Insulin and Sugars

Diabetes

Diabetes was recognized as early as 1500 BCE. Diabetes is a disease in which glucose cannot be utilized properly. There are basically two types of diabetes. Type 1 diabetes occurs, typically in the young, when the β-cells of the pancreas have been destroyed (by viruses or autoimmunity or both) so that insulin secretion cannot take place when the glucose level in the blood is high. Type 2 diabetes occurs when β-cells are at least somewhat compromised (insufficient insulin is produced) or when glucose utilization is impaired, perhaps by a fault in the insulin receptor (IR) (insulin resistance) or in the glucose transporter. Several variant genes contribute to both types of diabetes. Obesity is a major factor in the development of type 2 diabetes. About 5%–10% of diabetics are type 1 and the rest are type 2. About 4% of pregnant women (about 135,000 each year in the United States) experience gestational diabetes . Chronic stress can cause diabetes by the responsive release of cortisol from the adrenal cortex so that glucose is increased in the blood by about 10% (cortisol affects peripheral tissues to suppress glucose uptake in those tissues), enough to elicit insulin release from the β-cell. Continuous demand for insulin release can exhaust the β-cell over time and cause diabetes. About 20 million persons in the United States have a condition known as “ prediabetes ” where the blood glucose is elevated but not high enough to be classified as type 2 diabetes. An American-Caucasian with a blood glucose value of 200 mg/dL (1 dL is 10% of a liter or 100 mL) has a 20% chance of developing diabetes. Lack of exercise and a high sugar (and fat) diet are contributors. The glucose tolerance test is used to indicate diabetes or prediabetes. An oral dose of glucose (usually 75 g for adults ingested within 5 minutes) is followed by blood sampling at zero time (fasting) and at 2 hours (or an intervening measurement could be made at 30–90 minutes). The test is performed in the morning after an 8- to 12-hour fast. Typical results are shown in Table 6.1 .

The genetic backgrounds of type 1 and type 2 diabetes are complicated. Many genes seem to be involved in both cases. Of interest is the relationship between obesity and type 2 diabetes. Both obesity and diabetes have become epidemic in the United States. For adults as well as for children, 80% of type 2 diabetes is due to obesity. Recent research in Australia indicates that fat cells produce pigment epithelium–derived factor ( PEDF ) that generates events leading to type 2 diabetes. As a result of PEDF action, the response to insulin in liver and muscle is reduced (insulin resistance). The pancreas responds to this situation by producing more insulin, eventually leading to exhaustion of the β-cells so that insulin release is slowed or stopped altogether, generating type 2 diabetes. Thus the more fat tissue , the greater production of this factor and the greater likelihood of developing type 2 diabetes. Inhibition of the action of PEDF, interestingly, reverses its negative effects. Likely, there are other factors (e.g., reduction in circulating adiponectin , a protein hormone derived from fat cells; less adiponectin is secreted by fat cells in obesity) issuing from obesity (inflammation, chemokines, and others) that contribute to the development of type 2 diabetes.

The cellular utilization of glucose depends on the normal functioning of the IR and the IR–signaling pathway. Insulin binds and activates the IR , actions of which reduce circulating (glucose) by stimulating glycogen synthesis. In type 1 diabetes, owing to the destruction of the β-cells, insulin is not present and (glucose) increases in the blood. High blood glucose can cause all sorts of problems. For example, hemoglobin can become glycosylated, affecting its function and possibly contributing to heart disease (even insulin, itself, can become glycosylated). Ingestion of fructose (half of the sucrose molecule and the major component of corn syrup and honey) will be converted quickly to fatty acids and to fat (since its metabolism, in glycolysis, skips two enzymatic steps, compared to glucose, facilitating conversion of fructose to pyruvate, acetyl Coenzyme A, and fatty acids). In type 2 diabetes, insulin utilization is subnormal and blood glucose is not converted appropriately to glycogen storage leading to a rise in blood (glucose). This reduced glucose utilization could be the result of an altered or “masked” IR that functions poorly or due to some other defect in glucose utilization. Possible overall scenarios are summarized in Fig. 6.1 .

β- cells are located in the pancreas that is located below and behind the stomach. The pancreas is divided between the exocrine and the endocrine pancreas . The exocrine pancreas produces digestive enzymes that are secreted into the intestinal tract. The endocrine pancreas produces hormones, primarily insulin (β-cell), glucagon (α-cell), and somatostatin (δ-cell). Cells of the endocrine pancreas are located in islets of Langerhans . A drawing representing an islet of Langerhans is shown in Fig. 6.2 .

After insulin is secreted by the β-cell in response to signals (see p. 138 and Chapter 19), insulin circulates in the bloodstream and binds to IRs in the cell membranes of various tissues (e.g., liver and muscle) where, through signaling pathways, various effects can be generated, including those leading to the reduction in circulating (glucose). Depending on the pathway activated by the insulin–IR complex, there can be mitogenic effects (DNA synthesis, transcription) and important metabolic effects (protein synthesis, lipid synthesis, and glycogen synthesis). In Fig. 6.3 are shown models of the IR. On the left is the aporeceptor. On the right is the receptor after binding insulin ( red ), the holoreceptor. The insulin-induced conformational change in the receptor is shown. These effects occur through the activation of the IR and its cytoplasmic tyrosine kinase, followed by the phosphorylation of IRS-1 (IR substrate-1) and IRS-2 (IR substrate-2) and a phosphorylation cascade. IRS stimulates PI3-kinase that activates glucose transport by GLUT4 (glucose transporter 4) and the conversion of intracellular glucose to glucose-6-phosphate, a precursor of UDP-glucose (uridine diphosphate glucose). These effects are summarized in Fig. 6.4 .

As shown in Fig. 6.4C , phosphoinositol-3 kinase ( PI3K ) is activated through IRS-1 that was phosphorylated by the activated IR. The phosphorylated IRS-1 serves as the docking site for the SH2 domains (Src homology 2 domains) of the regulatory subunit (p85) of PI3K which leads to the formation of PI(3,4,5)P3 (phosphatidylinositol tris phosphate). In turn, PI3K stimulates glucose transport into the cell (more GLUT4 from the cytoplasm to the cell membrane) and glycogen synthesis. The activation of PI3-kinase by the IR is shown in Fig. 6.5 .

The SH2 domain is contained in the Src oncoprotein and in some 115 human proteins. It has 2 α-helices and 7 β-strands and is about 100 amino acids long. It has a high affinity for phosphorylated tyrosine in proteins and the optimal motif it recognizes to which it binds is Tyr–P–Met–Glu–Pro (in general, the binding motif is Tyr–hydrophilic residue–hydrophilic residue–hydrophobic residue).

Following the binding of insulin to the IR and IR activation leading to phosphorylation of the component tyrosine kinase , multiple signaling pathways can be activated. These include glycogen synthesis, signal transduction, and growth regulation (insulin can act as a mitogen ), and these effects are summarized in Fig. 6.6 .

A mitogen is a substance that stimulates cell division. These signaling pathways are complex and involve other kinases [mitogen-activated protein kinase kinase (MEK), mitogen-activated protein kinase, PI3K, phosphoinositide-dependent kinase, protein kinase C (PKC), glycogen synthase kinase-3, and others]. The pathway on the left describes the growth stimulation that can be affected by the activated IR and pertains to cells that are capable of dividing. On the right the pathway is described in which the activated IR leads to glucose utilization. GLUT4 , a glucose transporter, is moved from the cytoplasm to the cell membrane (muscle and adipose tissues) where it facilitates the movement of glucose from outside of the cell to its interior. Once inside, glucose can be acted on by hexokinase to form glucose-6-phosphate, an intermediate in glycogen formation. If there is an immediate demand for energy, glucose will transit through glycolysis and the citric acid cycle.

Insulin

Insulin is synthesized in the β-cells of the pancreas as the preproprotein, preproinsulin . At the N -terminal end, there is a signal peptide of 24 amino acids that is connected to the B chain at its N -terminus (signal-peptide Ala to B-chain Phe). The signal peptide is first cleaved at Ala–Phe to produce proinsulin (connecting peptide, B chain and A chain). Three disulfide bonds are formed (B7–A7, B19–A20, and A6–A11). This is transported into vesicles budded from the Golgi apparatus. In this location the connecting peptide (containing 33 amino acids) that has cleavable amino acids on either end, –R–R– (–Glu–Arg–Arg–Thr–) on the amino end next to amino acid 31 (Glu) and –K–R– (–Gln–Lys–Arg–Gly–) at the N -terminal end next to amino acid 1 (Gln) is removed by convertases 1 and 2 at the indicated cleavage sites ( Fig. 6.7 ).

The released, active monomeric form of insulin is shown in Fig. 6.8 .

Insulin exists in solution as monomers, dimers, or as crystals of hexamers in the presence of zinc ions. Acidity favors the formation of dimers. It is primarily in the monomeric form in the blood. However, in the storage form in the β-cell of the pancreas, insulin may be in the hexameric form with two zinc ions per hexamer and it may be secreted in this form. The two zinc ions are in the center of a doughnut-like shape and are chelated by three His residues in the 10th position of the B chain (see Fig. 6.8A ). A molecular three-dimensional model of the insulin hexamer is shown in Fig. 6.9 .

Insulin is in the monomeric form at physiological concentrations of about 1 ng/mL. At higher concentrations, it forms dimers and in the presence of zinc, it forms hexamers. Two insulin monomers bind to the α-subunits of the IR. Pockets in the α-subunits of the IR interact with two regions of the insulin molecule ( legend , Fig. 6.4 ) generating a conformational change in which the extracellular portions of the IR approach each other ( Figs. 6.4 and 6.5 ). This results in the activation of the receptor’s tyrosine kinase which autophosphorylates the β-subunits. The β-subunit tyrosine kinase, in turn, phosphorylates various IR substrates in the cell, such as IRS-1 and IRS-2 and associated binding protein Grb that are intermediates in the signal transduction pathway ( Fig. 6.6 ). A motif in the juxtamembrane (inner cell membrane region) is NPEY (Asn–Pro–Glu–Tyr) contacts the receptor tyrosine kinase to various signaling molecules. Thus IRS-1 and Shc both interact with this motif and mutation of this sequence impairs the phosphorylation of these molecules and interferes with the normal movements of the IR and of insulin. Aberrant NPEY motif may cause type 2 diabetes .

The Pancreatic β-Cell and Insulin Secretion

Insulin is produced only in the β-cell. Elevated levels of circulating glucose can trigger the β-cell to secrete insulin. Elucidation of the pathway connecting elevated glucose to the release of insulin by the β-cell has been laborious and difficult. All of the answers may not yet be known; however, there is now a good working model to explain the phenomenon. The signaling pathway leads to an increase in the intracellular concentration of calcium ions that cause the release ( exocytosis ) of insulin from the secretory granule. An increase in the level of glucose in the blood of 10% or more is sufficient to evoke the release of insulin from the β-cell. Electron micrographs show the release of insulin from an insulin-containing granule in Fig. 6.10 .

The overall mechanism of insulin secretion in response to elevated extracellular glucose is shown in Fig. 6.11 . Glucose is transported into the β-cell by GLUT2 and phosphorylated by glucokinase to glucose-6-phosphate that is metabolized through glycolysis (to pyruvate) and the citric acid cycle to produce adenosine triphosphate (ATP). The elevated ATP/adenosine diphosphate ratio inhibits the ATP-sensitive K+ channel which causes depolarization of the cell membrane leading to the uptake of Ca ²⁺ from the outside. Calcium ions are the trigger for fusion of the insulin-containing granules to the plasma membrane.

An important glycoprotein in the granule membrane is 64-kDa Phogrin . The interaction of the insulin-containing granule and the inner cell membrane occurs in three phases, docking, priming, and fusion. Other proteins involved in docking are SNAP-25 and SNARE . Phogrin is phosphorylated in response to secretory stimuli and this phosphorylation inhibits its activity as a phosphatidylinositol phosphatase , dephosphorylating PIP2. Phosphorylated Phogrin in the insulin granule membrane attaches to the inner plasma cell membrane. The events of the secretion mechanism involve several components. After docking, α-SNAP activates N -ethylmaleimide sensitive factor that hydrolyzes ATP and activates SNARE proteins. A second ATP-dependent step generates PIP2. Synaptotagmin is the proposed Ca ²⁺ sensor and a fusion clamp that prevents fusion of the granule until there is a Ca ²⁺ signal. Thus after granule priming, Ca ²⁺ is the limiting signal for granule fusion with the inner plasma membrane. Autoantibodies to Phogrin are apparent in most prediabetics and these antibodies are diagnostic of the prediabetic state .

A powerful system supporting the release of insulin from the β-cell is the activity of the intestine following a meal. It is known that measuring the increase in plasma (insulin) with time after parenteral injection of glucose compared to oral ingestion of glucose results in far greater levels of plasma insulin after oral ingestion. The agents responsible for the higher plasma insulin levels after ingesting glucose are the incretins that consist of the glucose-dependent insulinotropic polypeptide ( GIP ) and glucagon-like peptide-1 ( GLP-1 ). In response to ingested nutrients in the gut, the K cells of the duodenum and proximal jejunal linings as well as the L cells of the ileum and colon linings secrete GLP-1 and GIP. These hormones are absorbed through the intestine, enter the bloodstream, and bind to their respective receptors on the membranes of the β-cells. They stimulate the production of IP3 and diacylglycerol that result in an increase in the secretion of Ca ²⁺ from the internal storage sites of Ca ²⁺ in the endoplasmic reticulum (ER) (IP3 activity) to the cytosol and the activation of protein kinase A (PKA). Uptake of Ca ²⁺ is increased from the outside. The increase of intracellular Ca ²⁺ causes the immediate secretion of insulin from the storage granules. The activation of PKA results in the phosphorylation of MEK–ERK that stimulates the production of preproinsulin messenger ribonucleic acid (RNA) in the nucleus, accounting for an increase in the contents of the insulin storage granules. These events are summarized in Fig. 6.12 .

In addition to the direct effects of incretins on the β-cells, there may be effects of GLP-1 on neuronal pathways. Thus there are two pathways, at least, contributing to the secretion of insulin from the β-cells. The direct effects of glucose ( Fig. 6.11 ) are supported by the effects of the incretins . The sequences of GIP and GLP-1 are shown in Fig. 6.13 .

Cleavage and inactivation of the incretins occurs by the action of dipeptidyl peptidase IV ( DPP IV ), cutting between the amino acids, A–E, in the N -terminus of GIP and between the amino acids A–E near the N -terminus of GLP-1. Inhibitors of DPP IV become important for the potential treatment of type 2 diabetes since inhibiting the inactivating enzyme and prolonging activity of the incretins increases the secretion of insulin from the pancreas. Some of the inhibitors being tested clinically are shown in Fig. 6.14 .

These inhibitors could be important because, in addition to stimulating the glucose-dependent insulin secretion from the β-cell, incretins induce β- cell proliferation and enhanced resistance to apoptosis and GLP-1 is known to inhibit glucagon secretion . In addition to inhibitors of this enzyme, direct treatment of type 2 diabetes patients with incretins is being tested.

Glucagon-like peptides 1 and 2 are encoded by the proglucagon gene (on the long arm of chromosome 2; Fig. 6.15A ) and GIP is encoded by the ProGIP gene (on the long arm of chromosome 17; Fig. 6.15B ).

Detrimental Effects of Diabetes

Both type 1 and type 2 diabetes may lead to serious medical conditions as shown in Table 6.2 .

Although a number of experimental approaches have been tried in animals to restore functioning β-cells, the potential use of adult progenitor/stem cells seems most encouraging. Future clinical research in humans will determine this possibility.

Synthetic Sweeteners

Sucrose is cane sugar and the common sweetener. Emphasis on dieting has spawned a number of synthetic sweeteners. Although they contain no calories, experimentally there are downsides to their ingestion. Most of the studies showing pathology have utilized animals, especially rodents. It is not clear what the tolerance level is in terms of amounts ingested in the human before harmful effects may occur. Clearly, the usage of artificial sweeteners is now common. Some of these are derived from sugars and some of their structures are shown in Fig. 6.16 .

In experimental models, many using rodents, some of the artificial sweeteners have pathological effects; however, it is difficult to translate the amount of intake required to generate pathologies in humans. Saccharin was discovered in 1879 and is 300 times sweeter than sucrose and does not affect blood insulin level. Saccharin in rodents causes cancer, presumably at levels that would be unrealistically high if translated to the human. Sorbitol is metabolized more slowly than sucrose. In Fig. 6.17A and B are shown the location of sweet receptors near the tip of the tongue and details of the taste pore in part (A). In part (B) a surface model of the taste receptor is shown. It is thought that there is a three-point attachment of the sweet molecule to the receptor. In Fig. 6.17C is shown the structure of lugduname that is reported to be 225,000 times sweeter than sucrose.

Chemistry of Simple Sugars

Simple sugars are single molecules, whereas more complex sugars are more than one molecule joined together by chemical bonds. Sugars are made up of carbon, oxygen, and hydrogen; no nitrogen, except for amino sugars. The simplest sugars are monosaccharides . Six-carbon monosaccharides are prevalent but monosaccharides can have from three to seven carbons in their structures (three carbons, triose ; four carbons, tetrose ; five carbons, pentose , six carbons, hexose ; and seven carbons, heptose ). The hexoses are common and important. Glucose is the most representative member of the hexoses. The structure of glucose is shown in Fig. 6.18 in a simple stick model ( Fischer projection ) and how it is closed into a ring structure ( Haworth projection ). Sugars contain chiral carbons that are those carbons having four different substituents. If two of the substituents of a carbon atom are the same (e.g., double bond to an oxygen or single bonds to two hydroxyls or to two hydrogens) that carbon is achiral. Inspecting the open stick model of glucose, for example, there are four chiral centers at C2, C3, C4, and C5. The number of chiral carbons determines the number of stereoisomers , thus, for an aldotetrose with 2 chiral centers, the number of stereoisomers is 2 ² or 4 stereoisomers; for glucose with 4 chiral centers, it has 2 ⁴ or 16 stereoisomers.

There are a number of simple sugars ranging from three carbons (triose) to seven carbons (heptose), although sugars of six-carbon length (hexose) are considered here. A five-carbon sugar is a pentose and a four-carbon sugar is a tetrose. Sugars are either aldoses or ketoses; an aldose sugar has an aldehyde group (e.g., the C1 of d -glucose seen clearly in the open stick model); a ketose has a ketone group (e.g., the C2 of d -fructose clearly seen in the open stick model). The simple stick structures (Fischer projections) are shown in Fig. 6.19 .

The number of chiral carbons determines the number of stereoisomers. The four stereoisomers of a tetrose are shown in Fig. 6.20 .

In the process of cyclization of glucose, the carbon-1 carbonyl can be attacked from two sides generating the possibility of α- or β-forms, with the rearrangement of a proton in the process as shown in Fig. 6.21 .

The α- and β-forms in the ringed form of glucose are named based on the position of the hydroxyl on carbon-1 as shown in Fig. 6.22 .

β- d -glucose has the C-1 hydroxyl on the left and α- d -glucose has the C-1 hydroxyl on the right. “ d ” refers to dextro , or right (as with the amino acids). When the sugar is in the form of a ring, the carbon bonds can bend into either one of two forms: a “chair” or a “boat” configuration . One form may be favored over the other, depending on hydroxyl substituents in the ring. The general forms of these structures for a six-carbon sugar skeleton are shown in Fig. 6.23 .

Sugar rings do not form a flat ring like benzene; the benzene ring contains three double bonds that are shorter (1.34 Å) than carbon-to-carbon single bonds (1.40 Å). Simple sugars can be represented in four ways (not including the boat structure) as shown in Fig. 6.24 .

Disaccharides are formed using the α-(axial bond down) or the β-(equatorial bond up) hydroxyl on the ring as shown in Fig. 6.25 . The location of a specific substituent (in this case, hydroxyl group) as up or down stems from the stereoisomeric center ( anomeric carbon ) of the sugar. In the ring forms (e.g., chair) the anomeric carbon can be located as the carbon next to the ring oxygen not attached to CH ₂ OH. In the chair configuration, one hydrogen on each carbon is equatorial and one hydrogen is axial. The equatorial hydrogens radiate from around the ring, while the axial hydrogens point along an axis or parallel to an axis; axial bonds are upward or downward along an axis through the center of the ring. Also the hydroxyl substituent of the anomeric carbon can be either up or down. If it is down (axial), the sugar is in the α form, while if it is up (equatorial), the sugar is in the β form (e.g., α- d -glucopyranose or β- d -glucopyranose).

In Fig. 6.26 are shown some common monosaccharides and disaccharides in chair configuration.

There are three common disaccharides: sucrose, lactose, and maltose ( Fig. 6.27 ). Sucrose is a nonreducing disaccharide of glucose and fructose having a glucosyl α-1-fructosyl β-2 linkage ( Fig. 6.27 ). Sucrose is commonly used as a natural sweetener. The ingestion of sucrose is followed by a rise in serum glucose that subsides in the normal individual. In a diabetic the level of glucose in the serum is abnormally elevated ( glycemia ) because there is insufficient insulin made available or because insulin is not functioning properly ( insulin resistance ). Sucrose is also cariogenic (operating with oral bacteria to form dental cavities). Interestingly, honey from a stingless bee (as well as in a few other species) produces trehalulose , a relatively rare disaccharide of glucose and fructose that differs from sucrose ( Fig. 6.27 ). The bond between glucose and fructose in sucrose is a C1–C2 linkage, whereas in trehalulose the bond between glucose and fructose is C1–C1. Trehalulose is noncariogenic and nonglycemic and it has 60% of the sweetness of sucrose. If it could be produced commercially and economically, it would be a great improvement over sucrose for use as a sweetener .