Piece Found in Puzzle of Autophagy Control

A delicate balance of signals controls autophagy, a cell’s homeostatic degradation of its own cytoplasmic components. The process accelerates to cope with stressful situations such as scarcity of nutrients or microbial invasions. But it can also contribute to tumor development if not constrained within its normal range of activity.

Several proteins interact to speed or slow autophagy as the cell’s needs change. About a year ago researchers discovered that the oncogene Bcl-2 inhibits autophagy. Molecules promoting autophagy remained unknown, however, until postdoctoral fellow Chengyu Liang with Jae Jung, HMS professor of microbiology and molecular genetics at the New England Primate Research Center, and colleagues borrowed from the world of virology to identify the novel autophagic UV irradiation resistance–associated gene (UVRAG) protein. Their findings appeared online on June 25 ahead of print in Nature Cell Biology.

Balancing act. A small group of proteins regulates autophagy, the homeostatic degradation of cytoplasmic components, by controlling production of the specialized vesicles, autophagosomes, that carry out this task. The proteins UVRAG and PI(3)KC3, shown in magenta, and Beclin1, in blue, colocalize with autophagosomes (green), indicating their interdependent involvement in controlling autophagy. Jae Jung’s research implicates UVRAG specifically as the factor promoting increased autophagosome formation.

Jung and colleagues used a viral version of Bcl-2, rather than the cellular version, to fish for the elusive autophagy-promoting protein. The viral Bcl-2 interacted with three proteins in living cells. Other researchers had previously identified two of these—Beclin1 and a PI(3) kinase class III lipid kinase complex [PI(3)KC3]—as components of the autophagy-controlling system. UVRAG is now the third.

Cellular Bcl-2 “has a long negative regulatory loop in its amino terminus that prevents tight binding to the Beclin1–PI(3)KC3 complex,” said Jung, which may be why researchers had not found UVRAG earlier. Viral Bcl-2 circumvents this problem, in part, by minimizing the loop region.

Having identified UVRAG and examined how it binds to the other components of the system, Jung can now propose a revised understanding of how cells control autophagy. In healthy cells, Bcl-2 and UVRAG each bind to a different region of the large Beclin1–PI(3)KC3 complex to maintain a normal autophagic balance. When cells become stressed, Bcl-2 loses its ability to bind, but UVRAG does not. The system tilts into a controlled imbalance, ramping up autophagy.

The interdependence of the four autophagy-controlling proteins creates a dangerous scenario if any one component is defective. In breast cancer, a mutation makes Beclin1 scarce. Without it, autophagy occurs at very low levels. Similarly, in colon cancer, a monoallelic mutation in UVRAG decreases its presence in cells, abnormally slowing the process. By adding functional UVRAG to colon cancer cells, Jung showed a restoration of autophagy and 10-fold reduction in tumor mass in mice, suggesting UVRAG functions as a tumor suppressor.

Based on the results, it appears that low auto-phagy promotes tumorigenicity. Yet according to Jung, “There’s still debate over whether autophagy is a tumor promotor or suppressor.” A current theory suggests that autophagy in early stage cancer acts as a tumor suppressor, which Jung’s results support. In later stage cancer, however, amplified autophagy may promote tumor growth by allowing cells deep within a tumor to survive in a low-nutrient environment.

—Kathleen Fink

Early Indicator Identified for Insulin Resistance

A protein secreted by fat cells may represent a new early identifier of insulin resistance in a variety of clinical scenarios. Research published in the June 15 New England Journal of Medicine reveals that serum levels of retinol-binding protein 4 (RBP4) correlate with insulin resistance in obese, nondiabetic subjects; those with impaired glucose tolerance or type 2 diabetes; and in nonobese, healthy individuals with a family history of diabetes.

The problem of diabetes has grown to such proportions that “this epidemic threatens to shorten life span in the U.S. for the first time in a century,” said Barbara Kahn, HMS professor of medicine and chief of the Division of Endocrinology, Diabetes, and Metabolism at Beth Israel Deaconess Medical Center.

Senior author Kahn, HMS instructor in medicine Tim Graham, postdoctoral fellow Qin Yang, and colleagues showed that RBP4 is an early marker for type 2 diabetes. Serum RBP4 levels may help identify high-risk patients earlier than currently possible and could also represent a target for diabetes therapies.

First the Kahn lab and their collaborators evaluated RBP4 levels in nondiabetic and diabetic obese men, finding that RBP4 levels in both groups correlated with insulin resistance. Interestingly, “the magnitude of elevation of RBP4 is as great for obesity alone as it is for obesity with diabetes,” said Kahn. “We think RBP4 is really tracking with insulin resistance.” RBP4 levels also correlate with components of the metabolic syndrome, a constellation of abnormalities, including many cardiovascular-disease risk factors often associated with insulin resistance.

The researchers next evaluated the impact of exercise on subjects with diabetes or impaired glucose tolerance—an intermediate condition that often progresses to type 2 diabetes. Those with the greatest improvement in insulin sensitivity after a four-week exercise regimen experienced the greatest drop in serum RBP4 levels. “The lowering of serum RBP4 correlated better with improvement in insulin sensitivity than any other marker we measured,” said Kahn. Other markers changed with exercise, but not in concert with improvements in insulin sensitivity. They simply showed exercise had occurred, not whether the exercise had any therapeutic benefit on sensitivity levels, which RBP4 showed.

A third set of subjects included healthy, nonobese men with a family history of type 2 diabetes. Serum RBP4 levels were elevated in the insulin-resistant family members, but not in the insulin-sensitive ones.

Taken together, these results indicate that “it may be possible to identify insulin resistance and diabetes risk early in people who otherwise would be overlooked in a clinical setting,” Kahn explained.

Though these studies reveal correlations, not causation, the Kahn lab previously showed that insulin resistance develops in mice following chronic injection of RBP4 or transgenic elevation of RBP4. If further study in humans reveals a causative relationship between elevated RBP4 and insulin resistance, this “may open up a whole new mechanistic pathway in terms of the pathogenesis of type 2 diabetes,” said Kahn.

—Kathleen Fink

Search to Promote Insulin Secretion Proves Fruitful

An extract of the gardenia fruit, used in traditional Chinese medicine, contains a compound potentially useful for the treatment of diabetes. In a screen of traditional compounds, Chen-Yu Zhang, at the time an instructor in medicine working with HMS associate professor of medicine Bradford Lowell, found the gardenia candidate promising because of its inhibition of a protein that controls insulin release. Isolation of the pharmacologically active component, with assistance from John Porco’s group in Boston University’s Department of Chemistry, revealed the molecule genipin.

The researchers report in the June Cell Metabolism that genipin inhibits function of uncoupling protein 2 (UCP2), a transmembrane protein that controls leakage of protons across the inner mitochondrial membrane. This flow indirectly controls insulin secretion from pancreatic beta cells. Inhibiting UCP2 slows the stream of protons, improving membrane potential and allowing mitochondria to produce greater quantities of ATP. As a result, potassium channels close, depolarizing the cell, which in turn causes calcium channels to open and release insulin.

“There’s been tremendous desire to have a UCP inhibitor,” said Lowell, from the Department of Endocrinology at Beth Israel Deaconess Medical Center. No known UCP2 inhibitors existed until this discovery, with the exception of purine nucleotides such as ATP and GTP, which have little utility in research because they lack cell permeability. Lowell and colleagues established that genipin directly inhibits UCP2 by first testing pancreatic islets obtained from normal mice, in which insulin secretion increased with genipin, followed by tests in pancreatic islets from UCP2-deficient mice, in which genipin induced no changes in insulin secretion.

Normally, pancreatic beta cells react to fluctuating levels of glucose in the body, releasing proportional levels of insulin. In type 2 diabetes, however, this dose-dependent relationship is lost; beta cells do not respond to changes in glucose, and the cells elevate their basal level of insulin secretion. By blocking UCP2 with genipin in cells mimicking this diabetic state, “glucose-sensing is now restored and basal insulin secretion goes down,” said Lowell. “Why the basal secretion comes down, we don’t know,” but a point of interest remains that inhibiting UCP2 corrects both of these defects.

The genipin molecule also harbors some less desirable activities, such as nonspecific cross-linking of proteins. The researchers established that an altered version of genipin, made by Porco at Boston University, lacks this cross-linking activity, but retains its ability to inhibit UCP2. In addition, genipin may inhibit proteins similar to UCP2, such as UCP3. “I don’t expect inhibition of UCP3 to cause adverse consequences,” Lowell said, though it warrants further study.

Genipin represents a “potential new lead compound for a novel treatment for type 2 diabetes,” he said. He also points to the importance of genipin as a research tool, saying scientists in several disparate fields are already making use of its UCP2-inhibitory activity for their research.

—Kathleen Fink

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