Susan C. Frost

DEVELOPMENT OF INSULIN RESISTANCE IN 3T3-L1 ADIPOCYTES

Insulin resistance is a manifestation of both diabetes mellitus and obesity. However, the mechanism is still not clearly identified. Herein, we describe a procedure that allows us to evaluate the development of insulin resistance in 3T3-L1 adipocytes. Under these conditions, we show that the concentration of insulin required for 50% desensitization of glucose transport activity is 100 pM; maximal desensitization could be achieved with 1 nM. This demonstrates for the first time that 3T3-L1 adipocytes develop insulin resistance in response to physiologically relevant concentrations of insulin. Glucose (or glucosamine), in addition to insulin, was required to establish desensitization. The expression of GLUT4 protein decreased by 50% with exposure to 10 nM insulin. The dose-dependent loss of GLUT4 was similar to the dose dependence for insulin-resistant transport activity. Translocation in the presence of acute insulin was apparent, but the extent of recruitment directly reflected the decrease in GLUT4 protein. GLUT4 mRNA also declined, but the ED50 was approximately 5 nM. Together, these data suggest that the loss of GLUT4 protein likely underlies the cause of desensitization. However, the loss of GLUT4 protein did not correlate with the loss in GLUT4 mRNA suggesting post-translational control of GLUT4 expression.

Carbonic anhydrase: mechanism, regulation, links to disease, and industrial applications

Preface. Part I Introduction. 1 Overview of the Carbonic Anhydrase Field. Part II Carbonic Anhydrases: Ancient but Relevant. Physiological Functions of the Alpha Class of Carbonic Anhydrases. 3 Catalytic Mechanism of alpha-Class Carbonic Anhydrases: CO2 Hydration and Proton Transfer. 4 Structure and Catalytic Mechanism of b-Carbonic Anhydrases. 5 Prokaryotic Carbonic Anhydrases of Earths Environment. 6 Carboxysomal Carbonic Anhydrases. 7 Carbonic Anhydrases and their Interplay with Acid/base-coupled Membrane Transporters. 8 Carbonic Anhydrase Related Proteins: Molecular Biology and Evolution. 9 Membrane Associated Carbonic Anhydrase IV (CA IV): A Personal and Historical Perspective. 10 Carbonic Anhydrase Expression in Kidney and Renal Cancer: Implications for Diagnosis and Treatment. 11 Carbonic Anhydrase IX: Regulation and Role in Cancer. 12 Carbonic Anhydrase IX as an Imaging and Therapeutic Target for Tumors and Metastases. 13 Carbonic Anhydrase IX (CAIX) as a Mediator of Hypoxia-induced Stress Response in Cancer Cells. 14 Carbonic Anhydrases and Brain pH in the Control of Neruronal Excitability. 15 Carbonic Anhydrase Inhibitors: Drug Design. 16 Natural Products that Inhibit Carbonic Anhydrases. 17 Glaucoma and the Application of Carbonic Anhydrase Inhibitors. 18 Carbonic Anhydrase Inhibitors and High Altitude Illnesses. 19 Thermal-Stable Carbonic Anhydrases: A Structural Overview. 20 Carbonic Anhydrases in Industrial Applications. Index.

Effect of Alternative Glycosylation on Insulin Receptor Processing

The mature insulin receptor is a cell surface heterotetrameric glycoprotein composed of two α- and two β-subunits. In 3T3-L1 adipocytes as in other cell types, the receptor is synthesized as a single polypeptide consisting of uncleaved α- and β-subunits, migrating as a 190-kDa glycoprotein. To examine the importance of N-linked glycosylation on insulin receptor processing, we have used glucose deprivation as a tool to alter protein glycosylation. Western blot analysis shows that glucose deprivation led to a time-dependent accumulation of an alternative proreceptor of 170 kDa in a subcellular fraction consistent with endoplasmic reticulum localization. Co-precipitation assays provide evidence that the alternative proreceptor bound GRP78, an endoplasmic reticulum molecular chaperone. N-Glycosidase F treatment shows that the alternative proreceptor contained N-linked oligosaccharides. Yet, endoglycosidase H insensitivity indicates an aberrant oligosaccharide structure. Using pulse-chase methodology, we show that the synthetic rate was similar between the normal and alternative proreceptor. However, the normal proreceptor was processed into α- and β-subunits (t 1 2 = 1.3 ± 0.6 h), while the alternative proreceptor was degraded (t 1 2 = 5.1 ± 0.6 h). Upon refeeding cells that were initially deprived of glucose, the alternative proreceptor was processed to a higher molecular weight form and gained sensitivity to endoglycosidase H. This “intermediate” form of the proreceptor was also degraded, although a small fraction escaped degradation, resulting in cleavage to the α- and β-subunits. These data provide evidence for the first time that glucose deprivation leads to the accumulation of an alternative proreceptor, which can be post-translationally glycosylated with the readdition of glucose inducing both accelerated degradation and maturation.

Translocation of GLUT1 Does Not Account for Elevated Glucose Transport in Glucose-deprived 3T3-L1 Adipocytes

Glucose deprivation increases the rate of glucose transport in 3T3-L1 adipocytes in a protein synthesis-dependent fashion. To determine if translocation of either GLUT1 or GLUT4 is responsible for this phenomenon, we adapted existing fractionation procedures toward isolating 3T3-L1 adipocyte membranes. By Western blot analysis of equal protein, GLUT1 was distributed between plasma membranes, high density “microsomal” membranes, and low density “microsomal” membranes isolated from control cells. GLUT4 comigrated with high density and low density membranes. Glucose deprivation for 12 h did not alter the distribution of either GLUT1 or GLUT4, despite an 8-10-fold increase in glucose transport activity in intact cells. Importantly, increased transport activity was retained in plasma membrane vesicles isolated from glucose-deprived cells. These data show for the first time that the increase in transport activity associated with glucose deprivation does not result from the translocation of either of the glucose transporters known to exist in 3T3-L1 adipocytes. As GLUT4 is excluded from the plasma membrane, these data provide evidence for activation of GLUT1.

Expression and activity of carbonic anhydrase IX is associated with metabolic dysfunction in MDA-MB-231 breast cancer cells.

The expression of carbonic anhydrase IX (CAIX), a marker for hypoxic tumors, is correlated with poor prognosis in breast cancer patients. We show herein that the MDA-MB-231 cells, a “triple-negative,” basal B line, express exclusively CAIX, while a luminal cell line (T47D) expresses carbonic anhydrase XII (CAXII). CAIX expression in the basal B cells is both density- and hypoxia-dependent and is correlated with carbonic anhydrase activity. Evidence is provided that CAIX contributes to extracellular acidification through studies on pH, lactic acid production, and CAIX inhibition. Together, these studies suggest that CAIX expression and activity is associated with metabolic dysfunction in MDA-MB-231 cells.

Antibody-specific detection of CAIX in breast and prostate cancers.

Carbonic anhydrase IX (CAIX) is frequently expressed in human tumors and serves as a marker for hypoxia. Further, CAIX expression is considered a predictor of poor survival in many, but not all, cancer types. Herein, we compare the specificity of two CAIX antibodies: the M75, monoclonal antibody which recognizes an epitope in the N-terminus and a commercially available polyclonal antibody generated against a C-terminal peptide (NB100-417). Western blot analysis of multiple breast cell lines revealed that the polyclonal antibody detected both membrane-bound and soluble proteins. The M75 antibody recognized only the membrane-bound species, which is presumed to be CAIX. These data were confirmed in an aggressive prostate cell line. We further compared these antibodies in prostate tumors by immunohistochemistry. Staining with NB100 was comparable to that of the M75 antibody, but only at high dilution. Otherwise, cytoplasmic staining was also noted. Two-dimensional gel electrophoresis followed by mass spectrometric analysis revealed that the cytoplasmic protein detected by NB100 is beta-tubulin. This cross-reactivity could lead to false-positives for CAIX expression in samples where cytosolic proteins are present.

Physiological Functions of the Alpha Class of Carbonic Anhydrases

Carbonic anhydrases are ubiquitous enzymes that catalyze the reversible hydration of carbon dioxide. These enzymes are of ancient origin as they are found in the deepest of branches of the evolutionary tree. Of the five different classes of carbonic anhydrases, the alpha class has perhaps received the most attention because of its role in human pathology. This review focuses on the physiological function of this class of carbonic anhydrases organized by their cellular location.

Metabolic effects of different protein intakes after short term undernutrition in artificially reared infant rats.

BACKGROUND Early postnatal nutrition is involved in metabolic programming. Small for gestational age and premature babies commonly receive insufficient dietary protein during the neonatal period due to nutrition intolerance, whereas high protein formulas are used to achieve catch up growth. Neither the short term, nor the long term effects of such manipulation of protein intake are known. AIM We hypothesized that high or low protein intake during infancy would induce metabolic alterations both during early-life and in adulthood. METHODS Gastrostomized neonatal rat pups received either 50% (P50%), 100% (P100%), or 130% (P130%) of the normal protein content in rat milk from the 7th to the 15th day of life (D7 to D15), when they were either sacrificed or placed with mothers for the long term study. Glucose tolerance tests (GTT) were performed at D230. Long term rats were sacrificed at D250. RESULTS At D15, weight of P50% pups was lower than P100% and P130% pups. Neither liver and kidney mass, nor islet beta-cell areas were altered. Brain weight (adjusted to body weight) was higher in P50% vs. P130% (p<0.05). Insulin/glucose ratio was lower in P50% vs. P130%. Expression of GLUT4 on adipocyte cell membrane and GLUT2 in liver cytosol was significantly enhanced in P50% vs. P130%. Long term, neither GTT results nor body nor organ weights differed between groups. CONCLUSION In neonatal rats, higher protein intakes via the enteral route led to enhanced short term weight gain, insulin resistance, and modified expression of glucose transporters. However, these differences were not sustained.

Catalysis and pH Control by Membrane-associated Carbonic Anhydrase IX in MDA-MB-231 Breast Cancer Cells

Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme whose expression is often considered a marker for hypoxia, an indicator of poor prognosis in the majority of cancer patients, and is associated with acidification of the tumor microenvironment. Here, we describe for the first time the catalytic properties of native CAIX in MDA-MB-231 breast cancer cells that exhibit hypoxia-inducible CAIX expression. Using 18O exchange measured by membrane inlet mass spectrometry, we determined catalytic activity in membrane ghosts and intact cells. Exofacial carbonic anhydrase activity increases with exposure to hypoxia, an activity which is suppressed by impermeant sulfonamide CA inhibitors. Inhibition by sulfonamide inhibitors is not sensitive to reoxygenation. CAIX activity in intact cells increases in response to reduced pH. Data from membrane ghosts show that the increase in activity at reduced pH is largely due to an increase in the dehydration reaction. In addition, the kinetic constants of CAIX in membrane ghosts are very similar to our previous measurements for purified, recombinant, truncated forms. Hence, the activity of CAIX is not affected by the proteoglycan extension or membrane environment. These activities were measured at a total concentration for all CO2 species at 25 mm and close to chemical equilibrium, conditions which approximate the physiological extracellular environment. Our data suggest that CAIX is particularly well suited to maintain the extracellular pH at a value that favors the survival fitness of tumor cells.

The promise of nanotechnology for solving clinical problems in breast cancer

Approaches for breast cancer treatment are invasive, disfiguring, have significant side‐effects, and are not always curative. Nanotechnology is an emerging area which is focused on engineering of materials <100 × 10−9 m. There is significant promise for advancing nanotechnology to improve breast cancer diagnosis and treatment including non‐invasive therapy, monitoring response to therapy, advanced imaging, treatment of metastatic disease, and improved nodal staging. Current approaches and important future directions are discussed. J. Surg. Oncol. 2011; 103:317–325.