Metabolic Functions Research Articles

Spatiotemporal Modeling of Mitochondrial Network Architecture

In many cell types, mitochondria undergo extensive fusion and fission to form dynamic, responsive network structures that contribute to a number of homeostatic, metabolic, and signaling functions. The relationship between the dynamic interactions of individual mitochondrial units and the cell-scale network architecture remains an open area of study. In this work, we use coarse-grained simulations and approximate analytic models to establish how the network morphology is governed by local mechanical and kinetic parameters. The transition between fragmented structures and extensive networks is controlled by local fusion-to-fission ratios, network density, and geometric constraints. Similar fusion rate constants are found to account for the very different structures formed by mammalian networks (poised at the percolation transition) and well-connected budding yeast networks. Over a broad parameter range, the simulated network structures can be described by effective mean-field association constants that exhibit a nonlinear dependence on the microscopic nonequilibrium fusion, fission, and transport rates. Intermediate fusion rate constants are shown to result in the highest rates of network remodeling, with mammalian mitochondrial networks situated in a regime of high turnover. This spatially resolved modeling and simulation framework helps elucidate the emergence of cellular scale network structures, and allows for the quantitative extraction of microscopic kinetic parameters from past and future experimental data. Published by the American Physical Society 2024

Effects of Prebiotic Dietary Fibers on the Stimulation of the Mucin Secretion in Host Cells by In Vitro Gut Microbiome Consortia.

The gastrointestinal microbiota are important for human health. Dietary intake may modulate the composition and metabolic function of the gut microbiome. We examined how the breakdown of prebiotic dietary fibers by the gut microbiome affects mucin secretion by intestinal epithelial cells. Metagenomic analyses of in vitro gut microbiome consortia revealed taxonomic profiles and genetic diversity of carbohydrate-active enzymes that digest polysaccharides. Two independent consortia exhibited different abilities to produce acetic acid, propionic acid, and butyric acid via the fermentation of polysaccharides derived from dietary fibers of grains and mushrooms. Although acetic acid generally had the highest concentration, the ratios of butyric acid and propionic acid to acetic acid varied depending on the polysaccharide source. These short-chain fatty acids affected morphological differentiation and mucin secretion in HT-29 human intestinal epithelial cells. These results suggest that prebiotic dietary fibers can be digested and metabolized by the gut microbiome to short-chain fatty acids, which can affect gut epithelial cells both directly and indirectly via the modulation of the gut microbiota and their enzymes.

Elucidating the power of arginine restriction: taming type I interferon response in breast cancer via selective autophagy

BackgroundType I interferons (IFN-I) are potent alarm factors that initiate cancer cell elimination within tumors by the immune system. This critical immune response is often suppressed in aggressive tumors, thereby facilitating cancer immune escape and unfavorable patient outcome. The mechanisms underpinning IFN-I suppression in tumors are incompletely understood. Arginase-1 (ARG1)-expressing immune cells that infiltrate tumors can restrict arginine availability by ARG1-mediated arginine degradation. We hypothesized that arginine restriction suppresses the IFN-I response in tumors.MethodsComprehensive, unbiased open approach omics analyses, various in vitro techniques, including microscopy, qPCR, immunoblotting, knock-down experiments, and flow cytometry were employed, as well as ex vivo analysis of tumor tissue from mice. Several functional bioassays were utilized to assess metabolic functions and autophagy activity in cancer cells.ResultsArginine restriction potently induced expression of selective autophagy receptors, enhanced bulk and selective autophagy and strongly suppressed the IFN-I response in cancer cells in an autophagy-dependent manner.ConclusionOur study proposes a mechanism for how tumor-infiltrating immune cells can promote cancer immune escape by dampening the IFN-I response. We suggest ARG1 and autophagy as putative therapeutic targets to activate the IFN-I response in tumors.

Effect of Moniezia Benedeni infection on ileal transcriptome profile characteristics of sheep

BackgroundThe intestinal mucosal immune system, renowned for its precise and sensitive regulation, can provide comprehensive and effective protection for the body, among which the ileum is a critical induction site for regulating mucosal immune homeostasis. Moniezia benedeni parasitizes the small intestine of sheep and can cause serious pathological damage or even death to the host when the infection is severe. In this study, 5 sheep infected with Moniezia benedeni were selected as the infected group, and 5 uninfected sheep were selected as the control group. The ileal transcriptome profile characteristics of Moniezia benedeni infection were analyzed based on RNA-seq sequencing technology, aiming to lay a foundation for further exploring the perception mechanism of sheep intestines to Moniezia benedeni infection and formulating effective prevention and control strategies.ResultsThe results showed that a total of 3,891 differentially expressed genes (DEGs) were detected in the ileum tissues of sheep between the infected and control groups with 2,429 up-regulated genes and 1,462 down-regulated genes. GO and KEGG pathway enrichment analysis of differential genes, as well as Clue GO analysis showed that differential genes were significantly enriched in immune and metabolic-related biological processes and signaling pathways. Particularly, in immune-related signaling pathways, the B cell receptor signaling pathway was significantly down-regulated, while in metabolic regulation related signaling pathways, Bile secretion, Fat digestion and absorption and Vitamin digestion and absorption were notably up-regulated. On this basis, the differential core genes related to immune metabolism were verified by qRT-PCR method. The results showed that OVAR, CD3E, CD8A, CD4 and CD28 were significantly up-regulated (P < 0.05), while CIITA, BLNK, BCL6 and CD79A were significantly down-regulated (P < 0.05), which were consistent with transcriptome sequencing data.ConclusionsThe results demonstrated that Moniezia benedeni infection significantly affected the immune and metabolic processes in sheep ileum, particularly, it significantly inhibited the activation process of host B cells, and also led to an overactive function of bile acid metabolism. This finding provides a solid foundation for further elucidating the response mechanism of Peyer's patches in sheep ileum to Moniezia tapeworm infection.

Effectiveness of a fixed combination of empagliflozin with metformin in reduction of cardiovascular risk in patients with uncontrolled arterial hypertension and decompensated type 2 diabetes mellitus

Objective is to study the effectiveness of a fixed combination of empagliflozin and metformin in reducing cardiovascular risk in patients with uncontrolled arterial hypertension (AH) and decompensated type 2 diabetes mellitus (DM). Materials and methods. 36 patients with uncontrolled AH and decompensated type 2 diabetes mellitus aged 45 to 60 were examined. Before inclusion in the study, all patients received standard antihypertensive (angiotensin‑converting enzyme (ACE) inhibitor or angiotensin‑II receptor blocker (ARB) in combination with a calcium channel blocker and a thiazide‑like diuretic), glucose‑lowering (metformin) and hypolipidemic (atorvastatin) therapy. However, target levels of blood pressure (BP), glycated haemoglobin (HbA1c) and lipids were not achieved. After the initial examination, all patients were prescribed a fixed combination of empagliflozin and metformin at a dose of 12.5 mg/1000 mg 1—2 times a day, and antihypertensive and hypolipidemic therapy was continued. Re‑examination was carried out after 20 weeks. In the work, a statistical analysis of the obtained data was carried out using standard methods using Microsoft Excel 7.0 and SPSS 19.0 application packages. Results. The transfer of the examined patients from previous ineffective hypoglycemic therapy to a fixed combination of empagliflozin with metformin against the background of standard antihypertensive and hypolipidemic therapy made it possible to achieve the target levels of HbA1c in 69% of patients and target BP levels in 64%. The frequency of reaching target BP levels in the subgroup of patients who received ARB was significantly higher than in the subgroup of patients who received ACE inhibitors. The specified therapy contributed to a significant reduction in body weight, reaching the target levels of low‑density lipoprotein cholesterol (LDL‑C) and non‑high‑density lipoprotein cholesterol (Non‑HDL‑C) in more than a third of patients, and reducing the severity of albuminuria in some patients. The tested therapy, in general, led to reduction in cardiovascular risk and did not cause side effects that could lead to discontinuation of treatment. Conclusions. The fixed combination of empagliflozin with metformin against the background of standard antihypertensive and hypolipidemic therapy in patients with uncontrolled hypertension and decompensated type 2 diabetes mellitus contributed to the achievement in the vast majority of patients of the target levels of HbA1c in the blood and BP levels, reduced body weight, positive changes in lipid metabolism and kidney function, and demonstrated the possibility of a significant reduction in cardiovascular risk. A more pronounced decrease in BP was detected in the case of a fixed combination of empagliflozin and metformin with ARB compared to ACE inhibitors in this category of patients.

The Combined Effects of Temperature and pH to the Toxicity of the Water-Soluble Fraction of Gasoline (WSFG) to the Neotropical Yellow-Tail Tetra, Astyanax altiparanae.

Continental aquatic environments have undergone chemical pollution due to increased anthropogenic activities. Among those substances, petroleum hydrocarbons are a potential hazard for the aquatic animals. Additionally, alterations in the abiotic characteristics of the water, such as temperature and pH, can impose additional stress when those substances are present. We evaluate how alterations in water temperature and pH modified the acute (96h) toxicity of the water-soluble fraction of gasoline (WSFG) to Astyanax altiparanae through physiological analysis. We also investigated the physiological responses after the fish recovery from exposure (96h) in clean water. Both isolated and combined exposures to WSFG resulted in significant physiological changes. Alone, WSFG altered energetic metabolism and haematopoietic functions, potentially due to metabolic hypoxia. When combined with changes in water temperature (30°C) and pH (4.0), A. altiparanae activated additional physiological mechanisms to counterbalance osmoregulatory and acid-base imbalances, likely exacerbated by severe metabolic hypoxia. In both isolated and combined exposure scenarios, A. altiparanae maintained cellular hydration, suggesting a robust capacity to uphold homeostasis under environmental stress conditions. Following a recovery in clean water, energetic metabolism returned to control levels. Nevertheless, plasmatic Na+ and Cl- levels and haematological parameters remained affected by WSFG exposure. Our findings underscore the impact of interactions between WSFG contaminants, temperature and pH, leading to additional biological damage in A. altiparanae.

Utilization of a Human Liver Tissue Chip for Drug-Metabolizing Enzyme Induction Studies of Perpetrator and Victim Drugs.

Polypharmacy-related drug-drug interactions (DDIs) are a significant and growing healthcare concern. An increasing number of therapeutic drugs on the market underscores the necessity to accurately assess new drug combinations during pre-clinical evaluation for DDIs. In vitro primary human hepatocytes (PHH) models are only applicable for short term induction studies due to their rapid loss of metabolic function. Though co-culturing non-human stromal cells with PHH has been shown to stabilize metabolic activity long-term, there are concerns about human specificity for accurate clinical assessment. In this study, we demonstrate a PHH-only liver microphysiological system (MPS) in the Liver Tissue Chip (LTC) is capable of maintaining long-term functional and metabolic activity of PHH from three individual donors, and thus a suitable platform for long-term DDI induction studies. The responses to rifampicin induction of three PHH donors were assessed using CYP activity and mRNA changes. Additionally, victim PK studies were conducted with midazolam (high clearance) and alprazolam (low clearance) following perpetrator drug treatment, rifampicin-mediated induction, which resulted in a 2-fold and a 2.6-fold increase in midazolam and alprazolam intrinsic clearance values respectively compared to the untreated liver MPS. We also investigated the induction effects of different dosing regimens of the perpetrator drug (rifampicin) on CYP activity levels, showing minimal variation in the intrinsic clearance of the victim drug (midazolam). This study illustrates the utility of the LTC for in vitro liver-specific DDI induction studies, providing a translational experimental system to predict clinical clearance values of both perpetrator and victim drugs. Significance Statement This study demonstrates the utility of the Liver Tissue Chip (LTC) with primary human hepatocyte (PHH)-only liver microphysiological system (MPS) for drug-drug interaction (DDI) induction studies. This unique in vitro system with continuous recirculation maintains long-term functionality and metabolic activity for up to 4 weeks, enabling the study of perpetrator and victim drug pharmacokinetics, quantification of drug-induced CYP mRNA and activity levels, investigation of patient variability, and ultimately clinical predictions.

Harnessing the potential of copper-based nanoparticles in mitigating abiotic and biotic stresses in crops.

The demand for crops production continues to intensify with the rapid increase in population. Agricultural crops continue to encounter abiotic and biotic stresses, which can substantially hamper their productivity. Numerous strategies have been focused to tackle the abiotic and biotic stress factors in various plants. Nanotechnology has displayed great potential to minimize the phytotoxic impacts of these environmental constraints. Copper (Cu)-based nanoparticles (NPs) have displayed beneficial effects on plant growth and stress tolerance. Cu-based NPs alone or in combination with plant growth hormones or microorganisms have been documented to induce plant tolerance and mitigate abiotic or biotic stresses in different plants. In this review, we have comprehensively discussed the uptake and translocation of Cu-based NPs in plants, and beneficial roles in improving the plant growth and development at various growth stages. Moreover, we have discussed how Cu-based NPs mechanistically modulate the physiological, biochemical, metabolic, cellular, and metabolic functions to enhance plant tolerance against both biotic (viruses, bacterial and fungal diseases, etc.) and abiotic stresses (heavy metals or metalloids, salt, and drought stress, etc.). We elucidated recent advancements, knowledge gaps, and recommendations for future research. This review would help plant and soil scientists to adapt Cu-based novel strategies such as nanofertilizers and nanopesticides to detoxify the abiotic or biotic stresses. These outcomes may contribute to the promotion of healthy food production and food security, thus providing new avenues for sustainable agriculture production.

The Potential Role of Boron in the Modulation of Gut Microbiota Composition: An In Vivo Pilot Study

Background/Objectives: The role of the gut microbiome in the development and progression of many diseases has received increased attention in recent years. Boron, a trace mineral found in dietary sources, has attracted interest due to its unique electron depletion and coordination characteristics in chemistry, as well as its potential role in modulating the gut microbiota. This study investigates the effects of inorganic boron derivatives on the gut microbiota of mice. Methods: For three weeks, boric acid (BA), sodium pentaborate pentahydrate (NaB), and sodium perborate tetrahydrate (SPT) were dissolved (200 mg/kg each) in drinking water and administered to wild-type BALB/c mice. The composition of the gut microbiota was analyzed to determine the impact of these treatments. Results: The administration of BA significantly altered the composition of the gut microbiota, resulting in a rise in advantageous species such as Barnesiella and Alistipes. Additionally, there was a decrease in some taxa associated with inflammation and illness, such as Clostridium XIVb and Bilophila. Notable increases in genera like Treponema and Catellicoccus were observed, suggesting the potential of boron compounds to enrich microbial communities with unique metabolic functions. Conclusions: These findings indicate that boron compounds may have the potential to influence gut microbiota composition positively, offering potential prebiotic effects. Further research with additional analyses is necessary to fully understand the interaction between boron and microbiota and to explore the possibility of their use as prebiotic agents in clinical settings.

8515 Role of Insulin Receptor in Trpv1-positive Adipocyte Progenitor Cellsin Adipocyte Differentiation and Metabolic Function

Abstract Disclosure: C. Sahin: None. H. Camara: None. F. Shamsi: None. M. Lynes: None. Y. Tseng: None. Brown adipose tissue (BAT), a specialized fat that dissipates energy to produce heat, is an important endocrine organ in the regulation of energy balance. Stimulating BAT activation and beiging of white adipose tissue (WAT) are new strategies to combat metabolic diseases, such as obesity and diabetes. Traditionally, thermogenic adipocytes are known to originate from mesenchymal Pdgfrα-expressing adipocyte progenitor cells (APCs). A previous study conducted by our lab identified vascular smooth muscle cells as a novel cellular origin of APCs. These cells are characterized by the expression of transient receptor potential cation channel subfamily V member 1 (Trpv1+). With cold exposure, the Trpv1+ APC-derived adipocytes are increased in BAT and WAT and differentiated into highly thermogenic adipocytes. These findings indicate that the Trpv1+-APCs may play an important role in the development of brown and beige adipocytes in response to cold. This study was designed to explore the function of the Trpv1+ APC-derived adipocytes in thermogenic adipocyte differentiation and metabolic regulation. We generated a mouse model in which insulin receptor (IR), a well-known player in adipocyte differentiation, was deleted in the Trpv1+ APCs to impair their adipogenic potential. Trpv1-Cre::mTmG::IRflox transgenic and IRwt control mice were housed at cold temperature (5°C) for 7 days. Because the lineage tracing mTmG system was included, we were able to trace the fate of Trpv1+ APCs in the presence or absence of IR. We first examined the adipogenic capacity of the IR-deficient Trpv1+ cells in BAT and WAT tissues using whole-mounted adipose tissue imaging. In control mice, approximately 30% of all mature adipocytes were derived from Trpv1+ APCs in adipose tissue in response to cold. With ablation of IR in Trpv1+ APCs, this contribution was significantly reduced in BAT (by 11.7 % in females; 6.1% in males), inguinal WAT (by 16.8% in females; 19.3% males), and perigonadal WAT (by 11.4% in females; 14.8% in males) in transgenic mice compared to control mice. These results showed that IR-deficiency in the Trpv1-expressing cells impaired their adipogenesis in both male and female mice. In contrast, thermogenic genes (e.g., Ucp1, Pparγc1a, Cox7a1) were not significantly altered in BAT and inguinal WAT of the IR-deficient Trpv1+ APCs mice. Despite the changes in the adipocyte populations, we observed that deletion of IR in Trpv1+ APCs has no impact on body weight, food intake, fat mass, glucose tolerance, and insulin sensitivity relative to control mice. Taken together, blocking the contribution of Trpv1+ APCs to the mature adipocyte population results in no metabolic changes in mice, suggesting that the other APCs, such as PdgfRα+ APCs, may potentially compensate for impaired adipogenesis in different adipose tissues. Presentation: 6/1/2024

7975 TLR4 Signaling Modulates Reproductive and Metabolic Outcomes in a Mouse Model of Polycystic Ovary Syndrome

Abstract Disclosure: K.D. Wiggins: None. Z. Del Mundo: None. J.A. Ayala Angulo: None. M. Lopez: None. C. Garcia: None. C. Nguyen: None. J. De La Torre: None. A. Saba: None. D. Trinh: None. D. Nicholas: None. Polycystic Ovarian Syndrome (PCOS), characterized by disruptions in both reproductive and metabolic functions, is associated with abnormal immune system activity and chronic inflammation. Although the precise mechanistic drivers of chronic inflammation in PCOS are not fully elucidated, elevated serum levels of lipopolysaccharide (LPS) have been observed. We propose that Toll-like receptor 4 (TLR4) signaling, influenced by dysregulated LPS levels, is a central driver of chronic inflammation affecting both reproductive and metabolic pathways. To investigate this hypothesis, we subcutaneously implanted a nonsteroidal aromatase inhibitor, Letrozole (LET), in pubertal TLR4 knockout mice (TLR4KO) and C57BL/6J (wild type) mice. Our five-week LET treatment revealed that TLR4KO mice successfully entered all phases of the estrous cycle, with normalized Luteinizing Hormone (LH) and increased Follicle Stimulating Hormone (FSH) levels. These findings highlight TLR4's role in shaping cycling patterns and regulating fundamental hormonal pathways vital for reproductive health. Despite the genetic manipulation of the inflammatory pathway leading to a reduction in overall body weight gain, it had no discernible impact on the overall body composition of fat and lean mass. Additionally, our study found that dysregulation of blood glucose levels over time was independent of reduced body weight. TLR4KO mice demonstrated inefficient glucose clearance compared to wild-type mice, hinting at potential increases in insulin resistance, imbalances in adipose tissue inflammation, or possible alterations in the gut microbiota. Our study underscores the significance of disrupting the pro-inflammatory TLR4 pathway in shaping the reproductive and metabolic phenotypes in Letrozole-induced PCOS. The investigation into TLR4 becomes pivotal for comprehending the role of chronic inflammation in PCOS development or progression, offering valuable insights into potential therapeutic targets. Presentation: 6/2/2024

7996 Neuroanatomic Characterization of Mouse Kisspeptin Neurons in the Bed Nucleus of the Stria Terminalis

Abstract Disclosure: S.T. Zdon: None. M.S. Silva: None. V.M. Navarro: None. Kisspeptin (Kiss1) neurons are essential for reproductive function as they are strong regulators of the hypothalamic-pituitary-gonadal axis (HPG). Mammalian Kiss1 neurons are mainly located in the hypothalamus, controlling gonadotropin-releasing hormone (GnRH) secretion, where the anteroventral periventricular nucleus (AVPV) population is morphologically and functionally sexually differentiated. Kiss1 neurons are also found in limbic areas, such as the bed nucleus of the stria terminalis (BNST). This nucleus is involved in the integration of the HPG axis with sex-dependent social behaviors and metabolic functions. However, it remains unclear whether these populations show sexually dimorphic features. The present study focused on the neuroanatomical characterization of Kiss1BNST neurons in mice on both sexes. A Kiss1cre;salsa6f mouse strain carrying the expression of tdTomato specifically in Kiss1 neurons was used to study the distribution of Kiss1BNST neurons. The BNST is divided into the anterior division of the BNST (aBNST), and a more posterior division called the principal component of the BNST (prBNST). Results indicate that male mice have significantly more Kiss1prBNST neurons than females (Male: 139.4 ± 23.32 vs. Female: 43.70 ± 5.33 neurons; **P &amp;lt; 0.01). This sex-biased distribution was absent in Kiss1aBNST neurons. Additionally, stereotaxically injecting a Cre-dependent recombinant adenovirus carrying the expression of mCherry reporter into the aBNST or prBNST in adult mice was performed to observe neuronal projections. We observed fibers from aBNST-injected Kiss1 neurons projecting widely to areas that include the AVPV, medial preoptic area (MPO), lateral preoptic area (LPO), and periventricular hypothalamic nucleus (PVH). Moreover, fibers and cell bodies from male prBNST-injected Kiss1 neurons projected to dorsal lateral BNST (dlBNST), juxta-periventricular lateral hypothalamic area (JPLH), and the posterolateral stria terminalis (STMPL). This study observed a sexual dimorphic Kiss1 neuronal population in the prBNST, with males having more Kiss1prBNST neurons present and prospective postsynaptic areas in the brain where these neurons project to, helping to aid future studies on characterizing the functional role of these neurons. Presentation: 6/1/2024

7143 Near Term Cardiometabolic Outcomes in Transgender Boys and Men in the First Year of Gender-Affirming Testosterone Therapy

Abstract Disclosure: J. Dey: None. M. Widlansky: None. S. Cabrera: None. Background: Gender affirming testosterone (T) therapy is used to masculinize transgender adolescent men (TMs), resulting in elevated T and suppressed estradiol (E2). In cis-females, hypoestrogenism reduces vaso-protective E2-mediated estrogen receptor expression, decreasing nitric oxide vasodilation and increasing susceptibility to future vascular complications. Whether T promotes similar changes in TMs remains poorly understood. Methods: A longitudinal observational study is conducted in TMs aged 12-30yrs over the 1st yr of T, with visits at baseline (BL; pre-T), 6mo, and 1yr, compared to cis-males (CMs) with endogenous T. Primary outcome is change in brachial artery flow-mediated dilation (FMD%), an in vivo measure of vascular endothelial health, after 1yr of T. To detect a significant relative change in FMD%, 60 participants are needed (40 TMs, 20 CMs). Metabolic profile is assessed by fasting lipids, glucose, and insulin. Body composition is assessed by BMI and DEXA. Differences in mean values between and within a cohort were analyzed using the unpaired two-tailed T-test assuming unequal and equal variances, respectively. Results: To date, 51 participants are enrolled; the 21 participants who fully completed the study are included here (13 TMs, 8 CMs). At BL, TMs were post-menarchal with a mean age of 15.6yrs, ht 162.6cm, and BMI z-score 0.47. At BL, 46% were on progestin-only menstrual suppression. CMs had a mean age of 18.4yrs, ht 170.7cm, and BMI z-score 0.13. Cohorts were similar for age, height, and BMI.At BL, FMD% was higher in TMs than CMs (8.2 vs 4.6, p=0.01) but not at 1yr (6.9 vs 6.2, p=0.6). FMD% did not change in TMs receiving T over 1yr (8.2 vs 6.9, p=0.3). At BL, the brachial artery resting diameter (mm) was lower in TMs than CMs (2.6 vs 3.5, p&amp;lt;0.01) and remained such at 1-yr. Resting diameter increased in TMs over 1yr of T (2.6 vs 3.1, p&amp;lt;0.01) but not in CMs. There were no differences in resting or hyperemic flow velocity, or FMD (mm) between or within groups. At BL, % lean body mass (%LBM) was lower in TMs than CMs (60 vs 72, p&amp;lt;0.01) and estimated percent visceral adiposity tissue (%VAT) was higher (26 vs 14, p&amp;lt;0.01). In TMs, %LBM increased (60 vs 66, p &amp;lt;0.01) and %VAT decreased (26 vs 21, p&amp;lt;0.01) over 1yr of T, such that TMs and CMs were similar at 1yr. At BL, TMs had lower T but similar E2 (46 vs 27 pg/mL, p=0.2) to CMs. At 1-yr, TMs and CMs had similar T (556 vs 574 ng/dL, p=0.9) and E2 (52 vs 29, p=0.1). In TMs, T did not change E2. Lipid and glycemic indices did not change over 1yr in TMs. Conclusion: Initial analyses suggest that T does not affect FMD; however, FMD% interpretation is confounded by the small number of participants, progestins at BL in TMs, and the lower resting brachial artery diameter in TMs. The stable E2 in TMs on T may explain the stable FMD%. Overall, our findings suggest T results in relative stability of vascular and metabolic function and a beneficial shift in body composition in TMs. Presentation: 6/2/2024

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

6509 IGF-1 receptor and insulin receptor in LepRb neurons coordinates body growth, reproduction, and metabolism

Abstract Disclosure: M. Wang: None. J.W. Hill: None. Growth and reproduction are tightly linked. Growth hormone (GH) deficiency results in a profound suppression of postnatal growth accompanied by severely delayed puberty and reproductive senescence, while GH excess is correlated with the reverse. The specific mechanism underlying this delay is undefined. Although leptin receptor (LepRb)-expressing neurons are known to link body growth and reproduction, the role of insulin-like growth factor 1 receptor (IGF1R) signaling in LepRb neuron function is unknown. Previous studies have shown that IGF-1 and insulin can bind to each other’s receptor, permitting IGF-1 signaling in the absence of IGF1R. Therefore, we created mice lacking IGF1R exclusively in LepRb neurons (IGF1RLepRb mice) and mice simultaneously lacking IGF1R and IR in LepRb neurons (IGF1R/IRLepRb mice) to test if IGF1R and insulin receptor (IR) cooperatively regulate metabolic and reproductive functions. IGF1RLepRb and IGF1R/IRLepRb mice displayed delayed onset of puberty and impaired fertility in both sexes, which suggested that both IGF1R and IR in LepRb neurons are required for normal reproduction. Additionally, we found transient growth retardation in IGF1RLepRb and IGF1R/IRLepRb mice. Loss of IGF1R in LepRb neurons led to lower serum IGF-1 and GH levels in male mice and impaired trabecular and cortical bone development in mice of both sexes. Only transient and mild changes were noticed in the body weight. However, we found IGF1R and IR in LepRb neurons jointly regulate body mass composition, energy homeostasis and glucose homeostasis, as shown by elevated fat mass composition, low energy expenditure and low physical activity in IGF1R/IRLepRb mice. Taken together, these studies identify the unique and cooperative role of LepRb IGF1R and IRs in the regulation of body growth, reproduction, and metabolism. Presentation: 6/3/2024

12468 Loss Of Enterococcus And Faecalibacterium Associate With Polycystic Ovary Syndrome And Hirsutism In A Sample Of Hispanic Patients

Abstract Disclosure: L.A. Gonzalez-Rodriguez: Research Investigator; Self; Eli Lilly &amp; Company. C.M. Casas Loyola: None. C.I. Martinez-Hernandez: None. L. El Musa Penna: None. J. Romaguera: None. F. Godoy-Vitorino: None. Polycystic Ovary Syndrome (PCOS) is a common endocrine disorder affecting women of reproductive age. It is characterized by symptoms such as irregular menstrual periods, excess androgen levels and polycystic ovaries. Women with PCOS often display insulin resistance independent of fat and have been associated with an increased risk of diabetes mellitus, cardiovascular disease, and mood disorders. The gut microbiota, the community of microbes living in the human gastrointestinal tract, plays a crucial role in human health and has been implicated in various diseases, including metabolic and inflammatory conditions. We aimed to characterize the gut microbiota in a sample of Hispanic women living in Puerto Rico (n=50). Of these 38 had confirmed PCOS as per Rotterdam criteria and 12 were controls. A stool sample was collected for genomic DNA extractions followed by amplification and sequencing of 16S rRNA genes (V4 region) with Illumina MiSEQ. Data analyses were performed with standard pipelines with variables sample group (PCOS vs Controls), insulin resistance assessed using HOMA-IR and hirsutism status according to modified-Ferriman Gallwey (mFG) score and controlled for age and other clinical variables. Despite there were no changes in alpha or beta diversity among the microbiota of these groups, composition was significantly different. Women with PCOS had significantly lower levels of Enterococcus species as well as Hungatella, taxa that are part of a physiologically normal gut flora. Enterococcus species contribute to the balance of the microbial community and play roles in the metabolism and immune function and are very resilient. Hungatella species are a known source of catabolic enzymes that degrade glycosaminoglycans which are important in tissue repair. These dominant taxa in controls was substituted by other taxa in PCOS. Women with prominent hirsutism had significantly lower levels of Faecalibacterium while those with insulin resistance had increased levels of Clostridium. We found that higher levels Clostridium, and lower levels of protective anti-inflammatory taxa such as Faecalibacterium or Enterococci, which are important for gut health and metabolic regulation, are linked to this metabolic disturbance. Even though this is a preliminary report, it opens potential therapeutic strategies, such as the use probiotics, and dietary modifications to modulate the gut microbiome and improve PCOS outcomes. Presentation: 6/1/2024

7310 System-level Analysis in Polycystic Ovary Syndrome on a Panel of Genetically Diverse Strains of Mice

Abstract Disclosure: L.M. Velez: None. C. Johnson: None. I. Tamburrini: None. M. Zhou: None. C. Viesi: None. N. Ujagar: None. D. Ashbrook: None. M. Nelson: None. A. Senior: None. D. James: None. R. Williams: None. D. Nicholas: None. M. Seldin: None. Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women, with a prevalence of ∼4-20% in women of reproductive age. The diagnosis of the syndrome generally occurs when the patient consults for fertility issues and is only based on a reproductive criterion, which includes (1) hyperandrogenism, (2) oligo-anovulation, and (3) polycystic ovary morphology. However, the overlap of PCOS with cardiometabolic diseases is significant. To put in numbers; up to 75% of PCOS women present some degree of insulin insensitivity, 38-88% present obesity or overweight, 20-50% develop type 2 diabetes by age 40, and PCOS women are at increased risk of cardiovascular disease. Despite these facts, shared reproductive/metabolic mechanisms are largely underexplored. Moreover, studies addressing the genetic architecture of PCOS are missing. Here, we induced a PCOS-like condition in 25 recombinant and classical inbred female strains and matched placebo controls over 6 weeks. Comprehensive in vivo and terminal reproductive/metabolic analyses were performed, as well as ovary and adipose RNA-Seq. These strains varied in PCOS response in a number of key metabolic and reproductive traits, including circulating hormone levels, glucose metabolism, and cardiac function. We applied a linear mixed-effects model to estimate heritability, and genetic (h2), PCOS, and gene-by-PCOS interactions. High h2 was observed for lean and fat mass, glucose, and AUC, whereas PCOS effects were high for the BW change, testosterone, and AUC. Substantial gene-by-PCOS interactions were found for reproductive hormones. Undirected network construction and centrality estimates showed that the reproductive hormones LH and LH/FSH ratio were the strongest central traits connecting metabolic phenotypes. We also showed select strains represent subtypes of human PCOS-metabolism interaction with varied susceptibilities to disease in a PCOS setting. Ovarian RNA-seq analysis of PCOS DEGs showed strong enrichments with human disease settings such as hyperandrogenism, inflammation, and pregnancy hypertension. Similar analyses in GWAT RNA-seq showed enrichments in weight gain, liposarcoma, inflammation, and reproductive diseases were at the top, with adipose genes connecting these diseases and potentially involved with PCOS. In conclusion, we established a PCOS model to study relevant mechanisms intersecting reproduction with metabolism in the context of genetic variation. Presentation: 6/2/2024

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

12468 Loss Of Enterococcus And Faecalibacterium Associate With Polycystic Ovary Syndrome And Hirsutism In A Sample Of Hispanic Patients

Abstract Disclosure: L.A. Gonzalez-Rodriguez: Research Investigator; Self; Eli Lilly &amp; Company. C.M. Casas Loyola: None. C.I. Martinez-Hernandez: None. L. El Musa Penna: None. J. Romaguera: None. F. Godoy-Vitorino: None. Polycystic Ovary Syndrome (PCOS) is a common endocrine disorder affecting women of reproductive age. It is characterized by symptoms such as irregular menstrual periods, excess androgen levels and polycystic ovaries. Women with PCOS often display insulin resistance independent of fat and have been associated with an increased risk of diabetes mellitus, cardiovascular disease, and mood disorders. The gut microbiota, the community of microbes living in the human gastrointestinal tract, plays a crucial role in human health and has been implicated in various diseases, including metabolic and inflammatory conditions. We aimed to characterize the gut microbiota in a sample of Hispanic women living in Puerto Rico (n=50). Of these 38 had confirmed PCOS as per Rotterdam criteria and 12 were controls. A stool sample was collected for genomic DNA extractions followed by amplification and sequencing of 16S rRNA genes (V4 region) with Illumina MiSEQ. Data analyses were performed with standard pipelines with variables sample group (PCOS vs Controls), insulin resistance assessed using HOMA-IR and hirsutism status according to modified-Ferriman Gallwey (mFG) score and controlled for age and other clinical variables. Despite there were no changes in alpha or beta diversity among the microbiota of these groups, composition was significantly different. Women with PCOS had significantly lower levels of Enterococcus species as well as Hungatella, taxa that are part of a physiologically normal gut flora. Enterococcus species contribute to the balance of the microbial community and play roles in the metabolism and immune function and are very resilient. Hungatella species are a known source of catabolic enzymes that degrade glycosaminoglycans which are important in tissue repair. These dominant taxa in controls was substituted by other taxa in PCOS. Women with prominent hirsutism had significantly lower levels of Faecalibacterium while those with insulin resistance had increased levels of Clostridium. We found that higher levels Clostridium, and lower levels of protective anti-inflammatory taxa such as Faecalibacterium or Enterococci, which are important for gut health and metabolic regulation, are linked to this metabolic disturbance. Even though this is a preliminary report, it opens potential therapeutic strategies, such as the use probiotics, and dietary modifications to modulate the gut microbiome and improve PCOS outcomes. Presentation: 6/1/2024

Evaluating the effectiveness of Toxfin and Novasil as dietary aflatoxin-binding agents in broilers for sustaining hepatic antioxidant capacity and intestinal health status during aflatoxin B1 exposure.

To assess the efficacy of Toxfin and Novasil as aflatoxin-binding agents in broilers exposed to aflatoxin B1 (AFB1) from 11 to 30days, 288 mixed-sex Ross 308 broiler chickens were randomly allocated to four dietary groups: control feed, control feed + 0.25mg/kg AFB1, AFB1 feed + 0.3% Toxfin, and AFB1 feed + 0.3% Novasil. The evaluation encompassed growth performance for the grower (11-20days), finisher (21-30days), and overall (11-30days) phases, carcass characteristics, serum biochemical components, liver function enzymes, hepatic antioxidant capacity, AFB1 residue in the liver and kidney, and ileal morphology at 30days, and apparent nutrient digestibility during 29-30days. Exposure to AFB1 significantly resulted in reduced growth efficiency, lowered carcass yields, liver hypertrophy, impaired metabolic and hepatic functions, liver oxidative stress, disrupted ileum architecture, diminished nutrient digestibility, and accumulated AFB1 in the liver and kidney. Conversely, supplementation of Toxfin or Novasil significantly augmented body weight gain (BWG) and reduced feed conversion ratio (FCR) during the finisher and overall phases, elevated BWG in the grower phase, heightened levels of glucose, hepatic protein, and glutathione peroxidase, declined malondialdehyde content, improved apparent metabolizable energy, and lowered AFB1 residues in the liver and kidney. Furthermore, Toxfin inclusion significantly reduced FCR during the grower phase, enhanced European production efficiency factor during the grower and overall phases, augmented dressing percentage, declined proportional liver weight, elevated concentrations of total protein, albumin, and total antioxidant capacity, heightened villus surface area, and boosted crude protein digestibility. To conclude, incorporating 0.3% Toxfin into broilers' feeds confers a more effectual safeguard than Novasil against the deleterious consequences of AFB1 exposure.