BACKGROUND:Refractory hypoglycemia is common in malignant insulin-secreting tumors and is associated with substantial morbidity. Ersodetug is a fully human monoclonal antibody that allosterically attenuates insulin receptor signaling with the potential to treat various forms of hyperinsulinism (HI). We retrospectively report outcomes from compassionate use of ersodetug in eight individuals with refractory hypoglycemia due to tumor HI. METHODS:Ersodetug was administered intravenously at 6 or 9 mg/kg every 1-2 weeks initially, followed by a frequency of every 2-5 weeks, as appropriate. Glycemic, functional, and safety outcomes were analyzed. RESULTS:Eight adults (4 M/4F; 24-74 years; Eastern Cooperative Oncology Group (ECOG) 1-3) received ersodetug for insulin-secreting tumors (metastatic insulinoma, n = 7; cervical neuroendocrine carcinoma, n = 1). No drug-related serious adverse events were observed. Most patients experienced improved glycemic control, including discontinuation of parenteral glucose in 6 of the 7 applicable patients (median, 4.5 days) and a 35.6% relative reduction from baseline in time in hypoglycemia (<70 mg/dL) by continuous glucose monitoring (mean 12.4% to 8.0%; pseudo-median paired change of -3.8 percentage points [90% CI, -8.2 to -1,1; P = 0.02). These glycemic improvements permitted hospital discharge and reductions in other antihypoglycemic therapies beyond glucose infusion rate (pseudo-median change, -2.5; 90% CI, -3.5 to -1.0; P = 0.02), with associated improvements in ECOG status. The median treatment duration with ersodetug (11.5 months, range 5-17) tended to correspond to patient lifespan in the setting of metastatic disease. CONCLUSIONS:Ersodetug therapy resulted in reduced hypoglycemia burden, ability to discontinue parenteral glucose infusion, allowing discharge from hospital, use of fewer antihypoglycemic therapies, and improved ECOG performance status in individuals with severe, refractory tumor HI.

Replenishment of pancreatic beta cells is a key to the cure for diabetes. Beta cells regeneration is achieved predominantly by self-replication especially in rodents, but it was also shown that pancreatic duct cells can transdifferentiate into beta cells. How pancreatic duct cells undergo transdifferentiated and whether we could manipulate the transdifferentiation to replenish beta cell mass is not well understood. Using a genome-wide CRISPR screen, we discovered that loss-of-function of ALDH3B2 is sufficient to transdifferentiate human pancreatic duct cells into functional beta-like cells. The transdifferentiated cells have significant increase in beta cell marker genes expression, secrete insulin in response to glucose, and reduce blood glucose when transplanted into diabetic mice. Our study identifies a novel gene that could potentially be targeted in human pancreatic duct cells to replenish beta cell mass for diabetes therapy.

Innate immunity predictors of HIV-1 risk and pathogenesis vary with reproductive hormones, pregnancy, and lactation, yet the underlying mechanisms remain unclear. We hypothesized that pregnancy-associated physiological adaptations alter systemic microRNA (miRNA) expression, thereby regulating immunity, pathogenesis and susceptibility to infection. We analyzed 174 serum samples from 88 participants in a longitudinal cohort from Uganda and Zimbabwe across pre-pregnancy (PP), pregnancy (P), and postpartum breastfeeding (BF). Cell-free peripheral blood miRNAs (n = 2083) were profiled using HTG EdgeSeq. Pregnancy-specific miRNAs were identified by intersecting differentially expressed (DE) miRNAs from P vs. PP and P vs. BF comparisons. miRNA targets and pathways were analyzed using miRWalk, Cytoscape/ClueGO, and cytoHubba. Pregnancy was associated with DE miRNAs (29 upregulated and 131 downregulated) targeting 2733 validated genes. Enriched pathways (FDR < 0.05) included adaptive immune response, Hippo Signaling, Cellular Senescence, HSV-1 infection, and two cancer-related pathways. Pregnancy-enriched targets within each pathway overlapped with the HIV-host interactome by 37-88%. Network analysis identified 47 hub genes interacting with 18 HIV-1 proteins, with Tat and gp120 being most connected viral and HLA-A being the most connected host protein. These findings indicate that pregnancy-driven systemic miRNAs target the HIV-host interactome and specifically identify pregnancy-enriched central hub genes involved in cell cycle control, viral immune evasion and replication to be further investigated for their predictive value in HIV acquisition and pathogenesis in longitudinal cohorts and experimental settings.

Adipocytes exist along a functional spectrum: white adipocytes are energy storing, and brown adipocytes have thermogenic capacity such that activation may counteract obesity-related disease. In between are UCP1-expressing beige adipocytes, which can transition between these two energetic states. We previously showed that bone morphogenetic protein 7 (BMP7), a member of the TGF-β superfamily, enables differentiation of brown preadipocytes to mature thermogenic cells. To see whether immortalized, clonal human white and brown preadipocytes (hWAs and hBAs, respectively) would become more thermogenic in response to BMP exposure, we treated them with BMP7 or BMP4 for the first 7 days of a 30-day differentiation protocol. In hBAs, absence of either BMP7 or BMP4 led to lower expression of brown-specific markers and oxygen consumption relative to 7 days with either BMP. hWAs treated for 7 days with either BMP did not increase expression of thermogenic protein UCP1 nor induce a brown-like transcription profile. However, BMP-treated hWAs produced adipocytes that had higher basal and drug-induced maximal oxygen consumption, which was UCP1-independent and due substantially to the futile creatine cycle. Our results demonstrate that energetically quiescent hWAs can be pushed into an energy-expending phenotype without transdifferentiation into beige adipocytes, providing a new approach to treat obesity-related metabolic disease.

Somatically acquired mitochondrial DNA (mtDNA) mutations accumulate with age, but the mechanisms and consequences of this accumulation are poorly understood. Here we show that transient injuries induce a burst of persistent mtDNA mutations that impair resilience to future injuries. mtDNA mutations suppressed energy-intensive nucleotide metabolism. Repletion of adenosine, but not other nucleotides, restored adenosine triphosphate generation, which required a nuclear-encoded purine biosynthetic enzyme, adenylate kinase 4 (AK4). Analysis of 369,912 UK Biobank participants revealed a graded association between mutation burden and chronic kidney disease severity as well as an independent increase in the risk of future acute kidney injury events (P < 10-7). Heteroplasmic mtDNA mutations may therefore reflect the cumulative effect of acute injuries to metabolically active cells, impairing major functions in a fashion amenable to nuclear-controlled purine biosynthesis.

Understanding the molecular mechanisms underlying the ability of vaginal dysbiosis to alter the mucosal barrier to HIV acquisition is an essential step toward prevention. We hypothesized that micro(mi)-RNAs dysregulated by vaginal pathobiont bacteria epigenetically control host pathways exploited by the virus. The impact of these endogenous non-coding short RNAs on the anti-viral mucosal barrier function in the female reproductive tract is largely unknown. This study utilized cervicovaginal specimens collected during the luteal and follicular phase of the menstrual cycle along with data on age, race, ethnicity, education, and body mass index from 141 healthy reproductive-age women confirmed negative for sexually transmitted infections. Vaginal microbiota was classified by Nugent scoring. Shot-gun vaginal microbiome sequencing and metagenome taxonomic classification was performed on a subset of 21 women. Levels of miRNAs in exosomes isolated from cervicovaginal secretions were quantified using the EdgeSeq-NextGen global transcriptome platform. Differential expression (DE) was determined using R. Epigenetic target prediction was performed using MirTarBase. MiRNA profiles varied by both Nugent score categories (0-3 scores = normal, 4-6 = intermediate, and 7-10 = bacterial vaginosis, BV) and by metagenome classification. Higher microbiome diversity was associated with higher number of significantly dysregulated miRNAs (588 in BV compared to Nugent 0-3 versus 42 in Nugent 4-6 compared to Nugent 0-3, false discovery rate FDR<0.01) affecting over 400 experimentally validated genes targeted for post-transcriptional regulation. The miRNAs dysregulated by G. vaginalis -dominated compared to L. crispatus -dominated metagenomes included 24 DE miRNAs (92% overlap with BV by Nugent score) and 112 validated target genes. BV-dysregulated miRNA mediated the immunosuppressive effects of BV on cytokine levels previously associated with HIV acquisition risk. The gene ontology predictions based on BV-dysregulated miRNAs identified enrichment for 445 downregulated and 50 upregulated genes previously validated as part of the HIV-host interactome. miRNAs mediation revealed a mechanism of suppressed immunity by BV predictive of HIV risk. In conclusion, miRNAs dysregulated by vaginal dysbiosis may facilitate immune imbalance and cellular pathways associated with HIV risk.

Introduction and Objective: Breast milk is a dynamic substance rich in both nutritive and nonnutritive compounds that may influence infant growth, development, and obesity risk. We aimed to identify metabolites in human milk that are associated with infant growth and body composition. Methods: We analyzed breast milk metabolomics from 350 mother-infant pairs at 1 month postpartum using high-sensitivity LC/GC-MS and assessed infant anthropometrics (BMI percentile) and body composition [percent body fat (%BF), Fat Free Mass Index (FFMI)] using air displacement plethysmography (at 1-month) and DXA (at 6-months). We tested associations between milk metabolites and infant anthropometric and body composition measures using linear regressions adjusting for covariates including maternal age, parity, delivery mode, gestational age, infant sex, birth weight, race, ethnicity, and enrollment site. We used Benjamini-Hochberg procedure to adjust for multiple comparisons (FDR <0.05). Results: Semi-quantitative concentrations were obtained for 458 metabolites. A greater number of milk metabolites were associated with %BF (9 with FDR<0.05) and BMI percentile (4 with FDR <0.05) than with FFMI (0 with FDR <0.05). Highest ranking metabolites associated with %BF included gamma-glutamylglutamine (beta=0.056, FDR=0.004) and 7-methylguanine (beta=0.061, FDR=0.03) while docosahexaenoate (beta = -0.020, FDR= 0.02) and p-hydroxybenzoate (beta = -0.012, FDR= 0.02) were the highest ranking metabolites associated with BMI percentile. The purine metabolite 7-methylguanine has been implicated previously in adipogenesis. Conclusion: Infant adiposity measures, but not measures of fat free mass, are associated with differences in the human milk metabolome. Further studies are needed to determine whether differences in human milk metabolites play a mechanistic role in infant growth and obesity risk. A. Uniyal: None. C. Lu: None. J.M. Dreyfuss: None. E.M. Nagel: None. A. Pena: None. M. Rudolph: None. D.A. Fields: None. E.W. Demerath: None. E.M. Isganaitis: None. NIH/NICHD (R01HD080444, R01HD109830)

Leptin receptor-expressing hypothalamic neurons (LepR Hypo ) are key regulators of energy balance, yet a comprehensive, cell type-resolved, chromatin accessibility map of these neurons is lacking. We profiled ∼20,000 LepR Hypo nuclei using single-nucleus multiome (snRNA-seq/snATAC-seq), identifying 39 transcriptionally and epigenetically distinct clusters, including AgRP (two subtypes), Pomc (two subtypes), Foxb1, Irx5/3 (three subtypes), Nts, PNOC (two subtypes), Kiss1/Pdyn (KNDy, two subtypes), Ghrh, Tcf7l2, and Sf1/Nr5a1 (three subtypes) populations. We also identified three Glp1r-expressing clusters with the highest Lepr enrichment, each marked by distinct molecular signatures. Cluster-specific open chromatin regions (OCRs) delineated putative cis-regulatory elements unique to each LepR Hypo subtype. Mouse cell-type specific OCRs conserved in the human genome were identified; a subset were proximal to genes with high Human Genetic Evidence (HuGE) scores for obesity-related traits, overlapped obesity-associated GWAS loci, and/or coincided with eQTLs, including variants with the potential to influence human energy balance. Together, these data provide cell type-specific cis-regulatory atlas of LepR Hypo neuronal subtypes, including Glp1r/Lepr-enriched populations, and highlight evolutionarily conserved, subtype-specific regulatory elements, associated candidate genes, and putative functional variants that may modulate LepR Hypo subtype function and influence energy homeostasis and obesity susceptibility in humans.

Introduction and Objective: DNA methylation (DNAm) changes have been associated with diabetic retinopathy (DR), but primarily in circulating white blood cells (WBCs). It is critical to determine whether DNAm patterns in WBCs are reflective of those in the retina. We compared DNAm patterns in the retina and WBCs of same individuals of the Joslin Medalist Study with ≥50 years of type 1 diabetes (T1D), 35% with no-mild DR and 50% with severe DR. Methods: DNAm was assessed by whole genome bisulfite sequencing (WGBS, 32.8×coverage, Illumina NovaSeq) of retina and WBCs within the same individuals with no-mild or severe DR (n=5 per group). Retinal proteomics (mass spectrometry) was done in those with or without severe DR (n=10). Principal component analysis (PCA) and linear regression models adjusted by age and sex tested the differential DNAm patterns in severe DR vs. no-mild DR. Results: WGBS output a total of 105M CpG sites. There was dramatic differentiation of DNAm patterns between retina and WBCs on PCA. Within the retina or WBCs, there were clear differential DNAm patterns in people with DR vs. those without, with 487 and 1960 differentially methylated regions (DMR) in the retina and WBCs, respectively (Bonferroni p<1E-8), and 14 and 32 changed KEGG pathways (p<0.05). However, only 23-upregulated and 3-downregulated DMR-related genes and 6 pathways overlapped between retina and WBCs, suggesting DNAm patterns are significantly different between these tissues. In proteomics, 543 proteins were changed (p<0.05) in the retina comparing DR vs. no DR. Three upregulated and 15 downregulated protein targets overlapped with those detected by DMR analysis, some with essential functions in neurovascular retina. Conclusion: DNAm changes induced by T1D and associated with DR severity are very different between retina and leukocytes. Future epigenetic studies on DR need to use retina, rather than surrogate tissues. M. Ma: None. S. Jangolla: None. H. Shah: None. K. Park: None. W. Fickweiler: None. J. Dreyfuss: None. H. Pan: None. I. Wu: None. J.K. Sun: Research Support; Optovue, Boehringer-Ingelheim. Other Relationship; Boehringer-Ingelheim. Research Support; Novo Nordisk, Roche Pharmaceuticals. Other Relationship; Roche Pharmaceuticals. Research Support; Physical Sciences, Inc, Boston Micromachines, Adaptive Sensory Technologies. L.P. Aiello: Consultant; Boehringer-Ingelheim, Ceramedix, Inc. Advisory Panel; Novo Nordisk. Stock/Shareholder; KalVista. G.L. King: None.

Context: Exercise is recommended for postpartum health, but its effects on breast milk composition and offspring are understudied. Objective: This work aimed to test whether the breast milk metabolome is altered with (i) acute exercise and/or (ii) habitual physical activity, and (iii) whether exercise-altered metabolites are associated with infant adiposity. Methods: Milk metabolites were assessed before and after acute exercise and in association with habitual activity score in 2 independent cohorts at 2 academic medical centers. The acute exercise cohort had 15 mother-infant dyads. The habitual activity nested case-control analysis had 84 physically active "cases" and 35 inactive "controls," and was conducted in a subset of the Mothers and Infants Linked for Healthy Growth (MILk)/4M study (N = 348). The acute exercise exposure was a 30-minute moderate-intensity treadmill session. The habitual activity exposure was based on Physical Activity Recall questionnaire scores. Main outcome measures included milk metabolite relative abundance at 1-month post partum by liquid chromatography-gas chromatography mass spectrometry, and infant anthropometric and body composition measures at 1, 3, and 6 months. Results: An acute exercise bout altered milk concentrations in 28 of 511 detectable metabolites (false discovery rate [FDR] < .05). In the habitual activity analysis, 4 of 454 detectable metabolites differed between active cases vs inactive controls (FDR < .05). Ten metabolites were altered (P < .05) by both exercise exposures. Of these, 4 were positively associated with fat mass index at 1 month, and 2 were associated with greater increase in body mass index z score between 1 and 3 months. Conclusion: Maternal exercise was associated with differences in the breast milk metabolome. Metabolites that were associated both with acute exercise and habitual activity correlated with infant adiposity measures.

Introduction and Objective: Environmental stimuli can impact the epitranscriptome to regulate cellular function; however, reversibility is not fully explored. We aimed to identify glucose-responsive changes in liver epitranscriptome with potential to be reversed by improving systemic metabolism. Methods: Two distinct inbred strain mouse models of hyperglycemia and reversal by sodium-glucose transporter 2 inhibitors (SGLT2i) were analyzed: (1) hyperglycemia with relative insulin deficiency (streptozotocin, STZ): control (CTRL), STZ, STZ+canagliflozin (STZ+CANA), (2) dietary hyperglycemia: low-fat diet (LFD), high-fat diet (HFD), HFD+CANA. Liver RNA-seq, m6A-seq and DNA methylation (DNAm, Illumina EPIC v2.0) were analyzed (n=5-7/group). Results: We identified 2695 glucose-responsive genes (FDR<0.05); downregulated genes (1350) enriched for translation and OXPHOS; of these, 11% reversed with CANA. Genes upregulated with hyperglycemia (1345) were enriched in lipid metabolism, Rho-GTPase signaling, H3K4me3 sites, and regulation by MLL3/4 H3K4me1 methyltransferases; 18% were reversed with CANA. m6A-seq identified 4374 glucose-responsive m6A-methylated mRNA peaks (FDR<0.05). Genes corresponding to downregulated peaks (2729) were enriched in RNA/protein metabolism; 18% were reversed with CANA. Upregulated peak genes (1625) were enriched in chromatin modification, coagulation and complement; 11% were reversed. Pathway analysis of genes associated with differential DNAm (250 probes, FDR<0.05) identified lipid metabolism, with only 2 probes reversed by CANA. Glucose-responsive changes in m6A methylation were more associated with mRNA than DNA methylation changes, and m6A alterations were associated with dysregulation of lipid metabolism and translation-related gene expression. Conclusion: Treatment with CANA in two distinct models of hyperglycemia reveals glucose-responsive, reversible changes in the liver epitranscriptome. B. Ozturk: None. D.F. De Jesus: None. J.M. Dreyfuss: None. H. Pan: None. R. Ferraz-Bannitz: None. H.S. Saifeldin: None. S. Lessard: None. R. Kulkarni: Advisory Panel; Novo Nordisk, Biomea Fusion, REDD Pharma, Inversago Pharma. Research Support; Inversago Pharma. Stock/Shareholder; Biomea Fusion. M. Patti: Research Support; Dexcom, Inc. Other Relationship; Recordati, Fractyl Health, Inc. Consultant; Spruce Biosciences, Premier, Cello, Alpha sight, Boxer Capital. Other Relationship; Amylyx. BILH Epigenetics Initiative

Introduction and Objective: DNA methylation (DNAm) has been implicated in “metabolic memory” of hyperglycemia and diabetes complication risk. Due to potential reversibility, epigenetic dysregulation may be targeted for therapy. We aimed to identify DNAm associated with type 1 (T1D) or type 2 (T2D) diabetes vs. non-diabetic individuals (NDM), and the impact of poor glycemic control in these groups. Methods: Buffy coat samples from people with long-duration T1D (Joslin Medalist Study, n=59), T2D (Joslin Heart Study, n=59), and respective age-matched NDM controls (n=46) were used for DNAm analysis (Illumina Methylation EPIC v2.0 935K). Samples were selected from those with poor (HbA1c ≥9%) and good (<7%) glycemic control (1:1 ratio). Using R/Bioconductor, per-probe differential DNAm between groups was determined, adjusting for age, sex, and white blood cell (WBC) type proportions. Results: T1D and T2D groups had mean age 64 years, 64% and 33% females, respectively, and diabetes duration 56 and 14 years. Compared to NDM, T1D had lower BMI (25 kg/m2) and better lipids, while T2D had higher BMI (34 kg/m2) and lipids. No differences in DNAm were observed between HbA1c strata in T1D or T2D. However, significant differences in DNAm were observed in T1D (23K CpGs) and, to a lesser extent, in T2D (298 CpGs) vs. NDM (FDR<0.05). In both T1D and T2D, the majority of differentially methylated sites had decreased DNAm. Differentially methylated probes were enriched (FDR<0.001) in transcription start sites (TSS) and quiescent sites in T1D vs controls, with similar TSS enrichment in T2D. Conclusion: Hyperglycemia (captured by HbA1c) was not associated with significant DNAm differences in WBCs of people with diabetes. However, significant differences in DNAm at TSS were observed in T1D and, to a lesser extent, in T2D. Further studies are needed to understand whether these differences are related to diabetes treatment, disease duration, autoimmunity (T1D), or insulin resistance (T2D). H. Shah: None. J. Dreyfuss: None. B. Ozturk: None. R. Ferraz-Bannitz: None. G.L. King: None. A. Doria: Research Support; Abbott, Lexicon Pharmaceuticals, Inc, Dexcom, Inc. M. Patti: Research Support; Dexcom, Inc. Other Relationship; Recordati, Fractyl Health, Inc. Consultant; Spruce Biosciences, Premier, Cello, Alpha sight, Boxer Capital. Other Relationship; Amylyx. Beth Israel Lahey Health (BILH) Epigenetics Initiative

Posttraumatic stress disorder (PTSD), a severe psychiatric disorder that predominantly affect women, is characterized by heightened inflammation and perturbations of the stress-buffering endocannabinoid system. However, whether these alterations contribute to PTSD pathophysiology in both men and women is largely unknown. This case-control study examined sex-differences in circulating levels of endocannabinoids (eCBs) and pro-inflammatory markers in a cohort of individuals with PTSD and non-psychiatric controls. Eighty-eight patients with PTSD and 85 sex- and age- matched healthy controls (HCs) were retrospectively selected from the Mass General Brigham Biobank. Serum samples were assayed to measure circulating levels of eCBs [N-arachidonoylethanolamine (AEA), 2-arachidonoylglycerol (2-AG), oleoylethanolamide (OEA), and arachidonic acid (AA)] and pro-inflammatory markers [interleukin-1β (IL-1β), IL-6, IL-8, IL-18, tumor necrosis factor-alpha (TNF-α), and C-reactive Protein (CRP)]. Our results showed distinct molecular profiles based on sex and PTSD diagnosis. Male PTSD patients exhibited decreased levels of AEA, AA and OEA compared to both male controls (p's < 0.001 to 0.05) and to the female subgroups (PTSD and HCs) (p < 0.01). In contrast, female PTSD patients showed elevated levels of IL-6 and IL-8 compared to the other subgroups (p's < 0.010), although only a trend-level effect in IL-6 levels persisted when examining the magnitude of group differences (PTSD vs HCs) across sexes. Similar results were obtained after controlling for the FAAH 385 A genotype and in the subgroup of individuals with comorbid MDD. These findings suggest that distinct neurobiological mechanisms may underlie PTSD in men and women and highlight the need for sex-based therapeutic approaches.

Introduction and Objective: Type 1 diabetes (T1D) can impact reproductive health in males. Given the potential impact of environmental stimuli, including hyperglycemia, on epigenetic marks, we assessed DNA methylation (DNAm) in sperm from men with T1D. Methods: We recruited 29 weight and age-matched men, 19 with T1D and 10 normoglycemic controls (CON). Samples were collected at baseline and after 3 months. We analyzed reproductive hormones, sperm quality, and sperm DNA methylation (Infinium MethylationEPIC), analyzed using SeSAMe pipeline. Results: Men with T1D had higher levels of luteinizing hormone (LH) (46%, p=0.03) and sex hormone binding globulin (SHBG) (91%, p=0.01), but similar testosterone levels and sperm quality vs. CON. Sperm DNAm remained stable in repeated samples over 3-months in both T1D and CON. Of the 755,827 CpG on the array, 97,723 (12.9%) were differentially methylated (FDR<0.05) in men with T1D vs. CON, adjusted for BMI; DNAm at these sites was increased in 91.7% and reduced in 8.3%. Differential methylation was attenuated after adjustment for HbA1c, suggesting glucose impact on methylation. Ontology analysis of differentially methylated loci revealed annotation to genes regulating helper T cell differentiation, natural killer cell chemotaxis, lysine deacetylation and lipoprotein lipase activity. Among differentially methylated sites, methylation was increased at multiple CpG sites within the human leukocyte antigen (HLA) complex, including HLA-DQA2, DQB2, DRA, DRB1, DPA1, DMB, and C (FDR<0.05). Conclusion: Men with T1D exhibited significantly higher levels of DNA methylation in sperm vs. CON. Whether differential DNA methylation in sperm could alter early post-fertilization phenotypes in offspring and contribute to disease risk remains unknown. Given the association between HLA genotype and T1D risk, ongoing analysis will determine whether differentially methylated loci within the HLA complex are associated with specific HLA risk alleles. R. Ferraz-Bannitz: None. B. Ozturk: None. V. Efthymiou: None. H. Wang: None. A. Cruz: None. C.J. Cummings: None. J. Patel: None. H. Pan: None. J. Dreyfuss: None. E.M. Isganaitis: None. M. Patti: Research Support; Dexcom, Inc. Other Relationship; Recordati, Fractyl Health, Inc. Consultant; Spruce Biosciences, Premier, Cello, Alpha sight, Boxer Capital. Other Relationship; Amylyx. Beatson FoundationIacocca Foundation

Hepatic insulin resistance is central to type 2 diabetes (T2D) and metabolic syndrome, but defining the molecular basis of this defect in humans is challenging because of limited tissue access. Utilizing inducible pluripotent stem cells differentiated into hepatocytes from control individuals and patients with T2D and liquid chromatography with tandem mass spectrometry-based (LC-MS/MS-based) phosphoproteomics analysis, we identified a large network of cell-intrinsic alterations in signaling in T2D. Over 300 phosphosites showed impaired or reduced insulin signaling, including losses in the classical insulin-stimulated PI3K/AKT cascade and their downstream targets. In addition, we identified over 500 phosphosites of emergent, i.e., new or enhanced, signaling. These occurred on proteins involved in the Rho-GTPase pathway, RNA metabolism, vesicle trafficking, and chromatin modification. Kinome analysis indicated that the impaired phosphorylation sites represented reduced actions of AKT2/3, PKCθ, CHK2, PHKG2, and/or STK32C kinases. By contrast, the emergent phosphorylation sites were predicted to be mediated by increased action of the Rho-associated kinases 1 and 2 (ROCK1/2), mammalian STE20-like protein kinase 4 (MST4), and/or branched-chain α-ketoacid dehydrogenase kinase (BCKDK). Inhibiting ROCK1/2 activity in T2D induced pluripotent stem cell-derived hepatocytes restored some of the alterations in insulin action. Thus, insulin resistance in the liver in T2D did not simply involve a loss of canonical insulin signaling but the also appearance of new phosphorylations representing a change in the balance of multiple kinases. Together, these led to altered insulin action in the liver and identified important targets for the therapy of hepatic insulin resistance.

Disorders of the reproductive system, including hypogonadism and reduced fertility, are an under-recognized complication of diabetes. Based on experimental data in mice, hyperglycemia and obesity may modify epigenetic marks in sperm and impact health and development of offspring, but data are more limited in humans. Thus, we sought to study the impact of type 2 diabetes and glycemic control on sperm quality and DNA methylation. In this prospective cohort study, we recruited 40 men with BMI greater than 25 kg/m2 including 18 with type 2 diabetes, 6 with prediabetes, and 16 normoglycemic controls. Assessments were repeated after 3 months in 9 men with type 2 diabetes and 7 controls. We analyzed reproductive hormones, sperm concentration and motility, and sperm DNA methylation (MethylationEPIC BeadChip). Men with type 2 diabetes had higher levels of follicle-stimulating hormone (FSH), but similar testosterone levels and sperm quality as controls. Sperm DNA methylation was stable with repeat sampling at 3 months in men with and without type 2 diabetes. We identified differential methylation at 655 of 745,804 CpG sites in men with type 2 diabetes versus controls (FDR < 0.05). Of these, 96.5

Diet and obesity contribute to insulin resistance and type 2 diabetes, in part via the gut microbiome. To explore the role of gut-derived metabolites in this process, we assessed portal/peripheral blood metabolites in mice with different risks of obesity/diabetes, challenged with a high-fat diet (HFD) + antibiotics. In diabetes/obesity-prone C57BL/6J mice, 111 metabolites were portally enriched and 74 were peripherally enriched, many of which differed in metabolic-syndrome-resistant 129S1/129S6 mice. Vancomycin treatment of HFD-fed C57BL/6J mice modified the microbiome and the portal/peripheral ratio of many metabolites, including upregulating tricarboxylic acid (TCA) cycle-related metabolites, like mesaconate, in portal blood. Treatment of isolated hepatocytes with mesaconate, itaconate, or citraconate improved insulin signaling and transcriptionally regulated genes involved in gluconeogenesis, fatty acid oxidation, and lipogenesis in vitro and in vivo. In humans, citraconate levels are inversely correlated with plasma glucose. Thus, portal versus peripheral metabolites play important roles in mediating effects of the microbiome on hepatic metabolism and the pathogenesis of HFD-related insulin resistance.

Transfer of regulatory molecules, such as microRNAs (miRNAs), via small extracellular vesicles (sEVs) called exosomes represents a newly recognized mode of inter-organ communication that provides an important mechanism of metabolic regulation. We have previously shown that adipose tissue is a major contributor to the pool of circulating exosomal miRNAs and that these adipose-derived exosomal miRNAs can be taken up by target tissues and suppress gene expression. We have also shown that biogenesis, secretion, and exosomal cargo sorting is a highly regulated process and that miRNAs contain 4-8 nucleotide motifs which contribute to exosomal secretion. Several exosomal miRNAs have been linked to obesity and diabetes, yet their regulation by physiological stimuli such as insulin remains unknown. To identify the role of insulin in regulation of adipose miRNAs and their secretion in exosomes, we performed small RNA-seq on exosomes isolated from differentiated 3T3-L1 cells with and without insulin stimulation. We find that out of the 460 miRNAs detected, 39 were up- and 75 were down-regulated in exosomes in response to insulin, whereas only 14 were up- and 39 were down-regulated in cells. In some cases, exosomal secretion correlated with cellular expression of the miRNA. More interestingly, 103 of the 114 miRNAs (90.3%) that were regulated in exosomes had no significant change at the cellular level or even a change in the opposite direction, demonstrating insulin’s ability to regulate miRNA exosomal secretion independent of cellular level. These insulin-regulated exosomal miRNAs included members of the let-7 family, which have known functions in adipogenesis, as well miRNAs predicted to target mRNAs of molecules involved in fatty acid biosynthesis and insulin signaling. Thus, insulin can independently regulate miRNA expression and secretion in adipocyte-derived exosomes. These can serve as adipokines and create a novel mechanism for regulation of cell-cell and inter-organ communication. Disclosure M.Lino: None. H.Pan: None. J.Dreyfuss: None. C.Kahn: None. Funding National Institutes of Health (5R01DK082659)

Breastfeeding reduces childhood obesity risk, though the mechanisms remain unclear. We recently reported that maternal exercise significantly increases breastmilk levels of lipids and metabolites involved in brown adipocyte thermogenesis. It is unclear whether differences in these milk constituents may play a functional role in infant adipose tissue metabolism. Thus, we tested whether consumption of human milk after an acute bout of maternal exercise alters infant thermogenesis, a surrogate for brown adipose tissue function. Exclusively breastfeeding mothers (n=31, 1-mo postpartum) completed a supervised exercise session (45 minutes, heart-rate reserve 65-75%) and, on a separate day, a control period of rest, each followed by an infant feeding session 1h afterwards. We analyzed human milk (metabolomics, lipidomics) before and after exercise. We compared infant anterior neck surface temperature (infrared thermography, IRT) following feeding of either “exercise milk” or “control milk”. We analyzed resting metabolic rate (RMR) and body composition (DXA) in all infants; a subset (n=10) also underwent fat-fraction MRI to assess sub-clavicular beige fat depot volume. Infant temperature trended higher (+0.245°C, P=0.1) after consumption of “exercise milk” vs. “control milk”; higher delta temperature was associated with higher infant trunk fat free mass (P<0.05). Of 213 metabolites detected in breastmilk; 37 were nominally changed (P<0.05) and 3 significantly changed (FDR<0.05) by exercise. For 16 metabolites, the change in abundance with maternal exercise correlated with change in infant temperature after "exercise-" vs. "control-milk" consumption. Top-ranking positively correlated metabolites included signaling lipids, lactate, guanine, and spermine (P<0.05). Our findings indicate that exercise-induced changes in human milk are associated with activation of infant metabolism via thermogenesis. Disclosure E.M. Isganaitis: Research Support; Dexcom, Inc., Tandem Diabetes Care, Inc., Insulet Corporation, MannKind Corporation. C. Lu: None. J. Dreyfuss: None. G. Kyere-Davies: None. K.R. Short: None. D.A. Fields: None. M. Rudolph: None. Funding Harold Hamm Pilot and Feasibility Grant; NIDDK P30 Pilot and Feasibility Grant