Isoform-selective inhibition of facilitative glucose transporters: elucidation of the molecular mechanism of HIV protease inhibitor binding.

Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design.

Effects of the HIV protease inhibitor ritonavir on GLUT4 knock-out mice.

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.

Liver regeneration is impaired in lipodystrophic fatty liver dystrophy mice.

UNLABELLED: We previously reported that mice subjected to partial hepatectomy exhibit rapid development of hypoglycemia followed by transient accumulation of fat in the early regenerating liver. We also showed that disrupting these metabolic alterations results in impaired liver regeneration. The studies reported here were undertaken to further characterize and investigate the functional importance of changes in systemic adipose metabolism during normal liver regeneration. The results showed that a systemic catabolic response is induced in each of two distinct, commonly used experimental models of liver regeneration (partial hepatectomy and carbon tetrachloride treatment), and that this response occurs in proportion to the degree of induced hepatic insufficiency. Together, these observations suggest that catabolism of systemic adipose stores may be essential for normal liver regeneration. To test this possibility, we investigated the hepatic regenerative response in fatty liver dystrophy (fld) mice, which exhibit partial lipodystrophy and have diminished peripheral adipose stores. The results showed that the development of hypoglycemia and hepatic accumulation of fat was attenuated and liver regeneration was impaired following partial hepatectomy in these animals. The fld mice also exhibited increased hepatic p21 expression and diminished plasma levels of the adipose-derived hormones adiponectin and leptin, which have each been implicated as regulators of liver regeneration. CONCLUSION: These data suggest that the hypoglycemia that develops after partial hepatectomy induces systemic lipolysis followed by accumulation of fat derived from peripheral stores in the early regenerating liver, and that these events may be essential for initiation of normal liver regeneration.

Diacylglycerol enrichment of endoplasmic reticulum or lipid droplets recruits perilipin 3/TIP47 during lipid storage and mobilization.

Fatty acid-induced triacylglycerol synthesis produces triacylglycerol droplets with a protein coat that includes perilipin 3/TIP47 and perilipin 4/S3-12. This study addresses the following two questions. Where do lipid droplets emerge, and how are their coat proteins recruited? We show that perilipin 3- and perilipin 4-coated lipid droplets emerge along the endoplasmic reticulum (ER). Blocking membrane trafficking with AlF(4)(-) during fatty acid-induced triacylglycerol synthesis drove perilipin 3 to the tubular ER. Forskolin, which like AlF(4)(-) activates adenylate cyclase, did not redistribute perilipin 3, but when added together with AlF(4)(-) perilipin 3 was recruited to lipid droplets rather than the ER. Thus inhibiting trafficking with AlF(4)(-) redistributed perilipin 3 differently under conditions of triacylglycerol synthesis (fatty acid addition) versus hydrolysis (forskolin) suggesting a shared acylglycerol-mediated mechanism. We tested whether diacylglycerol (DG), the immediate precursor of triacylglycerol and its first hydrolytic product, affects the distribution of perilipin 3. Stabilizing DG with the DG lipase inhibitor RHC80267 enhanced the perilipin 3 recruited to lipid droplets and raised DG levels in this fraction. Treating cells with a membrane-permeable DG recruited perilipin 3 to the ER. Stabilizing DG, by blocking its hydrolysis with RHC80267 or its acylation with triacsin C, enhanced recruitment of perilipin 3 to the ER. Expressing the ER enzyme DGAT1, which removes DG by converting it to triacylglycerol, attenuated perilipin 3 DG-induced ER recruitment. Membrane-permeable DG also drove perilipin 4 and 5 onto the ER. Together the data suggest that these lipid droplet proteins are recruited to DG-enriched membranes thereby linking lipid coat proteins to the metabolic state of the cell.

OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization.

Lipid droplet proteins of the PAT (perilipin, adipophilin, and TIP47) family regulate cellular neutral lipid stores. We have studied a new member of this family, PAT-1, and found that it is expressed in highly oxidative tissues. We refer to this protein as "OXPAT." Physiologic lipid loading of mouse liver by fasting enriches OXPAT in the lipid droplet tissue fraction. OXPAT resides on lipid droplets with the PAT protein adipophilin in primary cardiomyocytes. Ectopic expression of OXPAT promotes fatty acid-induced triacylglycerol accumulation, long-chain fatty acid oxidation, and mRNAs associated with oxidative metabolism. Consistent with these observations, OXPAT is induced in mouse adipose tissue, striated muscle, and liver by physiological (fasting), pathophysiological (insulin deficiency), pharmacological (peroxisome proliferator-activated receptor [PPAR] agonists), and genetic (muscle-specific PPARalpha overexpression) perturbations that increase fatty acid utilization. In humans with impaired glucose tolerance, PPARgamma agonist treatment induces adipose OXPAT mRNA. Further, adipose OXPAT mRNA negatively correlates with BMI in nondiabetic humans. Our collective data in cells, mice, and humans suggest that OXPAT is a marker for PPAR activation and fatty acid oxidation. OXPAT likely contributes to adaptive responses to the fatty acid burden that accompanies fasting, insulin deficiency, and overnutrition, responses that are defective in obesity and type 2 diabetes.

Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR.

Although the thermophilic bacterium Thermus aquaticus grows optimally at 70 degrees C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37 degrees C. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37 degrees C yet retain apparently normal activity at 68 degrees C and resistance at 95 degrees C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold-sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity.

Exenatide improves glucose homeostasis and prolongs survival in a murine model of dilated cardiomyopathy.

BACKGROUND: There is growing awareness of secondary insulin resistance and alterations in myocardial glucose utilization in congestive heart failure. Whether therapies that directly target these changes would be beneficial is unclear. We previously demonstrated that acute blockade of the insulin responsive facilitative glucose transporter GLUT4 precipitates acute decompensated heart failure in mice with advanced dilated cardiomyopathy. Our current objective was to determine whether pharmacologic enhancement of insulin sensitivity and myocardial glucose uptake preserves cardiac function and survival in the setting of primary heart failure. METHODOLOGY/PRINCIPAL FINDINGS: The GLP-1 agonist exenatide was administered twice daily to a murine model of dilated cardiomyopathy (TG9) starting at 56 days of life. TG9 mice develop congestive heart failure and secondary insulin resistance in a highly predictable manner with death by 12 weeks of age. Glucose homeostasis was assessed by measuring glucose tolerance at 8 and 10 weeks and tissue 2-deoxyglucose uptake at 75 days. Exenatide treatment improved glucose tolerance, myocardial GLUT4 expression and 2-deoxyglucose uptake, cardiac contractility, and survival over control vehicle-treated TG9 mice. Phosphorylation of AMP kinase and AKT was also increased in exenatide-treated animals. Total myocardial GLUT1 levels were not different between groups. Exenatide also abrogated the detrimental effect of the GLUT4 antagonist ritonavir on survival in TG9 mice. CONCLUSION/SIGNIFICANCE: In heart failure secondary insulin resistance is maladaptive and myocardial glucose uptake is suboptimal. An incretin-based therapy, which addresses these changes, appears beneficial.

OP9 mouse stromal cells rapidly differentiate into adipocytes: characterization of a useful new model of adipogenesis.

Much knowledge of adipocyte biology has been learned from cell culture models, most notably 3T3-L1 cells. The 3T3-L1 model has several limitations, including the requirement of 2 weeks to generate adipocytes and the waning of adipogenic potential in culture. We have investigated the capacity of OP9 cells, a line of bone marrow-derived mouse stromal cells, to recapitulate adipogenesis. When OP9 cells are given any one of three adipogenic stimuli, they rapidly accumulate triacylglycerol, assume adipocyte morphology, and express adipocyte late marker proteins, including glucose transporter 4 and adiponectin. OP9 cells can differentiate into adipocytes within 2 days. This rapid rate of differentiation allows for the detection of transiently expressed proteins in mature OP9 adipocytes. Adipogenesis in OP9 cells involves the master transcriptional regulator of adipocyte differentiation, peroxisome proliferator-activated receptor gamma (PPARgamma). OP9 cells are late preadipocytes in that, before the addition of adipogenic stimuli, they express the adipocyte proteins CCAAT/enhancer binding proteins alpha and beta, PPARgamma, sterol-regulatory element binding protein-1, S3-12, and perilipin. OP9 differentiation is not diminished by maintenance in culture at high cell density or by long periods in continuous culture, thereby facilitating the generation of stable cell lines that retain adipogenic potential. Thus, the unique features of OP9 cells will expedite the study of adipocyte biology.

S3-12, Adipophilin, and TIP47 package lipid in adipocytes.

Animals have evolved mechanisms to maintain circulating nutrient levels when energy demands exceed feeding opportunities. Mammals store most of their energy as triacylglycerol in the perilipin-coated lipid droplets of adipocytes. How newly synthesized triacylglycerol is delivered to perilipin-coated lipid droplets is poorly understood. Perilipin is a member of the evolutionarily related family of PAT proteins (Perilipin, Adipophilin, TIP47), which is defined by sequence similarity and association with lipid droplets. We previously showed that S3-12, which is also a member of this family, associates with a separate pool of lipid droplets that emerge when triacylglycerol storage is driven by adding oleate to the culture medium of adipocytes. Our current data extend these findings to demonstrate that nascent lipid droplets emerge with a coat composed of S3-12, TIP47, and adipophilin. After 100 min of oleate treatment, the nascent lipid droplets are more heterogeneous: S3-12 and TIP47 coat smaller, peripheral droplets and adipophilin coats a more medial population of droplets. Fractionation of untreated and oleate-treated adipocytes shows oleate-dependent redistribution of TIP47 and adipophilin from cytosolic fractions to the lipid droplet fraction. Inhibition of protein synthesis with cycloheximide does not block the oleate-induced formation of the nascent lipid droplets, nor does it prevent TAG accumulation. We suggest that the non-lipid droplet pools of S3-12, adipophilin, and TIP47 constitute a ready reservoir of coat proteins to permit rapid packaging of newly synthesized triacylglycerol and to maximize energy storage during nutrient excess.

Adipocyte protein S3-12 coats nascent lipid droplets.

Most animals store lipid intracellularly in protein-coated droplets. The protein coat usually contains at least one member of the PAT (perilipin, adipose differentiation-related protein, and TIP47) family. Evidence suggests that PAT proteins control access to the lipid they enclose. The protein S3-12, which has sequence similarity to the PAT proteins, was found in a screen for adipocyte-specific proteins. The adipocyte expression of S3-12 and its similarity to the PAT proteins suggest that S3-12 is involved in adipocyte lipid storage. To test this hypothesis, we supplemented 3T3-L1 adipocytes with fatty acids and assessed the distribution of S3-12 by immunofluorescence microscopy. Prior to fatty acid incubation, S3-12 was distributed diffusely throughout the cytoplasm on punctate structures of heterogeneous size. After 10 min of lipid loading, S3-12 localized to 500-nm structures concentrated at the adipocyte periphery. After longer incubations, S3-12 coated the surface of lipid droplets up to several micrometers in diameter. Initially, these droplets were distinct from those droplets surrounded by perilipin; but by 240 min, most perilipin-coated droplets had some S3-12 on the surface as well. We additionally report that the formation of S3-12-coated droplets 1) required glucose and fatty acids that can be incorporated into triacylglycerol, 2) was blocked by an inhibitor of triacylglycerol synthesis, and 3) was insulin-dependent. This study reports for the first time the early morphological events in the genesis and maturation of adipocyte lipid droplets.