Tag: Byron Hetrick

High Fat Diet In Utero Exposure Disrupts Circadian Rhythms in Mice Offspring

Presenter: Mai’ana Feuerborn

Faculty Mentor: Carrie McCurdy, Byron Hetrick

Presentation Type: Poster 61

Primary Research Area: Science

Major: Human Physiology

Funding Source: University of Oregon Undergraduate Mini-grant, $1,000

Maternal obesity and excessive gestational weight are linked to increased risk of obesity in offspring, suggesting that in utero exposure programs an organism’s metabolism for life. Studies in mice have shown exposure to a high calorie environment in utero causes more weight gain in offspring fed a high fat diet (HFD). Circadian rhythms create an internal temporal clock that coordinates behavior and metabolism to daily cycles. Disruption of circadian cycles leads to increased weight gain, suggesting that metabolic dysregulation observed in offspring of obese mothers may be due to altered circadian cycles. We hypothesized that offspring exposed to a high fat environment in utero will be more sensitive to postnatal HFD, with dampened circadian cycles, than mice exposed to a lean maternal environment in utero. To study the effect of diet on fetal programming, mice were subjected to either a HFD or control diet during gestation. Post weaning, the offspring were fed a HFD or control diet. Insulin controlled tissues were then collected at different times of the day. We propose to measure the effect of maternal diet on offspring by measuring the expression levels of key circadian genes by quantitative PCR. Understanding the role of fetal programming in metabolic regulation of metabolism will better help us understand and combat obesity in western society, and potentially understand the diseases increasing incidence.

Assay of Insulin-Stimulated Signaling by Flow Cytometry: Key Points of Regulation

Presenter(s): Shawn Melendy − Biochemistry

Faculty Mentor(s): Carrie McCurdy, Byron Hetrick

Poster 75

Research Area: Natural/Physical Science

Funding: UROP mini-grant, American Physiological Society Undergraduate Summer Research Fellowship Program

Type 2 Diabetes, an increasingly prevalent disease worldwide, is partially caused by a progressive loss of insulin response in adipose tissue and skeletal muscle. Multicolor flow cytometry is a powerful tool that can be used to measure multiple signaling events simultaneously in specific cell types within mixed populations. The objective of this study is to design a sensitive and high-throughput assay to measure key points of regulation in the insulin signaling pathway for myocytes using flow cytometry. We have developed a multicolor flow cytometry panel to measure the insulin stimulated phosphorylation of Akt(S473) and the transport of the insulin responsive glucose transporter, GLUT4, to the plasma membrane. C2C12 myoblasts were stained with primary conjugated antibodies for pAkt(S473) and an extracellular region of GLUT4, indicative of translocated GLUT4 present
in the plasma membrane. Both C2C12 myoblasts, an immortalized cell line, and primary myoblasts, isolated from non-human primate muscle, responded to insulin with increased pAkt(S473) and plasma membrane GLUT4 with an EC50 of <10nM, similar to physiological response. Future work will expand the panel to measure phosphorylation of insulin receptor substrate and phosphoinosital 3-kinase (PI3K) activity by quantitating phosphoinosital (3,4,5) phosphate (PIP3) production. The sensitivity of the assay will be demonstrated by inhibiting key insulin-activated kinases including PI3K by Wortmannin, and Akt activation by MK- 2206 and measuring insulin signaling at points up and downstream of inhibition. We anticipate that this will provide a powerful method to rapidly dissect the insulin signaling cascade for a specific cell type within mixed populations of cells.

Adipocyte-Specific p85a Overexpression in Mice: Insight into Type-II Diabetes Pathogenesis

Presenter(s): Shawn Melendy

Faculty Mentor(s): Carrie McCurdy & Byron Hetrick

Poster 7

Session: Sciences

Type 2 Diabetes is an increasingly prevalent disease worldwide that is partially caused by a progressive loss of insulin response in adipose tissue and skeletal muscle, two essential insulin target tissues. The class 1A phosphatidylinositol-3-kinase (PI3K) plays a central role in the insulin signal transduction cascade, as it controls the first point of signal propagation. It has been previously shown that the PI3K regulatory subunits (p85α/p55α/p50α) are upregulated in adipose tissue from high-fat diet (HFD) fed obese mice, concurrent with insulin resistance. Obese, insulin resistant adipose tissue is also characterized by chronic, low grade inflammation. This elevated inflammation attenuates signaling through the PI3K/Akt signaling pathway contributing to insulin resistance. The objective of this study is to determine how adipocyte specific overexpression of p85α affects insulin signaling in adipose tissue, independent of obesity. We have generated a lean mouse model designed to overexpress p85α in an adipocyte- specific manner, and measured insulin response in white adipose tissue (WAT) via Simple Western probing for phosphorylated Akt (pAkt). No significant difference in pAkt has been observed compared to wild-type, though the data trends towards increased signaling in p85α overexpressing (OX) mice. Additionally, p85α OX mice show no significant change in insulin sensitivity, as observed by an oral glucose tolerance test (OGTT). These results prompt the need for further validation of the overexpression of p85α in the transgenic mice. Future work will include measuring WAT p85α abundance via Simple Western, p85α transcript abundance via RT- qPCR, and detecting the presence of the p85α-inserted transgene with PCR.

Intergenerational Effects of Maternal Obesity on Offspring Mitochondrial Reactive Oxygen Species Production and DNA Damage

Presenter(s): Maurisa Rapp—Human Physiology

Faculty Mentor(s): Carrie McCurdy, Byron Hetrick

Session: Prerecorded Poster Presentation

Epidemiological studies have shown that offspring from pregnancies complicated by maternal obesity have a 4-fold greater risk for developing childhood obesity and symptoms of metabolic syndrome . The developmental origins of health and disease (DOHaD) hypothesis states that certain environmental exposures during critical windows of development may have consequences for an individuals long term health . DOHaD may explain a portion of the continual increase in obesity rates among children . In a non-human primate model, offspring of obese dams become sensitized to obesity-induced metabolic disruptions, including insulin resistance and mitochondrial disfunction . Increased reactive oxygen species (ROS) production contributes to mitochondrial defects observed in obesity . Oxidative stress, which is caused by overproduction of ROS, can lead to mitochondrial DNA (mtDNA) mutations, decreased copy number, reduced membrane permeability and subsequent suppression of mitochondrial respiratory chain activity . Therefore, I hypothesize that maternal obesity increases offspring mitochondrial ROS production leading to mtDNA damage without loss of mtDNA abundance . To study the effect of maternal obesity, we used a previously established Japanese macaque model of fetal programming . Dams were fed either a control (CON) diet or western style diet (WSD) prior to and during pregnancy and lactation . Offspring were then weaned at 8 months and fed a healthy CON diet . Skeletal muscle biopsies from offspring were collected at 3 years of age and relative mtDNA abundance was measured using quantitative PCR (qPCR) amplification of short regions of mtDNA . No differences were measured in the amount of mtDNA between offspring groups . Moving forward, I will test for elevations in ROS-induced mtDNA damage by qPCR amplification . Overall, these data indicate that exposure to maternal obesity and WSD during fetal development does not reduce mitochondrial abundance in skeletal muscle of adolescent offspring .