Ovarian Cancer and Diabetes:

Metformin’s Metabolic Effects

LACEY GIBSON, SOUTHERN ILLINOIS

UNIVERSITY, CLASS OF 2015
DR. BUCK HALES, DEPARTMENT CHAIR OF
PHYSIOLOGY, SOUTHERN ILLINOIS
UNIVERSITY SCHOOL OF MEDICINE
 Introduction: Objectives

Objectives
Our study was undertaken to:
 compare the ability of Metformin and flaxseed in reducing
hyperglycemic, pro-inflammatory environment
 and to set the stage for a long term study of their direct
comparison in reducing ovarian cancer incidence and severity,
using a hen model.
 Introduction: How are Ovarian Cancer & Diabetes Related?

Ovarian Cancer & Diabetes

 Ovarian cancer & type II
diabetes are devastating
diseases that share a
variety of risk factors;
individuals diagnosed with
type II diabetes are more
likely than by chance to be
diagnosed with cancer
(Giovannuci et al 2010)
 Introduction: How are Ovarian Cancer & Diabetes Related?

 Warburg Effect describes ability of

fast-growing cancer cells to
metabolize glucose via glycolysis in
addition to oxidative phosphorylation;
More glucose needed for
proliferation (Ladley 2012).
 Diabetes provides a nutrient-rich
metabolic environment for ovarian
cancer cells, where tumor formation
and growth is encouraged by free
radical-induced DNA damage.
(Kellenberger et al 2010).
Introduction: What are Metformin and Flaxseed?

Flaxseed and Metformin as Mediators of Proliferative Metabolic

Environment
 Flaxseed and Metformin both may reduce incidence and severity of
cancer by reducing hyperglycemic, pro-inflammatory environment that
feeds cancer proliferation.
Introduction: What are Metformin and Flaxseed?

Metformin: Anti-diabetes drug Metformin (1-

carbamimidamido-N,N-dimethylmethanimidamide,
C4H11N5 ) activates fuel sensing enzyme AMP-
activated protein kinase.
 -> alters expression/location of
enzymes/transporters associated with glucose
metabolism: glucose-6-phosphatase (G6PC2),
glucose family transporter-2 (GLUT-2), histone
deacetylase 7 (HDAC7), phosphoenolpyruvate
carboxykinase (PCK1), and pyruvate kinase
(PKM2) and…
 -> glucose homeostasis is improved
(Fulgencio et al 2001; Ait-Omar et al 2011; Hardie
2013; Yuan et al 2002).
Introduction: What are Metformin and Flaxseed?

Flaxseed: Dietary supplement ALA

flaxseed rich in ALA, which is
converted to EPA and DHA
 ->decreases expression of
inflammatory markers COX-1 and
COX
COX-2 EPA
 -> reduction in inflammatory
markers correlates to reduction in
severity of ovarian cancer in hens
(Eilati et al 2013).

DHA
 Introduction: What Endpoints Can We Use to Compare the
Metabolic Effects of Metformin & Flaxseed?

Diabetes, Ovarian Cancer, and Metabolic Syndrome

 Metabolic Syndrome: Clustering of risk factors for type II diabetes, cardiovascular
disease, hypertension, lipid problems, obesity, NAFLD, cancers, and ovarian
distress.
 Metabolic distress indicated by the following measurements: low albumin; high
aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and
glutamate dehydrogenase (GLDH), triglycerides, cholesterol, blood glucose.
 Methods

Metformin animal study: 3 week dose finding: Animal

management and procedures were reviewed and approved by IACUC at
Southern Illinois University Carbondale. 28 hens of 1.5 years were obtained
and housed at SIUC vivarium in Life Science II. 7 hens per group were treated
daily with either capsules containing 10, 30, or 100 mg/kg of Metformin or a
control rice bran pill. On day 21 of treatment, hens were sacrificed.
Biochemical serum analysis: 12 hens were bled on day zero,
and all hens were bled on day 21 of the study. Blood was spun at 20º C for 10
minutes at 1200 rpm. Serum was collected and shipped on dry ice to University
of Illinois Veterinarian Diagnostic Laboratory for analysis of cholesterol,
triglycerides, glucose, AST, albumin, GGT, and GLDH. Statistical analysis of
data was performed using ANOVA using standard parametric methods to
identify significant differences (p<.05). A principal components analysis (PCA)
test was then performed to compare variables.
 Methods

Analysis of gene expression: Liver and ovary tissue were

collected from all hens during necropsy and stored at -80º C. RNA was isolated
from the tissue samples followed by cDNA synthesis. PCR primers were
identified by literature review and obtained through a commercial vendor
(Eurofins). Fold differences in gene expression were validated through
semiquantitative RT-PCR using the BioRad CFX96 PCR system. mRNA levels
were normalized to GAPDH and relative expression was calculated using the
comparative Ct method. Statistical analysis of data was performed using
ANOVA using standard parametric methods to identify significant differences
(p<.05).
Comparison of data to previous flaxseed
findings: Results were compared to biochemical analysis and gene
expression of hens fed control or flaxseed diets during a series of long term
studies
 Results: General Health: Metformin

General health:
Hens treated with 30 mg/kg Metformin lost weight during the trial (Figure 1). Hens of all
treatments maintained egg-laying ability, and hens treated with 30 mg/kg Metformin were
the most fertile on day 21 compared to hens of other treatments (Figure 2).
 Results: Serum Analysis: Metformin

Biochemical serum analysis:

Significant increases in serum glucose and GGT were seen in post-treated
hens compared to pre-treated hens (Figures 3, 4). No significant changes in
cholesterol, triglycerides, albumin, or GLDH were seen (Table 1).
250 18
16
200
Glucose (mg/dL)

14

GGT (U/L)
12
150 10
8
100 6
4
50 2
0
0 Baseline (n=14) Metformin (n=14)
Baseline (n=14) Metformin (n=14)
Treatment
Treatment

Figure 3(left) Mean values for glucose in serum of hens with and without Metformin treatment. Mean values connected with an
asterisk are significantly different (P<0.05, ANOVA).
Figure 4(right) Mean values for GGT in serum of hens with and without Metformin treatment. Mean values connected with an
asterisk are significantly different (P<0.05, ANOVA).
 Results: Serum Analysis: Metformin
Variability in glucose and GGT caused by living conditions, not by
Metformin treatment; Variability in cholesterol, triglycerides, and albumin
unrelated to treatment or conditions (Figure 5).
Figure 5
PCA analysis Increasing
of
glucose,
biochemical
serum GGT
variables in
hens of all
treatments
on day 0
(blue points)
and day 21
(yellow
points) of
treatment.

Increasing Cholesterol,
Triglycerides, Albumin
 Results: Serum Analysis: Flaxseed

In long-term flaxseed studies,

cholesterol, triglycerides, and AST 300
A
were decreased in hens fed a 250
flaxseed-based diet compared to

Avg AST (U/L)

200
hens fed a control diet (Figures 6, 7, 150 B
B B

8). No significant changes in GGT, 100

C
albumin, or GLDH were seen (Table 50
C
C
C

1)
0
Control Flax D Contol Flax E Control Flax F Contol Flax G
D (n=16) E (n=11) F (n=16) G (n=17)
(n=16) (n=12) (n=15) (n=17)

Figure 6 Mean AST levels of serum from hens fed flaxseed or

control diets of age groups D (youngest, 2.5 years) through G
(oldest, 4 years) in a long term study. Mean values sharing the same
letter are not significantly different (P<0.05, Tukey’s test).
 Results: Serum Analysis: Flaxseed
4000 250
A A
3500

Cholesterol (mg/dL)
AB
3000 AB AB AB 200
AB
Average Triglyceride Level

2500 AB AB
BC

Average
2000 150
BC B AB AB
1500 C C
1000 100
(mg/dL)

500
0 50

0
Control Flax D Contol Flax E Control Flax F Contol Flax G
D (n=16) E (n=11) F (n=16) G (n=17)
(n=16) (n=12) (n=15) (n=17)
Group
Group

Figure 7 (left) Mean triglyceride levels of serum from hens fed flaxseed or control diets of age groups D (youngest, 2.5 years)
through G (oldest, 4 years). Mean values sharing the same letter are not significantly different (P<0.05, Tukey’s test).
Figure 8 (right) Mean cholesterol levels of serum from hens fed flaxseed or control diets of age groups D (youngest, 2.5 years)
through G (oldest, 4 years). Mean values sharing the same letter are not significantly different (P<0.05, Tukey’s test).
 Results: Serum Analysis: Summary

Flax Metformin
Glucose ? -*
Cholesterol +* NC
Triglycerides +* NC
Albumin NC NC
AST +* NC
GGT NC -*
GLDH NC NC
Table 2 Summary of the effects of a long-term flaxseed diet or short-term Metformin treatment in hens on biochemical metabolic
markers in serum analysis. (- = negative effect, + = positive effect, NC = no change; * = statistical significance: p<.05)
 Results: Gene Expression: Metformin

Gene expression: Liver:

Expression of GLUT-2 was significantly increased in hens treated with
30 mg/kg Metformin. No significant changes in expression of COX-2,
G6PC2, HDAC7, PCK1, or PKM2 were seen (Figure 9).

Figure 9 (left) Relative

normalized expression of
metaboolic genes in liver
tissue of control or Metfromin-
treated hens. Mean values
connected with an asterisk
are significantly different
(P<0.05, Tukey’s test).
 Results: Gene Expression: Metformin

Gene expression: Ovary:

No significant changes in expression of COX-2, HDAC7, or PKM2 were
seen in ovary tissue (Figure 10).

Figure 10 (left) Relative

normalized expression of
metaboolic genes in ovary tissue
of control or Metfromin-treated
hens. Mean values connected
with an asterisk are significantly
different (P<0.05, Tukey’s test).
 Results: Gene Expression: Flaxseed

Gene expression: Liver:

In long-term flaxseed studies, expression of PCK1 was significantly
decreased in hens treated with 15 g/kg flaxseed. No changes in
expression of COX-2, G6PC2, GLUT-2, HDAC7, or PKM2 were seen
(Figure 11, Table 3).
Figure 11 (left) Relative normalized
expression of metaboolic genes in
liver tissue of control or flaxseed-fed
hens. Mean values connected with
an asterisk are significantly different
(P<0.05, Tukey’s test).
 Results: Gene Expression: Summary

Flax Metformin
COX-2 NC NC
G6PC2 NC NC
GLUT-2 NC +*
PCK1 +* NC
PKM2 NC NC

Table 3 Summary of the effects of a long-term flaxseed diet or short-term Metformin treatment in hens on expression of metabolic
genes. (- = negative effect, + = positive effect, NC = no change; * = statistical significance: p<.05)
 Conclusions: Metformin

 Flax and Metformin differentially effect biochemical and genetic

markers of metabolic syndrome, liver disease, and insulin
resistance.

 Our short-term Metformin study has shown that Meformin is related

to:
 up-regulation in GLUT-2. Previous studies have found that Metformin may
alter the location and functioning of this transporter (Ait-Omar et al 2011);

 Increase in GGT and glucose due to conditions rather than treatment. 3 weeks
was not long enough for Metformin to change biochemical serum markers.
 Conclusion: Metformin

 Literature has shown that a single oral dose of 300 mg/kg Metformin can
reduce blood glucose and feed intake (Ashwell and McMurty 2003).

 However, consistent with our findings, another study has shown that daily
diets of Metformin of 250-10,000 mg/kg reduce feed intake and increase
lactate in highest doses without reducing plasma glucose (Rosebrough
and Ashwell 2005).

 Thus, in the hen, Metformin may reduce symptoms of Metabolic

Syndrome, thereby reducing cancer risk through an undefined form of
feedback inhibition that increases satiety without altering long-term
plasma glucose levels
 Conclusions: Flaxseed

 Our long-term flaxseed studies have shown that flax is related to:
 reduction in cholesterol, triglycerides, AST.
 decreased expression of PCK1, consistent with previous studies
that have found SDG from flaxseed to reduce expression of this gene
(Prasad 2002).
.
 Conclusions: Future Directions

 Despite lack of change in blood glucose levels, our short term study has
shown that most positive metabolic health effects (up-regulation of
GLUT-2, decrease in body weight, egg laying ability) occur in hens
treated with 30 mg/kg. This is therefore our optimal dose for future
studies for which this study has laid a foundation.

 Future long-term studies must be performed to directly compare the

benefits of flaxseed and Metformin in reducing incidence and severity of
ovarian cancer as well as its associated pro-inflammatory,
hyperglycemic environment.
 Acknowledgements
 I would like to graciously thank the following:
 All members of my lab for aiding in data collection and interpretation
 The REACH program at SIUC for financial support
 My family for moral support
 The selected sources below for providing background and discussion
information
 And YOU for your time and attention! Thanks!!
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