Mechanistically linking age-related diseases and dietary carbohydrate via autophagy and the ubiquitin proteolytic systems

Abstract

Epidemiological data indicate that consuming diets that deliver sugar to the blood rapidly (called high glycemic index, GI) is associated with enhanced risk for age-related diseases such as cardiovascular disease, type 2 diabetes, cataract and age-related macular degeneration (AMD). These debilities are associated with accumulation of toxic protein aggregates as observed in other protein precipitation or amyloid diseases including Alzheimer, Parkinson and Huntington diseases and encephalopathies. Barriers to recommending lower-GI diets to promote health include the absence of established intracellular biochemical mechanisms that link high-GI diets to compromised homeostasis. The data herein corroborate the epidemiological findings and provide platforms to elucidate additional mechanistic aspects of salutary effects of consuming diets of different GIs. They are also useful for testing drugs, including autophagy enhancers, glycemia regulators, or nutraceuticals, which can be exploited to extend health.

Autophagy, a cellular process in which cells degrade intracellular constituents, including organelles in lysosomes, is particularly activated under conditions of nutrient deprivation. Lysosomal proteolysis is also involved in processing proteins generated by toxic stresses. Ironically, the current carbohydrate-intense American diet appears to stress cells and tissues, even as it provides a rich source of many nutrients. Most Americans now consume a high-GI diet with 200 calories/day more than only 40 years ago, and much of that is in the form of sugars that enter the bloodstream rapidly.

Glycoxidation (here called glycation) is the sum of nonenzymatic reaction of sugars, or their reactive metabolites such as methyl glyoxal (MGO), with proteins along with oxidative sequele. We hypothesized that chronic consumption of higher-GI diets sets up a vicious cycle of higher levels of toxic intracellular glycated proteins and glycation-induced compromises to the proteolytic machinery leading to insufficient capacity to remove damaged proteins and further cytotoxic accumulation. This might be of particular concern in retinal pigmented epithelial cells (RPE). These cells have among the highest degradative burden in the body because each RPE services >30 adjacent photoreceptors and must degrade the outer 10% of photoreceptors (equivalent to 10 erythrocytes, or close to its own weight) that are shed every night.

In our paper, we describe the first mammalian model corroborating that consuming a lower-GI diet is associated with delayed appearance of AMD-like lesions in the retina, including deposition of RPE basal deposits and loss of basal infoldings. These are precursors of AMD. Furthermore, we observed higher levels of intracellular age-related glycation end products (AGEs) and protein carbonyls in diverse tissues from animals of the same age that consumed the higher-GI diet. The phenomenon is systemic, affecting tissues with the highest levels of oxygenation, rapid metabolism and protein turnover (retina and brain) as well as the lowest oxygen tension, slowest metabolism and rates of overall protein turnover (lens). Importantly, levels of AGEs appear to be elevated in the areas of RPE pathology. However, the animals were not diabetic.

The accelerated and coincident accumulation of lesions and AGEs, including carbonyls, in animals that consumed the higher-GI diet suggests that these are etiologically related. Specifically, the data inform that animals that consume the higher-GI diets confront elevated glycative stress and, conversely, that diminishing such stress can be used to confer protection.

To begin to elucidate mechanisms that link dietary glycemia to lesions, we used glucose- or MGO-exposed RPE to show that the ubiquitin-proteasome systems (UPS) and a lysosomal proteolytic system (LPS) are involved in the degradation of AGEs (Fig. 1). AGEs, many of which appear to be ubiquitinated, accumulate rapidly at rates that are proportionate to the extent of glycative stress. The AGEs are cleared after removal of MGO. Their accumulation suggests that the protein degradation machinery that is involved in removing AGEs cannot keep pace with the rates of AGE formation.

Figure 1. This scheme proposes that under normal homeostatic conditions, the UPS and LPS, functioning independently or in concert, maintain the proteome (top). Protein editing is unperturbed, and AGEs and ubiquitin conjugates do not accumulate. However, upon chronic glycative stress and ⁄or aging, proteolytic capacities are insufficient to maintain the proteome and may themselves be diminished (bottom). Sugar metabolites promote protein modification resulting in accumulation of ubiquitin conjugates, aggregation and cross-linking. Amino acid residues that encode susceptibility for the degradation of substrates may be blocked. Glycated ubiquitin may be incorporated into conjugates, rendering them less susceptible to degradation. This situation is exacerbated by the accumulation of damaged proteins, which may diminish proteolytic efficacy or render it insufficient, and induce oxidative stress, setting up a vicious cycle that results in additional stress and limited cellular function. The stress may also alter the interaction between the UPS or LPS as one or both pathways are attenuated and proteopoise (the balance of protein synthesis, modification and degradation) is perturbed.

Several experiments confirmed this notion. First exposure of RPE to >1.7 mM MGO for 1 h reduces the rate of intracellular protein degradation by 60%, coincident with the observed accumulation of AGEs. Glycative stress is also related to accumulation of ubiquitin conjugates. This raised concern because conjugates increase in neurodegenerative disease and upon aging. Second, diminished susceptibility of established UPS substrates after glycative modification limited UPS-dependent proteolysis. Third, glycated ubiquitin is less effectively incorporated into high mass conjugates that are usual substrates for degradation. Glycation of UBE2D2/UBC4, a ubiquitin-conjugating enzyme required for removal of altered proteins, catalyzes less ubiquitination and slower proteolysis. Surprisingly, activity of the proteasome is unaffected. The glycation-induced reduced degradation was observed with multiple substrates and in many types of cells, suggesting this is a generalizable phenomenon.

Next, we used inhibitors in live cells, cell fractionation, and immunohistochemical techniques to demonstrate that an autophagic LPS collaborates with the UPS to remove AGEs. In cells that are exposed briefly to MGO, UPS and LPS inhibitors delay degradation of endogenous proteins. There is a period of time (≈8 h) before which UPS or LPS inhibitors have a substantial stabilizing effect on AGEs. This appears to be because of continued formation of AGEs along with destruction of these moieties. The more AGEs and ubiquitin conjugates accumulate, the greater the proportion of these moieties that is of high mass and is directed to the insoluble fraction, where they are physiologically dysfunctional. The relationship between soluble and insoluble moieties is dynamic, with clearance first from the soluble, and only subsequently from the insoluble, compartment. As expected, the UPS has the greatest effect on levels of the soluble substrates. Clearance of AGEs and ubiquitin conjugates is markedly delayed when cells are exposed to inhibitors of the UPS and/or LPS, including an autophagic LPS. A role for the LPS was emphasized by findings of elevated levels of AGEs, some of which are ubiquitinated, in isolated lysosomes in MGO-exposed RPE, particularly when inhibitors of autophagy, as well as the UPS and LPS, are included. This was corroborated by colocalizaton of MG-H1-positive puncta with enlarged lysosomes; MG-H1 is an antibody directed against AGEs.

When the AGEs are chased to the lysosome, they appear to collect at the membranes, comparable to tau, rather than being effectively internalized where they might be more readily degraded. This inability of damaged proteins to reach a functional degradative compartment (the UPS in the cytosol, or the lysosome) provides a novel additional explanation for the accumulation of insoluble ubiquitin conjugates and AGEs in many age-related syndromes. Additional support for crosstalk between the pathways, specifically involving autophagy, is indicated by the formation of LC3-II when ubiquitin conjugates accumulate because of inhibition of the UPS by the expression of conjugation-competent but degradation-incompetent ubiquitin.

Taken together, these data establish synergistic roles for, and indicate a dynamic flux between, the UPS and LPS in the removal of AGEs and ubiquitinated species. It appears that AGE modification converts UPS substrates into inhibitors of the UPS, in effect rendering insufficient the amount of available proteasome even when the proteasome retains its activity. This may also overburden LPS capacities, further exacerbating cellular stress. New reagents that enhance or that protect proteins and/or the proteolytic machinery proteolysis may be useful to avoid protein insolubilization that is associated with pathology.

Glossary

Abbreviations:

GI

high glycemic index

MGO

methyl glyoxal

RPE

retinal pigmented epithelial cells

AGEs

age-related glycation end products

UPS

ubiquitin-proteasome systems

LPS

lysosomal proteolytic system

Uchiki T, Weikel KA, Jiao W, Shang F, Caceres A, Pawlak D, et al. Glycation-altered proteolysis as a pathobiologic mechanism that links dietary glycemic index, aging, and age-related disease (in nondiabetics) Aging Cell. 2012;11:1–13. doi: 10.1111/j.1474-9726.2011.00752.x.