Metformin and Glucose Concentration as Limiting Factors in Retinal Pigment Epithelial Cell Viability and Proliferation.

Metformin is a well-established drug for the treatment of type 2 diabetes; however, the mechanism of action has not been well described and many aspects of how it truly acts are still unknown. Moreover, regarding in vitro experiments, the glycaemic status when metformin is used is generally not considered, which, added to the suprapharmacological drug concentrations that are commonly employed in research, has resulted in gaps of its mechanism of action. The aim of this study was to determine how glucose and metformin concentrations influence cell culture. Considering that diabetic retinopathy is one of the most common complications of diabetes, a retinal pigment epithelial cell line was selected, and cell viability and proliferation rates were measured at different glucose and metformin concentrations. As expected, glucose concentration by itself positively influenced cell proliferation rates. When the metformin was considered, results were conditioned, as well, by metformin concentration. This conditioning resulted in cell death when high concentrations of metformin were used under physiological concentrations of glucose, while this did not happen when clinically relevant concentrations of metformin were used independently of glucose status. Our study shows the importance of in vitro cell growth conditions when drug effects such as metformin's are being analysed.

Type 2 Diabetes in Obesity: A Systems Biology Study on Serum and Adipose Tissue Proteomic Profiles.

Obesity is associated with metabolic disorders such as insulin resistance and type 2 diabetes mellitus (T2DM), further increasing an already heightened cardiovascular risk. Here, amongst obese class III bariatric surgery patients, we have investigated the effect of T2DM in serum and in two, same patient, adipose tissue (AT) depots through proteomic profile expression analyses. Serum and AT samples from subcutaneous (SAT) and visceral (VAT) fat were collected during bariatric surgery. Bead-based targeted multiplex assay systems were used to simultaneously detect and quantify multiple targets in serum samples (targeted proteomics) and analyze changes in adipokine serum composition. AT samples were assessed through an untargeted proteomics approach. Through a systems biology analysis of the proteomic data, information on the affected biological pathways was acquired. In obese class III individuals, the presence of T2DM induced a significantly higher systemic release of ghrelin, GLP-1, glucagon, MMP3, BAFF, chitinase 3-like 1, TNF-R1 and TNF-R2, and a lower systemic release of IL-8. SAT and VAT proteomes belonging to the same patient showed significant differences in local protein content. While the proteins upregulated in VAT were indicative of metabolic dysregulation, SAT protein upregulation suggested adequate endocrine regulation. The presence of T2DM significantly affected VAT protein composition through the upregulation of dysregulating metabolic pathways, but SAT protein composition was not significantly modified. Our results show that T2DM induces metabolic dysregulation in obese individuals with changes in systemic marker levels and impairment of proteostasis in VAT but not in SAT.

miR-24-3p and Body Mass Index as Type 2 Diabetes Risk Factors in Spanish Women 15 Years after Gestational Diabetes Mellitus Diagnosis.

Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance that is diagnosed for the first time during pregnancy. The objective of this study is to know the glucose tolerance status after 15 years of pregnancy in patients diagnosed with gestational diabetes and to assess the long-term effect of GDM on the circulating miRNA profile of these women. To answer these, 30 randomly selected women diagnosed with GDM during 2005-2006 were included in the study, and glucose tolerance was measured using the National Diabetes Data Group criteria. Additionally, four miRNAs (hsa-miR-1-3p, hsa-miR-24-3p, hsa-miR-329-3p, hsa-miR-543) were selected for their analysis in the plasma of women 15 years after the diagnosis of GDM. In our study we discovered that, fifteen years after the diagnosis of GDM, 50% of women have some degree of glucose intolerance directly related to body weight and body mass index during pregnancy. Dysglycemic women also showed a significantly increased level of circulating hsa-miR-24-3p. Thus, we can conclude that initial weight and BMI, together with circulating expression levels of hsa-miR-24-3p, could be good predictors of the future development of dysglycemia in women with a previous diagnosis of GDM.

100 Years since the Discovery of Insulin, from Its Discovery to the Insulins of the Future.

The term diabetes first emerged in the 3rd century BC, in a reference by Demetrius of Apamea, who described the disease as a dropsy in which any liquid ingested is eliminated in the form of urine. However, the great discovery that revolutionized this field came from the Canadian doctor Frederick Banting, who together with his student and assistant Charles Best, managed to isolate insulin and treat a patient with diabetes on 23 January 1922. This patient was Leonard Thompson, and the results obtained from him were surprising. His glycosuria and ketonuria disappeared and his blood glucose returned to normal. He received daily injections and lived 13 more years. Advances in the treatment of diabetes have been numerous in the 100 years since its discovery. In this review, we recapitulate the most important events that have occurred, and where research is progressing today.

Hypoglycemia during hyperosmolar hyperglycemic crises is associated with long-term mortality.

BACKGROUND: Previous research has indicated that hypoglycemia during hospitalization is a predictor of unfavorable outcomes in patients with diabetes. However, no studies have examined the long-term impact of hypoglycemia in adults admitted for hyperglycemic crises. The study was aimed to investigate the long-term implications of hypoglycemia during hyperosmolar hyperglycemic crises, particularly in terms of all-cause mortality. METHODS: This retrospective cohort study included 170 patients (82 men [48.2%], median age 72 years) admitted to a university hospital for hyperosmolar hyperglycemic crises, including pure hyperosmolar hyperglycemic states and hyperosmolar diabetic ketoacidoses. We separately investigated the prognostic significance of hypoglycemia on mortality during the initial intravenous insulin therapy phase and during the later subcutaneous insulin therapy phase, both during hospitalization and in the long term (median follow-up, 652 days; range 2-3460 days). RESULTS: Both hypoglycemia during the initial intravenous insulin therapy phase (observed in 26.5% of patients) and hypoglycemia during the later subcutaneous insulin therapy phase (observed in 52.7% of patients) were associated with long-term mortality. After adjusting for potential confounders, hypoglycemia during the initial intravenous insulin therapy phase remained associated with mortality (hazard ratio 2.10, 95% CI 1.27-3.46, p = 0.004). CONCLUSIONS: Hypoglycemia during hyperosmolar hyperglycemic crises is a marker of long-term mortality, especially when it occurs during the initial intravenous insulin therapy phase.

Use of electronic health records for the management of diabetes and its risk factors in the Principality of Asturias from 2014 to 2018.

BACKGROUND: In recent years, the implementation of electronic health records across all hospitals and primary care centres within the National Health System has significantly enhanced access to patients' clinical data. This study aims to estimate the prevalence of type 2 diabetes (T2DM) in primary care settings and to outline its associated cardiovascular risk factors (CVRF) and epidemiological characteristics. METHODS: An observational cross-sectional study was conducted including 89,679 patients diagnosed with T2DM who attended the primary health care system from 2014 to 2018. Data was provided by the Primary Health Care System of the Principality of Asturias (SESPA). RESULTS: The estimated prevalence of diagnosed T2DM was 8.01% (95% Confidence Interval [CI]: 7.96-8.06) of the total population. Additionally, it was more prevalent in males compared to females (9.90% [95% CI: 9.81-9.99] vs. 6.50% [95% CI: 6.44-6.57]) and increased with age in both sexes. People with T2DM had an average age of 74 years, 52.3% were male, and the most frequently associated CVRF were: dyslipidaemia (47.90%) and hypertension (62.20%). Glycaemic control improved during the 2014-2018 period (31.69%), as did lipid control (23.66%). However, the improvement in blood pressure control (9.34%) was less pronounced for the same period. Regarding the multifactorial control of diabetes (measured by LDL-cholesterol, HbA1C and blood pressure) the overall degree of control improved by 11.55% between 2014 and 2018. CONCLUSION: In this 5-year retrospective population-based study, the utilisation of data from electronic medical records provides insights into the prevalence of T2DM in a large population, as well as real-time CVRFs. Leveraging this data facilitates the development of targeted health policies.

Reclassification of type 2 diabetes to type 1 diabetes in Asturias (Spain) between 2011 and 2020.

BACKGROUND: Differentiating between type 1 diabetes (T1D) and type 2 diabetes (T2D) can be difficult in adults. The aim of this study was to determine the frequency of diagnostic reclassification from T2D to T1D, the characteristics of the patients and the impact on the management of the disease. METHODS: Observational and descriptive study including patients diagnosed with T1D in Asturias (Spain) between 2011 and 2020 who had been considered as T2D for at least 12 months. RESULTS: A total of 205 patients were included, representing 45.3% of those diagnosed with T1D over 30 years of age. Median time of evolution as T2D was 7,8 years. The age was 59.1 ± 12.9 years. BMI was > 25 kg/m2 in 46.8% of patients. HbA1c was 9.1 ± 2.1%, 77 ± 22 mmol/mol, and 56.5% were using insulin. Pancreatic antibodies were present in 95.5%, the most frequent being GAD, 82.6%. At 6 months, basal insulin use increased from 46.9 to 86.3%, and HbA1c decreased, 9.2 ± 2.0%vs7.7 ± 1.2%, 77 ± 22vs60 ± 13 mmol/mol; p < 0.0001. CONCLUSIONS: Diagnosis as T2D in patients with T1D in adults is common. Age, BMI, insulin use and other clinical features are not definitely discriminatory. GAD is the antibody of choice in case of diagnostic suspect. Reclassification has important implications for metabolic control.

Hypoglycemia in patients with type 2 diabetes mellitus during hospitalization: associated factors and prognostic value.

BACKGROUND: The risk factors for hypoglycemia during hospital admission and its consequences in patients with diabetes are not entirely known. The present study aimed to investigate the risk factors for hypoglycemia, as well as the potential implications of hypoglycemia in patients with type 2 diabetes mellitus admitted to the hospital. METHODS: This retrospective cohort study included 324 patients (214 [66.0%] men; median age 70 years, range 34-95 years) with type 2 diabetes admitted to a university hospital who were consulted the Endocrinology Department for glycemic control during a 12-month period. We investigated the potential role of demographic factors, metabolic factors, therapy, and comorbidities on the development of in-hospital hypoglycemia. We explored the prognostic value of hypoglycemia on mortality (both in-hospital and in the long-term), hospital readmission in the following year, and metabolic control (HbA1c value) after discharge (median follow-up, 886 days; range 19-1255 days). RESULTS: Hypoglycemia occurred in 154 (47.5%) patients during their hospitalization and was associated with advanced age, previous insulin therapy, higher Charlson Comorbidity Index, lower body mass index and lower baseline HbA1c values. Hypoglycemia was associated with greater in-hospital and long-term mortality, longer hospital stays, higher readmission rates, and poorer metabolic control after discharge. These negative consequences of hypoglycemia were more frequent in patients with severe (≤ 55 mg/dL) hypoglycemia and in patients who had hypoglycemia during a greater percentage of hospitalization days. CONCLUSIONS: Hypoglycemia during hospital admission is a marker of a poor prognosis in patients with type 2 diabetes.

[Cetoacidosis diabética al diagnóstico de diabetes mellitus tipo 1 en Asturias entre 2011 y 2020: influencia de la duración de los síntomas en la prevalencia de cetoacidosis y en la pérdida de peso.] / Diabetic ketoacidosis at diagnosis of type 1 diabetes in Asturias (Spain) between 2011 and 2020: influence of symptom duration on prevalence of ketoacidosis and weight loss.

OBJECTIVE: Diabetic ketoacidosis (DKA) is a serious complication that usually occurs at diagnosis of type 1 diabetes mellitus (T1D). However, the prevalence of DKA at diagnosis of T1D is heterogeneous in different regions of the world. The aim of this study was to determine the prevalence of DKA at diagnosis of T1D in Asturias. METHODS: This study included all patients under nineteen years of age diagnosed with T1D in Asturias between 2011 and 2020. Retrospective review of medical records was performed to analyse DKA and other characteristics at diagnosis. A log binary regression model was constructed to obtain an estimate of the prevalence ratio of DKA to diagnosis in the years studied. RESULTS: A total of 267 people were diagnosed with a mean age of 9.85±4.46 years. The prevalence of DKA at diagnosis during this period was 38.63%. There was an increasing trend, with a prevalence ratio over the years studied of 1.015 (95%CI: 0.96-1.07; p=0.61). Duration of symptoms before diagnosis was 4.57±7.64 weeks. Weight loss was 7.56±7.26%, being more than 10% of previous weight in almost half of the patients who loosed weight. There was a positive relationship between symptoms duration and prevalence of DKA and between time to diagnosis and weight loss. CONCLUSIONS: Asturias has a high prevalence of DKA at diagnosis of T1D, slightly higher than observed in other studies at national level and higher than in other similar countries, with a tendency to increase. Delayed diagnosis is a key factor in the prevalence of DKA and weight loss. Thus, health actions are needed for the early detection of T1D to avoid DKA at diagnosis.

OBJETIVO: La cetoacidosis diabética (CAD) es una complicación grave que puede producirse al diagnóstico de la diabetes mellitus tipo 1 (DM1). La prevalencia de CAD al diagnóstico de DM1 es desigual en las distintas regiones del mundo. El objetivo de este estudio fue conocer la prevalencia de CAD al diagnóstico de DM1 en Asturias. METODOS: Se incluyeron los pacientes menores de diecinueve años diagnosticados de DM1 en Asturias entre 2011 y 2020. Mediante revisión de historia clínica se analizó la prevalencia de CAD así como otras características al diagnóstico. Se construyó un modelo de regresión log binaria para obtener una estimación de la razón de prevalencia de CAD al diagnóstico en los años estudiados. RESULTADOS: Se diagnosticaron 267 personas con edad media de 9,85±4,46 años. La prevalencia de CAD al diagnóstico fue del 38,63%. Se apreció una tendencia al aumento, con una razón de prevalencia en los años estudiados de 1,015 (IC95%:0,96-1,07; p=0,61). La duración de los síntomas hasta el diagnóstico fue de 4,57±7,64 semanas. La pérdida de peso fue de 7,56±7,26%, siendo superior al 10% en casi la mitad de los pacientes que perdieron peso. Se apreció relación entre la duración de los síntomas y la prevalencia de CAD, y entre el tiempo de evolución y la pérdida de peso. CONCLUSIONES: Asturias presenta una alta prevalencia de CAD al diagnóstico de DM1, levemente superior a otros estudios a nivel nacional y superior a otros países de nuestro entorno, con tendencia al aumento. El retraso diagnóstico es clave en la prevalencia de CAD y en la pérdida de peso. Son necesarias actuaciones sanitarias para la detección precoz de la DM1.

Circulating miRNA expression in long-standing type 1 diabetes mellitus.

Type 1 diabetes is a chronic autoimmune disease which results in inefficient regulation of glucose homeostasis and can lead to different vascular comorbidities through life. In this study we aimed to analyse the circulating miRNA expression profile of patients with type 1 diabetes, and with no other associated pathology. For this, fasting plasma was obtained from 85 subjects. Next generation sequencing analysis was firstly performed to identify miRNAs that were differentially expressed between groups (20 patients vs. 10 controls). hsa-miR-1-3p, hsa-miR-200b-3p, hsa-miR-9-5p, and hsa-miR-1200 expression was also measured by Taqman RT-PCR to validate the observed changes (34 patients vs. 21 controls). Finally, through a bioinformatic approach, the main pathways affected by the target genes of these miRNAs were studied. Among the studied miRNAs, hsa-miR-1-3p expression was found significantly increased in patients with type 1 diabetes compared to controls, and positively correlated with glycated haemoglobin levels. Additionally, by using a bioinformatic approach, we could observe that changes in hsa-miR-1-3p directly affect genes involved in vascular development and cardiovascular pathologies. Our results suggest that, circulating hsa-miR-1-3p in plasma, together with glycaemic control, could be used as prognostic biomarkers in type 1 diabetes, helping to prevent the development of vascular complications in these patients.

A long non-coding RNA that harbors a SNP associated with type 2 diabetes regulates the expression of TGM2 gene in pancreatic beta cells.

Introduction: Most of the disease-associated single nucleotide polymorphisms (SNPs) lie in non- coding regions of the human genome. Many of these variants have been predicted to impact the expression and function of long non-coding RNAs (lncRNA), but the contribution of these molecules to the development of complex diseases remains to be clarified. Methods: Here, we performed a genetic association study between a SNP located in a lncRNA known as LncTGM2 and the risk of developing type 2 diabetes (T2D), and analyzed its implication in disease pathogenesis at pancreatic beta cell level. Genetic association study was performed on human samples linking the rs2076380 polymorphism with T2D and glycemic traits. The pancreatic beta cell line EndoC-bH1 was employed for functional studies based on LncTGM2 silencing and overexpression experiments. Human pancreatic islets were used for eQTL analysis. Results: We have identified a genetic association between LncTGM2 and T2D risk. Functional characterization of the LncTGM2 revealed its implication in the transcriptional regulation of TGM2, coding for a transglutaminase. The T2Dassociated risk allele in LncTGM2 disrupts the secondary structure of this lncRNA, affecting its stability and the expression of TGM2 in pancreatic beta cells. Diminished LncTGM2 in human beta cells impairs glucose-stimulated insulin release. Conclusions: These findings provide novel information on the molecular mechanisms by which T2D-associated SNPs in lncRNAs may contribute to disease, paving the way for the development of new therapies based on the modulation of lncRNAs.

Cardiovascular Risk Factors and Differential Transcriptomic Profile of the Subcutaneous and Visceral Adipose Tissue and Their Resident Stem Cells.

BACKGROUND: The increase in the incidence of obesity and obesity-related cardiovascular risk factors (CVRFs) over the last decades has brought attention on adipose tissue (AT) pathobiology. The expansion of AT is associated with the development of new vasculature needed to perfuse the tissue; however, not all fat depots have the same ability to induce angiogenesis that requires recruitment of their own endothelial cells. In this study we have investigated the effect of different CVRFs, on the angiogenic capacity of the subcutaneous (SAT) and visceral (VAT) adipose tissue and on the function of their mesenchymal cell reservoir. METHODS: A transcriptomic approach was used to compare the different angiogenic and inflammatory profiles of the subcutaneous and visceral fat depots from individuals with obesity, as well as their resident stem cells (ASCs). Influence of other risk factors on fat composition was also measured. Finally, the microvesicles (MVs) released by ASCs were isolated and their regenerative potential analyzed by molecular and cellular methodologies. RESULTS: Obesity decreases the angiogenic capacity of AT. There are differences between SAT and VAT; from the 21 angiogenic-related genes analyzed, only three were decreased in SAT compared with those decreased in VAT. ASCs isolated from both fat depots showed significant differences; there was a significant up-regulation of the VEGF-pathway on visceral derived ASCs. ASCs release MVs that stimulate endothelial cell migration and angiogenic capacity. CONCLUSIONS: In patients with obesity, SAT expresses a greater number of angiogenic molecules than VAT, independent of the presence of other CVRFs.

Stem cells from human cardiac adipose tissue depots show different gene expression and functional capacities.

BACKGROUND: The composition and function of the adipose tissue covering the heart are poorly known. In this study, we have investigated the epicardial adipose tissue (EAT) covering the cardiac ventricular muscle and the EAT covering the left anterior descending artery (LAD) on the human heart, to identify their resident stem cell functional activity. METHODS: EAT covering the cardiac ventricular muscle was isolated from the apex (avoiding areas irrigated by major vessels) of the heart (ventricular myocardium adipose tissue (VMAT)) and from the area covering the epicardial arterial sulcus of the LAD (PVAT) in human hearts excised during heart transplant surgery. Adipose stem cells (ASCs) from both adipose tissue depots were immediately isolated and phenotypically characterized by flow cytometry. The different behavior of these ASCs and their released secretome microvesicles (MVs) were investigated by molecular and cellular analysis. RESULTS: ASCs from both VMAT (mASCs) and the PVAT (pASCs) were characterized by the expression of CD105, CD44, CD29, CD90, and CD73. The angiogenic-related genes VEGFA, COL18A1, and TF, as well as the miRNA126-3p and miRNA145-5p, were analyzed in both ASC types. Both ASCs were functionally able to form tube-like structures in three-dimensional basement membrane substrates. Interestingly, pASCs showed a higher level of expression of VEGFA and reduced level of COL18A1 than mASCs. Furthermore, MVs released by mASCs significantly induced human microvascular endothelial cell migration. CONCLUSION: Our study indicates for the first time that the resident ASCs in human epicardial adipose tissue display a depot-specific angiogenic function. Additionally, we have demonstrated that resident stem cells are able to regulate microvascular endothelial cell function by the release of MVs.

Effects of a Carob-Pod-Derived Sweetener on Glucose Metabolism.

BACKGROUND: Patients with type 2 diabetes mellitus (T2DM) have a higher incidence of cardiovascular (CV) events. The ingestion of high-glycemic index (GI) diets, specially sweetened beverage consumption, has been associated with the development of T2DM and CV disease. OBJECTIVE: We investigated the effects of the intake of a sweetened beverage, obtained from natural carbohydrates containing pinitol (PEB) compared to a sucrose-enriched beverage (SEB) in the context of impaired glucose tolerance (IGT) and diabetes. METHODS: The study was divided in three different phases: (1) a discovery phase where the plasma proteomic profile was investigated by 2-DE (two-dimensional electrophoresis) followed by mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight-MALDI-TOF/TOF) in healthy and IGT volunteers; (2) a verification phase where the potential mechanisms behind the observed protein changes were investigated in the discovery cohort and in an additional group of T2DM volunteers; and (3) the results were validated in a proof-of-concept interventional study in an animal model of diabetic rats with complementary methodologies. RESULTS: Six weeks of pinitol-enriched beverage (PEB) intake induced a significant increase in two proteins involved in the insulin secretion pathway, insulin-like growth factor acid labile subunit (IGF1BP-ALS; 1.3-fold increase; P = 0.200) and complement C4A (1.83-fold increase; P = 0.007) in IGT subjects but not in healthy volunteers. Changes in C4A were also found in the serum samples of Zucker diabetic fatty (ZDF) rats after four weeks of PEB intake compared to basal levels (P = 0.042). In addition, an increased expression of the glucose transporter-2 (GLUT2) gene was observed in the jejunum (P = 0.003) of inositol-supplemented rats when compared to sucrose supplementation. This change was correlated with the observed change in C4A (P = 0.002). CONCLUSIONS: Our results suggest that the substitution of a common sugar source, such as sucrose, by a naturally-based, pinitol-enriched beverage induces changes in the insulin secretion pathway that could help to reduce blood glucose levels by protecting ß-cells and by stimulating the insulin secretion pathway. This mechanism of action could have a relevant role in the prevention of insulin resistance and diabetes progression.

P2Y12 antagonists and cardiac repair post-myocardial infarction: global and regional heart function analysis and molecular assessments in pigs.

Aims: P2Y12 antagonists are the standard in antiplatelet therapy but their potential effects on functional myocardial recovery and cardioprotection post-myocardial infarction (MI) are unknown. We investigated in a preclinical model of MI whether ticagrelor and clopidogrel differently affect cardiac repair post-MI. Methods and results: Pigs either received: (i) clopidogrel (600 mg; 75 mg/qd); (ii) ticagrelor (180 mg; 90 mg/bid); and (iii) placebo control. MI was induced by mid-left anterior descending coronary artery balloon occlusion (60 min) and animals received the maintenance doses for the following 42 days. Serial cardiac magnetic resonance was performed at Day 3 and Day 42 for the assessment of global and regional cardiac parameters. We determined cardiac AMP-activated protein kinase (AMPK), Akt/PKB, aquaporin-4, vascular density, and fibrosis. In comparison to controls, both P2Y12 antagonists limited infarct expansion at Day 3, although ticagrelor induced a further 5% reduction (P < 0.05 vs. clopidogrel) whereas oedema was only reduced by ticagrelor (≈23% P < 0.05). Scar size decreased at Day 42 in ticagrelor-treated pigs vs. controls but not in clopidogrel-treated pigs. Left ventricular ejection fraction was higher 3 days post-MI in ticagrelor-treated pigs and persisted up to Day 42 (P < 0.05 vs. post-MI). Regional analysis revealed that control and clopidogrel-treated pigs had severe and extensive wall motion abnormalities in the jeopardized myocardium and a reduced myocardial viability that was not as evident in ticagrelor-treated pigs (χ2P < 0.05 vs. ticagrelor). Only ticagrelor enhanced myocardial AMPK and Akt/PKB activation and reduced aquaporin-4 levels (P < 0.05 vs. control and clopidogrel). No differences were observed in vessel density and fibrosis markers among groups. Conclusions: Ticagrelor is more efficient than clopidogrel in attenuating myocardial structural and functional alterations post-MI and in improving cardiac healing. These benefits are associated with persistent AMPK and Akt/PKB activation.

Phytosterols and Omega 3 Supplementation Exert Novel Regulatory Effects on Metabolic and Inflammatory Pathways: A Proteomic Study.

Cardiovascular disease (CVD) remains one of the major causes of death and disability worldwide. In addition to drug treatment, nutritional interventions or supplementations are becoming a health strategy for CVD prevention. Phytosterols (PhyS) are natural components that have been shown to reduce cholesterol levels; while poly-unsaturated fatty acids (PUFA), mainly omega-3 (ω3) fatty acids, have shown to reduce triglyceride levels. Here we aimed to investigate whether the proteins in the main lipoproteins (low density lipoproteins (LDL) and high density lipoproteins (HDL)) as well as proteins in the lipid free plasma fraction (LPDP) were regulated by the intake of PhyS-milk or ω3-milk, in overweight healthy volunteers by a proteomic based systems biology approach. The study was a longitudinal crossover trial, including thirty-two healthy volunteers with body mass index (BMI) 25-35 kg/m² (Clinical Trial: ISRCTN78753338). Basal samples before any intervention and after 4 weeks of intake of PhyS or ω3-milk were analyzed. Proteomic profiling by two dimensional electrophoresis (2-DE) followed by mass spectrometry-(MALDI/TOF), ELISA, Western blot, conventional biochemical analysis, and in-silico bioinformatics were performed. The intake of PhyS-milk did not induce changes in the lipid associated plasma protein fraction, whereas ω3-milk significantly increased apolipoprotein (Apo)- E LDL content (p = 0.043) and induced a coordinated increase in several HDL-associated proteins, Apo A-I, lecitin cholesterol acyltransferase (LCAT), paraoxonase-1 (PON-1), Apo D, and Apo L1 (p < 0.05 for all). Interestingly, PhyS-milk intake induced a reduction in inflammatory molecules not seen after ω3-milk intake. Serum amyloid P component (SAP) was reduced in the LPDP protein fraction (p = 0.001) of subjects taking PhyS-milk and C-C motif chemokine 2 (CCL2)expression detected by reverse transcription polymerase chain reaction (RT-PCR) analysis in white blood cells was significantly reduced (p = 0.013). No changes were observed in the lipid-free plasma proteome with ω3-milk. Our study provides novel results and highlights that the PhyS-milk induces attenuation of the pro-inflammatory pathways, whereas ω3-milk induces improvement in lipid metabolic pathways.