Veterinary Endocrinology and Physiology

VPHY143

Exercise 10
Insulin-Induced Convulsion

Caeba, Judennesse Atria

del Mundo, Jona Marie
Nicolas, Halcyon Dawn

Dr. Mark Desamero

VPHY143 Laboratory Instructor

Introduction
Insulin is synthesized in the ribosomes of the rough endoplasmic reticulum as a
larger precursor peptide called proinsulin. It is then later on converted into a smaller
peptide, insulin in the golgi apparatus. In presence of increased glucose concentration in
the blood, insulin is secreted by the beta cells of the pancreas to lower the blood
glucose level by increasing the permeability of the cell membrane to glucose specifically
by all muscle cells and adipocytes. This occurs by binding of insulin to the receptors on
the cell membrane which will then trigger the autophosphorylation of tyrosine kinase
resulting in the expression of metabolic effects of insulin. When this happens, the cell
membrane of mycocytes and adipocytes, becomes highly permeable to glucose by
translocation of glucose transporter protein-4 (GLUT-4) from the cytosol to the cell
membrane. The increase in GLUT-4 then increases the transport of glucose into the cell.

Figure 1. Mechanism of action of insulin myocytes and adipocytes

(Chhabra, 2012)
Insulin is also degraded in the liver once it enters the portal venous system while
unextracted insulin enters the systemic circulation where it binds to receptors in target
sites. That is, the myocytes and adipocytes (Chhabra, 2012). In the liver, insulin inhibits
glucose production, this then, together with the increase in glucose uptake of myocytes
and adipoctyes would lead to decrease in the concentration of plasma glucose
concentration.

Figure 2. Glucose homeostasis mediated by insulin

(Chhabra, 2012)
However, in fasted state, there is a low insulin level. This then causes decreased
glucose uptake by myocytes and adipocytes, mobilization of amino acids and free fatty
acids and increased glucose production of the liver by gluconeogenesis and
glycogenolysis. These two processes are stimulated by glucagon, a secretion of the alpha

pancreatic cells. On the other hand, postprandially, there would be an increased glucose
concentration which would cause insulin levels to rise and glucagon levels to fall.
In this experiment, the effect of iatrogenic hyperinsulinism was demonstrated as
well as the comparison of the effectiveness of glucose and epinephrine in the treatment
of insulin-induced hypoglycemia was done.

Methodology
Materials
Four adult mice (1 control, 3 treatment)
distilled water
tuberculin syringe
50% sterile glucose solution
glucometer
regular insulin
epinephrine
gavage needle

Methods
Mice were labeled as A, B, C, and D. Two mice should have been fasted 24 hours
before the experiment. Blood glucose level will be initially measured using blood
collected from the tail vein. The behavior and respiratory rate will be observed.
After observation, four units of insulin will be injected intraperitoneally (IP) into
each mouse. Changes in respiratory rate and behavior will be observed again. The time
of the onset of tremors and convulsions will be documented by taking pictures and

videos. As soon as the tremors and convulsions are observed, blood glucose level would
be measured.
After the BGL measurement, either 0.5mL 50% glucose solution by gavage or 0.1
mL epinephrine intravenous (IV) should be administered. Further observations on the
time when tremors and convulsions ceased after the administration of glucose or
epinephrine.

Results
Table 1. Observed changes in RR after SC injection of Insulin.
Animal

RR

before

insulin RR after insulin injection

injection
A

156 bpm

166 bpm

150 bpm

92 bpm

148 bpm

134 bpm

172 bpm

135 bpm

A & B Non-fasted animals; C & D Fasted animals

Note: The normal respiratory rate of mice is 94-163 breaths per minute.

All fasted and non-fasted animals have upper range and high respiratory rate
before insulin injection. Then after insulin was injected, all animal subjects have lower
than the former recorded respiratory rate except for non-fasted animal B which has a
higher but within the normal range respiratory rate.

Table 2. Blood glucose level (mmol/L) after SC injection of Insulin

Animal

Just before insulin Time when tremors Time when tremors

injection

& convulsions was &

observed.

convulsions

ceased after admin

of

glucose

or

epinephrine.
A

6.3

1.8

3.1*

7.1

1.4

1.3*

5.7

1.2

(*)

4.2

(*)

(*)

(*) death just before blood glucose level was about to be tested and admin of Glu or E
NORMAL BGL OF MICE: 60-130 mg/dl (3.33 7.22 mmol/L)
A & B Non-fasted animals
A 0.1 ml Epinephrine
B 0.5 ml glucose
C & D Fasted animals
C 0.1 ml Epinephrine
D 0.5 ml glucose
Non fasted animals have upper range blood glucose level while fasted animals
have middle range blood glucose level. After tremors and convulsions were observed,
the blood glucose levels dropped less than the normal range. However, blood glucose
level fasted animal D was not recorded because of immediate death. After the tremors
and convulsions ceased in non-fasted animal A (epinephrine induced), blood glucose
elevated but still lower than the normal range while non-fasted animal B (glucose
induced), and the blood glucose level slightly fall by 0.1 mmol/L. blood glucose levels of
fasted animals C and D were not recorded because of sudden death.

Table 3. Time when tremors and convulsions observed and time when tremors and
convulsions ceased after administration of glucose or epinephrine
Animal

Time when tremors & Time when tremors &

convulsions observed

convulsions ceased after

admin

of

glucose

or

epinephrine
A

After 36 min

After 15 min

After 35 min

After 10 min

After 59 min

After 20 min

After 55 min

(*)

(*) death just before blood glucose level was about to be tested and admin of Glu or E
A & B Non-fasted animals
A 0.1 ml Epinephrine
B 0.5 ml glucose
C & D Fasted animals
C 0.1 ml Epinephrine
D 0.5 ml glucose
Non-fasted animals A and B convulsed approximately half an hour after insulin
was induced while non-fasted animals C and D took almost an hour before tremors and
convulsions were observed. The earliest time for cessation of convulsions was 10
minutes and was observed in non-fasted animal B treated with glucose then next was
non-fasted animal A treated with epinephrine and last was fasted animal C treated with
epinephrine. However, duration before cessation of convulsions was not observed in
fasted animal D treated with glucose due to sudden death.

Discussion
Insulin produced by the beta cells of the pancreas functions mainly in facilitating
cellular uptake of amino acids, potassium, phosphate and magnesium and by increasing
the permeability of cells to glucose.

Figure 3. Major effects of insulin

(source: Pineda, M.H. 2003. Mcdonalds Veterinary Endocrinology & Reproduction.
IOWA State Press. 212 State Ave. IOWA, USA. Pp. 144-156)

For fasted animals since the blood glucose level is low, lipolysis occur. In this
case, the muscle tissue depends on fatty acids as source of energy. Thus, there would be
increased consumption of oxygen since it is an important component of oxidation for
ATP production during catabolism. Ideally, fasted animals would have high respiratory
rate (upper range or higher) before insulin is induced since the body is trying to
compensate for the oxygen needed. The normal respiratory rate of mice is 94-163
breaths per minute.
For non-fasted animals, the pancreas is secreting large amount of insulin since
the blood glucose level is high. Sufficient amount of insulin is needed to provide the
body with its energy requirement while the extra insulin causes rapid transport of
glucose to muscle cells for glycogenesis. In this case, glucose is preferred over fatty acids

as source of energy. Ideally, non-fasted animals would have high respiratory rate (upper
range) before insulin is induced.
After insulin is administered, there would be interruption in the lipolysis through
the inhibition of hormone-sensitive lipase which is the enzyme that causes hydrolysis of
the triglycerides already stored in the fat cells. Also, there would be an increased cellular
uptake and utilization of glucose. In return, these anabolic processes would normalize
the oxygen requirement of the cells. Thus, there would be lower respiratory rate
compared to the previous respiratory rate before inducement of insulin.

Figure 4. The role of insulin on insulin-dependent and insulin-independent cells

(source: Pineda, M.H. 2003. Mcdonalds Veterinary Endocrinology & Reproduction.
IOWA State Press. 212 State Ave. IOWA, USA. Pp. 144-156)

The nervous system controls the body organ systems coordination with close
cooperation with the endocrine system. Glucose is the only energy substrate of the brain
specifically synthesized by the mitochondria of the neuronal cell bodies. Brain cells are
permeable to glucose and do not require insulin as intermediate. Therefore, it is
important to maintain the blood glucose level above the critical level because when it

falls down (in cases of hypoglycemia), central nervous system disorders such as seizures,
weakness, lethargy, ataxia and strange behaviours can occur.

In cases of mild

hypoglycemia, tremors and weakness are observed when the blood glucose level falls
down to 3.3 3.8 mmol/L. On the other hand, convulsions and shock can be observed
in cases of severe hypoglycemia when the blood glucose level drops to 0.83 2.70
mmol/L. (Cunningham and Klein; 2007) this is true for the obtained blood glucose
levels of animals B and C after convulsions were demonstrated where in the levels are
within the 0.83 2.70 mmol/L.

Figure 5. Sodium-potassium pump

(source: online.science.psu.edu)
Generally when there is sufficient amount of glucose in the brain, there would be
normal ion influx (Na+/K+ pump), osmolarity and maintained hyperpolarization of
neurons. The ATP driven sodium-potassium pump maintains an artificially low
concentration of sodium and high concentration of potassium in the intracellular space,

which generates a resting potential difference on the order of -75 mV. (Reece, 2004)
Normal motor control, sensory perception, behavior, and autonomic function are
observed.

However, when there is hypoglycemia or depletion in glucose levels, the ioninflux is impaired resulting to sodium leakage that in return would cause incontrollable
depolarization in the neurons. This leads to alteration in the motor control, sensory
perception, behavior, and autonomic function and even damage in the neurons. It is
when tremors, convulsions and shock occur. (Guyton and Hall, 2006)

Figure 5. Insulin released after induction of glucose

Figure 6. Factors affecting insulin secretion

(source: Pineda, M.H. 2003. Mcdonalds Veterinary Endocrinology & Reproduction.

IOWA State Press. 212 State Ave. IOWA, USA. Pp. 144-156)
Administration of 0.5 ml 50% glucose solution triggered the feedback mechanism
of insulin to normalize the blood glucose level and proceed with glycogenesis and
lipogenesis. There would be a direct response due to the presence of incretin in the
gastrointestinal tract. On the other hand, administration of 0.1 ml epinephrine has an
antagonizing effect on insulin. It is a potent inducer of glycogenolysis in the liver thus
increasing the glucose levels in the blood. As a result, there would be stabilization of the
blood glucose level. The net result of these events would be stabilization of the
osmolarity and ion-influx in the brain and therefore lead to cessation of convulsions.
However, cases of death occurred due to severe hypoglycemia that led to shock.

References
Cunningham, SJ & B.G. Klein. 2007. Textbook of Veterinary Physiology. 4th ed. US
Elsevier Saunders. Pp. 66, 68, 72-73
Guyton AC & Hall E. 2006. Textbook of Medical Physiology.11th ed. US Elsevier
Saunders Inc. pp 512, 962-973.
Pineda, M.H. 2003. Mcdonalds Veterinary Endocrinology & Reproduction. IOWA State
Press. 212 State Ave. IOWA, USA. Pp. 144-156
Reece, W.O. 2004. Dukes Physiology of Domestic Animals. 12thed. USA: Cornell
University Press. Pp. 657-659.
The Endocrine Pancreas, Daniel E. Peavy Ph.D Medical Physiology 2nd ed. p. 595-596.
Mauricio Pineda and Michael Dooley. 2003: McDonalds Veterinary Endocrinology and
Reproduction. 5th edition. PA Martin and MH Crump p. 148- 150