Ketosis

Q. Give the details of the energy yield from the complete oxidation of ketone bodies.

Answer-The energy provided to the peripheral tissues from acetoacetate and for beta-hydroxybutyrate are shown below:

Reaction	Energy product	Multiplier	ATP equivalents
Co A transferase	- GTP (No GTP formation  from Succinyl coA to Succinate in TCA cycle	1	-1
Acetyl co A OxidationIn TCA cycle	12 ATP	2	24
Acetoacetate oxidationTotal	-	-	23
β-OH ButyrateDehydrogenase	1NADH-3ATP	1	3
Total β-OH Butyrate oxidation	-	-	26

This may be appreciated when it is realized that complete oxidation of 1 mol of palmitate involves a net production of 129 mol of ATP via beta oxidation and CO₂ production in the citric acid cycle, whereas only 23 mol of ATP are produced when acetoacetate is the end product and only 26 mol when 3-hydroxybutyrate is the end product. Thus, ketogenesis may be regarded as a mechanism that allows the liver to oxidize increasing quantities of fatty acids within the constraints of a tightly coupled system of oxidative phosphorylation.

Q.- Discuss in brief about  the regulation of ketosis.

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What is the effect of insulin, glucagon, or epinephrine upon lipolysis in adipose tissue? How can a decrease in the insulin/glucagon ratio explain the increased production of ketone bodies during a fast?

Answer- Ketogenesis is regulated at three steps-

(1) Lipolysis in Adipose tissues- Ketosis does not occur unless there is an increase in the level of circulating free fatty acids that arise from lipolysis of triacylglycerol in adipose tissue. Fatty acid release from adipose tissue is controlled via the activity of hormone-sensitive lipase (HSL). When glucose levels fall, pancreatic glucagon secretion increases resulting in phosphorylation of adipose tissue HS (Figure-1), thus resulting in increased hepatic ketogenesis due to increased substrate (free fatty acids) delivery from adipose tissue. Conversely, insulin, released in the well-fed state, inhibits ketogenesis via the triggering dephosphorylation and inactivation of adipose tissue HSL.

Figure-1- Showing the degradation of Triglycerides in adipose cell by hormone sensitive lipase. Hormone sensitive lipase exists in two forms inactive- dephosphorylated (brought by Insulin) and active phosphorylated form (brought by glucagon, ACTH and catecholamines). Insulin promotes lipogenesis while the other hormones  promote lipolysis.

Free fatty acids are the precursors of ketone bodies in the liver. The liver, both in fed and in fasting conditions, extracts about 30% of the free fatty acids passing through it, so that at high concentrations (After high fat diet or in conditions of excessive lipolysis) the flux passing into the liver is substantial. Therefore, the factors regulating mobilization of free fatty acids from adipose tissue are important in controlling ketogenesis.

2) Fate of  Fatty acid- After uptake by the liver, free fatty acids are either oxidized to CO₂ or ketone bodies or esterified to triacylglycerol and phospholipid.  If the liver has sufficient supplies of glycerol-3-phosphate, most of the fats will be turned to the production of triacylglycerols.

There is regulation of entry of fatty acids into the oxidative pathway by carnitine Acyl transferase-I (CAT-I), and the remainder of the fatty acid taken up is esterified. CAT-I activity is low in the fed state, leading to depression of fatty acid oxidation. Malonyl-CoA, the initial intermediate in fatty acid biosynthesis (Figure-2), formed by acetyl-CoA carboxylase in the fed state, is a potent inhibitor of CAT-I . Under these conditions, free fatty acids enter the liver cell in low concentrations and are nearly all esterified to acylglycerols and transported out of the liver in very low density lipoproteins (VLDL).

Figure-2- Showing the inhibition of CAT-1 by Malonyl Co A

However, CAT-1 activity is higher in starvation, allowing fatty acid oxidation to increase. Since the concentration of free fatty acids increases with the onset of starvation, acetyl-CoA carboxylase is inhibited directly by acyl-CoA, and malonyl-CoA level decreases, releasing the inhibition of CAT-I and allowing more acyl-CoA to be oxidized. These events are reinforced in starvation by decrease in the[insulin]/[glucagon] ratio.  

Figure-3-Showing the regulation of Acetyl co A carboxylase by covalent modification. During starvation glucagon causes inhibition of Acetyl co A carboxylase by c AMP dependent phosphorylation. Reverse occurs in the presence of Insulin.

Glucagon In addition, results in phosphorylation and inhibition of acetyl-CoA carboxylase (ACC), the rate limiting enzyme of de novo fatty acid synthesis (Since the enzymes gets inactivated upon phosphorylation). Conversely, under conditions of insulin release, in fed state, hepatic ACC is activated, by dephosphorylation and the excess acetyl-CoA  is converted into malonyl-CoA and then to fatty acids, CAT-1 is inhibited and fatty acid oxidation is also inhibited. Thus, -oxidation from free fatty acids is controlled by the CAT-I gateway into the mitochondria.

(3) Fate of Acetyl co A- In turn, the acetyl-CoA formed in  beta-oxidation is oxidized in the citric acid cycle, or it enters the pathway of ketogenesis to form ketone bodies. If the hepatic demand for ATP is high the fate of acetyl-CoA is likely to be further oxidation to CO₂. This is especially true under conditions of hepatic stimulation by glucagon which results in increased gluconeogenesis and the energy for this process is derived primarily from the oxidation of fatty acids supplied from adipose tissue.

As the level of serum free fatty acids is raised, proportionately more free fatty acids are  converted to ketone bodies and less are oxidized via the citric acid cycle to CO₂. The partition of acetyl-CoA between the ketogenic pathway and the pathway of oxidation to CO₂ is so regulated that the total free energy captured in ATP which results from the oxidation of free fatty acids remains constant as their concentration in the serum changes.

Q.- Enlist the conditions causing ketosis,  Discuss the underlying defect in each of them responsible for causing ketosis.

Answer- The production of ketone bodies occurs at a relatively low rate during normal feeding and under conditions of normal physiological status. Ketosis is basically observed in conditions of glucose deprivation and excess lipolysis.

A) Conditions causing glucose deprivation- Normal physiological responses to carbohydrate shortages cause the liver to increase the production of ketone bodies from the acetyl-CoA generated from fatty acid oxidation. This allows the heart and skeletal muscles primarily to use ketone bodies for energy, thereby preserving the limited glucose for use by the brain. The common causes of glucose deprivation are as follows-

a) Starvation

b) Chronic alcoholism

c) Von- Gierke’s disease

d) Heavy exercise

e) Low carbohydrate diet- For weight loss

f) Glycogen storage disease type 6(Due to phosphorylase kinase deficiency)

g) Pyruvate carboxylase deficiency

B) Conditions causing excessive Lipolysis- All conditions causing hypoglycemia cause lipolysis to compensate for the energy needs, but in uncontrolled diabetes mellitus (Type 1 especially) glucose is available yet cannot be utilized due to insulin deficiency. There is an imbalance between Insulin to Glucagon ratio. Excess glucagon in such conditions induces a state of catabolism , causing lipolysis and thus enhanced ketogenesis. Similarly extreme stress and glucagon producing tumors can cause ketosis.

C) Prolonged ether anesthesia, toxaemia of pregnancy and certain conditions of alkalosis associated with excessive vomiting can also cause ketosis.

D) Nonpathologic forms of ketosis are found under conditions of high-fat feeding and after severe exercise in the post absorptive state.

Q. What is the biochemical basis of ketosis in prolonged fasting or starvation

Answer-Prolonged fasting may result from an inability to obtain food, from the desire to lose weight rapidly, or in clinical situations in which an individual cannot eat because of trauma, surgery, neoplasms, burns etc. In the absence of food the plasma levels of glucose, amino acids and triacylglycerols fall, triggering a decline in insulin secretion and an increase in glucagon release. The decreased insulin to glucagon ratio, and the decreased availability of circulating substrates, make this period of nutritional deprivation a catabolic state, characterized by degradation of glycogen, triacylglycerol and protein. This sets in to motion an exchange of substrates between liver, adipose tissue, muscle and brain that is guided by two priorities (i) the need to maintain glucose level to sustain the energy metabolism of brain ,red blood cells and other glucose requiring cells and (ii) to supply energy to other tissues by mobilizing fatty acids from adipose tissues and converting them to ketone bodies to supply energy to other cells of the body (Figure-4)

Figure-4 Showing the distribution of fuels in different tissues during starvation

After about 3 days of starvation, the liver forms large amounts of acetoacetate and d3- hydroxybutyrate. Their synthesis from acetyl CoA increases markedly because the citric acid cycle is unable to oxidize all the acetyl units generated by the degradation of fatty acids. Gluconeogenesis depletes the supply of oxaloacetate, which is essential for the entry of acetyl CoA into the citric acid cycle. Consequently, the liver produces large quantities of ketone bodies, which are released into the blood. At this time, the brain begins to consume appreciable amounts of acetoacetate in place of glucose. After 3 days of starvation, about a third of the energy needs of the brain are met by ketone bodies. The heart also uses ketone bodies as fuel. After several weeks of starvation, ketone bodies become the major fuel of the brain. Acetoacetate is activated by the transfer of CoA from succinyl CoA to give acetoacetyl CoA .Cleavage by Thiolase then yields two molecules of acetyl CoA, which enter the citric acid cycle.

In essence, ketone bodies are equivalents of fatty acids that can pass through the blood-brain barrier. Only 40 g of glucose is then needed per day for the brain, compared with about 120 g in the first day of starvation. The effective conversion of fatty acids into ketone bodies by the liver and their use by the brain markedly diminishes the need for glucose. Hence, less muscle is degraded than in the first days of starvation. The breakdown of 20 g of muscle daily compared with 75 g early in starvation is most important for survival.

A person’s survival time is mainly determined by the size of the triacylglycerol depot.

Q. Discuss in detail about the causes, clinical manifestations, laboratory diagnosis and treatment of diabetic ketoacidosis.

Answer- Explained in Complications of diabetes Mellitus.

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