The effects of the ß1-adrenergic receptor antagonist bisoprolol administration on mirabegron-stimulated human brown adipose tissue thermogenesis.

AIM: Pharmacological stimulation of human brown adipose tissue (BAT) has been hindered by ineffective activation or undesirable off-target effects. Oral administration of the maximal allowable dose of mirabegron (200 mg), a ß3-adrenergic receptor (ß3-AR) agonist, has been effective in stimulating BAT thermogenesis and whole-body energy expenditure. However, this has been accompanied by undesirable cardiovascular effects. Therefore, we hypothesized that combining mirabegron with a ß1-AR antagonist could suppress these unwanted effects and increase the stimulation of the ß3-AR and ß2-AR in BAT. METHODS: We performed a randomized crossover trial (NCT04823442) in 8 lean men. Mirabegron (200 mg) was administered orally with or without the ß1-AR antagonist bisoprolol (10 mg). Dynamic [11C]-acetate and 2-deoxy-2-[18F]fluoro-d-glucose PET/CT scans were performed sequentially after oral administration of mirabegron ± bisoprolol. RESULTS: Compared to room temperature, mirabegron alone increased BAT oxidative metabolism (0.84 ± 0.46 vs. 1.79 ± 0.91 min-1, p = 0.0433), but not when combined with bisoprolol. The metabolic rate of glucose in BAT, measured using [18F]FDG PET, was significantly higher with mirabegron than mirabegron with bisoprolol (24 ± 10 vs. 16 ± 8 nmol/g/min, p = 0.0284). Bisoprolol inhibited the mirabegron-induced increase in systolic blood pressure and heart rate. CONCLUSION: The administration of bisoprolol decreases the adverse cardiovascular effects of mirabegron. However, the provided dose also blunted the mirabegron-stimulated increase in BAT lipolysis, thermogenesis, and glucose uptake. The attenuation in BAT blood flow induced by the large dose of bisoprolol may have limited BAT thermogenesis.

High-fructose feeding suppresses cold-stimulated brown adipose tissue glucose uptake independently of changes in thermogenesis and the gut microbiome.

Diets rich in added sugars are associated with metabolic diseases, and studies have shown a link between these pathologies and changes in the microbiome. Given the reported associations in animal models between the microbiome and brown adipose tissue (BAT) function, and the alterations in the microbiome induced by high-glucose or high-fructose diets, we investigated the potential causal link between high-glucose or -fructose diets and BAT dysfunction in humans. Primary outcomes are changes in BAT cold-induced thermogenesis and the fecal microbiome (clinicaltrials.gov, NCT03188835). We show that BAT glucose uptake, but not thermogenesis, is impaired by a high-fructose but not high-glucose diet, in the absence of changes in the gastrointestinal microbiome. We conclude that decreased BAT glucose metabolism occurs earlier than other pathophysiological abnormalities during fructose overconsumption in humans. This is a potential confounding factor for studies relying on 18F-FDG to assess BAT thermogenesis.

Total Postprandial Hepatic Nonesterified and Dietary Fatty Acid Uptake Is Increased and Insufficiently Curbed by Adipose Tissue Fatty Acid Trapping in Prediabetes With Overweight.

Excessive lean tissue uptake of fatty acids (FAs) is important in the development of insulin resistance and may be caused by impaired dietary FA (DFA) storage and/or increased nonesterified FA (NEFA) flux from adipose tissue intracellular lipolysis. Cardiac and hepatic total postprandial FA uptake of NEFA+DFA has, however, never been reported in prediabetes with overweight. In this study, 20 individuals with impaired glucose tolerance (IGT) and 19 participants with normal glucose tolerance (NGT) and normal fasting glucose underwent postprandial studies with whole-body positron emission tomography/computed tomography (PET/CT) with oral [18F]fluoro-thia-heptadecanoic acid and dynamic PET/CT with intravenous [11C]palmitate. Hepatic (97 [range 36-215] mmol/6 h vs. 68 [23-132] mmol/6 h, P = 0.03) but not cardiac (11 [range 4-24] mmol/6 h vs. 8 [3-20] mmol/6 h, P = 0.09) uptake of most sources of postprandial FA (NEFA + DFA uptake) integrated over 6 h was higher in IGT versus NGT. DFA accounted for lower fractions of total cardiac (21% [5-47] vs. 25% [9-39], P = 0.08) and hepatic (19% [6-52] vs. 28% [14-50], P = 0.04) uptake in IGT versus NGT. Increased adipose tissue DFA trapping predicted lower hepatic DFA uptake and was associated with higher total cardiac FA uptake. Hence, enhanced adipose tissue DFA trapping in the face of increased postprandial NEFA flux is insufficient to fully curb increased postprandial lean organ FA uptake in prediabetes with overweight (ClinicalTrials.gov; NCT02808182).

Increased postprandial nonesterified fatty acid efflux from adipose tissue in prediabetes is offset by enhanced dietary fatty acid adipose trapping.

The mechanism of increased postprandial nonesterified fatty acid (NEFA) appearance in the circulation in impaired glucose tolerance (IGT) is due to increased adipose tissue lipolysis but could also be contributed to by reduced adipose tissue (AT) dietary fatty acid (DFA) trapping and increased "spillover" into the circulation. Thirty-one subjects with IGT (14 women, 17 men) and 29 with normal glucose tolerance (NGT, 15 women, 14 men) underwent a meal test with oral and intravenous palmitate tracers and the oral [18F]-fluoro-thia-heptadecanoic acid positron emission tomography method. Postprandial palmitate appearance (Rapalmitate) was higher in IGT versus NGT (P < 0.001), driven exclusively by Rapalmitate from obesity-associated increase in intracellular lipolysis (P = 0.01), as Rapalmitate from DFA spillover was not different between the groups (P = 0.19) and visceral AT DFA trapping was even higher in IGT versus NGT (P = 0.02). Plasma glycerol appearance was lower in IGT (P = 0.01), driven down by insulin resistance and increased insulin secretion. Thus, we found higher AT DFA trapping, limiting spillover to lean organs and in part offsetting the increase in Rapalmitate from intracellular lipolysis. Whether similar findings occur in frank diabetes, a condition also characterized by insulin resistance but relative insulin deficiency, requires further investigation (Clinicaltrials.gov: NCT04088344, NCT02808182).NEW & NOTEWORTHY We found higher adipose tissue dietary fatty acid trapping, limiting spillover to lean organs, that in part offsets the increase in appearance rate of palmitate from intracellular lipolysis in prediabetes. These results point to the adaptive nature of adipose tissue trapping and dietary fatty acid spillover as a protective mechanism against excess obesity-related palmitate appearance rate from intracellular adipose tissue lipolysis.

Human Brown Adipocyte Thermogenesis Is Driven by ß2-AR Stimulation.

Stimulation of brown adipose tissue (BAT) thermogenesis in humans has emerged as an attractive target to improve metabolic health. Pharmacological stimulations targeting the ß3-adrenergic receptor (ß3-AR), the adrenergic receptor believed to mediate BAT thermogenesis, have historically performed poorly in human clinical trials. Here we report that, in contrast to rodents, human BAT thermogenesis is not mediated by the stimulation of ß3-AR. Oral administration of the ß3-AR agonist mirabegron only elicited increases in BAT thermogenesis when ingested at the maximal allowable dose. This led to off-target binding to ß1-AR and ß2-AR, thereby increasing cardiovascular responses and white adipose tissue lipolysis, respectively. ADRB2 was co-expressed with UCP1 in human brown adipocytes. Pharmacological stimulation and inhibition of the ß2-AR as well as knockdown of ADRB1, ADRB2, or ADRB3 in human brown adipocytes all confirmed that BAT lipolysis and thermogenesis occur through ß2-AR signaling in humans (ClinicalTrials.govNCT02811289).

Seven-day overfeeding enhances adipose tissue dietary fatty acid storage and decreases myocardial and skeletal muscle dietary fatty acid partitioning in healthy subjects.

Increased myocardial partitioning of dietary fatty acids (DFA) and decreased left ventricular (LV) function is associated with insulin resistance in prediabetes. We hypothesized that enhanced myocardial DFA partitioning and reduced LV function might be induced concomitantly with reduced insulin sensitivity upon a 7-day hypercaloric (+50% in caloric intake), high-saturated fat (~11%energy), and simple carbohydrates (~54%energy) diet (HIGHCAL) versus an isocaloric diet (ISOCAL) with a moderate amount of saturated fat (~8%energy) and carbohydrates (~50%energy). Thirteen healthy subjects (7 men/6 women) underwent HIGHCAL versus ISOCAL in a randomized crossover design, with organ-specific DFA partitioning and LV function measured using the oral 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid and [11C]acetate positron emission tomography methods at the end of both interventions. HIGHCAL induced a decrease in insulin sensitivity indexes with no significant change in body composition. HIGHCAL led to increased subcutaneous abdominal (+4.2 ± 1.6%, P < 0.04) and thigh (+2.4 ± 1.2%, P < 0.08) adipose tissue storage and reduced cardiac (-0.31 ± 0.11 mean standard uptake value [(SUV), P < 0.03] and skeletal muscle (-0.17 ± 0.08 SUV, P < 0.05) DFA partitioning without change in LV function. We conclude that early increase in adipose tissue DFA storage protects the heart and skeletal muscles from potential deleterious effects of DFA.

Plasma Palmitoyl-Carnitine (AC16:0) Is a Marker of Increased Postprandial Nonesterified Incomplete Fatty Acid Oxidation Rate in Adults With Type 2 Diabetes.

OBJECTIVES: Enhanced mitochondrial fatty acid utilization is known to increase radical oxidative stress and induce insulin resistance. An increased level of plasma acylcarnitine (AC) has been proposed to indicate mitochondrial energy substrate overload, a possible mechanism leading to insulin resistance. The aim of our study was to determine fasting and postprandial plasma acetyl-carnitine (AC2:0), palmitoyl-carnitine (AC16:0), oleoyl-carnitine (AC18:1) and linoleoyl-carnitine (AC18:2) levels and their relationships with plasma nonesterified fatty acid appearance and oxidation rates and insulin sensitivity in participants with type 2 diabetes and normoglycemic offspring of 2 parents with type 2 diabetes (FH+) compared to healthy participants without family histories of type 2 diabetes (FH-). METHODS: All participants underwent 3 metabolic protocols: 1) a euglycemic hyperinsulinemic clamp at fasting; 2) a 6-hour steady-state oral standard liquid meal and 3) an identical 6-hour steady-state meal intake study with a euglycemic hyperinsulinemic clamp. AC levels were measured by liquid chromatography with tandem mass spectrometry, and fatty acid oxidation (FAO) rates were measured by stable isotopic tracer techniques with indirect respiratory calorimetry. RESULTS: During the insulin clamp at fasting, AC16:0 was significantly higher in the group with type 2 diabetes vs. FH- (p<0.05). In the postprandial state, AC2:0, AC16:0 and AC18:1 decreased significantly, but this reduction was blunted in type 2 diabetes, even during normalization of postprandial glucose levels during the insulin clamp. Fasting AC16:0 correlated with FAO (ρ=+0.604; p=0.0002); triacylglycerol (ρ=+0.427; p<0.02) and waist circumference (ρ=+0.416; p=0.02). CONCLUSIONS: Spillover of AC occurs in type 2 diabetes but is not fully established in FH+. AC16:0 can be a useful biomarker of excessive FAO.

Performance characteristics of a straight versus bent 25-gauge vitrector.

BACKGROUND AND OBJECTIVE: During vitrectomy in phakic patients, lens damage can occur while shaving the peripheral vitreous. Modifying the vitrector with a slight bend can diminish this risk. MATERIALS AND METHODS: To assess whether the bent vitrector performs to standard, the cutting and aspiration functionality of a straight and bent cutter was tested on both the vitreous from an enucleated porcine eye and balanced salt solution. Data were analyzed using a paired two-tailed t-test for comparison of two small sample means. RESULTS: The average time for removal of vitreous was 19.32 seconds with the straight cutter and 19.26 seconds for the bent cutter. There was no statistically significant difference between the removal rates (P = .87). The average time for aspiration of balanced salt solution was 14.3 seconds with the straight tip and 14.16 seconds with the bent tip. There was no statistically significant difference between the aspiration rates (P = .55). CONCLUSION: Both unmodified and bent vitrectors demonstrate an equally efficient rate of removal of vitreous and balanced salt solution in vitro.

A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding.

BACKGROUND: Streptomyces sp. N174 chitosanase (CsnN174), a member of glycoside hydrolases family 46, is one of the most extensively studied chitosanases. Previous studies allowed identifying several key residues of this inverting enzyme, such as the two catalytic carboxylic amino acids as well as residues that are involved in substrate binding. In spite of the progress in understanding the catalytic mechanism of this chitosanase, the function of some residues highly conserved throughout GH46 family has not been fully elucidated. This study focuses on one of such residues, the arginine 42. RESULTS: Mutation of Arg42 into any other amino acid resulted in a drastic loss of enzyme activity. Detailed investigations of R42E and R42K chitosanases revealed that the mutant enzymes are not only impaired in their catalytic activity but also in their mode of interaction with the substrate. Mutated enzymes were more sensitive to substrate inhibition and were altered in their pattern of activity against chitosans of various degrees of deacetylation. Our data show that Arg42 plays a dual role in CsnN174 activity. CONCLUSIONS: Arginine 42 is essential to maintain the enzymatic function of chitosanase CsnN174. We suggest that this arginine is influencing the catalytic nucleophile residue and also the substrate binding mode of the enzyme by optimizing the electrostatic interaction between the negatively charged carboxylic residues of the substrate binding cleft and the amino groups of GlcN residues in chitosan.

Biochemical and molecular characterization of a thermostable chitosanase produced by the strain Paenibacillus sp. 1794 newly isolated from compost.

Chitosan raises a great interest among biotechnologists due to its potential for applications in biomedical or environmental fields. Enzymatic hydrolysis of chitosan is a recognized method allowing control of its molecular size, making possible its optimization for a given application. During the industrial hydrolysis process of chitosan, viscosity is a major problem; which can be circumvented by raising the temperature of the chitosan solution. A thermostable chitosanase is compatible with enzymatic hydrolysis at higher temperatures thus allowing chitosan to be dissolved at higher concentrations. Following an extensive micro-plate screening of microbial isolates from various batches of shrimp shells compost, the strain 1794 was characterized and shown to produce a thermostable chitosanase. The isolate was identified as a novel member of the genus Paenibacillus, based on partial 16S rDNA and rpoB gene sequences. Using the chitosanase (Csn1794) produced by this strain, a linear time course of chitosan hydrolysis has been observed for at least 6 h at 70 °C. Csn1794 was purified and its molecular weight was estimated at 40 kDa by SDS-PAGE. Optimum pH was about 4.8, the apparent Km and the catalytic constant kcat were 0.042 mg/ml and 7,588 min⁻¹, respectively. The half-life of Csn1794 at 70 °C in the presence of chitosan substrate was >20 h. The activity of chitosanase 1794 varied little with the degree of N-acetylation of chitosan. The enzyme also hydrolyzed carboxymethylcellulose but not chitin. Chitosan or cellulose-derived hexasaccharides were cleaved preferentially in a symmetrical way ("3+3") but hydrolysis rate was much faster for (GlcN)6 than (Glc)6. Gene cloning and sequencing revealed that Csn1794 belongs to family 8 of glycoside hydrolases. The enzyme should be useful in biotechnological applications of chitosan hydrolysis, dealing with concentrated chitosan solutions at high temperatures.

Quantitative fluorometric analysis of the protective effect of chitosan on thermal unfolding of catalytically active native and genetically-engineered chitosanases.

We have taken advantage of the intrinsic fluorescence properties of chitosanases to rapidly and quantitatively evaluate the protective effect of chitosan against thermal denaturation of chitosanases. The studies were done using wild type chitosanases N174 produced by Streptomyces sp. N174 and SCO produced by Streptomyces coelicolor A3(2). In addition, two mutants of N174 genetically engineered by single amino acid substitutions (A104L and K164R) and one "consensus" (N174-CONS) chitosanase designed by multiple amino acid substitutions of N174 were analyzed. Chitosan used had a weight average molecular weight (Mw) of 220 kDa and was 85% deacetylated. Results showed a pH and concentration-dependent protective effect of chitosan in all the cases. However, the extent of thermal protection varied depending on chitosanases, suggesting that key amino acid residues contributed to resistance to heat denaturation. The transition temperatures (T(m)) of N174 were 54 degrees C and 69.5 degrees C in the absence and presence (6 g/l) of chitosan, respectively. T(m) were increased by 11.6 degrees C (N174-CONS), 13.8 degrees C (CSN-A104L), 15.6 degrees C (N174-K164R) and 25.2 degrees C (SCO) in the presence of chitosan (6 g/l). The thermal protective effect was attributed to an enzyme-ligand thermostabilization mechanism since it was not mimicked by the presence of anionic (carboxymethyl cellulose, heparin) or cationic (polyethylene imine) polymers, polyhydroxylated (glycerol, sorbitol) compounds or inorganic salts. Furthermore, the data from fluorometry experiments were in agreement with those obtained by analysis of reaction time-courses performed at 61 degrees C in which case CSN-A104L was rapidly inactivated whereas N174, N174-CONS and N174-K164R remained active over a reaction time of 90 min. This study presents evidence that (1) the fluorometric determination of T(m) in the presence of chitosan is a reliable technique for a rapid assessment of the thermal behavior of chitosanases, (2) it is applicable to structure-function studies of mutant chitosanases and, (3) it can be useful to provide an insight into the mechanism by which mutations can influence chitosanase stability.