Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic α- and β-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase β-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify α-, β-, and δ-cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the subpopulations by flow cytometry, and, using next-generation RNA sequencing, we report the detailed transcriptomes of fetal and adult α- and β-cells. We observed that human islet composition was not influenced by age, sex, or BMI, and transcripts for inflammatory gene products were noted in fetal β-cells. In addition, within highly purified adult glucagon-expressing α-cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet α- and β-cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes.

The facilitated diffusion of glucose, galactose, fructose, urate, myoinositol, and dehydroascorbic acid in mammals is catalyzed by a family of 14 monosaccharide transport proteins called GLUTs. These transporters may be divided into three classes according to sequence similarity and function/substrate specificity. GLUT1 appears to be highly expressed in glycolytically active cells and has been coopted in vitamin C auxotrophs to maintain the redox state of the blood through transport of dehydroascorbate. Several GLUTs are definitive glucose/galactose transporters, GLUT2 and GLUT5 are physiologically important fructose transporters, GLUT9 appears to be a urate transporter while GLUT13 is a proton/myoinositol cotransporter. The physiologic substrates of some GLUTs remain to be established. The GLUTs are expressed in a tissue specific manner where affinity, specificity, and capacity for substrate transport are paramount for tissue function. Although great strides have been made in characterizing GLUT‐catalyzed monosaccharide transport and mapping GLUT membrane topography and determinants of substrate specificity, a unifying model for GLUT structure and function remains elusive. The GLUTs play a major role in carbohydrate homeostasis and the redistribution of sugar‐derived carbons among the various organ systems. This is accomplished through a multiplicity of GLUT‐dependent glucose sensing and effector mechanisms that regulate monosaccharide ingestion, absorption, distribution, cellular transport and metabolism, and recovery/retention. Glucose transport and metabolism have coevolved in mammals to support cerebral glucose utilization. © 2012 American Physiological Society. Compr Physiol 2:863‐914, 2012.

The dolichol-linked oligosaccharide Glc3Man9GlcNAc2-PP-Dol is the in vivo donor substrate synthesized by most eukaryotes for asparagine-linked glycosylation. However, many protist organisms assemble dolichol-linked oligosaccharides that lack glucose residues. We have compared donor substrate utilization by the oligosaccharyltransferase (OST) from Trypanosoma cruzi, Entamoeba histolytica, Trichomonas vaginalis, Cryptococcus neoformans, and Saccharomyces cerevisiae using structurally homogeneous dolichol-linked oligosaccharides as well as a heterogeneous dolichol-linked oligosaccharide library. Our results demonstrate that the OST from diverse organisms utilizes the in vivo oligo saccharide donor in preference to certain larger and/or smaller oligosaccharide donors. Steady-state enzyme kinetic experiments reveal that the binding affinity of the tripeptide acceptor for the protist OST complex is influenced by the structure of the oligosaccharide donor. This rudimentary donor substrate selection mechanism has been refined in fungi and vertebrate organisms by the addition of a second, regulatory dolichol-linked oligosaccharide binding site, the presence of which correlates with acquisition of the SWP1/ribophorin II subunit of the OST complex.

GLUT1-catalyzed equilibrative sugar transport across the mammalian blood-brain barrier is stimulated during acute and chronic metabolic stress; however, the mechanism of acute transport regulation is unknown. We have examined acute sugar transport regulation in the murine brain microvasculature endothelial cell line bEnd.3. Acute cellular metabolic stress was induced by glucose depletion, by potassium cyanide, or by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which reduce or deplete intracellular ATP within 15 min. This results in a 1.7-7-fold increase in V max for zero-trans 3-Omethylglucose uptake (sugar uptake into sugar-free cells) and a 3-10-fold increase in V max for equilibrium exchange transport (intracellular [sugar] ‫؍‬ extracellular [sugar]). GLUT1, GLUT8, and GLUT9 mRNAs are detected in bEnd.3 cells where GLUT1 mRNA levels are 33-fold greater than levels of GLUT8 or GLUT9 mRNA. Neither GLUT1 mRNA nor total protein levels are affected by acute metabolic stress. Cell surface biotinylation reveals that plasma membrane GLUT1 levels are increased 2-3-fold by metabolic depletion, although cell surface Na ؉ ,K ؉ -ATPase levels remain unaffected by ATP depletion. Treatment with the AMP-activated kinase agonist, AICAR, increases V max for net 3-O-methylglucose uptake by 2-fold. Glucose depletion and treatment with potassium cyanide, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and AICAR also increase AMP-dependent kinase phosphorylation in bEnd.3 cells. These results suggest that metabolic stress rapidly stimulates blood-brain barrier endothelial cell sugar transport by acute upregulation of plasma membrane GLUT1 levels, possibly involving AMP-activated kinase activity.The cells of the mammalian brain do not contain large stores of glycogen. It is therefore essential that glucose uptake by the brain exceeds glucose utilization to maintain proper brain function. To enter the brain, serum glucose must cross the bloodbrain barrier, an epithelium comprising endothelial cells connected by tight junctions that prevent paracellular diffusion of glucose and other nutrients. Thus, glucose transport into the brain requires trans-endothelial cell transport. This process is catalyzed by the glucose transport protein GLUT1, which is expressed at both luminal and abluminal membranes of the endothelium (1-5).Endothelial cells of the blood-brain barrier (bEND) 2 differ from those of the peripheral circulatory system (pEND) in several important ways. 1) bEND cells contain 2-5-fold more mitochondria than pEND cells (6). 2) Brain capillary walls (comprising bEND cells) are 40% thinner than capillary walls of the peripheral circulation (7). 3) pEND cells present significantly fewer tight junctions than bEND cells (8). 4) bEND cell tight junction complexes result in polarized cell surface protein expression that is less marked or absent in pEND cells (8). The resulting bEND cell architecture may give rise to behaviors that differ from those of pEND cells but that resemble those of other metabolically active cells a...

Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.