GLUT1 mRNA Abundance Research Articles

256 LIVER GLUCONEOGENESIS REGULATION BY TNFα

Liver glycolysis/gluconeogenesis dysregulation during sepsis/septic shock has been suggested to be the cause of hypoglycemia in endotoxic shock. TNFα is a mediator in endotoxic shock. However, TNFα effects on liver gluconeogenesis is not well known. We have shown that TNFα increases glucose transporter GLUT mRNA abundance and decreases gluconeogenesis in heapatocytes. Therefore, we hypothesize that TNFα increases glucose entry into hepatocytes and causes the suppression of hepatocyte gluconeogenesis. Objective: To invesigate TNFα induced glucose entry into the hepatocyte. Materials and Methods: Hepatocytes (106cell/ml) were isolated from 24 hour fasted 10 day old Sprague-Dawley rats (23-27 grams) and incubated in RPMI 1640 plus 14C-2-deoxy-D-glucose (0.2μCi) and recombinant murine TNFα (4.5×105u/ml). Hepatocyte 14C-2-deoxy-D-glucose uptake and GLUT1 mRNA were determined after a 4 hour incubation. Results: TNFα increased 14C-2-deoxy-D-glucose uptake (0.12±0.02 vs 0.06±0.01mM, p<0.05)and GLUT1 mRNA abundance. Summary: Glucose transport and GLUT1 mRNA abundance was increased by TNFα. Conclusion: The increased glucose entry may cause suppression of hepatocyte gluconeogenesis.

Enhanced expression of the blood-brain barrier GLUT1 glucose transporter gene by brain-derived factors

The blood-brain barrier GLUT1 glucose transporter is localized in brain to the capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo. However, its expression is markedly downregulated in cultured bovine brain capillary endothelium (ECL cells), possibly due to the absence of brain-derived or astrocyte trophic factors in the tissue culture medium. To examine this hypothesis, we studied the effect of a bovine brain homogenate (BBH), and conditioned media and plasma membranes obtained from the rat C6 glioma cell line, on the abundance of the GLUT1 transcript in ECL cells. BBH induced a significant increase in the abundance of both GLUT1 and actin mRNAs, and this effect was dose and time dependent. The increase in the GLUT1 mRNA levels correlated with an increase in the transcriptional rate of this gene measured by nuclear run-on experiments. C6 conditioned media and C6 plasma membranes had no effect on the abundance of either GLUT1 or actin mRNA. To determine whether known growth factors cause BBH-like induction of GLUT1 and actin mRNAs, a series of growth factors was also tested. EGF and PDGF had no effect on the levels of these mRNAs. Basic FGF had a moderate effect and TNFα partially mimicked the effect of BBH on both GLUT1 and actin transcripts. The present data suggests that brain-derived trophic factors present in BBH stimulate BBB-GLUT1 glucose transporter gene expression in ECL cells through a transcriptional mechanism. Although this effect was partially mimicked by TNFα, C6 cell membranes or C6 conditioned media were unable to induce changes in the abundance of GLUT1 mRNA. Therefore, BBH may be a useful model to study the characterization of soluble brain-derived trophic factors involved in the induction of BBB-GLUT1 gene expression.

TNF ALTERS GLUCOSE PRODUCTION AND MEMBRANE GLUCOSE TRANSPORTER mRNA ABUNDANCE IN ISOLATED RAT BEPATOCYTES

Hypoglycemia is a common sign in newborn endotoxic shock. Our previous study showed that glucose production was decreased and membrane glucose transporter gene expression was altered in the liver of endotoxic suckling rats. TNFα, a key mediator of endotoxic shock, may contribute to altered liver glucose production and membrane glucose transporter gene expression. TNFα this study was performed to evaluate TNFα effects on isolated hepatocyte glucose production and glucose transporter mRNA abundance, Hepatocytes were isolated from 10 day old Sprague-Dawley rats and incubated in RPMI media for 3 hours with or without 4.5×105 unit/ml rMuTNFα. Glucose production and membrane glucose transporter GLUT1 and GLUT2 mRNA abundance were determined. Hepatocyte membrane mRNA abundance was expressed as percent of controls. TNF blunted hepatic glucose production (82.6 vs 0.8 μg/107 cells without and with TNFα, respectively. p<0.001). TNFα increased GLUT2 mRNA abundance to 207% (p<0.01) and decreased GLUT2 mRNA abundance to 19% (p<0.001). Therefore TNFα decreased liver glucose production and altered hepatocyte membrane glucose transporter gene expression.

Induction of GLUT1 mRNA in response to inhibition of oxidative phosphorylation.

In previous studies, we have shown that inhibition of oxidative phosphorylation in Clone 9 cells (a nontransformed rat liver cell line) by 5 mM azide results in a marked biphasic stimulation of glucose transport that is mediated by GLUT1 [M. Shetty, J. N. Loeb, and F. Ismail-Beige. Am.J. Physiol. 262 (Cell Physiol. 31): C527-C532, 1992]. The late phase of the response (at 8-24 h) is associated with a doubling of cell GLUT1 content and an 8- to 10-fold increment in GLUT1 mRNA abundance. To investigate the mechanisms mediating GLUT1 mRNA induction, we have examined the effect of incubation in the presence of azide on GLUT1 gene transcription. In nuclear run-on assays, the rate of GLUT1 gene transcription was increased 2.5 +/- 0.3-fold in nuclei from cells exposed to azide for 4 h. Additionally, GLUT1 mRNA turnover was decreased in cells treated with azide: upon inhibition of RNA synthesis by actinomycin D, GLUT1 mRNA content decreased with half-lives of 2.3 +/- 0.3 and 8.0 +/- 0.5 h in control cells and cells treated with azide for 4 h, respectively. GLUT1 mRNA half-life was most prolonged (> 12 h) when azide was added subsequent to the addition of actinomycin D, and the half-life continued to be prolonged (6.5 +/- 0.5 h) in cells exposed to azide for 16 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose transport stimulation by thyroid hormone in ARL 15 cells: partial role of increased GLUT1 glucose transporter gene transcription.

We have previously reported that the stimulation of glucose transport by thyroid hormone in the rat liver-derived ARL 15 cell line is attributable, at least in part, to increased abundance of cellular glucose transporters with a corresponding increase in the mRNA coding for the GLUT1 glucose transporter isoform. To elucidate further the mechanism by which thyroid hormone increases glucose transport, we examined the time-course of the effect of L-triiodothyronine (T3) on 3H-2-deoxyglucose uptake, GLUT1 protein abundance, and GLUT1 mRNA abundance in ARL 15 cells. At 6 h of T3 treatment, 3H-2-deoxyglucose uptake was increased by 40 +/- 11%, whereas the abundance of GLUT1 protein in cell extracts had not yet changed at this time. At 48 h, GLUT1 protein was increased by 58 +/- 10%, whereas 3H-2-deoxyglucose uptake at this time was increased by 116 +/- 14%. GLUT1 mRNA levels rose within 4 h of T3 treatment, preceding the increase in GLUT1 protein, and more than doubled by 24 h. In additional experiments to determine the mechanism by which T3 increases GLUT1 mRNA, T3 treatment for 48 h increased the rate of transcription of the GLUT1 gene, determined by nuclear run-on analysis, by 55 +/- 11%. T3 treatment did not significantly alter the half-life of GLUT1 mRNA. In the presence of inhibitors of protein synthesis, GLUT1 mRNA increased at 6 h (5-7-fold), but there was no further induction of this mRNA by T3 in the presence of these inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of mammalian facilitative glucose transporter messenger rna in bovine tissues

1.1. The complementary DNA for five human facilitative glucose transporters (GLUT1, GLUT2, GLUT3, GLUT4 and GLUT5) were used to determine the distribution of facilitative glucose transporter mRNA in bovine tissues by Northern blotting. Under high stringency hybridization conditions, a single 2.8 kb transcript of GLUT1 was seen in all bovine tissues examined except liver. Mammary gland had the highest abundance of GLUT1 mRNA.2.2. Four GLUT2 transcripts of 6.3, 3.8, 2.2 and 1.6 kb were observed to be most abundant in liver, with lower abundance in kidney and duodenum.3.3. Only a very low level of GLUT3 mRNA was detected in the mammary gland, skeletal muscle and duodenum.4.4. Skeletal muscle contained the greatest abundance of GLUT4 mRNA, which was barely detectable in omental fat, kidney and mammary gland.5.5. Transcripts of GLUT5 mRNA were detected in relatively high abundance in liver and kidney, and in lower abundance in the duodenum and mammary gland.6.6. With the exception of the mammary gland, for which human data have not been reported, the distributions of GLUT1, 2 and 4 mRNA for the bovine tissues examined are similar to that reported for humans. On the other hand, the distributions of GLUT3 and 5 mRNA in the bovine differ from those reported for humans.

Treatment of insulin-resistant mice with the oral antidiabetic agent pioglitazone: evaluation of liver GLUT2 and phosphoenolpyruvate carboxykinase expression.

Obese KKAy insulin-resistant mice represent a model for the human syndrome of noninsulin-dependent diabetes mellitus. As such, the animals are hyperglycemic and hyperinsulinenic. Treatment of KKAy mice with pioglitazone, a new antihyperglycemic agent, lowered elevated blood glucose and insulin levels to near normal. Since hepatic glucose overproduction is a key abnormality in noninsulin-dependent diabetes mellitus, the aim of the present study was to define the specific effects of pioglitazone on hepatic glucose metabolism and release. To do so, we evaluated the expression of the major liver glucose transporter, GLUT2, and examined the activity and expression of the major rate-limiting enzyme for gluconeogenesis, phosphoenolpyruvate carboxykinase. Our results showed that GLUT2 mRNA abundance was unchanged in diabetic KKAy mice compared to nondiabetic animals, and that no changes were elicited by pioglitazone treatment. Such unaltered GLUT2 levels were consistent with a role for liver GLUT2 in bidirectional transport of glucose during physiological states of uptake or release. In contrast, phosphoenolpyruvate carboxykinase activity and mRNA abundance were concordantly elevated 2-fold in diabetic animals and were returned to normal levels after treatment with pioglitazone. Given that pioglitazone therapy led to decreased hepatic gluconeogenesis while insulin levels were concomitantly lowered, it appeared that pioglitazone acted to restore sensitivity to insulin's normal inhibitory actions.

Regulation of glucose transport in clone 9 cells by thyroid hormone

Triiodothyronine (T 3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a ‘non-transformed’ rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T 3 is due to an increase in transport V max and occurs in the absence of a change in either the K m for glucose transport (∼ 3 mM) or the K i for inhibition of transport by cytochalasin B ((1–2) · 10 −7 M). Consistent with the observed K i for cytochalasin B, Northern blot analysis of RNA from control and T 3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T 3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T 3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.

Altered glucose transporter mRNA abundance in a rat model of endotoxic shock

To better understand molecular mechanisms of glucose transport in shock, we studied glucose transporter isoform mRNA abundance after injection of S. enteritidis endotoxin (40mg/kg) or saline. Six to 8 hours after injection, endotoxin-treated animals compared to controls became hypoglycemic (44±6 vs. 111±4 mg/dl) and lactacidemic (5.9±0.5 vs. 1.3±0.1). At such times, tissue RNA was isolated and hybridized to Riboprobes for GLUT1 (erythrocyte), GLUT2 (liver), and GLUT4 (muscle/fat) glucose transporter isoforms and expressed as percent of control. GLUT1 mRNA abundance was increased in fat (660%, p<.05), soleus muscle (314%, p<.05), and liver (871%, p<.001) of endotoxin-treated rats. Soleus muscle GLUT4 mRNA levels were increased (+33%, p<.02), while liver GLUT2 mRNA levels were markedly decreased (−58%, p<.01). The overall increase in GLUT1 mRNA abundance accompanied by lowered liver GLUT2 mRNA levels may either cause or reflect profoundly altered glucose transport.