Dottorato di Ricerca in Obesità e Patologie correlate - VIII Ciclo N. S.
Tesi di Dottorato di Francesca Raffaelli
ROLE OF NITRIC OXIDE METABOLISM IN OBESITY:
POSSIBILE MOLECULAR MECHANISMS AND FUTURE IMPLICATIONS
Background and aim:
Human obesity is a clinical condition characterized by an increased body mass index (BMI) resulting from
a positive energy balance. Obesity has a multifactorial pathogenesis: both genetic and environmental
factors, such as incorrect diet or sedentary life style, play a major role; even so, our knowledge on the role
of physiological and molecular mechanisms involved in this pathology is still limited. Obesity represents
an important risk factor for different pathologies, from damage by mechanical overload (articular
pathologies as gonarthrosis and feet arthrosis) to serious diseases as insulin-resistance, dislipidemia,
hypertension, inflammation, hormonal imbalance, diabetes, atherosclerosis, cancer and the ones which are
grouped under cardiometabolic pathologies.
In addition, obesity can be associated with gestational complication as gestational diabetes and
preeclampsia; gestational obesity implies a high risk for mother and foetus, proportional to both obesity
grade and metabolism alteration.
Furthermore, different studies have highlighted that in humans, oxidative stress is responsible of many
chronic diseases, often degenerative, including obesity. Moreover, numerous experimental evidences
demonstrate that obesity state is characterized by endothelial dysfunction and associated with altered
release on endothelial NO. Responsible of this alteration are free radicals which cause oxidative stress and
destroy NO. Different epidemiological studies have shown that obesity, dismetabolic pathology associated
with oxidative damage, is considered a risk factor for atherosclerosis and cardiovascular pathologies onset
and gestational complications. Thus, an altered NO metabolism and his reduced bioavailability , could
contribute to obesity onset, in pregnancy or not, and to complication associated.
Unfortunately, pathogenetic mechanisms of obesity are still to be clarified but it’s known that obesity is
associated with alterations of plasmatic lipids and lipoprotein metabolism. Previous studies have
demonstrated as functional and structural variations, whether quantitative or qualitative, of
lipid/lipoproteins and lipoproteins/platelets, play a key role in the onset of the major cardiovascular
diseases obesity-induced.
Thus, the aim of this research, during the first year, was to investigate such functional and structural
alterations, whether in lipoproteins or in platelets of obese patients compared to healthy controls, and
interactions between lipoproteins and platelets in such pathological condition. During the second year, the
aim was to asses oxidative and nitrosative stress in platelets and plasma of the same obese subjects
compared to controls. Finally, during the third year, the aim was to asses oxidative and nitrosative stress in
platelets and plasma of obese pregnant women at 33th gestational week compared to healthy pregnant
women. At this purpose, in this research has been assessed:
1) During the fist year:
- levels of triglycerides, total cholesterolemia, HDL- and LDL cholesterol, total cholesterol/HDL
cholesterol ratio in obese subjects compared to controls;
- levels of proteins, triglycerides, phospholipids, cholesterol and lipid hydroperoxide in LDL of obese
subjects compared to controls LDL;
- Ca2+ ATPase and Na+/K+ ATPase activity and membrane fluidity tested by fluorescent probe TMA-DPH
in platelets of obese subjects, controls and in control platelets incubated with LDL isolated from obese
patients.
2) During the second year:
- Nitric Oxide (NO) levels in plasma and platelets of obese subjects compared to controls;
- Peroxynitrite (ONOO-) levels in plasma and platelets of obese subjects compared to controls;
- Lipid hydroperoxide levels, assessed by oxidation with ferrous xylenol orange (FOX), in plasma and
platelets of obese subjects compared to controls;
- Lipoperoxide levels, assessed by thiobarbituric acid reactive substances (TBARs), in plasma and platelets
of obese subjects compared to controls;
- Conjugated dienes levels only in platelets of obese subjects compared to controls.
3) During the third year:
- Nitric Oxide (NO) levels in plasma and platelets of obese pregnant women compared to healthy pregnant
women;
- Peroxynitrite (ONOO-) levels in plasma and platelets of obese pregnant women compared to healthy
pregnant women;
- Lipid hydroperoxide levels, assessed by oxidation with ferrous xylenol orange (FOX), in plasma and
platelets of obese pregnant women compared to healthy pregnant women;
- Lipoperoxide levels, assessed by thiobarbituric acid reactive substances (TBARs), in plasma and platelets
of obese pregnant women compared to healthy pregnant women;
- Conjugated dienes levels only in platelets of obese pregnant women compared to healthy pregnant
women;
- Paraoxonase activity only in plasma of obese pregnant women compared to healthy pregnant women.
Results:
1) Fist year:
It has been observed a significant increase in triglycerides, total cholesterol/HDL cholesterol ratio and
LDL cholesterol in obese subjects compared to controls. It has been shown a non significant increase in
total cholesterol as well as a non significant decrease in HDL cholesterol obese subjects compared to
controls.
The analysis of the chemical composition of LDL from obese patients showed a significant increase in the
percent content of TC and TG and in the mean levels of lipids hydroperoxide compared to controls LDL.
The LDL of same patients have shown not significant increases in percent content of proteins and
phospholipids. Platelet Na+/K+ ATPase and Ca2+ ATPase activities showed respectively a significant
decrease and increase in patients compared to controls; minor significant respectively decreases and
increases are shown also in control platelets incubated with LDL from obese patients. Anisotropy tested
with TMA-DPH probe was significantly increased both in platelets from obese patients and in control
platelets incubated with LDL from obese patients compared to control platelets.
Finally, in obese patients positive significant correlations between Ca2+ ATPase activity and respectively
content of triglycerides, total cholesterol, lipid hydroperoxide in obese LDL have been observed; positive
significant correlations between TMA-DPH anisotropy and respectively content of triglycerides, total
cholesterol, lipid hydroperoxide in obese LDL have been observed. Moreover, negative significant
correlations between Na+/K+ ATPase activity and respectively content of triglycerides, total cholesterol,
lipid hydroperoxide in obese LDL have been observed. In obese patients, a negative significant
correlations between BMI and Na+/K+ ATPase activity, as well as a positive significant correlation
between BMI and respectively Ca2+ ATPase activity, TMA-DPH anisotropy, content of triglycerides, total
cholesterol, lipid hydroperoxide in obese LDL have been observed.
2) Second year:
It has been observed a significant decrease of NO levels in platelets and plasma of obese subjects
compared to controls, with a stronger decrease in platelets respect to plasma.
It has been observed a significant decrease of ONOO- levels in platelets and plasma of obese subjects
compared to controls, with a stronger decrease in platelets respect to plasma.
Regarding peroxidation levels assessed by FOX, TBARs, it has been observed a significant increase in
platelets and plasma of obese subjects compared to controls, with a stronger decrease in platelets respect to
plasma. In addition, peroxidation levels assessed by conjugated dienes have been shown significant
increase in platelets of obese subjects compared to controls.
Moreover, a significant negative correlation between BMI and NO in plasma and platelets of obese
subjects has been observed; positive significant correlations between BMI and respectively ONOO-, FOX,
TBARs and conjugated dienes in plasma and platelets of obese subjects have been observed.
3) Third year:
It has been observed a significant decrease of NO levels in platelets and plasma of obese pregnant women
compared to pregnant controls, with a stronger decrease in platelets respect to plasma.
It has been observed a significant decrease of ONOO- levels in platelets and plasma of obese pregnant
women compared to pregnant controls, with a stronger decrease in platelets respect to plasma.
Regarding peroxidation levels assessed by FOX, TBARs, it has been observed a significant increase in
platelets and plasma of obese pregnant women compared to pregnant controls, with a stronger decrease in
platelets respect to plasma. In addition, peroxidation levels assessed by conjugated dienes have been shown
significant increase in platelets of obese pregnant women compared to pregnant controls.
Finally, Paraoxonase activity has been shown a significant decrease in plasma of obese pregnant women
compared to pregnant controls.
Moreover, a significant negative correlation between BMI and respectively NO and PON activity in
plasma and platelets of obese pregnant women has been observed; positive significant correlations between
BMI and respectively ONOO-, FOX, TBARs and conjugated dienes in plasma and platelets of obese
pregnant women have been observed.
Conclusions:
At the end of the first year, our data evidenced that obesity induces important modifications in platelets and
lipoproteins; thus, it can be hypothesized that the increase in triglycerides, cholesterol and lipid
hydroperoxides content in LDL from obese patients, enhances platelet aggregability, through both the
release of arachidonic acid/augmented synthesis of thromboxane A2, and the increment in intracellular free
Ca2+ concentration which leads to an increase in Ca2+ ATPase activity of obese patients. Moreover, the
decrease in Na+/K+ ATPase activity observed in obese patients can be related to the diminished fluidity in
the platelet membranes of the same patients due to alterations related to obesity (i.e. the increased TG, TC
and lipid hydroperoxides content in LDL).
Moreover, in obesity we observed modified LDL which in turn, by interacting with platelets, could induce
relevant structural and functional modifications for the development of atherosclerotic lesion.
The effects of LDL from obese patients on enzymatic activities and structural organization of platelet
membranes from controls seem to mimic the alterations observed in platelets from obese subjects.
Both platelet hyperfunction and quantitative/qualitative alterations in plasma lipoproteins, as well as an
altered interaction between circulating lipoproteins and platelets, might play a relevant role in the increased
prevalence of the early atherosclerotic lesions development in obese subjects. Thus, the present data point
out that obesity might represent a major potentially modifiable risk factor for the onset of numerous
complications, in particular cardiovascular ones.
According to different studies existing till now, whether during the second year of research, which
involved adult obese subjects, and during the third year of research, which involved obese pregnant women,
it has been observed an increase in oxidative and nitrosative stress in obese patients compared to controls.
While presenting the same trend, levels of NO, ONOO-, FOX, TBARs and conjugated dienes were higher
in obese pregnant women, due to physiology of pregnancy.
In condition of oxidative stress, characteristic of obesity and as observed in our research, the induction of
platelet iNOS induces an NO hyperproduction which causes a shift of ONOO- hyper production, leading to
a reduction in NO bioavailability. ONOO- is an highly reactive specie able to cause lipid peroxidation, as
demonstrated by both the increase of lipoperoxidation levels (FOX, TBARs and conjugated dienes) and the
decrease of PON activity. This induces a platelet dysfunction. An hyperproduction and an uncontrolled
formation of ONOO- as well as an increase in free radical production, play an important role in tissue
damage mechanism during pathological condition as obesity.
Different studies have been demonstrated the involvement of oxidative stress in different pathologies,
including obesity, and many researches evidenced that the oxidative stress could be an important
mechanism whereby obesity increases the incidence of cardiovascular disease. In reality, only few studies
have highlighted the specific relationship among platelet alteration, NO pathway, obesity also in pregnancy,
and atherosclerosis as investigated in this research.
From data obtained at the end of the second and the third year highlighted that the pathological condition
related to obesity induces important modifications on NO metabolism, reducing its bioavailability and
engaging in ONOO- production; ONOO- amplify and propagate tissue oxidative damage, as demonstrated
by the increase in peroxidation levels. In addiction, major modifications observed in platelets compared to
plasma could lead to an higher reactivity and aggregability of platelets, thus inducing both the onset of
atherosclerotic process, and making more difficult maternal-foetal circulation with consequent gestational
complications onset.
In conclusion, our data lead us to hypothesize that oxidative and nitrosative stress could represent an
important and modifiable risk factor for the onset of obesity and its different complications, overall the
cardiovascular ones. From here, systems able to counterbalance alterations in NO metabolism and its
bioavailability, could contribute to prevention of the above-mentioned complication onset.