From the AGA Journals

Inducible nitric oxide synthase promotes insulin resistance in obesity

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A new understanding of insulin resistance

Understanding the mechanisms for how obesity affects cellular pathways is critical for identifying therapeutic targets to prevent its adverse consequences. The current study by Qian et al. identifies acquired lysosome dysfunction as a core cellular event that predisposes to insulin resistance in obesity. Lysosomes are degradative organelles that orchestrate cellular metabolism to facilitate homeostasis and confer stress resistance. Through a well-designed series of experiments conducted in a mouse model of diet-induced obesity, the authors demonstrate localization of inducible nitric oxide synthase (iNOS) to lysosomes in the livers of obese animals. This triggers excess local nitric oxide (NO) generation which leads to excessive nitrosylation of lysosomal proteins. A direct consequence of the resultant lysosome dysfunction is impaired autophagy, which is a critical cellular pathway for clearing away damaged organelles and proteins and generating energy under nutrient stress. Their studies also implicate lysosomal NO generation in suppressing the activity of transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis. Remarkably, genetic ablation of iNOS prevents the lysosome dysfunction and autophagy impairment, to attenuate obesity-induced insulin resistance.

Future studies will be required to assess the mechanisms for iNOS localization to the lysosomes and its interplay with the mammalian target of rapamycin (mTOR) signaling pathway in the face of sustained nutrient excess.

Dr. Diwan Abhinav Washington University and John Cochran VA in St Louis

Dr. Abhinav Diwan

These findings will spur future investigation into the role for lysosomal NO generation in a broad range of conditions that the obesity epidemic predisposes sufferers to, namely diabetes, fatty liver disease, atherosclerosis, and heart failure. Most importantly, these observations kindle the hope that therapies to stimulate the autophagy-lysosome pathway, which are being hotly pursued in the context of neurodegenerative and cardiovascular pathologies, may also be translated to prevent the adverse consequences of obesity.

Abhinav Diwan, MD, is an associate professor of medicine, cell biology, and physiology at Washington University and associate division chief of cardiology at the John Cochran VA Medical Center, both in St. Louis. He has no conflicts.



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.

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