Pharmacological inhibition of lipolysis prevents adverse metabolic outcomes during glucocorticoid administration.

OBJECTIVE: Glucocorticoids are one of the most commonly prescribed classes of anti-inflammatory drugs; however, chronic treatment promotes iatrogenic (drug-induced) diabetes. As part of their physiological role, glucocorticoids stimulate lipolysis to spare glucose. We hypothesized that persistent stimulation of lipolysis during glucocorticoid therapy plays a causative role in the development of iatrogenic diabetes. METHODS: Male C57BL/6J mice were given 100 µg/mL corticosterone (Cort) in the drinking water for two weeks and were fed either normal chow (TekLad 8640) or the same diet supplemented with an adipose triglyceride lipase inhibitor (Atglistatin - 2  g/kg diet) to inhibit the first step of lipolysis. RESULTS: Herein, we report for the first time that glucocorticoid administration promotes a unique state of substrate excess and energetic overload in skeletal muscle that primarily results from the rampant mobilization of endogenous fuels. Inhibiting lipolysis protected mice from Cort-induced gains in fat mass, excess ectopic lipid accrual, hyperinsulinemia, and hyperglycemia. The role lipolysis plays in Cort-mediated pathology appears to differ between tissues. Within skeletal muscle, Cort-induced lipolysis facilitated diversion of glucose-derived carbons toward the pentose phosphate and hexosamine biosynthesis pathways but contributed to <3% of the Cort-induced genomic adaptations. In contrast, Cort stimulation of lipolysis accounted for ∼35% of the genomic changes in the liver but had minimal impact on hepatic metabolites reported. CONCLUSIONS: These data support the idea that activation of lipolysis plays a causal role in the progression toward iatrogenic diabetes during glucocorticoid therapy with differential impact on skeletal muscle and liver.

Redefining and categorizing emergency medical service opioid-related incidents in Massachusetts.

AIMS: To create a novel emergency medical service (EMS) opioid-related incident (ORI) tiering framework to describe more accurately the opioid epidemic in Massachusetts. By classifying the data, we could more accurately detail differing trends among the new categories. DESIGN: Free-text fields of Massachusetts EMS reports, from 2013 through 2020, were analyzed to identify ORIs and then categorized into a five-tier severity cascade based on symptom presentation: 'dead on arrival,' 'acute overdose,' 'intoxication,' 'withdrawal' and 'other ORI.' As a validation of the new classification, an emergency medical technician, paramedic and emergency medical physician reviewed clinical reports and assigned a severity category to 100 randomly selected cases. The algorithm then assessed the same 100 cases to determine if it could accurately identify the severity category for each case. FINDINGS: Validation of the algorithm by clinical review indicated a substantial level of agreement between the algorithm and the reviewers. Over half of all ORIs were acute overdose (55%), 21% were intoxication, 20% were other ORI, 3% were withdrawal, and 1% were dead on arrival. Overall ORIs decreased in 2020, but the number of 'dead on arrival' increased 32% from 2019. Administration of naloxone also differed between the categories, with 95% of acute overdose and 29% of intoxication receiving naloxone. CONCLUSIONS: This novel categorization of emergency medical service opioid-related incidents in Massachusetts, United States, reveals new trend details and strains on the emergency medical service system. Using these categories also improves dataset linkage within the state and interstate rate comparisons.

Growth Substrate and Prophage Induction Collectively Influence Metabolite and Lipid Profiles in a Marine Bacterium.

Bacterial growth substrates influence a variety of biological functions, including the biosynthesis and regulation of lipid intermediates. The extent of this rewiring is not well understood nor has it been considered in the context of virally infected cells. Here, we used a one-host-two-temperate phage model system to probe the combined influence of growth substrate and phage infection on host carbon and lipid metabolism. Using untargeted metabolomics and lipidomics, we reported the detection of a suite of metabolites and lipid classes for two Sulfitobacter lysogens provided with three growth substrates of differing complexity and nutrient composition (yeast extract/tryptone [complex], glutamate and acetate). The growth medium led to dramatic differences in the detectable intracellular metabolites, with only 15% of 175 measured metabolites showing overlap across the three growth substrates. Between-strain differences were most evident in the cultures grown on acetate, followed by glutamate then complex medium. Lipid distribution profiles were also distinct between cultures grown on different substrates as well as between the two lysogens grown in the same medium. Five phospholipids, three aminolipid, and one class of unknown lipid-like features were identified. Most (≥94%) of these 75 lipids were quantifiable in all samples. Metabolite and lipid profiles were strongly determined by growth medium composition and modestly by strain type. Because fluctuations in availability and form of carbon substrates and nutrients, as well as virus pressure, are common features of natural systems, the influence of these intersecting factors will undoubtedly be imprinted in the metabolome and lipidome of resident bacteria. IMPORTANCE Community-level metabolomics approaches are increasingly used to characterize natural microbial populations. These approaches typically depend upon temporal snapshots from which the status and function of communities are often inferred. Such inferences are typically drawn from lab-based studies of select model organisms raised under limited growth conditions. To better interpret community-level data, the extent to which ecologically relevant bacteria demonstrate metabolic flexibility requires elucidation. Herein, we used an environmentally relevant model heterotrophic marine bacterium to assess the relationship between growth determinants and metabolome. We also aimed to assess the contribution of phage activity to the host metabolome. Striking differences in primary metabolite and lipid profiles appeared to be driven primarily by growth regime and, secondarily, by phage type. These findings demonstrated the malleable nature of metabolomes and lipidomes and lay the foundation for future studies that relate cellular composition with function in complex environmental microbial communities.

A Surface Exposed, Two-Domain Lipoprotein Cargo of a Type XI Secretion System Promotes Colonization of Host Intestinal Epithelia Expressing Glycans.

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISSOMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenorhabdus nematophila NilC is surface exposed. Biophysical data and structure predictions indicate that NilC is a two-domain protein with a C-terminal, 8-stranded ß-barrel. This structure has been noted as a common feature of TXISS effectors and may be important for interactions with the TXISSOMP. The NilC N-terminal domain is more enigmatic, but our results indicate it is ordered and forms a ß-sheet structure, and bioinformatics suggest structural similarities to carbohydrate-binding proteins. X. nematophila NilC and its presumptive TXISSOMP partner NilB are required for colonizing the anterior intestine of Steinernema carpocapsae nematodes: the receptacle of free-living, infective juveniles and the anterior intestinal cecum (AIC) in juveniles and adults. We show that, in adult nematodes, the AIC expresses a Wheat Germ Agglutinin (WGA)-reactive material, indicating the presence of N-acetylglucosamine or N-acetylneuraminic acid sugars on the AIC surface. A role for this material in colonization is supported by the fact that exogenous addition of WGA can inhibit AIC colonization by X. nematophila. Conversely, the addition of exogenous purified NilC increases the frequency with which X. nematophila is observed at the AIC, demonstrating that abundant extracellular NilC can enhance colonization. NilC may facilitate X. nematophila adherence to the nematode intestinal surface by binding to host glycans, it might support X. nematophila nutrition by cleaving sugars from the host surface, or it might help protect X. nematophila from nematode host immunity. Proteomic and metabolomic analyses of wild type X. nematophila compared to those lacking nilB and nilC revealed differences in cell wall and secreted polysaccharide metabolic pathways. Additionally, purified NilC is capable of binding peptidoglycan, suggesting that periplasmic NilC may interact with the bacterial cell wall. Overall, these findings support a model that NilB-regulated surface exposure of NilC mediates interactions between X. nematophila and host surface glycans during colonization. This is a previously unknown function for a TXISS.

Apex Predator Nematodes and Meso-Predator Bacteria Consume Their Basal Insect Prey through Discrete Stages of Chemical Transformations.

Microbial symbiosis drives physiological processes of higher-order systems, including the acquisition and consumption of nutrients that support symbiotic partner reproduction. Metabolic analytics provide new avenues to examine how chemical ecology, or the conversion of existing biomass to new forms, changes over a symbiotic life cycle. We applied these approaches to the nematode Steinernema carpocapsae, its mutualist bacterium, Xenorhabdus nematophila, and the insects they infect. The nematode-bacterium pair infects, kills, and reproduces in an insect until nutrients are depleted. To understand the conversion of insect biomass over time into either nematode or bacterium biomass, we integrated information from trophic, metabolomic, and gene regulation analyses. Trophic analysis established bacteria as meso-predators and primary insect consumers. Nematodes hold a trophic position of 4.6, indicative of an apex predator, consuming bacteria and likely other nematodes. Metabolic changes associated with Galleria mellonella insect bioconversion were assessed using multivariate statistical analyses of metabolomics data sets derived from sampling over an infection time course. Statistically significant, discrete phases were detected, indicating the insect chemical environment changes reproducibly during bioconversion. A novel hierarchical clustering method was designed to probe molecular abundance fluctuation patterns over time, revealing distinct metabolite clusters that exhibit similar abundance shifts across the time course. Composite data suggest bacterial tryptophan and nematode kynurenine pathways are coordinated for reciprocal exchange of tryptophan and NAD+ and for synthesis of intermediates that can have complex effects on bacterial phenotypes and nematode behaviors. Our analysis of pathways and metabolites reveals the chemistry underlying the recycling of organic material during carnivory. IMPORTANCE The processes by which organic life is consumed and reborn in a complex ecosystem were investigated through a multiomics approach applied to the tripartite Xenorhabdus bacterium-Steinernema nematode-Galleria insect symbiosis. Trophic analyses demonstrate the primary consumers of the insect are the bacteria, and the nematode in turn consumes the bacteria. This suggests the Steinernema-Xenorhabdus mutualism is a form of agriculture in which the nematode cultivates the bacterial food sources by inoculating them into insect hosts. Metabolomics analysis revealed a shift in biological material throughout progression of the life cycle: active infection, insect death, and conversion of cadaver tissues into bacterial biomass and nematode tissue. We show that each phase of the life cycle is metabolically distinct, with significant differences including those in the tricarboxylic acid cycle and amino acid pathways. Our findings demonstrate that symbiotic life cycles can be defined by reproducible stage-specific chemical signatures, enhancing our broad understanding of metabolic processes that underpin a three-way symbiosis.

Cross-Omics Analysis of Fenugreek Supplementation Reveals Beneficial Effects Are Caused by Gut Microbiome Changes Not Mammalian Host Physiology.

Herbal remedies are increasing in popularity as treatments for metabolic conditions such as obesity and Type 2 Diabetes. One potential therapeutic option is fenugreek seeds (Trigonella foenum-graecum), which have been used for treating high cholesterol and Type 2 diabetes. A proposed mechanism for these benefits is through alterations in the microbiome, which impact mammalian host metabolic function. This study used untargeted metabolomics to investigate the fenugreek-induced alterations in the intestinal, liver, and serum profiles of mice fed either a 60% high-fat or low-fat control diet each with or without fenugreek supplementation (2% w/w) for 14 weeks. Metagenomic analyses of intestinal contents found significant alterations in the relative composition of the gut microbiome resulting from fenugreek supplementation. Specifically, Verrucomicrobia, a phylum containing beneficial bacteria which are correlated with health benefits, increased in relative abundance with fenugreek. Metabolomics partial least squares discriminant analysis revealed substantial fenugreek-induced changes in the large intestines. However, it was observed that while the magnitude of changes was less, significant modifications were present in the liver tissues resulting from fenugreek supplementation. Further analyses revealed metabolic processes affected by fenugreek and showed broad ranging impacts in multiple pathways, including carnitine biosynthesis, cholesterol and bile acid metabolism, and arginine biosynthesis. These pathways may play important roles in the beneficial effects of fenugreek.

Opioid-related incident severity and emergency medical service naloxone administration by sex in Massachusetts, 2013-2019.

Background: A Cross-sectional study of all emergency ambulance runs reported by licensed Emergency Medical Services (EMS) providers between 2013 and 2019 was undertaken to determine if the sex of a patient experiencing opioid-related symptoms had an impact on their odds of receiving naloxone from EMS. Methods: All runs within Massachusetts for individuals 11 years and older with a reported sex between 2013 and 2019 (n = 5,533,704 runs) were included. Covariates modeled were patient age, year of the incident, and county of the incident. Runs were separated into those that were opioid-related versus not; opioid-related runs were further subdivided into five severity categories including dead on arrival, acute opioid overdose, opioid intoxicated, opioid withdrawal, and other opioid-related incident. Results: Among opioid-related runs, women had 24% lower odds (95% CI 0.68-0.86) of appearing in the dead on arrival category and 20% lower odds (95% CI 0.78-0.82) of appearing in the acute opioid overdose category than men. Among acute opioid overdoses, runs where patient symptoms met Massachusetts EMS guidelines for naloxone administration, women had 18% lower odds (95% CI 0.76-0.89) of receiving naloxone than men. Conclusions: Sex-related differences persist in the odds of naloxone administration by EMS providers when controlling for symptom presentation.

Identification of Substance-Exposed Newborns and Neonatal Abstinence Syndrome Using ICD-10-CM - 15 Hospitals, Massachusetts, 2017.

Opioid use disorder and neonatal abstinence syndrome (NAS) increased in Massachusetts from 1999 to 2013 (1,2). In response, in 2016, the state passed a law requiring birth hospitals to report the number of newborns who were exposed to controlled substances to the Massachusetts Department of Public Health (MDPH)* by mandating monthly reporting of International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) diagnostic codes related to maternal dependence on opioids (F11.20) or benzodiazepines (F13.20) and to newborns affected by maternal use of drugs of addiction (P04.49) or experiencing withdrawal symptoms from maternal drugs of addiction (P96.1) separately.† MDPH uses these same codes for monthly, real-time crude estimates of NAS and uses P96.1 alone for official NAS state reporting.§ MDPH requested CDC's assistance in evaluating the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of either maternal or newborn codes to identify substance-exposed newborns, and of newborn exposure codes (both exposure [P04.49] or withdrawal [P96.1]) and the newborn code for withdrawal alone (P96.1) to identify infants with NAS cases related to three exposure scenarios: 1) opioids, 2) opioids or benzodiazepines, and 3) any controlled substance. Confirmed diagnoses of substance exposure and NAS abstracted from linked clinical records for 1,123 infants born in 2017 and their birth mothers were considered the diagnostic standard and were compared against hospital-reported ICD-10-CM codes. For identifying substance-exposed newborns across the three exposure scenarios, the newborn exposure codes had higher sensitivity (range = 31%-61%) than did maternal drug dependence codes (range = 16%-41%), but both sets of codes had high PPV (≥74%). For identifying NAS, for all exposure scenarios, the sensitivity for either newborn code (P04.49 or P96.1) was ≥92% and the PPV was ≥64%; for P96.1 alone the sensitivity was ≥79% and the PPV was ≥92% for all scenarios. Whereas ICD-10-CM codes are effective for NAS surveillance in Massachusetts, they should be applied cautiously for substance-exposed newborn surveillance. Surveillance for substance-exposed newborns using ICD-10-CM codes might be improved by increasing the use of validated substance-use screening tools and standardized facility protocols and improving communication between patients and maternal health and infant health care providers.

One week of continuous corticosterone exposure impairs hepatic metabolic flexibility, promotes islet ß-cell proliferation, and reduces physical activity in male C57BL/6 J mice.

Clinical glucocorticoid use, and diseases that produce elevated circulating glucocorticoids, promote drastic changes in body composition and reduction in whole body insulin sensitivity. Because steroid-induced diabetes is the most common form of drug-induced hyperglycemia, we investigated mechanisms underlying the recognized phenotypes associated with glucocorticoid excess. Male C57BL/6 J mice were exposed to either 100ug/mL corticosterone (cort) or vehicle in their drinking water. Body composition measurements revealed an increase in fat mass with drastically reduced lean mass during the first week (i.e., seven days) of cort exposure. Relative to the vehicle control group, mice receiving cort had a significant reduction in insulin sensitivity (measured by insulin tolerance test) five days after drug intervention. The increase in insulin resistance significantly correlated with an increase in the number of Ki-67 positive ß-cells. Moreover, the ability to switch between fuel sources in liver tissue homogenate substrate oxidation assays revealed reduced metabolic flexibility. Furthermore, metabolomics analyses revealed a decrease in liver glycolytic metabolites, suggesting reduced glucose utilization, a finding consistent with onset of systemic insulin resistance. Physical activity was reduced, while respiratory quotient was increased, in mice receiving corticosterone. The majority of metabolic changes were reversed upon cessation of the drug regimen. Collectively, we conclude that changes in body composition and tissue level substrate metabolism are key components influencing the reductions in whole body insulin sensitivity observed during glucocorticoid administration.