Obesity induces PD-1 on macrophages to suppress anti-tumour immunity.

Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.

Microglial-specific knockdown of iron import gene, Slc11a2, blunts LPS-induced neuroinflammatory responses in a sex-specific manner.

Neuroinflammation and microglial iron load are significant hallmarks found in several neurodegenerative diseases. In in vitro systems, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and it has been shown that iron can augment cellular inflammation, suggesting a feed-forward loop between mechanisms involved in iron import and inflammatory signaling. However, it is not understood how microglial iron import mechanisms contribute to inflammation in vivo, or whether altering a microglial iron-related gene affects the inflammatory response. These studies aimed to determine the effect of knocking down microglial iron import gene Slc11a2 on the inflammatory response in vivo. We generated a novel model of tamoxifen-inducible, microglial-specific Slc11a2 knockdown using Cx3cr1Cre-ERT2 mice. Transgenic male and female mice were administered intraperitoneal saline or lipopolysaccharide (LPS) and assessed for sickness behavior post-injection. Plasma cytokines and microglial bulk RNA sequencing (RNASeq) analyses were performed at 4 h post-LPS, and microglia were collected for gene expression analysis after 24 h. A subset of mice was assessed in a behavioral test battery following LPS-induced sickness recovery. Control male, but not female, mice significantly upregulated microglial Slc11a2 at 4 and 24 h following LPS. In Slc11a2 knockdown mice, we observed an improvement in the acute behavioral sickness response post-LPS in male, but not female, animals. Microglia from male, but not female, knockdown animals exhibited a significant decrease in LPS-provoked pro-inflammatory cytokine expression after 24 h. RNASeq data from male knockdown microglia 4 h post-LPS revealed a robust downregulation in inflammatory genes including Il6, Tnfα, and Il1ß, and an increase in anti-inflammatory and homeostatic markers (e.g., Tgfbr1, Cx3cr1, and Trem2). This corresponded with a profound decrease in plasma pro-inflammatory cytokines 4 h post-LPS. At 4 h, male knockdown microglia also upregulated expression of markers of iron export, iron recycling, and iron homeostasis and decreased iron storage and import genes, along with pro-oxidant markers such as Cybb, Nos2, and Hif1α. Overall, this work elucidates how manipulating a specific gene involved in iron import in microglia alters acute inflammatory signaling and overall cell activation state in male mice. These data highlight a sex-specific link between a microglial iron import gene and the pro-inflammatory response to LPS in vivo, providing further insight into the mechanisms driving neuroinflammatory disease.

Adipose tissue macrophages: Unique polarization and bioenergetics in obesity.

Macrophages comprise a majority of the resident immune cells in adipose tissue (AT) and regulate both tissue homeostasis in the lean state and metabolic dysregulation in obesity. Since the AT environment rapidly changes based upon systemic energy status, AT macrophages (ATMs) must adapt phenotypically and metabolically. There is a distinct dichotomy in the polarization and bioenergetics of in vitro models, with M2 macrophages utilizing oxidative phosphorylation (OX PHOS) and M1 macrophages utilizing glycolysis. Early studies suggested differential polarization of ATMs, with M2-like macrophages predominant in lean AT and M1-like macrophages in obese AT. However, recent studies show that the phenotypic plasticity of ATMs is far more complicated, which is also reflected in their bioenergetics. Multiple ATM populations exist along the M2 to M1 continuum and appear to utilize both glycolysis and OX PHOS in obesity. The significance of the dual fuel bioenergetics is unclear and may be related to an intermediate polarization, their buffering capacity, or the result of a mixed population of distinct polarized ATMs. Recent evidence also suggests that ATMs of lean mice serve as a substrate buffer or reservoir to modulate lipid, catecholamine, and iron availability. Furthermore, recent models of weight loss and weight cycling reveal additional roles for ATMs in systemic metabolism. Evaluating ATM phenotype and intracellular metabolism together may more accurately illuminate the consequences of ATM accumulation in obese AT, lending further insight into obesity-related comorbidities in humans.

Deletion of complement factor 5 amplifies glucose intolerance in obese male but not female mice.

Complement factor 5 of the innate immune system generates C5a and C5b ligands, which initiate inflammatory and cell lysis events, respectively. C5 activation has been linked with obesity-associated metabolic disorders; however, whether it has a causative role is unclear. We generated a C5 null (C5-/-) mouse using CRISPR-Cas9 gene editing to determine whether loss of C5 improves obesity-linked metabolic dysfunction. Generation of a new mouse model was prompted in part by the observation of off-target gene mutations in commercially available C5-/- lines. Male and female wild-type (WT), heterozygous (Het), and C5-/- mice were fed low-fat diet (LFD) or high-fat diet (HFD) for 22 wk. Body weight gain did not differ between genotypes on LFD or HFD. In lean animals, male C5-/- mice had similar glucose tolerance compared with WT controls; however, in obese conditions, glucose tolerance was worsened in C5-/- compared with controls. In contrast, female mice did not exhibit differences in glucose tolerance between genotypes under either dietary paradigm. Fasting insulin was not different between genotypes, whereas diet-induced obese male C5-/- mice had lower fed insulin concentrations compared with WT controls. No differences in adipose tissue inflammation or adipocyte size were identified between groups. Similarly, susceptibility to fatty liver and hepatic inflammation was similar between WT and C5-/- mice. However, the systemic cytokine response to acute endotoxin exposure was decreased in C5-/- mice. Together, these data suggest that loss of C5 worsens glucose tolerance in obese male but not female mice. Additional work is required to pinpoint the mechanisms by which loss of C5 amplifies glucose intolerance in obesity.NEW & NOTEWORTHY We generated a new mouse model of complement factor 5 deficiency. This work was prompted by a need for improved transgenic mouse lines of C5, due to off-target gene mutations. We find that loss of C5 worsens glucose tolerance in a sex-dependent manner. Though the mechanisms evoking glucose intolerance are not clear, we are confident this model will be useful in interrogating complement activation in obesity-associated diseases.

Exon 2-mediated deletion of Trem2 does not worsen metabolic function in diet-induced obese mice.

Triggering receptor expressed on myeloid cells 2 (Trem2) is highly expressed on myeloid cells and is involved in cellular lipid homeostasis and inflammatory processes. Trem2 deletion in mice (Trem2-/- ) evokes adipose tissue dysfunction, but its role in worsening obesity-induced metabolic dysfunction has not been resolved. Here we aimed to determine the causal role of Trem2 in regulating glucose homeostasis and insulin sensitivity in mice. Nine-week-old male and female littermate wild-type (WT) and Trem2-/- mice were fed a low- or high-fat diet for 18 weeks and phenotyped for metabolic function. Diet-induced weight gain was similar between genotypes, irrespective of sex. Consistent with previous reports, we find that loss of Trem2 causes massive adipocyte hypertrophy and an attenuation in the lipid-associated macrophage transcriptional response to obesity. In contrast to published data, we find that loss of Trem2 does not worsen metabolic function in obese mice. No differences in intraperitoneal glucose tolerance (ipGTT), oral GTT or mixed meal substrate control, including postprandial glucose, non-esterified fatty acids, insulin or triglycerides, were found between WT and Trem2-/- animals. Similarly, no phenotypic differences existed when animals were challenged with stressors on metabolic demand (i.e. acute exercise or environmental temperature modulation). Collectively, we report a disassociation between adipose tissue remodelling caused by loss of Trem2 and whole-body metabolic homeostasis in obese mice. The complementary nature of experiments conducted gives credence to the conclusion that loss of Trem2 is unlikely to worsen glucose homeostasis in mice.

Immune Cell Activation in Obesity and Cardiovascular Disease.

PURPOSE OF REVIEW: In this review, we focus on immune cell activation in obesity and cardiovascular disease, highlighting specific immune cell microenvironments present in individuals with atherosclerosis, non-ischemic heart disease, hypertension, and infectious diseases. RECENT FINDINGS: Obesity and cardiovascular disease are intimately linked and often characterized by inflammation and a cluster of metabolic complications. Compelling evidence from single-cell analysis suggests that obese adipose tissue is inflammatory and infiltrated by almost all immune cell populations. How this inflammatory tissue state contributes to more systemic conditions such as cardiovascular and infectious disease is less well understood. However, current research suggests that changes in the adipose tissue immune environment impact an individual's ability to combat illnesses such as influenza and SARS-CoV2. Obesity is becoming increasingly prevalent globally and is often associated with type 2 diabetes and heart disease. An increased inflammatory state is a major contributor to this association. Widespread chronic inflammation in these disease states is accompanied by an increase in both innate and adaptive immune cell activation. Acutely, these immune cell changes are beneficial as they sustain homeostasis as inflammation increases. However, persistent inflammation subsequently damages tissues and organs throughout the body. Future studies aimed at understanding the unique immune cell populations in each tissue compartment impacted by obesity may hold potential for therapeutic applications.

An Iron Refractory Phenotype in Obese Adipose Tissue Macrophages Leads to Adipocyte Iron Overload.

Adipocyte iron overload is a maladaptation associated with obesity and insulin resistance. The objective of the current study was to determine whether and how adipose tissue macrophages (ATMs) regulate adipocyte iron concentrations and whether this is impacted by obesity. Using bone marrow-derived macrophages (BMDMs) polarized to M0, M1, M2, or metabolically activated (MMe) phenotypes, we showed that MMe BMDMs and ATMs from obese mice have reduced expression of several iron-related proteins. Furthermore, the bioenergetic response to iron in obese ATMs was hampered. ATMs from iron-injected lean mice increased their glycolytic and respiratory capacities, thus maintaining metabolic flexibility, while ATMs from obese mice did not. Using an isotope-based system, we found that iron exchange between BMDMs and adipocytes was regulated by macrophage phenotype. At the end of the co-culture, MMe macrophages transferred and received more iron from adipocytes than M0, M1, and M2 macrophages. This culminated in a decrease in total iron in MMe macrophages and an increase in total iron in adipocytes compared with M2 macrophages. Taken together, in the MMe condition, the redistribution of iron is biased toward macrophage iron deficiency and simultaneous adipocyte iron overload. These data suggest that obesity changes the communication of iron between adipocytes and macrophages and that rectifying this iron communication channel may be a novel therapeutic target to alleviate insulin resistance.

Myeloid-specific deletion of ferroportin impairs macrophage bioenergetics but is disconnected from systemic insulin action in adult mice.

Tissue iron overload is associated with insulin resistance and mitochondrial dysfunction in rodents and humans; however, the mechanisms or cell types that mediate this phenotype are not completely understood. Macrophages (Mɸs) are known to contribute to iron handling; thus, we hypothesized that perturbed iron handling by Mɸs impairs mitochondrial energetics and evokes systemic insulin resistance in mice. Male and female mice with myeloid-targeted (LysMCre) deletion of the canonical iron exporter, ferroportin (Fpn, encoded by Slc40a1), floxed littermates, and C57BL/6J wild-type mice were used to test our hypotheses. Myeloid-targeted deletion of Fpn evoked multitissue iron accumulation and reduced mitochondrial respiration in bone marrow-derived Mɸs, liver leukocytes, and Mɸ-enriched populations from adipose tissue (AT). In addition, a single bolus of exogenous iron administered to C57BL/6J mice phenocopied the loss of Fpn, resulting in a reduction in maximal and mitochondrial reserve capacity in Mɸ-enriched cellular fractions from liver and AT. In vivo exogenous iron chelation restored mitochondrial reserve capacity in liver leukocytes from Fpn LysMCre mice, but had no effect in AT myeloid populations. However, despite the impairments in mitochondrial respiration, neither loss of myeloid-specific Fpn nor exogenous iron overload perturbed glucose homeostasis or systemic insulin action in lean or obese mice, whereas aging coupled with lifelong loss of Fpn unmasked glucose intolerance. Together these data demonstrate that iron handling is critical for the maintenance of macrophage mitochondrial function, but perturbing myeloid iron flux via the loss of Fpn action is not sufficient to evoke systemic insulin resistance in young adult mice. These findings also suggest that if Mɸs are capable of storing iron properly, they have a pronounced ability to withstand iron excess without evoking overt collateral damage and associated insulin resistance that may be age dependent.NEW & NOTEWORTHY We used myeloid-specific knockout of ferroportin to determine whether macrophage iron enrichment alters systemic metabolism. We found that macrophages in several tissues showed mitochondrial defects such as a reduction in mitochondrial reserve capacity. However, insulin action in the mice was preserved. These findings also suggest that Mɸs have a pronounced ability to withstand iron excess without evoking overt collateral damage and associated insulin resistance, which appears to be age dependent.

CD8+ T cells regulate liver injury in obesity-related nonalcoholic fatty liver disease.

Nonalcoholic steatohepatitis (NASH) has increased in Western countries due to the prevalence of obesity. Current interests are aimed at identifying the type and function of immune cells that infiltrate the liver and key factors responsible for mediating their recruitment and activation in NASH. We investigated the function and phenotype of CD8+ T cells under obese and nonobese NASH conditions. We found an elevation in CD8 staining in livers from obese human subjects with NASH and cirrhosis that positively correlated with α-smooth muscle actin, a marker of hepatic stellate cell (HSC) activation. CD8+ T cells were elevated 3.5-fold in the livers of obese and hyperlipidemic NASH mice compared with obese hepatic steatosis mice. Isolated hepatic CD8+ T cells from these mice expressed a cytotoxic IL-10-expressing phenotype, and depletion of CD8+ T cells led to significant reductions in hepatic inflammation, HSC activation, and macrophage accumulation. Furthermore, hepatic CD8+ T cells from obese and hyperlipidemic NASH mice activated HSCs in vitro and in vivo. Interestingly, in the lean NASH mouse model, depletion and knockdown of CD8+ T cells did not impact liver inflammation or HSC activation. We demonstrated that under obese/hyperlipidemia conditions, CD8+ T cell are key regulators of the progression of NASH, while under nonobese conditions they play a minimal role in driving the disease. Thus, therapies targeting CD8+ T cells may be a novel approach for treatment of obesity-associated NASH.NEW & NOTEWORTHY Our study demonstrates that CD8+ T cells are the primary hepatic T cell population, are elevated in obese models of NASH, and directly activate hepatic stellate cells. In contrast, we find CD8+ T cells from lean NASH models do not regulate NASH-associated inflammation or stellate cell activation. Thus, for the first time to our knowledge, we demonstrate that hepatic CD8+ T cells are key players in obesity-associated NASH.

Links between Immunologic Memory and Metabolic Cycling.

Treatments for metabolic diseases, such as diet and therapeutics, often provide short-term therapy for metabolic stressors, but relapse is common. Repeated bouts of exposure to, and relief from, metabolic stimuli results in a phenomenon we call "metabolic cycling." Recent human and rodent data suggest metabolic cycling promotes an exaggerated response and ultimately worsened metabolic health. This is particularly evident with cycling of body weight and hypertension. The innate and adaptive immune systems have a profound impact on development of metabolic disease, and current data suggest that immunologic memory may partially explain this association, especially in the context of metabolic cycling. In this Brief Review, we highlight recent work in this field and discuss potential immunologic mechanisms for worsened disease prognosis in individuals who experience metabolic cycling.

Contributions of innate type 2 inflammation to adipose function.

A critical contributor to the health consequences of the obesity epidemic is dysregulated adipose tissue (AT) homeostasis. While white, brown, and beige AT function is altered in obesity-related disease, white AT is marked by progressive inflammation and adipocyte dysfunction and has been the focus of extensive "immunometabolism" research in the past decade. The exact triggering events initiating and sustaining AT inflammation are still under study, but it has been shown that reducing inflammation improves insulin action in AT. Scientific efforts seeking interventions to mitigate obesity-associated AT inflammation continue, and many groups are now determining how lean healthy AT homeostasis is maintained in order to leverage these mechanisms as therapeutic targets. Such studies have revealed that an elaborate network of immune cells, cytokines, and other cellular mediators coordinate AT function. Recent studies elucidated the involvement of the innate immune system in AT homeostasis (e.g., beiging and insulin sensitivity), including M2-like macrophages, eosinophils, innate lymphoid type 2 cells, and several others. In this review, we summarize the existing literature on innate type 2 inflammation in AT; additionally, we draw attention to areas of debate where seemingly conflicting data promises to yield more surprising and elegant biology as studies continue to dissect AT physiology.

Impaired insulin signaling in the B10.D2-Hc0 H2d H2-T18c/oSnJ mouse model of complement factor 5 deficiency.

Given the chemoattractant potential of complement factor 5 (C5) and its increased expression in adipose tissue (AT) of obese mice, we determined whether this protein of the innate immune system impacts insulin action. C5 control (C5cont) and spontaneously C5-deficient (C5def, B10.D2-Hc0 H2d H2-T18c/oSnJ) mice were placed on low- and high-fat diets to investigate their inflammatory and metabolic phenotypes. Adenoviral delivery was used to evaluate the effects of exogenous C5 on systemic metabolism. C5def mice gained less weight than controls while fed a high-fat diet, accompanied by reduced AT inflammation, liver mass, and liver triglyceride content. Despite these beneficial metabolic effects, C5def mice demonstrated severe glucose intolerance and systemic insulin resistance, as well as impaired insulin signaling in liver and AT. C5def mice also exhibited decreased expression of insulin receptor (INSR) gene and protein, as well as improper processing of pro-INSR. These changes were not due to the C5 deficiency alone as other C5-deficient models did not recapitulate the INSR processing defect; rather, in addition to the mutation in the C5 gene, whole genome sequencing revealed an intronic 31-bp deletion in the Insr gene in the B10.D2-Hc0 H2d H2-T18c/oSnJ model. Irrespective of the genetic defect, adenoviral delivery of C5 improved insulin sensitivity in both C5cont and C5def mice, indicating an insulin-sensitizing function of C5.

MFehi adipose tissue macrophages compensate for tissue iron perturbations in mice.

Resident adipose tissue macrophages (ATMs) play multiple roles to maintain tissue homeostasis, such as removing excess free fatty acids and regulation of the extracellular matrix. The phagocytic nature and oxidative resiliency of macrophages not only allows them to function as innate immune cells but also to respond to specific tissue needs, such as iron homeostasis. MFehi ATMs are a subtype of resident ATMs that we recently identified to have twice the intracellular iron content as other ATMs and elevated expression of iron-handling genes. Although studies have demonstrated that iron homeostasis is important for adipocyte health, little is known about how MFehi ATMs may respond to and influence adipose tissue iron availability. Two methodologies were used to address this question: dietary iron supplementation and intraperitoneal iron injection. Upon exposure to high dietary iron, MFehi ATMs accumulated excess iron, whereas the iron content of MFelo ATMs and adipocytes remained unchanged. In this model of chronic iron excess, MFehi ATMs exhibited increased expression of genes involved in iron storage. In the injection model, MFehi ATMs incorporated high levels of iron, and adipocytes were spared iron overload. This acute model of iron overload was associated with increased numbers of MFehi ATMs; 17% could be attributed to monocyte recruitment and 83% to MFelo ATM incorporation into the MFehi pool. The MFehi ATM population maintained its low inflammatory profile and iron-cycling expression profile. These studies expand the field's understanding of ATMs and confirm that they can respond as a tissue iron sink in models of iron overload.

A decade of progress in adipose tissue macrophage biology.

One decade has passed since seminal publications described macrophage infiltration into adipose tissue (AT) as a key contributor to inflammation and obesity-related insulin resistance. Currently, a PubMed search for 'adipose tissue inflammation' reveals over 3500 entries since these original reports. We now know that resident macrophages in lean AT are alternatively activated, M2-like, and play a role in AT homeostasis. In contrast, the macrophages in obese AT are dramatically increased in number and are predominantly classically activated, M1-like, and promote inflammation and insulin resistance. Mediators of AT macrophage (ATM) phenotype include adipokines and fatty acids secreted from adipocytes as well as cytokines secreted from other immune cells in AT. There are several mechanisms that could explain the large increase in ATMs in obesity. These include recruitment-dependent mechanisms such as adipocyte death, chemokine release, and lipolysis of fatty acids. Newer evidence also points to recruitment-independent mechanisms such as impaired apoptosis, increased proliferation, and decreased egress. Although less is known about the homeostatic function of M2-like resident ATMs, recent evidence suggests roles in AT expansion, thermoregulation, antigen presentation, and iron homeostasis. The field of immunometabolism has come a long way in the past decade, and many exciting new discoveries are bound to be made in the coming years that will expand our understanding of how AT stands at the junction of immune and metabolic co-regulation.

Retention of sedentary obese visceral white adipose tissue phenotype with intermittent physical activity despite reduced adiposity.

Regular physical activity is effective in reducing visceral white adipose tissue (AT) inflammation and oxidative stress, and these changes are commonly associated with reduced adiposity. However, the impact of multiple periods of physical activity, intercalated by periods of inactivity, i.e., intermittent physical activity, on markers of AT inflammation and oxidative stress is unknown. In the present study, 5-wk-old male C57BL/6 mice were randomized into three groups (n = 10/group): sedentary, regular physical activity, and intermittent physical activity, for 24 wk. All animals were singly housed and fed a diet containing 45% kcal from fat. Regularly active mice had access to voluntary running wheels throughout the study period, whereas intermittently active mice had access to running wheels for 3-wk intervals (i.e., 3 wk on/3 wk off) throughout the study. At death, regular and intermittent physical activity was associated with similar reductions in visceral AT mass (approximately -24%, P < 0.05) relative to sedentary. However, regularly, but not intermittently, active mice exhibited decreased expression of visceral AT genes related to inflammation (e.g., monocyte chemoattractant protein 1), immune cell infiltration (e.g., CD68, CD11c, F4/80, CD11b/CD18), oxidative stress (e.g., p47 phagocyte oxidase), and endoplasmic reticulum stress (e.g., CCAAT enhancer-binding protein homologous protein; all P < 0.05). Furthermore, regular, but not intermittent, physical activity was associated with a trend toward improvement in glucose tolerance (P = 0.059). Collectively, these findings suggest that intermittent physical activity over a prolonged period of time may lead to a reduction in adiposity but with retention of a sedentary obese white AT and metabolic phenotype.

Obesity induced by a high-fat diet is associated with increased immune cell entry into the central nervous system.

Obesity is associated with chronic low-grade inflammation in peripheral tissues caused, in part, by the recruitment of inflammatory monocytes into adipose tissue. Studies in rodent models have also shown increased inflammation in the central nervous system (CNS) during obesity. The goal of this study was to determine whether obesity is associated with recruitment of peripheral immune cells into the CNS. To do this we used a bone marrow chimerism model to track the entry of green-fluorescent protein (GFP) labeled peripheral immune cells into the CNS. Flow cytometry was used to quantify the number of GFP(+) immune cells recruited into the CNS of mice fed a high-fat diet compared to standard chow fed controls. High-fat feeding resulted in obesity associated with a 30% increase in the number of GFP(+) cells in the CNS compared to control mice. Greater than 80% of the GFP(+) cells recruited to the CNS were also CD45(+) CD11b(+) indicating that the GFP(+) cells displayed characteristics of microglia/macrophages. Immunohistochemistry further confirmed the increase in GFP(+) cells in the CNS of the high-fat fed group and also indicated that 93% of the recruited cells were found in the parenchyma and had a stellate morphology. These findings indicate that peripheral immune cells can be recruited to the CNS in obesity and may contribute to the inflammatory response.

Crucial role of a long-chain fatty acid elongase, Elovl6, in obesity-induced insulin resistance.

Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes the conversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high-fat diet or mated to leptin-deficient ob/ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia and hyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate-2 and suppression of hepatic protein kinase C epsilon activity resulting in restoration of Akt phosphorylation. Collectively, these data show that hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance and stress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes and cardiovascular risks, even in the presence of a continuing state of obesity.

Deficiency of the hemoglobin-haptoglobin receptor, CD163, worsens insulin sensitivity in obese male mice.

Excessive iron accumulation in metabolic organs such as the adipose tissue, liver, and skeletal muscle is associated with increased diabetes risk. Tissue-resident macrophages serve multiple roles including managing inflammatory tone and regulating parachymal iron homeostasis; thus protecting against metabolic dysfunction upon iron overload. The scavenger receptor CD163 is uniquely present on tissue-resident macrophages, and plays a significant role in iron homeostasis by clearing extracellular hemoglobin-haptoglobin complexes, thereby limiting oxidative damage caused by free hemoglobin in metabolic tissues. We show that the absence of CD163 exacerbates glucose intolerance and insulin resistance in male mice with obesity. Additionally, loss of CD163 reduced the expression of iron regulatory genes (Tfr1, Cisd1, Slc40a1) in adipose tissue macrophages and anti-inflammatory (M2-like) bone marrow-derived macrophages (BMDMs). Further, CD163 deficiency mediated a pro-inflammatory shift and limited hemoglobin scavenging specifically in M2-like BMDMs. To this end, iron buffering was diminished in inguinal white adipose tissue (iWAT) macrophages in vivo, which culminated in iron spillover into adipocytes and CD45+CD11B- non-myeloid immune cells in iWAT. These findings show that CD163 on tissue-resident macrophages is critical for their anti-inflammatory and hemoglobin scavenging roles, and its absence results in impaired systemic insulin action in an obese setting.

Knockdown of microglial iron import gene, DMT1, worsens cognitive function and alters microglial transcriptional landscape in a sex-specific manner in the APP/PS1 model of Alzheimer's disease.

Background Microglial cell iron load and inflammatory activation are significant hallmarks of late-stage Alzheimer's disease (AD). In vitro , microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2 ) in response to inflammatory stimuli, and excess iron can augment cellular inflammation, suggesting a feed-forward loop between iron import mechanisms and inflammatory signaling. However, it is not understood whether microglial iron import mechanisms directly contribute to inflammatory signaling and chronic disease in vivo . These studies determined the effects of microglial-specific knockdown of Slc11a2 on AD-related cognitive decline and microglial transcriptional phenotype. Methods In vitro experiments and RT-qPCR were used to assess a role for DMT1 in amyloid-ß-associated inflammation. To determine the effects of microglial Slc11a2 knockdown on AD-related phenotypes in vivo , triple-transgenic Cx3cr1 Cre - ERT2 ; Slc11a2 flfl ; APP/PS1 + or - mice were generated and administered corn oil or tamoxifen to induce knockdown at 5-6 months of age. Both sexes underwent behavioral analyses to assess cognition and memory (12-15 months of age). Hippocampal CD11b + microglia were magnetically isolated from female mice (15-17 months) and bulk RNA-sequencing analysis was conducted. Results DMT1 inhibition in vitro robustly decreased Aß-induced inflammatory gene expression and cellular iron levels in conditions of excess iron. In vivo, Slc11a2 KD APP/PS1 female, but not male, mice displayed a significant worsening of memory function in Morris water maze and a fear conditioning assay, along with significant hyperactivity compared to control WT and APP/PS1 mice. Hippocampal microglia from Slc11a2 KD APP/PS1 females displayed significant increases in Enpp2, Ttr , and the iron-export gene, Slc40a1 , compared to control APP/PS1 cells. Slc11a2 KD cells from APP/PS1 females also exhibited decreased expression of markers associated with disease-associated microglia (DAMs), such as Apoe, Ctsb, Csf1 , and Hif1α. Conclusions This work suggests a sex-specific role for microglial iron import gene Slc11a2 in propagating behavioral and cognitive phenotypes in the APP/PS1 model of AD. These data also highlight an association between loss of a DAM-like phenotype in microglia and cognitive deficits in Slc11a2 KD APP/PS1 female mice. Overall, this work illuminates an iron-related pathway in microglia that may serve a protective role during disease and offers insight into mechanisms behind disease-related sex differences.