A Molecular Whodunit in the Liver: Discovering What Molecules Cause Hepatic Insulin Resistance

March 17, 2021

A Molecular Whodunit in the Liver: Discovering What Molecules Cause Hepatic Insulin Resistance

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What happens to the carbs we eat?  Carbohydrates are one of the main sources of energy for cells in the human body. After eating a meal, carbohydrates pass through the digestive system, traveling from the stomach to the small intestines, and are broken down into their basic unit, the monosaccharide (such as glucose, fructose, and galactose), along the way. Upon their arrival in the small intestines, monosaccharides are transported into the bloodstream, increasing glucose concentrations in the blood and prompting the pancreas to secrete the essential hormone insulin to promote tissue glucose uptake and suppress endogenous glucose production. This process provides tissues access to glucose for energy production and storage as well as maintains a healthy concentration of blood glucose to prevent hyperglycemia. 

Hyperglycemia, or abnormally high blood sugar levels, can become dangerous since the body will turn to excessively breaking down fats when glucose cannot be accessed by tissues for energy production. This process of rapid fat breakdown produces excessive ketones, the buildup of which could be life-threatening. Additionally, hyperglycemia can cause damage to multiple tissues, such as the retina, kidneys, limb extremities, and cardiovascular system, which could lead to severe downstream complications, including vision loss, renal diseases, limb extremity necrosis and amputation, and cardiovascular diseases. Disruption of the insulin signaling pathway may lead to hyperglycemia in type 2 diabetes, a condition where the body not only exhibits lowered response to insulin but also does not produce enough insulin in chronic conditions. Researchers have conducted many studies investigating the pathways responsible for the development of insulin resistance. Recently, the Shulman Lab at Yale has come forth with a potential mechanism for the development of insulin resistance in the liver, also known as hepatic insulin resistance (HIR). 

Significance of the study 

The Shulman Lab, led by Gerald Shulman, the George R. Cowgill Professor of Medicine and Cellular & Molecular Physiology at Yale, has been extensively studying HIR and its contribution to type 2 diabetes for the past few years. “Insulin resistance is the primary determinant of whether or not someone develops type 2 diabetes. [Type 2 diabetes] is going to impact half a billion people within 10 years’ time and is the leading cause of blindness and end-stage renal disease, as well as a huge economic cost to society,” Shulman said. His research team has been dedicated to investigating the role of liver fat accumulation in insulin action disruption and hepatic insulin resistance. In a paper recently published in Cell Metabolism, the Shulman Lab presented its newest discovery: a possible pathway by which certain molecules, called diacylglycerols (DAGs), might be responsible for inducing HIR. Kun Lyu, a graduate student in the Shulman Lab and first author of the paper, explains that the pathway had been discovered step-by-step from decades of work, and that with its history, had had its fair share of debate and controversy. “Over the past two or three years, we have developed new tools and models to specifically address this controversy,” Lyu said.

Major results 

The pathway the team discovered describes how accumulation of plasma membrane sn-1,2-diacylglycerols (PM sn-1,2-DAGs) leads to HIR. These DAGs are a group of plasma (cell) membrane-bound stereoisomers (compounds composed of the same atoms differing only in their orientations) of DAG that was found to activate the Protein Kinase C- (PKCε) pathway, which has the ability to disrupt insulin signaling. PKCε activation results in phosphorylation—a type of chemical tagging—of a critical amino acid residue (a specific chemical building block of a protein) on insulin receptor kinase (IRK). By tagging this amino acid residue, PKCε then disrupts the downstream signaling pathway and can lead to insulin resistance.

The research team was able to establish the role of DAGs in inducing HIR by removing functioning copies of an enzyme called DGAT2, which converts DAGs to triglycerides, in mice—a model known as DGAT2 knockdown (KD). To determine the effects of high DAG content on liver insulin action, the researchers subjected regular chow-fed rats to a treatment that decreases DGAT2, allowing DAG to accumulate. They then subjected these acute hepatic DGAT2 KD rats to a hyperinsulinemic-euglycemic clamp, a method used to infuse high levels of insulin (“hyperinsulinemia”) to mimic insulin levels after ingestion of carbohydrates while maintaining normal blood-glucose concentrations (“euglycemia”). The researchers found that this model impaired insulin’s suppression of endogenous glucose production by impairing the insulin signaling pathway, suggesting that DAGs could play a role in HIR. 

The study cemented the association of high levels of PM sn-1,2-DAGs with HIR in both rats and humans. The researchers analyzed DAG stereoisomer content in multiple subcellular compartments (endoplasmic reticulum, plasma membrane, mitochondria, lipid droplets, and cytosol). The focus on examining these compartments is significant as past studies have shown that only DAG accumulation in certain compartments is associated with HIR. In rats subjected to acute hepatic DGAT2 KD, they found higher sn-1,2-DAG content, particularly in the plasma membrane. In the liver tissues of human individuals with HIR, researchers discovered about five times higher levels of liver PM sn-1,2-DAGs than in humans without HIR. These higher levels of PM sn-1,2-DAGs also correlated with about three times higher levels of IRK-T1160 phosphorylation. From these results, the researchers drew the association of high levels of PM sn-1,2-DAGs with HIR, revealing that targeting this particular stereoisomer could ameliorate the effects of HIR.  

Considering this potential target, the Shulman lab found that knocking down PKCε specifically in the liver ameliorated HIR caused by a high-fat diet and DGAT2 KD. After treating the rats to decrease PKCε content in the liver, the researchers fed the rats a high-fat diet for four days to induce acute hepatic steatosis (fat buildup in the liver) and HIR. During a hyperinsulinemic-hyperglycemic clamp, they found that the PKCε KD improved insulin’s suppression of endogenous glucose production and resulted in a two times higher rate of insulin-stimulated hepatic glycogen synthesis compared with the control. They also observed improved downstream signaling in the insulin signaling pathway and lower levels of IRK-T1160 phosphorylation in these rats. Essentially, the liver-specific PKCε KD ameliorated the effects of high fat feeding and acute DGAT2 KD on causing HIR. These results indicate that not only are PKCε and PM sn-1,2-DAG associated with HIR, but also that they are directly responsible in mediating HIR, increasing their promise as an effective target in therapies aiming to reverse insulin resistance. 

So, the researchers had shown that PKCε is necessary for PM sn-1,2-DAG-mediated HIR, but is it sufficient? To test this, researchers treated healthy and lean rats to overexpress constitutively active PKCε. The constitutively active nature of these particular PKCε allowed for total PKCε content in the liver to increase significantly with six times greater translocation, which induces directly observable, correlated results. This treatment resulted in two times higher IRK-T1160 phosphorylation, impairing downstream signaling. Thus, the conclusion was that hepatic PKCε activation is both necessary and sufficient in mediating HIR, leading to hyperglycemia and hyperinsulinemia. 

Novelty of approach

The Shulman lab developed a range of new techniques and tools in their pursuit of these discoveries. One technique crucial to this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed by the lab to quantify DAG stereoisomers, such as the sn-1,2-DAG. “It took the whole team a lot of manpower and troubleshooting. We had to optimize the conditions using new tools with thousands of data points of testing and eventually came to this final product that is reliable and efficient to distinguish and quantify different DAG stereoisomers. It reflects tremendous work,” Lyu said. This new technique, together with the novel method measuring molecule levels in subcellular compartments, allowed the team to measure DAG content in subcellular compartments in order to draw the associations between DAG content and distribution with HIR. 

Additionally, the team had to develop a way to recognize the phosphorylation of IRK-T1160, which is mediated by PKCε. Although mass spectrometry had been sufficient in previous studies using purified proteins, the process of detecting this phosphorylation event proved much more complicated in vivo, or in the cell. To address this issue of recognition, the team turned to monoclonal antibodies, which are proteins that are used to target and bind to specific substances in the body, mimicking the way the immune system normally targets foreign substances.  “The key tool was to develop a monoclonal antibody that would recognize the phosphorylation of this 1160 position in vivo… Now we have a tool that [Lyu] was able to show worked in both animal and human liver to show that this key site is the target for PKCε, to ascribing hepatic insulin resistance,” Shulman said.

What comes next?

Armed with this new information, the Shulman lab continues to investigate and develop this new model. The discoveries they made can be applied to other tissues, such as skeletal muscle, white adipose tissue, and others that are responsive to insulin. They also aim to investigate unknown factors in their model, including how the DAGs translocate to the plasma membrane and why similar accumulation is not observed in other subcellular compartments in acute short-term models.
With this deeper understanding of the molecular basis of insulin resistance, new therapies can be developed to better target and treat the symptoms of hyperglycemia. “Right now, every drug that we are using to treat diabetes is pretty much treating the symptom of hyperglycemia, not really the root cause of insulin resistance…If we understand the molecular basis, we can identify the best targets to treat it,” Shulman said. This study indicates that a significant root cause of hepatic insulin resistance is PM sn-1,2-DAG and PKCε activation, and suggests that new therapies that target these molecules in the liver may help reverse insulin resistance.

Citations

  1. Lyu, K., Zhang, Y., Zhang, D., …, Cline, G. W., Samuel, V. T., & Shulman, G. I. (2020). A Membrane-Bound Diacyclglycerol Species Induces PKCεepsilon-Mediated Hepatic Insulin Resistance. Cell Metabolism, 32(4), 654-664. https://doi.org/10.1016/j.cmet.2020.08.001 
  2. Shulman, G. I. & Lyu, K. (Personal interview, October 29, 2020).
  3. Medicine LibreTexts. (2020, August 14). Digestion and Absorption of Carbohydrates. https://med.libretexts.org/Courses/American_Public_University/APUS%3A_An_Introduction_to_Nutrition_(Byerley)/Text/03%3A_Carbohydrates/3.03%3A_Digestion_and_Absorption_of_Carbohydrates
  4. Ayala, J. E., Bracy, D. P., McGuinness, O. P., & Wasserman, D. H. (2006). Considerations in the Design of Hyperinsulinemic-Euglycemia Clamps in the Conscious Mouse. Diabetes: A Journal of the American Diabetes Association, 55(2), 390-397. https://doi.org/10.2337/diabetes.55.02.06.db05-0686
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