Transcript Exploration of IR Markers, Focusing on Interleukin-6, within Southwest Native American Student Population Katie Zortman, Gabriel Thom, and Dr.

Exploration of IR Markers, Focusing on Interleukin-6, within Southwest Native American Student Population
Katie Zortman, Gabriel Thom, and Dr. Sherell Byrd
Fort Lewis College Biology Department, Durango, Colorado
ABSTRACT
 Explore the relationships between inflammatory cytokines IL-6 and TNF-alpha
 Explore the data within the research project, in order to find new correlations between
different indicators of type II diabetes
 Discover whether or not there is a higher propensity for developing insulin resistance
among the Native American population versus the Caucasian population at Fort
Lewis College
 Implement an awareness campaign if Native Americans are found to be at greater risk of
developing diabetes
 Add on to the current knowledge of Native American risk for developing type II diabetes
2.5
Mean IL-6 Concentration (pg/mL)
This study determined Native American population, aged 17-30 years, at Fort Lewis
College shows a higher likelihood for developing insulin resistance than the Caucasian
population. High levels of molecular markers such as IL-6, TNF-α, and erythrocyte
sedimentation rate are indicative of inflammation which can be correlated to insulin
resistance. In order to determine IL-6 concentrations, serum was extracted from whole
blood and a sandwich ELISA was performed. The concentration of IL-6 was determined
by absorbance at 405 nm. The mean concentration of IL-6 in the Native American group
was 1.7885 pg/mL + 0.84 whereas mean concentration for the Caucasian group was 1.3465
pg/mL + 0.15. This indicates a significant difference among the populations studied.
There were moderately significant correlations for IL-6 with TNF-α and erythrocyte
sedimentation rate shown by r values equaling 0.43 and 0.50 respectively. For continued
research a larger sample population would be required to further correlate data.
STUDY OBJECTIVES
Mean IL-6 Concentration per
Population Group
IL-6 IS FOUND TO BE
SIGNIFICANTLY
DIFFERENT IN NATIVE
AMERICAN
POPULATION
2
1.5
1
0.5
0
Native American
Caucasian
Population Group
Figure 1. The average plasma IL-6
concentration was significantly
higher in NA population (p=0.037).
Figure 4. Interleukin-6
protein structure.
INTRODUCTION
50
45
45
Sedimentation Rate (mm/hr)
50
40
35
30
25
20
y = 5.3076x + 6.4361
R² = 0.2563
15
10
5
0
30
25
20
y = 15.731x - 3.2891
R² = 0.0491
15
10
1
2
3
IL-6 Concentration (pg/mL)
4
5
1
1.2
1.4
1.6
IL-6 Concentration (pg/mL)
1.8
2
Comparison of IL-6 to TNF-α in
Native American Subjects
ENDOTHELIAL
DYSFUNCTION
SERINE
PHOPHORYLATION
OF IRS-1
DECREASED
INSULIN SIGNAL
TRANSDUCTION
Comparison of IL-6 to TNF-α in
Caucasian Subjects
60
TNF-alpha Concentration (pg/mL)
INFLAMMATION
IL-6/TNF-α
Figure 2. Comparison of IL-6 plasma concentrations to erythrocyte sedimentation
rates. For Native Americans, the r-value was 0.508, which is moderately
correlated. For Caucasians the r-value was 0.2945, which is a low correlation.
60
50
40
30
y = 2.1394x + 36.109
R² = 0.1814
20
10
50
INSULIN
RESISTANCE
40
30
y = -11.913x + 54.159
R² = 0.1072
20
10
Figure 5. Possible pathway of the proinflammatory
cytokines IL-6 and TNF-alpha leading to insulin resistance.
0
0
SUBJECTS
Volunteers were randomly selected through the Native American and Caucasian student
population at Fort Lewis College who were aged 17-30. The only consideration was that
they could not have been diagnosed with diabetes or insulin resistance. Subject
participation was voluntary receiving only $10 Durango dollars in return for participation.
Patient volunteers excluded only by presence of known diabetes condition.
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1
2
3
IL-6 Concentration (pg/mL)
4
5
1
1.2
1.4
1.6
IL-6 Concentration (pg/mL)
1.8
2
Figure 3. Comparison of IL-6 plasma concentrations to TNF-α plasma
concentrations. Has shown to be moderately correlated in Native American subjects
with an r-value of 0.425. For Caucasians the r-value was found to be -0.181, which is
a low correlation.
Table 1. Anthropometric and biochemical characteristics of the study subjects.
SAMPLE COLLECTION AND STORAGE
Ten mL of blood were collected in EDTA tubes (Becton Dickinson; Franklin Lakes, NJ) by
venipuncture at Fort Lewis College using standard protocols by a licensed phlebotomist.
Immediately after collection, specimens were processed per variable protocol. All
specimens were centrifuged at 1000g within 30 minutes of collection, and serum separated
from whole cells. All serum samples were stored at -20°C until subsequent ELISA
analysis.
IL-6 ENZYME-LINKED IMMUNOSORBENT ASSAY
IL-6 was measured by a quantitative sandwich ELISA kit (R&D Systems Inc.;
Minneapolis, MN) in duplicate.
TNF-ΑLPHA ENZYME-LINKED IMMUNOSORBENT ASSAY
TNF-α was measured by a quantitative sandwich ELISA kit (R&D Systems Inc.;
Minneapolis, MN) in duplicate.
SEDIMENTATION RATE ANALYSIS
One mL of whole blood was added to a 1mL serological pipette with a 0.3cm width, coated
with a 1.25M solution of sodium citrate. After one hour the sedimentation rate was
determined by recording the distance the erythrocytes have fallen in mm per hour.
Evidence for the link between inflammation and IR is provided in this study which builds
upon current research. Systemic inflammation, as measured by serum IL-6 concentration
was significantly higher in Native American as compared to their matched Caucasian peers.
Furthermore, IL-6 positively correlated with other physiological indicators of
inflammation, TNF- α and erythrocyte sedimentation rate. TNF- α locally identifies
inflammation and moderately correlates ESR in the Native American population.
Sedimentation rate positively correlates with IL-6 collectively identifying inflammation as
prevalent in the Native American population. The goal of our study was to identify cellular
markers of IR in Native Americans. A high correlation in inflammatory cellular markers
IL-6, TNF- α, and sedimentation rate was present in the Native American population. This
indicates that underlying inflammation that is often a precursor to insulin resistance, is
already present in this college-aged population.
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0
0
EXPERIMENTAL DESIGN AND METHODS
40
5
TNF- α is found to have a direct role in metabolic syndrome (Petersen and Pedersen, 2005)
through blocking insulin function (Fernandez-Real and Ricart, 2003) and generating IR
(Coppack, 2001; Kershaw and Flier, 2004).
IL-6 and TNF- α share a mechanistic pathway in which they block the expression of
glucose transporter-4 (Glut-4) and insulin receptor substrate-1 (IRS-1). Glut-4 has been
found to aid in the insulin stimulated glucose transport system while IRS-1 is a protein that
aids in the binding of insulin to its receptors on cells (Rotter et al., 2003). Through
inhibition of integral protein function, the effectiveness of insulin to bind is decreased
(Rotter et al., 2003).
Comparison of IL-6 to
Sedimentation Rate in Caucasian
Subjects
TNF-alpha Concentration (pg/mL)
IL-6 is a pro-inflammatory cytokine (Coppack, 2001; Andreozzi et al., 2006) involved in
the inflammation process and hypothesized to lead to IR (Kopp et al., 2003). IR is
positively correlated with high levels of IL-6 in the bloodstream (Kern et al., 2001;
Fernandez-Real and Ricart, 2003; Kershaw and Flier, 2004).
Sedimentation Rate (mm/hr)
Insulin resistance (IR) is a growing problem for Native Americans that carries into a high
propensity for developing diabetes leading to a shortened lifespan (Carter et al.,
2000). Chronic inflammation has been identified as a contributor to IR and metabolic
syndrome (Fernandez-Real and Ricart, 2003; Dandona et al., 2005; Petersen and Pedersen,
2005; Andreozzi et al., 2006). Inflammatory markers indicate the presence of IR which
progresses into type II diabetes (Fernandez-Real and Ricart, 2003). Adipocytes in visceral
fat are responsible for releasing proinflammatory cytokines such as Interleukin-6 (IL-6)
and Tumor Necrosis Factor-α (TNF- α) (Pedersen et al., 2001; Shoelson et al., 2006), thus
increased adiposity correlates with increased inflammation.
Comparison of IL-6 to
Sedimentation Rate in Native
American Subjects
CONCLUSIONS
Characteristics
Sex (M/F)
Native American
3/11
Caucasian
3/11
Waist-to-hip ratio
0.81 ± 0.08 (0.69-0.96)
0.79 ± 0.07 (0.90-.68)
BMI (kg/m2)
24.94 ± 3.58 (19.8-32.9)
23.87 ± 3.50 (19.2-32.1)
% Body Fat
22.29 ± 5.16 (16-29.8)
19.31 ± 5.56 (13-27.9)
Fasting Glucose (mg/dL)
78.43 ± 20.46 (33-109)
80.71 ± 10.74 (61-102)
Sedimentation Rate (mm/hr) 15.93 ± 8.82 (5-34.5)
17.89 ± 10.59 (4-41)
TNF-α (pg/mL)
Adiponectin (pg/mL)
39.94 ± 4.23 (32.29-49.42)
2265.43 ± 1384.47 (9156000)
38.12 ± 5.43 (27.87-47.37)
2261.79 ± 1389.96 (9156000)
IL-6 (pg/mL)
1.80 ± 0.84 (1.22-3.05)
1.34 ± 0.15 (1.12-1.69)
Data are means ± SD (range) and median (range).
Figure 6. 2010 Diabetes Research Team. From
left to right: Leon Clah, Katie Zortman, Gabe
Thom, Chelsea Bonfiglio, Samantha Johnson,
Brittany Walters, Hannah Meinking, Heather
Dahm, Dr. Sherell Byrd, Edlin Jara-Molinar.
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ACKNOWLEDGMENTS
Thank you to Fort Lewis College for assisting us in our research through a research grant
from the Department of Natural and Behavioral Sciences. We acknowledge the Student
Health Center staff for doing all of the blood draws.