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Identifying children with insulin resistance (IR) is difficult due to insulin assay reliability/reproducibility. We examined if the triglyceride/high-density lipoprotein ratio (TG/HDL) can identify children with IR and if it is important to use age- & sex-specific cutoffs. In a representative sample of 3,464 US adolescents 12-18 yrs participating in NHANES (2003-2020) , we calculated the fasting TG/HDL ratio & homeostasis model assessment-insulin resistance level (HOMA-IR) . We established age- & sex-specific & universal percentiles (10th, 25th, 50th, 75th, 95th) for TG/HDL ratio based on a subset of normal weight adolescents; IR was defined as having a HOMA-IR >4.39. We created receiver operating characteristic (ROC) curves predicting IR comparing age- & sex-specific, universal TG/HDL ratios, and raw TG/HDL cutpoints. Half were female, 14% were black, 13% were Mexican-American, and 24% had IR. AUC was similar for age-,sex-specific (0.69 (SE 0.01)) , universal (0.69 (SE 0.01)) percentiles and raw TG/HDL ratios (0.69 (SE 0.01)) (p=0.22) . Age and sex cutoffs did not provide further discrimination for identifying adolescents with IR. A universal TG/HDL ratio cutoff of 1.0 conferred a sensitivity of 84% and specificity of 42%. A TG/HDL ratio of 1.0 may be a reasonable test cutoff for identifying adolescents with IR, given that higher detection rates may improve identification of high risk children. Disclosure D.Eng: None. B.Gregg: None. K.Singer: None. J.M.Lee: Advisory Panel; GoodRx, Consultant; Tandem Diabetes Care, Inc. Funding Caswell Diabetes Institute at the University of Michigan

Obesity-induced inflammation is a risk factor for metabolic impairment leading to diabetes and cardiovascular disease. We, and others, have shown that sex differences exist in adipose tissue (AT) and myeloid inflammatory responses to high-fat diet (HFD) and lipolysis. However, the causal cellular mechanisms behind this sexual dimorphism are not well defined. Our objective was to investigate sex differences in lipid storage and metabolism that drive sexually dimorphic inflammatory responses. C57Bl/6J mice were fed a normal diet or 60%kcal%fat (HFD) for 16 weeks. AT macrophages (ATMs) were sorted for RNA sequencing. Animals were gonadectomized (GX) prior to starting HFD and gonadal white AT (GWAT) was collected for lipidomics. To directly model androgen receptor (AR) activation, GX mice were given dihydrotestosterone (DHT) to study AR-specific effects. RNA sequencing identified upregulated genes (Lpl, Ctsb, and Cd36) linked to a metabolically-activated macrophage only in male HFD ATMs. Furthermore, pathways associated with mitochondrial depolarization and fatty acid (FA) metabolism differed significantly by sex. GWAT lipidomics identified shifts in lipid species both by sex and hormone status, and differences were also observed in acylcarnitines and saturation of ceramide species that are implicated in inflammation. In addition, males showed elevated levels of key precursor free FAs (palmitic, linoleic, and arachidonic acid) when compared to female and GX mice. DHT treatment did not completely mimic male pro-inflammatory responses but induced GWAT crown-like structures in both sexes. DHT failed to upregulate inflammatory markers (Il6 and Mcp1) but showed impaired expression of adipogenic genes (Pparg and Pgc1α) in GX mice. Altogether, our data suggest there are sex-specific alterations in GWAT lipid storage and metabolism that may drive sexually dimorphic ATM polarization, which is only partially reversed by AR activation. Disclosure R. Mohan: None. M. Varghese: None. S. Abrishami: None. K. Singer: None. Funding National Institutes of Health - NIDDK (R01DK115583)

Obesity causes dramatic proinflammatory changes in the adipose tissue immune environment, but relatively little is known regarding how this inflammation responds to weight loss (WL). To understand the mechanisms by which meta-inflammation resolves during WL, we examined adipose tissue leukocytes in mice after withdrawal of a high-fat diet. After 8 weeks of WL, mice achieved similar weights and glucose tolerance values as age-matched lean controls but showed abnormal insulin tolerance. Despite fat mass normalization, total and CD11c+ adipose tissue macrophage (ATM) content remained elevated in WL mice for up to 6 months and was associated with persistent fibrosis in adipose tissue. ATMs in formerly obese mice demonstrated a proinflammatory profile, including elevated expression of interferon-γ, tumor necrosis factor-α, and interleukin-1β. T-cell–deficient Rag1−/− mice showed a degree of ATM persistence similar to that in WT mice, but with reduced inflammatory gene expression. ATM proliferation was identified as the predominant mechanism by which ATMs are retained in adipose tissue with WL. Our study suggests that WL does not completely resolve obesity-induced ATM activation, which may contribute to the persistent adipose tissue damage and reduced insulin sensitivity observed in formerly obese mice.

Men have a higher risk of metabolic and cardiovascular disease than pre-menopausal women, but the mechanisms of these differences are elusive. Chronic myeloid inflammation during obesity contributes to metabolic disease risk and is significantly more robust in males. Given the paucity of data on male sex hormones contributing to macrophage responses in obesity, our objective was to understand the role of androgens in promoting obesity-induced myeloid inflammation. Male and female C57Bl/6J mice were fed a 60% high fat diet for 24 weeks. Glucose tolerance and insulin sensitivity were measured. Monocyte transfer experiments assessed sex differences in bone marrow myeloid responses to obesity independent of host sex. Gonadectomy, mice deficient of androgen receptor signaling (ARtfm) and monocyte specific androgen receptor (AR) knockout mice (LysMAR-/y) were used to model androgen deficiency. Compared to males, females had dampened inflammatory responses with reduced CD11c+ ATMs and cytokines, even with increased adiposity. RNASeq demonstrated sex differences in gene pathways of both metabolic and inflammatory activation in ATMs. Male monocytes transferred into females remained primed for pro-inflammatory response and induced an increase in adiposity. Castrated males exposed to high fat diet had impaired glucose tolerance and increased insulin sensitivity with increased adiposity and lower numbers of CD11c+ ATMs. These same findings were seen in ARtfm mice. Testosterone enhanced palmitate-stimulated myeloid colony production, and this response was dampened in castrated, ARtfm, and LysMAR-/y mice. These studies demonstrate that androgens play a critical role in enhanced metabolic dysfunction and enhanced myeloid inflammation in response to obesogenic cues. Androgen driven myeloid inflammation leads to metabolic dysfunction in a cell-autonomous manner driving sex-differences in obesity induced meta-inflammation. Disclosure C.A. Griffin: None. S. Abrishami: None. M. Varghese: None. K. Singer: None.

Men and post-menopausal women are at greater risk for cardiovascular diseases and diabetes than younger women. In obese rodent models, males exhibit profound adipose tissue macrophage (ATM) accumulation in gonadal white adipose tissue (GWAT) linked to insulin resistance, while females are protected even with enhanced adiposity. We hypothesize that these inflammatory differences may be a result of altered lipolysis responses and that females would have enhanced ATM accumulation when lipolysis is induced. Lipolysis was stimulated with a beta3 adrenergic receptor (ADRB3) agonist CL-316, 243 in C57Bl6/j male and female mice fed 16 weeks of 60% high fat diet (HFD). GWAT leukocytes were isolated after 18 h and evaluated by flow cytometry. Lipogenic, lipolytic, oxidative and inflammatory gene expression along with immunofluorescence were performed. ADRB3 activation did not alter adipose tissue weights, but significantly reduced liver weights in obese females. Obese CL treated female mice showed elevated serum free fatty acid (FFA) and triglyceride (TG) levels compared to levels induced in males. Lipid overload impaired TG storage and utilization genes- Lpl, Fasn, Acsl1, Hsl, and Glut4 expression in obese females. Lipolysis promoted appearance of crown like structures (CLS) in males and females but only induced Il6 proinflammatory cytokine and Mcp1 chemokine expression in obese female GWAT. Activated lipolysis triggered CD11c- ATM accumulation in lean females but in both obese male and female GWAT. Obese males showed a marked reduction in GWAT dendritic cells (DC) but a significant reduction in both GWAT and inguinal WAT DCs of obese females. While obese females are resistant to inflammation, this study shows that induced lipolysis elevates FFA levels, alters lipid metabolism and increased CD11c- ATMs and CLSs in obese females. Elucidating the link between lipolysis and inflammation can better explain sex differences in overall metabolic health leading to targeted treatment strategies. Disclosure M. Varghese: None. S. Abrishami: None. K. Singer: None.

Despite the well-known efficacy of bariatric surgery in reducing body mass and improving metabolic outcomes, the mechanisms underlying these dramatic effects are not fully understood. Nearly 80% of patients that receive bariatric surgery are women, yet mechanistic pre-clinical studies have focused exclusively on males. Premenopausal women have a reduced risk for obesity-associated co-morbidities and enhanced lipid metabolism. Although both sexes lose weight and benefit metabolically after bariatric surgery, clear physiological sex differences in metabolism suggest the mechanistic pathway to those outcomes are dependent upon sex and hormone status. The goal of this study was to determine the impact of diet- and surgery-induced weight loss on sex differences in glucose regulation and hepatic lipid metabolism. Male and female mice were fed a 60% high-fat (HF) diet 12 weeks before undergoing either vertical sleeve gastrectomy (VSG) or sham surgery. After surgery, mice either remained on a HF diet or were switched to a standard chow diet. Both males and females decreased fat-mass after VSG, yet males showed further body mass loss on a chow diet. Both sexes improved fasting glucose and oral glucose tolerance on a HF diet after VSG, while only males showed further benefit from surgery and chow diet. Despite continued HF diet feeding, male but not female mice show significant improvements in hepatic triglyceride levels after VSG relative to their sham surgery counterparts. However, when mice were switched to a low-calorie chow diet both males and females reduced hepatic triglycerides after VSG. Furthermore, removing female gonadal hormones, via ovariectomy, did not impact hepatic triglyceride levels after VSG. Overall, these data suggest that the impact of surgery on hepatic triglycerides is diet-dependent in females and support the hypothesis that males and females achieve similar metabolic outcome, at least within the liver, via distinct mechanisms. Disclosure C. Hutch: None. D. Stelmak: None. K. Singer: None. D.A. Sandoval: Research Support; Self; Novo Nordisk A/S, Zafgen, Ethicon US, LLC..

The proinflammatory activation of leukocytes in adipose tissue contributes to metabolic disease. How crosstalk between immune cells initiates and sustains adipose tissue inflammation remains an unresolved question. We have examined the hypothesis that adipose tissue macrophages (ATMs) interact with and regulate the function of T cells. Dietary obesity was shown to activate the proliferation of effector memory CD4+ T cells in adipose tissue. Our studies further demonstrate that ATMs are functional antigen-presenting cells that promote the proliferation of interferon-γ–producing CD4+ T cells in adipose tissue. ATMs from lean and obese visceral fat process and present major histocompatibility complex (MHC) class II–restricted antigens. ATMs were sufficient to promote proliferation and interferon-γ production from antigen-specific CD4+ T cells in vitro and in vivo. Diet-induced obesity increased the expression of MHC II and T-cell costimulatory molecules on ATMs in visceral fat, which correlated with an induction of T-cell proliferation in that depot. Collectively, these data indicate that ATMs provide a functional link between the innate and adaptive immune systems within visceral fat in mice.