Peptide-encoding gene transfer to modulate intracellular protein-protein interactions.

Peptide drug discovery has great potential, but the cell membrane is a major obstacle when the target is an intracellular protein-protein interaction (PPI). It is difficult to target PPIs with small molecules; indeed, there are no intervention tools that can target any intracellular PPI. In this study, we developed a platform that enables the introduction of peptides into cells via mRNA-based gene delivery. Peptide-length nucleic acids do not enable stable ribosome binding and exhibit little to no translation into protein. In this study, a construct was created in which the sequence encoding dihydrofolate reductase (DHFR) was placed in front of the sequence encoding the target peptide, together with a translation skipping sequence, as a sequence that meets the requirements of promoting ribosome binding and rapid decay of the translated protein. This enabled efficient translation from the mRNA encoding the target protein while preventing unnecessary protein residues. Using this construct, we showed that it can inhibit Drp1/Fis1 binding, one of the intracellular PPIs, which governs mitochondrial fission, an important aspect of mitochondrial dynamics. In addition, it was shown to inhibit pathological hyperfission, normalize mitochondrial dynamics and metabolism, and inhibit apoptosis of the mitochondrial pathway.

ZLN005 improves the survival of polymicrobial sepsis by increasing the bacterial killing via inducing lysosomal acidification and biogenesis in phagocytes.

Polymicrobial sepsis still has a high mortality rate despite the development of antimicrobial agents, elaborate strategies to protect major organs, and the investment of numerous medical resources. Mitochondrial dysfunction, which acts as the center of energy metabolism, is clearly the basis of pathogenesis. Drugs that act on PGC1α, the master regulator of mitochondrial biosynthesis, have shown useful effects in the treatment of sepsis; therefore, we investigated the efficacy of ZLN005, a PGC1α agonist, and found significant improvement in overall survival in an animal model. The mode of action of this effect was examined, and it was shown that the respiratory capacity of mitochondria was enhanced immediately after administration and that the function of TFEB, a transcriptional regulator that promotes lysosome biosynthesis and mutually enhances PGC1α, was enhanced, as was the physical contact between mitochondria and lysosomes. ZLN005 strongly supported immune defense in early sepsis by increasing lysosome volume and acidity and enhancing cargo degradation, resulting in a significant reduction in bacterial load. ZLN005 rapidly acted on two organelles, mitochondria and lysosomes, against sepsis and interactively linked the two to improve the pathogenesis. This is the first demonstration that acidification of lysosomes by a small molecule is a mechanism of action in the therapeutic strategy for sepsis, which will have a significant impact on future drug discovery.

Maternal high-fat diet promotes calcified atherosclerotic plaque formation in adult offspring by enhancing transformation of VSMCs to osteochondrocytic-like phenotype.

Aim: Maternal high-fat diet (HFD) is associated with the development of cardiovascular disease (CVD) in adult offspring. Atherosclerotic vascular calcification is well documented in patients with CVD. We examined the effect of maternal HFD on calcified plaque formation. Methods and results: Seven-week-old female apo-E-/- mice (C57BL6/J) were nourished either an HFD or a normal diet (ND) a week before mating, and during gestation and lactation. Offspring of both the groups were fed a high-cholesterol diet (HCD) from 8 weeks of age. Osteogenic activity of the thoracic aorta, assessed using an ex vivo imaging system, was significantly increased after 3 months of HCD in male offspring of HFD-fed dams (O-HFD) as compared with those of ND-fed dams (O-ND). Alizarin-red-positive area in the aortic root was significantly increased after 6 months of HCD in male O-HFD as compared to that of O-ND. Plaque size and Oil Red O-positive staining were comparable between the two groups. Primary cultured vascular smooth muscle cells (VSMCs) of the thoracic aorta were treated with phosphate and interleukinL-1ß (IL-1ß) to transform them into an osteochondrocytic-like phenotype. Intracellular calcium content and alkaline phosphatase activity were markedly higher in the VSMCs of O-HFD than in O-ND. IL-1ß concentration in the supernatant of bone marrow-derived macrophages was markedly higher in O-HFD than in O-ND. Conclusion: Our findings indicate that maternal HFD accelerates the expansion of atherogenic calcification independent of plaque progression. In vitro phosphate- and IL-1ß-induced osteochondrocytic transformation of VSMCs was augmented in O-HFD. Inhibition of VSMCs, skewing toward osteochondrocytic-like cells, might be a potential therapeutic strategy for preventing maternal HFD-associated CVD development.

A novel mRNA decay inhibitor abolishes pathophysiological cellular transition.

In cells, mRNA synthesis and decay are influenced by each other, and their balance is altered by either external or internal cues, resulting in changes in cell dynamics. We previously reported that it is important that an array of mRNAs that shape a phenotype are degraded before cellular transitions, such as cellular reprogramming and differentiation. In adipogenesis, the interaction between DDX6 and 4E-T had a definitive impact on the pathway in the processing body (PB). We screened a library of α-helix analogs with an alkaloid-like backbone to identify compounds that inhibit the binding between DDX6 and 4E-T proteins, which occurs between the α-helix of structured and internally disordered proteins. IAMC-00192 was identified as a lead compound. This compound directly inhibited the interaction between DDX6 and 4E-T. IAMC-00192 inhibited the temporal increase in PB formation that occurs during adipogenesis and epithelial-mesenchymal transition (EMT) and significantly suppressed these cellular transitions. In the EMT model, the half-life of preexisting mRNAs in PBs was extended twofold by the compound. The novel inhibitor of RNA decay not only represents a potentially useful tool to analyze in detail the pathological conditions affected by RNA decay and how it regulates the pathological state. The identification of this inhibitor may lead to the discovery of a first-in-class RNA decay inhibitor drug.

Amelioration of Endotoxemia by a Synthetic Analog of Omega-3 Epoxyeicosanoids.

Sepsis, a systemic inflammatory response to pathogenic factors, is a difficult to treat life-threatening condition associated with cytokine and eicosanoid storms and multi-organ damage. Omega-3 polyunsaturated fatty acids, such as eicosapentaenoic (EPA) and docosahexaenoic acid, are the precursors of potent anti-inflammatory lipid mediators, including 17,18-epoxyeicosatetraenoic acid (17,18-EEQ), the main metabolite of EPA generated by cytochrome P450 epoxygenases. Searching for novel therapeutic or preventative agents in sepsis, we tested a metabolically robust synthetic analog of 17,18-EEQ (EEQ-A) for its ability to reduce mortality, organ damage, and pro-inflammatory cytokine transcript level in a mouse model of lipopolysaccharide (LPS)-induced endotoxemia, which is closely related to sepsis. Overall survival significantly improved following preventative EEQ-A administration along with decreased transcript level of pro-inflammatory cytokines. On the other hand, the therapeutic protocol was effective in improving survival at 48 hours but insignificant at 72 hours. Histopathological analyses showed significant reductions in hemorrhagic and necrotic damage and infiltration in the liver. In vitro studies with THP-1 and U937 cells showed EEQ-A mediated repression of LPS-induced M1 polarization and enhancement of IL-4-induced M2 polarization of macrophages. Moreover, EEQ-A attenuated the LPS-induced decline of mitochondrial function in THP-1 cells, as indicated by increased basal respiration and ATP production as well as reduction of the metabolic shift to glycolysis. Taken together, these data demonstrate that EEQ-A has potent anti-inflammatory and immunomodulatory properties that may support therapeutic strategies for ameliorating the endotoxemia.

Repeated Social Defeat Enhances CaCl2-Induced Abdominal Aortic Aneurysm Expansion by Inhibiting the Early Fibrotic Response via the MAPK-MKP-1 Pathway.

Depression is an independent risk factor for cardiovascular disease and is significantly associated with the prevalence of abdominal aortic aneurysm (AAA). We investigated the effect of repeated social defeat (RSD) on AAA development. Eight-week-old male wild-type mice were exposed to RSD by being housed with larger CD-1 mice in a shared cage. They were subjected to vigorous physical contact. After the confirmation of depressive-like behavior, calcium chloride was applied to the infrarenal aorta of the mice. At one week, AAA development was comparable between the defeated and control mice, without any differences being observed in the accumulated macrophages or in the matrix metalloproteinase activity. At two weeks, the maximum diameter and circumference of the aneurysm were significantly increased in the defeated mice, and a significant decrease in periaortic fibrosis was also observed. Consistently, the phosphorylation of the extracellular signal-regulated kinase and the incorporation of 5-bromo-2'-deoxyuridine in the primarily cultured aortic vascular smooth muscle cells were significantly reduced in the defeated mice, which was accompanied by a substantial increase in mitogen-activated protein kinase phosphatase-1 (MKP-1). The MKP-1 mRNA and protein expression levels during AAA were much higher in the defeated mice than they were in the control mice. Our findings demonstrate that RSD enhances AAA development by suppressing periaortic fibrosis after an acute inflammatory response and imply novel mechanisms that are associated with depression-related AAA development.

A tissue-engineered, decellularized, connective tissue membrane for allogeneic arterial patch implantation.

BACKGROUND: We have previously applied in vivo tissue-engineered vascular grafts constructed in patients' subcutaneous spaces. However, since the formation of these vascular grafts depends on host health, their application is challenging in patients with suppressed regenerative ability. Therefore, the allogeneic implantation of grafts from healthy donors needs to be evaluated. This study aimed to fabricate allogeneic cardiovascular grafts in animals. MATERIALS AND METHODS: Silicone rod molds were implanted into subcutaneous pouches in dogs; the implants, along with surrounding connective tissues, were harvested after four weeks. Tubular connective tissues were decellularized and stored before they were cut open, trimmed to elliptical sheets, and implanted into the common carotid arteries of another dog as vascular patches (n = 6); these were resected and histologically evaluated at 1, 2, and 4 weeks after implantation. RESULTS: No aneurysmal changes were observed by echocardiography. Histologically, we observed neointima formation on the luminal graft surface and graft wall cell infiltration. At 2 and 4 weeks after implantation, α-SMA-positive cells were observed in the neointima and graft wall. At 4 weeks after implantation, the endothelial lining was observed at the grafts' luminal surfaces. CONCLUSION: Our data suggest that decellularized connective tissue membranes can be prepared and stored for later use as allogeneic cardiovascular grafts.

Genetically Modified Cell Transplantation Through Macroencapsulated Spheroids with Scaffolds to Treat Fabry Disease.

Cell transplantation is expected to be another strategy to treat lysosomal diseases, having several advantages compared to enzyme replacement therapy, such as continuous enzyme secretion and one-time treatment to cure diseases. However, cell transplantation for lysosomal diseases holds issues to be resolved for the clinical field. In this study, we developed a new ex vivo gene therapy platform using a transplant pack, which consists of a porous membrane made of ethylene-vinyl alcohol in the pack-type and spheroids with scaffolds. These membranes have countless pores of less than 0.1 µm2 capable of secreting proteins, including alpha-galactosidase enzyme, and segregating the contents from the host immune system. When the packs were subcutaneously transplanted into the backs of green fluorescent protein (GFP) mice, no GFP-positive cells migrated to the transplanted pack in either autogenic or allogenic mice. The transplanted cells in the pack survived for 28 days after transplantation. When cells overexpressing alpha-galactosidase were used as donor cells for the packs and implanted into Fabry disease model mice, the accumulation of the alpha-galactosidase enzyme was also observed in the livers. In this study, we reported a new ex vivo therapeutic strategy combining macroencapsulation and cellular spheroids with scaffolds. This pack, macroencapsulated spheroids with scaffolds, can also be applied to other types of lysosomal diseases by modifying genes of interest.

Repeated Social Defeat Exaggerates Fibrin-Rich Clot Formation by Enhancing Neutrophil Extracellular Trap Formation via Platelet-Neutrophil Interactions.

Depression is an independent risk factor for cardiovascular disease (CVD). We have previously shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular trap (NET) formation. In this study, we investigated the impact of RSD on arterial thrombosis. Eight-week-old male wild-type mice (C57BL/6J) were exposed to RSD by housing with larger CD-1 mice in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. After confirming depression-like behaviors, mice underwent FeCl3-induced carotid arterial injury and were analyzed after 3 h. Although the volume of thrombi was comparable between the two groups, fibrin(ogen)-positive areas were significantly increased in defeated mice, in which Ly-6G-positive cells were appreciably co-localized with Cit-H3-positive staining. Treatment with DNase I completely diminished exaggerated fibrin-rich clot formation in defeated mice. Flow cytometric analysis showed that neutrophil CD11b expression before FeCl3 application was significantly higher in defeated mice than in control mice. In vitro NET formation induced by activated platelets was significantly augmented in defeated mice, which was substantially inhibited by anti-CD11b antibody treatment. Our findings demonstrate that RSD enhances fibrin-rich clot formation after arterial injury by enhancing NET formation, suggesting that NET can be a new therapeutic target in depression-related CVD.

Maternal High-Fat Diet Promotes Abdominal Aortic Aneurysm Expansion in Adult Offspring by Epigenetic Regulation of IRF8-Mediated Osteoclast-like Macrophage Differentiation.

Maternal high-fat diet (HFD) modulates vascular remodeling in adult offspring. Here, we investigated the impact of maternal HFD on abdominal aortic aneurysm (AAA) development. Female wild-type mice were fed an HFD or normal diet (ND). AAA was induced in eight-week-old pups using calcium chloride. Male offspring of HFD-fed dams (O-HFD) showed a significant enlargement in AAA compared with the offspring of ND-fed dams (O-ND). Positive-staining cells for tartrate-resistant acid phosphate (TRAP) and matrix metalloproteinase (MMP) activity were significantly increased in O-HFD. The pharmacological inhibition of osteoclastogenesis abolished the exaggerated AAA development in O-HFD. The in vitro tumor necrosis factor-α-induced osteoclast-like differentiation of bone marrow-derived macrophages showed a higher number of TRAP-positive cells and osteoclast-specific gene expressions in O-HFD. Consistent with an increased expression of nuclear factor of activated T cells 1 (NFATc1) in O-HFD, the nuclear protein expression of interferon regulatory factor 8 (IRF8), a transcriptional repressor, were much lower, with significantly increased H3K27me3 marks at the promoter region. The enhancer of zeste homolog 2 inhibitor treatment restored IRF8 expression, resulting in no difference in NFATc1 and TRAP expressions between the two groups. Our findings demonstrate that maternal HFD augments AAA expansion, accompanied by exaggerated osteoclast-like macrophage accumulation, suggesting the possibility of macrophage skewing via epigenetic reprogramming.

Generation of somatic mitochondrial DNA-replaced cells for mitochondrial dysfunction treatment.

Mitochondrial diseases currently have no cure regardless of whether the cause is a nuclear or mitochondrial genome mutation. Mitochondrial dysfunction notably affects a wide range of disorders in aged individuals, including neurodegenerative diseases, cancers, and even senescence. Here, we present a procedure to generate mitochondrial DNA-replaced somatic cells with a combination of a temporal reduction in endogenous mitochondrial DNA and coincubation with exogeneous isolated mitochondria. Heteroplasmy in mitochondrial disease patient-derived fibroblasts in which the mutant genotype was dominant over the wild-type genotype was reversed. Mitochondrial disease patient-derived fibroblasts regained respiratory function and showed lifespan extension. Mitochondrial membranous components were utilized as a vehicle to deliver the genetic materials into endogenous mitochondria-like horizontal genetic transfer in prokaryotes. Mitochondrial DNA-replaced cells could be a resource for transplantation to treat maternal inherited mitochondrial diseases.

Modifications of the mechanical properties of in vivo tissue-engineered vascular grafts by chemical treatments for a short duration.

In vivo tissue-engineered vascular grafts constructed in the subcutaneous spaces of graft recipients have functioned well clinically. Because the formation of vascular graft tissues depends on several recipient conditions, chemical pretreatments, such as dehydration by ethanol (ET) or crosslinking by glutaraldehyde (GA), have been attempted to improve the initial mechanical durability of the tissues. Here, we compared the effects of short-duration (10 min) chemical treatments on the mechanical properties of tissues. Tubular tissues (internal diameter, 5 mm) constructed in the subcutaneous tissues of beagle dogs (4 weeks, n = 3), were classified into three groups: raw tissue without any treatment (RAW), tissue dehydrated with 70% ET (ET), and tissue crosslinked with 0.6% GA (GA). Five mechanical parameters were measured: burst pressure, suture retention strength, ultimate tensile strength (UTS), ultimate strain (%), and Young's modulus. The tissues were also autologously re-embedded into the subcutaneous spaces of the same dogs for 4 weeks (n = 2) for the evaluation of histological responses. The burst pressure of the RAW group (1275.9 ± 254.0 mm Hg) was significantly lower than those of ET (2115.1 ± 262.2 mm Hg, p = 0.0298) and GA (2570.5 ± 282.6 mm Hg, p = 0.0017) groups. Suture retention strength, UTS or the ultimate strain did not differ significantly among the groups. Young's modulus of the ET group was the highest (RAW: 5.41 ± 1.16 MPa, ET: 12.28 ± 2.55 MPa, GA: 7.65 ± 1.18 MPa, p = 0.0185). No significant inflammatory tissue response or evidence of residual chemical toxicity was observed in samples implanted subcutaneously for four weeks. Therefore, short-duration ET and GA treatment might improve surgical handling and the mechanical properties of in vivo tissue-engineered vascular tissues to produce ideal grafts in terms of mechanical properties without interfering with histological responses.

RNA decay in processing bodies is indispensable for adipogenesis.

The RNA decay pathway plays key regulatory roles in cell identities and differentiation processes. Although adipogenesis is transcriptionally and epigenetically regulated and has been thoroughly investigated, how RNA metabolism that contributes to the stability of phenotype-shaping transcriptomes participates in differentiation remains elusive. In this study, we investigated Ddx6, an essential component of processing bodies (PBs) that executes RNA decay and translational repression in the cytoplasm and participates in the cellular transition of reprogramming. Upon adipogenic induction, Ddx6 dynamically accumulated to form PBs with a binding partner, 4E-T, at the early phase prior to emergence of intracellular lipid droplets. In contrast, preadipocytes with Ddx6 knockout (KO) or 4E-T knockdown (KD) failed to generate PBs, resulting in significant suppression of adipogenesis. Transcription factors related to preadipocytes and negative regulators of adipogenesis that were not expressed under adipogenic stimulation were maintained in Ddx6-KO and 4E-T-KD preadipocytes under adipogenic induction. Elimination of Dlk1, a major negative regulator of adipogenesis, in 3T3L1 Ddx6-KO cells did not restore adipogenic differentiation capacity to any extent. Similar to murine cells, human primary mesenchymal stem cells, which can differentiate into adipocytes upon stimulation with adipogenic cocktails, required DDX6 to maturate into adipocytes. Therefore, RNA decay of the entire parental transcriptome, rather than removal of a strong negative regulator, could be indispensable for adipogenesis.

Effects of Short-Duration Ethanol Dehydration on Mechanical Properties of Porcine Pericardium.

PURPOSE: Autologous pericardium is an ideal material for cardiovascular reconstruction including pulmonary artery plasty. Despite the fact that dehydration by ethanol has been used to improve its surgical handling, the effects of the ethanol on mechanical properties of the pericardium have not been previously investigated. The effects of short-duration ethanol dehydration on the mechanical properties of porcine pericardium were evaluated. METHODS: Porcine pericardia (n = 3) were separated into three groups: the raw group with no treatments (RAW), the group immersed in 70% ethanol for 10 min (ET group), and the group immersed in 0.6% glutaraldehyde for 10 min (GA). We measured five parameters of mechanical properties as specified in ISO 7198. RESULTS: ET treatment improved surgical handling as well as GA treatment. There were no significant differences in burst pressure (P = 0.639), suture retention strength (P = 0.529), ultimate tensile strength (UTS; P = 0.486), or Young's modulus (P = 0.408). Only the ultimate strain of the GA group was significantly higher among the three groups (RAW: 33.34% ± 2.02%, ET: 37.48% ± 1.84%, GA: 44.74% ± 2.87%; P = 0.046). CONCLUSIONS: Short-duration ethanol dehydration did not compromise its mechanical properties while maintaining its surgical handling improvements.

Cell Transplantation Combined with Recombinant Collagen Peptides for the Treatment of Fabry Disease.

Fabry disease is caused by a decrease in or loss of the activity of alpha-galactosidase, which causes its substrates globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) to accumulate in cells throughout the body. This accumulation results in progressive kidney injury due to glomerulosclerosis and in heart failure due to hypertrophy. Enzyme replacement therapy (ERT) has been used as the standard therapy for Fabry disease, but it causes a significant financial burden, and regular administration is inconvenient for patients. Because of the short half-life of alpha-galactosidase in vivo, therapeutic methods that can supplement or replace ERT are expected to involve continuous release of alpha-galactosidase, even at low doses. Cell transplantation therapy is one of these methods; however, its use has been hindered by the short-term survival of transplanted cells. CellSaic technology, which utilizes cell spheroids that form after cells are seeded simultaneously with a recombinant collagen peptide scaffold called a µ-piece, has been used to improve cell survival upon implantation. In this study, syngeneic murine embryonic fibroblasts were used to generate CellSaic that were transplanted into Fabry mice. These spheroids survived for 28 days in the renal subcapsular space with forming blood vessels. These results indicate CellSaic technology could be a platform to promote cellular graft survival and may facilitate the development of cell transplantation methods for lysosomal diseases.

From Cell Entry to Engraftment of Exogenous Mitochondria.

Mitochondrial transfer has been recognized to play a role in a variety of processes, ranging from fertilization to cancer and neurodegenerative diseases as well as mammalian horizontal gene transfer. It is achieved through either exogeneous or intercellular mitochondrial transfer. From the viewpoint of evolution, exogeneous mitochondrial transfer is quite akin to the initial process of symbiosis between α-protobacterium and archaea, although the progeny have developed more sophisticated machinery to engulf environmental materials, including nutrients, bacteria, and viruses. A molecular-based knowledge of endocytosis, including macropinocytosis and endosomal escape involving bacteria and viruses, could provide mechanistic insights into exogeneous mitochondrial transfer. We focus on exogeneous mitochondrial transfer in this review to facilitate the clinical development of the use of isolated mitochondria to treat various pathological conditions. Several kinds of novel procedures to enhance exogeneous mitochondrial transfer have been developed and are summarized in this review.

High throughput single cell analysis of mitochondrial heteroplasmy in mitochondrial diseases.

Mitochondrial heteroplasmy, which fundamentally means intracellular heterogeneity of mitochondrial DNA (mtDNA), has been measured in a group of cells, regardless of intercellular heterogeneity. Ordinal methods for mitochondrial heteroplasmy cannot discriminate between an intercellular homogenic population composed of cells with similar intracellular heterogeneity for mtDNA and an intercellular heterogenic population composed of cells with different rates of mutated mtDNA. A high-throughput method to determine mitochondrial heteroplasmy in a single cell was developed by using droplet digital PCR with TaqMan polymerase in this study. This technique revealed that there are three different cell populations of cultured fibroblasts derived from patients with mitochondrial disease carrying a mutation in the mtDNA; cells with homoplasmy of either mutated or healthy mtDNA; and cells mixed with mutated and healthy mtDNA. The presence of intercellular heterogeneity, even in uniformed cultured fibroblasts, suggests that heterogeneity should exist among different kinds of cells. The diagnosis of intercellular heterogeneity with respect to mitochondrial heteroplasmy by this methodology could provide novel insight into developing a treatment strategy for mitochondrial diseases.

Social Stress Increases Vulnerability to High-Fat Diet-Induced Insulin Resistance by Enhancing Neutrophil Elastase Activity in Adipose Tissue.

Social stress (SS) has been linked to the development of cardiovascular disease (CVD), which is closely associated with insulin resistance (IR); however, the causal effect of SS on IR remains unclear. The 8-week-old male C57BL/6 mice were exposed to SS by housing with a larger CD-1 mouse in a shared home cage without physical contact for 10 consecutive days followed by high-fat diet (HFD) feeding. Control mice were housed in the same cage without a CD-1 mouse. After 6 weeks of HFD, insulin sensitivity was significantly impaired in stressed mice. While the percentage of classically activated macrophages in epididymal white adipose tissue (eWAT) was equivalent between the two groups, the percentage of lymphocyte antigen 6 complex locus G6D (Ly-6G)/neutrophil elastase (NE)-double positive cells markedly increased in stressed mice, accompanied by augmented NE activity assessed by ex vivo eWAT fluorescent imaging. Treatment with an NE inhibitor completely abrogated the insulin sensitivity impairment of stressed mice. In vitro NE release upon stimulation with a formyl peptide receptor 1 agonist was significantly higher in bone marrow neutrophils of stressed mice. Our findings show that SS-exposed mice are susceptible to the development of HFD-induced IR accompanied by augmented NE activity. Modulation of neutrophil function may represent a potential therapeutic target for SS-associated IR.