Author Correction: Design of amidobenzimidazole STING receptor agonists with systemic activity.

Change history: In this Letter, author Ana Puhl was inadvertently omitted; this error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Design of amidobenzimidazole STING receptor agonists with systemic activity.

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.

Human gut microbiota after bariatric surgery alters intestinal morphology and glucose absorption in mice independently of obesity.

OBJECTIVE: Bariatric surgery is an effective treatment for type 2 diabetes (T2D) that changes gut microbial composition. We determined whether the gut microbiota in humans after restrictive or malabsorptive bariatric surgery was sufficient to lower blood glucose. DESIGN: Women with obesity and T2D had biliopancreatic diversion with duodenal switch (BPD-DS) or laparoscopic sleeve gastrectomy (LSG). Faecal samples from the same patient before and after each surgery were used to colonise rodents, and determinants of blood glucose control were assessed. RESULTS: Glucose tolerance was improved in germ-free mice orally colonised for 7 weeks with human microbiota after either BPD-DS or LSG, whereas food intake, fat mass, insulin resistance, secretion and clearance were unchanged. Mice colonised with microbiota post-BPD-DS had lower villus height/width and crypt depth in the distal jejunum and lower intestinal glucose absorption. Inhibition of sodium-glucose cotransporter (Sglt)1 abrogated microbiota-transmissible improvements in blood glucose control in mice. In specific pathogen-free (SPF) rats, intrajejunal colonisation for 4 weeks with microbiota post-BPD-DS was sufficient to improve blood glucose control, which was negated after intrajejunal Sglt-1 inhibition. Higher Parabacteroides and lower Blautia coincided with improvements in blood glucose control after colonisation with human bacteria post-BPD-DS and LSG. CONCLUSION: Exposure of rodents to human gut microbiota after restrictive or malabsorptive bariatric surgery improves glycaemic control. The gut microbiota after bariatric surgery is a standalone factor that alters upper gut intestinal morphology and lowers Sglt1-mediated intestinal glucose absorption, which improves blood glucose control independently from changes in obesity, insulin or insulin resistance.

Inflammation promotes adipocyte lipolysis via IRE1 kinase.

Obesity associates with inflammation, insulin resistance, and higher blood lipids. It is unclear if immune responses facilitate lipid breakdown and release from adipocytes via lipolysis in a separate way from hormones or adrenergic signals. We found that an ancient component of ER stress, inositol-requiring protein 1 (IRE1), discriminates inflammation-induced adipocyte lipolysis versus lipolysis from adrenergic or hormonal stimuli. Our data show that inhibiting IRE1 kinase activity was sufficient to block adipocyte-autonomous lipolysis from multiple inflammatory ligands, including bacterial components, certain cytokines, and thapsigargin-induced ER stress. IRE1-mediated lipolysis was specific for inflammatory triggers since IRE1 kinase activity was dispensable for isoproterenol and cAMP-induced lipolysis in adipocytes and mouse adipose tissue. IRE1 RNase activity was not associated with inflammation-induced adipocyte lipolysis. Inhibiting IRE1 kinase activity blocked NF-κB activation, interleukin-6 secretion, and adipocyte-autonomous lipolysis from inflammatory ligands. Inflammation-induced lipolysis mediated by IRE1 occurred independently from changes in insulin signaling in adipocytes, suggesting that inflammation can promote IRE1-mediated lipolysis independent of adipocyte insulin resistance. We found no role for canonical unfolded protein responses or ABL kinases in linking ER stress to IRE1-mediated lipolysis. Adiponectin-Cre-mediated IRE1 knockout in mice showed that adipocyte IRE1 was required for inflammatory ligand-induced lipolysis in adipose tissue explants and that adipocyte IRE1 was required for approximately half of the increase in blood triglycerides after a bacterial endotoxin-mediated inflammatory stimulus in vivo. Together, our results show that IRE1 propagates an inflammation-specific lipolytic program independent from hormonal or adrenergic regulation. Targeting IRE1 kinase activity may benefit metabolic syndrome and inflammatory lipid disorders.

MALT1 Protease Plays a Dual Role in the Allergic Response by Acting in Both Mast Cells and Endothelial Cells.

The signaling protein MALT1 plays a key role in promoting NF-κB activation in Ag-stimulated lymphocytes. In this capacity, MALT1 has two functions, acting as a scaffolding protein and as a substrate-specific protease. MALT1 is also required for NF-κB-dependent induction of proinflammatory cytokines after FcεR1 stimulation in mast cells, implicating a role in allergy. Because MALT1 remains understudied in this context, we sought to investigate how MALT1 proteolytic activity contributes to the overall allergic response. We compared bone marrow-derived mast cells from MALT1 knockout (MALT1-/-) and MALT1 protease-deficient (MALTPD/PD) mice to wild-type cells. We found that MALT1-/- and MALT1PD/PD mast cells are equally impaired in cytokine production following FcεRI stimulation, indicating that MALT1 scaffolding activity is insufficient to drive the cytokine response and that MALT1 protease activity is essential. In addition to cytokine production, acute mast cell degranulation is a critical component of allergic response. Intriguingly, whereas degranulation is MALT1-independent, MALT1PD/PD mice are protected from vascular edema induced by either passive cutaneous anaphylaxis or direct challenge with histamine, a major granule component. This suggests a role for MALT1 protease activity in endothelial cells targeted by mast cell-derived vasoactive substances. Indeed, we find that in human endothelial cells, MALT1 protease is activated following histamine treatment and is required for histamine-induced permeability. We thus propose a dual role for MALT1 protease in allergic response, mediating 1) IgE-dependent mast cell cytokine production, and 2) histamine-induced endothelial permeability. This dual role indicates that therapeutic inhibitors of MALT1 protease could work synergistically to control IgE-mediated allergic disease.

NOD2 in hepatocytes engages a liver-gut axis to protect against steatosis, fibrosis, and gut dysbiosis during fatty liver disease in mice.

Obesity promotes nonalcoholic fatty liver disease (NAFLD). The intestinal microbiota contributes to NAFLD progression through a gut-to-liver pathway that promotes inflammation and fibrosis. Gut microbiota-derived factors can travel to the liver and activate immune responses in liver resident cells to promote inflammation and NAFLD. Little is known about bacterial sensors or immune responses that can protect against NAFLD. We tested whether the bacterial cell wall sensor nucleotide-binding oligomerization domain-containing (NOD)2 protects against diet-induced NAFLD in mice. Whole body deletion of NOD2 exacerbated liver steatosis and fibrosis in mice fed a NAFLD-promoting diet. Mice with a hepatocyte-specific deletion of NOD2 (Nod2-/-HKO) also had higher liver steatosis and fibrosis compared with littermate wild-type mice (WT) fed a NAFLD-promoting diet. Hepatocyte-specific NOD2 deletion altered the composition of the gut microbiome. Nod2-/-HKO mice had increased relative abundance of Clostridiales and lower Erysipelotrichaceae among other changes in cecal bacteria compared with littermate WT mice. Hepatocyte-specific NOD2 deletion altered a transcriptional program of liver inflammation, metabolism, and fibrosis. Nod2-/-HKO mice had higher levels of transcripts involved in lipid and cholesterol metabolism. Nod2-/-HKO mice had higher transcript levels of transforming growth factor-ß and collagen isoforms, which coincided with higher levels of liver collagen compared with WT mice. These data show that bacterial cell wall sensing within hepatocytes can engage retrograde cross-talk from the liver to the gut, where liver immunity communicates with the gut to influence the intestinal host-microbe relationship during diet-induced NAFLD, and NOD2 within the hepatocyte confers protection from liver steatosis and fibrosis.

Postbiotics for NOD2 require nonhematopoietic RIPK2 to improve blood glucose and metabolic inflammation in mice.

Defining the host receptors and metabolic consequences of bacterial components can help explain how the microbiome influences metabolic diseases. Bacterial peptidoglycans that activate nucleotide-binding oligomerization domain-containing (NOD)1 worsen glucose control, whereas NOD2 activation improves glycemia. Receptor-interacting serine/threonine-protein kinase 2 (RIPK2) is required for innate immunity instigated by NOD1 and NOD2. The role of RIPK2 in the divergent effects of NOD1 versus NOD2 on blood glucose was unknown. We found that whole body deletion of RIPK2 negated all effects of NOD1 or NOD2 activation on blood glucose during an acute, low level endotoxin challenge in mice. It was known that NOD1 in hematopoietic cells participates in insulin resistance and metabolic inflammation in obese mice. It was unknown if RIPK2 in hematopoietic cells is required for the glucose-lowering and anti-inflammatory effects of NOD2 activation. We hypothesized that RIPK2 in nonhematopoietic cells dictated the glycemic effects of NOD2 activation. We found that whole body deletion of RIPK2 prevented the glucose-lowering effects of repeated NOD2 activation that were evident during a glucose tolerance test (GTT) in high-fat diet (HFD)-fed wild-type (WT) mice. NOD2 activation lowered glucose during a GTT and lowered adipose tissue inflammation in mice with RIPK2 deleted in hematopoietic cells. We conclude that RIPK2 in nonhematopoietic cells mediates the glucose lowering and anti-inflammatory effects of NOD2-activating postbiotics. We propose a model where lipopolysaccharides and NOD1 ligands synergize in hematopoietic cells to promote insulin resistance but NOD2 activation in nonhematopoietic cells promotes RIPK2-dependent immune tolerance and lowering of inflammation and insulin resistance.

S100A12 and S100A8/9 proteins are biomarkers of articular disease activity in Blau syndrome.

OBJECTIVE: To identify biomarkers of articular and ocular disease activity in patients with Blau syndrome (BS). METHODS: Multiplex plasma protein arrays were performed in five BS patients and eight normal healthy volunteers (NHVs). Plasma S100A12 and S100A8/9 were subsequently measured by ELISA at baseline and 1-year follow-up in all patients from a prospective multicentre cohort study. CRP was measured using Meso Scale Discovery immunoassay. Active joint counts, standardization uveitis nomenclature for anterior uveitis cells and vitreous haze by Nussenblatt scale were the clinical parameters. RESULTS: Multiplex Luminex arrays identified S100A12 as the most significantly elevated protein in five selected BS vs eight NHVs and this was confirmed by ELISA on additional samples from the same five BS patients. In the patient cohort, S100A12 (n = 39) and S100A8/9 (n = 33) were significantly higher compared with NHVs (n = 44 for S100A12, n = 40 for S100A8/9) (P = 0.0000004 and P = 0.0003, respectively). Positive correlations between active joint counts and S100 levels were significant for S100A12 (P = 0.0008) and S100A8/9 (P = 0.015). CRP levels did not correlate with active joint count. Subgroup analysis showed significant association of S100 proteins with active arthritis (S100A12 P = 0.01, S100A8/9 P = 0.008). Active uveitis was not associated with increased S100 levels. CONCLUSION: S100 proteins are biomarkers of articular disease activity in BS and potential outcome measures in future clinical trials. As secreted neutrophil and macrophage products, S100 proteins may reflect the burden of granulomatous tissue in BS.

The NLRP3 inflammasome contributes to sarcopenia and lower muscle glycolytic potential in old mice.

The mechanisms underpinning decreased skeletal muscle strength and slowing of movement during aging are ill-defined. "Inflammaging," increased inflammation with advancing age, may contribute to aspects of sarcopenia, but little is known about the participatory immune components. We discovered that aging was associated with increased caspase-1 activity in mouse skeletal muscle. We hypothesized that the caspase-1-containing NLRP3 inflammasome contributes to sarcopenia in mice. Male C57BL/6J wild-type (WT) and NLRP3-/- mice were aged to 10 (adult) and 24 mo (old). NLRP3-/- mice were protected from decreased muscle mass (relative to body mass) and decreased size of type IIB and IIA myofibers, which occurred between 10 and 24 mo of age in WT mice. Old NLRP3-/- mice also had increased relative muscle strength and endurance and were protected from age-related increases in the number of myopathic fibers. We found no evidence of age-related or NLRP3-dependent changes in markers of systemic inflammation. Increased caspase-1 activity was associated with GAPDH proteolysis and reduced GAPDH enzymatic activity in skeletal muscles from old WT mice. Aging did not alter caspase-1 activity, GAPDH proteolysis, or GAPDH activity in skeletal muscles of NLRP3-/- mice. Our results show that the NLRP3 inflammasome participates in age-related loss of muscle glycolytic potential. Deletion of NLRP3 mitigates both the decline in glycolytic myofiber size and the reduced activity of glycolytic enzymes in muscle during aging. We propose that the etiology of sarcopenia involves direct communication between immune responses and metabolic flux in skeletal muscle.

Blau syndrome: cross-sectional data from a multicentre study of clinical, radiological and functional outcomes.

OBJECTIVE: To report baseline articular, functional and ocular findings of the first international prospective cohort study of Blau syndrome (BS). METHODS: Three-year, multicentre, observational study on articular, functional (HAQ, Childhood HAQ and VAS global and pain), ophthalmological, therapeutic and radiological data in BS patients. RESULTS: Baseline data on the first 31 recruited patients (12 females and 19 males) from 18 centres in 11 countries are presented. Of the 31 patients, 11 carried the p.R334W NOD2 mutation, 9 the p.R334Q and 11 various other NOD2 missense mutations; 20 patients were sporadic and 11 from five BS pedigrees. Median disease duration was 12.8 years (1.1-57). Arthritis, documented in all but one patient, was oligoarticular in 7, polyarticular in 23. The median active joint count was 21. Functional capacity was normal in 41%, mildly impaired in 31% and moderate-severe in 28% of patients. The most frequently involved joints at presentation were wrists, ankles, knees and PIPs. On radiographs, a symmetrical non-erosive arthropathy was shown. Previously unknown dysplastic bony changes were found in two-thirds of patients. Ocular disease was documented in 25 of 31 patients, with vitreous inflammation in 64% and moderate-severe visual loss in 33%. Expanded manifestations (visceral, vascular) beyond the classic clinical triad were seen in 52%. CONCLUSION: BS is associated with severe ocular and articular morbidity. Visceral involvement is common and may be life-threatening. Bone dysplastic changes may show diagnostic value and suggest a previously unknown role of NOD2 in bone morphogenesis. BS is resistant to current drugs, suggesting the need for novel targeted therapies.

Signal transduction meets vesicle traffic: the software and hardware of GLUT4 translocation.

Skeletal muscle is the major tissue disposing of dietary glucose, a function regulated by insulin-elicited signals that impart mobilization of GLUT4 glucose transporters to the plasma membrane. This phenomenon, also central to adipocyte biology, has been the subject of intense and productive research for decades. We focus on muscle cell studies scrutinizing insulin signals and vesicle traffic in a spatiotemporal manner. Using the analogy of an integrated circuit to approach the intersection between signal transduction and vesicle mobilization, we identify signaling relays ("software") that engage structural/mechanical elements ("hardware") to enact the rapid mobilization and incorporation of GLUT4 into the cell surface. We emphasize how insulin signal transduction switches from tyrosine through lipid and serine phosphorylation down to activation of small G proteins of the Rab and Rho families, describe key negative regulation step of Rab GTPases through the GTPase-activating protein activity of the Akt substrate of 160 kDa (AS160), and focus on the mechanical effectors engaged by Rabs 8A and 10 (the molecular motor myosin Va), and the Rho GTPase Rac1 (actin filament branching and severing through Arp2/3 and cofilin). Finally, we illustrate how actin filaments interact with myosin 1c and α-Actinin4 to promote vesicle tethering as preamble to fusion with the membrane.

Stress biology: Complexity and multifariousness in health and disease.

Preserving and regulating cellular homeostasis in the light of changing environmental conditions or developmental processes is of pivotal importance for single cellular and multicellular organisms alike. To counteract an imbalance in cellular homeostasis transcriptional programs evolved, called the heat shock response, unfolded protein response, and integrated stress response, that act cell-autonomously in most cells but in multicellular organisms are subjected to cell-nonautonomous regulation. These transcriptional programs downregulate the expression of most genes but increase the expression of heat shock genes, including genes encoding molecular chaperones and proteases, proteins involved in the repair of stress-induced damage to macromolecules and cellular structures. Sixty-one years after the discovery of the heat shock response by Ferruccio Ritossa, many aspects of stress biology are still enigmatic. Recent progress in the understanding of stress responses and molecular chaperones was reported at the 12th International Symposium on Heat Shock Proteins in Biology, Medicine and the Environment in the Old Town Alexandria, VA, USA from 28th to 31st of October 2023.

The vascular disrupting agent STA-9584 exhibits potent antitumor activity by selectively targeting microvasculature at both the center and periphery of tumors.

Vascular disrupting agents (VDAs) are an emerging class of therapeutics targeting the existing vascular network of solid tumors. However, their clinical progression has been hampered because of limited single-agent efficacy, primarily caused by the persistence of surviving cells at the well perfused "viable rim" of tumors, which allows rapid tumor regrowth to occur. In addition, off-target adverse events, including cardiovascular toxicities, underscore a need for compounds with improved safety profiles. Here, we characterize the mechanism of action, antitumor efficacy, and cardiovascular safety profile of (S)-2-amino-N-(2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)phenyl)-3-phenylpropanamide hydrochloride (STA-9584), a novel tubulin-binding VDA. In vitro, 2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)aniline (STA-9122) (active metabolite of STA-9584) displayed increased potency relative to other tubulin-binding agents and was highly cytotoxic to tumor cells. STA-9584 induced significant tumor regressions in prostate and breast xenograft models in vivo and, in an aggressive syngeneic model, demonstrated superior tumor growth inhibition and a positive therapeutic index relative to combretastatin A-4 phosphate (CA4P). It is noteworthy that histological analysis revealed that STA-9584 disrupted microvasculature at both the center and periphery of tumors. Compared with CA4P, STA-9584 induced a 73% increase in central necrotic area, 77% decrease in microvasculature, and 7-fold increase in tumor cell apoptosis in the remaining viable rim 24 h post-treatment. Ultrasound imaging confirmed that STA-9584 rapidly and efficiently blocked blood flow in highly perfused tumor regions. Moreover, cardiovascular effects were evaluated in the Langendorff assay and telemetered dogs, and cardiovascular toxicity was not predicted to be dose-limiting. This bioactivity profile distinguishes STA-9584 from the combretastatin class and identifies the compound as a promising new therapeutic VDA candidate.

Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias.

The aberrant overexpression of Wilms tumor 1 (WT1) in myeloid leukemia plays an important role in blast cell survival and resistance to chemotherapy. High expression of WT1 is also associated with relapse and shortened disease-free survival in patients. However, the mechanisms by which WT1 expression is regulated in leukemia remain unclear. Here, we report that heat shock protein 90 (Hsp90), which plays a critical role in the folding and maturation of several oncogenic proteins, associates with WT1 protein and stabilizes its expression. Pharmacologic inhibition of Hsp90 resulted in ubiquitination and subsequent proteasome-dependant degradation of WT1. RNAi-mediated silencing of WT1 reduced the survival of leukemia cells and increased the sensitivity of these cells to chemotherapy and Hsp90 inhibition. Furthermore, Hsp90 inhibitors 17-AAG [17-(allylamino)-17-demethoxygeldanamycin] and STA-9090 significantly reduced the growth of myeloid leukemia xenografts in vivo and effectively down-regulated the expression of WT1 and its downstream target proteins, c-Myc and Bcl-2. Collectively, our studies identify WT1 as a novel Hsp90 client and support the crucial role for the WT1-Hsp90 interaction in maintaining leukemia cell survival. These findings have significant implications for developing effective therapies for myeloid leukemias and offer a strategy to inhibit the oncogenic functions of WT1 by clinically available Hsp90 inhibitors.

Synergistic activity of the Hsp90 inhibitor ganetespib with taxanes in non-small cell lung cancer models.

Systemic chemotherapy using two-drug platinum-based regimens for the treatment of advanced stage non-small cell lung cancer (NSCLC) has largely reached a plateau of effectiveness. Accordingly, efforts to improve survival and quality of life outcomes have more recently focused on the use of molecularly targeted agents, either alone or in combination with standard of care therapies such as taxanes. The molecular chaperone heat shock protein 90 (Hsp90) represents an attractive candidate for therapeutic intervention, as its inhibition results in the simultaneous blockade of multiple oncogenic signaling cascades. Ganetespib is a non-ansamycin inhibitor of Hsp90 currently under clinical evaluation in a number of human malignancies, including NSCLC. Here we show that ganetespib potentiates the cytotoxic activity of the taxanes paclitaxel and docetaxel in NSCLC models. The combination of ganetespib with paclitaxel, docetaxel or another microtubule-targeted agent vincristine resulted in synergistic antiproliferative effects in the H1975 cell line in vitro. These benefits translated to improved efficacy in H1975 xenografts in vivo, with significantly enhanced tumor growth inhibition observed in combination with paclitaxel and tumor regressions seen with docetaxel. Notably, concurrent exposure to ganetespib and docetaxel improved antitumor activity in 5 of 6 NSCLC xenograft models examined. Our data suggest that the improved therapeutic indices are likely to be mechanistically multifactorial, including loss of pro-survival signaling and direct cell cycle effects resulting from Hsp90 modulation by ganetespib. Taken together, these findings provide preclinical evidence for the use of this combination to treat patients with advanced NSCLC.

Life-long exercise training and inherited aerobic endurance capacity produce converging gut microbiome signatures in rodents.

High aerobic endurance capacity can be acquired by training and/or inherited. Aerobic exercise training (AET) and aging are linked to altered gut microbiome composition, but it is unknown if the environmental stress of exercise and host genetics that predispose for higher exercise capacity have similar effects on the gut microbiome during aging. We hypothesized that exercise training and host genetics would have conserved effects on the gut microbiome across different rodents. We studied young sedentary (Y-SED, 2-month-old) mice, old sedentary (O-SED, 26-month-old) mice, old mice with life-long AET (O-AET, 26-month-old), and aged rats selectively bred for high (HCR [High Capacity Runner], 21-month-old) and low (LCR [Low Capacity Runner], 21-month-old) aerobic capacity. Our results showed that O-SED mice had lower running capacity than Y-SED mice. The fecal microbiota of O-SED mice had a higher relative abundance of Lachnospiraceae, Ruminococcaceae, Turicibacteriaceae, and Allobaculum, but lower Bacteroidales, Alistipes, Akkermansia, and Anaeroplasma. O-AET mice had a higher running capacity than O-SED mice. O-AET mice had lower fecal levels of Lachnospiraceae, Turicibacteriaceae, and Allobaculum and higher Anaeroplasma than O-SED mice. Similar to O-AET mice, but despite no exercise training regime, aged HCR rats had lower Lachnospiraceae and Ruminococcaceae and expansion of certain Bacteroidales in the fecal microbiome compared to LCR rats. Our data show that environmental and genetic modifiers of high aerobic endurance capacity produce convergent gut microbiome signatures across different rodent species during aging. Therefore, we conclude that host genetics and life-long exercise influence the composition of the gut microbiome and can mitigate gut dysbiosis and functional decline during aging.

Effects of Obesity-Associated Chronic Inflammation on Peripheral Blood Immunophenotype Are Not Mediated by TNF in Female C57BL/6J Mice.

Chronic low-grade systemic inflammation in obesity contributes to the development and progression of aspects of metabolic syndrome. In obese male mice, expanded adipose tissue releases proinflammatory cytokines, including TNF, which promotes an increase in immature, proinflammatory, peripheral blood Ly-6Chigh monocytes. The aim of this study was to characterize how TNF alters circulating cellular immunity in female mice with diet-induced obesity. We initially quantified peripheral blood immune cells by flow cytometry in female wild-type C57BL/6J mice after 3-30 wk of allocation to a high-fat (HF) or standard chow diet. We assessed effects of diet and time on neutrophil, monocyte, B cell, NK cell, CD4+ T cell, and CD8+ T cell populations. There was a significant interaction of the effects of diet type and time on the numbers and prevalence of circulating total monocytes and Ly-6Chigh, Ly-6Clow, and Ly-6C- subsets. Circulating monocytes, in particular Ly-6Chigh monocytes, were increased in HF-fed mice compared with chow-fed mice. Ly-6Chigh monocytes from HF-fed mice also had a more immature phenotype yet were highly responsive to the chemotactic ligand CCL2 and had greater intracellular production of TNF. Comparisons of the effects of HF diet feeding in littermate wild-type (TNF+/+) and TNF-/- female mice showed that genetic ablation of TNF did not protect from higher adiposity or an increase in circulating, immature, proinflammatory Ly-6Chigh monocytes during HF diet-induced obesity. These data emphasize the importance of considering biological sex when determining the mechanisms of TNF action in obesity-induced cellular inflammation and in other chronic inflammatory conditions.

Lower brown adipose tissue activity is associated with non-alcoholic fatty liver disease but not changes in the gut microbiota.

In rodents, lower brown adipose tissue (BAT) activity is associated with greater liver steatosis and changes in the gut microbiome. However, little is known about these relationships in humans. In adults (n = 60), we assessed hepatic fat and cold-stimulated BAT activity using magnetic resonance imaging and the gut microbiota with 16S sequencing. We transplanted gnotobiotic mice with feces from humans to assess the transferability of BAT activity through the microbiota. Individuals with NAFLD (n = 29) have lower BAT activity than those without, and BAT activity is inversely related to hepatic fat content. BAT activity is not related to the characteristics of the fecal microbiota and is not transmissible through fecal transplantation to mice. Thus, low BAT activity is associated with higher hepatic fat accumulation in human adults, but this does not appear to have been mediated through the gut microbiota.

Contraction-related stimuli regulate GLUT4 traffic in C2C12-GLUT4myc skeletal muscle cells.

Muscle contraction stimulates glucose uptake acutely to increase energy supply, but suitable cellular models that faithfully reproduce this complex phenomenon are lacking. To this end, we have developed a cellular model of contracting C(2)C(12) myotubes overexpressing GLUT4 with an exofacial myc-epitope tag (GLUT4myc) and explored stimulation of GLUT4 traffic by physiologically relevant agents. Carbachol (an acetylcholine receptor agonist) induced a gain in cell surface GLUT4myc that was mediated by nicotinic acetylcholine receptors. Carbachol also activated AMPK, and this response was sensitive to the contractile myosin ATPase inhibitor N-benzyl-p-toluenesulfonamide. The gain in surface GLUT4myc elicited by carbachol or by the AMPK activator 5-amino-4-carboxamide-1 beta-ribose was sensitive to chemical inhibition of AMPK activity by compound C and partially reduced by siRNA-mediated knockdown of AMPK catalytic subunits or LKB1. In addition, the carbachol-induced gain in cell surface GLUT4myc was partially sensitive to chelation of intracellular calcium with BAPTA-AM. However, the carbachol-induced gain in cell surface GLUT4myc was not sensitive to the CaMKK inhibitor STO-609 despite expression of both isoforms of this enzyme and a rise in cytosolic calcium by carbachol. Therefore, separate AMPK- and calcium-dependent signals contribute to mobilizing GLUT4 in response to carbachol, providing an in vitro cell model that recapitulates the two major signals whereby acute contraction regulates glucose uptake in skeletal muscle. This system will be ideal to further analyze the underlying molecular events of contraction-regulated GLUT4 traffic.

RIPK2 Dictates Insulin Responses to Tyrosine Kinase Inhibitors in Obese Male Mice.

Tyrosine kinase inhibitors (TKIs) used in cancer are also being investigated in diabetes. TKIs can improve blood glucose control in diabetic cancer patients, but the specific kinases that alter blood glucose or insulin are not clear. We sought to define the role of Receptor Interacting Serine/Threonine Kinase 2 (RIPK2) in mouse models of insulin resistance. We tested the TKI gefitinib, which inhibits RIPK2 activity, in wild-type (WT), Nod1-/-, Nod2-/-, and Ripk2-/- mice fed an obesogenic high-fat diet. Gefitinib lowered blood glucose during a glucose tolerance test (GTT) in a nucleotide-binding oligomerization domain (NOD)-RIPK2-independent manner in all obese mice. However, gefitinib lowered glucose-stimulated insulin secretion only in obese Ripk2-/- mice. Gefitinib had no effect on insulin secretion in obese WT, Nod1-/-, or Nod2-/- mice. Hence, genetic deletion of Ripk2 promoted the insulin-sensitizing potential of gefitinib, since this TKI lowered both blood glucose and insulin only in Ripk2-/- mice. Gefitinib did not alter the inflammatory profile of pancreas, adipose, liver, or muscle tissues in obese Ripk2-/- mice compared with obese WT mice. We also tested imatinib, a TKI that does not inhibit RIPK2 activity, in obese WT mice. Imatinib lowered blood glucose during a GTT, consistent with TKIs lowering blood glucose independently of RIPK2. However, imatinib increased glucose-stimulated insulin secretion during the glucose challenge. These data show that multiple TKIs lower blood glucose, where actions of TKIs on RIPK2 dictate divergent insulin responses, independent of tissue inflammation. Our data show that RIPK2 limits the insulin sensitizing effect of gefitinib, whereas imatinib increased insulin secretion.