Glucose Transporter Protein Levels Research Articles

Effects of environmental enrichment on GLUT expression in the visual cortex of amblyopic rats

ObjectiveTo investigate the potential changes of glucose metabolism and glucose transporter protein (GLUT) in the visual cortex of formally deprived amblyopic rats, as well as the effects of enriched environments on the levels of nerve conduction and glucose metabolism in the visual cortex of amblyopic rats. Methods36 rats were randomly divided into three groups: CON + SE (n = 12), MD + SE (n = 12) and MD + EE (n = 12). The right eyelids of both MD + SE and MD + EE groups were sutured. After successful modelling, the MD + EE group was maintained in an enriched environment, and the other two groups were kept in the same environment. Pattern visual evoked potentials (PVEP) was used to confirm models' effect, glucose metabolism was analyzed by Micro-PET/CT (18F-FDG), and the protein as well as mRNA expression levels of GLUT were detected by Western Blot and quantitative RT-PCR (quantitative Reverse Transcription-Polymerase Chain Reaction) analyses, site of GLUT expression by immunofluorescence (IF). ResultsAfter suture modelling, both the MD + EE and MD + SE groups objective visual nerve conduction function decreased, the glucose metabolism in the visual cortex was markedly lower. After the enriched environment intervention, it recovered in the MD + EE group. The expression levels of GLUT1 and GLUT3 were increased in the MD + EE group in comparison with the MD + SE group. GLUT1 was primarily expressed on astrocytes and endothelial cells, but GLUT3 was mainly expressed on neurons. ConclusionEnrichment of the environment exhibited a therapeutic effect on amblyopia, which could be related to the enhancement of glucose metabolism and GLUT expression in the visual cortex.

Alterations of whole body glucose metabolism in a feline SARS-CoV-2 infection model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been especially devastating to patients with comorbidities, including metabolic and cardiovascular diseases. Elevated blood glucose during SARS-CoV-2 infection increased mortality of patients with COVID-19, although the mechanisms are not well understood. It has been previously demonstrated that glucose transport and utilization is a crucial pathway for other highly infectious RNA viruses. Thus, we hypothesized that SARS-CoV-2 infection could lead to alterations in cellular and whole body glucose metabolism. Specific pathogen-free domestic cats were intratracheally inoculated with USA-WA1/2020 (wild-type) SARS-CoV-2 or vehicle-inoculated, then euthanized at 4- and 8-days postinoculation (dpi). Blood glucose and cortisol concentrations were elevated at 4 and 8 dpi. Blood ketones, insulin, and angiotensin II concentrations remained unchanged throughout the experimental timeline. SARS-CoV-2 RNA was detected in the lung and heart, without changes in angiotensin-converting enzyme 2 (ACE2) RNA expression. In the lung, SARS-CoV-2 infection increased glucose transporter 1 (GLUT1) protein levels at 4 and 8 dpi, whereas GLUT4 level was only upregulated at 8 dpi. In the heart, GLUT-1 and -4 protein levels remained unchanged. Furthermore, GLUT1 level was upregulated in the skeletal muscle at 8 dpi, and AMPK was activated in the hearts of infected cats. SARS-CoV-2 infection increased blood glucose concentration and pulmonary GLUT protein levels. These findings suggest that SARS-CoV-2 infection induces metabolic reprogramming primarily in the lung to support viral replication. Furthermore, this translational feline model mimicked human COVID-19 and could be used to explore novel therapeutic targets to treat metabolic disease during SARS-CoV-2 infection.NEW & NOTEWORTHY Our study on a feline model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, mirroring human COVID-19, revealed alterations in whole body and cellular glucose metabolism. Infected cats developed mild hyperglycemia, increased protein levels of glucose transporters in the lung, and AMPK activation in the heart. These findings suggest that SARS-CoV-2 infection induces metabolic reprogramming in the cardiorespiratory system to support viral replication. Understanding these mechanisms could lead to novel antiviral therapeutic strategies.

Effect and mechanism of Yueju Pills on breast cancer induced by DMBA in rats

This paper aims to explore the inhibitory effect of Yueju Pills on breast cancer and decipher the underlying mechanism. A total of 92 SPF-grade SD female rats were involved in this study, and 14 of them were randomly selected into control group. The remaining 78 rats were administrated with 7,12-dimethylbenzanthracene(DMBA) by gavage to establish the breast cancer model. The modeled rats were randomized into model, tamoxifen(1.9 mg·kg~(-1)·d~(-1)), and low-and high-dose(17, 34 g·kg~(-1)·d~(-1)) Yueju Pills groups. The mental state, food intake, and activities of the rats were observed daily, and the body weight was measured on alternate days. After 12 weeks of administration, the rats were sacrificed and the tumor weight was measured. The serum estrogen and progeste-rone levels were determined by enzyme-linked immunosorbent assay. The histopathological changes of the breast and tumor were observed by hematoxylin-eosin staining. Western blot was employed to measure the protein levels of glucose transporter 1(GLUT1), lactate dehydrogenase A(LDHA), phosphofructokinase muscle(PFKM), pyruvate kinase isozyme type M2(PKM2), hexokinase 2(HK2), nuclear factor-kappaB(NF-κB), and phosphorylated NF-κB. The intestinal microbiome was examined by 16S rRNA high-throughput sequencing. The results showed that compared with the model group, high and low-dose Yueju Pills showed the tumor inhibition rate of 15.8% and 64.5%, respectively, and the low dose group had stronger inhibitory effect. Compared with the control group, the model group presented elevated the levels of estrogen and progesterone in serum. The administration of Yueju Pills lowered such ele-vation, and the low-dose group showed stronger lowering effect(P<0.05). Compared with the model group, Yueju Pills reduced the glands with increased breast tissue, the degree of breast duct expansion, the number and area of acinar cavity, the secretions, and the layers of mammary epithelial cells. Furthermore, Yueju Pills down-regulated the expression of GLUT1, LDHA, PFKM, PKM2, HK2, and NF-κB(P<0.05) and altered the diversity, composition, structure, and abundance of intestinal flora. The results showed that Yueju Pills could inhibit breast cancer by regulating the secretion of estrogen and progesterone, glycolysis, inflammatory cytokines, and intestinal flora.

BTF3 promotes proliferation and glycolysis in hepatocellular carcinoma by regulating GLUT1

ABSTRACT Hepatocellular carcinoma (HCC) is a grievous tumor with an increasing incidence worldwide. Basic transcription factor 3 (BTF3) is discovered to regulate the expression of glucose transporter 1 (GLUT1), which benefits glycolysis, a momentous signature of tumors, through transactivation of the forkhead box M1 (FOXM1) expression. BTF3 is highly expressed in HCC. However, whether BTF3 promotes GLUT1 expression through FOXM1 to modulate glycolysis in HCC remains unclear. The expression profile of BTF3 were determined by online database, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. The role and mechanism of BTF3 in the proliferation and glycolysis of HCC cells were examined by cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) incorporation, XF96 Extracellular Flux analyzer, spectrophotometry and western blot analysis. In addition, the direct interaction between BTF3 and FOXM1 was verified by dual-luciferase reporter and co-immunoprecipitation assays. Moreover, the role of BTF3 was also explored in a xenografted mice model. The expression of BTF3 was increased in HCC cells and tumor tissues. Knockdown of BTF3 reduced the cell viability, Edu positive cells, extracellular acidification rate (ECAR), glucose consumption and lactate production in both Huh7 and HCCLM3 cells. The expressions of FOXM1 and GLUT1 were increased in HCC tissues, which were positively correlated with the BTF3 expression. Moreover, a direct interaction existed between BTF3 and FOXM1 in HCC cells. Downregulation of BTF3 decreased the relative protein levels of FOXM1 and GLUT1, which were rescued with overexpression of FOXM1 in both cells. More importantly, overexpression of FOXM1 restored the cell viability, ECAR, glucose consumption and lactate production in both Huh7 and HCCLM3 cells transfected with siBTF3#1. Furthermore, inhibition of BTF3 decreased tumor weight and volume, and the relative level of BTF3, FOXM1, GLUT1 and Ki-67 in tumor tissues from mice xenografted with Huh7 cells. BTF3 enhanced the cell proliferation and glycolysis through FOXM1/GLUT1 axis in HCC.

APOE e4 dependent deficits in brain DHA phospholipids and mfsd2a in Alzheimer’s disease patients with severe cerebral amyloid angiopathy

AbstractBackgroundThe apolipoprotein E e4 allele (APOE4) is a major risk factor, contributing to vascular pathologies in AD which include CAA, BBB dysfunction and reduced cerebral vascular integrity. The diminished capacity of APOE4 to transport docosahexaenoic acid (DHA), an essential fatty acid required for the structural and functional maintenance and vascular integrity of the brain, could also contribute to AD pathogenesis. However, it remains undetermined if changes in the brain DHA content of phospholipids (PL) and DHA transporters exist in relation to APOE4, AD diagnosis and CAA pathology.MethodWe performed liquid chromatography/mass spectrometry‐based PL analysis of the cerebrovascular and parenchymal fractions from autopsied human brain of pathologically confirmed AD cases and controls. We performed an antibody‐based examination of the major facilitator superfamily domain containing 2A (mfsd2a) protein in the cerebrovasculature from these subjects and brain homogenates from transgenic mice with human APOE (APOE‐TR) and APOE‐TR crossed with mice with five AD mutations (EFAD). We performed proteomic analyses of the cerebrovascular and parenchymal fractions of the brains from 50‐week‐old APOE‐TR.ResultIn the cerebrovasculature and parenchyma, DHA containing PL species were lower in ε4 carriers with AD compared to control ε4 carriers. We observed an ε4‐dependent decrease in mfsd2a level in the cerebrovasculature of ε4 carriers compared to non‐carriers. Stratification of DHA containing PL by CAA showed that these PL levels were reduced in ε4 positive AD patients with severe CAA. In addition, brain levels of DHA containing PC species and mfsd2a were lower in APOE4‐TR and E4FAD mice compared to other genotypes in each mouse model. Furthermore, protein levels of glucose transporter 1 (Glut1) and a metabolite transporter, monocarboxylate transporter 1 (MCT1), were lower in the cerebrovasculature of APOE4‐TR relative to APOE3‐TR and APOE2‐TR mice.ConclusionThese findings demonstrate that brain DHA deficiencies in ε4 carriers may be due to reduced mfsd2a expression and partly associated with severe CAA. Thus, targeting this transport mechanism may improve the bioavailability of DHA into the brains of ε4 carriers who are at risk of developing AD.

Effects of Rosa roxburghii on insulin resistance in obese rats and its mechanisms

Objective: To investigate the effects of Rosa roxburghii on insulin resistance in obese rats and the regulation of phosphatidylinositol 3-kinase (PI3K)/ protein kinase Bβ(PKBβ/Akt2)/ glucose transporter 4(GLUT4) signaling pathway. Methods: Five-week-old male SD rats were randomly divided into normal control group (NC), model group (M), positive control group (PC), low-dose rosa roxburghii group (LD) and high-dose rosa roxburghii group (HD), with 10 rats in each group. The rats in the NC group were fed with normal diet, while those in the M, PC, LD and HD groups were fed with high-fat diet. From the 13th week, according to the dose standard of 6 ml/kg, rats in the LD group were intragastrically administered with 100 mg/kg Rosa roxburghii Tratt, the HD group were treated with 300 mg/kg Rosa roxburghii Tratt, the PC group were treated with 0.11 g/kg Chiglitazar sodium, and the NC and M groups were intragastrically administered with the same volume of normal saline. The body weight was measured every week until 20 weeks. The rats were sacrificed 24 h after the last experiment. Blood and skeletal muscle were collected. Serum total cholesterol (TC) and triglyceride (TG) contents were detected by colorimetric method, serum superoxide dismutase (SOD) activity was detected by xanthine oxidase method, serum malondialdehyde (MDA) content was detected by thiobarbituric acid method, blood glucose (FBG) value was detected by glucose oxidase method, insulin (FINS) content was detected by ELISA, and PI3K, Akt2, and GLUT4 protein and gene expressions were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Results: Compared with the NC group, the body weight, serum MDA, TG, TC, FBG, FINS, HOMA-IR levels in the M group were significantly increased (P＜0.01), while SOD activity, PI3K、Akt2、GLUT4 protein and mRNA expression levels were significantly increased(P＜ 0.01). Compared with group M, the body weight, serum MDA, TG, TC, FBG, FINS, and HOMA-IR were decreased significantly in LD group, HD group and PC group (P＜0.05 or P＜0.01), while SOD activity, PI3K, Akt2, GLUT4 protein and mRNA expression levels were increased significantly (P＜0.05 or P＜0.01). Conclusion: Rosa roxburghii can improve insulin resistance in obese rats by antioxidant stress and up-regulating the expressions of PI3K, Akt2, and GLUT4 proteins and genes, which may be related to the PI3K/Akt2/GLUT4 signaling pathway.

ApoE isoform-dependent effects of xanthohumol on high fat diet-induced cognitive impairments and hippocampal metabolic pathways.

Consumption of a high fat diet (HFD) is linked to metabolic syndrome and cognitive impairments. This is exacerbated in age-related cognitive decline (ACD) and in individuals with a genetic risk for Alzheimer’s disease (AD). Apolipoprotein E (apoE) is involved in cholesterol metabolism. In humans, there are three major isoforms, E2, E3, and E4. Compared to E3, E4 increases ACD and AD risk and vulnerability to the deleterious cognitive effects of a HFD. The plant compound Xanthohumol (XN) had beneficial effects on cognition and metabolism in C57BL/6J wild-type (WT) male mice put on a HFD at 9 weeks of age for 13 weeks. As the effects of XN in the context of a HFD in older WT, E3, and E4 female and male mice are not known, in the current study male and female WT, E3, and E4 mice were fed a HFD alone or a HFD containing 0.07% XN for 10 or 19 weeks, starting at 6 months of age, prior to the beginning of behavioral and cognitive testing. XN showed sex- and ApoE isoform-dependent effects on cognitive performance. XN-treated E4 and WT, but not E3, mice had higher glucose transporter protein levels in the hippocampus and cortex than HFD-treated mice. E3 and E4 mice had higher glucose transporter protein levels in the hippocampus and lower glucose transporter protein levels in the cortex than WT mice. In the standard experiment, regardless of XN treatment, E4 mice had nearly double as high ceramide and sphingomyelin levels than E3 mice and male mice had higher level of glycosylated ceramide than female mice. When the differential effects of HFD in E3 and E4 males were assessed, the arginine and proline metabolism pathway was affected. In the extended exposure experiment, in E3 males XN treatment affected the arginine and proline metabolism and the glycine, serine, and threonine metabolism. Myristic acid levels were decreased in XN-treated E3 males but not E3 females. These data support the therapeutic potential for XN to ameliorate HFD-induced cognitive impairments and highlight the importance of considering sex and ApoE isoform in determining who might most benefit from this dietary supplement.

KLF8 enhances acute myeloid leukemia cell growth and glycolysis via AKT/mTOR pathway

Purpose: To determine the role and mechanisms of Krüppel-like transcription factor 8 (KLF8) in acute myeloid leukemia (AML).Methods: The transcriptional and translational levels of KLF8 in AML cell lines were determined by quantitative real time-polymerase chain reaction (qRT-PCR) and western blotting. Two RNAs targeting KLF8 were transfected into KG-1 and HL-60 cells. The growth and apoptosis of AML cells were determined using CCK-8, 5-ethynyl-2-deoxyuridine (EdU), flow cytometry, and western blot assays. Glycolysis was evaluated by relative glucose consumption, lactate generation, ATP levels, and hexokinase II (HK2), while glucose transporter 1 (GLUT1) protein expression levels. AKT, phosphorylated AKT (p-AKT), mammalian target of rapamycin (mTOR), and p-mTOR expression levels were assessed by western blot.Results: KLF8 mRNA and protein expression levels were elevated in AML cells (p < 0.01). KLF8 knockdown in AML cells decreased cell viability, EdU-positivity, relative glucose consumption, lactate generation, ATP levels, and HK2 and GLUT1 protein levels (p < 0.01). Apoptosis increased in KG-1 and HL-60 cells, with enhanced Bax and reduced Bcl-2 protein levels, after transfection with sh-KLF8. The relative expression levels of p-AKT/AKT and p-mTOR/mTOR were reduced in KG-1 and HL-60 cells transfected with sh-KLF8.Conclusion: Downregulation of KLF8 inhibits proliferation and glycolysis, and also promotes apoptosis in AML cells via AKT/mTOR pathway.

CNN1 Represses Bladder Cancer Progression and Metabolic Reprogramming by Modulating HIF-1α Signaling Pathway.

Bladder cancer (BC) is the second most common urinary system malignant tumor around the whole world. It has been reported that CNN1 was lowly expressed in BC tissues. However, the mechanisms of CNN1 on BC cells were unclear. Herein, we aimed to probe the specific influences of CNN1 on BC pathogenesis. First, the expression level and prognostic ability of CNN1 in BC patients were surveyed. Then, CNN1 overexpression was executed to exhibit the influences of CNN1 on BC cells. The real-time PCR and Western blotting were employed to detect by the mRNA and protein expression levels. CCK8 assay was utilized to examine cell proliferation, and transwell assay was executed to test cell invasion and migration. The corresponding kits were utilized to detect glucose absorption, lactate secretion, and ATP level. BC cells overexpressing CNN1 were utilized to establish a nude mouse xenograft tumor model, and the tumor volume and tumor weight were detected. Nude mouse tumor tissues were used for immunohistochemical experiments to test the expression levels of Ki-67 and CNN1. The outcomes indicated that CNN1 was significantly lowly expressed in BC tissues and cells. Besides, low expression of CNN1 might be concerned with poor prognosis. Moreover, overexpression of CNN1 repressed the proliferation, invasion, and migration of BC cells. Furthermore, CNN1 overexpression decreased the protein levels of glycolysis-related protein GLUT1 (glucose transporter 1), pyruvate kinase M2 (PKM2), and LDHA (lactate dehydrogenase A). Then, the decreased mRNA and protein levels of HIF-1α and PDK1 were identified after CNN1 overexpression. The in vivo assays verified the effects of aberrant expression of CNN1 in mice with BC. In conclusion, these findings suggested that CNN1 might modulate BC progression through activating HIF-1α pathway and CNN1 might be a promising marker for BC diagnosis.

Tetrahydrocurcumin Ameliorates Acute Hypobaric Hypoxia-Induced Cognitive Impairment in Mice.

Ma, Xuexinyu, Yang Pan, Yuye Xue, Yao Li, Yan Zhang, Yani Zhao, Xingzhao Xiong, Jianbo Wang, and Zhifu Yang. Tetrahydrocurcumin ameliorates acute hypobaric hypoxia-induced cognitive impairment in mice. High Alt Med Biol. 23:264-272, 2022. Background: Hypobaric hypoxia (HH) impairs spatial learning and increases oxidative stress in rodents. We hypothesized that tetrahydrocurcumin (THC) may attenuate HH-induced neurobehavioral deficits by reducing HH-induced lipid peroxidation and increasing glycolytic activity. Materials and Methods: A C57BL/6 mouse model of acute high altitude brain injury was established using an animal decompression chamber for 24 hours. Cognitive and behavioral assessments were conducted using the Y-maze, open field, and Rotarod tests. We measured superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity; malondialdehyde (MDA) and reactive oxygen species levels; anti-neuronal core antigen (NeuN) immunoreactivity; and active occludin, hypoxia-inducible factor-1α, glucose transporter 1 (GLUT1), and GLUT3 expression levels in mice brain tissue. Results: The mice in the THC group showed improved cognitive impairment compared with those in the HH group in cognitive and behavioral tests, but failed to show improvement in the decline in coordination. The mice in the THC group were more effected than those in the HH group in demonstrating alleviation of hyperemia in cortical vessels and cell voids, and cells in the CA1 region were more closely arranged. Compared with those in the mice of the HH group, the concentration of MDA decreased significantly, the expression of occludin, NeuN immunoreactivity, and the activities of SOD and GSH-Px significantly increased in the mice of the THC group. An increase in GLUT1 expression was observed in HH-exposed animals (N group vs. HH group: 0.4 ± 0.08 vs. 0.60 ± 0.07, p < 0.05), and this increase was enhanced in animals treated with THC (HH group vs. THC group: 0.60 ± 0.07 vs. 0.82 ± 0.08, p < 0.05). Conclusions: THC improved cognitive impairment in mice, accompanied by reduced oxidative stress and increased GLUT1 protein levels.

Bmi-1 directly upregulates glucose transporter 1 in human gastric adenocarcinoma.

This study aimed to investigate whether and how Moloney murine leukemia virus integration site 1 (Bmi-1) plays a role in the regulation of glucose transporter 1 (GLUT1) in gastric adenocarcinoma (GAC). GAC and matched noncancerous tissues were obtained from GAC patients who underwent surgical treatment at the China-Japan Union Hospital, Jilin University (Changchun, Jilin, China). The human GAC cell line AGS and the gastric epithelial cell line GES-1 were used for in vitro studies. BALB/c nude mice were used for in vivo studies. The Bmi-1 and GLUT1 protein levels were significantly greater in human tissues from GAC patients and AGS cells in comparison with controls. Silencing of Bmi-1 resulted in significant decrease in glucose uptake, lactate levels, and GLUT1 expression. In vivo 18F-deoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) imaging studies indicated that the nude mice bearing xenografts of AGS cells treated with Bmi-1-specific small interfering RNA (siRNA) had a significantly lower maximum standardized uptake value (SUVmax) in comparison with the control mice. Thus, Bmi-1 directly upregulates GLUT1 gene expression, through which it is involved in enhancing glucose uptake in GAC. The results also provide scientific evidence for 18F-FDG PET/CT imaging to evaluate Bmi-1 and glucose uptake in GAC.

Dysregulated GLUT1 may be involved in the pathogenesis of preeclampsia by impairing decidualization

Preeclampsia (PE), a hypertensive complication in pregnancy, is a major contributor to maternal and fetal morbidity and mortality. PE has long been regarded a heterogeneous disorder with a pathogenesis that involves multiple genes and factors. Glucose transporter 1 (GLUT1) is a central rate-limiting pump that is involved in glucose uptake and subsequent utilization. Our previous RNA-seq results demonstrated GLUT1 was significantly downregulated in deciduas from patients with severe PE. Therefore, in this study, we aimed to explore the role of GLUT1 in the occurrence of PE. Our data showed that mRNA and protein levels of GLUT1 were significantly downregulated in the deciduas from patients with severe PE. Additionally, GLUT1 levels were substantially upregulated in human endometrial stromal cells (HESCs) during in vitro decidualization. Moreover, GLUT1 knockdown significantly reduced the mRNA levels of decidualization markers (IGFBP1 and PRL) and aerobic glycolysis-related genes (LDHA and MCT4), as well as decreased glucose uptake and lactate production. Furthermore, upon GLUT1 knockdown, the levels of apoptotic genes P53, P21, and BAX increased whereas the level of BCL2 decreased. Target prediction results and luciferase analysis showed that GLUT1 is one of the targets of miR-140-5p, which is partly responsible for downregulated GLUT1 levels. Collectively, these results demonstrate that GLUT1 exerts a pivotal role in human decidualization by participating in glycolysis, and that GLUT1 deficiency may trigger aberrant glycolysis, thereby leading to destructive decidualization that may impede blastocyst implantation, trophoblast invasion, and subsequent placental development, which are associated with PE. Taken together, these data suggest that GLUT1 might be a promising target for PE therapy.

Ursolic acid ameliorates adipose tissue insulin resistance in aged rats via activating the Akt-glucose transporter 4 signaling pathway and inhibiting inflammation.

Ageing often results in insulin resistance (IR) and chronic inflammation, and adipose is one of the tissues in which inflammation and IR occur earliest during this process. The present study investigated the effect and underlying mechanisms of ursolic acid (UA) on adipose IR and inflammation in ageing rats. Specific pathogen-free male Sprague-Dawley rats were randomly divided into 4 groups: i) Young normal (young); ii) untreated ageing (aged); and groups supplemented with UA either iii) low-UA 10 mg/kg (UA-L) or iv) high-50 mg/kg (UA-H). Animals in the UA-treated groups received 10 or 50 mg/kg UA (suspended in 5% Gum Arabic solution). The rats in the corresponding aged group and young groups received vehicle (5% Gum Arabic) alone. All rats were intragastrically treated once daily by oral gavage for 7 weeks. The day before the experiment terminated, overnight fasting blood (~700 µl) was collected and plasma was prepared to measure biochemical indicators; western blotting was performed to analyze the expression of insulin signaling proteins [(insulin receptor substrate 1 (IRS-1), phosphorylated (p)-IRS-1, PI3K, glucose transporter 4 (GLUT4), Akt and p-Akt)] and inflammatory factors (NF-κB, IL-6 and IL-1β) in the epididymis white adipose tissue (eWAT). The results revealed that treatment with UA-H decreased eWAT weight, the ratio of eWAT weight/body weight, fasted insulin and triglyceride levels, the homeostasis model assessment of insulin resistance and adipose tissue insulin resistance index in ageing rats, indicating the amelioration of systemic and adipose tissue IR, compared with the aged group. Mechanistically, UA-H administration upregulated p-protein kinase B, the ratio of p-Akt to protein kinase B and total and cellular membrane GLUT4 protein levels in eWAT of ageing rats. Conversely, UA inhibited the increase in NF-κB expression and proinflammatory cytokines IL-6 and IL-1β. However, these alterations were not observed in the rats of the aged group. Taken together, the findings of the present study indicated that UA may ameliorate adipose IR, which is associated with activation of the Akt-GLUT4 signaling pathway and inhibition of inflammation in ageing rats. These data provide a basis for the development of effective and safe drugs or functional substances, such as UA, for the prevention and treatment of metabolic diseases.

Decarboxylated osteocalcin, a possible drug for type 2 diabetes, triggers glucose uptake in MG63 cells.

BACKGROUNDUncarboxylated osteocalcin (GluOC) has been reported to improve glucose metabolism, prevent type 2 diabetes, and decrease the severity of obesity in mice with type 2 diabetes. GluOC can increase glucose uptake in a variety of cells. Glucose metabolism is the main source of energy for osteoblast proliferation and differentiation. We hypothesized that decarboxylated osteocalcin (dcOC), a kind of GluOC, can increase glucose uptake in MG63 cells (osteoblast-like osteosarcoma cells) and influence their proliferation and differentiation.AIMTo investigate the effects of dcOC on glucose uptake in human osteoblast-like osteosarcoma cells and the possible signaling pathways involved.METHODSMG63 cells (human osteoblast-like osteosarcoma cells) were treated with dcOC (0, 0.3, 3, 10, or 30 ng/mL) for 1 and 72 h, and glucose uptake was measured by flow cytometry. The effect of dcOC on cell proliferation was measured with a CCK-8 assay, and alkaline phosphatase (ALP) enzyme activity was measured. PI3K was inhibited with LY294002, and hypoxia-inducible factor 1 alpha (HIF-1α) was silenced with siRNA. Then, GPRC6A (G protein-coupled receptor family C group 6 subtype A), total Akt, phosphorylated Akt, HIF-1α, and glucose transporter 1 (GLUT1) levels were measured by Western blot to elucidate the possible pathways by which dcOC modulates glucose uptake.RESULTSThe glucose uptake of MG63 cells was significantly increased compared with that of the paired control cells after short-term (1 h) treatment with dcOC at different concentrations (0.3, 3, and 10 ng/mL groups, P < 0.01; 30 ng/mL group, P < 0.05). Glucose uptake of MG63 cells was significantly increased compared with that of the paired control cells after long-term (72 h) treatment with dcOC at different concentrations (0.3, 3, and 10 ng/mL groups, P < 0.01; 30 ng/mL group, P < 0.05). DcOC triggered Akt phosphorylation in a dose-dependent manner, and the most effective stimulatory concentration of dcOC for short-term (1 h) was 3 ng/mL (P < 0.01). LY294002 abolished the dcOC-mediated (1 h) promotion of Akt phosphorylation and glucose uptake without affecting GLUT1 protein expression. Long-term dcOC stimulation triggered Akt phosphorylation and increased the protein levels of HIF-1α, GLUT1, and Runx2 in a dose-dependent manner. Inhibition of HIF-1α with siRNA abolished the dcOC-mediated glucose uptake and substantially decreased GLUT1 protein expression. DcOC intervention promoted cell proliferation in a time- and dose-dependent manner as determined by the CCK-8 assay. Treatment with both 3 ng/mL and 10 ng/mL dcOC affected the ALP activity in MG63 cells after 72 h (P < 0.01).CONCLUSIONShort- and long-term dcOC treatment can increase glucose uptake and affect proliferation and ALP activity in MG63 cells. This effect may occur through the PI3K/Akt, HIF-1α, and GLUT1 signaling factors.

LRPPRC regulates metastasis and glycolysis by modulating autophagy and the ROS/HIF1-α pathway in retinoblastoma

Retinoblastoma (RB) is the most common intraocular tumor among children. Leucine-rich pentatricopeptide repeat (PPR)-motif-containing protein (LRPPRC), a suppressor gene of autophagy, has been proven to play a regulatory role in tumor progression. However, little is known about functional roles and mechanisms of LRPPRC in RB progression. First, we performed a detailed analysis for RB and normal control. The expression of LRPPRC in the RB tissues was significantly higher than that in normal tissues. Moreover, LRPPRC suppression could repress tumor cell migration, invasion, glycolysis, and reactive oxygen species (ROS)/hypoxia-inducible factor-1α (HIF1-α) pathway activation by mediating autophagy. Furthermore, overexpression of HIF1-α partially reversed the above changes induced by LRPPRC knockdown. The regulation of LRPPRC on tumor metastasis and glycolysis was also validated by a xenograft tumor assay. In summary, LRPPRC could regulate metastasis and glycolysis of RB by mediating autophagy suppression and further activating the ROS/HIF1-α pathway, and LRPPRC could be a promising prognostic biomarker for RB.

High Carbohydrate Diet Increased Glucose Transporter Protein Levels in Jejunum but Did Not Lead to Enhanced Post-Exercise Skeletal Muscle Glycogen Recovery.

We examined the effect of dietary carbohydrate intake on post-exercise glycogen recovery. Male Institute of Cancer Research (ICR) mice were fed moderate-carbohydrate chow (MCHO, 50%cal from carbohydrate) or high-carbohydrate chow (HCHO, 70%cal from carbohydrate) for 10 days. They then ran on a treadmill at 25 m/min for 60 min and administered an oral glucose solution (1.5 mg/g body weight). Compared to the MCHO group, the HCHO group showed significantly higher sodium-D-glucose co-transporter 1 protein levels in the brush border membrane fraction (p = 0.003) and the glucose transporter 2 level in the mucosa of jejunum (p = 0.004). At 30 min after the post-exercise glucose administration, the skeletal muscle and liver glycogen levels were not significantly different between the two diet groups. The blood glucose concentration from the portal vein (which is the entry site of nutrients from the gastrointestinal tract) was not significantly different between the groups at 15 min after the post-exercise glucose administration. There was no difference in the total or phosphorylated states of proteins related to glucose uptake and glycogen synthesis in skeletal muscle. Although the high-carbohydrate diet significantly increased glucose transporters in the jejunum, this adaptation stimulated neither glycogen recovery nor glucose absorption after the ingestion of post-exercise glucose.

Insulin Resistance Is Not Sustained Following Denervation in Glycolytic Skeletal Muscle.

Denervation rapidly induces insulin resistance (i.e., impairments in insulin-stimulated glucose uptake and signaling proteins) in skeletal muscle. Surprisingly, whether this metabolic derangement is long-lasting is presently not clear. The main goal of this study was to determine if insulin resistance is sustained in both oxidative soleus and glycolytic extensor digitorum longus (EDL) muscles following long-term (28 days) denervation. Mouse hindlimb muscles were denervated via unilateral sciatic nerve resection. Both soleus and EDL muscles atrophied ~40%. Strikingly, while denervation impaired submaximal insulin-stimulated [3H]-2-deoxyglucose uptake ~30% in the soleus, it enhanced submaximal (~120%) and maximal (~160%) insulin-stimulated glucose uptake in the EDL. To assess possible mechanism(s), immunoblots were performed. Denervation did not consistently alter insulin signaling (e.g., p-Akt (Thr308):Akt; p-TBC1D1 [phospho-Akt substrate (PAS)]:TBC1D1; or p-TBC1D4 (PAS):TBC1D4) in either muscle. However, denervation decreased glucose transporter 4 (GLUT4) levels ~65% in the soleus but increased them ~90% in the EDL. To assess the contribution of GLUT4 to the enhanced EDL muscle glucose uptake, muscle-specific GLUT4 knockout mice were examined. Loss of GLUT4 prevented the denervation-induced increase in insulin-stimulated glucose uptake. In conclusion, the denervation results sustained insulin resistance in the soleus but enhanced insulin sensitivity in the EDL due to increased GLUT4 protein levels.

Mechanisms Underlying the Altered Bioenergetics of Aged Brain Microvasculature

Objective Aging impairs the cerebral microvascular function leading to increased risk of stroke and cognitive decline. The objective of this study was to identify the mechanisms underlying the aging-induced alterations in cellular energetics of brain microvessels (BMVs). Methods BMVs were isolated from young (2-4 months) and aged (24 months) male mice (C57Bl/6) using a combination of filters with pore sizes of 300μm and 40μm followed by gradient centrifugation. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured from real time ATP rate assay utilizing Seahorse XFe24 analyzer. Subsequently, total ATP production rate, GlycoATP and MitoATP levels were calculated. Mitochondrial fuel dependency of BMVs was assayed. Immunoblotting measured the protein levels of glucose transporter 1 (GLUT1), carnitine palmitoyltransferases (CPTs), 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PKKFB3), and ETC complexes (ETCs). Enzyme assays measured the activity of lactate dehydrogenase (LDH), citrate synthase (CS), and glutamate dehydrogenase (GDH). Results We found that oxidative phosphorylation (OxPhos) is the major contributor of cellular energy in the BMVs instead of glycolysis (67.0±2.1% vs 32.9±2.1%, n=7-10 mice/age group). Aged BMVs exhibited decreased total ATP production (49.7%, 8.8±2.0 vs 17.6±6.7 pico moles of ATP/µg/min, p=0.004, n=7-10 mice/age group). ATP generation from the glycolysis was more diminished in aged BMVs (74.7%, 1.5±0.3 vs 5.9±1.1 pico moles of ATP/µg/min, p=0.004, n=7-10 mice/group) than OxPhos (55.6%, 5.2±0.53 vs 11.7±1.8 pico moles of ATP/µg/min, p=0.01, n=7-10mice/group) when compared with young BMVs. GLUT1 levels were decreased in the aged BMVs when compared with the young BMVs (32%, 0.19±0.01 vs 0.28±0.02 A.U., p=0.01, n=12 mice/group). Glycolysis-stimulating enzyme, PFKFB3 expression was diminished (51.4%, 0.69±0.08 vs 1.42±0.13 A.U., n=6 mice/group) in the aged BMVs. Neither CPT nor ETC protein levels were affected by aging. Activity of Krebs cycle enzyme, CS, was reduced in the aged versus young BMVs (25.7%, 74.3±6.1% vs 100.0±9.9% nanomoles of substrate/min/mg, p=0.04, n=11-12 mice/group). Although relative utilizations of glucose and fatty acids were not affected by aging, glutamine utilization was enhanced in aged BMVs compared to young BMVs which was accompanied by increased activity of GDH, an enzyme involved in the mobilization of glutamine through Krebs cycle (24.7%, 116.8±4.3% vs 93.6±7.3% pico moles of NADH/min/mg, p=0.01, n=11-12 mice/group). Conclusions The results suggest that 1). diminished energy production in the aged BMVs is related to impaired glycolysis and OxPhos, and 2). multiple defects involving the expression of proteins and activity of enzymes related to glycolysis, OxPhos, and energy substrate utilization mediate the aging-induced impaired microvascular bioenergetics.

Placental Glucose Uptake in a Nonhuman Primate Model of Western-Style Diet Consumption and Chronic Hyperandrogenemia Exposure.

We reported that consumption of a western-style diet (WSD) with and without hyperandrogenemia perturbed placental perfusion and altered levels of glucose transporter proteins in rhesus macaques. Based on that result, we hypothesized that placental glucose uptake would be dysregulated in this model. In this study, female rhesus macaques were assigned at puberty to one of four groups: subcutaneous cholesterol implants + standard chow diet (controls, C); testosterone implants + chow (T); cholesterol implants + a high-fat, WSD; and T+WSD. After ~6 years of treatment, animals were mated, and pregnancies were delivered by cesarean section at gestational day (G) 130 (the term is G168). Placental villous explants were immediately prepared for radiolabeled glucose assay. Linear glucose uptake was observed between 0 and 30 s. At 20 s, glucose uptake in placental villous explants did not differ across the four treatment groups with values as follows: C: 25.5 ± 6.33, T: 22.9 ± 0.404, WSD: 26.9.0 ± 3.71, and T+WSD: 33.0 ± 3.12 (mean ± SD expressed in pmol/mg). Unlike our prior experiment, glucose transporter expression was reduced in WSD placentas, and our in vitro functional assay did not demonstrate a difference in glucose uptake across the transporting epithelium of the placenta. Notably, maternal blood glucose levels were significantly elevated in animals chronically fed a WSD. This disparity may indicate differences in glucose utilization and metabolism by the placenta itself, as glucose transporter expression and circulating fetal glucose concentrations were comparable across all four groups in this pregnancy cohort.

Dysregulated GLUT1 is Involved in the Pathogenesis of Preeclampsia by Impairing Decidualization

Background: Preeclampsia (PE), a hypertensive complication in pregnancy, is a major contributor to maternal and fetal morbidity and mortality. Thus far, the molecular mechanism underlying PE has not been investigated thoroughly. Glucose transporter 1 (GLUT1) is a central rate-limiting pump for glucose uptake and subsequent utilization. Our previous RNA-seq results demonstrated it was significantly downregulated in the decidua of severe preeclampsia (sPE) patients. Therefore, we aimed to explore the role of GLUT1 in the occurrence of PE. Methods: GLUT1 levels were evaluated by quantitative PCR, Western blotting and immunohistochemical staining in severe preeclamptic decidua. The regulatory role of GLUT1 in decidualization was further studied in human endometrial stromal cells (hESCs). We also explored the regulatory role of miRNA in GLUT1 expression. Findings: We observed a significant downregulation of GLUT1 mRNA and protein levels in the decidual tissues of women with sPE. Additionally, the expression of GLUT1 was substantially induced during in vitro decidualization. Moreover, GLUT1 knockdown in human endometrial stromal cells resulted in significant reduction in the mRNA levels of decidualization markers (IGFBP1 and PRL) and aerobic glycolysis-related genes (LDHA and MCT4), and decreased glucose uptake and lactate production. Furthermore, the expression of apoptotic signaling genes P53, P21 and BAX increased whereas the expression of BCL2 decreased after GLUT1 knockdown. Target prediction results and luciferase analysis showed that GLUT1 is one of the targets of miR-140-5p, which is partly responsible for the impaired GLUT1 level. Interpretation: Collectively, these results demonstrate that GLUT1 exerts pivotal role in human decidualization by participating in glycolysis, and its deficiency may trigger aberrant glycolysis and thus leads to destructive decidualization, which may be a pathogenetic mechanism of PE. These data suggest GLUT1 might be a promising target for PE therapy. Funding: This study was supported by grants from the National Key R&D Program of China (2019YFA0802600 and 2017YFC1001403), and National Natural Science Foundation of China (NSFC: 31871512 and 31671199) to C. Zhang. Support was also obtained by the Shanghai Commission of Science and Technology (17DZ2271100). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: This study was approved by the Ethics Committee of Ji’nan Maternity and Child Care Hospital and informed written consent forms were obtained from all pregnant women.