ENDOPLASMIC RETICULUM STRESS INCREASES GLUCOSE PRODUCTION IN
VIVO VIA EFFECTS ON LIVER GLYCOGENOLYSIS AND GLUCOSEGLUCOSE-6PHOSPHATASE ACTIVITY
(Cont.): Dong Wang, Yuren Wei, and Michael
Jon C.Methods
Gonzales,
J. Pagliassotti,
ti, Colorado
State
University.
FortWe next investigated whether TUN activated glucoseglucose-6Pagliassot
Figure
5. Plasma Glucose
Levels Prior
to and
During
G6Pase:
phophatase, an ER localized enzyme responsible for
G6Pase: G6Pase activity was determined on whole liver
Pancreatic Clamp
homogenates at G6P concentrations of 0, 2.5, and 10 mM as Collins, CO
dephosphorylation of glucoseglucose-6-phosphate to glucose.
ATF6
Basa
l
Insulin (µU/ml)
50
Glucose (mg/dl)
Glucose Production and/or
Blood
Liver
Kidney
Blood
30
20
C O N
TU N
20
40
Corticosterone (ng/ml)
Pancreatic
Clamp
Basa
l
100
Aim: Recent evidence suggests that the ER stress response can
lead to impairments in insulin secretion and insulin resistance.
Type 2 diabetes is characterized by impairments in insulin
secretion, insulin resistance, and overproduction of glucose by the
liver. The aim of this study was to examine the effects of ER stress
stress
on glucose production. We hypothesized that ER stress would
increase glucose production.
Methods: Male rats were 44-8 hours fasted. Rats were
anesthetized with 50 mg/kg of sodium pentabarbitol.
pentabarbitol. Catheters
were placed in the carotid artery (blood sampling), jugular vein
(infusions), and jejunal vein (treatment). Experiments were 90
minutes in duration.
Isotope Dilution: 6,6 2H2- Glucose was infused for the duration of
the experiment to estimate glucose production.
Pancreatic Clamp Technique: In all rats, somatostatin was
infused (2 µg/kg/min) to inhibit pancreatic insulin and glucagon
secretion. Insulin and glucagon were then replaced at basal
levels.
Treatment: Six rats were infused with tunicamycin (inhibits
protein glycosylation to induce ER stress) as the treatment
group. Four rats were infused with saline as the control group.
Sampling:
Sampling: Blood samples were taken throughout the 90 minute
experiment. A liver sample was taken prior to and following
experiments. A kidney sample was taken following experiments.
80
60
40
CO N
TUN
20
0
-2 0
0
20
40
T im e ( m in )
60
Clamp
300
200
CON
TUN
100
0
-20
0
20
Time (min)
40
60
500
l
20
40
0
60
20
40
Time (min)
10.0
7.5
5.0
2.5
CON
TUN
0.0
0
20
40
60
1. Increased expression of genes/proteins involved in
glycogenolysis,
gluconeogenesis,
gluconeogenesis, and/or glucose
release.
400
300
200
CON
TUN
100
0
-20
0
20
40
Time (min)
Values are mean±
mean±SDEV for both TUN and CON groups. TUN, n=6;
CON, n=4.
60
4
2
0
Liver
Kidney
Figure 8. Terminal Liver
Glycogen
CON
TUN
Increased glucose production in response to Tunicamycin could
result from:
Clamp
6
0
Values are mean±
mean±SDEV for both TUN and CON groups. TUN,
n=6; CON, n=4. *, significantly different from CON (p<0.05)
Figure 4. Plasma Corticosterone and Free Fatty
Acid Levels Prior to and During Pancreatic
Clamps
Basa
Pancreatic
Basa
Pancreatic
l
5
Time (min)
Values are mean±
mean±SDEV for both TUN and CON groups. TUN, n=6;
CON n=4.
400
CON
TUN
8
To determine whether the supply of glucoseglucose-6-phosphate
might also be increased we measured liver glycogen
concentrations in terminal liver samples.
10
Time (min)
60
P
Transcription
5
0
0
T im e ( m in )
XBP
1
10
*
*
15
0
0
-2 0
*
*
*
15
CON
TUN
*
Values are mean±
mean±SDEV for both TUN and CON groups.
TUN, n=4; CON, n=4. *, significantly different from
CON (p<0.05)
20
20
Pancreatic
Clamp
10
Removal
Figure 6. Glucose Production (Ra), Glucose
Uptake (Rd), and Glucose Clearance
40
IR E1
Glucose-6-Phosphatase
(nmoles/mg protein/30min)
Blood
45
Figure 3. Plasma Insulin and Glucagon Levels
Prior to and During Pancreatic Clamps
Folde
d
Protei
n
P
Blood
10
With the increase in glucose levels, we then examined whether this
this
was due to an increase in glucose production, decrease in glucose
glucose
removal, or a combination of both.
Rd (mg/kg/min)
Blood
Liver
30
GlucoseGlucose-6Phosphatase
12
60
2. Acute activation of glycogenolysis, gluconeogenesis, and/or
glucose
release.
Real Time
PCR analysis of phosphoenol pyruvate carboxykinase
(PEPCK, a rate limiting protein in gluconeogenesis) and glucoseglucose-6phosphatase (G6Pase, responsible for glucose release from
hepatocyte) demonstrated that these two genes were not increased
over the time course of the experiment.
60
25
Glycogen (nmole/mg)
Translation
Gol
gi
Blood
15
40
T im e (m in )
Tunicamycin+ (TUN, experimental group; n=6)
0
20
Values are mean±
mean±SDEV for both TUN and CON groups. TUN,
n=6; CON, n=4. *, significantly different from CON (p<0.05)
Glucose Clearance (ml/kg/min)
eIF2α
eIF2α
P
0
Insulin*(basal)
Free Fatty Acids (µmol/L)
P
P
CON
TUN
-2 0
Saline + (CON, control group; n=4)
Glucos
e
Figure 7. GlucoseGlucose-6-Phosphatase
Activity Following Pancreatic Clamps
100
0
Glucagon*(basal)
-30
60
GlucoseGlucose-6Phosphate
*
*
*
200
Somatostatin*
ER
Chapero
ne
Pancreatic
Clamp
Basa
l
300
6,6 2H2- Glucose*
60
Unfolde
d
Protein
PERK
Figure 2. Experimental Design
Infusions: (*, Jugular Vein; +, Portal Vein)
Glucagon (ng/L)
Figure 1. ER Stress
Response2
described by Nordlie and Arion4.
Glycogen:
Glycogen: Was determined on liver homogenates by alkaline
hydrolysis and alcohol precipitation5.
RealIQReal-Time PCR:
PCR: PCR was performed on transcribed cDNA using IQSYBR green master mix (Bio(Bio-Rad) and primer sets designed by the
Beacon designer program version 3.1.
Analysis:
Analysis: Plasma levels of glucose, free fatty acids, corticosterone,
glucagon, and insulin were measured using standard techniques.
Ra (mg/kg/min)
Abstract: Recent evidence suggests that endoplasmic reticulum
(ER) stress can induce impairments in both insulin secretion and
insulin action. The aim of the present study was to examine the
effects of ER stress on glucose production in vivo. Fasted rats
were anesthetized and catheters were placed in the carotid
artery, jugular vein, and jejunal vein. A pancreatic clamp was
performed in which somatostatin was infused to inhibit
pancreatic insulin and glucagon secretion. These hormones were
then replaced at basal levels. To examine the effects of ER stress
stress
on glucose production, 6,66,6-2H2 Glucose was infused in the
absence (CON, n =4) or presence of jejunal vein tunicamycin
delivery (TUN, n =6). TUN induces ER stress through inhibition
of protein glycosylation. Arterial insulin, glucagon,
corticosterone, and free fatty acid concentrations were constant
throughout experiments and were not different between groups.
Glucose concentration and production increased by 76.2±
76.2±24.2
mg/dl and 2.6±
±SDEV),
2.6±1.2 mg/kg/min (mean
(mean±
SDEV), respectively, in TUN,
but did not change in CON. Liver glucoseglucose-6-phosphatase
(G6Pase) and phosphoenolpyruvate carboxykinase mRNA were
not different between groups. Liver, but not kidney, G6Pase
activity (nmoles
/mg protein/30min) was increased in TUN
(nmoles/mg
(7.2±
(7.2±2.1) vs. CON (0.2±
(0.2±0.3). Liver glycogen concentration was
reduced by 62% in TUN vs. CON. These data suggest that
experimental induction of ER stress can increase the production
of glucose in vivo, in part, via activation of hepatic
glycogenolysis
and
G6Pase. Reticulum (ER) has an important
Background:
The
Endoplasmic
role in balancing protein load and protein folding. Pathological
Pathological
stresses can disrupt homeostasis, leading to the accumulation of
unfolded proteins, which are toxic to the cell. This imbalance, or
“ER stress,”
stress,” leads to an “ER stress response”
response” (Figure 1). This
response activates three membrane bound proteins which
ultimately leads to the acute inhibition of protein translation and
increased transcription of chaperone proteins and proteins
involved in protein degradation1.
CON
TUN
20
15
10
*
5
0
Liver Glycogen
Values are mean±
mean±SDEV for both TUN and CON groups.
TUN, n=4; CON, n=4. *, significantly different from CON
(p<0.05)
Summary: These data suggest that experimentally induced ER
stress increased glucose production in vivo. The data also
suggest that the increase in glucose production was due, in
part, to an increase in hepatic glucoseglucose-6-phosphatase activity,
and perhaps increased hepatic glycogenolysis.
Relevant References
1. Ozcan, U. et al. Science 306: 457-461, 2004.
2. Rutkowski & Kaufman TRENDS in Cell Biology, 14.1:20,
2004
3. Wang, D. et al. Endocrinology, 147:350-358,2006
4. Nordlie RC and Arion WJ. Methods Enzymol 9: 619-625,
1966
5. Good, C. et al. J. Biol. Chem. 100:485-491, 1933