To examine the physiological role of the GLUT4/muscle-fat specific facilitative glucose transporter in regulating glucose homeostasis, we have generated transgenic mice expressing high levels of this protein in an appropriate tissue-specific manner. Examination of two independent founder lines demonstrated that high-level expression of GLUT4 protein resulted in a marked reduction of fasting glucose levels (=70 mg/dl) compared to wild-type mice ("'130 mg/dl). Surprisingly, 30 min following an oral glucose challenge the GLUT4 transgenic mice had only a slight elevation in plasma glucose levels (Q"90 mg/dl), whereas wild-type mice displayed a typical 2-to 3-fold increase (==250-300 mg/dl). In parallel to the changes in plasma glucose, insulin levels were --2-fold lower in the transgenic mice compared to the wild-type mice. Furthermore, isolated adipocytes from the GLUT4 transgenic mice had increased basal glucose uptake and subcellular fractionation indicated elevated levels of cell surface-associated GLUT4 protein. Consistent with these results, in situ immunocytochemical localization of GLUT4 protein in adipocytes and cardiac myocytes indicated a marked increase in plasma membrane-associated GLUT4 protein in the basal state. Taken together these data demonstrate that increased expression of the human GLUT4 gene in vivo results in a constitutively high level of cell surface GLUT4 protein expression and more efficient metabolic control over fluctuations in plasma glucose concentrations.The GLUT4/muscle-fat glucose transporter is one member of the facilitative glucose transporter super-gene family that is specifically expressed in muscle and adipose tissues (1, 2). In contrast to the other glucose transporter isoforms, GLUT4 contains specific amino acid targeting sequences (3-5) responsible for its localization to unique intracellular vesicular compartments found in adipocytes and muscle cells (6)(7)(8)(9)(10)(11). In response to acute insulin stimulation, these preformed GLUT4-containing vesicles rapidly translocate to the plasma membrane in a GTP-dependent process, resulting in a large increase in plasma membrane-associated GLUT4 protein (9-17).In contrast to this acute pathway of insulin action, catabolic states such as fasting and non-insulin-dependent diabetes are directly associated with a marked resistance of adipose and muscle tissue to insulin-stimulated glucose uptake (18)(19)(20). Recently, several studies have suggested that a decrease in GLUT4 expression may be the initial cause of insulin resistance in adipose tissue, which contributes to the maintenance ofinsulin resistance in muscle (21)(22)(23)(24)(25)(26)(27)(28)(29). Since the pathophysiological mechanisms responsible for insulin resistance are poorly understood, we have recently generated transgenic mice expressing high levels of the human GLUT4The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indi...
We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-lepr db/؉ (db/؉) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(D-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10 -12 weeks), both lower (two-to threefold) and higher (four-to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/؉ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo. Diabetes 50:593-600, 2001
Marked overexpression of the glucose transporter GLUT4 in skeletal muscle membrane fractions of GLUT4 transgenic (TG) mice is accompanied by disproportionately small increases in basal and insulin-stimulated glucose transport activity. Thus we have assessed cell surface GLUT4 by photolabelling with the membrane-impermeant reagent 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1, 3-bis(D-mannos-4-yloxy)-2-propylamine (ATB-BMPA) and measured the corresponding glucose transport activity using 2-deoxyglucose in isolated extensor digitorum longus (EDL) muscles from non-transgenic (NTG) and GLUT4 TG mice in the absence and presence of 13.3 nM (2000 mu units/ml) insulin, without or with hypoxia as a model of muscle contraction. TG mice displayed elevated rates of glucose transport activity under basal and insulin-stimulated conditions, and in the presence of insulin plus hypoxia, compared with NTG mice. Photoaffinity labelling of cell surface GLUT4 indicated corresponding elevations in plasma membrane GLUT4 in the basal and insulin-stimulated states, and with insulin plus hypoxia, but no difference in cell surface GLUT4 during hypoxia stimulation. Subcellular fractionation of hindlimb muscles confirmed the previously observed 3-fold overexpression of GLUT4 in the TG compared with the NTG mice. These results suggest that: (1) alterations in glucose transport activity which occur with GLUT4 overexpression in EDL muscles are directly related to cell surface GLUT4 content, regardless of the levels observed in the corresponding subcellular membrane fractions, (2) while overexpression of GLUT4 influences both basal and insulin-stimulated glucose transport activity, the response to hypoxia/ contraction-stimulated glucose transport is unchanged, and (3) subcellular fractionation provides little insight into the subcellular trafficking of GLUT4, and whatever relationship is demonstrated in EDL muscles from NTG mice is disrupted on GLUT4 overexpression.
Northern blot analysis of human tissues has demonstrated the expression of the brain-type glucose transporter isoform (GLUT 3) in liver, muscle and fat, raising the possibility that this transporter isoform may play a role in the regulation of glucose disposal in these tissues in response to insulin. We have raised an anti-peptide antibody against the C-terminal 13 amino acids of the murine homologue of this transporter isoform, and determined its tissue distribution in mouse tissues and murine-derived cell lines. The antibodies recognise a glycoprotein of about 50 kilodaltons, expressed at high levels in murine brain. In contrast to human tissues, the expression of GLUT 3 in mice is restricted to the brain, and no immunoreactivity was observed in either liver, fat or muscle membranes, or in murine 3T3-L1 fibroblasts or adipocytes. In contrast, high levels of expression of this isoform were observed in the NG 108 neuroblastoma x glioma cell line, a hybrid cell derived from rat glioma and mouse neuroblastoma cells. Taken together, these data suggest that the expression of GLUT 3 in rodents is restricted to non-insulin responsive neuronal cells and hence it is likely that the factors regulating the expression of this transporter in rodents differ to those in humans.
In drug discovery, establishing a correlation between in vitro potency and in vivo activity is critical for the validation of the selected target and for developing confidence in the in vitro screening strategy. The present study developed a competition equilibrium dialysis assay using a 96-well dialysis technique to determine the intrinsic K d for 13 inhibitors of human liver glycogen phosphorylase a (GPa) in the presence of liver homogenate to mimic the physiological environment. The results provided evidence that binding of an inhibitor to GPa was affected by extra cofactors present in the liver homogenate. A good correlation was demonstrated between the in vitro K d determined under liver homogenate environment and free liver concentration of an inhibitor at the minimum efficacious dose in diabetic ob/ob mice. This study revealed important elements (such as endogenous cofactors missing from the in vitro assay and free concentration at the target tissue) that contributed to a better understanding of the linkage between in vitro and in vivo activity. The approach developed here may be applied to many drugs in pharmacology studies in which the correlation between in vitro and in vivo activities for the target tissue (such as solid tumors, brain, and liver) is critical.In the drug discovery process within the pharmaceutical industry, initial lead compounds are usually identified from high-throughput in vitro biological screens, for example, an inhibition assay against a target enzyme. It is often hoped that the in vitro potency (such as IC 50 ) can be used to predict the in vivo pharmacological activity (such as EC 50 ). However, discovery scientists often face a question: why doesn't an in vitro potent inhibitor work in vivo or only work at a much higher in vivo concentration? Establishment of a correlation between in vitro potency and in vivo activity is crucial for validation of the target enzyme and for achieving confidence in an in vitro screening strategy.According to the fundamental free ligand hypothesis, the average free efficacious concentration at the steady-state in vivo should correlate with the intrinsic (unbound) potency determined from an in vitro assay. This hypothesis has been supported by many researchers (Wagner et al., 1965;Wagner, 1976;DeGuchi et al., 1992;Wright et al., 1996). In practice, however, this relationship is often obscured or confounded because of a variety of factors. For example, nonphysiological conditions, involvement of nonspecific binding within in vitro systems, or a combination may yield an inaccurate estimate of the true intrinsic potency. In addition, complex pharmacokinetic/pharmacodynamic relationships arising from indirect effects or target site disequilibrium may result in the inappropriate determination of in vivo potency.Several of the variables stated above confounded the initial establishment of a correlation between in vitro potency and This work was supported by Pfizer Inc. Article, publication date, and citation information can be found at http://jpe...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.