Week 7 Lecture Part II Biochemistry of Nutrition 560B Dr.

Charles Saladino Gluconeogenesis (the formation of glucose) At this point, first look at your text or the internet to find and learn the structures of lactate and pyruvate for the next exam. You will note that lactate with an OH group on the number 2 carbon has an OH group and is the reduced form of pyruvate (which has a carbonyl group on that same carbon). You will also note from your text that the reaction is catalyzed by lactate dehydrogenase; and you need to understand that the enzyme is bidirectional in its function. When conditions favor anaerobic metabolism (such as in heavilty exercised muscle with an oxygen deficit), pyruvate to lactate conversion is favored, which in fact is partly responsible for heavily-exercised-muscle cramps, where the pH drops due to increased levels of lactate. (In contrast, aerobic conditions favor pyruvate to acetyl CoA conversion for the Krebs cycle.) About the most important factor that determines the shift of the equilibrium of the interconversion of lactate and pyruvate is the NADH/NAD+ ratio. When this ratio is low, then pyruvate formation from lactate is favored, as in the conditions which favor gluconeogenesis, the formation of glucose from amino acids, or pyruvate, or lactate or glycerol, etc. Also, and specifically, alanine is easily converted into pyruvate by a transamination reaction (which we discussed earlier in the semester) when conditions require an amino acid for gluconeogenesis. As we begin to discuss the steps of gluconeogenesis, we can use the simple logic that if glycolysis yields some ATP, then gluconeogenesis overall will cost us ATP. The first step is to take pyruvate (from whatever source) and transport it into the mitochondria. The reason why we do not just reverse pyruvate into phosphoenolpyruvate (PEP) (at the last step in glycolysis) is for the simple reason that the pyruvate kinase of glycolysis is not reversible, and no enzyme exists in the cytosol for that reversal process. However, within the mitochondrial matrix is the enzyme pyruvate carboxylase, which adds CO2 to the three-carbon pyruvate to form the four carbon oxaloacetate (OAA). That is the same OAA that is part of the Krebs cycle. This costs an ATP, and the enzyme requires the vitamin derivative biotin as a coenzyme. Your text on pages 621 and 624 diagram the overall gluconeogenic process, although it shows no structural detail. Once OAA is formed it can be converted to PEP by the enzyme phosphoenolpyruvate carboxy kinase (PEPCK), which decarboxylates OAA. This step requires energy in the the form of GTP hydrolysis to GDP. The OAA can not cross the mitochondrial membrane to gain access to the cytotosol. Hence, we have the need for OAA conversion to PEP within the mitochondria. The PEP can then leave the mitochondria. However, also shown in the figure is an alternate gluconeogenic route. That is, OAA within the

mitochondria can form malate (same as the malate of the Krebs cycle), which can leave that organelle and enter the cytosol, where it is converted to OAA by a cytosolic isoenzyme of PEPCK. Further, OAA can be converted to the amino acid aspartate, which can leave the mitochondria, be reconverted to OAA, and then to PEP by a cytosolic PEPCK. Why all this conversion from and back to OAA? There are two answers. First there is backup.more than one way to produce PEP, a critical precursor for gluconeogenesis. Second, OAA can obviously not cross the mitochondrial membranes, in or out. By whatever route PEP is formed, it can be converted into 2-phosphoglycerate in the cytosol. From that point on, gluconeogenesis is essentially a reversal of glycolysis. Your logic would tell you that wherever a phosphate was added by a kinase enzyme to a substrate in glycolysis, a phosphatase enzyme would remove that phosphate by a phosphatase enzyme in the gluconeogenic pathway. The ATPs that are required for gluconeogensis will be supplied mostly by fatty acid oxidation (which is the subject of a coming lecture). This would make sense to use lipids for fuel, when mandatory gluconeogenesis is telling us that carbohydrate availability is insufficient to supply adequate amounts of glucose. In the gluconeogenic process, when we reach fructose-6-P generated by the enzymatic action of fructose-1,6-bisphosphatase, that fructose-6-P is easily converted into glucose6-P. However, in most tissues, that is the end point of gluconeogenesis. Free glucose is not generated, but rather glucose-6-P is the product. Here I make an interesting distinction. Glucose-6-phosphatase (catalyzing the conversion of glucose-6-phosphate to glucose is only present in tissues whose metabolic role is to maintain homeostasis in other words, tissues that release glucose into the blood. That means the liver, and to a smaller extent, the kidney, so that glucose can be supplied to the brain, as well as to muscle, the red blood cells, etc. By contradistinction, muscle contains no glucose-6phosphatase, and thus can not be converted to glucose for export. Rather, the glucose-6P is converted to glucose-1-P for storage as glycogen use and eventually retrieved as an energy sourse for muscle use only. In addition, tissues like muscle and the red blood cells, lactate from the muscle and alanine from both the muscle and red cells can be sent back to the liver, having been formed anaerobically from pyruvate. There, they are routinely used as gluconeogenic precursors. Then the glucose formed from gluconeogenesis can leave the liver and be sent back to those tissues in a cycle. These are referred to respectively as the Cori cycle and the alanine cycle. Finally, when muscle is heavily exercised, and lactate builds up, that this sugar does not build up to the point of dangerously lowering the pH in muscle is assured by the export of the lactate as a gluconeogenic precursor for the liver, as mentioned in the above paragraph. Regulation of Carbohydrate Metabolism (Topic covered here instead of next week).

I have already mentioned the importance of the NADH/NAD+ ratio in determining the direction of equilibrium between lactate and pyruvate. In our discussion of glycogen formation and breakdown, I have also asked you to refer to the information in your text regarding the regulation of glycogen phosphorylase. You can safely assume that those factors that favor activation of this enzyme inhibit directly or indirectly the action of glycogen synthase. Please read and understand the main points of metabolic regulation discussed on pages 852-862 in your Devlin text for the next exam. The main take home message that I want you to understand regarding hormones is that concerning insulin and glucagon. (You are not responsible for all the details of enzyme regulation by hormones, and allosteric regulators etc, but you need to know the main points. I leave that to your judgement.) We must, however, understand that insulin levels normally rise in the blood in response to a meal with carbohydrates. Glucagon levels concomitantly fall. Common sense would tell us then that increased insulin levels would not favor high glucose production from gluconeogenesis or from glycogen breakdown. Rather, glycolysis would be favored, as would the Krebs cycle. However, after a point where ATP levels were high, the pyruvate dehydrogenase enzyme (that forms acetyl CoA from pyruvate) would be deactivated by abundant ATP. This would necessitate a shift toward triglyceride formation the subject of the future lipid lecture material. Indeed, insulin favors triglyceride formation in the adipose tissue, especially when carbohydrate intake is excessive beyond need. Obviously, what I just noted about the effects of increased insulin levels would be generally antagonized by high glucagon levels. For example, insulin simulates glucose transporters in many tissues, whereas glucagon does not. Also, two important enzymes that help drive glycolysis are glucokinase (step # 1) and pyruvate kinase (where pyruvate is formed from PEP). Both enzymes are activated by way of the action of insulin. Glucagon has the opposite effect upon their activity. Some further points on the reciprocity of gluconeogenesis and glycolysis and Their Inter-related Control Systems These two pathways are coordinated so that they roughly reciprocally active. Now with both pathways balancing out regarding energy usage vs. energy given off (ex two ATP produced in glycolysis), there is no thermodynamic impediment to then not going on a full speed simultaneously. In fact, both are exergonic. However, the amounts and the activities of the enzymes of both pathways are so controlled that they do not activate fully at the ame time. Remember, the rate of glycolysis is also controlled by the amount of glucose and the rate of gluconeogenesis by the amount of its available precursors, such as lactate. We have not discussed in lecture how stringently controlled the conversion of fructose-1-P to fructose-1,6-bisphosphate (F-1,6-BP) is during glycolysis. Whereas AMP stimulates phosphofructokinase (the enzyme that puts the second phosphate on fructose during glycolysis), ATP and citrate inhibit it. For example, high AMP levels indicate that energy levels are low, and that there is need for ATP production from glycolysis. The converse is also true for high levels of ATP and citrate.

Phosphofructokinase and fructose-1,6-bisphosphatase (F-1,6-BPase) are also reciprocally controlled in the liver by F-1,6-BP. (Remember, the F-1,6-bisphosphatase removes a phosphate from F-1,6-BP) during gluconeogenesis.) The level of fructose-1,6-BP is obviously low during starvation (no glycolysis) and high in the fed state. This is due to the antagonistic effects of glucagon and insulin on the production and degradation of this important signal molecule. Thus glycolysis accelerates in the fed state. In starvation, gluconeogenesis is predominant, because the level of F-2,6-BP is low. Under these conditions, the liver-derived glucose becomes essential for brain and muscle viability. Without going into too much detail, the interconversion of PEP and pyruvate is also tightly regulated. The two counter-regulatory enzymes are PEP kinase and pyruvate carboxylase. You can review those, if you wish. One more major point: The activities and the amount of the above-mentioned enzymes are regulated hormonally. Here the hormones affect gene expression by regulating transcription levels, as well as by regulating the degradation of mRNA. As one example, after insulin rises subsequent to a meal, that hormone stimulates the expression of phosphofructokinase and pyruvate kinase. This obviously stimulates glycolysis. There is much more to the regulatory story, but I will save that as a semi-finale to the course, perhaps. I do think, however, you have some feel for the regulatory process. I have included a diagram below to help you understand my answer here. OK?

Conclusion These last few lectures have been involved (as is metabolism) for you to absorb and, frankly, for me to write. However, although we need to learn a good number of details, dont lose site of the big picture. I am asking you to think about possibilities of integration between pathways, as we have discussed with the feeder pathways into and from glycolysis, the relationship of glycolysis to gluconeogenesis, glycolysis to the pentose pathway, and eventually the relationship of carbohydrate metabolism to that of lipids and proteins