Endocrine Pancreas - Hormone Biosynthesis and Neoplasms

Question 1

Insulin

Answer 1

• The physiology and biochemistry of insulin are quite significant because insulin plays an important role in fuel metabolism by promoting anabolic processes and inhibiting catabolic ones predominantly in muscle, fat and liver tissue.
• Insulin promotes storage of fuels as glycogen, triacylglycerols and proteins.
• An acute action of insulin is the stimulation of amino acid and glucose uptake into muscle and fat cells; hence, blood glucose and amino acids are lowered by insulin.

Question 2

Insulin and its Precursor Forms

Answer 2

• Insulin is synthesized and released by the β-cells of the pancreas with glucose being the primary secretagogue (a compound that promotes secretion).
• The two chains of the insulin molecule are found initially within a single polypeptide chain, preproinsulin, held together by the C-peptide (connecting peptide) and containing a hydrophobic signal sequence (alias signal peptide or leader sequence) consisting of 23 amino acids, at the N-terminus.
• The signal sequence is removed by proteolysis to produce proinsulin in the ER.
• Proinsulin is proteolytically activated to insulin (Figure 1) in the Golgi through initial cleavage by a trypsin-like enzyme followed by several cleavages by carboxypeptidase-like enzymes.
• The proinsulin molecule has less than 5% of the bioactivity of insulin.
• The A and B chains of insulin are held together by disulfide bonds.

Question 3

Answer 3

•Insulin synthesis and the packaging of the hormone into secretory granules proceed in an orderly fashion.

Following transcription in the nucleus (step 1), preproinsulin is synthesized by ribosomes on the rough endoplasmic reticulum (RER) (step 2).

• The hydrophobic “pre” sequence of preproinsulin is a signal peptide sequence because it ‘signals’ the opening of a channel to allow the nascent peptide (destined for secretion) through the membrane of the endoplasmic reticulum (ER) into the lumen (cisternal space) of this organelle. Once the channel opens, the signal sequence guides the growing polypeptide chain into the lumen of the ER. Only the polypeptide scheduled for translocation has access to the channel, thus preventing other substances from arbitrarily entering the lumen of the ER. Once the signal peptide has entered the lumen, it is soon removed by a signal peptidase located on the inner surface (within the lumen) of the ER membrane.
• When synthesis of preproinsulin is completed and the protein is entirely within the lumen, the channel closes. Molecular chaperones supervise the proper folding of the molecule to proinsulin, and the free SH groups of cysteine residues are oxidized to form disulfide bonds.
• After proinsulin forms, small transfer vesicles pinch off from the RER and the proinsulin molecule is transported to the Golgi apparatus (step 3).
• In the Golgi, proinsulin is packaged into immature secretory granules with prohormone-converting enzymes that remove two pairs of basic amino acids that join C-peptide to the A and B chains of insulin. Thus insulin and C peptide are produced in the secretory granules (step 4).
• Granules continue to mature (step 5) as they traverse the cytoplasm along microtubules toward the plasma membrane (step 6).
• Upon stimulation of the cell by glucose or other secretagogues, there is entry into the cell of Ca2+. Calcium signals the mature granules to fuse with the plasma membrane and induces contraction of microfilaments causing the secretory granules to discharge their contents (i.e., insulin, C-peptide, protease and residual amounts of proinsulin) into the extracellular fluid by exocytosis (step 7).

Question 4

C-Peptide in Diagnosis

Answer 4

• C-peptide released into the blood is cleared more slowly than is insulin. Consequently, the concentration of C-peptide can be used to assess the rate of insulin secretion.
• For instance, after an overnight fast the concentrations of C-peptide would be barely detectable, whereas a patient with an insulinoma would show markedly elevated amounts of C-peptide.
• In patients with type I DM, because synthesis of insulin no longer occurs, no C-peptide is detectable.

Question 5

DM I

Answer 5

•One disorder associated with pancreatic beta-cells is type 1 diabetes mellitus, which is a result of a severe absolute lack of insulin caused by a reduction in beta-cell mass.

-Reduction in beta-cell mass is caused primarily by autoimmune destruction of these cells, where T-lymphocytes mount an immune response against pancreatic β-cell antigens. Histologically, an infiltration of small lymphocytes is seen attacking the islet cells (Insulinitis).

•As in all autoimmune diseases, patients have a genetic predisposition.

-Autoantibodies to β-cells are present in patients with type 1 DM and their family members.

•The HLA (human leukocyte antigen) locus is found on chromosome 6p21.

• Amongst Caucasians, 90-95% with Type 1 DM have HLA-DR3 or –DR4 haplotypes.
• Overall 40-50% of patients with type 1 DM are HLA-DR3/DR4.
• Other non-HLA factors may play a role including short nucleotide sequences in the promoter region of the insulin gene, genes that play a role in T-lymphocyte activity (CTLA4, PTPN22), and expression of cytokine receptors necessary for T-lymphocyte function (IL-2R).
• Viral infections (e.g., mumps, rubella, coxsackie B, CMV and others) often precede onset of type 1 DM.

Question 6

Rapid Acting Insulin

Answer 6

• lispro, Aspart, Glulisine
• They produce the typical anabolic effects on metabolism including increased storage of glucose as glycogen in liver and muscle and increased storage of triglycerides in fat cells.
• Additionally they boost muscle protein synthesis.
• Clinically they are used in treatment of type 1 DM but also are used in treatment of some patients with type 2 DM as well as gestational diabetes.
• Regular human insulin is considered a short-acting type and it provides postprandial glucose control as well as being used intravenously in the treatment of diabetic ketoacidosis (DKA).
• Additionally, this form can be used in patients with hyperkalemia or stress-induced hyperglycemia.

Question 7

Intermediate Acting Insulin

Answer 7

• NPH
• DM 1, DM 2, GDM

Question 8

Long Acting Insulin

Answer 8

• glargine, detemir
• mainatin basal glucose control
• DM 1, DM 2, GDM, basal GC

Question 9

DM 2

Answer 9

• Type 2 DM occurs when peripheral insulin resistance leads to ‘relative’ insulin deficiency.
• In a patient with type 2 DM, C-peptide may be elevated at the outset of the disease when the patient is hyperinsulinemic due to hyperglycemia.
• However, over time insulin secretion, and hence secretion of C-peptide, often diminishes because the pancreatic β-cells can become overtaxed and respond by decreasing their production of insulin.
• Hence measurements of C-peptide may help to monitor the progress of this disease.

Question 10

Glucagon Biosynthesis

Answer 10

• Glucagon is a peptide hormone that works to raise the concentration of glucose and fatty acids in the bloodstream, and is considered to be the main catabolic hormone of the body.
• Glucagon, like insulin, is translated as a pre-prohormone; it is synthesized in both pancreatic alpha-cells and in small intestinal L-cells of the duodenum
• The “pre” portion is a signal peptide sequence, and processing proceeds similarly to that of insulin.
• Glucagon, unlike insulin, contains only a single polypeptide chain.
• In the pancreatic alpha-cells, cleavage of proglucagon leads to secretion of glucagon as the most important product with additional pieces including a glicentin-related polypeptide (GRPP) and a large fragment containing glucagon-like peptides (GLP).
• The proteolytic cuts made in proglucagon in the intestinal cells yield a different mixture of products that include glicentin, and two glucagon-like peptides.

Question 11

Glicentin

Answer 11

• intestinal proglucagon —> glicentin and two glucagon like peptides
• Glicentin, a hormone, promotes insulin secretion (incretin effect), inhibits secretion of gastric acid and controls gut motility.
• Glicentin also may be cleaved to GRPP and oxyntomodulin.

-The release of oxyntomodulin after a meal may aid in reducing further food intake by suppressing appetite.

Question 12

GLP I

Answer 12

• intestinal proglucagon —> glicentin and two glucagon like peptides
• GLP-I is secreted from the L-cells in response to dietary essential amino acids derived from a high protein diet and in response to fatty acids.
• GLP-I enters the portal system for transport to the liver where it can promote glucose production akin to the effect of glucagon following food deprivation.
• GLP-I also acts as an incretin.

Question 13

Insulin Release Stimulation

Answer 13

• GLUCOSE
• glucagon
• leucine
• ketone bodies
• acetylcholine
• incretins
• GIP
• GLP-I

Question 14

Insulin Release Inhibition

Answer 14

• hypoglycemia
• symapthetic input during stress

-epinephrine

Question 15

Glucose Signal and Biphasic Release

Answer 15

• Increased plasma glucose concentration is the most important physiologic regulator of insulin secretion.
• Secretion is initiated at glucose concentrations greater than 100 mg/dL.
• Insulin secretion in response to glucose is biphasic.
• The immediate first-phase response begins within 1 min, peaks at 3-5 minutes and lasts about 10 minutes.
• When the blood glucose remains high, a second-phase ensues characterized by a more gradual, prolonged period that terminates soon after the glucose stimulus is removed.

•While the first-phase causes release of insulin containing granules at the cell surface, the second-phase involves secretion of both stored and newly synthesized insulin.

Question 16

Incretins

Answer 16

• The glucose-dependent secretion of insulin is enhanced by two hormones produced in the enteroendocrine cells of the small intestine. These hormones, glucose-dependent insulinotropic peptide (GIP) and glucagon like peptide-I (GLP-I) are termed incretins.
• This enhancement of insulin secretion is referred to as an incretin effect.
• Dietary glucose triggers the release of the incretins as a means of boosting insulin secretion, after a meal, to prevent hyperglycemia.
• As noted above, GLP-1 secretion can also be triggered by essential amino acids from dietary protein as a way to enhance gluconeogenesis using these precursors.

-On a high protein-low carbohydrate diet, dietary glucose is limited so that the dietary amino acids must be converted to glucose via gluconeogenesis to maintain blood glucose concentration.

• GIP originally was called gastric inhibitory peptide, however its inhibitory effect on gastric secretion is not physiologically important because the effect is seen only at a pharmacologic dose.
• The effects of GIP and GLP-I are additive.
• It is noteworthy that in patients with type 2 DM the effect of GIP is lost whereas the effectiveness of GLP-I is retained. Hence GLP-I receptor agonists have been a focus of potential treatment of patients with type 2 DM, as a way to boost insulin secretion to, in part, overcome effects of insulin resistance.

Question 17

How Soes Glucose Stimulate the Release of Insulin?

Answer 17

1. Glucose neters the cell and is phosporylated by glucokinase.
2. Glucose prevents Ca2+ from leaving the cell, and enhances its uptake.
   - An increased ratio of ATP to ADP cause the closing of channels that normally let K+ to enter —> depolarization! which generates action potentials that allow voltage sensitive Ca2+ channels to open.
3. Glucose indirectly causes mitochondrial cells to release Ca2+.
4. The increase in Ca2+ causes the immediate secretion of insulin by triggering the exocytosis of existing secretory granules.
5. Ca2+ also increases biosynthesis of insulin by
   - increasing production of cAMP –> protein kinase A
   - increasing activity of protein kinase C
   - binding to calmodulin

Question 18

Parasympathetic Effects

Answer 18

• Muscarinic agonists (e.g., acetylcholine, ACh) from the parasympathetic nervous system operate through activation of the ACh receptor that ultimately leads to activation of protein kinase C.
• Additionally, acetylcholine promotes release of Ca2+ from intracellular stores via production of inositol trisphosphate (IP3) from the cleavage of phosphatidylinositol bisphosphate, a membrane phospholipid, by activated phospholipase C.

Question 19

Sympathetic Effects

Answer 19

• Epinephrine generally has a net inhibitory effect on insulin release except under conditions of very low concentrations.
• The pancreatic beta-cell contains alpha2- adrenergic receptors that decrease secretion by inhibiting cyclic AMP formation via Gi.

The pancreatic beta-cell also contains beta2-adrenergic receptors that have a higher affinity for epinephrine but are much less abundant than the alpha2- receptors.

-For that reason a high concentration of circulating epinephrine, such as occurs following trauma, will lead to suppression of insulin secretion to sustain an elevated blood glucose concentration.

Question 20

Glucagon/GLP - 1

Answer 20

• Because glucagon and glucagon-like peptide-I increase intracellular cAMP, both hormones increase the synthesis and secretion of insulin, albeit to a much smaller extent than does glucose.
• The rationale for the effect of GLP-I, an incretin, relates to its production and release being linked to ingestion of oral nutrients, especially amino acids.

-On a high protein-low carbohydrate diet, dietary glucose is limited so that the dietary amino acids must be converted to glucose via gluconeogenesis to maintain blood glucose concentration. The small amount of insulin released promotes the uptake of amino acids by various tissues, especially liver.

•The reason that glucagon increases secretion of insulin is more subtle.

-Insulin and glucagon act in concert to maintain normoglycemia. This concomitant release of a small amount of insulin with glucagon provides fine tuning control of blood glucose homeostasis during food deprivation.

Question 21

Glucagon Secretion

Answer 21

• The most potent stimulant to glucagon secretion is hypoglycemia (reduced blood glucose concentration).
• This effect is in accord with the counterregulatory roles of glucagon versus insulin.

-Unlike insulin, glucagon promotes catabolic processes but diminishes anabolic ones.

• As glucose concentration falls, release of glucagon increases to first promote glycogenolysis and then increase gluconeogenesis.
• Glucagon secretion is also increased during stress, due to epinephrine, to aid in the excessive production of glucose needed to deal with the ‘fight or flight’ response or to facilitate stabilization following trauma.
• Amino acids promote glucagon secretion just as they increase release of GLP-I.
• While glucagon increases the release of insulin, high concentrations of insulin suppress the release of glucagon.

-Consequently, in a poorly controlled patient with type 1 DM or patients with type 2 DM exhibiting insulin resistance, the blood concentration of glucagon may be abnormally elevated thus exacerbating the hyperglycemia associated with the disease.

Question 22

Answer 22

Question 23

Insulinoma

Answer 23

• Insulinoma, is the most common pancreatic endocrine neoplasm, is a tumor of pancreatic beta- cells.
• They produce enough insulin to cause clinical hypoglycemia (glucose concentration <70 mg/dL).
• The clinical presentation of an insulinoma is the Whipple triad that consists of

1) fasting hypoglycemia (<50 mg/dL),
2) hypoglycemic symptoms (e.g., lethargy, syncope and diplopia, and
3) resolution of symptoms once blood glucose is normalized by administration of IV glucose.

• Additionally, patients can exhibit central nervous system manifestations including confusion, stupor, or loss of consciousness.
• As noted above the amount of circulating C-peptide increases significantly.

-This finding differentiates this cause of acute hypoglycemia from exogenous injection of insulin.

•About 10% of cases are associated with Multiple Endocrine Neoplasia Type 1 (MEN-1) syndrome.

-MEN-1 is an inherited syndrome associated with tumors of various endocrine glands. Besides the pancreatic islets, these tumors primarily originate in the parathyroid glands and pituitary gland.

• Histologically this tumor is characterized by monotonous islands and cords of cells resembling a normal islet that may have amyloid deposition in the surrounding extracellular tissue.
• Treatment is surgical excision

Question 24

Answer 24

• Histological insulinoma (H&E stain x200).
• Trabeculae of epitheloid cells surrounding a central nuclei showing pleomorphism.
• Cells stained positively for chromogranin (tumor marker in neuroendocrine cells), synaptophysin (marker protein of neuroendocrine cells) and insulin but negative for somatostatin, glucagon, and gastrin

Question 25

Glucagonoma

Answer 25

• Glucagonoma is a tumor of pancreatic alpha-cells.
• This tumor causes overproduction of glucagon.
• Patients present with dermatitis (necrolytic migratory erythema), mild diabetes (hyperglycemia), deep vein thrombosis (DVT), declining weight and depression.
• Treatment includes octreotide (Sandostatin), a somatostatin analog, and surgery.

Question 26

Somatostatinoma

Answer 26

• Somatostatinoma is a tumor of pancreatic delta-cells.
• This tumor overproduces somatostatin leading to decreased secretion of multiple hormones including cholecystokinin, gastric inhibitory peptide (GIP), gastrin, glucagon, insulin and secretin.
• Consequently patients may present with diabetes/glucose intolerance, gallstones, steatorrhea (fat in the stool), and hypochlorhydria (low or even absent gastric acid secretion).
• Treatment includes surgical resection and octreotide for symptom control.

Question 27

VIPoma

Answer 27

• VIPoma is an islet cell tumor that induces a characteristic syndrome (associated with MEN 1 syndrome) caused by excess release of vasoactive intestinal peptide (VIP).
• Patients exhibit watery diarrhea, hypokalemia, and achlorhydria (absent gastric acid secretion).
• Pancreatic tail histology (H&E staining) - well differentiated neuroendocrine tumor. Biopsy shows solid sheets of neoplastic cells composed of relatively uniform cells with finely granular amphophilic and eosinophilic cytoplasm and a centrally located round to oval nucleus without distinct nucleoli.