DIABETES MELLITUS

Presentation by:
Dr. Petrescu Elena
Dr. Stanescu Raluca

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 Biosynthesis of insulin

• Insulin is a peptide hormone (51 amino acids) composed of two polypeptide

chains (A and B) linked by two interchain disulfide bonds
• It is synthesized in the β cells of the pancreas as an inactive single-chain precursor,
preproinsulin
• The “pre” signal sequence at the N-terminal end is cleaved in the ER lumen
• Proinsulin folds into the proper conformation, and disulfide bonds are formed → it
is transported to the Golgi complex
• In the Golgi complex proinsulin is cleaved by peptidases → the C-peptide is
removed and insulin is formed: A and B chains connected by the disulfide bonds
• The C-peptide has no hormonal activity, but its measurement in blood is a good
indicator of insulin endogenous production and secretion (it is released in
equimolar amounts to insulin)
• Insulin is included in storage vesicles together with zinc ions

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 Secretion of insulin
•Insulin secretion is stimulated by elevated glycemia
•Glucose enters the β cell via the GLUT2 transporter → it is metabolized through
glycolysis and Krebs cycle → the ATP levels within the cell increase → closing of the
ATP-sensitive K+ channels in the plasma membrane → membrane depolarization
•This leads to the opening of the voltage-gated Ca2+ channels in the plasma
membrane → influx of Ca2+ and increase in intracellular Ca2+ → this triggers the
release of insulin by exocytosis

•Plasma half-life = 5-6 min → degraded

by insulinase in liver and kidneys

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 Factors that affect insulin secretion

1. Increase of glycemia after carbohydrate meals → the rate of insulin release is

proportional to the glucose concentration
2. Certain amino acids (especially arginine) - can stimulate insulin secretion, after
protein ingestion
3. Epinephrine - decreases the release of insulin (through α2 receptors)

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4. Intestinal hormones (incretins) - secreted by the intestinal cells after contact with
food
• GLP-1 (glucagon-like peptide 1) and GIP (glucose-dependent insulinotropic
peptide)
• They cause a rise in insulin levels in the blood before the rising of glycemia
• GLP-1 causes a transient decline in glycemia level after meals → its plasma half-life
is only 2 min because it is degraded by dipeptidyl peptidase-4 (DPP-4 - a peptidase
that removes proline-containing dipeptides from the N-terminus of polypeptides)
• Inhibitors of DPP-4 (known as gliptins) are a class of oral hypoglycemic drugs that
can be used to treat diabetes mellitus type 2

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Glucose homeostasis

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 Metabolic actions of insulin
1. Effects on carbohydrate metabolism (exerted mainly in liver, muscle, and adipose)

•It increases glucose uptake (muscle, adipose) – by increasing the number of GLUT4
transporters in the plasma membrane
•It stimulates glucose degradation through glycolysis (liver, muscle)
•It stimulates glycogen synthesis (liver, muscle)
•It inhibits glycogen breakdown (liver, muscle)
•It inhibits gluconeogenesis (liver)

All these actions lead to the hypoglycemiant effect of insulin

2. Effects on lipid metabolism

•It stimulates fatty acid synthesis (liver)

•It promotes triglyceride synthesis (adipose)
•It inhibits lipolysis (adipose)
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 In summary, the effect of insulin is to favor
- the use of glucose as an energy source
- the conversion of excess blood glucose to two storage forms:
- glycogen (in the liver and muscle)
- triglycerides (in the adipose tissue)

3. Effects on protein synthesis

• In most tissues, insulin stimulates the entry of amino acids into cells, and protein
synthesis

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The actions of insulin

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Combined effects of insulin and glucagon on substrate flow between
liver, adipose tissue and muscle

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 Classification of diabetes mellitus
• Type 1 diabetes (insulin-dependent)
- results from β-cell destruction → insufficient insulin secretion
- it accounts for 5-10% of the diagnosed cases of DM
- it manifests before 20 years of age
- patients are prone to ketoacidosis

• Type 2 diabetes (non-insulin-dependent) - progressive insulin secretory defect on

the background of insulin resistance
- patients are not prone to ketoacidosis

• Other types of diabetes:

- genetic defects in β-cell function or in insulin action (receptor defects)
- hormonal – hypersecretion of hyperglycemiant hormones (cortisol, epinephrine,
glucagon, growth hormone)
- pancreatic diseases (inflammatory, hemochromatosis, etc.)
- iatrogenic (therapy with glucocorticoids, diuretics, etc.)

• Gestational diabetes mellitus – diabetes diagnosed during pregnancy 12

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Type 1 DM

Type 2 DM

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Progression of blood glucose and insulin levels in patients with
type 2 DM

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 Criteria for the diagnosis of diabetes mellitus

• Fasting plasma glucose ≥ 126 mg/dL (fasting = no caloric intake for at least 8h) at
more than one determination
or
• 2-h plasma glucose ≥ 200 mg/dL during an OGTT
or
• Hb A1c (glycated Hb) ≥ 6.5%

 Prediabetes (impaired glucose tolerance - IGT):

• Fasting plasma glucose 111-125 mg/dL
• 2-h plasma glucose during OGTT between 140-199 mg/dL
• Hb A1c 5.7-6.4%

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 Metabolic disturbances in DM
• The insulin deficiency leads to a decreased insulin/glucagon ratio

1. Carbohydrate metabolism
• ↓ glucose uptake in muscle and adipose tissue
• ↑ glucose production in the liver – stimulation of glycogenolysis and
gluconeogenesis
• => hyperglycemia; when it exceeds the renal threshold (180 mg/dL) → glycosuria
with osmotic diuresis and electrolyte depletion (polyuria→ polydipsia)

2. Lipid metabolism
• ↑ lipolysis in the adipose tissue => ↑ release of fatty acids => ↑ level of plasma
FFA (free fatty acids)
• ↑ uptake of FA in the liver => ↑ synthesis of triglycerides (TG) and VLDL =>
↑ level of plasma VLDL
• ↓ lipoprotein lipase (LPL) activity => ↓ catabolism of TG from chylomycrons and
VLDL => ↑ level of plasma TG
• From ↑ VLDL => ↑ LDL => ↑ level of plasma cholesterol 18
• ↑ activity of CETP (cholesterol ester transfer protein) => ↑ transfer of TG from
VLDL to HDL and of cholesterol esters (CE) from HDL to VLDL =>
- ↓ level of plasma HDL => ↑ atherogenic risk
- formation of LDL with ↑ CE (small dense LDL), which are more atherogenic
• Mainly in type 1 DM: the ↑ level of FA in the liver => ↑ amounts of acetyl-CoA
resulted from their oxidation => ↑ conversion to ketone bodies
- ↑ ketogenesis leads to ketonemia, ketonuria and ketoacidosis

3. Protein metabolism
• ↓ cellular uptake of amino acids and ↓ protein synthesis
• ↑ utilization of amino acids in gluconeogenesis
• Non-enzymatic glycosylation of proteins → negatively affects their structure and
function

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Metabolic disturbances in type 1 DM

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Metabolic disturbances in type 2 DM

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 Markers for monitoring DM
• There is now clear evidence that in both type 1 and type 2 diabetes, the incidence
of long-term complications can be reduced by achieving tight control → this
requires meticulous monitoring of glycemic control

 Fasting glucose
 OGTT
 Hb A1c, fructosamine
 Microalbuminuria
 Ketone bodies
 Insulin, C peptide
 Autoantibodies – to: insulin, β-cell of pancreatic islets, GAD
 Total cholesterol, LDL-C, HDL-C, TG

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 Fasting glycemia

• N = 70-110 mg/dL
• Monitoring of therapy is performed according to this parameter
• Especially in type 1 diabetic patients, insulin doses need to be frequently and
carefully adjusted on the basis of multiple daily blood glucose measurements

 OGTT (oral glucose tolerance test)

• Evaluates glucose clearance from the circulation after glucose loading under
defined conditions

• Recommended in the following conditions:

- to assess individuals who have borderline fasting glucose levels and are at risk for the
development of diabetes (relatives with DM, dyslipidemia, obesity, hypertension)
- hyperglycemia during pregnancy
- patients with nephropathy, neuropathy or retinopathy of unexplained origin

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• OGTT → standard conditions for the patient:
- a minimum carbohydrate intake of 150 g/day for 3 days before the test
- a minimum 8-hour fast before testing
- exercise, smoking and emotional stress should be avoided during the test

• The test:
- blood and urine are collected for glycemia and glycosuria determination
- the glucose load: 75 g of glucose in 250 mL of water
- after 2 h blood is collected again for glycemia measurement

• Interpretation of results:
Glycemia
Fasting 2h
Normal < 110 < 140
IGT < 126 140-199
(prediabetes)

DM ≥ 126 ≥ 200
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 Glycated hemoglobin (Hb A1c)

• A minor Hb derivative that is produced by the nonenzymatic covalent binding of

glucose to Hb
• The extent of glycation depends on the average plasma glucose concentration to
which the RBCs are exposed during the 120-day life span
• It represents a measure of the glycemic status over a period of approx. 2 months
• Normal values = 3 - 5.7 %
• It should be repeated once every 2-3 months
• Several trials have demonstrated that achieving lower levels of Hb A1c, ideally
towards or into the nondiabetic range, reduces the incidence of micro- and
macrovascular complications

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 Glycated albumin (fructosamine)

• The albumin molecule can be glycated at lysine residues

• Glycated albumin test reflects control of blood [glucose] over the previous 10-15
days (albumin has a plasma half-life of 20 days)
• Useful in gestational DM - when the glycemic status should be monitored more
frequently (once every 2-3 weeks)
• Glycation of structural proteins (e.g. the vascular and the glomerular basement
membrane) might be responsible for some of the long-term complications of
diabetes

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 Glycosuria
• Is a poor marker for DM
• The normal renal threshold for glucose is 180 mg/dL → in diabetic patients may be
increased
• Glycosuria may be associated with normal glycemia (renal glycosuria: decreased
renal threshold)

 Microalbuminuria

• Represents an urinary excretion of albumin above the normal level (which is ≤ 30

mg/24 h), but undetectable by the urinalysis dipstick tests

• It allows the detection of nephropathy onset – when the level is > 300 mg/24 h
• Monitoring patients with DM for microalbuminuria is recommended – a rigorous
control of diabetes delays the onset and progression of nephropathy

• Microalbuminuria is also defined as an urinary albumin/creatinine ratio ≥ 30 mg/g

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 Ketone bodies

• The KB are: acetoacetate, β-hidroxybutyrate, and acetone

• Testing with dipstick tests (nitroprusside test) – allows only the detection of
acetoacetate and acetone (that have a keto group)
• The acetoacetate:β-hydroxybutyrate ratio is normally 1:3 → in severe ketoacidosis
the β-hydroxybutyrate level increases significantly (AcAc:β-HB changes up to 1:30)
• In these conditions the nitroprusside test underestimates the severity of
ketoacidosis → the measurement of β-hidroxybutyrate in serum is recommended

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 Insulin and C-peptide

• Recommendations:
- to estimate the endogenous insulin secretion in a diabetic patient
- to detect the cause of hypoglycemia (e.g. insulinoma)
- to detect insulin resistance – when the subject has borderline or slightly increased
glycemia, or IGT at the OGTT, and insulin + C-peptide are increased

• Fasting plasma insulin levels:

- in type 1 DM - are low
- in type 2 DM - are normal or even elevated → in time they decrease

• The C-peptide measurement is more accurate than that of insulin due to a longer
plasma half-life (20-30 min), and it is imposed in patients who are on insulin
therapy or have insulin autoantibodies
• Insulin produced by the pancreas is extensively (approx. 50%) first-pass
metabolized by the liver → peripheral insulin levels may not accurately reflect
portal insulin secretion
• Peripheral C-peptide levels more accurately reflect portal insulin secretion (C-
peptide has negligible extraction by the liver)
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 Autoantibodies

• Insulin autoantibodies (IAA)

- present in 100% of diabetic children less than 5 years of age and in only 20% of
diabetic adults
- their presence imposes the measurement of C-peptide and not insulin

• Islet cell autoantibodies (ICA)

- appear in 90% of the patients with type 1 DM, frequently several years before
diabetes onset (predictive value)

• Autoantibodies to glutamic acid decarboxylase (GAD)

- present in 80% of the patients with type 1 DM, being detected several months/years
before diabetes onset
- their testing is recommended for first-grade relatives of type 1 diabetic patients → if
the subject is positive, he will usually develop insulin-dependent DM (predictive
value)

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 Acute complications of DM

1. Diabetic ketoacidosis (DKA)

•Complication of type 1 DM
•Caused by conditions that increase the need for insulin: acute stress, infection,
trauma, excessive physical exercise – these are associated with an increased release of
hyperglycemiant hormones
•It can be the first manifestation of type 1 DM

•Marked hyperglycemia ( > 250-300 mg/dL) → glycosuria with osmotic diuresis → loss
of Na+, K+, and water → hypovolemia
•Water and electrolyte loss due to vomiting increases fluid depletion
•Significant dehydration

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• Increased lipolysis → ↑ plasma FFA →
↑ ketogenesis in the liver → ketonemia,
ketoacidosis and ketonuria, the odor of
acetone on the breath
• ↑ [H+] → they enter the cells by exchange
with K+ → hyperpotassemia
• Acidosis is partially compensated by
hyperventilation (Kussmaul’s respiration)
• ↓ pH, ↓ [HCO3-]
• ↑ anion gap – caused by the
accumulation of sodium salts of keto-
acids
• ↑ plasma urea – due to dehydration
(with decreased GFR) and protein
hypercatabolism

Mechanism of increased ketogenesis in DKA

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Metabolic and clinical abnormalities in DKA

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2. Hyperglycemic hyperosmolar nonketotic coma (HHNC)

•Occurs in elderly patients with type 2 DM and with a compromised renal function →
they have important losses of water and electrolytes; usually is determined by stress
factors or major diseases

•Severe hyperglycemia (> 600 mg/dL)

•Dehydration and ↑ plasma osmolality (> 350 mOsm/kg)
•Normal or slightly low blood pH, normal ketone body levels, absent ketonuria

•The absence of KB is explained by the differential sensitivity of glucose and lipid

metabolism to insulin: lipid metabolism is much more sensitive (10 times) to insulin
action compared to glucose metabolism
•The insulin concentration required to oppose the ketogenic actions of glucagon is
lower than that required to prevent increased glucose production
•In type 1 DM, severe insulin deficiency leads to increased lipolysis and KB production
•In type 2 DM there is sufficient insulin to limit lipolysis and thus KB production, but
insufficient to prevent hyperglycemia

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 Diabetic HHNC
ketoacidosis
Glycemia > 300 mg/dL > 600 mg/dL
Glycosuria positive positive
Anion gap (N: 8-16 > 16 < 16
mmol/l)
Plasma osmolality ≤ 320 > 350
(N ≤ 320 mOsm/kg)
Ketonemia /-uria ↑ / positive normal / negative
HCO3- (mmol/L) < 15 > 20
pH < 7.35 7.35-7.45
C-peptide < 0.7 normal/↑
(N: 0.7-1.8 μg/L)

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3. Hypoglycemia

•Reactive hypoglycemia – is most commonly caused by accidental over-administration of

insulin or hypoglycemic drugs
•Alternatively - the patient may have missed a meal or performed excessive physical
exercise after the usual dose of insulin or oral hypoglycemic drugs
•Fasting hypoglycemia – may have several causes, including hyperinsulinism associated
with insulinoma, and some endocrine disorders
•Differential diagnosis may require measurement of blood glucose, insulin and C- peptide

•Hypoglycemia is particularly dangerous, and some patients lack awareness of this →

they lose warning signs such as sweating, dizziness and headaches
•This is probably the most common cause of coma seen in diabetic patients

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Major causes and clinical features of hypoglycemia

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 Bibliography

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Biochemistry. 5th edition, Lippincot Williams & Wilkins, 2011.
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University Press, Târgu-Mureș, 2015.
Lawrence A. Kaplan, Amadeo J. Pesce. Clinical chemistry;theory,analysis,
correlation, 5th ed. Elsevier Mosby, 2010

Simon W Walker, Geoffrey J. Beckett, Peter Rae, Peter Ashby. Lecture Notes:
Clinical Biochemistry 9th Edition, Wiley- Blackwell, New Jersey, 2013

Martin A Crook. Clinical biochemistry and metabolic medicine, 8th Edition,

Hodder Arnold, London, 2012.

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