Changes in the gene expression of adiponectin and glucose transporter 12 (GLUT12) in lactating and non-lactating cows

Glucose delivery and uptake by the mammary gland is a rate‐limiting step in milk synthesis. Insulin resistance is believed to increase throughout the body following the onset of lactation. To study glucose metabolism in peak‐, late‐, and non‐lactating cows we analyzed the expression of an adipokine, namely, adiponectin, decreased insulin resistance, leptin, and a novel insulin‐responsive glucose transporter (GLUT12) in the adipose tissue and mammary gland by using real‐time polymerase chain reaction. Our results demonstrated that the mRNA level of adiponectin in the adipose tissue was greater in non‐lactating cows than in peak‐lactating cows. In the adipose tissue, there were no significant differences in the abundance of GLUT12 mRNA between the peak‐, late‐, and non‐lactating cows. In contrast, in the mammary gland, the mRNA level of GLUT12 was greater in non‐lactating cows than in peak‐ and late‐lactating cows. In the adipose tissue, the mRNA level of leptin and peroxisome proliferator‐activated receptor gamma 2 (PPARγ2) was greater in non‐lactating cows than in peak‐lactating cows. The results of the present study suggest that in lactating cows adiponectin plays an important role in insulin resistance in the adipose tissue; in the mammary gland, GLUT12 expression is believed to be an important factor for insulin‐dependent glucose metabolism.     

Lactogenic hormones stimulate expression of lipogenic genes but not glucose transporters in bovine mammary gland

During the onset of lactation, there is a dramatic increase in the expression of glucose transporters (GLUT) and a group of enzymes involved in milk fat synthesis in the bovine mammary gland. The objective of this study was to investigate whether the lactogenic hormones mediate both of these increases. Bovine mammary explants were cultured for 48, 72, or 96 h with the following hormone treatments: no hormone (control), IGF-I, insulin (Ins), Ins + hydrocortisone + ovine prolactin (InsHPrl), or Ins + hydrocortisone + prolactin + 17β-estradiol (InsHPrlE). The relative expression of β-casein, α-lactalbumin, sterol regulatory element binding factor 1 (SREBF1), fatty acid synthase (FASN), acetyl-CoA carboxylase α (ACACA), stearyol-CoA desaturase (SCD), GLUT1, GLUT8, and GLUT12 were measured by real-time PCR. Exposure to the lactogenic hormone combinations InsHPrl and InsHPrlE for 96 h stimulated expression of β-casein and α-lactalbumin mRNA by several hundred-fold and also increased the expression of SREBF1, FASN, ACACA, and SCD genes in mammary explants (P < 0.01). However, those hormone combinations had no effect on GLUT1 or GLUT8 expression and inhibited GLUT12 expression by 50% after 72 h of treatment (P < 0.05). In separate experiments, the expression of GLUTs in the mouse mammary epithelial cell line HC11 or in bovine primary mammary epithelial cells was not increased by lactogenic hormone treatments. Moreover, treatment of dairy cows with bovine prolactin had no effect on GLUT expression in the mammary gland. In conclusion, lactogenic hormones clearly stimulate expression of milk protein and lipogenic genes, but they do not appear to mediate the marked up-regulation of GLUT expression in the mammary gland during the onset of lactation.     

GLUT4 but not GLUT1 expression decreases early in the development of feline obesity

The increase in obesity in people and pets has been phenomenal. As in man, obesity in pets is a risk factor for many diseases including diabetes mellitus. Recently, tissue-specific regulation of glucose metabolism in fat and muscle tissue has been identified as an important factor for insulin sensitivity and it has been hypothesized that glucose uptake into tissues is altered in obesity causing insulin resistance. The purpose of this study was to determine the expression of the glucose transporter proteins GLUT4 and GLUT1 in muscle and fat from lean and obese cats. Seventeen domestic felines were tested in the lean state and again after a 6-month period of ad libitum food intake which led to a significant increase in weight (P<0.0001). Obese cats showed a significantly higher area under the curve (AUC) for glucose, AUC for insulin and a significant decrease in glucose percentage disappearance per min (K-value) (P=0.013, 0.018 and 0.017, respectively) during an intravenous glucose tolerance test, but no change in baseline glucose or glycosylated hemoglobin concentrations. GLUT4 expression was decreased in biopsies of both muscle (P=0.002) and fat (P=0.001) in the obese animals. GLUT4 in muscle and fat significantly and negatively correlated with the insulin AUC (r²=0.36, P=0.004 and r²=0.18, P=0.040, respectively). GLUT1 expression showed no significant change in the obese cats in either tissue. It is concluded that the changes in GLUT4 are early derangements in obesity and occur before glucose intolerance is clinically evident.     

Characterisation of glucose transporter (GLUT) gene expression in broiler chickens

1. Glucose transporter (GLUT) proteins, one of which is the major insulin-responsive transporter GLUT4, play a crucial role in cellular glucose uptake and glucose homeostasis in mammals. The aim of this study was to identify the extent of mRNA expression of GLUT1, GLUT2, GLUT3 and GLUT8 in chickens intrinsically lacking GLUT4. 2. GLUT1 mRNA was detected in most tissues of 3-week-old broiler chickens, with the highest expression measured in brain and adipose tissue. GLUT2 was expressed only in the liver and kidney. GLUT3 was highly expressed in the brain. GLUT8 was expressed ubiquitously, with expression in kidney and adipose tissue relatively higher than that of other tissues. 3. Expression levels of GLUT isoforms 1, 3 and 8 in skeletal muscle tissue were very low compared to the other tissues tested. 4. [3H]Cytochalasin B binding assays on tissue from 3-week-old chickens showed that the number of cytochalasin B binding sites in skeletal muscle plasma membranes was higher than in liver plasma membranes. These results suggest that GLUT proteins and/or GLUT-like proteins that bind cytochalasin B are expressed in chicken skeletal muscles. 5. It is proposed that GLUT expression and glucose transport in chicken tissues are regulated in a manner different from that in mammals.     

Postnatal development of glucose transporter proteins in bovine skeletal muscle and adipose tissue.

Facilitated diffusion of glucose across the plasma membrane is mediated by a family of glucose transporter (GLUT). GLUT1 is ubiquitously present in all tissues and involved in cellular glucose uptake, while GLUT4 plays a key role in cellular glucose uptake stimulated by insulin in skeletal muscles and adipose tissue. To examine the postnatal change in the GLUTs of ruminants, the protein levels of GLUT1 and GLUT4 were measured by Western blot analysis of skeletal muscles, adipose tissue and brain of Holstein male calves aged from 0 to 12 months. Analysis of rumen short chain volatile fatty acids revealed that rumen fermentation increased around 2-3 months old. The GLUT1 level did not change in all tissues examined during the postnatal period, while the GLUT4 levels in skeletal muscle and subcutaneous adipose tissue decreased gradually, and at 12 month old, it was about 40% of those seen at 0 month old. These results are contrast to those in non-ruminant species, in which GLUT4 increases during postnatal development, and may be related to the insulin-resistance seen in adult ruminants.     

Study on the Intervention Effect and Mechanism on Insulin Resistant Mice of Total Flavone from Melastoma dodecandrum Lour.

This experiment was conducted to explore the intervention effect and mechanism on insulin resistance (IR) mice of total flavonoids from Melastoma dodecandrum Lour.(TFMD).The model of IR mice was established by intragastric administration of high-fat emulsion,while 600,300 and 150 mg/kg TFMD were administered once daily for 30 days.After the end of the experiment the mices' fasting blood glucose (FBG),fasting serum insulin (FINS),serum total cholesterol(TC),triglyceride (TG),high density lipoprotein(HDL) were determined.The mRNA expression level of liver insulin receptor(InsR),fat peroxisome proliferator-activated receptor-γ(PPAR-γ),and glucose transporter 4 gene (GLUT4) in skeletal muscle were detected by Real-time quantative PCR,and the protein levels were detected by immunohistoche mical method.The results showed that TDMF could reduce the body weight of IR mice,decrease the level of serum FBG,FINS and HOMA-IR,increase the level of ISI,decrease the content of serum TG,TC and LDL,and increase the content of HDL(P<0.01,P<0.05);And the mRNA expression of INSR,PPAR-γ in liver and GLUT4 in skeletal muscle of IR mice were increased(P<0.01,P<0.05),and the protein expression level of InsR,PPAR-γ in liver and GLUT4 in skeletal muscle of IR mice were increased(P<0.01,P<0.05).The results confirmed that the TFMD could relieve experimental insulin resistance in mice,and the activity was related to the regulation of glucose and lipid metabolism and the enhancement of insulin sensitivity.     

地菍总黄酮对胰岛素抵抗小鼠的干预作用及机制研究

本研究旨在探讨地菍总黄酮（TFMD）对胰岛素抵抗（IR）小鼠的干预作用及其机制。试验以灌胃高脂乳剂建立胰岛素抵抗小鼠模型，同时灌胃给予地菍总黄酮混悬液（高剂量（600 mg/kg）、中剂量（300 mg/kg）及低剂量（150 mg/kg））进行治疗，治疗周期为30 d。试验结束后测定小鼠血清中空腹血清胰岛素（FINS）、空腹血糖（FBG）、胰岛素抵抗指数（HOMA-IR）、胰岛素敏感指数（ISI）、血清甘油三酯（TG）、总胆固醇（TC）、低密度脂蛋白（LDL）及高密度脂蛋白（HDL）水平；利用实时荧光定量PCR法检测各组小鼠肝脏中胰岛素受体（InsR）、过氧化物酶体增殖物激活受体-γ（PPAR-γ）及骨骼肌中葡萄糖转运体4（GLUT4）的mRNA表达水平；利用免疫组化技术检测各组小鼠肝脏中InsR、PPAR-γ和骨骼肌中GLUT4的蛋白表达水平。结果显示，与模型组相比，地菍总黄酮能降低IR小鼠的体重，降低血清FBG、FINS及HOMA-IR水平，升高ISI水平（P<0.01，P<0.05）；降低血清TG、TC及LDL含量，升高HDL含量（P<0.01，P<0.05）；同时提高IR小鼠肝脏中InsR、PPAR-γ及骨骼肌中GLUT4的mRNA表达量，提高小鼠肝脏InsR、PPAR-γ及骨骼肌中GLUT4的蛋白表达水平（P<0.01，P<0.05）。以上试验结果证实，地菍总黄酮能有效缓解小鼠试验性胰岛素抵抗症状，该活性与调节糖脂代谢、增强胰岛素敏感性有关。     

Partial sequencing and expression of genes involved in glucose metabolism in adipose tissues and skeletal muscle of healthy cats

Impaired insulin sensitivity is increasingly recognised in cats, but sequences of genes involved in insulin-signalling are largely undetermined in this species. In this study, extended feline mRNA sequences were determined for the adiponectin, glucose transporter-1 (GLUT1), GLUT4, peroxisome proliferative activated receptor-gamma1 (PPARgamma1), PPARgamma2, plasminogen activator inhibitor-1 (PAI-1), monocyte chemoattractant protein-1 (MCP-1) and insulin receptor genes. Conserved dog-specific primers identified from human-dog mRNA alignments were used to amplify feline cDNA in the polymerase chain reaction (PCR). The feline sequences determined by this method were used to design feline-specific primers suitable for real-time PCR for quantification of gene expression in insulin sensitive tissues of healthy cats. Partial sequences of feline mRNAs had 86-95% identity with dog and human genes. Expression of adiponectin, GLUT1, GLUT4, PPARgamma1, PPARgamma2, PAI-1 and insulin receptor mRNA was detected and quantified in subcutaneous and visceral fat and skeletal muscle, whereas MCP-1 mRNA was detected in adipose tissue but not in skeletal muscle. Further characterisation of genes related to glucose metabolism in cats will provide additional insights into insulin-signalling mechanisms in this species.     

Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS‐induced mastitis in dairy cows

In dairy cows, glucose is essential as energy source and substrate for milk constituents. The objective of this study was to investigate effects of long‐term manipulated glucose and insulin concentrations in combination with a LPS‐induced mastitis on mRNA abundance of glucose transporters and factors involved in milk composition. Focusing on direct effects of insulin and glucose without influence of periparturient endocrine adaptations, 18 dairy cows (28 ± 6 weeks of lactation) were randomly assigned to one of three infusion treatments for 56 h (six animals each). Treatments included a hyperinsulinemic hypoglycaemic clamp (HypoG), a hyperinsulinemic euglycaemic clamp (EuG) and a control group (NaCl). After 48 h of infusions, an intramammary challenge with LPS from E. coli was performed and infusions continued for additional 8 h. Mammary gland biopsies were taken before, at 48 (before LPS challenge) and at 56 h (after LPS challenge) of infusion, and mRNA abundance of genes involved in mammary gland metabolism was measured by RT‐qPCR. During the 48 h of infusions, mRNA abundance of glucose transporters GLUT1, 3, 4, 8, 12, SGLT1, 2) was not affected in HypoG, while they were downregulated in EuG. The mRNA abundance of alpha‐lactalbumin, insulin‐induced gene 1, κ‐casein and acetyl‐CoA carboxylase was downregulated in HypoG, but not affected in EuG. Contrary during the intramammary LPS challenge, most of the glucose transporters were downregulated in NaCl and HypoG, but not in EuG. The mRNA abundance of glucose transporters in the mammary gland seems not to be affected by a shortage of glucose, while enzymes and milk constituents directly depending on glucose as a substrate are immediately downregulated. During LPS‐induced mastitis in combination with hypoglycaemia, mammary gland metabolism was more aligned to save glucose for the immune system compared to a situation without limited glucose availability during EuG.     

Mammary cell proliferation and catabolism of adipose tissues in nutrition-restricted lactating sows were associated with extracellular high glutamate levels

Background: Persistent lactation,as the result of mammary cellular anabolism and secreting function,is dependent on substantial mobilization or catabolism of body reserves under nutritional deficiency.However,little is known about the biochemical mechanisms for nutrition-restricted lactating animals to simultaneously maintain the anabolism of mammary cells while catabolism of body reserves.In present study,lactating sows with restricted feed allowance(RFA)(n = 6),24% feed restriction compared with the control(CON) group(n = 6),were used as the nutrition-restricted model.Microdialysis and mammary venous cannulas methods were used to monitor postprandial dynamic changes of metabolites in adipose and mammary tissues.Results: At lactation d 28,the RFA group showed higher(P 0.05) loss of body weight and backfat than the CON group.Compared with the CON group,the adipose tissue of the RFA group had higher(P 0.05) extracellular glutamate and insulin levels,increased(P 0.05) lipolysis related genes(HSL and ATGL) expression,and decreased(P 0.05) glucose transport and metabolism related genes(VAMP8,PKLR and LDHB) expression.These results indicated that under nutritional restriction,reduced insulin-mediated glucose uptake and metabolism and increased lipolysis in adipose tissues was related to extracellular high glutamate concentration.As for mammary glands,compared with the CON group,the RFA group had up-regulated(P 0.05) expression of Notch signaling ligand(DLL3) and receptors(NOTCH2 and NOTCH4),higher(P 0.05) extracellular glutamate concentration,while expression of cell proliferation related genes and concentrations of most metabolites in mammary veins were not different(P 0.05) between groups.Accordingly,piglet performance and milk yield did not differ(P 0.05) between groups.It would appear that activation of Notch signaling and adequate supply of glutamate might assist mammogenesis.Conclusions: Mammary cell proliferation and catabolism of adipose tissues in nutrition-restricted lactating sows were associated with extracellular high glutamate levels.     

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

Although the functions of adiponectin, a differentiated adipocyte‐derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry‐off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.     

LPS-induced inflammation downregulates mammary gland glucose, fatty acid, and l-carnitine transporter expression at different lactation stages

Glucose, fatty acids, and l-carnitine are important substrates that support mammary epithelial cell metabolism, biosynthetic capacity, and milk yield and composition. Our study investigated the effects of LPS-induced inflammation on the expression of several glucose, fatty acid, and l-carnitine transporters in the lactating rat mammary gland at different lactation stages. Day 4, 11, and 18 lactating rats (n = 3/treatment) were administered LPS (1 mg/kg) or saline by intraperitoneal (IP) injection. Fold differences in the mRNA expression of glucose transporters Glut1, Glut8 and Sglt1, fatty acid transporters Fatp1, Fatp4 and Fabp3, and l-carnitine transporters Octn1, Octn2, and Octn3 were determined using the Comparative C_T method. The mRNA expression levels of all transporters evaluated, except Fatp4 and Octn2 were markedly higher in mammary gland at lactation day 11 compared to lactation day 4. LPS caused a marked decrease in transporter mRNA expression at each lactation stage except for Octn3 and Fatp1, which were markedly increased with LPS administration at lactation day 4, and Sglt1, which was slightly increased at day 11 of lactation. Our results suggest LPS-induced inflammation generally downregulates glucose, fatty acid, and l-carnitine transporter expression. Whether such changes lead to reductions in transporter substrate availability to the lactating mammary epithelial cell requires investigation since decreases in the availability of these nutrients may significantly impact mammary epithelial function and milk quality and yield.     

Insulin responsiveness to glucose and tissue responsiveness to insulin in lactating, pregnant, and nonpregnant, nonlactating beef cows

Insulin responsiveness to glucose and tissue responsiveness to insulin, using the hyperglycemic clamp and the hyperinsulinemic euglycemic clamp techniques, were measured in lactating, late pregnant, and nonpregnant, nonlactating (NPNL) beef cows. The glucose infusion rate (GIR) in the hyperglycemic clamp technique was higher (P less than .05). in lactating cows than in NPNL cows. The plateau in plasma insulin concentration (insulin responsiveness) was higher (P less than .05) in lactating cows than in late pregnant and NPNL cows. Pregnant cows tended to have higher GIR and lower plateau in plasma insulin concentration than NPNL cows. In the hyperinsulinemic euglycemic clamp technique, GIR (tissue responsiveness to insulin) was higher (P less than .05) in lactating cows than in late pregnant cows; values for NPNL cows were intermediate. We conclude that insulin responsiveness to glucose and tissue responsiveness to insulin were enhanced during lactation but tended to be decreased during late pregnancy in beef cows.     

Expression and subcellular localization of efflux transporter ABCG2/BCRP in important tissue barriers of lactating dairy cows,sheep and goats

Expression of efflux transporter ABCG2/BCRP in tissues barriers has shown to be associated with altered pharmaco‐ and toxicokinetics of xenobiotics. Until now, little is known about the functional expression of this transporter in dairy animals. We therefore systematically examined the expression and subcellular localization of ABCG2/BCRP in small intestine, colon, lung, liver, kidney and mammary gland in lactating cows, sheep and goats. Carrier expression was investigated by RT‐PCR and Western blot analysis showing highest expression of ABCG2/BCRP in small intestine and mammary gland, high levels in liver and moderate amounts of protein in lung, colon and kidney. Regarding subcellular localization, BCRP was predominantly found at the apical plasma membrane of small intestine, colon, bronchial epithelium, bile ducts and overall in endothelial structures in all tested species. In the mammary gland, there was strong apical staining of the alveolar epithelial cells and most of the ducts in all dairy ruminants. We also detected significantly elevated protein expression in lactating mammary gland compared with nonlactating cows, sheep and goats. Our results contribute to the role of BCRP in cytoprotection and disposition in important tissue barriers and may have important implications for veterinary pharmacotherapy of dairy animals using drugs identified as BCRP substrates.     

Insulin resistance selectively alters cell-surface glucose transporters but not their total protein expression in equine skeletal muscle

Background: Insulin resistance (IR) has been widely recognized in humans, and more recently in horses, but its underlying mechanisms are still not well understood. The translocation of glucose transporter 4 (GLUT4) to the cell surface is the limiting step for glucose uptake in insulin‐sensitive tissues. Although the downstream signaling pathways regulating GLUT translocation are not well defined, AS160 recently has emerged as a potential key component. In addition, the role of GLUT12, one of the most recently identified insulin‐sensitive GLUTs, during IR is unknown. Hypothesis/Objectives: We hypothesized that cell‐surface GLUT will be decreased in muscle by an AS160‐dependent pathway in horses with IR. Animals: Insulin‐sensitive (IS) or IR mares (n = 5/group). Methods: Muscle biopsies were performed in mares classified as IS or IR based on results of an insulin‐modified frequently sampled IV glucose tolerance test. By an exofacial bis‐mannose photolabeled method, we specifically quantified active cell‐surface GLUT4 and GLUT12 transporters. Total GLUT4 and GLUT12 and AS160 protein expression were measured by Western blots. Results: IR decreased basal cell‐surface GLUT4 expression (P= .027), but not GLUT12, by an AS160‐independent pathway, without affecting total GLUT4 and GLUT12 content. Cell‐surface GLUT4 was not further enhanced by insulin stimulation in either group. Conclusions and Clinical Importance: IR induced defects in the skeletal muscle glucose transport pathway by decreasing active cell‐surface GLUT4.