BIOL1181 Biochemistry and Molecular Biology: The Mechanism Of Diabetes

The following short answer questions relate to the lecture material presented in the metabolism lectures.
Q1. Using the illustration provided below systematically explain in detail the mechanism of diabetes mellitusThe illustration provided explains the mechanisms for Type 1 Diabetes mellitus, or insulin dependent diabetes.
Following the steps in the illustration:
1.    The insulin is now no longer available or the concentration of insulin is too low to regulate the glucose levels in the blood. This could be due to the cells that produce insulin such as the β cells in the pancreas have been damaged or a reduction in their activity resulting in a decline in insulin production.
2.    Insulin dependent GLUT4 is then unable to diffuse into the plasma membrane as the receptor proteins for insulin are no longer activated by insulin and therefore there is an inhibition of the signalling pathway for GLUT4 vesicles to release their GLUT transporters. 
3.    Due to GLUT4 being unavailable for the transport of glucose across the plasma membrane, there will be no metabolism of glucose i.e citric acid cycle, glycolysis, ETC. This inability to metabolise glucose will cause a surge in blood glucose levels resulting in diabetes mellitus or hyperglycaemia 
4.    Our cells require energy and tri-glycerides are an optimal fuel source. Within the hepatic cells, Acetyl Co-A is a product of fatty acids that is converted into ketone bodies, B-hydroxybutyrate and acetoacetate. These by-products will then be distributed to various cells to provide energy to them.
5.    The resultant metabolism of these ketone bodies reduces the pH of the cells causing ketoacidosis- untreated will be life threatening. 

This syndrome is known to arise from two types of conditions: Wernicke disease and Korsakoff syndrome. WKS is primarily caused by chronic and excessive alcoholism. Poor diet and nutrition intake is also a contributing factor. This limitation of nutritional absorption from alcoholism and poor diet can inhibit and or cut off vitamin B1 absorption.

Alcohol depletes thiamine stores within the liver and reduces its absorption, as well as inhibits the diphosphotransferase enzyme from converting thiamine from its provitamin from into its active state. Starvation and anorexia and prolonged/chronic emesis from disorders such as hyperemesis gravidarum can also deplete vitamin B1 and cause WKS. Cancer, AIDS and gastropathies as well as kidney disease may also cause WKS.

There are also hereditary factors causing a genetic predisposition to WKS, but further research is required to fully comprehend how. The SLC19A2 gene which codes for thiamine transporter 1 plays a role in those who develop this condition. Variants in this gene have been linked to patients presenting with WKS.

Q3. Explain how phosphocreatine is biosynthesised and where and why is it used. You may wish to draw a diagram to help you explain your answer (5 marks).
Phosphocreatine is synthesised from creatine and is used in the nervous system and skeletal muscle alongside ADP and ATP to create creatinine, the nitrogenous waste product. Phosphocreatine is muscles energy buffer helping to maintain the homeostasis of ATP in the muscle during exercise that would usually deplete the ATP concentration within the cells. Creatine is synthesised in the liver and transported to skeletal and heart muscles via the blood stream. Creatine enters the cells mitochondria which is where it is then phosphorylated into phosphocreatine. Creatine kinase is the catalyst for this reversible addition of phosphate. 
 

Q4. Explain in detail how fatty acids are biosynthesised. You may wish to draw a diagram to help you explain your answer (6 marks).
Fatty acids are biosynthesised via NADPH and acetyl-CoA as well as several enzymes that are within the cytoplasm alongside the glycolytic pathway.
The following steps shows how fatty acids are biosynthesise:
1.    Acetyl-CoA is transported from the mitochondria
2.    Acetyl-CoA joins with oxaloacetate in the mitochondria to create citrate which then moves across the membrane 
3.    These molecules then form malonyl-ACP and acetyl-ACP (acyl is the carrier protein)
4.    NADPH reduces the ketone into a hydroxyl
5.    This process is repeated with another malonyl-ACP being added growing the chain until a 16-carbon compound 

The following questions will assess your understanding of the main concepts you learned from the Analysis of Milk practical.Preparation of whey from skim milk
1. Label a microfuge ‘Sample M.’ Pipette 200 µL of undiluted skim milk into the tube.
2. To the Sample M tube, add 1000 µL of acetate buffer (0.2 M, pH 4.7). Mix well by inversion. Leave the tube on ice for 10 minutes.
3. Spin the microfuge at 13,000 rpm for 3 mins to precipitate the casein present in the skim milk.
4. Transfer the casein-free supernatant (whey) into a single clean microfuge tube using a pipette. Label this Sample M2.
5. Dilute Sample M2. Add 200 uL of Sample M2 to 800 uL of water in a test tube and mix well. This will be used for the Lowry Assay.

Q1. Complete the following Table including appropriate concentrations, volumes of standards and test samples, corrected absorbances and include an appropriate table caption.  You are to create a 6-point standard curve from 0 µg/mL – 250 µg/mL. Each standard is performed in duplicate (Tubes 1 – 12) (2 marks).              
 

Q2. Using Excel or any other statistical package, plot the standard curve for BSA (Tubes 1 – 12) based on the absorbance readings provided in Table 1. Label this Figure 1 and provide an appropriate title.  Do not forget to use suitable labels for the axes and read off values for Tubes 13 – 16 (4 marks).          

Q3. Show full calculations and determine the concentration (g/100 mL) of total protein in the original skim milk and whey (non-casein protein) samples (3 marks).    

Q4. Calculate the casein content as a percentage of the total protein in milk (2 marks).

Q5. How do your results compare to the expected value.

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