Trial Information

Calorie Restriction Retards the Aging Process by Inducing Sirtuins and Uncoupling Protein 3, Thereby Reducing the Production of Reactive Oxygen Species.

HYPOTHESIS: Calorie restriction delays the ageing process and thus telomere erosion, due to
a reduction of oxidative stress, via expression modulation of uncoupling proteins and
sirtuins.

RETROSPECTIVE STUDY:

Approximately 1000 adults aged 40 to 55 years, that had participated in nutritional
assessments studies during which their body weight was recorded, and had a body mass index
(BMI) of less than 30 kg/m2 at that time will be considered eligible. Other sources of
subject recruitment will be health records from armed forces personnel and our institutional
data base. Those that accept and sign a written informed consent, will be subjected to:

1. A complete clinical history, recording past and present diseases, medication use,
history of smoking, drinking and illicit drug use.

2. A dietary questionnaire to assess nutrient intake

3. A complete clinical examination including the measurement of body weight, height, blood
pressure.

4. A physical activity questionnaire previously validated by us 1

5. Measurement of body composition by DEXA

6. Withdrawal of a 10 hours fasting blood sample of 30 ml to measure

- Packed red cell volume, blood glucose, creatinine, serum lipids, TSH, thyroid
hormones and insulin

- Telomere length in peripheral blood mononuclear cells (PBMC)

- Sirt1 and 6 gene expression in PBMC.

- Plasma 8‐ isoprostanes

7. Measurement of carotid intima media thickness by ultrasound, performed by a trained
professional using previously reported protocols.

Once evaluated, subjects will be classified as:

1. Weight maintainers if their actual weight is, at the most within 10% of the weight
recorded at least 10 years before

2. Weight gainers, if their actual weight is over 20% of the weight previously recorded

The following will be excluded from further analysis:

1. Subjects with a history of chronic debilitating diseases such as renal failure, cancer,
autoimmune diseases,hypothyroidism, diabetes mellitus or AIDS.

2. Subjects engaged in extremely high physical activity (more than 10 hour per week of
sports or work requiring extreme physical activity such as shoveling or hard manual
work.

3. Subjects with a history of drug abuse or alcoholism

4. Subjects using anorexigenic or anabolic drugs, or chronic users of non steroidal
anti‐inflammatory drugs.

5. Subjects smoking more than 5 cigarettes per day (91 packs per year)

6. Overly malnourished subjects, defined as a body mass index below 18 kg/m2.

PROSPECTIVE STUDY:

Main objective:

To study the effects of a three month period of calorie restriction in normal volunteers on
the expression of expression of SIRT1 and UCP3 in muscle, SIRT6 in PBMC, brown adipose
tissue activity and the accumulation of markers of oxidative damage.

Specific goals:

1. To develop a prospective model of calorie restriction in normal human volunteers.

2. To study changes associated with calorie restriction on:

- Serum markers of inflammation and free fatty acids during fasting.

- The uptake of 18fluorodexoyglucose by brown adipose tissue measured by PET‐CT

- The expression of mRNA of UCP3 and SIRT1 in muscle biopsies.

- The expression of mRNA for SIRT1 and 6 in muscle and peripheral blood mononuclear
cells

- The accumulation of 4 hydroxynonenal and 8 hydroxyguanosine in muscle, as markers
of oxidative damage

- The production of ROS by peripheral neutrophils as an overall measure of the
capacity to generate free radicals

- Insulin sensitivity, measured using formula derived from glucose tolerance.
METHODOLOGY Premenopausal women, aged 25 to 40 years, with a BMI between 27 and 32
kg/m2, interested in losing weight, will be studied. Inclusion criteria will be:
complete high school, stable weight (± 2 kg) in the last six months, and absence
of diabetes, medication intake that could influence energy expenditure such as
beta blockers (except oral contraceptives) and lack of engagement in competitive
or exhausting physical activities. Women will be required to use an effective
contraceptive method, and a pregnancy test will be performed every month while the
study lasts.

After signing an informed consent form, total energy expenditure will be measured through
calorimetry and actigraphy or doubly labeled water. Subjects will be considered to be in
energy balance, taking into account that weight is stable (the lack of weight change will be
assessed during the two weeks required for inclusion studies). Therefore, it can be assured
that energy intake at that moment is equal to total energy expenditure .

Afterwards, subjects subjected to a 25% restriction of their measured total energy
expenditure (this level of restriction has been well tolerated by volunteers, even during
one year). Restriction will be balanced, using a mediterranean type diet, maintaining a
proportion of 50% carbohydrates, 25-30% of lipids, 25% of proteins and a provision of 100%
of micronutrient requirements. Patients will attend to dietician reinforcement every week.
To ease the restriction period, one extra meal will be allowed once a week. According to
weight change, volunteers will be considered as energy restricted (group 1) is they loose at
least 3 kg or 5 % weight during the study period. Instead those subjects that do not reach
these goals will be considered as the control group (Group 2).

The intervention will last three months and will be carried out during a period in which
temperature remains relatively stable and is lower, in Santiago Chile, to avoid an effect of
climate on brown adipose tissue activity. The ideal will be between May and August. Both
groups will be evaluated on a weekly basis by a nutritionist to assess compliance with the
diet, reinforce indications and be weighed. Subjects will receive a monthly monetary
remuneration in order to permit the purchase of specific foods and brands prescribed. Each
subject will be educated about healthy dietary and physical activity lifestyle. Eventual
changes in physical activity which will be ascertained using portable actigraphs
(Actiheart®).

Apart from weight change, volunteers will be considered as calorie restricted if
triglyceride levels decrease at least 15% and T3 levels are reduced by at least 8 ng/dl.

At baseline, and the end of the three month intervention period in which controls should be
in zero energy balance and adherent subjects in negative energy balance, the following
determinations will be carried out:

1. Measurement of routine clinical laboratory parameters to discard concomitant diseases.

2. Measurement of fasting serum free fatty acids.

3. Measurement of ultrasensitive C reactive protein and carboxymethyllisine (CML) as an
inflammation marker and glycotoxin respectively, using commercial ELISA kits.

4. Measurement of triglyceride and T3 levels to assess compliance with calorie
restriction.

5. Glucose tolerance curve to measure glucose and insulin and calculate insulin
sensitivity using Matsuda formula

6. Measurement of oxidative burst of peripheral neutrophils by Fluorescence Activated Cell
Sorting (FACS) as an expression of ROS production capacity of the organism

7. Measurement of SIRT1 and 6 gene expression in PBMC by RT‐PCR according to the technique
reported by Crujeiras et al

8. PET CT to measure 18fluorodeoxyglucose uptake by supraclavicular fat in thermoneutral
conditions and after immersing one foot in water at 8°C during five minutes, according
to Virtanen et al.

9. Vastus lateralis muscle biopsy, performed by a physician using a Bard needle using a
technique previously reported buy our group (this is a safe and simple procedure but
the biopsy sample obtained is small, therefore the analyses that can be performed are
limited), to measure:

- mRNA for UCP3 by RT‐PCR , using the technique reported by Millet et al in biopsies
obtained in a similar way in humans.

- mRNA for SIRT1 by RT‐PCR using the technique reported by Heilbronn et al.

- Immunohistochemical expression of 8 hydroxyguanosine and 4 hydroxynonenal as
oxidation markers, using methods previously reported by us.

10. Body composition measurement by double energy X ray absorptiometry (DEXA).

11. Resting energy expenditure measurement in and open‐circuit indirect calorimeter (model
2900, Sensor Medics Corp., Yerba Linda, CA, USA).

12. Actigraphy using Actiheart actigraphs to assess eventual changes in spontaneous
physical activity induced by calorie restriction.

Telomere length will not be measured in the prospective protocol, considering that not
changes will be detected in the short study period.