Sex and disease severity-based analysis of steroid hormones in ME/CFS.

PURPOSE: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disease characterized by persistent fatigue and decreased daily activity following physical and/or cognitive exertion. While ME/CFS affects both sexes, there is a higher prevalence in women. However, studies evaluating this sex-related bias are limited. METHODS: Circulating steroid hormones, including mineralocorticoids (aldosterone), glucocorticoids (cortisol, corticosterone, 11-deoxycortisol, cortisone), androgens (androstenedione, testosterone), and progestins (progesterone, 17α-hydroxyprogesterone), were measured in plasma samples using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Samples were obtained from mild/moderate (ME/CFSmm; females, n=20; males, n=8), severely affected patients (ME/CFSsa; females, n=24; males, n=6), and healthy controls (HC, females, n=12; males, n=17). RESULTS: After correction for multiple testing, we observed that circulating levels of 11-deoxycortisol, 17α-hydroxyprogesterone in females, and progesterone in males were significantly different between HC, ME/CFSmm, and ME/CFSsa. Comparing two independent groups, we found that female ME/CFSsa had higher levels of 11-deoxycortisol (vs. HC and ME/CFSmm) and 17α-hydroxyprogesterone (vs. HC). In addition, female ME/CFSmm showed a significant increase in progesterone levels compared to HC. In contrast, our study found that male ME/CFSmm had lower circulating levels of cortisol and corticosterone, while progesterone levels were elevated compared to HC. In addition to these univariate analyses, our correlational and multivariate approaches identified differential associations between our study groups. Also, using two-component partial least squares discriminant analysis (PLS-DA), we were able to discriminate ME/CFS from HC with an accuracy of 0.712 and 0.846 for females and males, respectively. CONCLUSION: Our findings suggest the potential value of including steroid hormones in future studies aimed at improving stratification in ME/CFS. Additionally, our results provide new perspectives to explore the clinical relevance of these differences within specific patient subgroups.

Cytosolic phosphoenolpyruvate carboxykinase is expressed in α-cells from human and murine pancreas.

The pancreatic islets of Langerhans, mainly formed by glucagon-producing α-cells and insulin-producing ß-cells, are critical for glucose homeostasis. Insulin and glucagon oppositely modulate blood glucose levels in health, but a combined decline in insulin secretion together with increased glucagon secretion contribute to hyperglycemia in diabetes. Despite this bi-hormonal dysregulation, most studies have focused on insulin secretion and much less is known about glucagon secretion. Therefore, a deeper understanding of α-cell metabolism and glucagon secretion is of great interest. Here, we show that phosphoenolpyruvate carboxykinase (PCK1), an essential cataplerotic enzyme involved in metabolism and long considered to be absent from the pancreatic islet, is expressed in pancreatic α-cells of both murine and human. Furthermore, PCK1 transcription is induced by fasting and diabetes in rat pancreas, which indicates that the PCK1 activity is required for α-cell adaptation to different metabolic states. To our knowledge, this is the first evidence implicating PCK1 expression in α-cell metabolism.

Sodium tungstate: Is it a safe option for a chronic disease setting, such as diabetes?

Diabetes is a complex metabolic disorder triggered by the deficient secretion of insulin by pancreatic ß cells, the resistance of peripheral tissues to the action of the hormone, or both, and is characterized by chronic hyperglycemia leading to organ damage and failure. Tight glycemic control represents the best therapy to delay or stop progression of diabetes, with many antidiabetic drugs being commercially available nowadays. However, no ideal normoglycemic agent has been developed as yet, and those already available still induce hypoglycemia and/or weight gain as major side effects, worsening glycemic control. In this respect, the inorganic salt sodium tungstate (Na2 WO4 ) has been proven to offer a good antidiabetic alternative in different animal models of diabetes, reducing body weight and normalizing glycemia without causing hypoglycemic episodes. The mechanisms of action mediating the potent antidiabetic actions but also the spectrum of undesirable effects of Na2 WO4 are still poorly understood. In fact, along with its beneficial effects, Na2 WO4 has been consistently reported to be toxic and even carcinogenic. Given that Na2 WO4 is accumulated in the kidneys for elimination, here, we discuss a possible association between long-term Na2 WO4 treatment and a higher risk of renal carcinogenesis in diabetic individuals.

Anti-Diabetic Agent Sodium Tungstate Induces the Secretion of Pro- and Anti-Inflammatory Cytokines by Human Kidney Cells.

Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (NaW) exerts anti-diabetic and immunomodulatory activities in diabetic animal models. Here, we used primary cultures of renal proximal tubule epithelial cells derived from type-2-diabetic (D-RPTEC) and non-diabetic (N-RPTEC) subjects as in vitro models to study the effects of NaW on cytokine secretion, as these factors participate in intercellular regulation of inflammation, cell growth and death, differentiation, angiogenesis, development, and repair, all processes that are dysregulated during DKD. In basal conditions, D-RPTEC cells secreted higher levels of prototypical pro-inflammatory IL-6, IL-8, and MCP-1 than N-RPTEC cells, in agreement with their diabetic phenotype. Unexpectedly, NaW further induced IL-6, IL-8, and MCP-1 secretion in both N- and D-RPTEC, together with lower levels of IL-1 RA, IL-4, IL-10, and GM-CSF, suggesting that it may contribute to the extent of renal damage/repair during DKD. Besides, NaW induced the accumulation of IκBα, the main inhibitor protein of one major pathway involved in cytokine production, suggesting further anti-inflammatory effect in the long-term. A better understanding of the mechanisms involved in the interplay between the anti-diabetic and immunomodulatory properties of NaW will facilitate future studies about its clinical relevance. J. Cell. Physiol. 232: 355-362, 2017. © 2016 Wiley Periodicals, Inc.

Defective insulin signaling and mitochondrial dynamics in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a common consequence of longstanding type 2 diabetes mellitus (T2DM) and encompasses structural, morphological, functional, and metabolic abnormalities in the heart. Myocardial energy metabolism depends on mitochondria, which must generate sufficient ATP to meet the high energy demands of the myocardium. Dysfunctional mitochondria are involved in the pathophysiology of diabetic heart disease. A large body of evidence implicates myocardial insulin resistance in the pathogenesis of DCM. Recent studies show that insulin signaling influences myocardial energy metabolism by impacting cardiomyocyte mitochondrial dynamics and function under physiological conditions. However, comprehensive understanding of molecular mechanisms linking insulin signaling and changes in the architecture of the mitochondrial network in diabetic cardiomyopathy is lacking. This review summarizes our current understanding of how defective insulin signaling impacts cardiac function in diabetic cardiomyopathy and discusses the potential role of mitochondrial dynamics.

Insulin requires normal expression and signaling of insulin receptor A to reverse gestational diabetes-reduced adenosine transport in human umbilical vein endothelium.

Reduced adenosine uptake via human equilibrative nucleoside transporter 1 (hENT1) in human umbilical vein endothelial cells (HUVECs) from gestational diabetes mellitus (GDM) is reversed by insulin by restoring hENT1 expression. Insulin receptors A (IR-A) and B (IR-B) are expressed in HUVECs, and GDM results in higher IR-A mRNA expression vs. cells from normal pregnancies. We studied whether the reversal of GDM effects on transport by insulin depends on restoration of IR-A expression. We specifically measured hENT1 expression [mRNA, protein abundance, SLC29A1 (for hENT1) promoter activity] and activity (adenosine transport kinetics) and the role of IR-A/IR-B expression and signaling [total and phosphorylated 42 and 44 kDa mitogen-activated protein kinases (p44/42(mapk)) and Akt] in IR-A, IR-B, and IR-A/B knockdown HUVECs from normal (n = 33) or GDM (n = 33) pregnancies. GDM increases IR-A/IR-B mRNA expression (1.8-fold) and p44/42(mapk):Akt activity (2.7-fold) ratios. Insulin reversed GDM-reduced hENT1 expression and maximal transport capacity (V(max)/K(m)), and GDM-increased IR-A/IR-B mRNA expression and p44/42(mapk):Akt activity ratios to values in normal pregnancies. Insulin's effect was abolished in IR-A or IR-A/B knockdown cells. Thus, insulin requires normal IR-A expression and p44/42(mapk)/Akt signaling to restore GDM-reduced hENT1 expression and activity in HUVECs. This could be a protective mechanism for the placental macrovascular endothelial dysfunction seen in GDM.

Dexmedetomidine protects the heart against ischemia-reperfusion injury by an endothelial eNOS/NO dependent mechanism.

The alpha2-adrenergic receptor agonist Dexmedetomidine (Dex) is a sedative medication used by anesthesiologists. Dex protects the heart against ischemia-reperfusion (IR) and can also act as a preconditioning mimetic. The mechanisms involved in Dex-dependent cardiac preconditioning, and whether this action occurs directly or indirectly on cardiomyocytes, still remain unclear. The endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway and endothelial cells are known to play key roles in cardioprotection against IR injury. Therefore, the aims of this work were to evaluate whether the eNOS/NO pathway mediates the pharmacological cardiac effect of Dex, and whether endothelial cells are required in this cardioprotective action. Isolated adult rat hearts were treated with Dex (10nM) for 25min and the dimerization of eNOS and production of NO were measured. Hearts were then subjected to global IR (30/120min) and the role of the eNOS/NO pathway was evaluated. Dex promoted the activation of eNOS and production of NO. Dex reduced the infarct size and improved the left ventricle function recovery, but this effect was reversed when Dex was co-administered with inhibitors of the eNOS/NO/PKG pathway. In addition, Dex was unable to reduce cell death in isolated adult rat cardiomyocytes subjected to simulated IR. Cardiomyocyte death was attenuated by co-culturing them with endothelial cells pre-treated with Dex. In summary, our results show that Dex triggers cardiac protection by activating the eNOS/NO signaling pathway. This pharmacological effect of Dex requires its interaction with the endothelium.

Novel players in cardioprotection: Insulin like growth factor-1, angiotensin-(1-7) and angiotensin-(1-9).

Insulin-like growth factor-1, angiotensin-(1-7) and angiotensin-(1-9) have been proposed to be important mediators in cardioprotection. A large body of evidence indicates that insulin like growth factor-1 has pleotropic actions in the heart (i.e., contractility, metabolism, hypertrophy, autophagy, senescence and cell death) and, conversely, its deficiency is associated with impaired cardiac function. Recently, we reported that insulin like growth factor-1 receptor is also located in plasma membrane invaginations with perinuclear localization, highlighting the role of nuclear Ca(2+) signaling in the heart. In parallel, angiotensin-(1-7) and angiotensin (1-9) acting through Mas receptor and angiotensin type 2 receptor have emerged as a novel anti-hypertensive molecules promoting vasodilatation and preventing heart hypertrophy. In this review we discuss the scientific evidence available regarding insulin-like growth factor-1, angiotensin-(1-7) and angiotensin-(1-9) in cardioprotection and its potential application as novel therapeutic targets for treating cardiac diseases.

Myristate induces mitochondrial fragmentation and cardiomyocyte hypertrophy through mitochondrial E3 ubiquitin ligase MUL1.

Introduction: Cardiovascular diseases, especially metabolic-related disorders, are progressively growing worldwide due to high-fat-containing foods, which promote a deleterious response at the cellular level, termed lipotoxicity, or lipotoxic stress. At the cardiac level, saturated fatty acids have been directly associated with cardiomyocyte lipotoxicity through various pathological mechanisms involving mitochondrial dysfunction, oxidative stress, and ceramide production, among others. However, integrative regulators connecting saturated fatty acid-derived lipotoxic stress to mitochondrial and cardiomyocyte dysfunction remain elusive. Methods: Here, we worked with a cardiomyocyte lipotoxicity model, which uses the saturated fatty acid myristate, which promotes cardiomyocyte hypertrophy and insulin desensitization. Results: Using this model, we detected an increase in the mitochondrial E3 ubiquitin ligase, MUL1, a mitochondrial protein involved in the regulation of growth factor signaling, cell death, and, notably, mitochondrial dynamics. In this context, myristate increased MUL1 levels and induced mitochondrial fragmentation, associated with the decrease of the mitochondrial fusion protein MFN2, and with the increase of the mitochondrial fission protein DRP1, two targets of MUL1. Silencing of MUL1 prevented myristate-induced mitochondrial fragmentation and cardiomyocyte hypertrophy. Discussion: These data establish a novel connection between cardiomyocytes and lipotoxic stress, characterized by hypertrophy and fragmentation of the mitochondrial network, and an increase of the mitochondrial E3 ubiquitin ligase MUL1.

Association analysis between symptomology and herpesvirus IgG antibody concentrations in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and multiple sclerosis.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and multiple sclerosis (MS) are two complex and multifactorial diseases whose patients experience persistent fatigue, cognitive impairment, among other shared symptoms. The onset of these diseases has also been linked to acute herpesvirus infections or their reactivations. In this work, we re-analyzed a previously-described dataset related to IgG antibody responses to 6 herpesviruses (CMV - cytomegalovirus; EBV - Epstein-Barr virus; HHV6 - human herpesvirus-6; HSV1 and HSV2 - herpes simplex virus-1 and -2, respectively; VZV - varicella-zoster virus) from the United Kingdom ME/CFS biobank. The primary goal was to report the underlying symptomology and its association with herpesvirus IgG antibodies using data from 4 disease-trigger-based subgroups of ME/CFS patients (n = 222) and patients with MS (n = 46). The secondary objective was to assess whether serological data could distinguish ME/CFS and its subgroup from MS using a SuperLearner (SL) algorithm. There was evidence for a significant negative association between temporary eye insight disturbance and CMV antibody concentrations and for a significant positive association between bladder problems and EBV antibody concentrations in the MS group. In the ME/CFS or its subgroups, the most significant antibody-symptom association was obtained for increasing HSV1 antibody concentration and brain fog, a finding in line with a negative impact of HSV1 exposure on cognitive outcomes in both healthy and disease conditions. There was also evidence for a higher number of significant antibody-symptom associations in the MS group than in the ME/CFS group. When we combined all the serological data in an SL algorithm, we could distinguish three ME/CFS subgroups (unknown disease trigger, non-infection trigger, and an infection disease trigger confirmed in the lab at the time of the event) from the MS group. However, we could not find the same for the remaining ME/CFS group (related to an unconfirmed infection disease). In conclusion, IgG antibody data explains more the symptomology of MS patients than the one of ME/CFS patients. Given the fluctuating nature of symptoms in ME/CFS patients, the clinical implication of these findings remains to be determined with a longitudinal study. This study is likely to ascertain the robustness of the associations during natural disease course.

Understanding, diagnosing, and treating Myalgic encephalomyelitis/chronic fatigue syndrome - State of the art: Report of the 2nd international meeting at the Charité Fatigue Center.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a devastating disease affecting millions of people worldwide. Due to the 2019 pandemic of coronavirus disease (COVID-19), we are facing a significant increase of ME/CFS prevalence. On May 11th to 12th, 2023, the second international ME/CFS conference of the Charité Fatigue Center was held in Berlin, Germany, focusing on pathomechanisms, diagnosis, and treatment. During the two-day conference, more than 100 researchers from various research fields met on-site and over 700 attendees participated online to discuss the state of the art and novel findings in this field. Key topics from the conference included: the role of the immune system, dysfunction of endothelial and autonomic nervous system, and viral reactivation. Furthermore, there were presentations on innovative diagnostic measures and assessments for this complex disease, cutting-edge treatment approaches, and clinical studies. Despite the increased public attention due to the COVID-19 pandemic, the subsequent rise of Long COVID-19 cases, and the rise of funding opportunities to unravel the pathomechanisms underlying ME/CFS, this severe disease remains highly underresearched. Future adequately funded research efforts are needed to further explore the disease etiology and to identify diagnostic markers and targeted therapies.

Decreased NO production in endothelial cells exposed to plasma from ME/CFS patients.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disease characterized by severe and persistent fatigue. Along with clinical studies showing endothelial dysfunction (ED) in a subset of ME/CFS patients, we have recently reported altered ED-related microRNAs in plasma from affected individuals. Inadequate nitric oxide (NO), mainly produced by the endothelial isoform of nitric oxide synthase (eNOS) in endothelial cells (ECs), is a major cause of ED. In this study, we hypothesized that plasma from that cohort of ME/CFS patients induces eNOS-related ED in vitro. To test this, we cultured human umbilical vein endothelial cells (HUVECs) in the presence of plasma from either ME/CFS patients (ME/CFS-plasma, n = 11) or healthy controls (HC-plasma, n = 12). Then, we measured the NO production in the absence and presence of tyrosine kinase and G protein-coupled receptors agonists (TKRs and GPCRs, respectively), well-known to activate eNOS in ECs. Our data showed that HUVECs incubated with ME/CFS-plasma produced less NO either in the absence or presence of eNOS activators compared to ones in presence of HC-plasma. Also, the NO production elicited by bradykinin, histamine, and acetylcholine (GPCRs agonists) was more affected than the one triggered by insulin (TKR agonist). Finally, inhibitory eNOS phosphorylation at Thr495 was higher in HUVECs treated with ME/CFS-plasma compared to the same treatment with HC-plasma. In conclusion, this study in vitro shows a decreased NO production in HUVECs exposed to plasma from ME/CFS patients, suggesting an unreported role of eNOS in the pathophysiology of this disease.

Revisiting IgG Antibody Reactivity to Epstein-Barr Virus in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Its Potential Application to Disease Diagnosis.

Infections by the Epstein-Barr virus (EBV) are often at the disease onset of patients suffering from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). However, serological analyses of these infections remain inconclusive when comparing patients with healthy controls (HCs). In particular, it is unclear if certain EBV-derived antigens eliciting antibody responses have a biomarker potential for disease diagnosis. With this purpose, we re-analyzed a previously published microarray data on the IgG antibody responses against 3,054 EBV-related antigens in 92 patients with ME/CFS and 50 HCs. This re-analysis consisted of constructing different regression models for binary outcomes with the ability to classify patients and HCs. In these models, we tested for a possible interaction of different antibodies with age and gender. When analyzing the whole data set, there were no antibody responses that could distinguish patients from healthy controls. A similar finding was obtained when comparing patients with non-infectious or unknown disease trigger with healthy controls. However, when data analysis was restricted to the comparison between HCs and patients with a putative infection at their disease onset, we could identify stronger antibody responses against two candidate antigens (EBNA4_0529 and EBNA6_0070). Using antibody responses to these two antigens together with age and gender, the final classification model had an estimated sensitivity and specificity of 0.833 and 0.720, respectively. This reliable case-control discrimination suggested the use of the antibody levels related to these candidate viral epitopes as biomarkers for disease diagnosis in this subgroup of patients. To confirm this finding, a follow-up study will be conducted in a separate cohort of patients.

Insulin-stimulated L-arginine transport requires SLC7A1 gene expression and is associated with human umbilical vein relaxation.

Insulin causes endothelium-derived nitric oxide (NO)-dependent vascular relaxation, and increases L-arginine transport via cationic amino acid transporter 1 (hCAT-1) and endothelial NO synthase (eNOS) expression and activity in human umbilical vein endothelium (HUVEC). We studied insulin effect on SLC7A1 gene (hCAT-1) expression and hCAT-transport activity role in insulin-modulated human fetal vascular reactivity. HUVEC were used for L-arginine transport and L-[(3) H]citrulline formation (NOS activity) assays in absence or presence of N-ethylmaleimide (NEM) or L-lysine (L-arginine transport inhibitors). hCAT-1 protein abundance was estimated by Western blot, mRNA quantification by real time PCR, and SLC7A1 promoter activity by Luciferase activity (-1,606 and -650 bp promoter fragments from ATG). Specific protein 1 (Sp1), and total or phosphorylated eNOS protein was determined by Western blot. Sp1 activity (at four sites between -177 and -105 bp from ATG) was assayed by chromatin immunoprecipitation (ChIP) and vascular reactivity in umbilical vein rings. Insulin increased hCATs-L-arginine transport, maximal transport capacity (V(max) /K(m) ), and hCAT-1 expression. NEM and L-lysine blocked L-arginine transport. In addition, it was trans-stimulated (∼7.8-fold) by L-lysine in absence of insulin, but unaltered (~1.4-fold) in presence of insulin. Sp1 nuclear protein abundance and binding to DNA, and SLC7A1 promoter activity was increased by insulin. Insulin increased NO synthesis and caused endothelium-dependent vessel relaxation and reduced U46619-induced contraction, effects blocked by NEM and L-lysine, and dependent on extracellular L-arginine. We suggest that insulin induces human umbilical vein relaxation by increasing HUVEC L-arginine transport via hCATs (likely hCAT-1) most likely requiring Sp1-activated SLC7A1 expression.

The SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: analysis of high-throughput epigenetic and gene expression studies.

Patients affected by Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) show specific epigenetic and gene expression signatures of the disease. However, it is unknown whether these signatures include abnormal levels of the human angiotensin-converting enzymes, ACE and ACE2, the latter being the main receptor described for the host-cell invasion by SARS-CoV-2. To investigate that, we first re-analyzed available case-control epigenome-wide association studies based on DNA methylation data, and case-control gene expression studies based on microarray data. From these published studies, we found an association between ME/CFS and 4 potentially hypomethylated probes located in the ACE locus. We also found another disease association with one hypomethylated probe located in the transcription start site of ACE2. The same disease associations were obtained for women but not for men after performing sex-specific analyses. In contrast, a meta-analysis of gene expression levels could not provide evidence for a differentially expression of ACE and ACE2 in affected patients when compared to healthy controls. In line with this negative finding, the analysis of a new data set on the gene expression of ACE and ACE2 in peripheral blood mononuclear cells did not find any differences between a female cohort of 37 patients and 34 age-matched healthy controls. Future studies should be conducted to extend this investigation to other potential receptors used by SARS-CoV-2. These studies will help researchers and clinicians to improve the understanding of the health risk imposed by this virus when infecting patients affected by this debilitating disease.

Herpesviruses Serology Distinguishes Different Subgroups of Patients From the United Kingdom Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Biobank.

The evidence of an association between Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and chronic herpesviruses infections remains inconclusive. Two reasons for the lack of consistent evidence are the large heterogeneity of the patients' population with different disease triggers and the use of arbitrary cutoffs for defining seropositivity. In this work we re-analyzed previously published serological data related to 7 herpesvirus antigens. Patients with ME/CFS were subdivided into four subgroups related to the disease triggers: S0-42 patients who did not know their disease trigger; S1-43 patients who reported a non-infection trigger; S2-93 patients who reported an infection trigger, but that infection was not confirmed by a lab test; and S3-48 patients who reported an infection trigger and that infection was confirmed by a lab test. In accordance with a sensitivity analysis, the data were compared to those from 99 healthy controls allowing the seropositivity cutoffs to vary within a wide range of possible values. We found a negative association between S1 and seropositivity to Epstein-Barr virus (VCA and EBNA1 antigens) and Varicella-Zoster virus using specific seropositivity cutoff. However, this association was not significant when controlling for multiple testing. We also found that S3 had a lower seroprevalence to the human cytomegalovirus when compared to healthy controls for all cutoffs used for seropositivity and after adjusting for multiple testing using the Benjamini-Hochberg procedure. However, this association did not reach statistical significance when using Benjamini-Yekutieli procedure. In summary, herpesviruses serology could distinguish subgroups of ME/CFS patients according to their disease trigger, but this finding could be eventually affected by the problem of multiple testing.

The SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) in myalgic encephalomyelitis/chronic fatigue syndrome: A meta-analysis of public DNA methylation and gene expression data.

People with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) often report a high frequency of viral infections and flu-like symptoms during their disease course. Given that this reporting agrees with different immunological abnormalities and altered gene expression profiles observed in the disease, we aimed at answering whether the expression of the human angiotensin-converting enzyme 2 (ACE2), the major cell entry receptor for SARS-CoV-2, is also altered in these patients. In particular, a low expression of ACE2 could be indicative of a high risk of developing COVID-19. We then performed a meta-analysis of public data on CpG DNA methylation and gene expression of this enzyme and its homologous ACE protein in peripheral blood mononuclear cells and related subsets. We found that patients with ME/CFS have decreased methylation levels of four CpG probes in the ACE locus (cg09920557, cg19802564, cg21094739, and cg10468385) and of another probe in the promoter region of the ACE2 gene (cg08559914). We also found a decreased expression of ACE2 but not of ACE in patients when compared to healthy controls. Accordingly, in newly collected data, there was evidence for a significant higher proportion of samples with an ACE2 expression below the limit of detection in patients than healthy controls. Altogether, patients with ME/CFS can be at a higher COVID-19 risk and, if so, they should be considered a priority group for vaccination by public health authorities. To further support this conclusion, similar research is recommended for other human cell entry receptors and cell types, namely, those cells targeted by the virus.

Gluconeogenic Enzymes in ß-Cells: Pharmacological Targets for Improving Insulin Secretion.

Pancreatic ß-cells express the gluconeogenic enzymes glucose 6-phosphatase (G6Pase), fructose 1,6-bisphosphatase (FBP), and phosphoenolpyruvate (PEP) carboxykinase (PCK), which modulate glucose-stimulated insulin secretion (GSIS) through their ability to reverse otherwise irreversible glycolytic steps. Here, we review current knowledge about the expression and regulation of these enzymes in the context of manipulating them to improve insulin secretion in diabetics. Because the regulation of gluconeogenic enzymes in ß-cells is so poorly understood, we propose novel research avenues to study these enzymes as modulators of insulin secretion and ß-cell dysfunction, with especial attention to FBP, which constitutes an attractive target with an inhibitor under clinical evaluation at present.

Neonates from women with pregestational maternal obesity show reduced umbilical vein endothelial response to insulin.

OBJECTIVE: Pregestational maternal obesity (PGMO) associates with foetoplacental vascular endothelial dysfunction and higher risk for insulin resistance in the neonate. We characterised the PGMO consequences on the insulin response of the human foetoplacental vasculature. METHODS: Umbilical veins were from pregnancies where the mother was with PGMO (body mass index 30-42.3 kg/m2, n = 33) or normal pregestational weight (PGMN) (body mass index 19.5-24.4 kg/m2, n = 21) with total gestational weight gain within the physiological range. Umbilical vein ring segments were mounted in a myograph for isometric force measurements. Primary cultures of human umbilical vein endothelial cells were used in passage 3. Vessel rings and cells were exposed to 1 nmol/L insulin (20 min) in the absence or presence of 100 µmol/L NG-nitro-l-arginine methyl ester (inhibitor of nitric oxide synthase, NOS). RESULTS: Vessel rings from PGMO showed reduced nitric oxide synthase-activity dependent dilation to insulin or calcitonin-gene related peptide compared with PGMN. PGMO associated with higher inhibitor phosphorylation of the insulin receptor substrate 1 (IRS-1) and lower activator phosphorylation of protein kinase B/Akt (Akt). Cells from PGMO also showed lower nitric oxide level and reduced activator serine1177 but increased inhibitor threonine495 phosphorylation of endothelial nitric oxide synthase (eNOS) and saturable transport of l-arginine. HUVECs from PGMO were not responsive to insulin. CONCLUSION: The lack of response to insulin by the foetoplacental endothelium may result from reduced IRS-1/Akt/eNOS signalling in PGMO. These findings may result in higher risk of insulin resistance in neonates to PGMO pregnancies.