Obesity promotes the localization of inducible nitric oxide synthase (iNOS) in hepatic lysosomes, leading to a cascade of downstream effects that include excess lysosomal nitric oxide production, reduced hepatic autophagy, and insulin resistance, investigators reported.
“It is well known that in the context of obesity, chronic inflammation and lysosome dysfunction coexist in the liver,” wrote Qingwen Qian, PhD, of the University of Iowa in Iowa City and associates in Cellular and Molecular Gastroenterology and Hepatology. “Our studies suggest that lysosomal iNOS-mediated nitric oxide signaling disrupts hepatic lysosomal function, contributing to obesity-associated defective hepatic autophagy and insulin resistance.” They noted that the findings could hasten the development of new treatments for metabolic diseases.
Lysosomes recycle autophagocytosed intracellular and extracellular material, which is crucial to maintain several types of homeostasis within the liver. Each hepatocyte has about 250 lysosomes, which help regulate nutrient sensing, glycogen metabolism, cholesterol trafficking, and viral defense.
Activation of iNOS is a hallmark of inflammation, and iNOS levels are known to be elevated in the livers of patients with hepatitis C, alcoholic cirrhosis, and alpha 1-anti-trypsin disorder, the researchers wrote. “At the cellular level, iNOS produces pathological nitric oxide [NO], which triggers downstream effects, such as aberrant S-nitrosylation. These downstream effects can disrupt the function of organelles such as the mitochondria and the endoplasmic reticulum.”
Studies indicate that pathologic NO impairs lysosomal function in neurodegenerative diseases, cardiovascular disease, nonalcoholic fatty liver disease, and kidney disease, Dr. Qian and associates noted. But it was unclear whether NO in hepatocytes was generated by local iNOS or localized to lysosomes.
The researchers therefore studied cell cultures of primary murine hepatocytes by measuring their lysosomal activity, autophagy levels, and NO levels. They also studied a murine model of diet-induced obesity in which 60% of calories were from fat. They performed glucose tolerance tests by means of intraperitoneal glucose injections and studied the effects of insulin infusion. Finally, they performed immunohistology, immunohistochemistry, electron microscopy, and measurements of nitrosylated proteins and lysosomal arginine in frozen liver sections from the mice. Lysosomal arginine is required to catalyze NO production in the setting of inflammation as observed in obesity. In fact, concomitant stimulation of lysosomal arginine transport and activation of mTOR (an enzyme which tightly regulates transcription factor EB) was sufficient to stimulate lysosomal NO production in hepatocytes even in the absence of an inflammatory stimulus; pointing to a central role for these processes.
The researchers found that a NO scavenger diminished lysosomal NO production, while overexpression of both mTOR and a lysomal arginine transporter upregulated lysosomal NO production and suppressed autophagy. In mice with diet-induced obesity, deleting iNOS also improved nitrosative stress in hepatic lysosomes, promoted lysosomal biogenesis by activating transcription factor EB, enhanced lysosomal function and autophagy, and improved hepatic insulin sensitivity. Improved insulin sensitivity diminished, however, when the researchers suppressed transcription factor EB or autophagy-related 7 (Atg7).
Usually, iNOS is primarily expressed in hepatic Kupffer cells, but obesity increases the expression of iNOS in hepatocytes, which promotes hepatic insulin resistance and inflammation, the researchers commented. Unpublished data indicate that deleting iNOS initially protects against obesity-linked fatty liver steatosis and insulin resistance, but that these benefits weaken over time. “Nevertheless, our data showed that liver-specific iNOS suppression has a protective role,” they wrote. “Specifically, we showed that iNOS inactivates transcription factor EB, and that suppression of transcription factor EB and Atg7 diminishes the improved hepatic insulin sensitivity by iNOS deletion.” Transcription factor EB both regulates autophagy and is a “key player in lipid metabolism,” they added. It remains unclear whether the metabolic effects of iNOS solely relate to autophagy, they noted.
Funders included the American Heart Association, American Diabetes Association, and National Institutes of Health. The researchers reported having no conflicts of interest.
SOURCE: Qingwen Qian, et al. Cell Molec Gastroenterol Hepatol. 2019;8(1):95-110.
