Exam 2: Biochemistry

Which of the following regulatory mechanisms helps increase net glucose catabolism in the liver after a meal?

Passage The liver plays a central role in maintaining blood glucose homoeostasis.  It contains the highest concentration of glycogen of any organ in the body and is one of few organs that can regenerate glucose from metabolic byproducts.  The liver can replenish glucose in muscle and brain tissue during times of fasting, and it can help prevent lactate accumulation during intense physical activity.Von Gierke disease is a rare autosomal recessive disorder that arises from inactivating mutations in the liver enzyme glucose 6-phosphatase (G6Pase).  It affects roughly 1 in 100,000 individuals, and the resulting loss of both hormonal sensitivity and enzymatic activity leads to hypoglycemia (low blood sugar).  Von Gierke can also cause an increase in blood acidity concurrent with lactate build-up.  Patients typically become symptomatic shortly after birth with convulsions, hyperventilation, and tremors.  Left untreated, patients may develop gout, osteoporosis, and life-threatening complications such as kidney failure and liver tumors.In an effort to examine the biochemical consequences of this disorder, scientists generated a genetically engineered mouse model with liver G6Pase expression under the control of the Tet-Off system at both alleles.  Under this system, addition of the small molecule tetracycline in the water source of the mouse rapidly shuts off G6Pase gene expression and therefore can recapitulate von Gierke disease.  Blood glucose and pH levels were measured daily in two groups of genetically engineered mice over 20 days: group A mice received no tetracycline and group B mice were given tetracycline starting at day 10.  All mice were fed identical low-sugar diets throughout the experiment. Adapted from Froissart R, Piraud M, Boudjemline AM, et al. Glucose-6-phosphatase deficiency. Orphanet J Rare Dis. 2011;6:27. -Inactivation of which enzyme is most likely to result in physiological effects similar to those of von Gierke disease?

Passage Glycolysis and gluconeogenesis are tightly regulated, opposing metabolic pathways that help control blood glucose levels.  Glycolysis converts glucose to two pyruvate molecules, whereas gluconeogenesis consumes 6 ATP equivalents to convert two pyruvate molecules back to glucose.  When glycolysis is upregulated, gluconeogenesis is downregulated, and vice versa.As shown in Figure 1, glycolysis and gluconeogenesis in the liver are largely regulated by the allosteric action of the small molecule fructose-2,6-bisphosphate (F2,6BP) on the enzymes phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F1,6BPase).  PFK-1 is a kinase that uses ATP to phosphorylate fructose-6-phosphate (F6P) in an irreversible step of glycolysis, forming fructose-1,6-bisphosphate (F1,6BP) and ADP.  During gluconeogenesis, F1,6BPase removes the phosphate group by hydrolysis. Figure 1  Activities of (A) PFK-1 and (B) F1,6BPase in the presence (solid lines) and absence (dashed lines) of F2,6BPA bifunctional enzyme that contains a phosphofructokinase-2 (PFK-2) domain and a fructose-2,6-bisphosphatase (F2,6BPase) domain controls F2,6BP levels in the liver.  The PFK-2 domain converts F6P to F2,6BP, and the F2,6BPase domain converts F2,6BP back to F6P.  When blood glucose levels are low, the enzyme becomes phosphorylated.  This phosphorylation event simultaneously activates the F2,6BPase domain and inactivates the PFK-2 domain.  Under high blood glucose conditions, the enzyme becomes dephosphorylated, activating the PFK-2 domain and inactivating the F2,6BPase domain. -If one glucose molecule is converted to pyruvate in the muscle and then the resulting pyruvate molecules are converted back to glucose in the liver, what is the net number of ATP equivalents consumed?

Passage Oxidative stress occurs in cells when reactive oxygen species (ROS) such as H₂O₂, superoxide (O₂⁻), and hydroxyl radicals (^•OH) interact destructively with biological molecules.  Several studies have indicated that histidine and certain peptides that contain histidine can decrease oxidative stress.A common marker of oxidative stress in cells is leakage of the enzyme lactate dehydrogenase (LDH) into the extracellular space.  Researchers incubated cultured rat cells (PC12 cells) with H₂O₂ in the presence of different tripeptides, each containing one alanine, one glycine, and one histidine residue.  Every possible sequence was tested, but the researchers found that the sequence GAH was the most effective in limiting extracellular LDH activity.  After obtaining these preliminary results, the following experiments were conducted.Experiment 1PC12 cells were incubated with or without 5 mM H₂O₂ and with varying concentrations of GAH for 4 hours.  Cells were centrifuged, and the supernatant (ie, extracellular material) was extracted and assayed for LDH activity (Figure 1). Figure 1  Extracellular LDH activity from PC12 cells exposed to H₂O₂ and varying concentrations of GAH.  p-values are relative to 5 mM H₂O₂ and 0 mg/mL GAH.Experiment 2LDH was purified and assayed for activity in the presence of 5 mM H₂O₂ and varying concentrations of GAH (Figure 2). Figure 2  Activity of purified LDH with varying concentrations of GAHExperiment 3PC12 cells were incubated with or without 5 mM H₂O₂ in the presence and absence of 1 mg/mL GAH.  Cells were then stained with the fluorescent marker TUNEL, which binds strongly to apoptotic cells.  The percentage of apoptotic cells in each condition is shown in Figure 3. Figure 3  Percentage of apoptotic cells in the presence or absence of H₂O₂ and GAH -The purpose of measuring purified LDH activity with various amounts of GAH was most likely:

During glycolysis, phosphofructokinase converts fructose 6-phosphate (F6P) into fructose 1,6-bisphosphate (F1,6BP), as shown below.  Aldolase then converts F1,6BP into glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Respectively, phosphofructokinase and aldolase are examples of:

An enzyme was assayed in the absence (−) or presence (+) of an allosteric effector, and the following Lineweaver-Burk plot was obtained: Which statement about the allosteric effector is correct?

Researchers quantified the amount of protein produced by cultured cells during a one-hour period by detecting the radioisotope ³⁵S.  Prior to this period, the cells were most likely provided with radiolabeled:

Passage Mitochondria are double membrane-bound cellular organelles that house several connected metabolic reactions.  The citric acid cycle takes place inside the mitochondrial matrix and produces two reduced electron carriers, NADH and FADH₂.  These two carriers then pass their electrons to ubiquinone (UQ), which is reduced to ubiquinol (UQH₂).  UQH₂ passes its electrons to oxidized cytochrome C (cyt-C_ox), regenerating UQ and forming reduced cytochrome C (cyt-C_red).  Finally, cyt-C_red transfers electrons to oxygen, regenerating cyt-C_ox and forming water.  Each reaction is facilitated by one of four protein complexes that are collectively known as the electron transport chain (ETC).Researchers isolated mitochondria and treated them with a novel ETC inhibitor, "drug X."  After an overnight incubation, they measured levels of several ETC-reduced electron carriers in the presence of drug X relative to untreated mitochondria.  Their results are shown in Figure 1. Figure 1  Relative levels of reduced electron carriers in the presence and absence of drug XNext, the researchers exposed mitochondria to FCCP, a chemical that can transport protons across membranes.  They observed that in the presence of FCCP, the citric acid cycle was fully active, and all reduced cofactors were present at the same levels as in untreated mitochondria.  However, the rate of ATP synthesis was significantly reduced.  ATP synthesis was immediately restored when FCCP was washed away. -Which of the following molecules is a precursor to the citric acid cycle?

Passage The tumor suppressor p53 is a homotetrameric protein with an N-terminal transactivation domain (TAD), a proline-rich domain, a DNA-binding domain (DBD), a tetramerization domain (TD), and a C-terminal regulatory domain (REG) on each subunit (Figure 1). Figure 1  Organization of the p53 domains (1-393 amino acids)Human double minute 2 (Hdm2) is a lysine-specific ubiquitin protein ligase that can change the half-life of some proteins, including p53, by signaling for their proteolytic cleavage.  Under normal circumstances, Hdm2 binds p53 at the AD1 region within the TAD and attaches ubiquitin to a lysine residue.  However, errors in DNA replication induce the phosphorylation of residue T18 on p53, which inhibits p53's association with Hdm2.  The overexpression of Hdm2 has been correlated with the downregulation of tumor suppressor p53 in certain types of cancers.  Changes in the NMR chemical shift (ppm) of amino acids in the AD1 region shown in Figure 2 provide evidence for the formation of a p53-Hdm2 complex. Figure 2  ¹H NMR spectrum of the AD1 region in ¹⁵N-labeled p53 during G₀ and G₂ phases of the cell cycleHdm2 also binds to Nedd8 ultimate buster 1 (NUB1), which enhances signaling for the degradation of Nedd8 proteins.  Preliminary data suggest that Hdm2 interacts differently with wild-type NUB1 (WTNUB1) than with mutant K159RNUB1.  To assess the effect of Hdm2 on NUB1 and p53, the half-lives of p53 and Nedd8 protein variants were compared in cells that overexpress Hdm2 (Figure 3). Figure 3  Half-lives of p53, phosphorylated p53 (p53-PO₄), and Nedd-8 proteins in cells with wild-type or mutant NUB1 over time Adapted from Ferreon JC, Lee CW, Arai M, Martinez-yamout MA, Dyson HJ, Wright PE. Cooperative regulation of p53 by modulation of ternary complex formation with CBP/p300 and HDM2. Proc Natl Acad Sci USA. 2009;106(16):6591-6. -Based on the data in Figure 2, which statement best supports the formation of a p53-Hdm2 complex in the absence of DNA errors?

Passage Porphyrin biosynthesis is a multistep process that is required for production of porphyrin derivatives such as heme, which is a crucial component of hemoglobin, myoglobin, and the cytochrome cofactors of the electron transport chain.  In the first step of this pathway, the enzyme porphobilinogen synthase catalyzes the production of porphobilinogen (PBGN) from two δ-aminolevulinate molecules, as shown in Reaction 1. Reaction 1In the next step, the monomeric enzyme hydroxymethylbilane synthase (HMBS) combines four PBGN molecules to form hydroxymethylbilane (Reaction 2).  Several additional steps are required to convert hydroxymethylbilane to heme. Reaction 2Defects in HMBS activity can lead to accumulation of δ-aminolevulinate and porphobilinogen in the liver and blood plasma.  The accumulation of these molecules can cause acute intermittent porphyria, a disorder characterized by episodes of severe abdominal pain.  Researchers expressed, purified, and assessed several variants of HMBS for enzymatic activity (Table 1) and thermal stability (Figure 1).Table 1  Michaelis-Menten equations generated from several HMBS variants, with V₀ and V_max measured in nmol/hr and [PBGN] and K_m measured in μM.  (Note: All reactions were carried out with 1 μM HMBS.) Figure 1  Thermal stability of several HMBS variants -Which HMBS mutant variant is LEAST likely to cause acute intermittent porphyria?

Passage Aldehyde dehydrogenase 2 (ALDH2) is essential for the oxidation of toxic aldehydes, and uses NAD⁺ as the oxidizing agent.  Mitochondrial ALDH2 exists as a 220-kD homotetrameric protein containing a catalytic, nucleotide-binding, and coenzyme-binding domain.  The most prevalent ALDH2 mutation (ALDH2*2) involves an E to K (E478K) substitution in the catalytic domain that leads to decreased enzymatic activity.Scientists hypothesize that Asian populations have higher rates of ALDH2*2 incidence.  The properties of ALDH2 variants in Caucasian and Japanese populations were analyzed in the following experiments.Experiment 1Mitochondrial extracts from both populations were combined in a single mixture and separated by native gel electrophoresis.  Proteins 210-230 kD in size were removed from the agarose gel and further isolated using affinity columns with antibodies designed to separate wild-type ALDH2 from ALDH2*2 by binding the catalytic domain of the wild-type but not the mutant protein.  Concentrated eluates E1 and E2 were produced by washing the affinity columns with buffers that remove unbound proteins and dislodge bound proteins from antibodies, respectively (Figure 1). Figure 1  Purification of ALDH2 proteinsALDH2 activity in each eluate was quantified by adding acetaldehyde and NAD⁺, and then monitoring the rate of NADH production.  The results are shown in Figure 2. Figure 2  Enzymatic activity of E1 and E2 eluatesExperiment 2Researchers predict that mutations at conserved amino acid sites containing S or T may prevent ALDH2 from being phosphorylated by protein kinase C epsilon (εPKC).  The activity of wild-type ALDH2 (WT) and ALDH2 mutants with substitutions at positions T185 and S279 in the presence or absence of εPKC are summarized in Table 1.Table 1  Effect of εPKC on ALDH2 Variants Adapted from Chen CH, Ferreira JC, Gross ER, Mochly-rosen D. Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiol Rev. 2014;94(1):1-34. -The method used to phosphorylate ALDH2 in Experiment 2 must include εPKC and which other component?

The physical properties of two isoforms of a protein, both of which bind the same ligand, are shown in the table: Which protocol would yield the best separation of the two isoforms?

Passage DNA polymerization is one of the most conserved mechanisms of genome replication.  Synthesis of a complete DNA strand requires a template, primers, a polymerase enzyme, and sufficient deoxyribonucleotide triphosphates (dNTPs).  The DNA polymerase enzyme binds consecutive base pairs on the template strand and extends the double helix by adding dNTPs to the primer.  The amino acid residues in the active site of DNA polymerase form hydrogen bonds with Watson-Crick donors and acceptors on incoming DNA nucleotides to facilitate base pairing.The formation of the DNA double helix creates opposing changes in entropy and enthalpy.  Favorable bonding interactions via hydrogen bonds during Watson-Crick base pairing results in negative enthalpy, and restricted rotation and flexibility of the DNA backbone generates negative entropy.  Scientists hypothesize that hydrogen bonding between bases not only stabilizes the double helix but is also crucial for selective and efficient replication.Analogs that are similar in size and shape to naturally occurring bases can be used to determine the influence of hydrogen bonding on base pair selectivity.  To mimic the structure of deoxythymidine triphosphate (dTTP), researchers synthesized dNTP derivatives of difluorotoluene (dFTP), a nonpolar analog that lacks Watson-Crick hydrogen bonding.  Klenow fragment (KF) polymerase, which has 3′-5′ but not 5′-3′ exonuclease activity, was incubated with a mixture of DNA template, primers, and dNTPs, including dFTP derivatives.  The efficiency of dFTP and natural dTTP nucleotide incorporation into a growing primer strand by KF is shown in Figure 1. Figure 1  Template-specific selection of dFTP and dTTP by the KF enzyme Adapted from Moran S, Ren RX, Kool ET. A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity. Proc Natl Acad Sci USA. 1997;94(20):10506-11. -In the DNA polymerase active site, which nucleotide base would provide the most Watson-Crick donors for interactions with amino acid residues?

Passage ATP synthase, also known as Complex V, produces the majority of the ATP in eukaryotic cells.  It is a transmembrane protein encoded by both nuclear and mitochondrial genes in humans, and is found in the inner mitochondrial membrane.  Complex V consists of over 20 subunits, including three sets of heterodimers arranged in a circle in the mitochondrial matrix.  Each dimer contains an α subunit and a β subunit, and each β subunit exists in one of three conformations: one that binds ATP, one that binds ADP and inorganic phosphate, and one that binds neither.As protons flow from the intermembrane space through Complex V and into the matrix, they cause the transmembrane γ subunit to rotate and interact with each β subunit in turn.  Four protons provide enough energy for rotation from one β subunit to the next, and each rotation causes a β subunit to release a new ATP molecule as it changes from the ATP-binding to the nonbinding conformation (Figure 1).  Thus 12 protons produce 3 ATP molecules each time the γ subunit makes a complete revolution.  Every NADH molecule that enters the electron transport chain (ETC) pumps 10 protons into the intermembrane space for use by Complex V whereas only 6 protons are pumped per FADH₂ molecule. Figure 1  Depictions of ATP synthase viewed from the side (left) and the bottom (right)Researchers visualized the rotation of the γ subunit by attaching it to a fluorescently labeled actin filament.  The observed rotation over time is shown in Figure 2. Figure 2  Counterclockwise rotation of the γ subunit of ATP synthase (bottom view) over time in seconds as visualized by a fluorescent labelAn L156P mutation in subunit A of Complex V is linked to neuropathy, ataxia, and retinal degeneration.  Although the exact molecular bases of these pathological states are unknown, the following hypotheses are currently under study:Hypothesis 1L156P Complex V functions normally but is less abundant than wild-type Complex V.Hypothesis 2The mutation partially decouples proton flow from ATP synthesis.Hypothesis 3The mutation leads to increased oxidative stress from the ETC, resulting in apoptosis. Adapted from Kucharczyk R, Zick M, Bietenhader M, et al. Mitochondrial ATP synthase disorders: molecular mechanisms and the quest for curative therapeutic approaches. Biochim Biophys Acta. 2009;1793(1):186-99. -A decrease in the observed rate of ATP synthase rotation in mitochondria isolated from L156P cells relative to that observed in mitochondria isolated from wild-type cells would support which of the hypotheses given in the passage?

Passage Antimicrobial peptides are an important component of innate immunity.  Since their discovery in 1939, over 5,000 peptides with antimicrobial properties have been isolated from organisms in every domain of life.  They have demonstrated activity against enveloped viruses, bacteria, fungi, and even tumor cells and have recently gained interest for their potential in fighting antibiotic-resistant bacteria.  Most known antimicrobial peptides are positively charged, but some with negative charges have also been discovered.Frogs are known to secrete a variety of antimicrobial peptides as a major source of immunity.  Secretions from a new species of frog were collected and the peptides were separated by cation exchange chromatography.  Four of the isolated peptides demonstrated strong antibacterial activity and were characterized by isoelectric focusing and sequencing: Figure 1  Results of isoelectric focusing of four antimicrobial peptidesTable 1  Amino Acid Compositions of Four Antimicrobial Peptides Adapted from Wang Z, Wang G. APD: the Antimicrobial Peptide Database. Nucleic Acids Res. 2004;32(Database issue):D590-2. -Some strains of Staphylococcus aureus modify their membranes by attaching lysine to phospholipids on the extracellular side.  Given this information, which of the peptides tested is likely to be most effective against these strains?

Studies on the catalytic activity of hexokinase provided the following data: Which experiment would contain the greatest percentage of folded protein?

Passage The Ankyrin repeat and SOCS box containing protein 4 (Asb-4) is highly expressed in the cytosol of hypothalamus cells during the well-fed state but is downregulated during fasting.  In addition, the protein insulin receptor substrate 4 (IRS4), known to be involved in appetite regulation, is expressed in the cytosol of the same cells as Asb-4.  Accordingly, it was hypothesized that Asb-4 suppresses appetite through interactions with IRS4.Small peptides such as myc (EQKLISEEDL) and flag (DYKDDDDK) are often attached to recombinant proteins to facilitate their purification and detection.  HEK293 cells were transfected with plasmids encoding Asb-4 and IRS4 tagged with myc or flag at their N-termini (myc-Asb4 and flag-IRS4, respectively).  Cell lysates were incubated with beads linked to α-flag, an antibody that binds flag, and proteins that did not bind were washed away.  The bound proteins were then visualized by Western blot using α-myc antibodies.  The α-myc antibody detected the presence of myc, indicating that myc-Asb-4 binds to flag-IRS4.To better understand interactions between Asb-4 and IRS4, cells were transfected with 1 μg of flag-IRS4 plasmid and varying amounts of either myc-Asb-4 plasmid or a plasmid with the sb domain deleted (myc-Asb-4Δsb).  Flag-IRS4 levels from each set of cells were then quantified, as shown in Figure 1. Figure 1  Flag-IRS4 levels in cells with varying amounts of myc-Asb-4 or myc-Asb-4Δsb plasmid addedIn a follow-up experiment, lysates from transfected cells were again incubated with the α-flag beads, and a Western blot was performed on the precipitated proteins using α-flag, α-myc, and an antibody against ubiquitin (α-ub).  The results are shown in Figure 2. Figure 2  Ubiquitin, flag-IRS4, and myc-Asb-4 levels in cells transfected with flag-IRS4 and either an empty vector (control), myc-Asb-4 plasmid, or myc-Asb-4Δsb plasmid Adapted from Li JY, Chai B, Zhang W, et al. Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation. BMC Neurosci. 2011;12:95. -Which of the following characteristics do the flag and myc tags have in common?  They have the same:

Passage The tumor suppressor p53 is a homotetrameric protein with an N-terminal transactivation domain (TAD), a proline-rich domain, a DNA-binding domain (DBD), a tetramerization domain (TD), and a C-terminal regulatory domain (REG) on each subunit (Figure 1). Figure 1  Organization of the p53 domains (1-393 amino acids)Human double minute 2 (Hdm2) is a lysine-specific ubiquitin protein ligase that can change the half-life of some proteins, including p53, by signaling for their proteolytic cleavage.  Under normal circumstances, Hdm2 binds p53 at the AD1 region within the TAD and attaches ubiquitin to a lysine residue.  However, errors in DNA replication induce the phosphorylation of residue T18 on p53, which inhibits p53's association with Hdm2.  The overexpression of Hdm2 has been correlated with the downregulation of tumor suppressor p53 in certain types of cancers.  Changes in the NMR chemical shift (ppm) of amino acids in the AD1 region shown in Figure 2 provide evidence for the formation of a p53-Hdm2 complex. Figure 2  ¹H NMR spectrum of the AD1 region in ¹⁵N-labeled p53 during G₀ and G₂ phases of the cell cycleHdm2 also binds to Nedd8 ultimate buster 1 (NUB1), which enhances signaling for the degradation of Nedd8 proteins.  Preliminary data suggest that Hdm2 interacts differently with wild-type NUB1 (WTNUB1) than with mutant K159RNUB1.  To assess the effect of Hdm2 on NUB1 and p53, the half-lives of p53 and Nedd8 protein variants were compared in cells that overexpress Hdm2 (Figure 3). Figure 3  Half-lives of p53, phosphorylated p53 (p53-PO₄), and Nedd-8 proteins in cells with wild-type or mutant NUB1 over time Adapted from Ferreon JC, Lee CW, Arai M, Martinez-yamout MA, Dyson HJ, Wright PE. Cooperative regulation of p53 by modulation of ternary complex formation with CBP/p300 and HDM2. Proc Natl Acad Sci USA. 2009;106(16):6591-6. -In Figure 3, why was protein concentration measured only in human embryonic kidney cells that overexpressed Hdm2?

Passage The recent advent of multidrug-resistant Mycobacterium tuberculosis (Mtb) has led to the search for novel agents to combat tuberculosis.  Mtb secretes over 70 serine proteases that may be used as viable drug targets.  Hydrolase important for pathogenesis 1 (Hip1) is a serine protease found in Mtb with proteolytic activity that is required for dampening the host inflammatory response.  Given the role of Hip1 in Mtb virulence, analyzing Hip1 substrate specificity is a critical step in designing selective inhibitors for effective tuberculosis treatment.Fluorogenic substrates are nonfluorescent compounds that release a fluorescent reporter group when acted upon by an enzyme.  Such substrates are useful biomolecular imaging tools that allow monitoring of enzymatic activity in vitro and in vivo.  A large library of peptides was screened against Hip1, and enzymatic cleavage was analyzed using mass spectrometry to facilitate the design of fluorogenic substrates (Figure 1). Figure 1  Schematic design of Hip1 fluorogenic substrates.  P1, P2, P3, and P4 in Group 1 represent the positions of variable amino acids in the peptide.  Group 2 depicts the fluorescent compound released on Hip1 cleavage.Researchers reported Michaelis-Menten kinetic parameters for substrates from two peptide families: the WKLL substrates and the CSL substrates.Table 1  Kinetic Parameters of Fluorogenic Substrates for Mtb Hip1 Adapted from Lentz CS, Ordonez AA, Kasperkiewicz P, et al. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS Infect Dis. 2016;2(11):807-815. -The CSL substrates from Table 1 were tested in vitro at saturating substrate concentration in solutions containing 10 pM or 100 pM Hip1 enzyme.  The relative fluorescence of these substrates was then measured.  Which of the following graphs best reflects the expected results?

Passage Diabetic nephropathy (kidney disease) is characterized in part by a significant decrease in the glomerular filtration rate.  Podocytes are differentiated cells that surround the glomerular capillaries and counteract the pressure of blood filtration.  Hyperglycemia (elevated blood glucose) and capillary hypertension (high blood pressure), both of which are common in diabetic patients, can subject podocytes to mechanical stress.  This mechanical stress has been shown to regulate glucose uptake by podocytes, possibly by altering the production of glucose transport (GLUT) proteins.Researchers investigating GLUT2 and GLUT4 expression in rat podocytes measured the impact of mechanical stress on glucose uptake.  Cells were grown for 4 hours on membranes exposed to atmospheric (atm) or high air pressure (HP) to mimic mechanical stress.  Antibodies specific to GLUT2 and GLUT4 were used to visualize the expression of each under normal glucose (NG) and hyperglycemic (HG) conditions, and to assess the effect of mechanical stress on expression.  The uptake of a radiolabeled glucose analogue, [³H]-2-deoxy-D-glucose (3H-2DG), was then determined.  The quantity of 3H-2DG transported by each GLUT protein was directly proportional to the cell surface expression. Figure 1  Cell surface expression of GLUT transporters in response to mechanical stress and varying glucose concentration.In vivo studies have shown that transport via GLUT2 is noninducible and depends only on extracellular glucose concentrations.  However, GLUT4-mediated uptake is induced by insulin binding and occurs under high glucose conditions when insulin is released.  The standard K_m values for GLUT2 and GLUT4 are 19.4 and 4.3 mM, respectively, at normal blood glucose levels after a meal (~7.8 mM).  The data shown in Figure 2 represent the combined kinetic properties of both transporters. Figure 2  Plot of glucose uptake in podocytes with and without mechanical stress. Adapted from Lewko, B., Bryl, E., Witkowski, J. M., Latawiec, E., Angielski, S., & Stepinski, J. (2005). Mechanical stress and glucose concentration modulate glucose transport in cultured rat podocytes. Nephrology Dialysis Transplantation, 20(2), 306-311. -What stress-induced change in Figure 2 provides evidence that mechanical stress alters the binding properties of the GLUT transporters?