The anti-inflammatory agent 5-ASA reduces the level of specific tsRNAs in sperm cells of high-fat fed C57BL/6J mouse sires and improves glucose tolerance in female offspring.

INTRODUCTION: The prevalence of obesity and associated comorbidities have increased to epidemic proportions globally. Paternal obesity is an independent risk factor for developing obesity and type 2 diabetes in the following generation, and growing evidence suggests epigenetic inheritance as a mechanism for this predisposition. How and why obesity induces epigenetic changes in sperm cells remain to be clarified in detail. Yet, recent studies show that alterations in sperm content of transfer RNA-derived small RNAs (tsRNAs) can transmit the effects of paternal obesity to offspring. Obesity is closely associated with low-grade chronic inflammation. Thus, we evaluated whether the anti-inflammatory agent 5-aminosalicylic acid (5-ASA) could intervene in the transmission of epigenetic inheritance of paternal obesity by reducing the inflammatory state in obese fathers. METHODS: Male C57BL/6JBomTac mice were either fed a high-fat diet or a high-fat diet with 5-ASA for ten weeks before mating. The offspring metabolic phenotype was evaluated, and spermatozoa from sires were isolated for assessment of specific tsRNAs levels. RESULTS: 5-ASA intervention reduced the levels of Glu-CTC tsRNAs in sperm cells and improved glucose tolerance in female offspring fed a chow diet. Paternal high-fat diet-induced obesity per se had only a moderate impact on the metabolic phenotype of both male and female offspring in our setting. CONCLUSION: The results indicate that the low-grade inflammatory response associated with obesity may be an important factor in epigenetic inheritance of paternal obesity.

Lot variation and inter-device differences contribute to poor analytical performance of the DCA Vantage™ HbA1c POCT instrument in a true clinical setting.

OBJECTIVES: The glycated haemoglobin fraction A1c (HbA1c) is widely used in the management of diabetes mellitus, and the Siemens DCA Vantage™ point-of-care testing (POCT) instrument offers rapid HbA1c results even far from a clinical laboratory. However, the analytical performance has been questioned, and not much is known about effects of changing reagent lot, instrument and operator. We therefore compared the analytical performance of the DCA Vantage™ with established routine methods (Tosoh G8/G11 ion exchange HPLC) in a true clinical setting at two Danish hospitals. METHODS: We extracted all routine clinical HbA1c results incidentally drawn from the same patient within 48 h (n=960 pairs) and evaluated the effect of reagent lot, operator and instrument. We also performed a prospective method comparison in our diabetes out-patient clinic (n=97). RESULTS: The critical difference (CD) between two POCT results varied between 5.14 and 6.61 mmol/mol (0.47-0.55%), and the analytical imprecision of the DCA Vantage™ (CVA) was >3%. Significant effect of reagent lot and inter-instrument differences were found, whereas no effect of operator was seen. CONCLUSIONS: The DCA Vantage™ HbA1c analysis does not fulfil the prevailing analytical performance specifications, but rigorous validation of new reagent lots and continuous recalibration of instruments may potentially improve the precision substantially. Our findings, therefore, clearly emphasise the necessity of a close collaboration between clinicians and laboratory professionals in the POCT field. Finally, POCT HbA1c results should always be interpreted together with other measures of glycaemic control to avoid inappropriate change of patient treatments due to measurement uncertainty.

Gastric Emptying Time and Volume of the Small Intestine as Objective Markers in Patients With Symptoms of Diabetic Enteropathy.

BACKGROUND/AIMS: Patients with diabetes mellitus (DM) often suffer from gastrointestinal (GI) symptoms, but these correlate poorly to established objective GI motility measures. Our aim is to perform a detailed evaluation of potential measures of gastric and small intestinal motility in patients with DM type 1 and severe GI symptoms. METHODS: Twenty patients with DM and 20 healthy controls (HCs) were included. GI motility was examined with a 3-dimensional-Transit capsule, while organ volumes were determined by CT scans. RESULTS: Patients with DM and HCs did not differ with regard to median gastric contraction frequency (DM: 3.0 contractions/minute [interquartile range {IQR}, 2.9-3.0]; HCs: 2.9 [IQR, 2.8-3.1]; P = 0.725), amplitude of gastric contractions (DM: 9 mm [IQR, 8-11]; HCs: 11 mm (IQR, 9-12); P = 0.151) or fasting volume of the stomach wall (DM: 149 cm3 [IQR, 112-187]; HCs: 132 cm3 [IQR, 107-154]; P = 0.121). Median gastric emptying time was prolonged in patients (DM: 3.3 hours [IQR, 2.6-4.6]; HCs: 2.4 hours [IQR, 1.8-2.7]; P = 0.002). No difference was found in small intestinal transit time (DM: 5 hours [IQR, 3.7-5.6]; HCs: 4.8 hours [IQR, 3.9-6.0]; P = 0.883). However, patients with DM had significantly larger volume of the small intestinal wall (DM: 623 cm3 [IQR, 487-766]; HCs: 478 cm3 [IQR, 393-589]; P = 0.003). Among patients, 13 (68%) had small intestinal wall volume and 9 (50%) had gastric emptying time above the upper 95% percentile of HCs. CONCLUSION: In our study, gastric emptying time and volume of the small intestinal wall appeared to be the best objective measures in patients with DM type 1 and symptoms and gastroenteropathy.

A diet-induced gut microbiota component and related plasma metabolites are associated with depressive-like behaviour in rats.

It is well-established in preclinical studies that various probiotics may improve behaviours related to psychiatric disease. We have previously shown that probiotics protected against high-fat diet (HFD)-induced depressive-like behaviour in Flinders Sensitive Line (FSL) rats, whereas FSL rats on control (CON) diet were unaffected. Therefore, we hypothesised that a dysmetabolic component of depression may exist that involves the gut microbiota and that such component may be reflected in the plasma metabolome. The aims of the present study post hoc analyses were 1) to study the effect of probiotics on gut microbiota composition and its association with depressive-like behaviour in FSL rats, and 2) to identify plasma metabolites associated with gut microbiota and depressive-like behaviour. Forty-six FSL rats were fed CON or HFD and treated with multi-species probiotics (nine Bifidobacterium, Lactococcus and Lactobacillus species) for 12 weeks. Faecal samples were collected for 16S rRNA (VR4) gene amplicon sequencing (Illumina MiSeq), and an untargeted plasma metabolomics was performed. We found that probiotics increased the relative faecal abundance of the Bifidobacterium, Lactococcus and Lactobacillus genera in HFD-fed rats only. Also, a HFD-induced microbiota component associated with depressive-like behaviour was identified, and probiotics improved the component score. Finally, the plasma levels of 44 metabolites correlated with the depression-related microbiota component, and three such metabolites had good predictive ability for depressive-like behaviour. Potentially, our findings imply that a subtype of depression characterised by a diet-induced, pro-depressant gut microbiota may exist and that analysis of related plasma metabolites may reveal aberrant microbiota functioning related to depression.

[Symptoms of diabetic gastroenteropathy in patients with diabetes].

Abdominal pain, nausea, vomiting, diarrhoea, constipation, and faecal incontinence are common symptoms of diabetic gastroenteropathy and often have a major impact on quality of life. The symptoms are usually caused by widespread dysfunction of the gastrointestinal tract. Hence, diagnosis requires panenteric assessment. The general principles of management are glycaemic control, diet, prokinetics, laxatives, and in selected cases, gastric electrical stimulation, which is summarised in this review.

Colonic motility in patients with type 1 diabetes and gastrointestinal symptoms.

BACKGROUND: Gastrointestinal (GI) symptoms are common in patients with diabetes mellitus (DM). The electromagnetic 3D-Transit system allows assessment of regional transit times and motility patterns throughout the GI tract. We aimed to compare GI transit times and detailed motility patterns of the colon in patients with DM and GI symptoms to those of healthy controls (HC). We further aimed to determine whether any abnormalities in motility were reversible by cholinergic stimulation. METHODS: We compared 18 patients with DM with 20 HC by means of the 3D-Transit system. Patients were studied before and during oral administration of 60 mg pyridostigmine. KEY RESULTS: Compared to HC, patients had prolonged gastric emptying (DM: 3.3 hours (interquartile range (IQR) 2.6-4.6); HC: 2.3 hours (IQR 1.7-2.7) (P < .01)), colonic transit time (DM: 52.6 hours (IQR 23.3-83.0); HC: 22.4 hours (IQR 18.9-43.6) (P = .02)), and whole gut transit time (DM: 69.4 hours (IQR 32.9-103.6); HC: 30.3 hours (IQR 25.2-49.9) (P < .01)). In addition, compared to HC, patients had prolonged transit time in the ascending colon (DM: 20.5 hours (IQR 11.0-44.0); HC: 8.0 hours (IQR 3.8-21.0) (P < .05)) and more slow retrograde movements in the colon (DM: 2 movements (IQR 1-4); HC: 1 movement (IQR 0-1) (P = .01)). In patients, pyridostigmine increased the number of bowel movements (P < .01) and reduced small intestine transit times (P < .05). CONCLUSIONS: Patients with DM and GI symptoms have longer than normal GI transit times. This is only partly reversible by pyridostigmine. The increased number of retrograde colonic movements in patients could potentially explain the abnormally long transit time in proximal colon.

Enteric cholinergic neuropathy in patients with diabetes: Non-invasive assessment with positron emission tomography.

BACKGROUND: 11 C-Donepezil positron emission tomography (PET) allows non-invasive assessment of cholinergic innervation of visceral organs. We aimed to compare cholinergic innervation in the gut in patients with diabetes mellitus (DM) and in healthy controls (HC). METHODS: 11 C-Donepezil PET and computed tomography (CT) were performed in 19 patients with type 1 DM and gastrointestinal symptoms and in 19 age- and sex-matched HC in a cross-sectional design. KEY RESULTS: All patients had severe gastrointestinal symptoms when assessed by standard questionnaires. DM patients had significantly increased volume of the small intestinal wall (DM: median 557 cm3 [interquartile range [IQR] 446-697] vs HC median: 448 cm3 [IQR; 341-518; P < .01]), and the 11 C Donepezil PET uptake was reduced in patients (DM: median 7.08 standardized uptake value [SUV] [IQR; 5.94-8.43] vs HC: median 9.18 SUV [IQR; 8.57-10.11; P < .01]). A similar pattern was found in colon (DM: median volume 1064 cm3 [IQR; 882-1312] vs HC: median 939 cm3 [IQR; 785-1081; P = .13] and DM: median 1.22 SUV (IQR; 1.08-1.36) vs HC: median 1.42 SUV (IQR; 1.32-1.53; P = .03). Furthermore, patients had significantly reduced pancreatic volume (DM: median 53 cm3 [IQR; 41-69] vs HC: median 98 cm3 [IQR;82-110; P < .01]) and reduced PET uptake of the pancreas (DM: median 13.14 SUV [IQR;9.58-15.82] vs HC: median 21.46 SUV [IQR;18.97-24.06; P < .01]) as well as the adrenal gland (DM: median 7.62 SUV [IQR;7.61;15.82] vs HC: median 15.51 SUV [IQR;12.22;19.49; P = .03]). CONCLUSION AND INFERENCES: Assessed with 11 C-Donepezil PET/CT, patients with DM and severe bowel symptoms have reduced cholinergic innervation of the gut indicative of parasympathetic denervation.

Grandmaternal high-fat diet primed anxiety-like behaviour in the second-generation female offspring.

The health consequences of maternal obesity during pregnancy are disturbing as they may contribute to mental disorders in subsequent generations. We examine the influence of suboptimal grandmaternal diet on potential metabolic and mental health outcome of grand-progenies with a high-fat diet (HFD) manipulation in adulthood in a rat HFD model. Grandmaternal exposure to HFD exacerbated granddaughter's anxiety-like phenotype. Grandmaternal exposure to HFD led to upregulated corticotropin-releasing hormone receptor 2 mRNA expression involved in the stress axis in the male F2 offspring. Thus, we demonstrate that suboptimal grandmaternal diet prior to and during pregnancy and lactation may persist across subsequent generations. These findings have important implications for understanding both individual rates of metabolic and mental health problems and the clinical impact of current global trends towards comorbidity of obesity and depression and anxiety. In conclusion, the effect of grandmaternal HFD consumption during pregnancy on stress axis function and mental disorders may be transmitted to future generations.

The antidepressant-like effect of probiotics and their faecal abundance may be modulated by the cohabiting gut microbiota in rats.

Numerous studies have been published describing the effect of various probiotics (PRO) on behaviours related to psychiatric disease. We have previously shown a robust antidepressant-like effect of PRO in rats, but over time, the treatment effect seems to vary significantly between different sets of rats from the same commercial vendor. Therefore, we hypothesised that the antidepressant-like response may be modulated by the cohabiting gut microbiota. The aims of the present study were (1) to investigate any differences in the gut microbiota composition between responders (Resp) and non-responders (Non-resp) to PRO with regards to depressive-like behaviour, and (2) to evaluate the effects of PRO on the microbiota composition. Two sets of 20 male Sprague-Dawley rats each were treated with multi-species PRO (nine Bifidobacterium, Lactococcus and Lactobacillus species) for eight weeks and subjected to a behavioural assessment. Faecal samples were collected for 16 s rRNA (VR4) gene amplicon sequencing (Illumina MiSeq). As previously reported, PRO-treated Resp animals showed a marked decrease in depressive-like behaviour, whereas no such response was seen in Non-resp. We observed profound differences in the gut microbiota composition between the two sets of rats, and the relative faecal abundance of the genera that comprised PRO was higher in Resp than in Non-resp although treated with the same dose of PRO. Particularly, the relative abundance of the Lactobacillus genus was not increased in PRO-treated Non-resp animals. In conclusion, the cohabiting microbiota and the faecal abundance of PRO may modulate the antidepressant-like effect of PRO in rats.

Maternal High-fat Diet Programs Offspring Emotional Behavior in Adulthood.

Prenatal exposure to high-fat diet (HFD) might predispose offspring to develop metabolic and mental disorders later in life. Insight into the molecular and behavioral consequences of maternal HFD on offspring is sparse but may involve both neuroinflammation and a dysregulated neuroendocrine stress axis. Thus, the aim of this work was to: (i) investigate the influence of maternal HFD on memory, anxiety and depression-like behavior in adult offspring and (ii) identify possible biological biomarkers related to neuroinflammation and stress responses. Seven-week-old, female Sprague-Dawley rats received a control diet or a HFD eight weeks prior to conception and during gestation and lactation. We investigated the phenotype of the offspring in the in elevated plus maze, forced swim test, novel object recognition and open field test. Furthermore, hippocampal gene expression related to neuroinflammation and the stress axis was quantitated by real-time qPCR. We found that maternal HFD led to an anxiogenic offspring phenotype in the elevated plus maze, independent of sex. This behavioral alteration was accompanied by significantly higher mRNA levels of the hippocampal pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) mRNA and monocyte-chemoattractant protein-1 (MCP-1), both of which correlated with degree of behavioral change. Maternal exposure to HFD increased the offspring's levels of hippocampal, corticosteroid releasing hormone receptor 2 (CRHR2) and kynurenine mono oxygenase (KMO) mRNA, whereas kynurenine aminotransferase I (KAT1) mRNA levels were decreased. The present results suggest that neuroinflammatory and stress axis pathways in the hippocampus may contribute to anxiogenic effects of maternal HFD in offspring.

Escitalopram Ameliorates Hypercortisolemia and Insulin Resistance in Low Birth Weight Men With Limbic Brain Alterations.

Context: Low birth weight (LBW; <2500 g) is linked to the development of insulin resistance and limbic-hypothalamic-pituitary-adrenal (LHPA) axis hyperactivity. Objective: Our first aim was to study insulin action, LHPA axis function, and limbic brain structures in young, healthy LBW men vs normal birthweight (NBW) controls (part 1). Our second aim was to investigate the effects of escitalopram vs placebo in LBW men in the LHPA axis and insulin sensitivity (part 2). Design Setting, Participants, and Intervention: The maximal (Rdmax) and submaximal (Rdsubmax) rates of insulin-stimulated glucose turnover, LHPA axis, and brain morphology were examined in 40 LBW men and 20 matched NBW men using two-stage hyperinsulinemic euglycemic clamp, 24-hour hormone plasma profiles, and magnetic resonance imaging. Subsequently, all LBW subjects underwent randomized and double-blind treatment with escitalopram 20 mg/d or placebo for 3 months followed by a complete reexamination. Main Outcome Measures (Part 2): Changes in Rdmax/Rdsubmax and plasma-free cortisol 24-hour area under the curve. Results: In LBW vs NBW, Rdsubmax and Rdmax were ∼16% (P = 0.01) and ∼12% (P = 0.01) lower, respectively, and 24-hour free cortisol levels were ∼20% higher (P = 0.02), primarily driven by a ∼99% increase at 05:00 am (P < 0.001). Furthermore, these changes were related to structural alterations within left thalamus and ventromedial prefrontal cortex. However, in LBW men, exposure to escitalopram normalized the free cortisol levels and improved the Rdsubmax by ∼24% (P = 0.04) compared with placebo. Conclusions: LBW vs NBW displayed alterations in key brain structures modulating the LHPA axis, elevated free cortisol levels, and insulin resistance. Escitalopram administration ameliorated these defects, suggesting a potential for LHPA axis modulation compounds to improve insulin action in LBW subjects.

DNA methylation in epigenetic inheritance of metabolic diseases through the male germ line.

The global rise in metabolic diseases can be attributed to a complex interplay between biology, behavior and environmental factors. This article reviews the current literature concerning DNA methylation-based epigenetic inheritance (intergenerational and transgenerational) of metabolic diseases through the male germ line. Included are a presentation of the basic principles for DNA methylation in developmental programming, and a description of windows of susceptibility for the inheritance of environmentally induced aberrations in DNA methylation and their associated metabolic disease phenotypes. To this end, escapees, genomic regions with the intrinsic potential to transmit acquired paternal epigenetic information across generations by escaping the extensive programmed DNA demethylation that occurs during gametogenesis and in the zygote, are described. The ongoing descriptive and functional examinations of DNA methylation in the relevant biological samples, in conjugation with analyses of non-coding RNA and histone modifications, hold promise for improved delineation of the effect size and mechanistic background for epigenetic inheritance of metabolic diseases.

The microbial metabolite indole-3-propionic acid improves glucose metabolism in rats, but does not affect behaviour.

CONTEXT: We have previously shown that an antidepressant-like effect of probiotics in rats was associated with a higher plasma level of the microbial tryptophan metabolite indole-3-propionic acid (IPA). OBJECTIVE: We therefore wanted to study the isolated effect of IPA on behaviour and glucose metabolism in rats. METHODS: Male Sprague-Dawley rats were fed control or IPA-enriched diet for six weeks (n = 12 per group) and assessed in the elevated plus maze, open field and forced swim test. Blood glucose, metabolic hormones and the white blood cell (WBC) composition were analysed. RESULTS: IPA (mean intake 27.3 mg/kg/day) significantly lowered fasting blood glucose level by 0.42 mM (95% CI 0.11-0.73). Similarly, fasting plasma insulin levels and the homeostatic model assessment (HOMA) index of insulin resistance were reduced, whereas plasma metabolic hormones, behaviour and WBC composition remained unaffected by IPA. CONCLUSIONS: Our findings highlight IPA as a promising candidate for treatment of metabolic disorders associated with insulin resistance.

[Workup and treatment of gastroparesis].

Gastroparesis is defined as impaired gastric emptying without mechanical obstruction and cardinal symptoms including vomiting, nausea, early satiety, and upper abdominal pain. Most cases of gastroparesis are diabetic, idiopathic or post-operative. The correlation between symptoms and objective measures of gastroparesis is poor. Basic treatment includes dietary advices and prokinetics. Selected patients not responding to basic treatment can be offered gastric electrical stimulation. In many cases, gastroparesis is present in combination with other motility disorders, especially constipation.

Inflammation Downregulates UCP1 Expression in Brown Adipocytes Potentially via SIRT1 and DBC1 Interaction.

Brown adipose tissue thermogenesis at the cost of energy is not only important for the development of obesity, but also possesses great promise in anti-obesity treatment. Uncoupling protein 1 (UCP1) expression has been reported to be under control of the intracellular deacetylase SIRT1. Here, we investigated the effect and mechanism of inflammation and sirtuin-1 (SIRT1) activation on the induction of thermogenic genes in immortalized brown adipocytes incubated with LPS or IL1ß and mice with elevated inflammatory tone. In vitro stimulation of brown adipocytes with dibutyryl cyclic adenosine monophosthate (dbcAMP) reduced the expression of deleted in breast cancer-1 (Dbc1) (SIRT1 inhibitor) and increased the Ucp1 expression. Silencing of SIRT1 attenuated dbcAMP induction of Ucp1. In contrast, IL1ß increased the expression of Dbc1 and greatly reduced the induction of Ucp1. Similarly, in vivo studies revealed decreased expression of Ucp1 in brown adipose tissue (BAT) in mice chronically infused with LPS. Resveratrol, a known SIRT1 activator, partly rescued the Ucp1 downregulation by inflammation in both the cell cultures and mice. Here, we describe how the expression of Ucp1 in BAT is controlled via SIRT1 and is reduced under inflammation and can be rescued by SIRT1 activation by resveratrol. We suggest the reduced UCP1 expression under inflammation is mediated by the increased expression of DBC1, which inhibits SIRT1 activity.

Probiotic treatment protects against the pro-depressant-like effect of high-fat diet in Flinders Sensitive Line rats.

Major depressive disorder (MDD) is highly associated with dysmetabolic conditions, such as obesity and diabetes mellitus type 2, and the gut microbiota may interact with both disease entities. We have previously shown that a high-fat diet (HFD) exacerbated depressive-like behaviour uniquely in Flinders Sensitive Line (FSL) rats that inherently present with an increased level of depressive-like behaviour compared with Flinders Resistant Line (FRL) rats. We therefore investigated whether multispecies probiotics possessed anti-depressant-like effect in FSL rats or protected against the pro-depressant-like effect of HFD. We also examined blood and cerebral T cell subsets as well as plasma cytokines. Lastly, we investigated the effect of HFD in outbred Sprague-Dawley (SD) rats to substantiate the association between depressive-like behaviour and any immunological measures affected by HFD. HFD exacerbated the depressive-like behaviour in FSL rats in the forced swim test, whereas SD rats remained unaffected. Probiotic treatment completely precluded the pro-depressant-like effect of HFD, but it did not affect FSL rats on control diet. Cerebral T lymphocyte CD4/8 ratios closely mirrored the behavioural changes, whereas the proportions of Treg and Th17 subsets were unaltered. No association between blood and brain CD4/8 ratios were evident; nor did plasma cytokine levels change as a consequence of HFD of probiotic treatment. Our findings suggest that MDD may hold a dysmetabolic component that responds to probiotic treatment. This finding has wide implications owing to the high metabolic comorbidity in MDD. Furthermore, the close association between depressive-like behaviour and cerebral T cell populations demonstrate lymphocyte-brain interactions as a promising future research area in the field of psychoneuroimmunology.

Probiotic treatment reduces depressive-like behaviour in rats independently of diet.

The gut microbiota has recently emerged as an important regulator of brain physiology and behaviour in animals, and ingestion of certain bacteria (probiotics) therefore appear to be a potential treatment for major depressive disorder (MDD). However, some conceptual and mechanistical aspects need further elucidation. We therefore aimed at investigating whether the habitual diet may interact with the effect of probiotics on depression-related behaviour and further examined some potentially involved mechanisms underlying the microbe-mediated behavioural effects. Forty male Sprague-Dawley rats were fed a control (CON) or high-fat diet (HFD) for ten weeks and treated with either a multi-species probiotic formulation or vehicle for the last five weeks. Independently of diet, probiotic treatment markedly reduced depressive-like behaviour in the forced swim test by 34% (95% CI: 22-44%). Furthermore, probiotic treatment skewed the cytokine production by stimulated blood mononuclear cells towards IFNγ, IL2 and IL4 at the expense of TNFα and IL6. In addition, probiotics lowered hippocampal transcript levels of factors involved in HPA axis regulation (Crh-r1, Crh-r2 and Mr), whereas HFD increased these levels. A non-targeted plasma metabolomics analysis revealed that probiotics raised the level of indole-3-propionic acid, a potential neuroprotective agent. Our findings clearly support probiotics as a potential treatment strategy in MDD. Importantly, the efficacy was not attenuated by intake of a "Western pattern" diet associated with MDD. Mechanistically, the HPA axis, immune system and microbial tryptophan metabolism could be important in this context. Importantly, our study lend inspiration to clinical trials on probiotics in depressed patients.

Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics.

Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.

A robust method to screen detergents for membrane protein stabilization, revisited.

This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907-4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of ß-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains useful in pre-screening assays testing various detergents.