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Objective: The liver is a central regulator of energy metabolism exerting its influence both through intrinsic processing of substrates such as glucose and fatty acid as well as by secreting endocrine factors, known as hepatokines, which influence metabolism in peripheral tissues. Human genome wide association studies indicate that a predicted loss-of-function variant in the Inhibin βE gene (INHBE), encoding the putative hepatokine Activin E, is associated with reduced abdominal fat mass and cardiometabolic disease risk. However, the regulation of hepatic Activin E and the influence of Activin E on adiposity and metabolic disease are not well understood. Here, we examine the relationship between hepatic Activin E and adipose metabolism, testing the hypothesis that Activin E functions as part of a liver-adipose, inter-organ feedback loop to suppress adipose tissue lipolysis in response to elevated serum fatty acids and hepatic fatty acid exposure., Methods: The relationship between hepatic Activin E and non-esterified fatty acids (NEFA) released from adipose lipolysis was assessed in vivo using fasted CL 316,243 treated mice and in vitro using Huh7 hepatocytes treated with fatty acids. The influence of Activin E on adipose lipolysis was examined using a combination of Inhbe knockout mice, a mouse model of hepatocyte-specific overexpression of Activin E, and mouse brown adipocytes treated with Activin E enriched media., Results: Increasing hepatocyte NEFA exposure in vivo by inducing adipose lipolysis through fasting or CL 316,243 treatment increased hepatic Inhbe expression. Similarly, incubation of Huh7 human hepatocytes with fatty acids increased expression of INHBE. Genetic ablation of Inhbe in mice increased fasting circulating NEFA and hepatic triglyceride accumulation. Treatment of mouse brown adipocytes with Activin E conditioned media and overexpression of Activin E in mice suppressed adipose lipolysis and reduced serum FFA levels, respectively. The suppressive effects of Activin E on lipolysis were lost in CRISPR-mediated ALK7 deficient cells and ALK7 kinase deficient mice. Disruption of the Activin E-ALK7 signaling axis in Inhbe KO mice reduced adiposity upon HFD feeding, but caused hepatic steatosis and insulin resistance., Conclusions: Taken together, our data suggest that Activin E functions as part of a liver-adipose feedback loop, such that in response to increased serum free fatty acids and elevated hepatic triglyceride, Activin E is released from hepatocytes and signals in adipose through ALK7 to suppress lipolysis, thereby reducing free fatty acid efflux to the liver and preventing excessive hepatic lipid accumulation. We find that disrupting this Activin E-ALK7 inter-organ communication network by ablation of Inhbe in mice increases lipolysis and reduces adiposity, but results in elevated hepatic triglyceride and impaired insulin sensitivity. These results highlight the liver-adipose, Activin E-ALK7 signaling axis as a critical regulator of metabolic homeostasis., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Genetic ablation of the voltage-gated potassium channel Kv1.3 improves insulin sensitivity and increases metabolic rate in mice. Inhibition of Kv1.3 in mouse adipose and skeletal muscle is reported to increase glucose uptake through increased GLUT4 translocation. Since Kv1.3 represents a novel target for the treatment of diabetes, the present study investigated whether Kv1.3 is functionally expressed in human adipose and skeletal muscle and whether specific pharmacological inhibition of the channel is capable of modulating insulin sensitivity in diabetic mouse models. Voltage-gated K(+) channel currents in human skeletal muscle cells (SkMC) were insensitive to block by the specific Kv1.3 blockers 5-(4-phenoxybutoxy)psoralen (PAP-1) and margatoxin (MgTX). Glucose uptake into SkMC and mouse 3T3-L1 adipocytes was also unaffected by treatment with PAP-1 or MgTX. Kv1.3 protein expression was not observed in human adipose or skeletal muscle from normal and type 2 diabetic donors. To investigate the effect of specific Kv1.3 inhibition on insulin sensitivity in vivo, PAP-1 was administered to hyperglycemic mice either acutely or for 5 days prior to an insulin tolerance test. No effect on insulin sensitivity was observed at free plasma PAP-1 concentrations that are specific for inhibition of Kv1.3. Insulin sensitivity was increased only when plasma concentrations of PAP-1 were sufficient to inhibit other Kv1 channels. Surprisingly, acute inhibition of Kv1.3 in the brain was found to decrease insulin sensitivity in ob/ob mice. Overall, these findings are not supportive of a role for Kv1.3 in the modulation of peripheral insulin sensitivity.

A PEGylated glucagon-like peptide-1 (GLP-1) agonist and glucagon antagonist hybrid peptide was engineered as a potential treatment for type 2 diabetes. To support preclinical development of this PEGylated dual-acting peptide for diabetes (DAPD), we developed a reproducible method for PEGylation, purification, and analysis. Optimal conditions for site-specific PEGylation with 22 and 43 kDa maleimide-polyethylene glycol (maleimide-PEG) polymers were identified by evaluating pH, reaction time, and reactant molar ratio parameters. A 3-step purification process was developed and successfully implemented to purify PEGylated DAPD and remove excess uncoupled PEG and free peptide. Five lots of 43 kDa PEGylated DAPD with starting peptide amounts of 100 mg were produced with overall yields of 53% to 71%. Analytical characterization by N-terminal sequencing, amino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and GLP-1 receptor activation assay confirmed site-specific attachment of PEG at the engineered cysteine residue, expected molecular weight, correct amino acid sequence and composition, and consistent functional activity. Purity and safety analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), analytical ion-exchange chromatography, reversed-phase high-performance liquid chromatography, and limulus amebocyte lysate test showed that the final products contained <1% free peptide, <5% uncoupled PEG, and <0.2 endotoxin units per milligram of peptide. These results demonstrate that the PEGylation and purification process we developed was consistent and effective in producing PEGylated DAPD preclinical materials at the 100 mg (peptide weight basis) or 1.2 g (drug substance weight basis) scale.

Type 2 diabetes is characterized by reduced insulin secretion from the pancreas and overproduction of glucose by the liver. Glucagon-like peptide-1 (GLP-1) promotes glucose-dependent insulin secretion from the pancreas, while glucagon promotes glucose output from the liver. Taking advantage of the homology between GLP-1 and glucagon, a GLP-1/glucagon hybrid peptide, dual-acting peptide for diabetes (DAPD), was identified with combined GLP-1 receptor agonist and glucagon receptor antagonist activity. To overcome its short plasma half-life DAPD was PEGylated, resulting in dramatically prolonged activity in vivo. PEGylated DAPD (PEG-DAPD) increases insulin and decreases glucose in a glucose tolerance test, evidence of GLP-1 receptor agonism. It also reduces blood glucose following a glucagon challenge and elevates fasting glucagon levels in mice, evidence of glucagon receptor antagonism. The PEG-DAPD effects on glucose tolerance are also observed in the presence of the GLP-1 antagonist peptide, exendin(9-39). An antidiabetic effect of PEG-DAPD is observed in db/db mice. Furthermore, PEGylation of DAPD eliminates the inhibition of gastrointestinal motility observed with GLP-1 and its analogues. Thus, PEG-DAPD has the potential to be developed as a novel dual-acting peptide to treat type 2 diabetes, with prolonged in vivo activity, and without the GI side-effects.

VPAC2P-PEG is a VPAC2 receptor agonist peptide that acts as a glucose-dependent insulin secretagogue. Proteolysis by DPPIV may contribute to the in vivo clearance of VPAC2P-PEG. Here, the N-terminus of VPAC2P-PEG is modified by N-terminal acetylation to impart DPPIV resistance. The acetylated peptide, Ac-VPAC2P-PEG, is a selective and potent VPAC2 agonist, resistant to DPPIV proteolysis, and exhibits substantially improved half-life and glucose disposal in rodents. Ac-VPAC2P-PEG has therapeutic potential for diabetes management.

The closely related peptides glucagon-like peptide (GLP-1) and glucagon have opposing effects on blood glucose. GLP-1 induces glucose-dependent insulin secretion in the pancreas, whereas glucagon stimulates gluconeogenesis and glycogenolysis in the liver. The identification of a hybrid peptide acting as both a GLP-1 agonist and a glucagon antagonist would provide a novel approach for the treatment of type 2 diabetes. Toward this end a series of hybrid peptides made up of glucagon and either GLP-1 or exendin-4, a GLP-1 agonist, was engineered. Several peptides that bind to both the GLP-1 and glucagon receptors were identified. The presence of glucagon sequence at the N terminus removed the dipeptidylpeptidase IV cleavage site and increased plasma stability compared with GLP-1. Targeted mutations were incorporated into the optimal dual-receptor binding peptide to identify a peptide with the highly novel property of functioning as both a GLP-1 receptor agonist and a glucagon receptor antagonist. To overcome the short half-life of this mutant peptide in vivo, while retaining dual GLP-1 agonist and glucagon antagonist activities, site-specific attachment of long chained polyethylene glycol (PEGylation) was pursued. PEGylation at the C terminus retained the in vitro activities of the peptide while dramatically prolonging the duration of action in vivo. Thus, we have generated a novel dual-acting peptide with potential for development as a therapeutic for type 2 diabetes.

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G(alpha)q or G(alpha)11 proteins. Here we report that murine G(alpha)11 is most abundant in fat and that expression of the constitutively active form of G(alpha)11 [G(alpha)11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G(alpha)11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G(alpha)11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G(alpha)11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways.

The GLUT4 glucose transporter resides mostly in perinuclear membranes in unstimulated 3T3-L1 adipocytes and is acutely translocated to the cell surface in response to insulin. Using a novel method to purify intracellular GLUT4-enriched membranes, we identified by mass spectrometry the intermediate filament protein vimentin and the microtubule protein alpha-tubulin as components of these membranes. Immunoelectron microscopy of the GLUT4-containing membranes also revealed their association with these cytoskeletal proteins. Disruption of intermediate filaments and microtubules in 3T3-L1 adipocytes by microinjection of a vimentin-derived peptide of the helix initiation 1A domain caused marked dispersion of perinuclear GLUT4 to peripheral regions of the cells. Inhibition of the microtubule-based motor dynein by brief cytoplasmic acidification of cultured adipocytes also dispersed perinuclear GLUT4 and inhibited insulin-stimulated GLUT4 translocation to the cell surface. Insulin sensitivity was restored as GLUT4 was again concentrated near the nucleus upon recovery of cells in physiological buffer. These data suggest that GLUT4 trafficking to perinuclear membranes of cultured adipocytes is directed by dynein and is required for optimal GLUT4 regulation by insulin.

Based on recent studies showing that phospholipase D (PLD)1 is associated with intracellular membranes and promotes membrane budding from the trans-Golgi, we tested its possible role in the membrane trafficking of GLUT4 glucose transporters. Using immunofluorescence confocal microscopy, expressed Myc epitope-tagged PLD1 was found to associate with intracellular vesicular structures by a mechanism that requires its N-terminal pleckstrin homology domain. Partial co-localization with expressed GLUT4 fused to green fluorescent protein in both 3T3-L1 adipocytes and Chinese hamster ovary cells was evident. Furthermore, microinjection of purified PLD into cultured adipocytes markedly potentiated the effect of a submaximal concentration of insulin to stimulate GLUT4 translocation to cell surface membranes. Insulin stimulated PLD activity in cells expressing high levels of insulin receptors but no such insulin effect was detected in 3T3-L1 adipocytes. Taken together, these results are consistent with the hypothesis that PLD1 associated with GLUT4-containing membranes acts in a constitutive manner to promote the mechanism of GLUT4 translocation by insulin.

The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.

Cellular levels of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) are rapidly elevated in response to activation of growth factor receptor tyrosine kinases. This polyphosphoinositide binds the pleckstrin homology (PH) domain of GRP1, a protein that also contains 200 residues with high sequence similarity to a segment of the yeast Sec7 protein that functions as an ADP ribosylation exchange factor (ARF) (Klarlund, J., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that dioctanoyl PtdIns(3,4,5)P3 binds the PH domain of GRP1 with a Kd = 0.5 microM, an affinity 2 orders of magnitude greater than dioctanoyl-PtdIns(4,5)P2. Further, the Sec7 domain of GRP1 is found to catalyze guanine nucleotide exchange of ARF1 and -5 but not ARF6. Importantly, PtdIns(3,4,5)P3, but not PtdIns(4,5)P2, markedly enhances the ARF exchange activity of GRP1 in a reaction mixture containing dimyristoylphosphatidylcholine micelles, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and a low concentration of sodium cholate. PtdIns(3,4,5)P3-mediated ARF nucleotide exchange through GRP1 is selectively blocked by 100 microM inositol 1,3,4,5-tetrakisphosphate, which also binds the PH domain of GRP1. Taken together, these data are consistent with the hypothesis that selective recruitment of GRP1 to PtdIns(3,4,5)P3 in membranes activates ARF1 and -5, known regulators of intracellular membrane trafficking.

Cellular mechanisms for regulating membrane movements appear to involve small GTPases of the Rab subfamily. Binding of GDP-bound Rab proteins to donor membranes and their release from target membranes appear to be regulated by GDP-dissociation inhibitor (GDI) protein isoforms. Previous work showed strikingly higher levels of GDI-2 than GDI-1 fractionate with total membranes of cultured cells and are visualized in the perinuclear region in 3T3-L1 adipocytes. Here we report that GDI-2-containing structural elements are concentrated predominantly in the pericentriolar area in interphase CHO-T cells and differentiated 3T3-L1 adipocytes based on colocalization of GDI-2 and the centrosomal marker pericentrin. This finding is documented by both immunofluorescence and immunoelectron microscopy. Expressed c-Myc-tagged GDI-2 in transfected COS-7 cells targets to the same region. During mitotic resolution of the centrosome into two identifiable foci in CHO-T cells, GDI-2 containing structures remain intact and also resolve into two regions surrounding the centrosome. Dissociation of pericentriolar GDI-2 from the Golgi markers beta-COP and lectin receptors was apparent upon brefeldin A treatment of 3T3-L1 adipocytes or CHO-T cells. The integrity of pericentriolar GDI-2-binding elements was not disrupted by either brief Triton X-100 extraction or microtubule cytoskeletal disassembly, achieved with nocodazole. These data demonstrate the presence of highly ordered, detergent-resistant GDI-2-specific structural elements around the centrosome and indicate functional differences for the GDI-1 and GDI-2 protein isoforms. The results suggest the presence of selective GDI-2 acceptors in this region and a possible role of pericentriolar GDI-2 in membrane recycling.

A novel method was developed to measure relative amounts of the GLUT4 glucose transporter on the surface of intact fat cells and to monitor the action of insulin on cell surface glucose transporters as they internalize into intracellular membranes. The method takes advantage of two predicted trypsin cleavage sites in the major exofacial loop of this transporter protein. Treatment of cyanide-poisoned rat adipocytes with 1 mg/ml trypsin at 37 degrees C for 30 min produced an immunoreactive GLUT4 protein species in subsequently isolated plasma membranes that migrated with higher mobility (apparent M(r) = 35,000) than native GLUT4 (apparent M(r) = 46,000) on SDS-polyacrylamide gel electrophoresis. This proteolyzed GLUT4 protein was absent in the intracellular low density microsomes. Insulin treatment of adipocytes for 20 min prior to sequential additions of cyanide and trypsin caused a 16-fold increase in the proteolytically cleaved GLUT4 species. Incubation of fresh fat cells with trypsin caused a rapid and progressive appearance of the proteolyzed GLUT4 species in the intracellular low density membranes as well as plasma membranes. After 5 min of trypsinization, 66% of the total cleaved GLUT4 in these cells had moved into the low density membranes. Insulin treatment markedly decreased the internalized cleaved GLUT4 to 20% of the total. These data indicate the following: 1) trypsinization of the GLUT4 transporter protein on intact fat cells is a convenient means to monitor the extent of transporter recruitment to the plasma membrane by insulin, as well as to estimate GLUT4 internalization rates; and 2) the action of insulin on glucose transporter redistribution to the cell surface is associated with a marked inhibition of the fraction of cell surface GLUT4 transporters internalized per unit time.

In this study we tested the hypothesis that functional erythrocyte-type glucose transporters (GLUT1) exist as oligomeric complexes by expressing chimeric transporter proteins in Chinese hamster ovary cells harboring endogenous GLUT1 transporters. The chimeric transporters were GLUT1-4c, in which the 29 C-terminal residues of human GLUT1 were replaced by the 30 C-terminal residues of rat skeletal muscle glucose transporter (GLUT4), and GLUT1n-4, containing the N-terminal 199 residues of GLUT1 and the 294 C-terminal residues of GLUT4. Endogenous GLUT1 was quantitatively co-immunoprecipitated by using an anti-GLUT4 C-terminal peptide antibody from detergent extracts of Chinese hamster ovary cells expressing either of the chimeric proteins, as detected by immunoblotting the precipitates with an anti-GLUT1 C-terminal peptide antiserum. No co-immunoprecipitation of native GLUT1 with native GLUT4 from extracts of 3T3-L1 adipocytes, which contain both these transporters, was observed with the same antibody. These data are consistent with the hypothesis that GLUT1 transporters exist as homodimers or higher order oligomers and that a major determinant of oligomerization is located within the first 199 residues of GLUT1.

Previous studies suggest that regulation of hexose uptake in Chinese hamster ovary fibroblasts can occur by alterations in glucose transporter intrinsic activity without changes in cell surface transporter number (Harrison, S. A., Buxton, J. M., Helgerson, A. L., MacDonald, R. G., Chlapowski, F. J., Carruthers, A., and Czech, M. P. (1990) J. Biol. Chem. 265, 5793-5801). We tested this hypothesis using 3T3-L1 fibroblasts and adipocytes which exhibit 5-6-fold increases in 2-deoxyglucose or 3-O-methylglucose uptake when exposed to low micromolar concentrations of cadmium for 18 h. Cadmium treatment decreased the apparent Km of 3T3-L1 fibroblasts for 3-O-methylglucose influx from approximately 28 to 9 mM and increased the apparent Vmax by 2-3-fold. These fibroblasts lack the skeletal muscle/adipocyte-type (GLUT4) transporter and showed only a small increase in total cellular immunoreactive HepG2 type (GLUT1) transporter in response to cadmium. Furthermore, cell surface GLUT1 levels did not change in 3T3-L1 fibroblasts exposed to cadmium, as assessed by the binding to intact cells of an antibody which recognizes an extracellular GLUT1 epitope. Insulin enhanced 2-deoxyglucose uptake 2-fold in 3T3-L1 fibroblasts, but did not further stimulate cadmium-activated transport rates. In contrast, insulin stimulated hexose transport 15-fold in 3T3-L1 adipocytes, which express both GLUT1 and GLUT4 proteins, and this effect was fully additive with the 5-fold effect of cadmium. Cadmium had little or no effect on immunoreactive GLUT1 or GLUT4 in isolated 3T3-L1 adipocyte plasma membranes. In contrast, insulin action led to marked recruitment (3-fold) of GLUT4 to the plasma membrane fraction in adipocytes treated with or without cadmium. Taken together, these data are consistent with the hypothesis that cadmium-activated sugar uptake is catalyzed by GLUT1, whereas insulin-stimulated sugar uptake is catalyzed predominantly by GLUT4 in 3T3-L1 adipocytes. Furthermore, the data suggest that the GLUT1 transporter can undergo significant increases in intrinsic catalytic activity in response to cadmium treatment of 3T3-L1 fibroblasts and adipocytes.

Previous studies indicated that the erythroidtype (GLUT1) glucose transporter isoform contributes to basal but not insulin-stimulated hexose transport in mouse 3T3-L1 adipocytes. In the present studies it was found that basal hexose uptake in 3T3-L1 adipocytes was about 50% lower than that in 3T3-L1 or CHO-K1 fibroblasts. Intrinsic catalytic activities of GLUT1 transporters in CHO-K1 and 3T3-L1 cells were compared by normalizing these hexose transport rates to GLUT1 content on the cell surface, as measured by two independent methods. Cell surface GLUT1 levels in 3T3-L1 fibroblasts and adipocytes were about 10- and 25-fold higher, respectively, than in CHO-K1 fibroblasts, as assessed with an anti-GLUT1 exofacial domain antiserum, delta. The large excess of cell surface GLUT1 transporters in 3T3-L1 adipocytes relative to CHO-K1 fibroblasts was confirmed by GLUT1 protein immunoblot analysis and by photoaffinity labelling (with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n) of glucose transporters in isolated plasma membranes. Thus, GLUT1 intrinsic activity is markedly reduced in 3T3-L1 fibroblasts compared with the CHO-K1 fibroblasts, and further reduction occurs upon differentiation to adipocytes. Intrinsic catalytic activities specifically associated with heterologously expressed human GLUT1 protein in transfected CHO-K1 versus 3T3-L1 cells were determined by subtracting appropriate control cell values for hexose transport and delta-antibody binding from those determined in the transfected cells expressing high levels of human GLUT1. The results confirmed a greater than 90% inhibition of the intrinsic catalytic activity of human GLUT1 transporters on the surface of mouse 3T3-L1 adipocytes relative to CHO-K1 fibroblasts. We conclude that a mechanism that markedly suppresses basal hexose transport catalyzed by GLUT1 is a major contributor to the dramatic insulin sensitivity of glucose uptake in 3T3-L1 adipocytes.

In this study, we tested the hypothesis that hexose transport regulation may involve proteins with relatively rapid turnover rates. 3T3-L1 adipocytes, which exhibit 10-fold increases in hexose transport rates within 30 min of the addition of 100 nM insulin, were utilized. Exposure of these cells to 300 microM anisomycin or 500 microM cycloheximide caused a maximal, 7-fold increase in 2-deoxyglucose transport rate after 4-8 h. The effects due to either insulin (0.5 h) or anisomycin (5 h) on the kinetics of zero-trans 3-O-methyl[14C]glucose transport were similar, resulting in 2.5-3-fold increases in apparent Vmax values (control Vmax = 1.6 +/- 0.3 x 10(-7) mmol/s/10(6) cells) coupled with approximately 2-fold decreases in apparent Km values (control Km = 23 +/- 3.3 mM). Insulin elicited the expected increases in plasma membrane levels of HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) transporters (1.6- and 2.8-fold, respectively) as determined by protein immunoblotting. In contrast, neither total cellular contents nor plasma membrane levels of these two transporter isoforms were increased when 3T3-L1 adipocytes were treated with either anisomycin or cycloheximide. 3-[125I]Iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n labeling of glucose transporters in plasma membrane fractions of similarly treated cells was also unaffected by these agents. Thus, a striking discrepancy was observed between the marked increase in cellular hexose transport rates due to these protein synthesis inhibitors and the unaltered amounts of glucose transporter proteins in the plasma membrane fraction. These data indicate that short-term protein synthesis inhibition in 3T3-L1 adipocytes leads to large increases in the intrinsic catalytic activity of one or both of the GLUT1 and GLUT4 transporter isoforms.

Complementary DNA encoding a HepG2 cell-facilitated glucose transporter (GLUT1) was subcloned into a metal-inducible, mammalian expression vector, pLEN. Mouse 3T3-L1 fibroblasts transfected with this new construct, pLENGT, exhibited zinc-inducible expression of human glucose transporter mRNA, protein, and glucose transport activity, before and after differentiation into adipocytes. Both mouse host GLUT1 and expressed human GLUT1 proteins distributed about equally between 3T3-L1 adipocyte plasma membranes and low density microsomal membranes, while host skeletal muscle/adipocyte-type glucose transporter (GLUT4) was concentrated in the latter fraction. Mouse GLUT1 and GLUT4 proteins and the constitutively expressed human GLUT1 protein in pLENGT adipocytes were all redistributed from low density microsomal membrane to plasma membrane fractions in response to insulin. Insulin stimulated 2-deoxyglucose uptake in untransfected fibroblasts about 2-fold, while untransfected adipocytes displayed a 14-fold increase in deoxyglucose uptake in response to insulin. Both the expression of human GLUT1 protein and basal 2-deoxyglucose uptake by 75 microM zinc-treated pLENGT fibroblasts and adipocytes were increased approximately 3-fold over untransfected cells. In such pLENGT fibroblasts expressing human GLUT1 protein, however, the absolute values for insulin-stimulated increases in sugar uptake were no different than in control fibroblasts. As was observed in pLENGT fibroblasts, the increased basal sugar uptake by pLENGT adipocytes was additive with the insulin-stimulated increase in the rate of sugar uptake and, therefore, the -fold stimulation by insulin was markedly reduced. These data indicate that: 1) the membrane distributions of a glucose transporter protein, which is not responsive to insulin in HepG2 cells, and both mouse GLUT1 and GLUT4 glucose transporter isoforms are regulated by insulin in mouse 3T3-L1 adipocytes, and 2) the expressed human GLUT1 appears to contribute significantly to the rate of basal uptake but not to the insulin-stimulated increase in 2-deoxyglucose uptake by 3T3-L1 fibroblasts and adipocytes.

Complementary DNA encoding a facilitative glucose transporter was isolated from a human hepatoma cell line (HepG2) cDNA library and subcloned into a metal-inducible mammalian expression vector, pLEN (California Biotechnology) containing human metallothionein gene II promoter sequences. Chinese hamster ovary (CHO) cells transfected with this transporter expression vector, pLENGT, exhibited a 2-17-fold increase in immunoreactive HepG2-type glucose transporter protein, as measured by protein immunoblotting with antipeptide antibodies directed against the HepG2-type glucose transporter C-terminal domain. Expression of the human glucose transporter was verified by protein immunoblotting with a mouse polyclonal antiserum that recognizes the human but not the rodent HepG2-type transporter. 2-Deoxy-D-glucose uptake was increased 2-7-fold in transfected cell lines. Polyclonal antisera directed against purified red blood cell glucose transporter were raised in several rabbits. Antiserum from one rabbit, delta, was found to bind to the surface of intact red cells but not to inside-out red cell ghosts. Using this delta-antiserum in intact cell-binding assays, 1.6-9-fold increases in cell surface expression of the human glucose transporter were measured in CHO-K1 cell lines transfected with the transporter expression vector. Measurements of total cellular glucose transporter immunoreactive protein using anti-HepG2 transporter C-terminal peptide serum, cell surface glucose transporter protein using delta-antiserum and 2-deoxyglucose uptake revealed proportional relationships among these parameters in transfected cell lines expressing different levels of transporter protein. Insulin increased 2-deoxyglucose uptake 40% in control CHO-K1 cells and in CHO-K1 cells expressing modest levels of the human glucose transporter protein. However, stimulation of sugar-uptake by insulin was only 10% in cells overexpressing human glucose transporter protein 9-fold, and no effect of insulin on sugar uptake was detected in several cell lines expressing very high levels (12-17-fold over controls) of human HepG2 glucose transporter protein. No insulin stimulation of anti-cell surface glucose transporter antibody binding was detected in any control or transfected CHO-K1 cell lines. These data indicate that a glucose transporter protein that is insensitive to insulin in HepG2 cells is regulated by insulin when expressed at low but not at high levels in insulin-response CHO-K1 cells. Additionally, the results suggest that insulin does not increase 2-deoxyglucose uptake by increasing the number of cell surface HepG2-type glucose transporters in CHO-K1 fibroblasts.