene (targeted microarray) and protein expression were analyzed to investigate underlying pathways. Antibiotics treatment did not significantly affect basal and maximal isoprenaline-mediated glycerol release from adipocytes. Adipose tissue ß-adrenoceptor expression or post-receptor signalling was also not different between groups. In conclusion, 7 d oral antibiotics treatment has no effect on ex vivo lipolysis in mature adipocytes derived from adipose tissue of obese insulin resistant men.

Celecoxib restores angiogenic factor expression at the maternal-fetal interface in the BPH/5 mouse model of preeclampsia.

Preeclampsia (PE), a hypertensive disease of pregnancy, is a leading cause of fetal and maternal morbidity/mortality. Early angiogenic and inflammatory disturbances within the placenta are thought to underlie the development of the maternal PE syndrome and poor pregnancy outcomes. However, the exact etiology remains largely unknown. Here, we use the BPH/5 mouse model of PE to elucidate the way in which inflammation early in pregnancy contributes to abnormal expression of angiogenic factors at the maternal-fetal interface. We have previously described improvement in maternal hypertension and fetal growth restriction in this model after treatment with the anti-inflammatory cyclooxygenase-2 (Cox2) specific inhibitor celecoxib. To further characterize the mechanisms by which celecoxib improves poor pregnancy outcomes in BPH/5 mice, we determined expression of angiogenic factors and complement pathway components after celecoxib. In BPH/5 implantation sites there was increased hypoxia inducible factor-1α ( Hif1α), heme oxygenase-1 ( Ho-1), and stem cell factor ( Scf) mRNA concomitant with elevated prostaglandin synthase 2 ( Ptgs2), encoding Cox2, and elevated VEGF protein. Angiopoietin 1 ( Ang1), tunica interna endothelial cell kinase-2 receptor ( Tie2), complement factor 3 ( C3), and complement factor B ( CfB) were increased in midgestation BPH/5 placentae. Whereas BPH/5 expression levels of VEGF, Ang1, and Tie2 normalized after celecoxib, placental C3 and CfB mRNA remained unchanged. However, celecoxib did reduce the pregnancy-specific circulating soluble fms-like tyrosine kinase-1 (sFlt-1) rise in BPH/5 mice at midgestation. These data show that elevated Cox2 during implantation contributes to placental angiogenic factor imbalances in the BPH/5 mouse model of PE.

Oral iron supplementation: Potential implications for the gut microbiome and metabolome in patients with CKD.

Patients with chronic kidney disease (CKD) and loss of kidney function are at increased risk for morbidity and mortality. The risks of CKD are attributed to "uremia," an increased concentration of uremic retention solutes (toxins) in the plasma. Recently, a colo-renal axis became clearly apparent and uremia has been associated with an altered gut microbiome composition and metabolism. There is a high prevalence of anemia in patients with CKD, for which patients are often treated with oral or intravenous iron. Recent in vivo and in vitro studies have reported adverse effects of oral iron supplementation on the gut microbiota composition, gut metabolome, and intestinal health, which in turn may result in an increased production of uremic toxins. It may also affect circulating levels of other microbe-derived molecules, that can act as mediators of immune regulation. Changes in body iron levels have also been reported to exert subtle effects on host immune function by modulating immune cell proliferation and differentiation, and by directly regulating cytokine formation and antimicrobial immune effector mechanisms. Based on the foregoing it is conceivable that oral iron supplementation in iron deficient predialysis CKD patients adversely changes gut microbiota composition, the gut and systemic metabolome, and host immunity and infection. Future studies are needed to confirm these hypotheses and to assess whether, compared to IV iron supplementation, oral iron supplementation negatively impacts on morbidity of CKD, and whether these adverse effects depend on the iron bioavailability of the iron formulation to the microbiota.

Effects of Gut Microbiota Manipulation by Antibiotics on Host Metabolism in Obese Humans: A Randomized Double-Blind Placebo-Controlled Trial.

The gut microbiota has been implicated in obesity and cardiometabolic diseases, although evidence in humans is scarce. We investigated how gut microbiota manipulation by antibiotics (7-day administration of amoxicillin, vancomycin, or placebo) affects host metabolism in 57 obese, prediabetic men. Vancomycin, but not amoxicillin, decreased bacterial diversity and reduced Firmicutes involved in short-chain fatty acid and bile acid metabolism, concomitant with altered plasma and/or fecal metabolite concentrations. Adipose tissue gene expression of oxidative pathways was upregulated by antibiotics, whereas immune-related pathways were downregulated by vancomycin. Antibiotics did not affect tissue-specific insulin sensitivity, energy/substrate metabolism, postprandial hormones and metabolites, systemic inflammation, gut permeability, and adipocyte size. Importantly, energy harvest, adipocyte size, and whole-body insulin sensitivity were not altered at 8-week follow-up, despite a still considerably altered microbial composition, indicating that interference with adult microbiota by 7-day antibiotic treatment has no clinically relevant impact on metabolic health in obese humans.

Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin sensitivity.

BACKGROUND & AIMS: Obesity has been associated with changes in the composition and function of the intestinal microbiota. Modulation of the microbiota by antibiotics also alters bile acid and glucose metabolism in mice. Hence, we hypothesized that short term administration of oral antibiotics in humans would affect fecal microbiota composition and subsequently bile acid and glucose metabolism. METHODS: In this single blinded randomized controlled trial, 20 male obese subjects with metabolic syndrome were randomized to 7 days of amoxicillin 500 mg t.i.d. or 7 days of vancomycin 500 mg t.i.d. At baseline and after 1 week of therapy, fecal microbiota composition (Human Intestinal Tract Chip phylogenetic microarray), fecal and plasma bile acid concentrations as well as insulin sensitivity (hyperinsulinemic euglycemic clamp using [6,6-(2)H2]-glucose tracer) were measured. RESULTS: Vancomycin reduced fecal microbial diversity with a decrease of gram-positive bacteria (mainly Firmicutes) and a compensatory increase in gram-negative bacteria (mainly Proteobacteria). Concomitantly, vancomycin decreased fecal secondary bile acids with a simultaneous postprandial increase in primary bile acids in plasma (p<0.05). Moreover, changes in fecal bile acid concentrations were predominantly associated with altered Firmicutes. Finally, administration of vancomycin decreased peripheral insulin sensitivity (p<0.05). Amoxicillin did not affect any of these parameters. CONCLUSIONS: Oral administration of vancomycin significantly impacts host physiology by decreasing intestinal microbiota diversity, bile acid dehydroxylation and peripheral insulin sensitivity in subjects with metabolic syndrome. These data show that intestinal microbiota, particularly of the Firmicutes phylum contributes to bile acid and glucose metabolism in humans. This trial is registered at the Dutch Trial Register (NTR2566).

Role of p-glycoprotein expression and function in cystinotic renal proximal tubular cells.

P-glycoprotein (P-gp) is an ATP-dependent transporter localized at the apical membrane of the kidney proximal tubules, which plays a role in the efflux of cationic and amphipathic endogenous waste products and xenobiotics, such as drugs, into urine. Studies in mice deficient in P-gp showed generalized proximal tubular dysfunction similar to the phenotype of patients with cystinosis, an autosomal recessive disorder caused by mutations in the lysosomal cystine transporter cystinosin. Renal disease in cystinosis is characterized by generalized dysfunction of the apical proximal tubular influx transporters (so-called renal Fanconi syndrome) developing during infancy and gradually progressing towards end-stage renal disease before the 10th birthday in the majority of patients that are not treated with the cystine-depleting drug cysteamine. Here, we investigated whether the proximal tubular efflux transporter P-gp is affected in cystinosis and whether this might contribute to the development of renal Fanconi syndrome. We used conditionally immortalized (ci) proximal tubular epithelial cells (ciPTEC) derived from cystinotic patients and healthy volunteers. P-gp-mediated transport was measured by using the P-gp substrate calcein-AM in the presence and absence of the P-gp-inhibitor PSC833. P-gp activity was normal in cystinotic cells as compared to controls. Additionally, the effect of cysteamine on P-gp transport activity and phosphate uptake was determined; demonstrating increased P-gp activity in cystinotic cells, and further decrease of proximal tubular phosphate uptake. This observation is compatible with the persistence of renal Fanconi syndrome in vivo under cysteamine therapy. In summary, P-gp expression and activity are normal in cystinotic ciPTEC, indicating that P-gp dysfunction is not involved in the pathogenesis of cystinosis.