Skeletal Muscle Diacylglycerol and Sphingolipids - Impact of Localization and Species on Insulin Resistance in Humans
Phase: NA
Conditions: Diabetes Mellitus, Type 2, Pre-diabetes, Obesity
Sponsor: University of Colorado, Denver

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COMIRB Protocol 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Protocol #: 
16-1404 
Project Title: 
Skeletal muscle diacylglycerol and sphingolipids – impact of localization and 
species on insulin resistance in humans 
 
 
 
 
Principal Investigator:  Bryan C. Bergman, PhD 
Version Date: 06/14/19 
 
 
 
 
 
  
 
I. Hypotheses and Specific Aims: The accumulation of muscle lipids are thought to promote insulin 
resistance and type 2 diabetes. While initially this was associated with triglycerides, bioactive lipids 
such as diacylglycerol (DAG) and sphingolipids have emerged as important mediators of insulin 
resistance. Recent data indicate these lipids are located in mixed membrane fractions, however, the 
exact membranes are unknown. This limited understanding of where DAG and sphingolipids promote 
insulin resistance is limiting our ability to prevent and treat lipid-induced insulin resistance. We 
developed a technique to isolate specific muscle membranes from human biopsies, and found DAG 
and sphingolipids accumulate in the sarcolemma and mitochondrial/ER in obese people with and 
without type 2 diabetes. DAG and sphingolipids are assumed to decrease insulin sensitivity in the 
sarcolemma, by activating protein kinase C (PKC) and protein phosphatase 2A (PP2A), respectively. 
The influence of DAG and sphingolipids on the function and signaling of the mitochondria and ER, 
which also regulate insulin sensitivity, is not known. There is a critical need to address these gaps in 
knowledge, as altering the cellular distribution of muscle lipids has the potential to ultimately prevent 
and treat insulin resistance and type 2 diabetes. 
 
Our long-term goal is to identify novel therapeutic targets to combat muscle insulin resistance and type 2 
diabetes, filling a need not met by current therapies. The overall objective for this project is to determine 
the impact of changes in subcellular DAG and sphingolipid species on cell signaling and metabolic 
function, before and after insulin sensitizing lifestyle interventions. Our central hypothesis is that DAG 
and sphingolipids in muscle promote insulin resistance via mechanisms that are unique to location, type 
of lipid, and species. This hypothesis is based on extensive preliminary data (see Research Strategy – 
Approach), showing insulin resistance is related to sarcolemmal and mitochondrial/ER accumulation of 
specific DAG and sphingolipids. Our pilot studies revealed changes in specific localized species after 
an insulin sensitizing lifestyle intervention, and that DAG and sphingolipids alter mitochondrial function 
in vitro. We propose to measure skeletal muscle DAG and sphingolipid isomers, species, localization, 
and de novo synthesis before and after diet-induced weight loss or exercise training interventions in 
obese men and women. Comparing the two interventions will uncover differences in lipid localization, 
cellular signaling, and mechanisms for improvements in insulin sensitivity. Insulin sensitivity will be 
measured using hyperinsulinemic/euglycemic clamps, muscle lipids using LC/MS, and mitochondrial 
function using an Oroboros respirometer. The rationale underlying the proposed research is that 
elucidating changes in localized DAG and sphingolipids predicting insulin sensitivity will reveal specific 
localized lipids to target in therapeutics to prevent or treat type 2 diabetes. 
 
To attain our overall objective, we 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 our working hypothesis that sarcolemmal 1,2-disaturated DAG and C18:0 
ceramide species will decrease after insulin sensitizing lifestyle interventions, leading to less PKC and 
PP2A activation, and enhanced insulin signaling.  
2.  
Determine the impact of mitochondrial/ER DAG and sphingolipids on mitochondrial 
function and ER stress in vivo, before and after insulin sensitizing lifestyle interventions. 
We 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 

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insulin sensitivity. Changes in sphingolipids will relate to increased mitochondrial function, less ER 
stress, ROS, and acyl-carnitine formation, while changes in DAG will associate with 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. 
Our working hypothesis is that DAG and sphingolipids will reduce mitochondrial respiration and increase 
ROS and acyl-carnitine content, yet the response to DAG will be attenuated after endurance exercise 
training.  
 
II. Background and Significance:  
Significance – The intracellular accumulation of DAG and sphingolipids are potential mechanism 
explaining skeletal muscle insulin resistance in the pathogenesis of type 2 diabetes. DAG and 
sphingolipids are greater in insulin resistant humans and animal models (1-9), and increase after high fat 
feeding and lipid infusions that acutely induce insulin resistance (7-9). However, the response of DAG 
and sphingolipids to insulin sensitizing interventions is variable (10-17), and there are paradoxical studies 
in the literature dissociating DAG and sphingolipid content from insulin sensitivity (9; 18; 19). These 
conflicting reports highlight that the current understanding of how and where DAG and sphingolipids 
induce insulin resistance is incomplete. A weakness of the above studies is analyzing whole muscle 
concentrations. Recent studies showed that DAG in membranes related to insulin resistance, which is 
also assumed for sphingolipids (20-23). However, a weakness of these studies is that all membranes 
were analyzed together and the exact membranes containing lipids promoting insulin resistance are 
unknown. Sub-membrane localization is important, as a recent study revealed DAG and ceramide in only 
certain membrane fractions promoted hepatic insulin resistance in mice (24). There are no studies in 
humans examining what membranes harbor lipids promoting insulin resistance, despite calls in the 
literature for clarity on this issue (25). Failure to identify bioactive lipid localization related to insulin 
resistance will prevent understanding how these lipids influence cellular signaling and metabolic function, 
and limit our ability to intervene in lipid-induced insulin resistance. 
The proposed studies will advance the field by identifying which subcellular membrane compartments of 
DAG and sphingolipids promote insulin resistance, and how these localized lipid species impact cell 
signaling and organelle function. These novel contributions will be significant because they will reveal the 
key species and locations of DAG and sphingolipids, as well as mechanisms explaining accumulation that 
could be modified by insulin sensitizing therapeutic interventions. Identifying specific localized DAG and 
sphingolipids species contributing to muscle insulin resistance will drive the field of lipid-induced insulin 
resistance forward in several ways. First, it will lay the foundation for more focused prevention and 
treatment therapies targeted to the localized species that promote insulin resistance. Second, these 
results will direct strategies to decrease localized DAG and sphingolipid content by identifying the extent 
to which de novo synthesis explains membrane accumulation. Third, this project will change the way 
pharmaceuticals are screened to reduce DAG and sphingolipids by focusing on changes in specific 
species and locations. This approach is expected to reveal novel therapeutic compounds that cannot be 
identified by focusing on whole cell content.  Together, these advances will address an unmet clinical 
need to improve muscle insulin sensitivity, and reveal novel treatment and prevention strategies for 
diabetes. 
 
 
III. Preliminary Studies/Progress Report:   
Preliminary Studies  
We can fractionate muscle into multiple membrane compartments. We are 
uniquely suited for this project because we spent 3 years developing a method to 
separate frozen human skeletal muscle into fractions enriched in sarcolemma, 
mitochondria/ER, nuclear, and cytosolic compartments (Figure 1). This technique 
is a substantial advance, as the most recent studies only separate cytosol from 
mixed membranes, which cannot discriminate specific organelles 
containing lipids promoting insulin resistance. Lipids are then extracted 
from these fractions and analyzed using our lipidomic platform.  
 
 

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Sarcolemmal 1,2-Disaturated DAG species relate 
to insulin resistance. The sum of all sarcolemmal di-
saturated 1,2-DAG species were significantly greater 
in T2D compared to lean volunteers (Figure 2A), and 
was inversely related to insulin sensitivity (Figure 2B). 
Notably, 1,3-disaturated DAG content was not 
different between groups revealing isomer specificity. 
We previously showed that membrane di-saturated 
DAG content predicted PKC activation and insulin 
resistance (26). These data agree and suggest that 
sarcolemmal DAG containing saturated acyl side 
chains represent membrane accumulation of DAG 
that is particularly negative for insulin sensitivity.  
  
PKCε activity is increased in obesity and T2D. We 
measured the membrane/cytosol ratio of PKCε, which is commonly used as a 
surrogate for PKC activity. We found significantly greater PKCε activation in obese 
and T2D subjects compared to lean controls (Figure 3), with no significant 
differences found for PKCβII, δ, and θ isoforms. These data are consistent with 
greater PKC activation from sarcolemmal DAG accumulation. 
 
Sarcolemmal C18:0 ceramide accumulates in insulin resistance. Of the 9 
sarcolemmal ceramide species measured, only C18:0 ceramide was significantly 
greater in T2D compared to lean (Figure 4A), and was inversely related to the rate 
of glucose disappearance during an insulin clamp (Figure 4B). In a pilot study 
sarcolemmal C18:0 ceramide decreased by 21% after a combined weight loss 
and exercise training intervention. These data parallel 
a recent publication by our lab showing C18:0 muscle 
ceramides significantly related to insulin resistance in 
a different cohort (27). C18:0 ceramide antagonizes an 
inhibitor of PP2A, resulting in increased PP2A activity 
(28). Combined, these data suggest that sarcolemmal 
C18:0 ceramide may promote insulin resistance, and 
due to the location may explain the canonical 
pathways of ceramide induced insulin resistance, 
including activation of PP2A activity, and inhibition of 
AKT phosphorylation in response to insulin.  
 
Mitochondrial/ER ceramide accumulates in insulin 
resistance. Mitochondrial ceramide content was 
greater in T2D compared to lean individuals, which is 
largely explained by C22:0 and C24:0 species (Figure 
7A). Total ceramide content was inversely related to 
insulin sensitivity (Figure 7B). These data are consistent with the hypothesis 
that mitochondrial ceramides promote insulin resistance.   
 
Mitochondrial/ER ceramides decrease after insulin sensitizing lifestyle 
intervention - Pilot data for a combined weight loss/exercise training 
intervention show decreased whole cell ceramide content, as shown before 
(11; 15) (Figure 8). However, the decrease in whole cell ceramide content 
comes only from a decrease in mitochondrial/ER ceramide. These data point 
to an important role of mitochondrial/ER ceramides on insulin sensitivity in 
vivo.  
 
 Mitochondrial/ER DAG accumulates in lean insulin sensitive 
individuals. Preliminary data show mitochondrial/ER 1,2- and 1,3-DAG were 
Figure 2.  Sarcolemmal di-saturated DAG content by group 
(A), and by insulin sensitivity (B) in lean, obese, and type 2 
diabetics. Values are means +/- SEM. #= significantly 
different than lean, p<0.05. 
A 
B 
Figure 4. Sarcolemmal C18:0 ceramide content by group (A), 
and by insulin sensitivity (B) in lean, obese, and type 2 
diabetics. Values are means +/- SEM. #= significantly 
different than lean, p<0.05. 
A 
B 
Figure 3. PKCε activation in 
lean, obese, and type 2 
diabetics. Values are means 
+/- SEM. #= significantly 
different than lean, p<0.05. 
 

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higher in lean individuals compared to obese 
or type 2 diabetic volunteers (Figure 9), with 
the greater DAG content distributed over 
most species. These data are 
counterintuitive, but may reflect the greater 
content of mitochondria in lean individuals 
compared to obese volunteers with and 
without diabetes, and/or increased 
mitochondrial fission/fusion (29; 30).  
  
Mitochondrial/ER DAG increase after 
weight loss and exercise training. 
Preliminary data from a weight loss and 
exercise training intervention show an 
increase in mitochondrial/ER 1,2-DAG after 
the intervention (Figure 10).  These data are 
consistent with some  reports in the literature 
where DAG concentration was increased in 
endurance trained athletes (13). These data 
agree with Figure 9 and suggest 
mitochondrial/ER DAG accumulation may be 
a previously unrecognized adaptation to 
exercise training and/or weight loss. 
 
Insulin resistant muscle accumulates long 
chain acyl-carnitines and shows oxidative 
stress. Basal acyl-carnitines revealed 
accumulation of many long chain acyl-CoA’s 
in type 2 diabetics including C18:0, C18:2-OH, and 
C20:4, which are thought to promote insulin 
resistance (Figure 11A). A significantly decreased 
GSH/GSSG ratio in type 2 diabetes individuals 
compared to lean volunteers suggest a more 
oxidized state, consistent with increased oxidative 
stress in insulin resistance (Figure 11B).  
 
DAG and ceramide decrease mitochondrial 
function. We administered 1,2-DAG, ceramide, 
dihydroceramide, and vehicle controls to isolated 
rat mitochondria in vitro to evaluate the impact on 
mitochondrial respiration using pyruvate and malate 
as substrates. DAG and ceramide decreased state 
3 respiration, with no changes observed with 
dihydroceramide administration (Figure 12A and B). 
State 4 respiration did not change for 1,2-DAG and 
dihydroceramide, but increased significantly with 
increasing doses of ceramide. Both 1,2-DAG and 
ceramide decreased ADP/O ratios (Figure 13A and 
B), while dihydroceramide had no effect. These data 
indicate 1,2-DAG and ceramide reduced 
mitochondrial respiration and coupling, and are 
consistent with accumulation of mitochondrial lipids 
altering mitochondrial function.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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IV. Research Methods 
 
A.  Outcome Measure(s):   
Primary outcomes:  
Aim 1: Change in insulin sensitivity, change in sarcolemmal Di-C18:0 DAG and C18:0 ceramide, 
change in PKCepsilon and PP2A activity 
Aim 2: Change in insulin sensitivity, change in mitochondrial/ER total DAG and ceramide content, 
change in state 3 mitochondrial respiration, change in GSH/GSSG, change in C18:0, C18:2-OH, and 
C20:4 acylcarnitines, change in GRP78 
Aim 3: Change in mitochondrial state 3 and state 4 respiration in response to DAG and ceramide 
before and after lifestyle interventions 
 
Secondary outcomes: Changes in other localized lipids and intracellular responses will be exploratory 
in nature. In addition, we will collect subjective and objective information about sleep and an exploratory 
analysis. 
 
B. Description of Population to be Enrolled:   
Obese men and women (BMI 30-40 kg/m2) will be recruited for this study who are between 30-50 
years of age and sedentary (<2 hours/week of planned physical activity). Individuals with normal 
glucose tolerance (NGT), pre-diabetes, and newly diagnosed type 2 diabetes will be enrolled. Normal 
glucose tolerance is defined as a HbA1c of <5.7%, pre-diabetes as a HbA1c of 5.7-6.4%, and type 2 
diabetes as a HbA1c of ≥6.5% (31). Oral contraceptive use is allowed as long as women don’t 
change their use, or lack thereof, during the course of the study. Exclusion criteria include: regular 
exercise more than 2 hours/week, medications that could affect glucose, lipid metabolism, thyroid 
disease, a history of lung disease, pregnancy, active cigarette smoking and severe plasma lipid 
disorders. 
 
Inclusion criteria:  
BMI: 30-40 kg/m2 
Age: 30-50 years 
Planned physical activity: <2 hrs/week 
Glucose tolerance: NGT, pre-diabetes, and Type 2 diabetes 
Normal glucose tolerance (NGT) is defined as HbA1c of <5.7%, pre-diabetes as HbA1c of 5.7-
6.4%, and type 2 diabetes as HbA1c of ≥6.5% (31). 
Oral contraceptive use: Yes or No as long as there is no change during the study 
Medications: not taking TZD’s, insulin 
Thyroid status: TSH between 0.5-5.0 mU/L 
Pregnancy status: Not pregnant 
Nonsmoker (tobacco and any form of marijuana use) 
Fasting TG <400mg/dl 
FSH < 20 IU/L 
 
C. Study Design and Research Methods   
Ninety-two individuals (30-50 years old) will be 
screened to recruit sixty-nine obese men and women 
into the study. Individuals with and without pre-
diabetes will be recruited to achieve a range of starting 
insulin sensitivities without confounding effects of 
medications used to treat type 2 diabetes. Volunteers 
will undergo an insulin clamp before and after block 
randomization by sex and pre-diabetes strata equally 
across intervention groups, into a three month 
intervention consisting of: 1) endurance exercise training without weight loss, 2) diet-induced weight loss, 
or 3) delayed intervention control (Figure 5). The three month intervention will result in significant gains in 
fitness, weight loss, and improvements in insulin sensitivity, but is short enough to maximize retention. 
 

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Exercise training and weight loss will differentially change DAG and sphingolipid content (11), revealing 
distinct changes in lipid localization, cellular signaling, metabolic function, and improvements in insulin 
sensitivity. We are expecting a 25% dropout rate, and are powered to complete testing on 17 individuals 
per group (51 total individuals). Subjects will be tested 48 hours after the last exercise bout to minimize 
acute exercise effects on insulin sensitivity. Women will be pre-menopausal and tested during the 
follicular phase (days 1-10) of the menstrual cycle to control for effects on insulin sensitivity. 
 
Preliminary screening- Subjects will arrive after an overnight fast to the outpatient Clinical Translational 
Research Center (CTRC), and a fasting blood sample will be taken for a standard lipid panel, fasting 
glucose, TSH, and HbA1c. Women will take a pregnancy test. A physician will perform a history and 
physical, and subjects will fill out a dietary questionnaire, the NIH Dietary History Questionnaire II 
(DHQII), and the IPAQ physical activity questionnaire (33). 
 
Strength and Exercise testing, body composition, maximal mitochondrial respiration, and MRI –
Eligible subjects will undergo a Kin-Kom isokinetic dynamometer quadriceps strength test, Balke treadmill 
test to measure maximal work capacity (VO2max) (34), a DEXA scan to determine body composition, a 
non-invasive test of maximal mitochondrial respiration, and MRI scan to measure leg muscle and fat 
mass, and liver fat content.  During the maximal mitochondrial respiration test, volunteers will lie down on 
a table, and we will have a near infrared spectrometer measurement unit strapped to their leg. This unit 
uses a laser to measure the oxygen levels in the muscle. A blood pressure cuff will be wrapped around 
their upper thigh. To start the test, we will ask volunteers to make a muscle contraction for 20 seconds, 
after which we will intermittently inflate and deflate the blood pressure cuff every 5-10 seconds over a 5 
minute period. This test will then be repeated. The total time for this test is 30 minutes. 
 
Dietary monitoring – We will provide food for all volunteers for 3 days prior to each insulin clamp study. 
The diet will be designed to replicate their typical diet.  An estimate of habitual fat consumption will be 
obtained from red blood cell phospholipid composition (35; 36), and the DHQII. Saturated fat intake will 
be used to determine if there is a relationship to DAG and sphingolipid saturation between individuals. 
 
Sleep monitoring – Subjects will wear small wrist activity and light exposure recorder (Actiwatch, Mini 
Mitter, Bend, OR) throughout the study. The Actiwatch is a small and light device about the size of a 
watch that uses accelerometer-derived information to estimate sleep and wake times. It should not be 
worn swimming or in the shower. 
 
 
Insulin clamp study - Subjects will spend the night on the inpatient CTRC to ensure compliance with the 
overnight fast. During the overnight stay we will administer standardized sleep questionnaires that ask 
about subjective sleep quality, sleep latency, number of awakenings, sleep duration, factors that may 
have disturbed sleep and a sleepiness rating. We will use Compumedics Inc Siesta digital sleep 
recorders to assess polysomnographic (PSG) data. Data will be stored and sampled at a rate of 256 
samples per second per channel with a 12-bit A-D board. High and low pass digital filters for 
electroencephalogram (EEG) and electroocculogram (EOG) will be set at 0.25 and 35 Hz. Monopolar 
EEGs-International 10-20 system using current guidelines (F3xM2, C3xM2, F4xM2, C4xM1, and O2xM1 
O1xM2). Sleep will be scored according to standard guidelines. Impedances below 5 kohms will be 
obtained. Sleep data will be scored to describe how the treatments influence sleep. Oronasal thermal 
sensor, nasal pressure, pulse oximiter, thorasic and abdominal plethysmography, ECG, and bilateral 
bipolar anterior tibialis electromyogram (EMG) recordings will be used to measure sleep disturbances 
(Apnea Hypopnea Index (AHI), Periodic Leg Movements (PLM)).  
 
The following morning, an antecubital catheter will be placed in one arm for infusions, and another placed 
in a distal hand vein in the contra-lateral arm for blood draws using the heated hand vein technique (37). 
After 30 minutes of rest, a baseline blood sample will be taken for metabolic substrates, hormones, and 
cytokines. Thereafter, a primed (0.03mg/kg), constant infusion (0.04mg/kg/min) of [6,6-2H2]glucose and 
[U-13C]glucose, as well as a constant infusion (0.03µmol/kg/min) of [U-13C]palmitate will begin.  The 
glucose and palmitate stable isotope infusions will continue through the end of the study and will be used 
to determine hepatic glucose production, peripheral glucose disposal, and muscle de novo DAG and 
sphingolipid synthesis. After 2 hours of infusion, a percutaneous needle biopsy will be taken midway 

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between the greater trochanter of the femur and patella. Then a standard 3 hour 
hyperinsulinemic/euglycemic clamp will follow as described by DeFronzo with insulin infused at 40 
mU/m2/min, and glucose clamped at 90mg/dl with a variable dextrose infusion (38). Dextrose infused to 
maintain euglycemia will be “spiked” with [6,6-2H2]glucose to minimize changes in isotopic enrichment. 
Blood will be sampled every 5 minutes to determine glucose concentration and the dextrose infusion 
adjusted as necessary. A second muscle biopsy will be taken from the contra-lateral limb 1 hour into the 
insulin clamp to measure insulin-stimulated insulin signaling.  Blood samples will be taken over the final 
30 minutes for substrate, hormone, and cytokine analyses. Isotopically measured glucose rate of 
disappearance will be measured during the last 30 minutes of the 3 hour clamp, normalized to plasma 
insulin concentration, and used as a measure of whole body insulin sensitivity (39).  
 
 
Muscle biopsy – Subcellular fractionation will be performed as described in Figure 1. 
Diacylglycerol (1,2- and 1,3- isomers), sphingolipids (dihydroceramide, ceramide, glucosylceramide, 
lactosylceramide, sphingosine, sphingosine-1-phosphate, sphingomyelin), triacylglycerol, phosphatidic 
acid, and long chain acyl-CoA species will be analyzed by Dr. Kathleen Harrison using the lipidomics 
platform in our laboratory. Subcellular fractions are fortified with internal standards (ISs), lipid extracted, 
and analyzed by an Agilent 1100 HPLC connected to an API 2000 triple quadrupole mass spectrometer 
(40). The 1,3- and 1,2- DAG isomers are separated chromatographically using a Hilic 2.1 micron, 
3x100mm column. Concentration is determined by comparing ratios of unknowns to odd chain or 
deuterated internal standards. Incorporation of [U-13C]glucose into DAG glycerol, and [U-13C] palmitate 
into the sphingolipid backbone will be used to estimate de novo synthesis of DAG and sphingolipids (41; 
42). Western blot analysis will quantify PKC translocation of  θ, ε, βII, and δ isoforms in basal biopsies, as 
well as insulin signaling in basal and insulin stimulated biopsies including AKTser473 /total, IRS-1Tyr612/total, 
and AS160Thr642/total using standard techniques (43).  AKTThr34 phosphorylation will be used to estimate 
PKCζ activation by ceramide. PP2A activity will be measured using manufacturer instructions (Ser/Thr 
Phosphatase Assay Kit 1, Billerica, MA). 
 
After the baseline clamp, subjects will be randomized into one of three interventions for three months: 
 
  1. Endurance exercise training - Participants will 
undergo supervised endurance exercise training 
administered by the Nutrition Obesity Research Center 
(NORC) Energy Balance Core (see attached letter of 
support from Dr. Kohrt) using well-established methods 
(44-48). Participants will perform supervised exercise 
4x/week as well as exercise 1x/week on their own. Each 60 
min session will include a short warm-up, 40 to 50 min of 
endurance exercise, and a cool-down. The exercise 
program will consist primarily of brisk walking or jogging, 
and will be supplemented with rowing, stepping, or elliptical 
exercise. The initial exercise prescription will be 30 minutes 
at 65% of maximal HR. During the first 2 weeks of training, 
exercise duration and intensity will be gradually increased 
to 45 min at 80 to 85% of maximal HR. This exercise training program resulted in a 9% increase in 
VO2max in our pilot study (Figure 6A). Participants will be weighed weekly, and will be instructed to 
consume more food to prevent weight loss during this intervention. Maximal exercise testing will be 
performed half way through the intervention, and exercise prescriptions updated every 2 weeks. 
 
2. Diet-Induced Weight loss - The weight loss program will be administered by the Clinical Core of the 
NORC (see attached letter of support from Dr. Bessesen). Participants will receive a low calorie diet 
consisting of 4-5 servings of vegetables per day and 4-5 meal replacements (Health Nutrition Technology 
Inc., Carmel California). No other food intake will be allowed.  To reduce the risk of gallstone formation 
subjects will consume 2 teaspoons (10g) per day of vegetable oil.  This diet will provide 890 kcal/d, 75 g 
of protein, 15 g fat and 110 g of carbohydrate and 100% of the DRI of all vitamins, minerals and 
micronutrients. This meal replacement program was used in our pilot study with an average 9% weight 
loss and 62% increase in insulin sensitivity in in 12 weeks (Figures 6B and 6C). Subjects will be weighed 
 
 
 
 

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weekly and will meet bi-weekly in group sessions to receive nutritional/behavioral counseling.  After the 3-
month intervention, subjects will transition to their normal diet for 2 weeks of weight maintenance, 
supervised by research dieticians with extensive experience helping subjects maintain a weight-reduced 
state. 
 
3. Delayed intervention control – This group of individuals will serve as our free-living control group, 
and ensure none of the changes observed in the diet-induced weight loss or exercise training 
interventions are due to receipt of health information, exercise testing, or contact with investigators. After 
finishing the study, they can choose to undergo the weight loss, exercise training, or combination 
intervention.  
 
After finishing the intervention, participants will repeat the maximal exercise testing, MRI scan, DEXA 
measurement of body composition, sleep recording and the insulin clamp study after 2 weeks of weight 
stability on their typical diet. 
 
Muscle biopsy specimens from specific aims 1 and 2 will be used for this aim. Fresh muscle biopsies 
samples will be permeabilized for mitochondrial respiration studies, as well as homogenized for 
mitochondrial respiratory chain complex activity. Measurements shown in Table 2A will be performed 
with and without the addition of increasing doses of specific 1,2- and 1,3-DAG and sphingolipid 
species, as shown in Table 2B, before and after each intervention. Ceramide and dihydroceramide 
will be administered as an equal mix of C18:0, C24:0, and C24:1 species, while 1,2- and 1,3-DAG will 
be an equal mix of Di-C18:0, C16:0/C18:0, and C16:0/C18:1 species. In addition to vehicle controls, 
we will also administer triolein to ensure responses observed are not due to a general effect of lipids 
on mitochondrial function.  
 
  
Mitochondrial Respiration - All experimental 
measurements of oxygen consumption will be determined 
by a high-resolution respirometry Oxygraph-2K 
(OROBOROS Instruments, Innsbruck, Austria, see 
attached letter of support from Dr. MacLean). 
Measurements will be performed at 37 °C and respiration 
rates expressed in nmolO2·min−1 ·mg·dry wt−1. 
Respiration will be normalized to vehicle control, since 
basal respiration rates will vary based on training status and quality of 
muscle fibers from the biopsy. Permeabilization will be carried out directly 
in an oxygraph chamber with saponin (20 μg/mL) for 5 min before starting 
the measurements as previously described (49). To evaluate the in vivo 
condition, mitochondrial respiration in permeabilized muscle will be 
determined with glutamate + malate (GM), octanoyl carnitine + 
malate(OM), and succinate as respiratory substrates (Table 2A). In the 
presence of either GM or OM, respiration is initiated with a saturating 
concentration of ADP (2 mM) and the Vmax of maximal ADP-stimulated 
respiration rate or state 3 respiration rate is determined. To evaluate the 
influence of DAG and sphingolipids on mitochondrial respiration, 
sphingolipids in isopropyl alcohol (1ul in 2ml respiration buffer), DAG in 
ethanol (1ul in 2ml respiration buffer), or vehicle controls are then added  
serially to measure affects of low, medium, and high lipid content. The 
intactness of mitochondrial membranes will then be validated by 
cytochrome c tests for each experiment. Only samples passing this quality 
test will be used further. Oligomycin is then given to measure mitochondrial 
proton leak. FCCP will then be titrated to uncouple the mitochondria and a 
maximal rate of the electron transport system rate determined.  Antimycin A 
(10 μM) is then added to determine the rate of residual oxygen 
consumption (Rox).  Respiration rates are then corrected for Rox. 
Respiration kinetics of GM and OM will also be determined by adding ADP 

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cumulatively and the maximum rates of respiration (Vmax) and values for apparent KmADP 
determined using Michelis-Menton kinetics.   
Mitochondrial Complex Activity – Respiratory chain enzyme activity for complexes I, II, II+III, III, IV, 
and citrate synthase will be assayed from muscle homogenates as previously described, modified for 
96 well plate format (50). Respiratory chain activity assays will be performed with addition of DAG 
and sphingolipids as shown in Table 2B to parallel mitochondrial respiration studies.  
GSH/GSSG and acyl-carnitines – These will be measured as described in specific aim 2. 
  
 
D.   Description, Risks and Justification of Procedures and Data Collection Tools: 
 
 
Potential Risks 
Radiation exposure. The DEXA measurement involves a very low dose of radiation 
(approximately 0.03mRem total equivalent dose per measurement) which is less than 1% of the 
radiation received from a chest x-ray.  There is no known risk from receiving this dose of radiation. 
 
IV risks.  A hollow needle/plastic tube will be placed for obtaining blood samples or giving fluids.  
This will be left in for 5-8 hours.  When the needle goes in the vein it may hurt for a short time, and 
there may be discomfort from having the hollow needle/plastic tube in the subjects’ arm.  There is a 
1/10 chance a small bruise forming in the area, 1/100 chance of a clot forming in the vein, and 1/1000 
chance of infection or significant blood loss. A total of ~250ml of blood will be taken from the subjects 
during the study in aims 1 and 2.  This is approximately 5% of total blood volume and is not 
associated with a risk of anemia. 
 
Venipuncture. Subjects will feel pain when the needle goes into the vein. Approximately 5% of 
people may faint, feel nauseous or dizzy.  Not uncommonly a bruise may form at the puncture site.  
 
Muscle strength testing. Subjects may feel muscular discomfort during the muscle strength test. 
They may have sore muscles the day after the test. 
 
Risk of maximal mitochondrial respiration test. You will experience frequent tightening of the blood 
pressure cuff on your leg, which may be uncomfortable.  
 
Risks of sleep recording. Measurement of the electrocardiogram (ECG-heart tracing) may cause 
some skin irritation from the sensors. Measurement of the electroencephalogram (EEG-brain wave 
activity), Electroocculogram (EOG-eye movement activity) Electromyogram (EMG-muscle activity on 
the chin and legs), nasal-oral air thermister (breathing in and out of the nose & mouth), and 
respiratory bands placed over the chest and abdomen, may cause some minor discomfort and/or skin 
irritation due to the paste used to attach the sensors.  In addition, the paste used to hold sensors to 
the scalp may leave a flaky residue for several days. The physiological recording device is electrically 
isolated and complies with hospital standards for electrical safety. 
 
MRI. Subjects may feel claustrophobic during the MRI. They may also experience flashing light in 
their eyes, and temporary warmth and reddening of the skin. 
 
Insulin clamp. The hyperinsulinemic/euglycemic clamp carries a 1 in 10 risk for symptoms of 
hypoglycemia (neuroglycopenic or autonomic), and less than 1 in 10,000 of serious sequellae of 
hypoglycemia such as seizure, coma or death. 
 
Muscle biopsies. Muscle biopsies are frequently uncomfortable and result is a bruise over the 
site.  Risks of bleeding, infection, damage to nerves or vessels is ~1 in 100 or less. There may be 
discomfort following the biopsies after the anesthetic wears off. The CTRC will provide pain relievers 
to minimize potential discomfort. 
 
Weight loss. During the weight loss phase, subjects could experience one or more of the 
following: bowel changes, cold sensation, and menstrual irregularities.  During the weight loss phase, 

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subjects could experience one or more of the following: dizziness or lightheadedness, fatigue, dry 
skin, and some temporary hair loss.  Not everyone will have these side effects. Eating high fat foods 
while on the low calorie diet has additional risk of triggering an acute gallbladder attack. Some 
research studies have found that during a low calorie diet, there is an increased risk of gallstone 
formation or gallbladder disease. 
 
Exercise testing and training. During the exercise testing and training subjects may experience a 
fall, as well as discomforts such as pain in muscles or joints. During the exercise testing and training 
subjects may feel dizzy or faint as well as experience irregular heartbeats. During the exercise testing 
and training subjects may experience a stroke or heart attack. The risk of death during or immediately 
after an exercise test is less than 1 in 10,000. The risk of a heart attack during or immediately after an 
exercise test is less than 4 in 10,000. The risk of a problem that would require hospitalization, such as 
chest pain, is less than 2 in 1000. 
 
Risks of losing confidentiality. Violation of privacy and loss of confidentiality are both risks to 
which research participants are exposed.  The possibility of these risks increases when protected 
health information is collected.  
 
Alternative treatments.  We see no psychological, social, or legal risks beyond those of 
participation in health-related research. There are no alternative methods available that would permit 
the collection of the required information for this research study.  
 
ADEQUACY OF PROTECTION AGAINST RISKS 
a. Recruitment and informed consent 
A member of the investigative team will obtain informed consent and HIPAA authorization in 
tandem.  Subjects will have a copy of the COMIRB-approved consent form sent to them my mail or 
email for their review prior to meeting with a team member.  Potential subjects will talk with a team 
member for a full explanation of the study logistics, rationale, risks and benefits, and also to 
ask/answer questions. Informed consent will be obtained at the start of the preliminary screening visit. 
This will be done in a private, quiet, and unhurried environment. Volunteers will be asked to explain 
the consent form in their own words to ensure understanding of the study and what is expected of 
them.  No tests or procedures will be performed prior to the subject signing the informed consent. 
Only volunteers capable of providing informed consent may participate in this research.  Informed 
consent will be obtained by a member of the research team who has been through COMIRB 101 and 
102 training (from our local institutional review board). Subjects will receive a copy of their signed 
consent.   
 
b. Protection against risk 
Every attempt will be made to minimize the risks involved to patients in this study. Subjects 
privacy, autonomy and confidentiality will be protected with redundant IRB & HIPAA-compliant 
measures.  Subjects may withdraw from the study at any time.  Only state-of-the-art techniques will 
be used in a NIH-funded, JCAHO accredited CTRC facility.  CTRC personnel are exquisitely trained, 
and emergency equipment is easily accessible on-site.  Universal precautions will be taken to protect 
everyone involved with subject testing.  Sterile technique is invoked where appropriate.  All infusion 
are prepared and stored by a licensed pharmacist and tested for sterility and pyrogenicity. We will 
also have an independent safety officer who will evaluate subject risk annually. Taken together, these 
measures have proven successful in the minimization of risk to subjects. Protected and de-identified 
information will be kept on password-protected computer systems with hard copies in locked filing 
cabinets in locked offices. 
 
    
E.   Potential Scientific Problems:   
We have extensive experience recruiting individuals across the metabolic spectrum (51-55), as well 
as performing insulin clamps (39; 51; 56; 57), and muscle biopsies (26; 43; 51; 53; 58-61) and therefore 
no problems are expected with the proposed studies. An alternative approach to our proposal would be to 
use a combined exercise and weight loss intervention, which may have more dramatic results compared 

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to exercise or weight loss only interventions. If we find minimal changes in insulin sensitivity with either 
intervention used in isolation, we will move to a combined intervention. Different degrees of success with 
the interventions are important to provide a spread in the data for changes in localized lipids and insulin 
sensitivity. An alternative approach would be to provide food for subjects to consume prior to both insulin 
clamps. We excluded this design because of the possibility of changing muscle lipid species and 
localization with changing dietary patterns. If individuals cannot maintain their weight within 2kg of starting 
weight in the exercise only intervention, they will be removed from the study. Our focus in this proposal is 
on DAG and sphingolipids, however, we will also measure changes in triglyceride, long chain acyl-CoA, 
and phosphatidic acid to determine other lipids related to changes in insulin sensitivity and/or cellular 
signaling.  If there are no changes in PKC, we will evaluate changes in non-PKC DAG targets such as 
PKD and RasGRP1/4 to determine if these less studied DAG targets relate to insulin resistance. 
 
Our typical muscle biopsy yield after removal of extra-muscular lipid, is approximately 160mg wet weight, 
and the tissue required for the assays in this project is 135mg wet weight, allowing all assays to be performed 
on the majority of biopsies. If muscle yield is limiting, we will prioritize analysis in the following order: 
subcellular fractionation (50mg) >western blot (15mg) >mitochondrial respiration (25mg) >PP2A activity 
(10mg) >PKC activity (35mg). We will also evaluate changes in other mitochondrial/ER lipids related to 
changes in mitochondrial function. In addition to those lipids listed under aim 1, we will also measure 
cardiolipin content as well as composition. 
 
 
F.   Data Analysis Plan:   
Aim 1: Our statistical methods are rigorous with an a priori statistical analysis plan to test our hypothesis 
in an unbiased manner, and power analysis based on our own preliminary data. Prior to analysis, data will 
be analyzed for normality, and any non-normally distributed variables will be log-transformed for use in 
analytic models. Correlations between the change in sarcolemmal DAG and sphingolipid species and the 
change in insulin sensitivity will be performed using Pearson’s correlation coefficients in the entire study 
group (n=51). Then, data comparing changes in sarcolemmal DAG and sphingolipid species to changes 
in insulin sensitivity before and after the intervention will be analyzed using a one-way Analysis of 
Variance (ANOVA) to examine overall differences between the intervention groups, and using Tukey’s 
procedure to make pair-wise comparisons (e.g control vs. exercise, control vs. diet only) while adjusting 
for multiple comparisons. Changes in individual DAG and ceramide species will be examined using the 
Benjamini Hochberg procedure to limit false discoveries (62), and then Principal Component Analysis 
(PCA) will be used to examine groups of DAG and ceramide species (13). Analyses will be conducted 
using SAS statistical software ver. 9.4 (Cary, NC). The power analysis is based on changes in 
sarcolemmal lipids assuming there will be no significant changes in the control intervention. With an 
overall n of 51, we will have 92% power to detect a Pearson correlation coefficient similar to our 
preliminary data (Figure 2B, r= -0.45), and 80% power to detect a correlation coefficient as small as -0.38.  
Pilot data showed sarcolemmal C18:0 ceramide decreased 0.041 ng/ug protein with a SD of .02 after the 
combined intervention. With an n of 17/group we will have 84.6% power to measure a similar change in 
sarcolemmal DAG and sphingolipids for individual group comparisons using the Tukey procedure. The 
type 1 error rate (alpha) will be set at 5% for all comparisons. 
 
Aim 2: Our statistical methods are rigorous with an a priori statistical analysis plan to test our hypothesis 
in an unbiased manner, and power analysis based on our own preliminary data. Data will be analyzed for 
normality, and any non-normally distributed variables will be log-transformed for use in analytic models. 
Correlations between the change in mitochondrial/ER DAG and sphingolipid species and the change in 
insulin sensitivity, mitochondrial respiration, ROS production, acyl-carnitine species, and ER stress will be 
performed using Pearson’s correlations coefficient in the overall study group. Data comparing changes in 
mitochondrial/ER DAG and sphingolipid species to changes in insulin sensitivity and parameters of 
mitochondrial respiration between groups before and after the intervention will be analyzed using a one-
way ANOVA (SAS statistical software ver. 9.4, Cary, NC). If overall differences are observed by group 
then individual group comparisons will be made using Tukey’s procedure, which corrects for family-wise 
error rates.  Changes in individual DAG and ceramide species will be examined using the Benjamini 
Hochberg procedure to limit false discoveries, and changes in groups of DAG and ceramide species will 
be examined using PCA as described in Aim #1. Our power analysis is based on our preliminary data for 

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the change in mitochondrial ceramide and DAG species after the pilot intervention shown in Figures 8 
and 10, and assuming there will be no significant changes in the control intervention. With an overall n of 
51, we will have 99% power to detect a Pearson correlation coefficient as large as that observed in our 
preliminary data (Figure 7B, r= -0.44), and we will have 80% power to detect a correlation coefficient as 
small as -0.38.  With an n of 17/group, we will have 95% power to measure a similar change in 
mitochondrial DAG and sphingolipids as we have observed in our pilot data after the weight loss or 
exercise interventions using pairwise comparisons in ANOVA models using the Tukey procedure.  The 
type 1 error rate (alpha) will be set at 5% for all comparisons. 
 
Data analysis plan for secondary data points regarding sleep component of the study: 
Sleep duration and markers of sleep quality will be obtained from Actiwatch recordings and scored using 
the provided Actiware Software. Data are collected from one week preceding the baseline clamp study 
visit and again from one week preceding the post-intervention clamp study visit. Actigraphy data will be 
averaged across 7 days to create average sleep duration and sleep quality scores for each participant 
before and after intervention. We will then examine the relationship between changes in sleep variables 
and changes in insulin sensitivity assessed by the clamp study. 
 
 
G.  Summarize Knowledge to be Gained:   
Achievement of these aims will yield the following expected outcomes: 1) sarcolemmal 1,2-
disaturated DAG and C18:0 ceramide promote insulin resistance through PKC and PP2A activation; 2) 
mitochondrial/ER sphingolipids correlate with mitochondrial function, ER stress, ROS, acyl-carnitine 
content, and insulin resistance; 3) DAG and sphingolipids reduce mitochondrial function and increase 
ROS and acyl-carnitine generation in vitro consistent with a cause and effect relationship. These 
outcomes will be clinically significant by identifying signaling pathways and mechanisms of accumulation 
of specific localized DAG and ceramide species which can ultimately be targeted for the prevention and 
treatment of diabetes. 
 
 
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