The metabolism of insulin and DDI between insulin and several anti-diabetes drugs
- Zhe Li
In this paper, I am going to study the metabolism of insulin in human body and briefly introduce the drug-drug interactions between insulin and some anti-diabetes drugs. In the first part of the paper, I will introduce background information of insulin. Then, in the second part, I will talk about the most important enzymes in insulin degradation, the mechanisms of insulin metabolism in different human tissues and the assays that were used in literature to study insulin metabolism. In the final part, I will briefly discuss how the DDI between insulin and some other anti-diabetes drugs may influence insulin production and metabolism and as a result, influence therapeutic efficacy.
Insulin is an endogenous peptide hormone, which plays a critical role in controlling blood sugar level. The relationship between pancreas and diabetes was first studied in Germany in the late 19th century. Since then, scientists kept working on isolating pure insulin. In 1921, Frederick Banting and J.J.R. Macleod first isolated insulin from dog pancreas. They were then awarded the Nobel Prize in Physiology or Medicine in 1923, for their great contribution in extracting insulin.
Insulin is produced by beta cells in pancreas. It shows great importance in regulating carbohydrates and fat level in the body. Human insulin has 51 amino acids. The molecular weight of it is 5808 Da. The secondary structure of human insulin is a dimer, containing chain A and chain B, which are connected by disulfide bonds. Figure 1 shows the primary and tertiary structure of human insulin.
The most important physiological role of insulin is to control cellular uptake of glucose into muscle and adipose cells. Malfunction of endogenous insulin production will leads to type-1 diabetes. Insulin resistance in insulin demanding cells is one of the reasons of type-2 diabetes. Since insulin is critical in maintaining blood glucose level and is highly related to diabetes, it has been extensively used as a major therapeutic agent for both type-1 and type-2 diabetes patients. Therefore, understanding the metabolism pathways of insulin in human body is essential in helping us determine proper doing strategies for type-1 diabetes treatments and benificial in developing drugs for type-2 diabetes.
- Enzymes for insulin degradation
There are several insulin-degrading enzymes in human body, including the insulin-degrading enzyme (IDE), protein disulfide isomerase (PDI) and acidic proteinases (Duckworth et al., 1998). These three enzymes are most important in insulin degradation in cells. I will talk about the mechanisms of these three enzymes and the in vitro assays that could be used to study their activity.
Insulin degradation enzyme (IDE) is the major enzyme that degrades insulin in human. It is a 110 KDa, zinc-binding proteinase of the M16A metalloprotease superfamily (Affholter et al., 1988). IDE degrades insulin with a relatively high specificity. First observation of the activity of IDE in degrading insulin was in 1949 by Mirsky and Broh-Kahn (Mirsky and Broh-Kahn, 1949). Since then, a lot of studies was conducted to look at its specificity and ability in insulin metabolism.
IDE is found in all cells and predominantly exist in cytosol (Duckworth et al., 1998). It degrades insulin by disconnecting amino acids in B chain. IDE can cause cleavages in several locations of the B chain. Duckworth et al performed in vitro study and determined the products of insulin degradation by IDE. Figure 2 shows how IDE break the B chain of insulin.
The apparent Km of IDE for insulin vary from 20 nM to 200 nM, as reported in the literature (Authier et al., 1996). The reason for the large range of afficinty is both because of the purification of IDE preparation, and the nature of this enzyme to form complexes (Duckworth et al., 1998).
The activity of IDE may be induced and inhibited, by the effect of some cations and chemical agents. Since IDE has a zinc binding site, some divalent cations, such as Ca2+, Mn2+ and Co2+, can induce the activity of IDE. Actually, increasing Ca2+ concentration in muscle has been shown to increase IDE activity in muscle (Ryan et al., 1985). IDE can be inhibited by p-chloromercuriphenyl-sulfonic acid (PCMBS). PCMBS is a sulfhydryl-active agents, which reduces the amount of sulfhydryl groups in cells, which is essential for IDE formation. Therefore, sulfhydryl-active agents, such as PCMBS show IDE inhibition activity (Duckworth et al., 1998).
After cleavage of peptide bonds between amino acids by IDE, the disulfide bonds that links A chain and B chain can be digested by protein disulfide isomerase (PDI). This process generates fragments of insulin in cells. Scientists have shown that these fragments are related with some biological functions of insulin (Varandani, 1980).
- Acidic proteinases
Some of the metabolized insulin fragments and undegraded insulin will finally goes into lysosome. The pH of lysosome is around 6, where IDE looses most of its activity (Duckworth et al., 1998). Acidic proteinases, however, are able to further digest insulin fragments in the lysosome. The identity of the specific acidic proteinase that degrades insulin is unknown and scientists suggest that it may be a combined effect of several enzymes.
- Assays for the metabolism of insulin
In the review paper by Duckworth et al., the author discussed two assays that were used to study insulin metabolism, which are TCA assay and HPLC method.
TCA assay uses [125I]iodo-insulin in trichloracetic acid (TCA) to study IDE property. As mentioned by Duckworth et al, the purification of IDE is crutial in getting reliable results. TCA method is widely used, despite the fact that this method greatly underestimates proteolysis of insulin (Duckworth et al., 1998). Nevertheless, TCA assay gives scientists a easy way to look at insulin metabolism.
HPLC method, on the other hand, gives a 2-3 times higher sensitivity, comparing to TCA method. Experiment results have shown that insulin could be degraded 6 times faster by using HPLC assay, comparing to TCA (Duckworth et al., 1998).
- Insulin uptake into cells
As I have talked above, insulin is synthesized in the pancreas by beta cells. They circulate in systemic circulation and have to enter target cells to have biological effects. The internalization of insulin into cells is critical in understanding insulin metabolism.
Insulin uptake is predominantly through receptor mediated endocytosis under physiological concentration. However, under high concentrations, pinocytosis becomes more important because insulin can self induce pinocytosis (Duckworth et al., 1998). Here I will introduce the endocytosis pathway mediated by insulin receptor.
The first step is binding to insulin receptor. After binding, insulin may be metabolized by IDE on the cell membrane, or internalized into endosome or even released intact into systemic circulation (HAMEL et al., 1986). In the endosome, insulin metabolism by IDE starts even before acidification (Duckworth et al., 1998). After acidification, insulin is released from insulin receptor and further metabolized by acidic proteinases in late endosome (Duckworth et al., 1998). It should be noticed that not all insulins that enters the endosome are metabolized. The amount of insulin degraded in the endosome is dependent on various factors, including insulin amount, enzyme level and others (Seabright and Smith, 1996). After that, degradation fragmentsof insulin are delivered into different parts of the cell, including nucleus (Harada et al., 1993), cytosol (Harada et al., 1995), Golgi or they can be sent back into systemic circulation. The biological functions of these fragments are not know, but they have been shown to be related with the activity of insulin (Duckworth et al., 1998). Finally, some fragments and intact insulins are completely degraded in the lysosome. This uptake pathway is also shown in figure 3.
- Hepatic clearance of insulin
Liver if one of the most important tissues in insulin clearance. First pass extraction ratio of insulin in the liver is approximately 50% after i.v. administration. The uptake of insulin into liver under physiological concentration is primarily through recepter mediated endocytosis, as I have talked above. Therefore, high concentration of insulin in the blood may saturate this active uptake process. Experiment results showed that with increased insulin level in blood, the fraction of insulin uptaken by liver was decreased, though total amount increased (Marshall, 1985). Mean residence time of endogenous insulin is estimated to be 71 min (Hovorka et al., 1993). In the paper by Hovorka et al., they used a five-compartment model, including a hepatic compartment and an intestinal compartment, to study insulin PK. They estimated the mean residence time ofinsulin in different tissues and found that insulin spent most of the time binding with liver receptors (61.6 min out of 70.8 min). Table 1 shows the MRT of insulin in different tissue compartments in the body. This finding highlights theimportance of liver in insulin disposition and metabolism.
Hepatic clearance of insulin can be affected by diet and nutrient factors. Glucose, for example, can induce liver uptake of insulin. Experiment results showed that with increasing doses of glucose, biliary secretion of insulin increased and the ratio of hepatic extraction decreased (Hennes et al., 1997). This effect may be due to gut signals, because no similar effect was found with intraportal glucose administration (Duckworth et al., 1998). Obesity and other hormones, such as GH will decrease hepatic clearance of insulin (Okuda et al., 1994). Liver diseases also show ability in decreasing insulin clearance and increase blood insulin level (Auletta et al., 1993).
- Renal clearance of insulin
Kidney is an important site for systemic insulin clearance. It removes approximatelyhalf of peripheral insulin (Rabkin et al., 1984). Renal clearance of insulin generally follow two pathways, glomerular filtration and reabsorption. About 99% of insulin that are filtered by glomerulus are eventually reabsorbed in the proximal tubule, mainly by endocytosis (Duckworth et al., 1998). The mechanism of insulin metabolism in kidney cells are very similar to that in liver cells. Degradation of insulin starts in the endosome by IDE, followed by PDI and acidic proteinases. Whole metabolism process completes in lysosome. Inhibition of IDE by bacitracin increases the amount of intact insulin that re-enters blood circulation, which demonstrates the importance of IDE in renal clearance of insulin (De Vries et al., 1989).
In diabetes patients, renal clearance plays a even more important role because insulin is not orally dosed and it can escape first pass elimination. Therefore, the fraction of insulin that are eliminated in the kidney increases. As a result, patients with renal malfuntion should reduce the dose of insulin, to avoid the potential of getting hypoglycemia because of reduced renal insulin clearance.
- Insulin clearance in other tissues
Other than liver and kidney, insulin can also be metabolized in all insulin sensitive tissues (Duckworth et al., 1998). This is consistent with the nature of the metabolism of peptides and proteins in human body. Muscle accounts for the largests amount insulin metabolism besides liver and kidney. The mechanism of insulin clearance in other tissues is similar to that in liver and kidney.
- Drug-drug interaction between insulin and other anti-diabetes drugs
All type-1 diabetes patients require insulin as medication. For type-2 diabetes patients, about 40% still need insulin as co-medication (Duckworth et al., 1998). In this section, I will introduce several classes anti-diabetes drugs that are related to insulin, briefly talk about their mechanisms and how their activities are related to insulin. First class of drug I want to introduce is insulin secretagogues. Insulin secretagogues is a class of anti type-2 diabetes drugs, that stimulate insulin secretion in the pancreas to have therapeutic efficacy. This class of drugs includes sulfonylureas and nonsulfonylurea secretagogues. When administrated, this class of drugs can block potassium channels on the cell surface of beta cells, thus increase production of endogenous insulin (Ripsin et al., 2009). Second class of drug is biguanides. Although the primary function of biguanides in type-2 diabetes patients is to reduce hepatic glucose output, this class of drugs may also influence endogenous insulin activity by sensitize peripheral tissues to insulin (Ripsin et al., 2009). The third anti type-2 diabetes drug is thiazolidinediones. Thiazolidinediones (TZDs), also called glitazones, can interact with nulear regulatory protein PPARγ, which controls glucose and fat metabolism and also induces peripheral tissue sensitivity to insulin (Ripsin et al., 2009). Finally, I want to include Incretin Mimetics and incretin inhancers. Incretins are also insulin secretagogues. They stimulate glucose-dependent insulin secretion (Ripsin et al., 2009).
In this paper, I discussed in detail how insulin is metabolized in human body. I talked about important enzymes, how insulin is internalized into cells and how it is metabolized inside cells. Then, I separately discussed insulin clearance in liver, kidney and other tissues. Finally, I introduced several anti type-2 diabetes drugs and briefly discussed how their therapeutic functions are related to their abilities to change insulin production and metabolism.
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