Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

Report from the HarmoSter study: inter-laboratory comparison of LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration. METHODS: Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation. RESULTS: Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from -5.6 to 12.3% for corticosterone, -14.6 to 12.4% for 11-deoxycortisol and -4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability. CONCLUSIONS: Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization.

[Hypoglycaemia in patients without diabetes mellitus]. / Hypoglykemie bij patiënten zonder diabetes mellitus.

A structured approach in the diagnostic process of hypoglycemia is important to find the right diagnosis. The first step is to recognize the symptoms of hypoglycemia, confirming the hypoglycemia during symptoms and dissolvement of complaints once the glucose level is restored to normal. This confirms the Whipple triad. The second step is to exclude common causes. The third, and most important, step is a diagnostic fasting test. Measurement of insulin and C-peptide during hypoglycemia will guide to exogenic or endogenic causes of hyperinsulinism. Targeted additional investigation is then required. Often the underlying cause is treatable. This justifies the need to measure a well-timed serum glucose when hypoglycemia is suspected to make a quick diagnosis.

Supercharging reagents in LC-MS/MS hormone analyses: Enhancing ionization, not limit of quantification.

One of the critical steps during LC-MS/MS hormone analyses that affects the sensitivity of the assay is the ionization process. Enhancing ionization efficiencies by the addition of supercharging reagents might be one way to improve sensitivity and reduce the limit of quantification (LOQ). Therefore, we investigated whether the addition of the supercharging reagents m-nitrobenzyl alcohol (m-NBA), sulfolane, propylene carbonate, and o-nitroanisole (o-NA) increased ionization efficiency and improved assay LOQ of insulin, oxytocin, sex steroids, and corticosteroids in test solutions. Additionally, the influence of the supercharging reagents was tested in serum samples after sample pretreatment to determine whether ionization would be enhanced similarly in routine analyses and, subsequently, lead to improved sensitivity. The screening experiments showed that the impact of the supercharging reagents varied for each hormone; although the addition of m-NBA increased the signal of all hormones, the other reagents only enhanced ionization efficiencies for some hormones. While the addition of 0.05 v/v% m-NBA and 0.05 v/v% o-NA did result in an increase in peak area in both test solutions and serum samples, it did not significantly improve the signal-to-noise ratio, as a simultaneous increase in noise was observed. In conclusion, even though supercharging reagents can enhance ionization efficiencies of hormones significantly, the addition of these reagents does not result in an improved LOQ for hormone measurements with LC-MS/MS.

Report from the HarmoSter study: impact of calibration on comparability of LC-MS/MS measurement of circulating cortisol, 17OH-progesterone and aldosterone.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. METHODS: Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. RESULTS: Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from -6.6 to 6.9%, -17.2 to 7.8% and -12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation. CONCLUSIONS: Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.

Vitamin D status in Armenian women: a stratified cross-sectional cluster analysis.

BACKGROUND: Vitamin D plays a critical role in skeletal development and maintenance, thus sufficiency is an important goal for public health programs. Given the absence of foods fortified in vitamin D in Armenia, we hypothesized that vitamin D insufficiency would be widespread. METHODS: We conducted a random modified cluster model survey of vitamin D status of women in the country. We measured 25-hydroxyvitamin D [25(OH)D] by liquid chromatography mass spectrometry in dried blood spot samples and utilized a questionnaire to assess lifestyle factors. RESULTS: In summer, we sampled 1206 participants from 40 communities in Armenia. Mean 25-OH D level among women aged 18-24 was 20 ± 8 ng/mL; aged 25-64 was 21 ± 7 ng/mL; and >65 was 18 ± 8 ng/mL. The country-wide mean of the entire female population was 20 ± 8 ng/mL. A majority (>54%) had 25-OH D levels <20 ng/mL with nearly 13% having 25-OH D levels <12 ng/mL. Participants who reported calcium, vitamin D, or multivitamin supplementation had higher levels of 25-OH D (p values 0.004, 0.0002, and 0.03 respectively) as did pre- vs. postmenopausal women (p = 0.01), pregnant vs. nonpregnant women (p = <0.0001), and women who had experienced a sunburn in the past year (p = 0.004). CONCLUSION: In Armenia, there is a high prevalence of vitamin D insufficiency. This information provides data that can be used to inform public health directives to address this pervasive threat to optimal health.

Beneficial effects of a plant-fish oil, slow carbohydrate diet on cardio-metabolic health exceed the correcting effects of metformin-pioglitazone in diabetic pigs fed a fast-food diet.

BACKGROUND: Lifestyle influences endocrine, metabolic and cardiovascular homeostasis. This study investigated the impact of diet and oral anti-diabetic medication on cardio-metabolic health in human-sized diabetic pigs. METHODS: After a growing pre-phase from ~30 to ~69 kg during which domestic pigs were fed either a low fat, low sucrose diet (group A) or a fast food-type diet elevated in lard (15%) and sucrose (40%) (group B), the pigs were subdivided in 5 groups (n = 7-8 pigs per group). Group 1, normal pigs from group A on a low fat, low sugar (L) pig diet and group 2, normal pigs from group B on a high lard (25%), sucrose-fructose (40%), cholesterol (1%) fast food-type (F) diet. Diabetes (D) was induced in group B pigs by streptozotocin and group 3 received the F diet (DF), group 4 received the F diet with Anti-diabetic medication metformin (2 g.day-1)-pioglitazone (40 mg.day-1) (DFA) and group 5 switched to a Plant-Fish oil (25%), Slowly digestible starch (40%) diet (DPFS). The F and PFS diets were identical for fat, carbohydrate and protein content but only differed in fat and carbohydrate composition. The 5 pig groups were followed up for 7 weeks until reaching ~120 kg. RESULTS: In normal pigs, the F diet predisposed to several abnormalities related to metabolic syndrome. Diabetes amplified the inflammatory and cardiometabolic abnormalities of the F diet, but both oral FA medication and the PFS diet partially corrected these abnormalities (mean±SEM) as follows: Fasting plasma TNF-ɑ (pg.ml-1) and NEFA (mmol.l-1) concentrations were high (p<0.02) in DF (193±55 and 0.79±0.16), intermediate in DFA (136±40 and 0.57±012) and low in DPFS pigs (107±31 and 0.48±0.19). Meal intolerance (response over fasting) for glucose and triglycerides (area under the curve, mmol.h-1) and for lactate (3-h postprandial, mmol.l-1) was high (p<0.03) in DF (489±131, 8.6±4.8 and 2.2±0.6), intermediate in DFA (276±145, 1.4±1.1 and 1.6±0.4) and low in DPFS (184±62, 0.7±1.8 and 0.1±0.1). Insulin-mediated glucose disposal (mg.kg-1.min-1) showed a numerical trend (p = NS): low in DF (6.9±2.2), intermediate in DFA (8.2±1.3) and high in DPFS pigs (10.4±2.7). Liver weight (g.kg-1 body weight) and liver triglyceride concentration (g.kg-1 liver) were high (p<0.001) in DF (23.8±2.0 and 69±14), intermediate in DFA (21.1±2.0 and 49±15) and low in DPFS pigs (16.4±0.7 and 13±2.0). Aorta fatty streaks were high (p<0.01) in DF (16.4±5.7%), intermediate in DFA (7.4±4.5%) and low in DPFS pigs (0.05±0.02%). CONCLUSION: This translational study using pigs with induced type 2 diabetes provides evidence that a change in nutritional life style from fast food to a plant-fish oil, slowly digestible starch diet can be more effective than sole anti-diabetic medication.

Optimizing oxytocin LC-MS/MS sensitivity by choosing the right column.

OBJECTIVE: Sensitivity is often an issue in bioanalytical LC-MS/MS applications. Commonly investigated parameters to improve it include additives to mobile phase, derivatization and sample-preparation. The nature of the column, however, is not frequently evaluated. DESIGN AND METHODS: The sensitivity is compared for 18 different reversed phase and 2 different HILIC columns using 2 different mobile phase compositions. Sensitivity was evaluated in terms of S/N for 1,5 pg oxytocin on column, using a scouting gradient. RESULTS: The measured signal to noise ranged from 55 to 1473, indicating a substantial difference in sensitivity. The most sensitive columns were the Synergi Hydro RP (reversed phase) and the Atlantis HILIC (HILIC). CONCLUSIONS: This study shows that choosing the right column contributes to the sensitivity of the method.

Solutions for hematocrit bias in dried blood spot hormone analysis.

Over the last years, dried blood spot (DBS) sampling has gained significant interest due to development of analytical techniques combined with DBS, the simplicity and low cost of the method. Despite its wide use, DBS sampling can lead to inaccurate results due to the impact of the hematocrit (Hct) on the analysis. Some analytes have shown to be hardly impacted by Hct values. However, in other cases, a significant impact of Hct is observed, which requires the use of alternative approaches to circumvent this issue. This review describes the possible impact of Hct-related bias in DBS sampling in the context of hormone analysis and discusses the different methodologies that can be used to overcome this bias to ensure accurate results.

Hematocrit and standardization in DBS analysis: A practical approach for hormones mainly present in the plasma fraction.

BACKGROUND AND AIM: Dried blood spot (DBS) sampling has many advantages over conventionally used blood samples, but is thought to suffer from hematocrit related issues. The aim of our research was to investigate whether reliable results can be obtained without bothering about hematocrit effects in DBS analysis of analytes that are mainly present in the plasma compartment. MATERIALS AND METHODS: Venous blood samples with variation in hematocrit and spotted volume were prepared. Spot diameter and 25-OH Vitamin D3 and testosterone concentrations were measured. Moreover, DBS and plasma concentrations of 25-OH Vitamin D3, testosterone and hematocrit were determined in random patient samples. RESULTS: DBS spot size was linearly related to hematocrit. Measured DBS concentrations of 25-OH Vitamin D3 and testosterone were independent of hematocrit and spotted volume. Determining the relation between plasma and DBS concentration resulted in a factor that can be used to convert DBS concentrations to standardized plasma concentrations. CONCLUSION: Addressing the hematocrit issue is not necessary for hormones that are mainly present in the plasma compartment. The relation between plasma and DBS concentration can be used to convert DBS concentrations to standardized plasma concentrations which makes interpretation of DBS concentrations easier.

The relation between postprandial glucagon-like peptide-1 release and insulin sensitivity before and after bariatric surgery in humans with class II/III obesity.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) receptor agonist treatment is beneficial for the human glucose metabolism, and GLP-1 secretion is greatly enhanced following Roux-en-Y gastric bypass (RYGB). OBJECTIVES: To elucidate the relationship between GLP-1 concentrations and insulin sensitivity in subjects with class II/III obesity without diabetes and to assess the relation between GLP-1 and the improvements in glucose metabolism following RYGB. SETTING: Clinical research facility in a university hospital. METHODS: We recruited 35 patients scheduled for RYGB and assessed their plasma GLP-1, insulin, and glucose responses to a high-fat mixed meal. Basal and insulin-mediated glucose fluxes were determined during a 2-step hyperinsulinemic-euglycemic clamp with stable isotope-labeled tracers. Out of 35 subjects, 10 were studied both before surgery and at 1 year of follow-up. RESULTS: Plasma GLP-1 increased following the high-fat mixed meal. Postprandial GLP-1 excursions correlated positively with hepatic and peripheral insulin sensitivity, but not with body mass index. At 1 year after RYGB, participants had lost 24% ± 6% of their body weight. Plasma GLP-1, insulin, and glucose levels peaked earlier and higher after the mixed meal. The positive association between the postprandial GLP-1 response and peripheral insulin sensitivity persisted. CONCLUSIONS: Postprandial GLP-1 concentrations correlate with insulin sensitivity in subjects with class II/III obesity without diabetes before and 1 year after RYGB. Increased GLP-1 signaling in postbariatric patients may, directly or indirectly, contribute to the observed improvements in insulin sensitivity and metabolic health.

Metabolite Profile of Treatment-Naive Metabolic Syndrome Subjects in Relation to Cardiovascular Disease Risk.

Metabolic syndrome (MetSyn) is an important risk factor for type 2 diabetes and cardiovascular diseases (CVD). This study aimed to find distinct plasma metabolite profiles between insulin-resistant and non-insulin resistant subjects with MetSyn and evaluate if MetSyn metabolite profiles are related to CVD risk and lipid fluxes. In a cross-sectional study, untargeted metabolomics of treatment-naive males with MetSyn (n = 132) were analyzed together with clinical parameters. In a subset of MetSyn participants, CVD risk was calculated using the Framingham score (n = 111), and lipolysis (n = 39) was measured by a two-step hyperinsulinemic euglycemic clamp using [1,1,2,3,3-2H5] glycerol to calculate lipolysis suppression rates. Peripheral insulin resistance was related to fatty acid metabolism and glycerolphosphorylcholine. Interestingly, although insulin resistance is considered to be a risk factor for CVD, we observed that there was little correspondence between metabolites associated with insulin resistance and metabolites associated with CVD risk. The latter mainly belonged to the androgenic steroid, fatty acid, phosphatidylethanolamine, and phophatidylcholine pathways. These data provide new insights into metabolic changes in mild MetSyn pathophysiology and MetSyn CVD risk related to lipid metabolism. Prospective studies may focus on the pathophysiological role of the here-identified biomarkers.

We need to talk about the analytical performance of our laboratory developed clinical LC-MS/MS tests, and start separating the wheat from the chaff.

With the upcoming EU regulation on the use of in-vitro diagnostic devices, a critical evaluation of the current status of our in-house developed LC-MS/MS methods is timely and of great relevance. Recently, much attention has been devoted to the need for better specification of analytical and clinical performance. Appropriate reporting of the actual achieved analytical performance is an important determinant of the clinical performance and subsequent clinical effectiveness of a test. We advocate for the application of CLSI C62-A guidelines for method validation and suggest some adaptations for analytical validation of in-house developed LC-MS/MS methods for endogenous substances. Additionally, we underline the importance of well-equipped reviewers and standardized method description, including the presentation of figural evidence of obtained method performance. Achieving this ensures future quality of our in-house developed LC-MS/MS methods.

Hepatic Insulin Resistance Is Not Pathway Selective in Humans With Nonalcoholic Fatty Liver Disease.

OBJECTIVE: Both glucose and triglyceride production are increased in type 2 diabetes and nonalcoholic fatty liver disease (NAFLD). For decades, the leading hypothesis to explain these paradoxical observations has been selective hepatic insulin resistance wherein insulin drives de novo lipogenesis (DNL) while failing to suppress glucose production. Here, we aimed to test this hypothesis in humans. RESEARCH DESIGN AND METHODS: We recruited obese subjects who met criteria for bariatric surgery with (n = 16) or without (n = 15) NAFLD and assessed 1) insulin-mediated regulation of hepatic and peripheral glucose metabolism using hyperinsulinemic-euglycemic clamps with [6,6-2H2]glucose, 2) fasting and carbohydrate-driven hepatic DNL using deuterated water (2H2O), and 3) hepatocellular insulin signaling in liver biopsy samples collected during bariatric surgery. RESULTS: Compared with subjects without NAFLD, those with NAFLD demonstrated impaired insulin-mediated suppression of glucose production and attenuated-not increased-glucose-stimulated/high-insulin lipogenesis. Fructose-stimulated/low-insulin lipogenesis was intact. Hepatocellular insulin signaling, assessed for the first time in humans, exhibited a proximal block in insulin-resistant subjects: Signaling was attenuated from the level of the insulin receptor through both glucose and lipogenesis pathways. The carbohydrate-regulated lipogenic transcription factor ChREBP was increased in subjects with NAFLD. CONCLUSIONS: Acute increases in lipogenesis in humans with NAFLD are not explained by altered molecular regulation of lipogenesis through a paradoxical increase in lipogenic insulin action; rather, increases in lipogenic substrate availability may be the key.

Infusion of donor feces affects the gut-brain axis in humans with metabolic syndrome.

OBJECTIVE: Increasing evidence indicates that intestinal microbiota play a role in diverse metabolic processes via intestinal butyrate production. Human bariatric surgery data suggest that the gut-brain axis is also involved in this process, but the underlying mechanisms remain unknown. METHODS: We compared the effect of fecal microbiota transfer (FMT) from post-Roux-en-Y gastric bypass (RYGB) donors vs oral butyrate supplementation on (123I-FP-CIT-determined) brain dopamine transporter (DAT) and serotonin transporter (SERT) binding as well as stable isotope-determined insulin sensitivity at baseline and after 4 weeks in 24 male and female treatment-naïve metabolic syndrome subjects. Plasma metabolites and fecal microbiota were also determined at these time points. RESULTS: We observed an increase in brain DAT after donor FMT compared to oral butyrate that reduced this binding. However, no effect on body weight and insulin sensitivity was demonstrated after post-RYGB donor feces transfer in humans with metabolic syndrome. Increases in fecal levels of Bacteroides uniformis were significantly associated with an increase in DAT, whereas increases in Prevotella spp. showed an inverse association. Changes in the plasma metabolites glycine, betaine, methionine, and lysine (associated with the S-adenosylmethionine cycle) were also associated with altered striatal DAT expression. CONCLUSIONS: Although more and larger studies are needed, our data suggest a potential gut microbiota-driven modulation of brain dopamine and serotonin transporters in human subjects with obese metabolic syndrome. These data also suggest the presence of a gut-brain axis in humans that can be modulated. NTR REGISTRATION: 4488.

Correlation of plasma metabolites with glucose and lipid fluxes in human insulin resistance.

OBJECTIVE: Insulin resistance develops prior to the onset of overt type 2 diabetes, making its early detection vital. Direct accurate evaluation is currently only possible with complex examinations like the stable isotope-based hyperinsulinemic euglycemic clamp (HIEC). Metabolomic profiling enables the detection of thousands of plasma metabolites, providing a tool to identify novel biomarkers in human obesity. DESIGN: Liquid chromatography mass spectrometry-based untargeted plasma metabolomics was applied in 60 participants with obesity with a large range of peripheral insulin sensitivity as determined via a two-step HIEC with stable isotopes [6,6-2H2]glucose and [1,1,2,3,3-2H5]glycerol. This additionally enabled measuring insulin-regulated lipolysis, which combined with metabolomics, to the knowledge of this research group, has not been reported on before. RESULTS: Several plasma metabolites were identified that significantly correlated with glucose and lipid fluxes, led by plasma (gamma-glutamyl)citrulline, followed by betaine, beta-cryptoxanthin, fructosyllysine, octanylcarnitine, sphingomyelin (d18:0/18:0, d19:0/17:0) and thyroxine. Subsequent machine learning analysis showed that a panel of these metabolites derived from a number of metabolic pathways may be used to predict insulin resistance, dominated by non-essential amino acid citrulline and its metabolite gamma-glutamylcitrulline. CONCLUSION: This approach revealed a number of plasma metabolites that correlated reasonably well with glycemic and lipolytic flux parameters, measured using gold standard techniques. These metabolites may be used to predict the rate of glucose disposal in humans with obesity to a similar extend as HOMA, thus providing potential novel biomarkers for insulin resistance.

Treatment with Anaerobutyricum soehngenii: a pilot study of safety and dose-response effects on glucose metabolism in human subjects with metabolic syndrome.

Dysbiosis of the intestinal microbiota has been implicated in insulin resistance, although evidence regarding causality in humans is scarce. We performed a phase I/II dose-finding and safety study on the effect of oral intake of the anaerobic butyrogenic strain Anaerobutyricum soehngenii on glucose metabolism in 24 subjects with metabolic syndrome. We found that treatment with A. soehngenii was safe and observed a significant correlation between the measured fecal abundance of administered A. soehngenii and improvement in peripheral insulin sensitivity after 4 weeks of treatment. This was accompanied by an altered microbiota composition and a change in bile acid metabolism. Finally, we show that metabolic response upon administration of A. soehngenii (defined as improved insulin sensitivity 4 weeks after A. soehngenii intake) is dependent on microbiota composition at baseline. These data in humans are promising, but additional studies are needed to reproduce our findings and to investigate long-term effects, as well as other modes of delivery.

RBP4 increases lipolysis in human adipocytes and is associated with increased lipolysis and hepatic insulin resistance in obese women.

Retinol-binding protein-4 (RBP4) is elevated in serum and adipose tissue (AT) in obesity-induced insulin resistance and correlates inversely with insulin-stimulated glucose disposal. But its role in insulin-mediated suppression of lipolysis, free fatty acids (FFA), and endogenous glucose production (EGP) in humans is unknown. RBP4 mRNA or protein levels were higher in liver, subcutaneous adipose tissue (SAT), and visceral adipose tissue (VAT) in morbidly obese subjects undergoing Roux-en-Y gastric bypass surgery compared to lean controls undergoing elective laparoscopic cholecystectomy. RBP4 mRNA expression in SAT correlated with the expression of several macrophage and other inflammation markers. Serum RBP4 levels correlated inversely with glucose disposal and insulin-mediated suppression of lipolysis, FFA, and EGP. Mechanistically, RBP4 treatment of human adipocytes in vitro directly stimulated basal lipolysis. Treatment of adipocytes with conditioned media from RBP4-activated macrophages markedly increased basal lipolysis and impaired insulin-mediated lipolysis suppression. RBP4 treatment of macrophages increased TNFα production. These data suggest that elevated serum or adipose tissue RBP4 levels in morbidly obese subjects may cause hepatic and systemic insulin resistance by stimulating basal lipolysis and by activating macrophages in adipose tissue, resulting in release of pro-inflammatory cytokines that impair lipolysis suppression. While we have demonstrated this mechanism in human adipocytes in vitro, and correlations from our flux studies in humans strongly support this, further studies are needed to determine whether this mechanism explains RBP4-induced insulin resistance in humans.

The effect of dapagliflozin on apolipoprotein B and glucose fluxes in patients with type 2 diabetes and well-controlled plasma LDL cholesterol.

AIM: To dissect the effects of the sodium-glucose linked transporter 2 inhibitor dapagliflozin on lipid metabolism and assess whether these effects could potentially offset cardiovascular benefit with this drug-class. MATERIALS AND METHODS: We assessed the effect of dapagliflozin on lipid metabolism in 11 adults with uncomplicated type 2 diabetes. After 4 weeks of statin wash-out and 4 weeks of rosuvastatin 10 mg treatment, participants were treated with dapagliflozin 10 mg once-daily for 5 weeks. Before and after dapagliflozin, plasma lipids were measured and very low-density lipoprotein (VLDL)-1 and VLDL-2 apolipoprotein (Apo)B fluxes were assessed using (5.5.5-2 H3 )-leucine tracer infusion. In addition, hepatic and peripheral insulin sensitivity as well as insulin-mediated inhibition of peripheral lipolysis were measured during a two-step hyperinsulinemic-euglycaemic clamp using (6,6-2 H2 )-glucose and (1,1,2,3,3-2 H5 )-glycerol tracers. RESULTS: Rosuvastatin decreased all plasma lipids significantly: total cholesterol from 4.5 (3.2-6.2) to 3.1 (2.5-3.8) mmol/L, LDL cholesterol from 2.6 (1.7-3.4) to 1.5 (1.1-2.2) mmol/L, HDL cholesterol from 1.34 (0.80-2.02) to 1.19 (0.74-1.89) mmol/L and triglycerides from 0.92 (0.31-3.91) to 0.79 (0.32-2.10) mmol/L. The addition of dapaglifozin to rosuvastatin did not raise either LDL cholesterol or total cholesterol, and only increased HDL cholesterol by 0.08 (-0.03-0.13) mmol/L (P = 0.03). In line with this, dapagliflozin did not affect VLDL-1 or VLDL-2 ApoB fluxes. Fasting endogenous glucose production tended to increase by 0.9 (-3.4-3.1) µmol kg-1 min-1 (P = 0.06), but no effect on hepatic and peripheral insulin sensitivity or on peripheral lipolysis was observed. CONCLUSIONS: Dapagliflozin has no effect on plasma LDL-cholesterol levels or VLDL-apoB fluxes in the context of optimal lipid-lowering treatment, which will thus not limit cardiovascular benefit when lipids are adequately controlled.

Downregulation of Type 3 Deiodinase in the Hypothalamus During Inflammation.

Background: Inflammation is associated with marked changes in cellular thyroid hormone (TH) metabolism in triiodothyronine (T3) target organs. In the hypothalamus, type 2 deiodinase (D2), the main T3 producing enzyme, increases upon inflammation, leading to an increase in local T3 availability, which in turn decreases thyrotropin releasing hormone expression in the paraventricular nucleus. Type 3 deiodinase (D3), the T3 inactivating enzyme, decreases during inflammation, which might also contribute to the increased T3 availability in the hypothalamus. While it is known that D2 is regulated by nuclear factor κB (NF-κB) during inflammation, the underlying mechanisms of D3 regulation are unknown. Therefore, the aim of the present study was to investigate inflammation-induced D3 regulation using in vivo and in vitro models. Methods: Mice were injected with a sublethal dose of bacterial endotoxin (lipopolysaccharide [LPS]) to induce a systemic acute-phase response. A human neuroblastoma (SK-N-AS) cell line was used to test the involvement of the thyroid hormone receptor alpha 1 (TRα1) as well as the activator protein-1 (AP-1) and NF-κB inflammatory pathways in the inflammation-induced decrease of D3. Results: D3 expression in the hypothalamus was decreased 24 hours after LPS injection in mice. This decrease was similar in mice lacking the TRα. Incubation of SK-N-AS cells with LPS robustly decreased both D3 mRNA expression and activity. This led to increased intracellular T3 concentrations. The D3 decrease was prevented when NF-κB or AP-1 was inhibited. TRα1 mRNA expression decreased in SK-N-AS cells incubated with LPS, but knockdown of the TRα in SK-N-AS cells did not prevent the LPS-induced D3 decrease. Conclusions: We conclude that the inflammation-induced D3 decrease in the hypothalamus is mediated by the inflammatory pathways NF-κB and AP-1, but not TRα1. Furthermore, the observed decrease modulates intracellular T3 concentrations. Our results suggest a concerted action of inflammatory modulators to regulate both hypothalamic D2 and D3 activities to increase the local TH concentrations.