GLUT4 mRNA Expression Research Articles

Distinct Impact of Doxorubicin on Skeletal Muscle and Fat Metabolism in Mice: Without Dexrazoxane Effect

The chemotherapeutic agent doxorubicin (DOX) leads to the loss of skeletal muscle and adipose tissue mass, contributing to cancer cachexia. Experimental research on the molecular mechanisms of long-term DOX treatment is modest, and its effect on both skeletal muscle and adipose tissue has not been studied in an integrative manner. Dexrazoxane (DEXRA) is used to prevent DOX-induced cancer-therapy-related cardiovascular dysfunction (CTRCD), but its impact on skeletal muscle and adipose tissue remains elusive. Therefore, this study aimed to investigate the long-term effects of DOX on adipose tissue and skeletal muscle metabolism, and evaluate whether DEXRA can mitigate these effects. To this end, 10-week-old male C57BL6/J mice (n = 32) were divided into four groups: (1) DOX, (2) DOX-DEXRA combined, (3) DEXRA and (4) control. DOX (4 mg/kg weekly) and DEXRA (40 mg/kg weekly) were administered intraperitoneally over 6 weeks. Indirect calorimetry was used to assess metabolic parameters, followed by a molecular analysis and histological evaluation of skeletal muscle and adipose tissue. DOX treatment led to significant white adipose tissue (WAT) loss (74%) and moderate skeletal muscle loss (Gastrocnemius (GAS): 10%), along with decreased basal activity (53%) and energy expenditure (27%). A trend toward a reduced type IIa fiber cross-sectional area and a fast-to-slow fiber type switch in the Soleus muscle was observed. The WAT of DOX-treated mice displayed reduced Pparg (p < 0.0001), Cd36 (p < 0.0001) and Glut4 (p < 0.05) mRNA expression—markers of fat and glucose metabolism—compared to controls. In contrast, the GAS of DOX-treated mice showed increased Cd36 (p < 0.05) and Glut4 (p < 0.01), together with elevated Pdk4 (p < 0.001) mRNA expression—suggesting reduced carbohydrate oxidation—compared to controls. Additionally, DOX increased Murf1 (p < 0.05) and Atrogin1 (p < 0.05) mRNA expression—markers of protein degradation—compared to controls. In both the WAT and GAS of DOX-treated mice, Ppard mRNA expression remained unchanged. Overall, DEXRA failed to prevent these DOX-induced changes. Collectively, our results suggest that DOX induced varying degrees of wasting in adipose tissue and skeletal muscle, driven by distinct mechanisms. While DEXRA protected against DOX-induced CTRCD, it did not counteract its adverse effects on skeletal muscle and adipose tissue.

Slowly digestible starch impairs growth performance of broiler chickens offered low-protein diet supplemental higher amino acid densities by inhibiting the utilization of intestinal amino acid

BackgroundThe synchronized absorption of amino acids (AAs) and glucose in the gut is crucial for effective AA utilization and protein synthesis in the body. The study investigated how the starch digestion rate and AA levels impact intestinal AA digestion, transport and metabolism, breast muscle protein metabolism, and growth in grower broilers. A total of 720 21-day-old healthy male Arbor Acres Plus broilers were randomly assigned to 12 treatments, each with 6 replicates of 10 birds. The treatments comprised 3 different starch [corn: control, cassava: rapidly digestible starch (RDS), and pea: slowly digestible starch (SDS)] with 4 different AA levels [based on standardized ileal digestible lysine (SID Lys), 0.92%, 1.02% (as the standard), 1.12% and 1.22%].ResultsAn interaction between dietary starch sources and SID Lys levels significantly affected breast muscle yield (P = 0.033). RDS and SDS diets, or SID Lys levels of 0.92%, 1.02%, or 1.22%, significantly decreased the breast muscle yield of broilers in contrast to the corn starch diet with 1.12% SID Lys (P = 0.033). The SID Lys levels of 1.12% and 1.22% markedly improved body weight (BW), body weight gain (BWG) from 22 to 42 days of age, and mRNA expression of y+LAT1 and mTOR while reducing feed intake (FI) and feed/gain ratio (F/G) compared to the 0.92% SID Lys level (P < 0.05). The SDS diet significantly decreased BW and BWG of broilers from 22 to 42 days of age, distal ileal starch digestibility, jejunal amylase and chymotrypsin activities, and mRNA expression of GLUT2 and y+LAT1 compared to the corn starch diet (P < 0.05). The RDS diet suppressed the breast muscle mass by down-regulating expression of mTOR, S6K1, and eIF4E and up-regulating expression of MuRF, CathepsinB, Atrogin-1, and M-calpain compared to the corn starch diet (P < 0.05). Targeted metabolomics analysis revealed that the SDS diet significantly increased acetyl-CoA and α-ketoglutaric acid levels in the tricarboxylic acid (TCA) cycle (P < 0.05) but decreased the ileal digestibility of Lys, Tyr, Leu, Asp, Ser, Gly, Pro, Arg, Ile, and Val compared to the corn starch group (P < 0.05).ConclusionThe SDS diet impaired broiler growth by reducing intestinal starch digestibility, which inhibited intestinal AA and glucose absorption and utilization, increased AA oxidation for energy supply, and lowered the efficiency of protein synthesis. Although the RDS diet resulted in growth performance similar to the corn starch diet, it reduced breast muscle mass by inhibiting protein synthesis and promoting degradation.Graphical

Placental expression of GLUT-1, GLUT-3, and GLUT-4 mRNA and transcriptome profiling in pregnant women with diabetes.

Placental glucose transport is regulated by glucose transporter proteins (GLUTs). The study aimed to examine placental expression of GLUT-1, GLUT-3, and GLUT-4 mRNA in patients with type 1 diabetes, early gestational diabetes (eGDM), and healthy controls, and to investigate correlations between GLUTs expression and clinical parameters. Additionally, we compared placental transcriptome profiles in recruited subgroups. We recruited 59 pregnant women: 23 with type 1 diabetes, 17 with eGDM, and 19 controls. Patients with diabetes attended follow-up visits at each trimester. Transcriptome studies were performed in 4 patients per subgroup. The mean age was similar across all subgroups. eGDM patients had significantly higher BMI and were predominantly obese. We observed a significant 2-fold (P = 0.009) decrease in placental GLUT-3 mRNA expression in the type 1 diabetes and eGDM groups. GLUT-4 mRNA expression was significantly lower in the eGDM group compared to type 1 diabetes (3-fold) and controls (6-fold) (P = 0.007). There was a significant negative correlation between GLUT-3 (R = -0.29) and GLUT-4 (R = -0.27) mRNA expression and neonatal birth weight. GLUT-4 expression was negatively correlated with 1st trimester HbA1c (R = -0.72) and OGTT 120' (R = -0.82) results in eGDM patients, and 3rd trimester glycemic variability (R = -0.49) in type 1 diabetes. Microarray analysis revealed significant transcriptomic changes, with 45 down-regulated and 365 up-regulated genes in type 1 diabetes, and 21 significant changes in eGDM. Placental samples from patients with diabetes exhibit changes in GLUTs expression, which correlates with neonatal growth and several glycemic parameters. Additionally, multiple changes in transcriptomic profiles are observed in hyperglycemic patients.

Exposure to Hypoxic Conditions Up-regulates HER2 in Breast Cancer Cell Lines.

Tissue specimen quality is becoming increasingly important for basic research and routine clinical results. Warm ischemia time (WIT) affects human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) scores. However, the role of WIT on HER2 modulation remains unclear. We hypothesized that the WIT-mediated increase in HER2 IHC scores was caused by hypoxia. Therefore, this study aimed to determine the mechanism by which WIT mediates the increase in HER2. HER2 mRNA expression was measured in 4T1, SKBR3, and HCC1954 breast cancer cell lines using real-time PCR following hypoxia exposure. The membrane proteins were isolated and extracted using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) or evaluated through non-permeabilized immunofluorescent analysis. Hypoxic conditions up-regulated GLUT1 mRNA expression but not HER2 expression. The HER2 membrane protein fraction increased in response to hypoxic conditions. Nonpermeabilized immunofluorescence analysis showed that membrane-bound HER2 was also promoted under hypoxic conditions. HER2 is not regulated at the mRNA level; however, the level of membrane-bound HER2 increases in response to hypoxia.

Differential effects of leptin on energy metabolism in murine cell models of metastatic triple negative breast cancer

BackgroundLeptin, an energy balance regulator secreted by adipocytes, increases metastatic potential of breast cancer cells. The impact on cancer cell metabolism remains unclear given that most studies of leptin and breast cancer cell metabolism utilize supraphysiological glucose concentrations.MethodsUsing two murine models of metastatic triple-negative breast cancer (TNBC) differing in genetic alterations (4T1: p53 and Pik3ca mutations; metM-Wntlung: increased Wnt signaling) and cultured in physiological (5 mM) glucose media, we tested the hypothesis that leptin increases migration of metastatic breast cancer cells through regulation of glucose metabolism.ResultsOur results showed that leptin treatment, compared with vehicle, increased cell migration in each cell line, with decreased leptin receptor (Ob-R) mRNA expression in 4T1, but not metM-Wntlung, cells. AMP-activated protein kinase (AMPK) was activated in 4T1 with leptin treatment but decreased in metM-Wntlung. Leptin decreased fatty acid synthase (Fasn) and carnitine palmitoyltransferase 1a (Cpt1a) mRNA expression in 4T1 cells but increased their expression in metM-Wntlung cells. Fatty acid oxidation was not necessary for leptin-induced migration in either cell line. Leptin increased palmitate synthesis from glucose in metM-Wntlung, but not 4T1 cells. Moreover, although leptin increased glucose transporter 1 (Glut1) mRNA expression in both cell lines and inhibition of glycolysis blocked leptin-induced migration in metM-Wntlung, but not 4T1 cells.ConclusionTaken together, these results demonstrate that at physiological glucose concentrations, leptin increases migration of 4T1 and metM-Wntlung cells via shared and distinct effects on energy metabolism, suggesting that the type of TNBC genetic alteration plays a role in differential metabolic regulation of leptin-induced migration.

A novel approach to regulate glucose uptake in an anaplastic thyroid cancer cell line.

Curcumin's function in affecting cancer metabolic reprogramming remains poorly understood. Herein, we aimed to elucidate a novel link between Curcumin and the glucose uptake metabolism and glucose transporters (GLUTs) status in SW1736 cell line derived from anaplastic thyroid cancer. TheMTT test and flow cytometry was employed to test cell viability and cell death. For glucose uptake detection, ''GOD-PAP'' enzymatic colorimetric assay was applied to measure the direct glucose levels inside of the cells. Determination of GLUT1 and GLUT3 mRNA and protein expression in SW1736 cells was performed by qRT-PCR and western blotting. Also, the scratch wound healing assay was conducted for cell migration. The data indicated that Curcumin-induced cell death is independent of apoptosis in this type of thyroid cancer cell line. Furthermore, significantly reduced GLUT1 and GLUT3 expression was observed after treatment with Curcumin, resulting in the inhibition of glucose uptake (p < 0.05). Scratch assay indicated the inhibition of cell migration in SW1736 cells treated by Curcumin (p < 0.05). It can be concluded that GLUTs as metabolic targets can be blocked specifically by Curcumin for thyroid cancer prevention. Curcumin, as a promising anti-cancer agent, inhibits the growth of SW1736 anaplastic thyroid cancer cell line by regulating glucose uptake pathway and cell death. Altogether, these results suggest that the glucose pathway may be an important target for therapeutic intervention to sensitize tumor cells to cell death process by inhibition of glucose transporters.

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.

Licochalcone D from Glycyrrhiza uralensis Improves High-Glucose-Induced Insulin Resistance in Hepatocytes.

This study investigated the therapeutic potential of licochalcone D (LicoD), which is derived from Glycyrrhiza uralensis, for improving glucose metabolism in AML12 hepatocytes with high-glucose-induced insulin resistance (IR). Ultra-high-performance liquid chromatography-mass spectrometry revealed that the LicoD content of G. uralensis was 8.61 µg/100 mg in the ethanol extract (GUE) and 0.85 µg/100 mg in the hot water extract. GUE and LicoD enhanced glucose consumption and uptake, as well as Glut2 mRNA expression, in high-glucose-induced IR AML12 cells. These effects were associated with the activation of the insulin receptor substrate/phosphatidylinositol-3 kinase signaling pathway, increased protein kinase B α phosphorylation, and suppression of gluconeogenesis-related genes, such as Pepck and G6pase. Furthermore, GUE and LicoD promoted glycogen synthesis by downregulating glycogen phosphorylase. Furthermore, LicoD and GUE mitigated the downregulated expression of mitochondrial oxidative phosphorylation proteins in IR hepatocytes by activating the PPARα/PGC1α pathway and increasing the mitochondrial DNA content. These findings demonstrate the potential of LicoD and GUE as therapeutic options for alleviating IR-induced metabolic disorders by improving glucose metabolism and mitochondrial function.

Tryptophan supplements in high-carbohydrate diets by improving insulin response and glucose transport through PI3K-AKT-GLUT2 pathways in blunt snout bream (Megalobrama amblycephala)

The aim of this experiment was to elucidate the metabolic ramifications of tryptophan supplementation in the context of high-carbohydrate diet-feeding, which is important for improving feeding strategies in aquaculture in order to improve fish carbohydrate metabolism. Juvenile blunt snout bream with an initial mean body mass of 55.0±0.5 g were allocated to consume one of three experimental diets: CN, a normal diet with carbohydrate content of 30% (w/w); HC, a diet with high carbohydrate content of 43% (w/w); and HL, a high-carbohydrate diet to which 0.8% L-tryptophan (L-trp) had been added. These diets were fed for 8 weeks, and the effects of the carbohydrate and tryptophan contents of the diets were assessed.Histological analysis using Hematoxylin and Eosin (H&E) and Oil Red O staining revealed that high-carbohydrate intake was associated with abnormal hepatocyte morphology and excessive liver lipid accumulation, which were notably ameliorated by tryptophan supplementation. A significant increase in plasma glucose, glucagon, AGEs (advanced glycation end products), triglycerides, total cholesterol, and a significant decrease in insulin and hepatic glycogen after a high-carbohydrate diet in terms of plasma indices, compared to the control group. Almost all of them were restored to the normal level in the HL group. The present study might preliminarily suggest that tryptophan supplementation ameliorates the imbalance in glucose metabolism of this species induced by a high-carbohydrate diet. Transcriptomics showed that glucose metabolism under high carbohydrate was mainly regulated by the PI3K-AKT signaling pathway. The mRNA expression and protein levels of GLUT2 also varied with this pathway, which would suggest that sustained activation of this pathway with the addition of tryptophan accelerates glucose transport and insulin secretion under high-carbohydrate diet. Subsequent GTT and ITT experiments have also demonstrated that tryptophan improves glucose tolerance and insulin tolerance in blunt snout bream on a high-carbohydrate diet.In conclusion, these findings elucidate the positive regulatory effect of tryptophan on the PI3K-AKT-GLUT2 pathway under a high carbohydrate diet and provide a theoretical basis for the subsequent rational application of high carbohydrate diets in the future.

Exploring the Anticancer Potential of Astragalin in Triple Negative Breast Cancer Cells by Attenuating Glycolytic Pathway through AMPK/mTOR.

Aerobic glycolysis is crucial for cancer cells to survive, grow, and progress. In the current study, the anti-cancer effects of astragalin (ASG) on breast cancer cells and in the glycolytic pathway through AMPK/mTOR have been evaluated. The objective of this study was to examine the impact of ASG, a natural flavonoid, on glycolysis via targeting AMPK/mTOR signalling in MDA-MB-231 breast cancer cells. The study utilized ASG, which was isolated from Haplophyllum tuberculatum. The cells were treated with different concentrations of ASG (20 and 40 μg/mL), and anti- glycolytic activities were measured through cell proliferation, expression of glycolytic enzymes (HK-2, LDH-A, GLUT-1), glucose uptake, and lactate concentration assays. The MTT assay was used to assess cellular proliferation, while the glucose uptake and lactate levels were determined by employing colorimetric assays. The mRNA expression of target glycolytic enzymes was determined by qRT-PCR. The protein levels of glycolytic targets, as well as that of AMPK and mTOR, were determined by western blot. in silico docking of ASG was done with mTOR and AMPK proteins. Astragalin exhibited dose- and time-dependent anti-proliferative effects in MDA-MB-231 cells. In breast cancer cells, the mRNA and protein expression of GLUT-1, LDH-A, and HK-2 were all significantly downregulated after receiving ASG treatments. Furthermore, after ASG treatments, MDA-MB231 cells showed a significant decrease in lactate and glucose uptake compared to control cells. Mechanistically, ASG increased AMPK activation and suppressed mTOR activation in these cells. The inhibitory role of ASG on aerobic glycolysis was prevented by treatments with compound C (an AMPK inhibitor). However, combined treatment of compound C and ASG could nullify the ASG-induced anti-glycolysis effect and restore the level of p-AMPK and p-mTOR in MDA-MB231 cells. The results from molecular docking predicted that ASG had the potential to bind AMPK and mTOR, with free energy for binding, -8.2 kcal/mol and -8.1 kcal/mol, respectively. Taken together, the findings from this study indicated that ASG might modulate the AMPK/mTOR pathway to inhibit aerobic glycolysis and proliferation of MDAMB231 breast cancer.

IL-17A Cytokine-Regulated Glut1 Expression in Placenta Cells.

Trophoblasts, the principal cellular component of the placenta, play an important role in nutrient and gas exchange. Previous studies have indicated that maternal immune activation (MIA) leads to an elevation in IL-17A cytokine levels in maternal serum, subsequently influencing fetal brain development during pregnancy. In this study, we aimed to elucidate the impact of the IL-17A cytokine on placental function. First, we treated JAR and JEG-3, which is a placenta cell line, with IL-17A in a concentration-dependent or time-dependent manner and observed cell morphology and viability. It was confirmed that treatment with IL-17A or a double-stranded RNA mimic (PolyI:C) had no effect on the morphology or cell viability. IL-17A treatment increased the expression of IL-17R at the mRNA and protein levels, and Poly(I:C) increased the levels of IFNγ and TNFα. Additionally, PPARγ, known as a metabolism regulator, was increased by IL-17A treatment. Also, we observed that the expression of Glut1 and Glut3 was increased by IL-17A treatment. To confirm this, we examined the expression of transporters in the placental tissue of the MIA rodent model, and we observed that mRNA expression of glut1 and glut3 was significantly increased. However, the expression of Gltu1 and Glut3 was observed to be significantly inhibited in the brains of MIA-induced offspring. This study suggests that IL-17A increases signaling through IL-17R in the placenta and fetal brain tissue; however, there is a mechanism for regulating the expression of glucose transporters by increased IL-17A in the placenta.

Upregulation of Transferrin Receptor 1 (TfR1) but Not Glucose Transporter 1 (GLUT1) or CD98hc at the Blood-Brain Barrier in Response to Valproic Acid.

Transferrin receptor 1 (TfR1), glucose transporter 1 (GLUT1), and CD98hc are candidates for targeted therapy at the blood-brain barrier (BBB). Our objective was to challenge the expression of TfR1, GLUT1, and CD98hc in brain capillaries using the histone deacetylase inhibitor (HDACi) valproic acid (VPA). Primary mouse brain capillary endothelial cells (BCECs) and brain capillaries isolated from mice injected intraperitoneally with VPA were examined using RT-qPCR and ELISA. Targeting to the BBB was performed by injecting monoclonal anti-TfR1 (Ri7217)-conjugated gold nanoparticles measured using ICP-MS. In BCECs co-cultured with glial cells, Tfrc mRNA expression was significantly higher after 6 h VPA, returning to baseline after 24 h. In vivo Glut1 mRNA expression was significantly higher in males, but not females, receiving VPA, whereas Cd98hc mRNA expression was unaffected by VPA. TfR1 increased significantly in vivo after VPA, whereas GLUT1 and CD98hc were unchanged. The uptake of anti-TfR1-conjugated nanoparticles was unaltered by VPA despite upregulated TfR expression. VPA upregulates TfR1 in brain endothelium in vivo and in vitro. VPA does not increase GLUT1 and CD98hc proteins. The increase in TfR1 does not result in higher anti-TfR1 antibody targetability, suggesting targeting sufficiently occurs with available transferrin receptors without further contribution from accessory VPA-induced TfR1.

Moderate aerobic training enhances the effectiveness of insulin therapy through hypothalamic IGF1 signaling in rat model of Alzheimer's disease

Alzheimer's disease (AD) is a neurological condition that is connected with a decline in a person's memory as well as their cognitive ability. One of the key topics of AD research has been the exploration of metabolic causes. We investigated the effects of treadmill exercise and intranasal insulin on learning and memory impairment and the expression of IGF1, BDNF, and GLUT4 in hypothalamus. The animals were put into 9 groups at random. In this study, we examined the impact of insulin on spatial memory in male Wistar rats and analyzed the effects of a 4-week pretreatment of moderate treadmill exercise and insulin on the mechanisms of improved hypothalamic glucose metabolism through changes in gene and protein expression of IGF1, BDNF, and GLUT4. We discovered that rat given Aβ25–35 had impaired spatial learning and memory, which was accompanied by higher levels of Aβ plaque burden in the hippocampus and lower levels of IGF1, BDNF, and GLUT4 mRNA and protein expression in the hypothalamus. Additionally, the administration of exercise training and intranasal insulin results in the enhancement of spatial learning and memory impairments, the reduction of plaque burden in the hippocampus, and the enhancement of the expression of IGF1, BDNF, and GLUT4 in the hypothalamus of rats that were treated with Aβ25–35. Our results show that the improvement of learning and spatial memory due to the improvement of metabolism and upregulation of the IGF1, BDNF, and GLUT4 pathways can be affected by pretreatment exercise and intranasal insulin.

In vitro effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the expression of genes related to sperm motility and energy metabolism and intracytoplasmic sperm injection outcomes in obstructive azoospermic patients.

The presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor in various testicular cells and spermatozoa suggests a potential role in enhancing spermatogonial and postmeiotic cell development. Moreover, GM-CSF activates the pivotal pathways implicated in sperm motility regulation and glucose metabolism. However, the impact of GM-CSF on testicular biopsies from patients with obstructive azoospermia (OA) remains unexplored. Therefore, this study aimed to investigate the in vitro effects of GM-CSF on the expression of genes related to glucose transporters and signaling pathways, sperm motility, and viability in testicular biopsies. Following testicular sperm extraction from 20 patients diagnosed with OA, each sample was divided into two parts: the experimental samples were incubated with medium containing 2ng/ml GM-CSF at 37°C for 60min, and the control samples were incubated with medium without GM-CSF. Subsequently, the oocytes retrieved from the partner were injected with sperm from the treatment and control groups. The sperm parameters (motility and viability), the expression levels of sperm motility-related genes (PIK3R1, PIK3CA, and AKT1), and the expression levels of sperm energy metabolism-related genes (GLUT1, GLUT3, and GLUT14) were assessed. Furthermore, the fertilization and day 3 embryo development rate and embryo quality were evaluated. Compared with those in the nontreated group, the motility parameters and the mRNA expression levels of PIK3R1, AKT1, and GLUT3 in testicular sperm supplemented with GM-CSF were significantly greater (p < 0.05). However, no significant differences in the mRNA expression of PIK3CA, GLUT1, or GLUT14 were detected. According to the ICSI results, compared with the control group, the GM-CSF treatment group exhibited significantly greater fertilization rates (p = 0.027), Day 3 embryo development rate (p = 0.001), and proportions of good-quality embryos (p = 0.002). GM-CSF increased the expression of genes related to motility and the energy metabolism pathway and effectively promoted the motility of testis-extracted spermatozoa, consequently yielding positive clinical outcomes.

Effects of dietary vitamin D3 and carbohydrate levels on growth performance, carbohydrate metabolism, and intestinal health of yellow catfish (Pelteobagrus fulvidraco)

The study was performed to determine effects of vitamin D3 (VD3) and carbohydrate levels in the diets on growth, carbohydrate absorption and metabolism, and intestinal health of yellow catfish (Pelteobagrus fulvidraco). Four groups of yellow catfish with an average weight of 4.12 ± 0.00 g were provided with diets containing varying proportions of VD3 and carbohydrate. These diets included two VD3 levels at 1000 IU/kg (basic VD3) and 16,100 IU/kg diet (high VD3), each containing carbohydrate levels of 25% and 35%, respectively. After analyzing dietary VD3 and carbohydrate contents, these groups were labeled as follows: the control (30.78% carbohydrates +1040 IU/kg), the VD3 group (30.67% carbohydrates +16,041 IU/kg), the high-carbohydrate group (HCD group, 39.65% carbohydrates +1017 IU/kg), and the HCD + VD3 group (39.67% carbohydrates +16,061 IU/kg). Each tank consisted of three replicates, with each replicate containing 30 fish. The experiment continued for 10 weeks. Compared with the control, HCD group reduced growth performance, the muscular layer thickness, the zonulae occludentes1 and 3 (zo1, zo3), junctional adhesion molecules 3a (jam3a), claudin1, occludin, phosphoglucomutase 1 (pgm1) mRNA expression, but increased intestinal villi height, glycogen content, glycogen synthase (gs), glycogen branching enzyme (gbe), sodium/glucose cotransporter 1 and 2 (sglt1, sglt2) and glucose transporter 2 (glut2) mRNA expression, solute carrier family 5 member 1 (SGLT1) protein expression, and dietary VD3 group alleviated HCD-induced variations of these indices mentioned above. Compared to the control, HCD group significantly suppressed pyruvate carboxylase b (pcxb), phosphoenolpyruvate carboxykinase (pepck), interleukin10 (il10), NFE2 like bZIP transcription factor 2 (nrf2), superoxide dismutase 2 (sod2) and catalase 1 (cat1) mRNA expression, and also inhibited the activities of glutathione peroxidase (GPX), catalase (CAT), total superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC), but increased hexokinase 1 (hk1), glucokinase (gk), 1-phosphofructokinase (pfkb), interleukin 1β (il1β), interleukin 6 (il6), tumor necrosis factor a and β (tnfa, tnfβ) mRNA expression, and malondialdehyde (MDA) content; dietary VD3 group further suppressed gluconeogenic genes (pcxb, pepck) expression, but significantly enhanced the expression of glycolytic genes (hk1, gk, pfkp). Dietary VD3 also alleviated HCD-induced variations of these indices involved in antioxidant and inflammatory responses in yellow catfish intestine. Based on these results, our study indicated that dietary VD3 contributed to alleviate HCD-induced adverse effects on intestinal carbohydrate utilization and health. Our study also elucidated the innovative insights into the interaction of HCD and VD3 in fish, and provided the potential strategies for improving dietary carbohydrate utilization of fish via VD3 addition in aquafeeds. These findings can potentially contribute to cost savings for businesses and serve as a reference for optimizing feed formulations.

Cuiru Keli Improves Postpartum Hypogalactia in Rats Through Secreted Frizzled-Related Protein 2-Wnt/β-catenin Signaling Pathway

Based on the secreted frizzled-related protein 2 (SFRP2)-Wnt/β-catenin signaling pathway, this study explored the effect and mechanism of Cuiru Keli (CRKL) in the treatment of postpartum hypogalactia. A rat model of postpartum hypogalactia was established by gavaging 2 mL of 1.6 mg/mL bromocriptine mesylate to female rats on the third day after delivery. Female rats with a delivery time difference of less than 48 hours were selected and randomly assigned to 7 groups, including a normal group (without any modeling or medication), a model group, a CRKL low-dose group of model group model rats receiving CRKL at the dose of 3 g/kg, a CRKL medium-dose group of model rats receiving CRKL at the dose of 6 g/kg, a CRKL high-dose group of model rats receiving CRKL at the dose of 9 g/kg, a positive drug group of model rats receiving domperidone at the dose of 3 mg/kg, and a negative control (NC) group of model rats receiving normal saline. Each group contained 6 rats. Except for the normal and model groups, the remaining 5 groups were continuously administered with the respective intervention drugs at the specified doses by gavage once a day for 10 days. Changes in the total litter mass of the offspring in the 7 groups within 10 days were measured, and HE staining was performed to identify pathological changes in the mammary tissue (MT). Six groups of rats (excluding the positive control group) were used to observe the pathological changes of eosinophils in pituitary tissue. ELISA was performed to determine the content of prolactin (PRL) in serum, immunohistochemical staining was used to determine the expression of prolactin receptor (PRLR) in MT, and RT-qPCR was used to determine the mRNA expression of genes related to lactation in MT. Network pharmacology and molecular docking were used to study the therapeutic effect and mechanism of CRKL on postpartum hypogalactia, particularly whether it acted through the SFRP2-Wnt/β-catenin signaling pathway. The mechanism of CRKL treatment was further validated by detecting mRNA (RT-qPCR) and protein expression (Western blot) of related pathway genes. Cell experiments were conducted using primary culture rat mammary epithelial cells (RMEC) from rat MT. RMEC were divided into four groups, including a normal group (primary culture RMEC, untreated), SFRP2 overexpression group (primary cultured RMEC treated with SFRP2 overexpression vector), SFRP2 overexpression+CRKL group (receiving treatment for SFRP2 overexpression group plus 10% drug-containing serum), and negative control group (primary culture RMEC treated with empty vector). The effect of CRKL on the expression of lactation-related genes FASN, CSN2, and GLUT1 mRNA after SFRP2 overexpression was detected by RT-qPCR. In this study, CRKL was administered at a dose of 3 g/kg in the CRKL low-dose group, 6 g/kg in the medium-dose group, and 9 g/kg in the high-dose group (P<0.05 or P<0.01). Compared with the model group, CRKL at all doses significantly increased the total litter weight gain of the offsprings within 10 days (P<0.05 or P<0.01), and effectively increased lactation (P<0.01), the area of mammary lobules, and the size and filling of acinar cavities. CRKL at all doses also increased the number of eosinophils that secreted PRL in the pituitary gland of the postpartum hypogalactia rat model, and increased the content of PRL in the serum (P<0.05 or P<0.01). CRKL promoted the secretion and expression of PRL in postpartum hypogalactic model rats. In addition, it significantly promoted the expression of genes related to milk fat, milk protein, and lactose synthesis in MT (P<0.05 or P<0.01). Network pharmacology predicted that the Wnt signaling pathway might be a key pathway for CRKL in treating postpartum hypogalactia. The molecular docking results showed that related chemical components in CRKL had good binding ability with CCND1 and SFRP2. Compared with the model group, CRKL at all doses inhibited the expression of SFRP2 gene in vivo (P<0.01) and activated the mRNA and protein expression of CCND1 and c-Myc in the Wnt/β-catenin signaling pathway in MT (P<0.05 or P<0.01). Cell experiments showed that, compared to the normal group, SFRP2 overexpression reduced the mRNA expression of milk synthesis-related genes FASN, CSN2, and GLUT1 in RMEC (P<0.01). The CCK8 results indicated that 10% of the drug-containing serum was the effective concentration administered to cells (P<0.01). After administering drug-containing serum, the expression of the lactation-related genes FASN, CSN2, and GLUT1 were up-regulated (compared with the SFRP2 overexpression group, P<0.01). CRKL alleviates postpartum hypogalactia through the SFRP2-Wnt/β-catenin signaling pathway. SFRP2 might be a potential new target for the diagnosis and treatment of postpartum hypogalactia. This reveals a new mechanism of CRKL in treating postpartum hypogalactia and promotes its clinical application.

Common food additive carrageenan inhibits proglucagon expression and GLP-1 secretion by human enteroendocrine L-cells

Proglucagon mRNA expression and GLP-1 secretion by cultured human L-cells (NCI-H716) were inhibited following exposure to λ-carrageenan, a commonly used additive in processed foods. Carrageenan is composed of sulfated or unsulfated galactose residues linked in alternating alpha-1,3 and beta-1,4 bonds and resembles the endogenous sulfated glycosaminoglycans. However, carrageenan has unusual alpha-1,3-galactosidic bonds, which are not innate to human cells and are implicated in immune responses. Exposure to carrageenan predictably causes inflammation, and carrageenan impairs glucose tolerance and contributes to insulin resistance. When cultured human L-cells were deprived overnight of glucose and serum and then exposed to high glucose, 10% FBS, and λ-carrageenan (1 µg/ml) for 10 minutes, 1 h, and 24 h, mRNA expression of proglucagon and secretion of GLP-1 were significantly reduced, compared to control cells not exposed to carrageenan. mRNA expression of proglucagon by mouse L-cells (STC-1) was also significantly reduced and supports the findings in the human cells. Exposure of co-cultured human intestinal epithelial cells (LS174T) to the spent media of the carrageenan-treated L-cells led to a decline in mRNA expression of GLUT-2 at 24 h. These findings suggest that ingestion of carrageenan-containing processed foods may impair the production of GLP-1, counteract the effect of GLP-1 receptor agonists and induce secondary effects on intestinal epithelial cells.

Dietary apidaecin Api-PR19 addition enhances growth performance by regulating gut health and microbiota in broilers.

This study investigated the effects of Apidaecin Api-PR19 as feed additive on growth performance, intestinal health, and small intestinal microbiota of broilers. A total of 360 1-d-old Arbor Acres broilers were randomly assigned to 3 groups with 6 replicates including control group with basal diet (CON), antibiotic growth promotor group with basal plus 10 mg/kg colistin sulfate and 50 mg/kg roxarsone (AGP), and antibacterial peptide group with basal diet plus 330 mg/kg Apidaecin Api-PR19 (ABP). The trial lasted 35 d. Results showed that dietary Api-PR19 addition increased (p<0.05) the average daily feed intake, average daily gain and decreased (p<0.05) feed conversion ratio (FCR) during 1 to 21 d compared with the CON group. The digestibility of dry matter and crude protein were higher in AGP and ABP groups (p<0.05) where greater trypsin activity was detected in duodenum (p<0.05). The ratio of villus height to crypt depth (V/C) in duodenum and jejunum was increased at 35 d when broilers were given diets with ABP or AGP (p<0.05). Besides, ABP treatments up-regulated (p<0.05) the mRNA expression of EAAT3, GLUT2, ZO-1, and Claudin-1 in duodenum of broilers at 35 d of age. The results of immunohistochemistry showed that ABP treatment significantly increased (p<0.05) duodenal secretory immunoglobulin A (sIgA) content. In addition, 16S rRNA gene sequencing revealed that there were differences in the intestinal microbiota diversity and composition among three groups. Notably, the linear discriminant analysis effect size showed that p_Firmicutes, g_Enterococcus, g_Carnobacterium, g_Kitasatospora, and g_Acidaminococcus were dominant in ABP group. Redundancy analysis showed that these changes in gut microbiota in ABP group had correlation with growth performance, intestinal morphology, and content of sIgA. In general, these results indicated that dietary 330 mg/kg Apidaecin Api-PR19 supplementation promoted growth performance of broilers by improving intestinal development, nutrients absorption, immune function and modulating intestinal microbiota.

Lactobacillus paracasei JY062 Alleviates Glucolipid Metabolism Disorders via the Adipoinsular Axis and Gut Microbiota.

Glycolipid metabolic disorders (GLMD) refer to a series of metabolic disorders caused by abnormal processes of glucose and lipid synthesis, decomposition, and absorption in the body, leading to glucose and lipid excess, insulin resistance, and obesity. Probiotic intervention is a new strategy to alleviate metabolic syndrome. Lactobacillus paracasei JY062 (L. paracasei JY062) was separated from the Tibet-fermented dairy products. The results demonstrated a strong ability to relieve blood glucose disorders, blood lipid disorders, and tissue damage. The LPH group had the best effect, significantly decreasing the total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), leptin, insulin, and free fatty acid (FFA) concentrations and increasing the high-density lipoprotein cholesterol, adiponectin, and GLP-1 level compared to HFD-group mice. L. paracasei JY062 could activate the APN-AMPK pathway, increased AdipoQ, AMPK GLUT-4, and PGC-1α mRNA expression and decreased SREBP-1c, ACC, and FAS mRNA expression. L. paracasei JY062 intervention decreased the relative abundance of harmful bacteria, increased the relative abundance of beneficial bacteria, and restored the imbalance of gut microbiota homeostasis caused by a high-glucose-fat diet. L. paracasei JY062 alleviated glucolipid metabolism disorders via the adipoinsular axis and gut microbiota. This study provided a theoretical basis for probiotics to ameliorate glucolipid metabolism disorders by regulating the adipoinsular axis.

Effects of late gestational nutrient restriction on uterine artery blood flow, placental size, and cotyledonary mRNA expression in primiparous beef females.

Fall-calving primiparous beef females [body weight (BW): 451 ± 28 (SD) kg; body condition score (BCS): 5.4 ± 0.7] were individually-fed either 100% (control; CON; n = 13) or 70% (nutrient restricted; NR; n = 13) of metabolizable energy and metabolizable protein requirements for maintenance, pregnancy, and growth from day 160 of gestation to parturition. Doppler ultrasonography of both uterine arteries was conducted pre-treatment and every 21 d from days 181 to 265 of gestation. Expelled placentas were collected, and ipsilateral cotyledonary tissue was sampled to assess relative messenger ribonucleic acid (mRNA) expression. Placentas were separated into ipsilateral and contralateral sides, dissected (cotyledonary vs. intercotyledonary), and dried. Data were analyzed with nutritional plane, treatment initiation date, and calf sex (when P < 0.25) as fixed effects. Uterine blood flow included day and nutritional plane × day as repeated measures. We previously reported that post-calving, NR dams weighed 64 kg less and were 2.0 BCS lower than CON, but calf birth weight was not affected. Maternal heart rate was less (P < 0.001) for NR dams than CON after nutritional planes began. Nutritional plane did not affect (P ≥ 0.20) uterine artery hemodynamics, but all variables were affected (P ≤ 0.04) by day. Contralateral cotyledonary and placental weight were less (P ≤ 0.04) and contralateral intercotyledonary weight and number of cotyledons tended to be less (P ≤ 0.10) for NR dams than CON, but ipsilateral and whole placental weights were not affected (P ≥ 0.13). Ipsilateral placental weight as a percentage of total placental weight was greater (P = 0.03) for NR dams than CON. Whole placental cotyledonary: intercotyledonary weight was less (P = 0.01) for NR dams than CON. Placental efficiency was not affected (P = 0.89) by nutritional plane. Cotyledonary relative mRNA expression of GLUT3 and SNAT2 was greater (P ≤ 0.05) and relative expression of GLUT1, GLUT4, and NOS3 tended to be greater (P ≤ 0.07) for NR dams than CON. Nutritional plane did not affect (P ≥ 0.13) relative mRNA expression of GLUT5, 4F2hc, CAT1, LAT1, LAT2, VEGFA, FLT1, KDR, GUCY1B3, and PAG2. Despite less contralateral placental growth, beef heifers experiencing late gestational nutrient restriction maintained uterine artery blood flow and total placental mass and had 4 nutrient transporters and 1 angiogenic factor upregulated in cotyledons, all of which likely contributed to conserving fetal growth.