Determining the Fatty Acid Substrate Preferences of Long-Chain Acyl-CoA Synthetase Isoforms

Before a fatty acid can be used in a cell, it must first be converted to its active form acyl-coenzyme A (acyl-CoA). This activation is catalyzed by a group of enzymes known as acyl-CoA synthetases, which use the energy of ATP to add a CoA group to the fatty acid to create fatty acyl-CoA. By controlling the synthesis of fatty acyl-CoAs, long-chain acyl-CoA synthetases (ACSL) can regulate fatty acid uptake and metabolism by selective activation of fatty acids. Activated fatty acids can be channeled to numerous downstream pathways after their conversion into acyl-CoA. The control over this fatty acid channeling towards different downstream pathways is not clear, but may vary depending on the isoform of the ACSL enzyme used to synthesize the acyl-CoA. Five different isoforms of ACSL (1,3,4,5,6) exist, each with varying roles in the body5. With each isoform, there is likely to be a distinct fatty acid preference and metabolic fate for the generated fatty acyl-CoA8,9,10,11. We expect each ACSL isoform to have differing chain-length and saturation preferences for its substrates. To better understand the substrate preferences of each isoform we used engineered expression vectors containing genes for each ACSL isoform along with a FLAG tag to produce purified recombinant enzyme. These expression vectors were transformed into E. coli and induced with IPTG to make recombinant protein. The FLAG-ACSL enzyme produced was affinity purified using a FLAG column and then used in an indirect spectrophotometric assay with different substrates to determine ACSL isoform substrate preference. The specific activity for each isoform was calculated with fatty acids of varying chain-length and saturation, to give quantitative values for the preferences of each isoform. Through troubleshooting and developing a protocol, we found that active isoforms were produced when induced at 25˚C for 16 hours. An indirect assay performed with purified ACSL5 showed activity with oleic acid and palmitic acid. ACSL5 showed a greater preference for palmitic acid, particularly at lower concentrations of purified protein. Due to time constrictions, and problems obtaining active ACSL isoforms, the protocol developed will have to be used in further studies to determine the substrate preferences of each ACSL isoform.Bachelor of Science in Public Healt

Long-chain acyl-CoA synthetase isoforms differ in preferences for eicosanoid species and long-chain fatty acids

Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS/MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain FAs were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification

Disparities in brain health comorbidity management in intracerebral hemorrhage

BackgroundIntracerebral hemorrhage (ICH) disproportionally affects underserved populations, and coincides with risk factors for cardiovascular events and cognitive decline after ICH. We investigated associations between social determinants of health and management of blood pressure (BP), hyperlipidemia, diabetes, obstructive sleep apnea (OSA), and hearing impairment before and after ICH hospitalization.MethodsSurvivors of the Massachusetts General Hospital longitudinal ICH study between 2016 and 2019 who received healthcare at least 6 months after ICH were analyzed. Measurements of BP, LDL and HbA1c and their management in the year surrounding ICH and referrals for sleep studies and audiology up to 6 months after ICH were gathered from electronic health records. The US-wide area deprivation index (ADI) was used as proxy for social determinants of health.ResultsThe study included 234 patients (mean 71 years, 42% female). BP measurements were performed in 109 (47%) before ICH, LDL measurements were performed in 165 (71%), and HbA1c measurements in 154 (66%) patients before or after ICH. 27/59 (46%) with off-target LDL and 3/12 (25%) with off-target HbA1c were managed appropriately. Of those without history of OSA or hearing impairment before ICH, 47/207 (23%) were referred for sleep studies and 16/212 (8%) to audiology. Higher ADI was associated with lower odds of BP, LDL, and HbA1c measurement prior to ICH [OR 0.94 (0.90–0.99), 0.96 (0.93–0.99), and 0.96 (0.93–0.99), respectively, per decile] but not with management during or after hospitalization.ConclusionSocial determinants of health are associated with pre-ICH management of cerebrovascular risk factors. More than 25% of patients were not assessed for hyperlipidemia and diabetes in the year surrounding ICH hospitalization, and less than half of those with off-target values received treatment intensification. Few patients were evaluated for OSA and hearing impairment, both common among ICH survivors. Future trials should evaluate whether using the ICH hospitalization to systematically address co-morbidities can improve long-term outcomes

Obestatin stimulates glucose-induced insulin secretion through ghrelin receptor GHS-R

AbstractOrexigenic hormone ghrelin and anorexic hormone obestatin are encoded by the same preproghrelin gene. While it is known that ghrelin inhibits glucose-stimulated insulin secretion (GSIS), the effect of obestatin on GSIS is unclear. Ghrelin’s effect is mediated by its receptor Growth Hormone Secretagogue Receptor (GHS-R), but the physiologically relevant receptor of obestatin remains debatable. Here we have investigated the effect of obestatin on GSIS in vitro, in vivo and ex vivo, and tested whether obestatin regulates insulin secretion through GHS-R. We found that under hyperglycemic condition, obestatin augments GSIS in rat insulinoma cells (INS-1) and in pancreatic islets from ghrelin−/− mice. Surprisingly, obestatin-induced GSIS was absent in β-cells in which GHS-R was suppressed. Obestatin-induced insulin secretion was abolished in the circulation of Ghsr−/− mice, and in pancreatic islets isolated from Ghsr−/− mice. We also found that obestatin-induced GSIS was attenuated in islets isolated from β-cell-specific Ghsr knockout MIP-Cre/ERT;Ghsrf/f mice. Our data collectively demonstrate that obestatin is a potent insulin secretagogue under hyperglycemic condition, and obestatin’s effect on insulin secretion is mediated by GHS-R in pancreatic β-cells. Our findings reveal an intriguing insight that obestatin and ghrelin have opposing effects on insulin secretion, and both are mediated through ghrelin receptor GHS-R.</jats:p

A Quantitative Systems Pharmacology Platform Reveals NAFLD Pathophysiological States and Targeting Strategies

Non-alcoholic fatty liver disease (NAFLD) has a high global prevalence with a heterogeneous and complex pathophysiology that presents barriers to traditional targeted therapeutic approaches. We describe an integrated quantitative systems pharmacology (QSP) platform that comprehensively and unbiasedly defines disease states, in contrast to just individual genes or pathways, that promote NAFLD progression. The QSP platform can be used to predict drugs that normalize these disease states and experimentally test predictions in a human liver acinus microphysiology system (LAMPS) that recapitulates key aspects of NAFLD. Analysis of a 182 patient-derived hepatic RNA-sequencing dataset generated 12 gene signatures mirroring these states. Screening against the LINCS L1000 database led to the identification of drugs predicted to revert these signatures and corresponding disease states. A proof-of-concept study in LAMPS demonstrated mitigation of steatosis, inflammation, and fibrosis, especially with drug combinations. Mechanistically, several structurally diverse drugs were predicted to interact with a subnetwork of nuclear receptors, including pregnane X receptor (PXR; NR1I2), that has evolved to respond to both xenobiotic and endogenous ligands and is intrinsic to NAFLD-associated transcription dysregulation. In conjunction with iPSC-derived cells, this platform has the potential for developing personalized NAFLD therapeutic strategies, informing disease mechanisms, and defining optimal cohorts of patients for clinical trials

Cell Replacement and Regeneration Therapy for Diabetes

Reduction of beta cell function and a beta cell mass is observed in both type 1 and type 2 diabetes. Therefore, restoration of this deficiency might be a therapeutic option for treatment of diabetes. Islet transplantation has benefits, such as reduced incidence of hypoglycemia and achievement of insulin independence. However, the major drawback is an insufficient supply of islet donors. Transplantation of cells differentiated in vitro or in vivo regeneration of insulin-producing cells are possible approaches for beta cell/islet regenerative therapy. Embryonic and adult stem cells, pancreatic ductal progenitor cells, acinar cells, and other endocrine cells have been shown to differentiate into pancreatic beta cells. Formation of fully functional beta cells and the safety of these cells are critical issues for successful clinical application

Transient expression of Ngn3 in Xenopus endoderm promotes early and ectopic development of pancreatic beta and delta cells

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in genesis 50 (2012): 271-285, doi:10.1002/dvg.20828.Promoting ectopic development of pancreatic beta cells from other cell types is one of the strategies being pursued for the treatment of diabetes. To achieve this, a detailed outline of the molecular lineage that operates in pancreatic progenitor cells to generate beta cells over other endocrine cell types is necessary. Here, we demonstrate that early transient expression of the endocrine progenitor bHLH protein Neurogenin 3 (Ngn3) favors the promotion of pancreatic beta and delta cell fates over an alpha cell fate, while later transient expression promotes ectopic development of all three endocrine cell fates. We found that short-term activation of Ngn3 in Xenopus laevis endoderm just after gastrulation was sufficient to promote both early and ectopic development of beta and delta cells. By examining gene expression changes four hours after Ngn3 activation we identified several new downstream targets of Ngn3. We show that several of these are required for the promotion of ectopic beta cells by Ngn3 as well as for normal beta cell development. These results provide new detail regarding the Ngn3 transcriptional network operating in endocrine progenitor cells to specify a beta cell phenotype and should help define new approaches to promote ectopic development of beta cells for diabetes therapy.National Institutes of Health (DK077197

TXNIP Regulates Peripheral Glucose Metabolism in Humans

BACKGROUND: Type 2 diabetes mellitus (T2DM) is characterized by defects in insulin secretion and action. Impaired glucose uptake in skeletal muscle is believed to be one of the earliest features in the natural history of T2DM, although underlying mechanisms remain obscure. METHODS AND FINDINGS: We combined human insulin/glucose clamp physiological studies with genome-wide expression profiling to identify thioredoxin interacting protein (TXNIP) as a gene whose expression is powerfully suppressed by insulin yet stimulated by glucose. In healthy individuals, its expression was inversely correlated to total body measures of glucose uptake. Forced expression of TXNIP in cultured adipocytes significantly reduced glucose uptake, while silencing with RNA interference in adipocytes and in skeletal muscle enhanced glucose uptake, confirming that the gene product is also a regulator of glucose uptake. TXNIP expression is consistently elevated in the muscle of prediabetics and diabetics, although in a panel of 4,450 Scandinavian individuals, we found no evidence for association between common genetic variation in the TXNIP gene and T2DM. CONCLUSIONS: TXNIP regulates both insulin-dependent and insulin-independent pathways of glucose uptake in human skeletal muscle. Combined with recent studies that have implicated TXNIP in pancreatic β-cell glucose toxicity, our data suggest that TXNIP might play a key role in defective glucose homeostasis preceding overt T2DM

Skeletal muscle munc18c and syntaxin 4 in human obesity

<p>Abstract</p> <p>Background</p> <p>Animal and cell culture data suggest a critical role for Munc18c and Syntaxin 4 proteins in insulin mediated glucose transport in skeletal muscle, but no studies have been published in humans.</p> <p>Methods</p> <p>We investigated the effect of a 12 vs. 48 hr fast on insulin action and skeletal muscle Munc18c and Syntaxin 4 protein in lean and obese subjects. Healthy lean (n = 14; age = 28.0 +/- 1.4 yr; BMI = 22.8 +/- 0.42 kg/m²) and obese subjects (n = 11; age = 34.6 +/- 2.3 yr; BMI = 36.1 +/- 1.5 kg/m²) were studied twice following a 12 and 48 hr fast. Skeletal muscle biopsies were obtained before a 3 hr 40 mU/m²/min hyperinsulinemic-euglycemic clamp with [6,6-²H₂]glucose infusion.</p> <p>Results</p> <p>Glucose rate of disappearance (Rd) during the clamp was lower in obese vs. lean subjects after the 12 hr fast (obese: 6.25 +/- 0.67 vs. lean: 9.42 +/- 1.1 mg/kgFFM/min, p = 0.007), and decreased significantly in both groups after the 48 hr fast (obese 3.49 +/- 0.31 vs. lean: 3.91 +/- 0.42 mg/kgFFM/min, p = 0.002). Munc18c content was not significantly different between lean and obese subjects after the 12 hour fast, and decreased after the 48 hr fast in both groups (p = 0.013). Syntaxin 4 content was not altered by obesity or fasting duration. There was a strong positive relationship between plasma glucose concentration and Munc18c content in lean and obese subjects during both 12 and 48 hr fasts (R²= 0.447, p = 0.0015). Significant negative relationships were also found between Munc18c and FFA (p = 0.041), beta-hydroxybutyrate (p = 0.039), and skeletal muscle AKT content (p = 0.035) in lean and obese subjects.</p> <p>Conclusion</p> <p>These data indicate Munc18c and Syntaxin 4 are present in human skeletal muscle. Munc18c content was not significantly different between lean and obese subjects, and is therefore unlikely to explain obesity-induced insulin resistance. Munc18c content decreased after prolonged fasting in lean and obese subjects concurrently with reduced insulin action. These data suggest changes in Munc18c content in skeletal muscle are associated with short-term changes in insulin action in humans.</p

Long-Term Outcomes in Patients With Spontaneous Cerebellar Hemorrhage: An International Cohort Study

International audienceBACKGROUND:Spontaneous intracerebral hemorrhage (ICH) in the cerebellum has a poor short-term prognosis, whereas data on the long-term case fatality and recurrent vascular events are sparse. Herewith, we aimed to assess the long-term case fatality and recurrence rate of vascular events after a first cerebellar ICH.METHODS:In this international cohort study, we included patients from 10 hospitals (the United States and Europe from 1997 to 2017) aged ≥18 years with a first spontaneous cerebellar ICH who were discharged alive. Data on long-term case fatality and recurrence of vascular events (recurrent ICH [supratentoria or infratentorial], ischemic stroke, myocardial infarction, or major vascular surgery) were collected for survival analysis and absolute event rate calculation.RESULTS:We included 405 patients with cerebellar ICH (mean age [SD], 72 [13] years, 49% female). The median survival time was 67 months (interquartile range, 23–100 months), with a cumulative survival rate of 34% at 10-year follow-up (median follow-up time per center ranged: 15–80 months). In the 347 patients with data on vascular events 92 events occurred in 78 patients, after initial cerebellar ICH: 31 (8.9%) patients had a recurrent ICH (absolute event rate, 1.8 per 100 patient-years [95% CI, 1.2–2.6]), 39 (11%) had an ischemic stroke (absolute event rate, 2.3 [95% CI, 1.6–3.2]), 13 (3.7%) had a myocardial infarction (absolute event rate, 0.8 [95% CI, 0.4–1.3]), and 5 (1.4%) underwent major vascular surgery (absolute event rate, 0.3 [95% CI, 0.1–0.7]). The median time to a first vascular event during follow-up was 27 months (interquartile range, 8.7–50 months), with a cumulative hazard of 47% at 10 years.CONCLUSIONS:The long-term prognosis of patients who survive a first spontaneous cerebellar ICH is poor and comparable to that of patients who survive a first supratentorial ICH. Further identification of patients at high risk of vascular events following the initial cerebellar ICH is needed. Including patients with cerebellar ICH in randomized controlled trials on secondary prevention of patients with ICH is warranted