Dmitri Samovski, Ph.D.
Project Overview:
Obesity is associated with continuous exposure of the liver to high levels of fats, which over time induces abnormal fat accumulation in liver cells and leads to fatty liver and tissue malfunction. Eventually, these changes can result in insulin resistance and type 2 diabetes. The molecular causes that initiate this cycle of negative events remain unclear.
I have discovered a novel molecular pathway that serves to adjust liver fat breakdown to fat supply, which maintains energy balance and normal function. I am proposing to examine if disruption of this pathway results in abnormal fat handling that is detrimental to liver health.
I have discovered that high fat regulates the function of AMPK and PI3K, intracellular proteins that are master coordinators of liver energy utilization. High fat alters the association between proteins important for proper function of AMPK and PI3K. The specific questions I will ask are:
- How does high fat regulate AMPK and PI3K?
- Does obesity alter communication between the proteins involved in AMPK and PI3K function?
- Does obesity cause disruption of cellular energy metabolism and insulin resistance?
I will use liver cells and mice with altered liver levels of proteins that are important for the regulation of AMPK and PI3K function. I anticipate my study will provide novel insights into the molecular mechanisms that link obesity to the development of abnormal liver function. The findings should allow the design of therapeutic approaches that can target the link between CD36 and AMPK to enhance fat breakdown by the liver and decrease liver fat accumulation.
Progress Report:
Obesity is a major cause of insulin resistance and type 2 diabetes (T2D), conditions that continue to increase in prevalence worldwide. Insulin resistance is at the core of metabolic syndrome (MetS), which involves more than 25% of the population with an even higher incidence in some subpopulations such as African Americans. CD36, the fatty acid (FA) receptor studied in this project, has been linked to a metabolic disease.
Our recent findings indicate that hepatocyte CD36 is involved in the regulation of the IR (insulin receptor) in the liver and this modulates insulin action on glucose and lipid metabolism, including de-novo lipogenesis (DNL) and hepatic glucose production (HGP). We also identify the influence of hepatic CD36 in transcriptional effects of insulin on Pcsk9 (Proprotein convertase subtilisin/kexin type 9) and Angptl3 (Angiopoietin-like 3), which are essential effectors of triglyceride (TG)/lipoprotein clearance. These findings suggest a link between CD36 signaling, FA flux and insulin action in regulation of liver metabolism and function in lipoprotein clearance. This is highly relevant to our understanding of hepatic insulin sensitivity and diabetic dyslipidemia and etiology of cardiovascular disease.
We anticipate that this study will provide novel insights into the molecular mechanisms that link obesity to the development of fatty liver, abnormal blood lipids, and heart disease. The findings would validate the use of novel diagnostic markers that would predict the risk for the development of fatty liver and heart disease.
Final Report:
Impact of impaired hepatic FAs sensing on steatosis and IR. CD36, the FA receptor studied in this project, has been linked to metabolic disease. Obesity and T2D were shown to associate with an upregulated hepatic expression of CD36. Using cells in culture and mouse models, we examined how CD36 senses FAs availability in the liver and adjusts their utilization and storage accordingly. We identified a novel molecular pathway that links FA supply to energy regulation by insulin and AMP Kinase. The mechanism involves the direct impact of FA on protein interactions within multiprotein signaling complexes. This effect is mediated by CD36 and is differentially modulated by FA availability and FA type. Our studies in cultured cells revealed that saturated FA (such as palmitic and myristic acids), but not unsaturated FA (such as linoleic and oleic acids) are capable, by modulating protein-protein interactions, to simultaneously enhance AMPK and suppress insulin signaling. These effects would result in FA targeting to oxidation and in minimizing FA storage into lipids. The operation of this mechanism should serve to limit excessive FA accumulation and generation of toxic metabolites. However, it is likely that this FA sensing mechanism is impaired by over-nutrition and obesity resulting in deleterious effects on AMPK and insulin signal transduction.
To validate these findings in vivo, we generated a mouse model with a hepatocyte-specific CD36 deficiency (hepCD36-/-). Using these mice, we confirmed in vivo our findings from cell culture studies. Our results showed that CD36 promotes insulin signaling in hepatocytes by facilitating the assembly of insulin receptor signaling complex while saturated, but not unsaturated, FAs suppressed the complex assembly, resulting in inhibition of insulin signaling.
The described mechanism provides means for insulin signaling regulation by FAs with differential effects of saturated and unsaturated FAs. Dysregulation of CD36-mediated FA sensing mechanisms, as it occurs in obesity, would lead to impaired lipid metabolism, accumulation of toxic lipid metabolites, hepatic IR and metabolic dysfunction. 2. Differences in circulating sEVs between people with low or high intrahepatic triglycerides Small extracellular vesicles (sEVs) (also known as exosomes) are nanoparticles with a diameter <200 nanometers that are secreted by most cells in a tissue and provide a pathway for communication with other tissues. sEVs are membrane-bound vesicles that are formed inside the cell and are released from the cell on fusion with the plasma membrane. The composition of the sEV cargo reflects the cell of origin and is believed to mediate paracrine and endocrine communication between the tissue of origin and target tissues. sEVs can exert specific effects on gene expression and function of recipient tissues by delivering miRNAs (the most studied sEV cargo) as well as bioactive lipids, and regulatory proteins.
Obesity and T2D were shown to associate with increased abundance of circulating sEVs. We postulated that sEVs secreted from liver of people with NAFLD transport disease-promoting signals to skeletal muscle and other tissues and facilitate the development of multi-organ IR and the associated metabolic diseases. To explore this hypothesis, we initiated a pilot human study where we recruited subjects with obesity and either low or high intrahepatic triglyceride (IHTG) content (n=10 subjects/group). Plasma samples and metabolic profiles of human subjects were obtained through a collaboration with the Center of Human Nutrition at Washington University in St. Louis. The following metabolic characteristics were used as inclusion criteria for the study: 1) low IHTG group: BMI 30.0-44.9 kg/m2, normal fasting blood glucose and oral glucose tolerance and IHTG <5% of total liver volume; and 2) high IHTG group: BMI 30.0-44.9 kg/m2, impaired oral glucose tolerance and IHTG content ≥10%. Each participant underwent comprehensive metabolic testing, including a state-of-the-art assessment of liver fat and insulin sensitivity. We collected blood from all participants and developed and optimized methods for reliable and reproducible isolation of circulating sEVs. Currently, we are conducting systematic, high-throughput analyses of the isolated sEVs, to identify the specific cargo molecules (miRNAs, proteins and lipids) carried by sEVs of people with high IHTG. The content of signaling molecules in sEVs of people with obesity and low or high IHTG is systematically characterized, to identify the factors that contribute to IR and metabolic dysfunction associated with NAFLD. In parallel, we have been examining the pathophysiological effects of sEVs on IR in cells in culture. Our early findings show that sEVs from people with high IHTG induce insulin resistance in human muscle cells. These findings indicate that plasma sEVs in people with obesity and NAFLD contain molecules that promote metabolic dysfunction in muscle. Later, we plan to examine the effects of sEVs on IR and metabolic diseases in mice infused with the different types of human sEVs that we are collecting.
