The Biochemical Cause for the Defective Muscle Glycogen Synthesis - Essay Example

1. An infant patient suffers from hypotonia, poor feeding, and low weight gain. In an effort to diagnose his condition, you examined mitochondria in muscle cells obtained from a biopsy and detect a deficiency in oxidative metabolism. The patient's mitochondria consumed oxygen normally when incubated with pyruvate and malate; succinate; or palmitoyl CoA and carnitine. Reduced oxygen consumption was observed when mitochondria were incubated with linoleoyl CoA and carnitine. The patient's blood levels of carnitine are lower than normal and an acylcarnitine derivative is present in blood and urine. Mass spectrometry analysis of urine identifies this acylcarnitine derivative as trans- 2-cis-4-decadienoyl-carnitine. Deduce the likely enzyme deficiency causing the infant's condition and explain how this deficiency can cause his symptoms. How might you treat this disorder Answer 1. Clearly our patient is suffering from an inability to metabolize linoleic fatty acids(LFA). The enzyme responsible for LFA is LFA desaturase1 The reaction is as follows: linoleoyl-CoA + AH2 + O2 gamma-linolenoyl-CoA + A + 2 H2O The lack of the denaturase enzyme results in a build up of unreduced LFA and a increased request of caritine to transport the accumulating waste products. The caritine is then becoming acylated with the LFA which results in low serum caritine and a disruption of fatty acid metabolism overall. Treating the disorder is a logical restriction of fat containing LFA from the diet. 2. Wakil's pioneering studies on the synthesis of fatty acids included the crucial observation that bicarbonate was required for the synthesis of palmitoyl CoA. He was surprised to find that very low levels of bicarbonate could sustain palmitate biosynthesis and that there is no correlation between the amount of bicarbonate required and the amount of palmitate produced. Explain these observations and describe how radiolabelled bicarbonate could be used to support your explanation. Answer 2. The following reactiom is the accepted mechanism for producing the long chain fatty acid palmitoyl-CoA. When acetyl coenzyme A, malonyl coenzyme A and reduced triphospopyridine nucleotide (TPNH) are used as substrates palmitic acid is the principal product of the reaction. Naturally the reaction is enzymatic. Obtained from metazoic and microbial cells. The proposed stoichiometric mechanism is: 7Malonyl-CoA + acetyl-CoA+ 14 TPNH- palmitate+14TPN+ 7CO2 +8CoA2 Wakil and Sterbi et al favored a simple condensation that would yield an alpha-caroxyl-beta-ketothioester, wheras Vagelos and Alberts and Lynenet al have favored a concerted condensation-decarboxylation that would yield a beta-ketothioester. Using two sources of radiolabelled Carbon can find out which is correct. Source 1 external from bicarbonate Source 2 internal from malonyl As was determined no external bicarbonate source was necessary. The decarboxylation reaction produces CO2. 3. I dreamt about studying fatty acid synthesis in two different experimental systems; cultured rat hepatocytes and Escherichia coli. In my dream I was able to covalently anchor the ketoacyl ACP synthase activities of the fatty acid synthase complexes from both experimental systems to gold nanoparticles (aka quantum dots) through a sulfur-gold bond involving the active site cysteine thiol of -ketoacyl ACP synthase. This resulted in a complete loss of fatty acid synthesis from 14C-1 radiolabelled acetyl CoA in Escherichia coli, but only about 50% reduction in palmitate biosynthesis in the rat liver cells in my dream. Explain the molecular basis for the observations I made in my dream. 3. Answer While it seems the quantum dots were sufficient in destroying the active site of the enzyme in the E.Coli, there appears to be a second active site from rat heptocytes enzyme not denatured by the quantum dots, but sufficient in halving activity. 4. Niemann-Pick Disease is an inherited disorder of sphingomyelin breakdown due to a deficiency of sphngomyelinase. This enzyme is found in all tissue and is easily assayed from white blood cells collected from a patient. Sphingomyelinase converts sphingomyelin to ceramide and phosphocholine. One of these hydrolytic products is highly soluble in water while the other is preferentially soluble in chloroform. You would like to measure sphingomyelinase activity from white blood cells of your patient using sphingomyelin labeled with 14C at any carbon to determine whether she has Niemann-Pick disease. Based on the information provided here and our discussions in class, design an experiment that would allow you to confirm the diagnosis of Niemann-Pick disease. Answer Experiment- The Sphingomyelinase Assay Test Materials-Cell Hydrolysis Agent(Lysozyme) 14C Spingomyelin Blood Sample (approx. !0ml) uncoagaulated in EDTA Chromomatic chloroform/water separation equipment Radiometric Isotope detection equipment Reaction Sphingomyelin*14C) phosphocholine* + Ceramide* Insoluble(HOH) Soluable(HOH) Insoluable(HOH) *=radioactive In slow centrifuge separate liquid from cell portion of blood. At interface of liquid and cell portion white cells can be obtained by carefully removing white cell (WBC)buffy coat and place in 2ml of distilled water. Add 3 drops of cell hydrolysis agent Lysozyme and centrifuge hard to obtain solute with cell products. Again pipette out solute leaving cell membranes and dilute in phosphate buffered saline. This is our test sample for Sphingomyelinase. Add WBC Solute + 14C Sphingomyelin* phosphocholine* + ceramide* For 1hour at 30 degrees centigrade incubate reaction Perform water/chloroform separation and test water fraction for radiolabel 14C phosphocholine*. Results Phosphocholine* present, WBC solute was positive for Sphingomyelinase Phosphocholine* Absent WBC Solute negative for Spingomyelinase. 5. Patients with the genetic disorder familial hypercholesterolemia (FH) present elevated serum cholesterol levels that contribute to atherosclerotic plaques early in the disease progression. These patients exhibit defective LDL receptors which impair receptor-mediated uptake of LDL-cholesterol. Treatment includes modification of diet, incorporation of edible resins to prevent intestinal reabsorption of bile acids, and administration of a pharmaceutical agent to lower serum cholesterol. Briefly explain how the following pharmaceutical agents would decrease the serum cholesterol levels in patients with FH. a) statin drugs which resemble mevalonate b) inhibitors of the proteases that release the sterol regulatory element binding protein (SREBP) Answer 5. 5a. Working by inhibiting the controlling enzyme HMG-CoA reductase Statins act by reducing the production of (sterol) cholesterol synthesis in the liver, blocking mevalonate and the chain of events that produce cholesterol along with other compounds.3 5b. Inside the liver cell, other enzymes of the protease class sense the decreased level of cholesterol produced. In response, they cleave a protein called "membrane-bound sterol regulatory binding protein(SREBP)", which then responds by migrating to the nucleus to increase production of various other proteins and enzymes, including the LDL receptor. The LDL receptor then relocates to the cell membrane of the liver cell, and binds to passing low density lipoprotein and very low density lipoprotein particles (both containing cholesterol in the undesired form). LDL and VLDL enter the liver and are digested.4 6. For several hours after birth, premature infants are particularly susceptible to hypoglycemia and are also unable to rapidly generate ketone bodies. Describe how each of the following characteristics would contribute to hypoglycemia, low circulating levels of ketone bodies, or both: a) A large brain to body ratio b) A small store of liver glycogen c) Low specific activity of cytosolic carnitine long-chain acyl CoA transferase in liver d) Very low levels of liver phosphoenolpyruvate carboxykinase. Answer 6. 6a. A large brain to body ratio will contribute to hypoglycemia due to the brain cells high consumption of glucose, depriving the body of its share and resulting hypoglycemia 6b. Glycogen ist he store house for glucose, mainly found in muscle and the liver.Without this backup it can be difficult in times of stress for the body to maintain adequate glucose levels. 6c. The low activity of these transferase enzymes would limit the liver from incorporating the fatty acid building blocks to make the glucose it needs. Without the ketone signal that activates these transferase enzymes the liver will be slow to produce the glucose as required and intravenous glucose needs to be supplied 6d. Phosphoenolpyruvate carboxykinase (or PEPCK) is an enzyme used in the natural process of glucogenisis It converts oxaloacetate into phophophenolpyrvate andcarbon dioxide.4 The abscense disrupts the liver production of glucose and is a major contributor to hypoglycemia. Ketones the normal waste product of glucose catabolism acts as a feedback mechanism to produce more glucose, without its presence the glucogenisis in the premature infant is stalled. 7. Read the attached paper entitled "Cellular Mechanisms of Insulin Resistance" and answer the following 3 questions;. a. How did Shulman and colleagues identify the biochemical cause for the defective muscle glycogen synthesis proposed to play a role in the development of insulin resistance in type 2 diabetics What do they propose is the defective metabolic step as supported by their results b. What is the biochemical basis proposed by Randle, et al. to explain the connection between elevated plasma free fatty acid concentrations and insulin resistance Explain it in your own words based on our discussions of metabolism. c. Shulman has proposed a different mechanism to explain fatty acid-induced insulin resistance that is consistent with their findings on defective muscle glycogen metabolism. Summarize the key pieces of evidence that refute Randle's model and support Shulman's model. Describe the salient features of Shulman's proposed model. Answers 7. 7a. Shulman et al5 using C13 nuclear magnetic resonance NMR to monitor the rate of C13 lablel glucose found that glycogen synthesis was approximately 50% lower in diabetic patient than in normal volunteers.In conditions of high glucose, high insulin levels(ie. Just ate 6 snickers candy bars) the normal and diabetic turn the excess glucose into glycogen. However in the diabetic the ability to make this glycogen is reduced causing insulin resistense. Is was then necessary to determine the rate-controlling defect in the biochemical mechanism, 3 candidates: Glycogen Synthase(GS) Hexokinase II(HXII) Glucose Transport(GT) C13 and P31 NMR studies were then used to measure glucose, glucose-6-phospate (the intermediary in glycogen synthesis) and glycogen. In the event of a GS defect it was expected that glucose-6- phosphate(G6P) would be elevated. The experiments did not reveal this result and HXII and transport hypothesis were further explored. The model for Glycogen synthesis is a follows: II II II HXII GS Plasma(Glucose)Glut 4#GlucoseG6PUDP-GlucoseGlycogen II II II## #Glucose membrane Transporter Receptor ##Cell Membrane A novel NMR method was used to test for intracellular glucose and G6P. If the defect was at the HXII enzyme an elevated intracellular glucose level was expected. However it was observed that relative glucose to G6P were equal and the expected defect in glycogen synthesis was cell transport. 7b. An Inverse relationship existed between a diabetic type 2 and normal control group for fatty acid levels and insulin sensitivity. Also a finding from intracellular triglycrides by muscle biopsy and intracellular triglyceride content using H1 NMR have shown a result of of accumulation of intramyocellular triglycerides. Randle et al all demonstrated that fatty acids compete with glucose and its metabolism. The consumption of fatty acids directly produce two products Acetyl CoA and Citrate which inhibit the two essential enzymes in glucose metabolism phosphofructrokinase and pyruvate dehydrogenase. The increase in Fatty acyl Coa alsostimulate the Serine Threonine Kinase cascade which final effect slows insulin to stimulate the phosphoinosistol3 kinase that activates the production of Glut4 transporters and plasma glucose is elevated, stimulating the cycle to produce fatty acids. 7c. Randle et al predicted an increase in intramyocellular G6P and subsequent interference in in enzyme metabolism by phosphofructokinase and pyruvate dehydrogenase, where Shulman intracellular findings show no elevation of intramyocellular glucose or G6P and blame the signal transductions and lack of mobilization of Glut4 transporters caused by increased fatty acid production and affecting a condition that causes insulin resistence. References 1. Web Site 05 May 2009http://en.wikipedia.org/wiki/Linoleoyl-CoA_desaturase Lineoyl-O-CoA desaturase Wikipedia 2. Fatty Acid Synthesis in Adipose Tissue Journal of Biological Chemistry Vol236 March 1961, March, Horning and Valegos 3. Web Site 06 May2009 http://en.wikipedia.org/wiki/Statin Statin Wikipedia 4. Ibid 5. Cellular mechanisms of insulin resistance The Journal of Clinical Investigation, July 2000 Vol106 Number 3 Gerald I Shuylman Read More