Glucose Transporter Research Articles

Knock-out of the major regulator Flo8 in Komagataella phaffii results in unique host strain performance for methanol-free recombinant protein production

Flo8 is a main transcriptional regulator of flocculation and pseudohyphal growth in yeast. Disruption of FLO8 in the popular recombinant protein production host Komagataella phaffii (Pichia pastoris) prevents pseudohyphal growth and reduces cell-to-surface adherence, making the mutant an interesting platform for research and industry. However, knowledge of the physiological impact of the mutation remained scarce. In-depth analysis of transcriptome data from FLO8-deficient K. phaffii revealed that Flo8 affects genes involved in cell cycle, mating, respiration, and catabolite repression additionally to flocculation targets. One gene with considerably increased expression in flo8 was GTH1, encoding a high-affinity glucose transporter in K. phaffii. Its promoter (PG1) was previously established as a strong, glucose-regulatable alternative to methanol-induced promoters. PG1 and its improved derivatives PG1-3, D-PGS4 and D-PGS5, proved to be promising candidates for controlling recombinant protein production in the FLO8-deficient background. In small-scale screenings, PG13-controlled intracellular EGFP levels were 2.8-fold higher, and yields of different secreted recombinant proteins were up to 4.8-fold increased. The enhanced productivity of the flo8 mutant in combination with the PG1 variants was transferrable to glucose-limited fed-batch processes and could largely be attributed to higher transcriptional activity of the promoter, leading to a much higher productivity per chromosomally integrated gene copy. K. phaffii flo8 has many advantageous characteristics, such as reduced surface growth and increased transcriptional strength of glucose-regulatable promoters. These features turn the flo8 strain into a valuable new base strain for various experimental designs and establish flo8 as an excellent strain background for methanol-free recombinant protein production processes.

Selenium polysaccharide form sweet corn cob mediated hypoglycemic effects in vitro and untargeted metabolomics study on type 2 diabetes

Type 2 diabetes mellitus (T2D) causes complications due to metabolic disorders besides increasing blood glucose. Sweet corn cob selenium polysaccharide (SeSCP) is a complex of Se with Sweet corn cob polysaccharide that has good hypoglycemic efficacy, but its effect on T2D metabolism has not been determined. In this study, the hypoglycemic effect of SeSCP was investigated by in vitro and in vivo experiments, and the levels of metabolites in feces were analyzed in a high-fat diet and STZ-induced T2D mouse model by Liquid chromatography-mass spectrometry (LC-MS). The results indicated that SeSCP regulates α-amylase and α-glucosidase through competitive reversible inhibition, and the reaction is spontaneous, driven by van der Waals forces and hydrogen bonding. In vivo, SeSCP modulates glucose transport decreasing glucose entry into the bloodstream. The metabolites mainly affected by SeSCP-MC were adenine, LysoPA (0:0/18:2(9Z, 12Z)), cysteine-S-sulfate, and demeclocycline (hydrochloride) metabolites. SeSCP interfered with β-alanine metabolism, starch and sucrose metabolism, ether lipid metabolism, glycerophospholipid metabolism, glyoxylate and dicarboxylate metabolism, pantothenate and CoA biosynthesis, etc. Additionally, SeSCP exhibited more effective metabolic interventions than metformin and SCP. Therefore, SeSCP can reduce complications while improving T2D blood glucose.

Safety and Efficacy of Dapagliflozin in Recurrent Ascites: A Pilot Study.

In cirrhosis, activation of renin-angiotensin-aldosterone system leads to sodium and water retention causing ascites. Dapagliflozin, a sodium glucose linked transporter-2 inhibitor, induces natriuresis in patients with heart failure. A similar natriuretic effect may improve ascites in patients with cirrhosis. In this pilot study, we evaluated the safety and efficacy of dapagliflozin in patients with cirrhosis and recurrent ascites. Forty patients with recurrent ascites and cirrhosis were randomized to 1:1 in a double blinded fashion to receive either dapagliflozin (10mg/day) with standard medical therapy (Group A) or placebo with standard medical therapy (Group B). The primary outcome was control of ascites at 6 months. Secondary outcomes were urine output, 24-h urinary sodium, Child Turcotte Pugh (CTP), model for end-stage liver disease (MELD) scores, survival at 6 months, incidence of acute kidney injury (AKI) and infections. The 2 groups were comparable at baseline. Control of ascites at 6 months was significantly better in group A than that in Group B (p = 0.04). Change in urinary sodium was significantly higher in Group A (p < 0.001]. However, there was no difference in change in urine output, CTP or MELD scores and survival (65% vs 72.2%, p = 0.75) between the groups at 6 months. Incidence of AKI (50% vs 15%, p = 0.04) and infections (55% vs 20%, p = 0.04) were significantly higher in Group A. Significantly better control of ascites and higher natriuresis are observed with dapagliflozin. However, it does not improve disease severity scores or survival, and is associated with increased AKI and infections (NCT05014594).

SGLT1 inhibition alleviates radiation-induced intestinal damage through promoting mitochondrial homeostasis

Radiation-induced intestinal injury (RIII) constitutes a challenge in radiotherapy. Ionizing radiation (IR) induces DNA and mitochondrial damage by increasing reactive oxygen species (ROS). Sodium–glucose cotransporter 1 (SGLT1) is abundant in the gastrointestinal tract and the protective effects of inhibited SGLT1 in kidney and cardiovascular disease have been widely reported. However, the function of SGLT1 in RIII remains unclear. Herein, we reported that IR induced intestinal epithelial cell damage along with upregulation of SGLT1 in vivo and in vitro, which was alleviated by inhibition of SGLT1. Specifically, maintaining intestinal cell homeostasis was detected through cellular proliferation, apoptosis, and DNA damage assays, promoting epithelial regeneration and lifespan extension. Considering the importance of mitochondrial function in cell fate, we next confirmed that SGLT inhibition maintains mitochondrial homeostasis through enhanced mitophagy in intestinal epithelial cells. Finally, based on the bioinformatics analysis and cell validation, we demonstrated that inhibition of SGLT1 suppresses the PI3K/AKT/mTOR pathway to enhance mitophagy activation post-irradiation. In addition, we preliminarily demonstrate that SGLT inhibitors do not affect the radiosensitivity of tumors. Hence, our findings suggest that inhibition of SGLT is a promising therapeutic strategy to protect against RIII. To the best of our knowledge, this is the first report on the potential effect of SGLT1 inhibition in RIII.

Mannose-Functionalized Chitosan-Coated PLGA Nanoparticles for Brain-Targeted Codelivery of CBD and BDNF for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disease causing cognitive and memory decline. AD is characterized by the deposition of amyloid-β and hypophosphorylated forms of tau protein. AD brains are found to be associated with neurodegeneration, oxidative stress, and inflammation. Cannabidiol (CBD) shows neuroprotective, antioxidant, and anti-inflammatory properties and simultaneously reduces amyloid-β production and tau hyperphosphorylation. The brain-derived neurotrophic factor (BDNF) plays a vital role in the development and maintenance of the plasticity of the central nervous system. A decline of BDNF levels in AD patients results in reduced plasticity and neuronal cell death. Current therapeutics against AD are limited to only symptomatic relief, necessitating a therapeutic strategy that reverses cognitive decline. In this scenario, combination therapy of CBD and BDNF could be a fruitful strategy for the treatment of AD. We designed mannose-conjugated chitosan-coated poly(d,l-lactide-co-glycolide (PLGA) (CHTMAN-PLGA) nanoparticles for the codelivery of CBD and BDNF to the brain. Chitosan is modified with mannose to specifically target the glucose transporter-1 (GLUT-1) receptor abundantly present in the blood-brain barrier for selectively delivering therapeutics to the brain. The CBD-encapsulated nanoparticles showed an average hydrodynamic diameter of 306 ± 8.12 nm and a zeta potential of 31.7 ± 1.53 mV. The coated nanoparticles prolonged encapsulated CBD release from the PLGA matrix. The coated nanoparticles exhibited sustained release of CBD for up to 22 days with 91.68 ± 2.91% release of the encapsulated drug. The coated nanoparticles, which had a high positive zeta potential (31.7 ± 1.53 mV), encapsulated the plasmid DNA. The qualitative transfection efficiency was investigated using CHTMAN-PLGA-CBD/pGFP in bEND.3, primary astrocytes, and primary neurons, while the quantitative transfection efficiency of the delivery system was determined using CHTMAN-PLGA-CBD/pBDNF. In vitro, the pBDNF transfection study revealed that the BDNF expression was 4-fold higher for CHTMAN-PLGA-CBD/pBDNF than for naked pBDNF in all of the cell lines. The cytotoxicity and hemocompatibility of the designed nanoparticles were tested in bEND.3 cells and red blood cells, respectively, and the nanoparticles were found to be nontoxic and hemocompatible. Hence, mannose-conjugated chitosan-coated PLGA nanoparticles could be useful as brain-targeting delivery vehicles for the codelivery of CBD and BDNF for possible AD treatment.

Envisioning Glucose Transporters (GLUTs and SGLTs) as Novel Intervention against Cancer: Drug Discovery Perspective and Targeting Approach.

Metabolic reprogramming and altered cellular energetics have been recently established as an important cancer hallmark. The modulation of glucose metabolism is one of the important characteristic features of metabolic reprogramming in cancer. It contributes to oncogenic progression by supporting the increased biosynthetic and bio-energetic demands of tumor cells. This oncogenic transformation consequently results in elevated expression of glucose transporters in these cells. Moreover, various cancers exhibit abnormal transporter expression patterns compared to normal tissues. Recent investigations have underlined the significance of glucose transporters in regulating cancer cell survival, proliferation, and metastasis. Abnormal regulation of these transporters, which exhibit varying affinities for hexoses, could enable cancer cells to efficiently manage their energy supply, offering a crucial edge for proliferation. Exploiting the upregulated expression of glucose transporters, GLUTs, and Sodium Linked Glucose Transporters (SGLTs), could serve as a novel therapeutic intervention for anti-cancer drug discovery as well as provide a unique targeting approach for drug delivery to specific tumor tissues. This review aims to discussthe previous and emerging research on the expression of various types of glucose transporters in tumor tissues, the role of glucose transport inhibitors as a cancer therapy intervention as well as emerging GLUT/SGLT-mediated drug delivery strategies that can be therapeutically employed to target various cancers.

Ameliorative effects of Penthorum chinense Pursh on insulin resistance and oxidative stress in diabetic obesity db/db mice.

Penthorum chinense Pursh (PCP), a medicinal and edible plant, has been reported to protect against liver damage by suppressing oxidative stress. Type 2 diabetes mellitus (T2DM) is associated with liver dysfunction and oxidative stress. In the present study, we aim to investigate the hypoglycemic effect of PCP on db/db mice and further explore the underlying mechanisms. Thirty-two db/db mice were randomized into four groups, including a diabetic model control group (MC) and three diabetic groups treated with low (LPCP, 300 mg/kg/d), medium (MPLP, 600 mg/kg/d), and high doses of PCP (HPCP, 1200 mg/kg/d), and the normal control group (NC) of eight db/m mice were included. Mice in the NC and MC groups received the ultrapure water. After four weeks of intervention, parameters of fasting blood glucose (FBG), insulin resistance (IR), blood lipid levels, hepatic oxidative stress, and enzymes related to hepatic glucose metabolism were compared in the groups. PCP administration significantly reduced FBG and IR in diabetic db/db mice, and improved hepatic glucose metabolism by increasing glucose transporter 2 (GLUT2) and glucokinase (GCK) protein expression. Meanwhile, PCP supplementation ameliorated hepatic oxidative stress by decreasing malonaldehyde content and increasing the activities of superoxide dismutase and glutathione peroxidase in db/db mice. Furthermore, PCP treatment reduced obesity and food intake in db/db mice, and improved dyslipidemia demonstrated by increasing high-density lipoprotein cholesterol (HDL-C) while decreasing total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (HDL-C). All doses of PCP treatment decreased the values of LDL-C/HDL-C in a dose-response relationship. PCP significantly alleviated hyperglycemia, hyperinsulinemia, hyperlipidemia, and obesity, inhibited hepatic oxidative stress, and enhanced hepatic glucose transport in T2DM mice. Based on the above findings, the hypoglycemic effect of PCP may be attributed to the activation of the GLUT2/GCK expression in the liver and the reduction of hepatic oxidative stress.

GC-MS SCRUTINY OF PHYTOCONSTITUENTS, IN VITRO EVALUATION OF ANTIHYPERGLYCEMIC, ANTIADIPOGENIC ACTIVITIES, AND CYTOTOXIC EFFECT USING 3T3 L1 ADIPOCYTE CELL LINE AND MOLECULAR DOCKING STUDIES OF PREMNA CORYMBOSA

Objective: The present study was intended to list out the phytochemical multiples and to investigate the antihyperglycemic effect of Premna corymbosa using in vitro assays and in silico molecular docking methods. Methods: The phytochemical multiples of methanol proportion of P. corymbosa leaves were appraised by Gas Chromatography-Mass Spectrometry (GC-MS) scrutiny to illustrate the attendance of phytochemical composites. Moreover, the in vitro antihyperglycemic, antiadipogenic activities, and cytotoxic effects of the extract were elucidated using a 3T3 L1 adipocyte cell line. Mode of action of phytochemical composites in methanol leaf extract of P. corymbosa was probed by Western blotting with IRS1, IRS2, mTOR, and glucose transporter type 4 (GLUT 4) receptors. At present, to probe the consequence of the aboriginal drugs, it is necessary to perform in silico docking on the diabetic receptor which could be useful for the progress of enhanced formulation for the psychoanalysis of diabetes. Results: The GC-MS scrutiny depicted the being there of thirty-five phytochemical multipart. Amid the thirty-five multipart’s recognized, focal composites were Phytol, acetate (RT-16.78), n-Hexadecanoic acid (RT-18.16), Phytol (RT-19.51), 9,12,15-octadecatrienoic acid (Z,Z,Z) (RT-19.85), octadecanoic acid (RT-20.04), and Bis (2-ethylhexyl) phthalate (RT-23.09). The results of the glucose conception assay, adipocyte differentiation assay, and MTT assay showed potent in vitro antihyperglycemic activity with methanol leaf extract of P. corymbosa in 3T3l1Cell line. The results attained from western blotting revealed good antihyperglycemic activity of P. corymbosa. The in silico molecular docking results illustrated that the selected herbal lead compound is an effective target against the receptors. The compound showed favorable interactions with the amino acid residues thereby substantiating their proven efficacy as an antihyperglycemic compound. Conclusion: The outcome of the current study substantiates the antihyperglycemic prospective of the methanol leaf extract of P. corymbosa on the hyperglycemic causal agents and its activity against diabetes by a molecular approach.

Glucose Transporter-Targeting Chimeras Enabling Tumor-Selective Degradation of Secreted and Membrane Proteins.

Tumor-selective degradation of target proteins has the potential to offer superior therapeutic benefits with maximized therapeutic windows and minimized off-target effects. However, the development of effective lysosome-targeted degradation platforms for achieving selective protein degradation in tumors remains a substantial challenge. Cancer cells depend on certain solute carrier (SLC) transporters to acquire extracellular nutrients to sustain their metabolism and growth. This current study exploits facilitative glucose transporters (GLUTs), a group of SLC transporters widely overexpressed in numerous types of cancer, to drive the endocytosis and lysosomal degradation of target proteins in tumor cells. GLUT-targeting chimeras (GTACs) were generated by conjugating multiple glucose ligands to an antibody specific for the target protein. We demonstrate that the constructed GTACs can induce the internalization and lysosomal degradation of the extracellular and membrane proteins streptavidin, tumor necrosis factor-alpha (TNF-α), and human epidermal growth factor receptor 2 (HER2). Compared with the parent antibody, the GTAC exhibited higher potency in inhibiting the growth of tumor cells in vitro and enhanced tumor-targeting capacity in a tumor-bearing mouse model. Thus, the GTAC platform represents a novel degradation strategy that harnesses an SLC transporter for tumor-selective depletion of secreted and membrane proteins of interest.

Individualized treatment of diabetes mellitus in older adults.

The population of older adults with diabetes mellitus is growing but heterogeneous. Because geriatric syndromes, comorbidity or multimorbidity, the complexity of glucose dynamics, and socioeconomic conditions are associated with the risk of severe hypoglycemia and mortality, these factors should be considered in individualized diabetes treatment. Because cognitive impairment and frailty have similar etiologies and risk factors, a common strategy can be implemented to address them through optimal glycemic control, management of vascular risk factors, diet, exercise, social participation, and support. To prevent frailty or sarcopenia, optimal energy intake, adequate protein and vitamin intake, and resistance or multi-component exercise are recommended. For hypoglycemic drug therapy, it is important to reduce hypoglycemia, to use sodium glucose cotransporter-2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1) receptor agonists, taking into account the benefits for cardiovascular disease and the risk of adverse effects, and to simplify treatment to address poor adherence. Glycemic control goals for older adults with diabetes should be set according to three categories, based on cognitive function and activities of daily living, using the Dementia Assessment Sheet for Community-based Integrated Care System 8-items. This categorization can be used to determine treatment strategies for diabetes when combined with the Comprehensive Geriatric Assessment (CGA). Based on the CGA, frailty prevention, treatment simplification, and social participation or services should be implemented for patients in Category II and above. Measures against hypoglycemia and for the prevention of cardiovascular disease and chronic kidney disease should also be promoted. Treatment based on categorization and CGA by multidisciplinary professionals would be an individualized treatment for older adults with diabetes. Geriatr Gerontol Int 2024; ••: ••-••.

Dietary supplementation with pterostilbene activates the PI3K-AKT-mTOR signalling pathway to alleviate progressive oxidative stress and promote placental nutrient transport

BackgroundProgressive oxidative stress easily occurs as a result of a gradual increase in the intensity of maternal metabolism due to rapid foetal development and increased intensity of lactation. However, studies on the effects of processive oxidative stress on nutrient transport in the placenta have received little attention. The present study was conducted on sows at 85 days of gestation to study the effects of pterostilbene (PTE) on maternal oxidative stress status and placental nutrient transport.ResultsPTE increased the antioxidant capacity and immunoglobulin content in mothers’ blood and milk, reduced the level of inflammatory factors, and improved the nutrient content of milk. PTE also reduced sow backfat loss and the number of weak sons, and increased piglet weaning weight and total weaning litter weight. We subsequently found that PTE enhanced placental glucose and fatty acid transport and further affected glycolipid metabolism by increasing the expression of LAL, PYGM, and Gbe-1, which activated the PI3K phosphorylation pathway. Moreover, PTE addition altered the relative abundance of the Firmicutes, Proteobacteria, Parabacillus, and Bacteroidetes-like RF16 groups in sow faeces. PTE increased the levels of acetate, propionate, butyrate and isovalerate in the faeces.ConclusionsThese findings reveal that the addition of PTE during pregnancy and lactation mitigates the effects of processive oxidative stress on offspring development by altering maternal microbial and placental nutrient transport capacity.Graphical

GLUT1 overexpression in CAR-T cells induces metabolic reprogramming and enhances potency

The intensive nutrient requirements needed to sustain T cell activation and proliferation, combined with competition for nutrients within the tumor microenvironment, raise the prospect that glucose availability may limit CAR-T cell function. Here, we seek to test the hypothesis that stable overexpression (OE) of the glucose transporter GLUT1 in primary human CAR-T cells would improve their function and antitumor potency. We observe that GLUT1OE in CAR-T cells increases glucose consumption, glycolysis, glycolytic reserve, and oxidative phosphorylation, and these effects are associated with decreased T cell exhaustion and increased Th17 differentiation. GLUT1OE also induces broad metabolic reprogramming associated with increased glutathione-mediated resistance to reactive oxygen species, and increased inosine accumulation. When challenged with tumors, GLUT1OE CAR-T cells secrete more proinflammatory cytokines and show enhanced cytotoxicity in vitro, and demonstrate superior tumor control and persistence in mouse models. Our collective findings support a paradigm wherein glucose availability is rate limiting for effector CAR-T cell function and demonstrate that enhancing glucose availability via GLUT1OE could augment antitumor immune function.

6421 Imaging Phenotype May Predict Genotype In Human Pheochromocytoma: Relationships Between Genetic Mutation Clusters, Metabolite Transporter Expression, And Radiotracer Uptake

Abstract Disclosure: I. Sakuma: None. M. Fujimoto: None. D.F. Vatner: None. K. Yokote: None. T. Tanaka: None. Context [1]23I-metaiodobenzylguanidine scintigraphy (MIBG) and [1]8F-fluoro-2-doxy-D-glucose positron emission tomography (FDG-PET) are helpful for the localization of pheochromocytoma. The norepinephrine transporter (NET) and the glucose transporter 1 (GLUT1) are thought to be critical for the uptake of these radioisotopes. NET expression is activated by Paired Like Homeobox (PHOX2) and glucocorticoid receptor (GR) in neuroblastoma cells. Around 70% of pheochromocytomas carry well-known mutations. Pheochromocytoma-associated genes are divided into two clusters: the pseudohypoxia-related cluster 1 and the kinase signaling-related cluster 2. Cluster 1 mutation are associated with noradrenergic pheochromocytomas with an elevated metastatic risk. Cluster 2 tumors demonstrate an adrenergic phenotype with a less aggressive course. How mutations of cluster 1 and cluster 2 impact the expression of NET/GLUT1 and on radiotracer uptake remains unclear. Hypothesis Cluster 1 and cluster 2 will have different effects on the expression of transcription factors associated with chromaffin cell differentiation with resultant differential effects on NET/GLUT1 expression. Methods 42 patients with pheochromocytoma were evaluated by MIBG and FDG-PET. Tumor gene expression was evaluated by real-time qPCR, and mutation analyses were performed by Sanger or next-generation sequencing. Results 88% of tumors were MIBG-avid, and 67% were FDG-avid. The expression of NET in MIBG-negative tumors was 70% lower than in MIBG-positive tumors. The expression of GLUT1 in FDG-PET-negative tumors was 75% lower than in FDG-PET-positive tumors. The expression of NET positively correlated with transcription factors involved in chromaffin cell maturation, including PHOX2A, PHOX2B, GATA binding protein 3 (GATA3), GR, catecholamine synthases, and insulinoma associated 1 (INSM1). The JASPAR database for promoter analysis revealed putative responsive regions of the transcription factors PHOX2A, PHOX2B, GATA3, GR, and INSM1 on the NET gene. Seven patients (16.7%) had mutations in cluster 1, and fourteen patients (33.3%) had mutations in cluster 2. Cluster 2 exhibited elevated NET, PHOX2A, PHOX2B, GATA3, GR, and INSM1 expression levels compared to cluster 1. Cluster 1 showed a higher expression level of GLUT1 and BMP4 that is associated with early chromaffin cell differentiation. Conclusions In pheochromocytomas, MIBG and FDG uptake correlated with the expression levels of NET and GLUT1, respectively. NET expression is associated with chromaffin cell differentiation transcription factor expression. We propose that pheochromocytomas that carry Cluster 1 mutations are more likely to be missed in MIBG due to low NET expression, while pheochromocytomas that carry Cluster 2 mutations are more likely to be missed on FDG-PET due to low GLUT1 expression with a more mature chromaffin cell phenotype. Presentation: 6/2/2024

8780 The Role of Nuclear Receptor Corepressors 1 and 2 on Intestinal Carbohydrate Transport in Mice

Abstract Disclosure: M.J. Ritter: None. I. Amano: None. A.H. van der Spek: None. H.E. Daniel: None. A.N. Hollenberg: None. The nuclear receptor corepressors NCoR1 and NCoR2 are two critical regulators of metabolism that are required for maintaining metabolic homeostasis. Both NCoR1 and NCoR2 (also known as the silencing mediator of retinoic acid and thyroid hormone receptors; SMRT) recruit histone deacetylase 3 (HDAC3) to their deacetylase domain (DAD) to repress nuclear receptor (NR) target gene expression. Interruption of the DAD of NCoR1 in mice leads to lower body weight, decreased whole-body fat mass, insulin sensitization with altered oscillatory patterns of key metabolic genes (1). On the other hand, total body knock-out (KO) of SMRT leads to weight gain, insulin resistance, and hepatic steatosis with an increase in hepatic lipogenesis (2). Previously, we showed that the KO of NCoR1 and SMRT in tandem from adult mice led rapidly to weight loss, hypoglycemia, hypothermia and was lethal. Reductions in intestinal carbohydrate transporters were seen, including sodium-glucose linked transporter-1 (SGLT1), glucose transporter 2 (GLUT2), and glucose transporter 5 (GLUT5) (3). Thus, we hypothesized that NCoR1 and SMRT play unique roles in regulating intestinal carbohydrate metabolism, in turn controlling metabolic parameters including body mass and glucose levels. We then generated a novel model to enable KO of NCoR1 and SMRT throughout intestinal epithelial cells (IECs) in adult mice using a tamoxifen inducible cre under control of the villin 1 promoter. Mice were injected with tamoxifen and body weight and glucose were monitored daily. This was followed by both oral and intraperitoneal glucose tolerance testing (GTT) and metabolic phenotyping. Preliminary data showed that NCoR1 and SMRT KO leads to sustained weight loss and hypoglycemia with 50% survivability. Metabolic phenotyping confirmed reduction in body mass without concomitant reduction in food intake. Oral GTT showed a blunted peak in response to glucose gavage compared to intraperitoneal administration. Gene expression analysis showed reduction in SGLT1, GLUT2, and GLUT5 in addition to changes in metabolic pathways associated with fatty acid metabolism. Here, we show that NCoR1 and SMRT are critical regulators of body mass and glucose in part via their actions on carbohydrate transporters in the intestine. With the global burden of metabolic diseases, including obesity and diabetes, ever-rising, NCoR1 and SMRT represent an overlooked but important area of study to better our understanding of disease processes and identify new targets for treatment. We next aim to determine the functional mechanism by which weight loss and hypoglycemia develop in addition to identifying molecular targets and pathways regulated by NCoR1 and SMRT in IECs.

12527 Determining The Impact Of Dampened Oxidative Stress On Islet Function And Gene Expression During Diabetes Onset In NOD Mice

Abstract Disclosure: K.V. Coutinho: None. H. Shinde: None. J. Feduska: None. K. Burnette: None. S.O. Poole: None. H.M. Tse: None. C.S. Hunter: None. Type 1 diabetes (T1D) arises from autoimmune destruction of insulin-producing pancreatic β-cells. Revealing the mechanisms underlying T1D onset are critical for therapeutic development but not completely understood. NADPH oxidase (NOX) generates superoxide, a precursor of reactive oxygen species (ROS), to regulate cell survival, differentiation, signaling, and autoimmune responses in T1D. NOD (Non-Obese Diabetic) mice spontaneously develop T1D, featuring increased cytokine/chemokine synthesis, inflammatory responsive macrophages and T cells, and ROS. Here, we harness NOD mice deficient in NOX-derived superoxide (NOD.Ncf1m[1]J), which have significantly lower T1D incidence. However, the impacts on islet β-cells in this T1D resistant model are unknown. We assessed a time course of glucose homeostasis between 6-20 weeks (W), spanning the onset T1D in NOD mice. Surprisingly, comparative glucose tolerance testing (GTT) in NOD.Ncf1m[1]J mice revealed increased glucose intolerance at 10W with no differences at 6, 14, and 20W as compared to NOD. Insulin tolerance was similar between the two models at all stages examined. This suggests superoxide production is necessary for β-cell function, despite that NOD.Ncf1m[1]J mice are T1D resistant. To understand islet transcriptomic changes between NOD and NOD.Ncf1m[1]J mice, we conducted comparative bulk islet RNA sequencing from 6 to 20W. Principal component analysis was applied to ensure differences between genotypes at each timepoint. Quality control methods within the Nextflow core pipeline were used to ensure sample quality. As expected, NOD.Ncf1m[1]J mice had decreased Ncf1 and immune response-related mRNA levels. Expression of T-cell exhaustion (Pdcd1, Lag3, and Tigit) genes remained level in NOD mice, but had significantly lower expression at 6W in NOD.Ncf1m[1]J mice. Interestingly, Ctla4, encoding a gene product that inhibits T-cell function, was upregulated at 14W in NOD.Ncf1m[1]J mice. Furthermore, GO analysis of downregulated NOD.Ncf1m[1]J gene sets include T-cell activation, response to IFN-β and IFN-γ at 6W, and decreased cytokine production at 20W. Supporting the dysregulation of β-cell function, Bace2, encoding a protease that limits β-cell function and mass, was upregulated with NOD. Ncf1m[1]J. Additionally, 14W β-cell function gene expression was decreased in the NOD.Ncf1m[1]J mice, including Slc2a2, which encodes the β-cell GLUT2 glucose transporter, and Glp1r, which is involved in preserving β-cell mass and function. Taken together, NOX-derived superoxide, whose loss decreases T1D incidence, may also dampen β-cell gene expression and function. Future directions will include comparative NOD and NOD.Ncf1m[1]J single cell transcriptomics to determine cell type specific transcriptional signatures between islet and immune cell subtypes. Presentation: 6/2/2024

7284 Ablation of hexose-6-phosphate dehydrogenase in mice causes skeletal muscle atrophy and dramatically impairs exercise capacity

Abstract Disclosure: M. Zogg: None. A.D. Grötsch: None. J. Birk: None. P. Pelczar: None. J. Bouitbir: None. A. Odermatt: None. Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two steps of the pentose-phosphate pathway (PPP) within the endoplasmic reticulum (ER), generating the redox equivalent NADPH. It has been shown that mice lacking H6PD develop defects mainly in skeletal muscles [1]. Nevertheless, the mechanisms underlying the myopathy in H6PD KO mice is unknown. Here, we further assessed muscle responses to H6PD deficiency and aimed to uncover the mechanisms that may cause myopathy in mice. We conducted a detailed characterization of the muscle structure and function as well as energy metabolism in oxidative and glycolytic muscles of H6PD KO mice. At the age of 15 weeks, H6PD KO mice displayed lower body weight than WT mice, despite similar food and water intake. Grip strength was significantly lower in H6PD KO than in WT mice. This was associated with decreased weight of the mixed (gastrocnemius, quadriceps) and fast fiber type rich muscles of the hind leg (extensor digitorum longus, tibialis anterior) in H6PD KO animals. Atrogin1 and Murf1 mRNA expression, markers of atrophy, increased only in the white (glycolytic) quadriceps. In this context, fast-twitch fibers in WT mice displayed higher H6pd mRNA expression than slow-twitch fibers. Electron microscopy analysis of red and white gastrocnemius muscles displayed the presence of large intramyofibrillar vacuoles, and morphologic changes of the sarcoplasmic reticulum and mitochondria in H6PD KO muscle. Metabolically, H6PD KO muscles presented an accumulation of glycogen granules and significantly decreased mRNA expression of glucose transporter Glut4, glucose-6-phosphate transporter G6pt and glycogen phosphorylase Pygm. The exercise capacity, measured by maximal running distance, decreased by more than 50% in H6PD KO mice. Upon exercise, both WT and H6PD KO animals were able to mobilize the majority of glycogen stored in their muscles. However, immediately after exercise, there was a significant increase in plasma lactate and glucose levels in H6PD KO animals. Disturbances in glucose metabolism were further supported by in vitro experiments using stable C2C12 H6PD KO myoblasts, revealing a shift in basal state mitochondrial fuel oxidation usage from glucose to fatty acid and glutamine in metabolic dependency assays. In conclusion, our findings revealed that H6PD seems to play a key role in proper function and energy metabolism of skeletal muscles.

8418 Association of GLP-1 Receptor Agonists and SGLT-2 Inhibitors with Major Adverse Cardiovascular Events and Serious Liver Events among Patients with Type 2 Diabetes and MASLD

Abstract Disclosure: A. Kutz: None. D.J. Wexler: None. J. Liu: None. J.M. Paik: None. E. Patorno: None. Introduction and study question Little is known about the cardiovascular (CV) and hepatic effectiveness of glucagon-like peptide 1 receptor agonists (GLP-1 RA) and sodium–glucose cotransporter 2 inhibitors (SGLT-2i) in people with type 2 diabetes (T2D) and known metabolic dysfunction-associated steatotic liver disease (MASLD) in clinical practice. We assessed the comparative CV and hepatic effectiveness and the safety of GLP-1 RA and SGLT-2i in people with T2D and prevalent MASLD. Methodology Using pooled data from Medicare fee-for-service (2013–2020) and a large U.S. health insurance database (2013-June 2022), we conducted two propensity-score fine stratification weighted cohort studies including adults with T2D and a diagnosis of MASLD who initiated GLP-1RA, SGLT-2i, or dipeptidyl peptidase 4 inhibitors (DPP-4i, reference). Using Cox proportional hazards models, we assessed the primary CV effectiveness composite outcome of acute myocardial infarction, ischemic stroke, hospitalization for heart failure, or all-cause mortality. The primary hepatic effectiveness outcome was a composite of serious liver events (hospitalization for ascites, spontaneous bacterial peritonitis, hepatorenal syndrome, bleeding esophageal varices, hepatic encephalopathy, or liver transplantation). The safety outcome was a composite of severe adverse events (lower limb amputation, non-vertebral fracture, emergency admission for hypoglycemia, or hospitalization for acute kidney injury, diabetic ketoacidosis [DKA], severe genital or urinary tract infection, acute pancreatitis, or biliary event). Major results Compared with DPP-4i (n=17,084 initiators), the hazard ratio (HR) for the primary CV outcome associated with GLP-1 RA (n=13,666 initiators) was 0.67 (95% CI, 0.56 to 0.81), corresponding to an incidence rate difference (IRD) per 1,000 person-years of -21.6 (95% CI, -26.7 to -16.6). The HR for the primary hepatic outcome was 0.47 (95% CI, 0.25 to 0.90), the IRD -2.1 (95% CI -3.4 to -0.9). Similar CV benefits were observed for SGLT-2i (n=11,108) vs DPP-4i (n=16,979), with a HR for the primary CV outcome of 0.82 (95% CI 0.70–0.96) and an IRD of -11.0 (95% CI -16.2 to -5.8). For the primary hepatic outcome, the HR was 0.81 (95% CI 0.45 to 1.44) and the IRD -1.1 (95% CI -2.4 to 0.3). Severe adverse events were not more frequent with GLP-1RA or SGLT-2i compared with DPP-4i. Interpretation and conclusion Among people with T2D and MASLD, the use of GLP-1 RA or SGLT-2i was associated with fewer CV events, vs DPP-4i, while GLP-1 RA also reduced serious liver events. Compared with DPP-4i, GLP-1RA or SGLT-2i were not associated with higher safety adverse event rates. Presentation: 6/2/2024

7578 SGLT2 Inhibitor Is Associated With A Decreased Risk Of Cerebrovascular Events And Dementia Compared To Thiazolidinedione In Subjects With Type 2 Diabetes

Abstract Disclosure: H. Park: None. S. Park: None. H. Kim: None. D. Byun: None. Subjects with type 2 diabetes (T2D) are at higher risk of developing cerebrovascular disease, cognitive decline, and dementia. Hyperinsulinemia and insulin resistance in the brain have been linked to amyloid beta metabolism, raising the risk of Alzheimer's dementia. Recently, several clinical studies have shown potential beneficial effects of sodium glucose co-transporter-2 (SGLT2) inhibitors, and thiazolidinediones, in the risk of cerebrovascular events and dementia in T2D. This study was aimed to investigate the comparative efficacy of SGLT2 inhibitors and thiazolidinediones in modifying the risk of cerebrovascular events, dementia, and Alzheimer's dementia. Using the common data model, we analyzed patients from December 31, 2011 to December 30, 2022 from 15 hospitals in South Korea. Clinical outcomes between SGLT2 inhibitors and thiazolidinediones were compared using hazard ratios (HRs), with large-scale propensity score matching and a random-effects model applied. Our analyses showed that subjects on SGLT2 inhibitors had a significantly reduced rate of cerebrovascular events (HR 0.2787, 95% CI 0.1882-0.4127, p&amp;lt;0.00001), dementia (HR 0.5395, 95% CI 0.3824-0.7614, p=0.0004), and Alzheimer's dementia (HR 0.586, 95% CI 0.4634-0.7411, p&amp;lt;0.00001), compared to those on thiazolidinediones. In summary, these results suggest that SGLT2 inhibitor administration is associated with a lower risk of cerebrovascular events, dementia, and Alzheimer's dementia compared to thiazolidinedione in subjects with T2D. Further studies are warranted to confirm our findings and elucidate the underlying mechanisms. Presentation: 6/3/2024

Expression of nuclear receptors and glucose metabolic pathway proteins in sebaceous carcinoma: Androgen receptor and monocarboxylate transporter 1 have a key role in disease progression.

Standard systemic treatments are not consistently effective for treating unresectable or advanced sebaceous carcinoma (SC). The present study investigated the pathogenic roles of nuclear receptors (NRs), glucose metabolic dysregulation and immune checkpoint proteins in SC as prognostic markers or therapeutic targets. Patients with pathologically confirmed SC between January 2002 and December 2019 at three university hospitals in South Korea were included in the present study. Immunohistochemistry was performed on paraffin-embedded tumor tissues for glucocorticoid receptors (GR), androgen receptors (AR), estrogen receptors (ER), progesterone receptors (PR), glucose transporter 1 (GLUT1), monocarboxylate transporters (MCT1 and MCT4), CD147, phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) and the immune checkpoint protein, programmed cell death-ligand 1 (PD-L1). The results were semi-quantitatively assessed and the associations of these proteins with various clinicopathological parameters were determined. A total of 39 cases of SC comprising 19 periocular and 20 extraocular tumors were enrolled. NRs were frequently detected in the tumor nuclei, with GR having the highest frequency (89.7%), followed by AR, ER (both 51.3%) and PR (41.0%). Regarding glucose metabolism, CD147, GLUT1 and MCT1 were highly expressed at 100, 89.7 and 87.2%, respectively, whereas MCT4 and pAMPK expression levels were relatively low at 38.5 and 35.9%, respectively. Membranous expression of PD-L1 was detected in five cases (12.8%), four of which were extraocular. In the multivariate analysis, advanced stage, low AR positivity and high MCT1 expression were independent poor prognostic factors for metastasis-free survival (all P<0.05). The present results suggested that hormonal and metabolic dysregulation may be associated with the pathogenesis of SC, and that AR and MCT1 in particular may serve as prognostic indicators and potential therapeutic targets. Additionally, ~10% of SC cases exhibited PD-L1 expression within the druggable range, and these patients are expected to benefit from treatment with immune checkpoint inhibitors.

In Vitro and In Silico Evaluation of Caffeic and Ferulic Acids Involvement in the Translocation of Glucose Transporter 4

Background: Insulin is a key hormone in our systems. Upon binding of insulin to its receptors in fat and muscle tissues, tens of proteins in the insulin signaling pathway are involved in the process of GLUT4 vesicle recruitment to the Plasma Membrane (PM) and the absorption of serum glucose. Deficits in the aforementioned pathway lead to insulin resistance and eventually to Type II Diabetes Mellitus. Objective: We appreciate the contribution of phytochemicals in the treatment of diabetes. Yet, in vitro and in silico studies are needed to validate the safety and efficacy of the phytochemicals, plus their action mechanisms. Methods: Herein, we tested two phytochemicals, caffeic acid and ferulic acid in vitro and in silico. We shed light on the insulin signaling proteins as plausible therapeutic targets using in silico studies, via AutoDock and SwissADME. Results: Results obtained in vitro indicate that Caffeic Acid (CA) increased GLUT4 translocation at 125μM by 31% in the absence of insulin, and 24.5% in presence of insulin, when compared to the control. Ferulic Acid (FA) was less potent as an enhancer of GLUT4 translocation. Best docking results were found for the binding of the phytochemicals CA and FA to PDK1, AKT, IRS1 and PTEN proteins of the insulin signaling, with comparable results. Conclusion: These findings indicate that CA and FA possess a limited anti-diabetic potency by increasing GLUT4 trafficking to the PM in skeletal muscles. These results suggest that these compounds are candidates for further investigation in pre-clinical and clinical stages of drug discovery.