An unusual cause of diabetes - how the pancreas senses a rise in blood glucose

Insulin stimulates uptake of glucose into muscle and adipose tissue. It achieves this by recruitment of glucose transporters from intracellular vesicles to the cell surface.

In the presence of insulin, glucose becomes major fuel for muscle contraction. There is increased synthesis of glycogen as a result of activation of glycogen synthase and inactivation of glycogen phosphorylase.

In adipose tissue, glucose is used for the synthesis of fatty acids and and the triacylglycerol for storage, as a result of increased activity of fatty acid synthase and decreased activity of hormone-sensitive lipase.

Liver uptake of glucose is not affected by insulin, but insulin stimulates the synthesis of glycogen from glucose, as in muscle, as a result of activation of glycogen synthase and inactivation of glycogen phosphorylase.

Either failure of insulin secretion or decreased sensitivity of insulin receptors leads to elevated blood glucose (hyperglycaemia). Glucose reacts non-enzymically with amino groups in proteins, including:

• collagen (hence increased arthritis and damage to epithelial basement membrane leading to damage to blood vessels, and contributing to circulatory problems, retinal damage and renal damage)
• proteins in low density lipoprotein (hence increased risk of developing atherosclerosis)
• crystallin in the lens of the eye ( hence the development of cataracts)
• proteins in myelin (hence nerve damage)
• haemoglobin – not a pathological problem, but diagnostically useful to monitor long-term control of blood glucose by measurement of glycated haemoglobin (haemoglobin A1c)
  

In addition, hyperglycaemia may lead to osmotic imbalance and dehydration. Glucose may also be reduced to sorbitol in nerve and other tissues, again leading to osmotic damage.

The beta-islet cells of the pancreas secrete insulin in response to an increase in blood glucose. This problem is concerned with the way in which the beta-cells detect the increased concentration of glucose.

One early hypothesis was that there is a glucose receptor on the outer surface of the beta -cell membrane, and when this binds glucose it initiates a series of intracellular events that lead to secretion of insulin.

Coore & Randle (1964) measured the secretion of insulin by rabbit pancreas incubated in vitro with two concentrations of glucose, with and without the addition of the 7-carbon sugar mannoheptulose, which they had previously shown to be an inhibitor of phosphorylation of glucose to glucose 6-phosphate. Their results are shown below.

Secretion of insulin (µg /minute /incubation) by rabbit pancreas incubated in vitro with 3.3 or 16.6mol /L glucose. (3.3 mmol /L is the glucose concentration that would be seen in relatively prolonged fasting; 16.6 mol /L is about the concentration that would be seen in the hepatic portal vein after a moderately carbohydrate-rich meal):

From data reported by Coore HG & Randle PJ, Biochemical Journal 93: 66 – 77, 1964.

In the control incubations there is the expected increase in insulin secretion in response to glucose; in the presence of mannoheptulose there is no increase in insulin secretion in response to glucose. This suggests that the way in which the pancreas senses increased blood glucose is not by binding to a receptor on the cell surface, but by increased formation and metabolism of glucose 6-phosphate.

his problem concerns a small number of families with a clear pattern of dominant inheritance of an unusual form of diabetes that develops in early childhood. It is generally referred to as maturity-onset diabetes of the young (MODY), although it is distinct from types 1 and 2 diabetes.

Dominant inheritance means that the disease is manifest in heterozygotes, with one normal and one abnormal gene. Commonly this is because the product of the abnormal gene has undesirable effects, or because having only half the normal amount of the enzyme or protein is not sufficient.

A recessive condition is where there must be two copies of the abnormal gene for the condition to be manifest because having only half the normal amount of enzyme does not have any effect.

As will be obvious by the time you have finished working through this problem, MODY is a dominant condition because having only half the normal amount of an enzyme does not permit adequate secretion of insulin in response to increasing blood glucose.

Glucose enters the cells of most tissues by active transport, which is stimulated in response to insulin. In the absence of insulin, glucose transporters are sequestered in intracellular vesicles, which migrate to the cell surface and become active in the presence of insulin. This means that insulin promotes the uptake and utilisation of glucose in most tissues. After entry, glucose is phosphorylated to glucose 6-phosphate before onward metabolism or use for synthesis of glycogen (in muscle) or fatty acids (in adipose tissue).

In the liver, glucose enters cells by passive diffusion and is then trapped by phosphorylation to glucose 6-phosphate, which cannot cross cell membranes. Glucose 6-phosphate is then either metabolised as a metabolic fuel or used to synthesise glycogen and fatty acids.

Two isoenzymes catalyse the formation of glucose 6-phosphate from glucose

Isoenzymes are enzymes that catalyse the same reaction, but encoded by different genes. They may differ in tissue or subcellullar distribution, reaction kinetics (i.e. a different value of Km), the range of substrate specificity or inhibitor sensitivity.

Two isoenzymes, hexokinase and glucokinase, catalyse the formation of glucose 6-phosphate from glucose. Hexokinase has a broad specificity for 6-carbon (hexose) sugars, while glucokinase is more specific for glucose.

The normal range of plasma glucose is between 3.5 - 5 mmol/L, rising in peripheral blood to 8 - 10 mmol/L after a moderately high intake of glucose. The concentration of glucose in the portal blood, coming from the small intestine to the liver, may be considerably higher than this after a moderately carbohydrate-rich meal.

Note that glucokinase shows cooperativity in glucose binding, and has sigmoid kinetics rather than hyperbolic, as for hexokinase.

Because its Km is so far below plasma concentration of glucose under all conditions, and glucose enters liver cells freely, hexokinase in the liver will always be acting at its Vmax – i.e. it will act at a constant rate, regardless of the concentration of glucose.

Note that in muscle and other tissues, glucose uptake is controlled by insulin, so hexokinase will not be significantly active except when insulin secretion is high.

Hexokinase acts at a constant rate in the liver, and so serves to maintain a constant supply of glucose 6-phosphate for the liver’s own metabolic needs.

The Km of glucokinase is higher than fasting concentrations of glucose, so it only has significant activity when there is a great deal of glucose coming into the liver from the gut after a meal. The more glucose there is available, the faster glucokinase will form glucose 6-phosphate.

The increased glucose 6-phosphate formed by glucokinase as glucose entering the liver increases is used for synthesis of glycogen and fatty acids.

Glucokinase provides the first stage in controlling the amount of glucose entering the peripheral circulation . The concentration of glucose in the hepatic portal vein may be 20 mmol/L or higher; in the blood leaving the liver (and hence in the peripheral circulation) the concentration of glucose is normally no more than about 10 mmol /L.

Froguel and coworkers (1993) reported studies of the glucokinase gene in a number of families affected by MODY, and also in unaffected families. They published a list of 16 variants of the glucokinase gene. All their patients with MODY had an abnormality of the glucokinase gene:

From data reported by Froguel P et al. 1993, ‘Familial hyperglycaemia due to mutations in glucokinase: definition of a subtype of diabetes mellitus’, New England Journal of Medicine, 328: 697-702

Click here for a copy of the genetic code to work out the amino acid changes

The mutation in codon 4 is a significant change, from aspartate (which is acidic) to asparagine (which is neutral). However, this is the 4th amino acid from the amino terminal of the protein, and is unlikely to be important for the folding of the protein to form the active site.

The mutations in codons 10 and 116 are silent, since they do not cause any change in the amino acid.

All of the other mutations lead to either premature termination of translation, and hence no active enzyme, or a radical change in the amino acid encoded, which may reduce the activity of the enzyme considerably, by reducing its Vmax or increasing its Km. Mutations that affect the amino acids that make up the catalytic or substrate-binding site may lead to complete loss of activity.

The results tell us that all patients with MODY had defective glucokinase, and the table shows us that all but one of the people with abnormal glucokinase also had MODY. This suggests very strongly that a defect in glucokinase is the underlying cause of MODY.

Since this study by Froguel et al., more than 130 MODY-associated mutations have been found in the glucokinase gene.

Froguel and coworkers (1993) studied the secretion of insulin in response to glucose infusion in patients with MODY and normal control subjects. They were given an intravenous infusion of glucose; the rate of infusion was varied so as to maintain a constant plasma concentration of 10 mmol/L.

Their plasma concentrations of glucose and insulin were measured in the fasting state and and after 60 minutes of glucose infusion:

From data reported by Froguel P et al. 1993, ‘Familial hyperglycaemia due to mutations in glucokinase: definition of a subtype of diabetes mellitus’, New England Journal of Medicine, 328: 697-702

In the fasting state the patients were able to secrete a more or less normal amount of insulin, although this was not sufficient to maintain a normal fasting blood glucose concentration. This suggests that although they can synthesise and secrete insulin (unlike type I diabetes), they cannot secrete an appropriate amount to reduce the abnormally high fasting blood glucose. This suggests that their problem may be an inability to secrete additional insulin in response to glucose. (This is unlike type II diabetes, where insulin secretion in response to glucose is normal or higher than normal, but tissues are insensitive to insulin).

In response to the glucose infusion, the patients are unable to increase their insulin secretion appropriately. This confirms that their problem is secreting insulin in response to increasing blood glucose.

From this information it seems most likely that the additional glucose 6-phosphate synthesised by glucokinase in response to increasing glucose entering the pancreas acts as the sensor of increased blood glucose and indirectly triggers secretion of insulin.

The patients can increase their secretion of insulin in response to glucose to some extent, but not as much as normal. This suggests that they are heterozygotes, with one normal and one abnormal gene for glucokinase. The result is about half the normal activity of glucokinase in the cells, and this is not enough to provide enough glucose 6-phosphate in response to increased glucose to stimulate full insulin secretion. Hence the dominant pattern of inheritance.

Glucose 6-phosphate is not the immediate signal for insulin secretion. Rather, it is metabolised, leading to increased formation of ATP. This leads to closure of an ATP-potassium channel, causing membrane depolarisation and opening of a voltage-gated calcium channel. The resultant influx of calcium ions leads to fusion of the insulin secretory granules with the cell membrane, and the release of insulin .

[The electron micrograph shows secretion of insulin by exocytosis from secretory granules that have fused with the cell membrane, from Alberts B, et al., Molecular Biology of the Cell (3rd Ed). Garland Publishing, New York, 1994]

There is some evidence that glucokinase is alsoexpressed in the alpha-islet cells of the pancreas, that secrete glucagon. Here the role of the additional glucose 6-phosphate produced in response to an increase in blood glucose is to inhibit the secretion of glucagon, presumably by the same mechanism as the stimulation of insulin secretion.

More about MODY

Altogether 11 types of MODY have been described, due to mutations in different genes (see http://omim.org/entry/606391 for these different types of MODY).

MODY 2, due to glucokinase deficiency, is characterised by moderate hyperglycaemia, but is not associated with the adverse effects of poor glycaemic control seen in other types of diabetes, and generally no treatment is required. More on MODY due to glucokinase deficiency at http://www.diabetesgenes.org/content/glucokinase and http://omim.org/entry/125851.

The following paper on mutations in glucokinase and MODY may be of interest. Note that when the paper was published MODY was considered to be a variant of type 2 diabetes, since patients were still able to secrete some insulin, but not enough to maintain normal glycaemia in response to a glucose load. MODY is now considered to be a separate type of diabetes, distinct from either type 1 or type 2.

Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1932-6.
Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships.
Gidh-Jain M, Takeda J, Xu LZ, Lange AJ, Vionnet N, Stoffel M, Froguel P, Velho G, Sun F, Cohen D, et al.

Abstract

The glycolytic enzyme glucokinase plays an important role in the regulation of insulin secretion and recent studies have shown that mutations in the human glucokinase gene are a common cause of an autosomal dominant form of non-insulin-dependent (type 2) diabetes mellitus (NIDDM) that has an onset often during childhood. The majority of the mutations that have been identified are missense mutations that result in the synthesis of a glucokinase molecule with an altered amino acid sequence. To characterize the effect of these mutations on the catalytic properties of human beta-cell glucokinase, we have expressed native and mutant forms of this protein in Escherichia coli. All of the missense mutations show changes in enzyme activity including a decrease in Vmax and/or increase in Km for glucose. Using a model for the three-dimensional structure of human glucokinase based on the crystal structure of the related enzyme yeast hexokinase B, the mutations map primarily to two regions of the protein. One group of mutations is located in the active site cleft separating the two domains of the enzyme as well as in surface loops leading into this cleft. These mutations usually result in large reductions in enzyme activity. The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft. These mutations show a small approximately 2-fold reduction in Vmax and a 5- to 10-fold increase in Km for glucose. The characterization of mutations in glucokinase that are associated with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinase catalysis and localized functionally important regions of the glucokinase molecule.

link to the abstract and free full text by clicking here and a recent review

Rev Endocr Metab Disord. 2010 Sep;11(3):179-83.
Mutations in pancreatic ß-cell glucokinase as a cause of hyperinsulinaemic hypoglycaemia and neonatal diabetes mellitus.
Hussain K.

Abstract

Glucokinase is a key enzyme involved in regulating insulin secretion from the pancreatic ß-cell. The unique role of glucokinase in human glucose physiology is illustrated by the fact that genetic mutations in glucokinase can either cause hyperglycaemia or hypoglycaemia. Heterozygous inactivating mutations in glucokinase cause maturity-onset diabetes of the young (MODY), homozygous inactivating in glucokinase mutations result in permanent neonatal diabetes whereas heterozygous activating glucokinase mutations cause hyperinsulinaemic hypoglycaemia.

link to the abstract and full text by clicking here

Key points from this exercise:

• If an enzyme has a Km that is significantly lower than the normal range of substrate concentration in the cell then it will act at a more or less constant rate regardless of changes in the availability of substrate.
• If an enzyme has a Km that is similar to, or higher than, the normal range of substrate concentration in the cell, it will show increasing activity as the concentration of substrate increases.
• In the liver, the low Km isoenzyme, hexokinase, always acts at a constant rate, since it is saturated at all normal concentrations of glucose in the liver. Its role is to provide a constant entry of glucose into glycolysis to meet the liver's metabolic needs.
• In the liver and pancreas, the high Km isoenzyme, glucokinase, permits increased metabolism of glucose when the plasma concentration rises above the normal fasting level. In the liver this provides the first step in the control of blood glucose concentration, trapping glucose in the liver as glucose 6-phosphate, which cannot cross the cell membrane, and leading to increased synthesis of glycogen and fatty acids.
• In the pancreas, glucokinase acts as the sensor of increased blood glucose, leading to increased formation and metabolism of glucose 6-phosphate. Metabolism of this glucose leads to an increase in ATP formation, which leads to closure of a potassium channel, depolarisation of the cell membrane and opening of a voltage-gated calcium channel. The increased intracellular calcium stimulates fusion of insulin secretory granules with the cell membrane and exocytosis - the secretion of insulin.