LACEY GIBSON, SOUTHERN ILLINOIS UNIVERSITY, CLASS OF 2015

DR. BUCK HALES, DEPARTMENT CHAIR OF PHYSIOLOGY, SOUTHERN ILLINOIS UNIVERSITY SCHOOL OF MEDICINE

Ovarian Cancer and Diabetes: Metformin’s Metabolic Effects

Introduction: Objectives

ObjectivesOur 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 & DiabetesOvarian 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)

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: How are Ovarian Cancer & Diabetes Related?

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 flaxseed rich in ALA, which is converted to EPA and DHA ->decreases expression of

inflammatory markers COX-1 and COX-2

-> reduction in inflammatory markers correlates to reduction in severity of ovarian cancer in hens (Eilati et al 2013).

ALA

COXEPA

DHA

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

Flaxseed?

Diabetes, Ovarian Cancer, and Metabolic SyndromeMetabolic 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).

Baseline (n=14) Metformin (n=14)145150155160165170175180185190195

Treatment

Glu

cose

(m

g/d

L)

Baseline (n=14) Metformin (n=14)02468

10121416

TreatmentG

GT

(U

/L)

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).

Increasing Cholesterol, Triglycerides, Albumin

Increasing glucose, GGT

Figure 5 PCA analysis of biochemical serum variables in hens of all treatments on day 0 (blue points) and day 21 (yellow points) of treatment.

Results: Serum Analysis: Flaxseed

In long-term flaxseed studies, cholesterol, triglycerides, and AST were decreased in hens fed a flaxseed-based diet compared to hens fed a control diet (Figures 6, 7, 8). No significant changes in GGT, albumin, or GLDH were seen (Table 1)

Control D

(n=16)

Flax D (n=16)

Contol E

(n=12)

Flax E (n=11)

Control F

(n=15)

Flax F (n=16)

Contol G

(n=17)

Flax G (n=17)

0

50

100

150

200

250

300

Avg

AS

T (

U/L

)

A

B

CC

C C

B B

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

Contro

l D (n

=16)

Flax D

(n=16

)

Conto

l E (n

=12)

Flax E

(n=11

)

Contro

l F (n

=15)

Flax F

(n=16

)

Conto

l G (n

=17)

Flax G

(n=17

)0

500100015002000250030003500

Group

Ave

rag

e T

rig

lyce

rid

e L

evel

(m

g/d

L)

AB

BC

AB

A

C C

ABBC

Control D

(n=16)

Flax D (n=16)

Contol E

(n=12)

Flax E (n=11)

Control F

(n=15)

Flax F (n=16)

Contol G

(n=17)

Flax G (n=17)

0

50

100

150

200

250

Group

Ave

rag

eC

ho

lest

ero

l (m

g/d

L)

B

ABAB

AB

AB

A

AB

AB

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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