Triglyceride-rich LIPoproteins and INflammatory Cytokines After Oral FAT Loading as Potential Early Biomarkers of the Risk of Progression Towards DIABETES and Development of Complications. LIPINFAT Diabetes Study.

Diabetes is a serious chronic disease characterized by elevated blood glucose levels resulting from abnormal pancreatic β-cell biology in relation to insulin action.

According to estimates from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD), diabetes is the eighth leading cause of death and disability worldwide, affecting nearly 460 million people of all ages and in all countries in 2019. It is currently estimated that approximately 529 million people of all ages worldwide are living with diabetes, with a global age-standardized prevalence of 6.1%.

Estimates from the International Diabetes Federation (IDF) indicate that global health expenditure on diabetes reached 910 billion euros in 2021 and is projected to exceed 1,000 billion euros by 2045.

Diabetes is also a major risk factor for ischemic heart disease and stroke, which according to GBD estimates are the first and second leading causes of disease globally. Diabetes itself is associated with increased mortality compared with non-diabetic individuals, worsens prognosis for all other diseases, increases premature mortality (Years of Life Lost, YLL), years lived with disability (Years Lived with Disability, YLD), and loss of healthy life years (Disability-Adjusted Life Years, DALY). Globally, diabetes accounted for 37.8 million YLL due to premature death and 41.4 million YLD, for a total of 79.2 million DALYs in 2021. Type 2 diabetes accounted for the vast majority of YLL, YLD, and DALYs attributable to diabetes. The global age-standardized DALY rate for diabetes was 915 per 100,000, with YLL and YLD rates of 437 and 478 per 100,000, respectively. In Italy, age-standardized DALYs in 2021 were 521.1 thousand, representing an 11.5% reduction compared with 2010, whereas increases were observed in the rest of Western Europe, in high-income countries, and globally.

Type 1 diabetes (T1D) and type 2 diabetes (T2D) are the most common forms of the disease and are diagnosed according to well-defined criteria reported in the operational manual. T1D often develops during childhood, whereas T2D has a strong genetic component and is strongly associated with obesity and a sedentary lifestyle. Cases of T2D account for approximately 96% of all diabetes cases.

Between 20% and 25% of adults with diabetes meet laboratory criteria for the diagnosis but have not been formally diagnosed (undiagnosed diabetes). Several studies have shown that individuals may spend 5-6 years in an asymptomatic phase of prediabetes and T2D prior to diagnosis, during which microvascular and macrovascular complications may already develop.

Prediabetes refers to individuals whose glucose levels do not meet diagnostic criteria for diabetes but who exhibit abnormal carbohydrate metabolism. Prediabetes is defined by impaired fasting glucose (IFG), and/or impaired glucose tolerance (IGT) following an oral glucose load, and/or HbA1c values between 5.7% and 6.4%, as reported in the operational manual.

Prediabetes should not be considered a distinct clinical entity, but rather a risk factor for progression to diabetes and cardiovascular disease (CVD). Prediabetes is associated with obesity, particularly abdominal or visceral obesity, dyslipidemia characterized by elevated triglycerides and/or low HDL cholesterol, and hypertension. The presence of prediabetes should prompt comprehensive cardiovascular risk factor screening.

Although prevention and management strategies differ across diabetes types, established approaches exist to reduce disease burden, including control of risk factors for the development and progression of T2D and improvement of healthcare system infrastructure.

T2D and prediabetes are characterized by insulin resistance (IR) in multiple cellular systems, including hepatocytes, adipocytes, and myocytes, excessive hepatic glucose production, altered lipid metabolism, and progressive impairment of insulin secretion. IR represents a central mechanism not only in diabetes but also in obesity and metabolic syndrome, and is associated with an increased risk of microvascular and macrovascular complications. While hyperglycemia, hypertension, kidney disease, and dyslipidemia are considered traditional CVD risk factors in diabetes, an association between IR and CVD has been increasingly recognized even in the absence of overt diabetes. IR is linked to alterations in lipid and lipoprotein metabolism, resulting in atherogenic dyslipidemia. Atherogenic dyslipidemia is highly prevalent in patients with T2D.

IR plays a major role in the metabolism of triglyceride-rich lipoproteins of hepatic origin, particularly very low-density lipoproteins (VLDL), including increased hepatic VLDL triglyceride synthesis. A key mechanism underlying increased VLDL triglyceride production is the accelerated lipolysis of stored triglycerides in adipose tissue, leading to increased free fatty acid flux to the liver. In insulin-resistant adipocytes, lipolysis is dysregulated, resulting in continuous release of free fatty acids into circulation. In hepatocytes, this promotes increased triglyceride synthesis, contributing both to intracellular lipid accumulation and hepatic steatosis, as well as enhanced VLDL production. Increased VLDL triglyceride synthesis is variably associated with increased hepatic production of apolipoprotein B-100. Overall, this results in hypertriglyceridemia, increased numbers of apoB-100-containing particles, and reduced HDL cholesterol concentrations. IR is also associated with increased hepatic triglyceride lipase activity, which may accelerate HDL clearance and reduce HDL cholesterol levels. In addition to increased VLDL synthesis, IR is associated with impaired VLDL clearance in skeletal muscle and adipose tissue due to reduced activity of lipoprotein lipases.

The lipid abnormalities of atherogenic dyslipidemia are not only quantitative but also qualitative and kinetic. IR is associated with changes in the average particle size of lipoproteins and likely in the lipoprotein lipidome. Lipids constitute a major and heterogeneous family of biomolecules within the metabolome. Complex lipids can be classified into multiple classes and subclasses, including triacylglycerols, diacylglycerols, phosphatidylcholines, phosphatidylethanolamines, ceramides, sphingomyelins, and cholesterol esters. Abnormal lipid profiles are associated with several diseases, including metabolic syndrome, T2D, cancer, nephropathy, and cardiovascular and neurodegenerative diseases.

A key feature of atherogenic dyslipidemia in T2D and IR is postprandial hyperlipidemia, which plays a fundamental role in the development of cardiovascular disease. Postprandial serum triglyceride levels vary considerably depending on meal composition and time elapsed after food intake. Due to this variability, assessment of postprandial hyperlipidemia requires an oral fat loading test (OFLT). Elevated postprandial triglyceride levels reflect increased concentrations of triglyceride-rich lipoproteins, including chylomicrons, VLDL, and their remnants. Intestinally synthesized chylomicrons transport dietary triglycerides to peripheral tissues in the postprandial state. Reduced lipoprotein lipase activity associated with IR impairs triglyceride hydrolysis from chylomicrons, leading to altered postprandial chylomicron responses. Measurement of apolipoprotein B48, non-fasting triglycerides, non-HDL cholesterol, and remnant cholesterol is essential for identifying postprandial hyperlipidemia.

Multiple factors contribute to chylomicron production during the postprandial phase. Substantial evidence supports the physiological role of glucagon-like peptides, microsomal triglyceride transfer protein, and the central role of apolipoprotein B48 in chylomicron synthesis and postprandial kinetics.

Just as postprandial triglycerides are an independent predictor of coronary artery disease, remnant lipoproteins possess multiple atherogenic properties and are associated with increased all-cause mortality in patients with ischemic heart disease and with the development of coronary artery disease, even after adjustment for major risk factors.

Diagnosis of postprandial hyperlipidemia requires an OFLT; however, there is no consensus on the optimal timing of postprandial measurements or on meal standardization. The test is time-consuming and requires prolonged rest. Despite these limitations, the OFLT remains the only available test for assessing postprandial hyperlipidemia.

Dyslipidemia and chronic inflammation are considered the main drivers of atherosclerotic plaque formation in diabetes. Atherosclerosis is accompanied by local inflammation within the vascular wall due to endothelial dysfunction and vascular smooth muscle cell involvement. Components of the diabetic milieu, oxidative stress, and other factors are believed to damage vascular endothelial cells, leading to increased expression of adhesion molecules and secretion of chemokines, promoting monocyte adhesion. Adherent monocytes migrate into the subendothelial space and differentiate into macrophages, which release cytokines such as interleukin-1β, interleukin-18, tumor necrosis factor-α, and interferon-γ, amplifying the inflammatory process. Compared with chylomicrons and VLDL, remnant lipoproteins can penetrate the arterial wall and do not require oxidation for macrophage uptake. Remnants enhance monocyte rolling, adhesion, and transmigration on endothelial cells and are involved in inflammation, platelet activation, and endothelial dysfunction through activation of transcription factors such as NF-κB. Adipocyte dysfunction associated with obesity is an integral component of T2D pathogenesis and, together with atherogenic dyslipidemia, promotes chronic systemic inflammation that contributes to insulin resistance, β-cell dysfunction, and ultimately T2D. This chronic inflammatory state contributes to long-term diabetic complications.

Despite substantial evidence supporting a strong relationship among insulin resistance, inflammation, and dyslipidemia, the determinants of progression from isolated insulin resistance to prediabetes, overt T2D, and T2D with complications remain unclear. Moreover, investigation of the postprandial phase, which occupies a large portion of the day, may provide critical insights into whether postprandial metabolism differentiates prediabetes from clinically overt diabetes, thereby elucidating pathophysiological mechanisms and informing dietary and pharmacological strategies to reduce disease progression.

The aim of the study is to evaluate whether the Oral Fat Loading Test (OFLT) determines a different response in terms of the quantity, quality, and kinetics of triglyceride-rich lipoproteins in subjects with T2D, prediabetics, and control subjects, and whether triglyceride-rich lipoproteins and inflammatory cytokines after OFLT are potential early biomarkers of the risk of progression to diabetes and the development of complications in a general practice setting.

To address these questions, a hybrid cohort study was designed by identifying three groups of subjects: T2D and prediabetics (exposed and near-exposed) and control subjects (unexposed). The subjects were recruited from the patient datasets of the Poggio Renatico Primary Care Unit - Poggio Rete Salute Group Medicine, Ferrara Local Health Authority, with the support of a propensity score matching analysis. In an initial cross-sectional phase, the differential response to OFLT will be studied in the three groups (TRL, TRL remnants, lipidoma, inflammatory cytokines, etc.). The second prospective phase will observe up to 36 months whether the response to OFLT of triglyceride-rich lipoproteins and inflammatory cytokines are potential early biomarkers of the risk of progression to diabetes and the development of complications.

Main Objectives:

Primary (Cross-sectional phase) To evaluate the odds ratio (OR) that TG after OFLT is higher in T2D, in good glyc

• Triglyceride-rich lipoprotein concentrations after oral fat loading test [Time frame: From enrollment after two years at the end of the enrollment]
• Chylomicron remnant concentrations after oral fat loading test [Time frame: From enrollment after two years at the end of the enrollment]
• Interleukin-1β concentration after oral fat loading test [Time frame: From enrollment after two years at the end of the enrollment]
• Interleukin-18 (IL-18) concentration after oral fat loading test [Time frame: From enrollment after two years at the end of the enrollment]
• Tumor necrosis factor-α (TNF-α) concentration after oral fat loading test [Time frame: From enrollment after two years at the end of the enrollment]
• Progression of diabetic nephropathy during follow-up [Time frame: 36-month follow-up]
• Major adverse cardiovascular events (5-point MACE) [Time frame: 36-month follow-up]