ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV.

Novel therapeutic strategies are needed to attenuate increased systemic and gut inflammation that contribute to morbidity and mortality in chronic HIV infection despite potent antiretroviral therapy (ART). The goal of this study is to use preclinical models of chronic treated HIV to determine whether the antioxidant and anti-inflammatory apoA-I mimetic peptides 6F and 4F attenuate systemic and gut inflammation in chronic HIV. We used two humanized murine models of HIV infection and gut explants from 10 uninfected and 10 HIV infected persons on potent ART, to determine the in vivo and ex vivo impact of apoA-I mimetics on systemic and intestinal inflammation in HIV. When compared to HIV infected humanized mice treated with ART alone, mice on oral apoA-I mimetic peptide 6F with ART had consistently reduced plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) that are products of ADAM17 sheddase activity. Oral 6F attenuated gut protein levels of ADAM17 that were increased in HIV-1 infected mice on potent ART compared to uninfected mice. Adding oxidized lipoproteins and endotoxin (LPS) ex vivo to gut explants from HIV infected persons increased levels of ADAM17 in myeloid and intestinal cells, which increased TNF-α and CX3CL1. Both 4F and 6F attenuated these changes. Our preclinical data suggest that apoA-I mimetic peptides provide a novel therapeutic strategy that can target increased protein levels of ADAM17 and its sheddase activity that contribute to intestinal and systemic inflammation in treated HIV. The large repertoire of inflammatory mediators involved in ADAM17 sheddase activity places it as a pivotal orchestrator of several inflammatory pathways associated with morbidity in chronic treated HIV that make it an attractive therapeutic target.

Mfn2 deletion in brown adipose tissue protects from insulin resistance and impairs thermogenesis.

BAT-controlled thermogenic activity is thought to be required for its capacity to prevent the development of insulin resistance. This hypothesis predicts that mediators of thermogenesis may help prevent diet-induced insulin resistance. We report that the mitochondrial fusion protein Mitofusin 2 (Mfn2) in BAT is essential for cold-stimulated thermogenesis, but promotes insulin resistance in obese mice. Mfn2 deletion in mice through Ucp1-cre (BAT-Mfn2-KO) causes BAT lipohypertrophy and cold intolerance. Surprisingly however, deletion of Mfn2 in mice fed a high fat diet (HFD) results in improved insulin sensitivity and resistance to obesity, while impaired cold-stimulated thermogenesis is maintained. Improvement in insulin sensitivity is associated with a gender-specific remodeling of BAT mitochondrial function. In females, BAT mitochondria increase their efficiency for ATP-synthesizing fat oxidation, whereas in BAT from males, complex I-driven respiration is decreased and glycolytic capacity is increased. Thus, BAT adaptation to obesity is regulated by Mfn2 and with BAT-Mfn2 absent, BAT contribution to prevention of insulin resistance is independent and inversely correlated to whole-body cold-stimulated thermogenesis.

Blood immune cells from people with HIV on antiviral regimens that contain tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF) have differential metabolic signatures.

BACKGROUND: Mitochondria regulate immune and organ function. It is unknown whether higher intracellular drug levels observed in peripheral blood mononuclear cells (PBMCs) treated with tenofovir alafenamide (TAF) compared to tenofovir disoproxil fumarate (TDF) may alter mitochondrial function and energy production in immune cells in HIV(+) patients. METHODS: Cellular bioenergetics were determined in PBMCs from HIV-1(-) participants exposed to TAF versus TDF in vitro, at a comparable concentration to a clinically relevant plasma exposure. A decrease in cellular oxygen consumption rate (OCR) at baseline (basal-OCR) and under cellular stress (max-OCR) may suggest mitochondrial dysfunction. We also assessed the in vivo impact of TAF vs TDF on OCR in PBMCs from 26 people with HIV (PWH) interchanged from TDF-based to TAF-based antiretroviral therapy (ART) over a 9-month period in the setting of an open label clinical trial. The Wilcoxon and Mann Whitney tests were used for comparison of continuous variables. RESULTS: PBMCs from HIV-1(-) participants exposed in vitro to a concentration of 0.12-3.3 µM for TAF and TDF at 2 and 24 h, reduced basal and maximal OCR compared to vehicle control. Switch studies of antivirals (TAF vs TDF) within the same PWH showed that TAF-based ART was associated with reduced OCR compared to TDF-based ART in PBMCs. We observed that TAF-treated PBMCs selectively relied more on glucose/pyruvate supply rather than fatty acid to fuel their mitochondria. CONCLUSIONS: Compared to TDF, TAF may alter bioenergetics in immune cells from PWH in vitro and in vivo. The clinical significance in terms of the differential impact caused by TAF versus TDF on mitochondrial function and energy production in immune cells, a regulator of immune function, requires further studied in HIV, preexposure prophylaxis and hepatitis B.

Mitoquinone mesylate targets SARS-CoV-2 infection in preclinical models.

To date, there is no effective oral antiviral against SARS-CoV-2 that is also anti-inflammatory. Herein, we show that the mitochondrial antioxidant mitoquinone/mitoquinol mesylate (Mito-MES), a dietary supplement, has potent antiviral activity against SARS-CoV-2 and its variants of concern in vitro and in vivo . Mito-MES had nanomolar in vitro antiviral potency against the Beta and Delta SARS-CoV-2 variants as well as the murine hepatitis virus (MHV-A59). Mito-MES given in SARS-CoV-2 infected K18-hACE2 mice through oral gavage reduced viral titer by nearly 4 log units relative to the vehicle group. We found in vitro that the antiviral effect of Mito-MES is attributable to its hydrophobic dTPP+ moiety and its combined effects scavenging reactive oxygen species (ROS), activating Nrf2 and increasing the host defense proteins TOM70 and MX1. Mito-MES was efficacious reducing increase in cleaved caspase-3 and inflammation induced by SARS-CoV2 infection both in lung epithelial cells and a transgenic mouse model of COVID-19. Mito-MES reduced production of IL-6 by SARS-CoV-2 infected epithelial cells through its antioxidant properties (Nrf2 agonist, coenzyme Q10 moiety) and the dTPP moiety. Given established safety of Mito-MES in humans, our results suggest that Mito-MES may represent a rapidly applicable therapeutic strategy that can be added in the therapeutic arsenal against COVID-19. Its potential long-term use by humans as diet supplement could help control the SARS-CoV-2 pandemic, especially in the setting of rapidly emerging SARS-CoV-2 variants that may compromise vaccine efficacy. One-Sentence Summary: Mitoquinone/mitoquinol mesylate has potent antiviral and anti-inflammatory activity in preclinical models of SARS-CoV-2 infection.

The ratio of SRPK1/SRPK1a regulates erythroid differentiation in K562 leukaemic cells.

SRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less studied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. Small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. A decreased SRPK1/SRPK1a ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells as well as in normal human erythroid progenitor cells. Mass spectrometric analysis of SRPK1a-associated proteins identified multiple classes of RNA-binding proteins including RNA helicases, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and mRNA-associated proteins. Several of the SRPK1a-copurifying proteins have been previously identified in ribosomal and pre-ribosomal complexes, thereby suggesting that SRPK1a may play an important role in linking ribosomal assembly and/or function to erythroid differentiation in human leukaemic cells.

DLST-dependence dictates metabolic heterogeneity in TCA-cycle usage among triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is traditionally considered a glycolytic tumor with a poor prognosis while lacking targeted therapies. Here we show that high expression of dihydrolipoamide S-succinyltransferase (DLST), a tricarboxylic acid (TCA) cycle enzyme, predicts poor overall and recurrence-free survival among TNBC patients. DLST depletion suppresses growth and induces death in subsets of human TNBC cell lines, which are capable of utilizing glutamine anaplerosis. Metabolomics profiling reveals significant changes in the TCA cycle and reactive oxygen species (ROS) related pathways for sensitive but not resistant TNBC cells. Consequently, DLST depletion in sensitive TNBC cells increases ROS levels while N-acetyl-L-cysteine partially rescues cell growth. Importantly, suppression of the TCA cycle through DLST depletion or CPI-613, a drug currently in clinical trials for treating other cancers, decreases the burden and invasion of these TNBC. Together, our data demonstrate differential TCA-cycle usage in TNBC and provide therapeutic implications for the DLST-dependent subsets.

The ApoA-I mimetic peptide 4F attenuates in vitro replication of SARS-CoV-2, associated apoptosis, oxidative stress and inflammation in epithelial cells.

An oral antiviral against SARS-CoV-2 that also attenuates inflammatory instigators of severe COVID-19 is not available to date. Herein, we show that the apoA-I mimetic peptide 4 F inhibits Spike mediated viral entry and has antiviral activity against SARS-CoV-2 in human lung epithelial Calu3 and Vero-E6 cells. In SARS-CoV-2 infected Calu3 cells, 4 F upregulated inducers of the interferon pathway such as MX-1 and Heme oxygenase 1 (HO-1) and downregulated mitochondrial reactive oxygen species (mito-ROS) and CD147, a host protein that mediates viral entry. 4 F also reduced associated cellular apoptosis and secretion of IL-6 in both SARS-CoV-2 infected Vero-E6 and Calu3 cells. Thus, 4 F attenuates in vitro SARS-CoV-2 replication, associated apoptosis in epithelial cells and secretion of IL-6, a major cytokine related to COVID-19 morbidity. Given established safety of 4 F in humans, clinical studies are warranted to establish 4 F as therapy for COVID-19.

Apolipoprotein A-I mimetics attenuate macrophage activation in chronic treated HIV.

OBJECTIVES: Despite antiretroviral therapy (ART), there is an unmet need for therapies to mitigate immune activation in HIV infection. The goal of this study is to determine whether the apoA-I mimetics 6F and 4F attenuate macrophage activation in chronic HIV. DESIGN: Preclinical assessment of the in-vivo impact of Tg6F and the ex-vivo impact of apoA-I mimetics on biomarkers of immune activation and gut barrier dysfunction in treated HIV. METHODS: We used two humanized murine models of HIV infection to determine the impact of oral Tg6F with ART (HIV+ART+Tg6F+) on innate immune activation (plasma human sCD14, sCD163) and gut barrier dysfunction [murine I-FABP, endotoxin (LPS), LPS-binding protein (LBP), murine sCD14]. We also used gut explants from 10 uninfected and 10 HIV-infected men on potent ART and no morbidity, to determine the impact of ex-vivo treatment with 4F for 72 h on secretion of sCD14, sCD163, and I-FABP from gut explants. RESULTS: When compared with mice treated with ART alone (HIV+ART+), HIV+ART+Tg6F+ mice attenuated macrophage activation (h-sCD14, h-sCD163), gut barrier dysfunction (m-IFABP, LPS, LBP, and m-sCD14), plasma and gut tissue oxidized lipoproteins. The results were consistent with independent mouse models and ART regimens. Both 4F and 6F attenuated shedding of I-FABP and sCD14 from gut explants from HIV-infected and uninfected participants. CONCLUSION: Given that gut barrier dysfunction and macrophage activation are contributors to comorbidities like cardiovascular disease in HIV, apoA-I mimetics should be tested as therapy for morbidity in chronic treated HIV.

Mitochondrial Proton Leak Regulated by Cyclophilin D Elevates Insulin Secretion in Islets at Nonstimulatory Glucose Levels.

Fasting hyperinsulinemia precedes the development of type 2 diabetes. However, it is unclear whether fasting insulin hypersecretion is a primary driver of insulin resistance or a consequence of the progressive increase in fasting glycemia induced by insulin resistance in the prediabetic state. Herein, we have discovered a mechanism that specifically regulates non-glucose-stimulated insulin secretion (NGSIS) in pancreatic islets that is activated by nonesterified free fatty acids, the major fuel used by ß-cells during fasting. We show that the mitochondrial permeability transition pore regulator cyclophilin D (CypD) promotes NGSIS, but not glucose-stimulated insulin secretion, by increasing mitochondrial proton leak. Islets from prediabetic obese mice show significantly higher CypD-dependent proton leak and NGSIS compared with lean mice. Proton leak-mediated NGSIS is conserved in human islets and is stimulated by exposure to nonesterified free fatty acids at concentrations observed in obese subjects. Mechanistically, proton leak activates islet NGSIS independently of mitochondrial ATP synthesis but ultimately requires closure of the KATP channel. In summary, we have described a novel nonesterified free fatty acid-stimulated pathway that selectively drives pancreatic islet NGSIS, which may be therapeutically exploited as an alternative way to halt fasting hyperinsulinemia and the progression of type 2 diabetes.

IAPP toxicity activates HIF1α/PFKFB3 signaling delaying ß-cell loss at the expense of ß-cell function.

The islet in type 2 diabetes (T2D) is characterized by amyloid deposits derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by ß-cells. In common with amyloidogenic proteins implicated in neurodegeneration, human IAPP (hIAPP) forms membrane permeant toxic oligomers implicated in misfolded protein stress. Here, we establish that hIAPP misfolded protein stress activates HIF1α/PFKFB3 signaling, this increases glycolysis disengaged from oxidative phosphorylation with mitochondrial fragmentation and perinuclear clustering, considered a protective posture against increased cytosolic Ca2+ characteristic of toxic oligomer stress. In contrast to tissues with the capacity to regenerate, ß-cells in adult humans are minimally replicative, and therefore fail to execute the second pro-regenerative phase of the HIF1α/PFKFB3 injury pathway. Instead, ß-cells in T2D remain trapped in the pro-survival first phase of the HIF1α injury repair response with metabolism and the mitochondrial network adapted to slow the rate of cell attrition at the expense of ß-cell function.

Publisher Correction: IAPP toxicity activates HIF1α/PFKFB3 signaling delaying ß-cell loss at the expense of ß-cell function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors.

OBJECTIVE: Islets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function. METHODS: We have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies). RESULTS: By increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 µm2 area (∼210 µm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time. CONCLUSIONS: We have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research.

Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis.

Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96) analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes.

Variant-specific patterns and humoral regulation of HP1 proteins in human cells and tissues.

We have examined the occurrence and distribution of HP1alpha and HP1beta under in vivo, ex vivo and in vitro conditions. Consistent with a non-essential role in heterochromatin maintenance, both proteins are diminished or undetectable in several types of differentiated cells and are universally downregulated during erythropoiesis. Variant-specific patterns are observed in almost all human and mouse tissues examined. Yet, the most instructive example of HP1 plasticity is observed in the lymph nodes, where HP1alpha and HP1beta exhibit regional patterns that are exactly complementary to one another. Furthermore, whereas HP1alpha shows a dispersed sub-nuclear distribution in the majority of peripheral lymphocytes, it coalesces into large heterochromatic foci upon stimulation with various mitogens and IL-2. The effect of inductive signals on HP1alpha distribution is reproduced by coculture of immortalized T- and B-cells and can be confirmed using specific markers. These complex patterns reveal an unexpected plasticity in HP1 variant expression and strongly suggest that the sub-nuclear distribution of HP1 proteins is regulated by humoral signals and microenvironmental cues.