Skeletal Muscle Diacylglycerol and Sphingolipids - Impact of Localization and Species on Insulin Resistance in Humans

Accumulation of bioactive lipids such as diacylglycerol (DAG) and sphingolipids are one
mechanism proposed to promote muscle insulin resistance. Recent data indicate these lipids
are located in membranes, but the distribution and signaling of DAG and sphingolipids in
specific cellular organelles which regulate insulin sensitivity is not known. There is a
critical need to address these gaps in knowledge to design appropriate interventions to
prevent and treat lipid-induced insulin resistance. The overall objective of this project is
to determine the impact of changes in subcellular DAG and sphingolipid species, signaling,
and metabolic function before and after insulin sensitizing lifestyle interventions. The
investigators central hypothesis is that DAG and sphingolipids in muscle promote insulin
resistance via mechanisms that are unique to location, type of lipid, and species. The
rationale for the proposed research is that elucidating changes in localized DAG and
sphingolipid species that predict insulin sensitivity will reveal specific localized lipids
to target in therapeutics for type 2 diabetes. To attain the overall objective, the
investigators propose three specific aims: 1. Identify the influence of sarcolemmal DAG and
sphingolipids on cell signaling and insulin sensitivity before and after insulin sensitizing
lifestyle interventions. Strong preliminary data shape the hypothesis that sarcolemmal
1,2-disaturated DAG and C18:0 ceramide species will decrease after insulin sensitizing
lifestyle interventions, leading to less Protein kinase C (PKC) and Protein phosphatase 2A
(PP2A) activation, and enhanced insulin signaling. Skeletal muscle DAG and sphingolipid
isomers, species, localization, and de novo synthesis will be measured before and after
diet-induced weight loss or exercise training interventions in obese men and women. Insulin
sensitivity will be measured using insulin clamps, and muscle lipids using Liquid
Chromatography Mass Spectrometry (LC/MS). 2. Determine the impact of mitochondrial/ER
(endoplasmic reticulum) DAG and sphingolipids on mitochondrial function and ER stress in
vivo, before and after insulin sensitizing lifestyle interventions. The investigators
hypothesize, again based on preliminary data, that mitochondrial/ER sphingolipids will
decrease, yet DAG will increase after insulin sensitizing lifestyle interventions, and each
will associate with increased insulin sensitivity. Changes in sphingolipids will relate to
increased mitochondrial function, less ER stress, reactive oxygen species (ROS), and
acyl-carnitine formation, while changes in DAG will relate to increased mitochondrial content
and dynamics. 3. Identify the effect of exogenous DAG and sphingolipids on mitochondrial
function in vitro, before and after insulin sensitizing lifestyle interventions. The working
hypothesis is that DAG and sphingolipids will reduce mitochondrial respiration and increase
ROS and acyl-carnitine content, but will be attenuated after endurance exercise training. The
proposed research is innovative because it represents a substantive departure from the status
quo by addressing cellular compartmentalization of bioactive lipids. The investigators
contribution will be significant by identifying key species and locations of DAG and
sphingolipids promoting insulin resistance, as well as mechanisms explaining accumulation
that could be modified by insulin sensitizing therapeutic interventions.

Inclusion Criteria:

- BMI: 30-40 kg/m2

- Planned physical activity: <2 hrs/week

- Glucose tolerance:

1. Normal glucose tolerance (NGT) defined as:

1. HbA1c of <5.7%,

2. pre-diabetes as HbA1c of 5.7-6.4%, and

3. type 2 diabetes as HbA1c of ≥6.5%

2. pre-diabetes, and

3. Type 2 diabetes

- Oral contraceptive use: Yes or No as long as there is no change during the study

- Thyroid status: TSH between 0.5-5.0 mU/L

Exclusion Criteria:

- Currently taking

1. Thiazolidinediones

2. Insulin

- Pregnant

- Smoker (tobacco and any form of marijuana use)

- Fasting triglycerides >400mg/dl