Targeting a microRNA shows potential to enhance effectiveness of diabetes drugs

Over the past 15 years, University of Alabama at Birmingham endocrinologist Anath Shalev, M.D., has unraveled a crucial biological pathway that malfunctions in diabetes.

Her latest discovery in this beta-cell pathway, published in the journal Diabetes, shows the potential to enhance the effectiveness of existing diabetes drugs, as well as reduce some of the unwelcome side effects of those drugs.

The need for improved treatment is great. Diabetes is a disorder characterized by elevated blood sugar that afflicts one of every 10 U.S. adults and doubles the risk of early death. More than 30 million people in the United States have diabetes, which is the seventh-leading cause of death and also leads to blindness and lower-limb amputations.

In 2013, the UAB researchers found that either diabetes or elevated production of the protein TXNIP induced beta-cell expression of microRNA-204, or miR-204, and this microRNA, in turn, blocked insulin production. The Shalev group has now found another vital role for miR-204 — regulating the cell surface receptor that is the target of many of the newer type 2 diabetes drugs, such as Byetta, Victoza, Trulicity, Januvia, Onglyza and Tradjenta. This drug target is the glucagon-like peptide 1 receptor, or GLP1R. Activation of GLP1R with these drugs helps the beta cell produce and secrete more insulin.

Shalev’s new work was performed in rat beta cells, genetically modified mice, mouse pancreatic islets and human pancreatic islets. Healthy beta cells, which are found in the pancreatic islets, produce insulin to control blood sugar levels; in diabetes the beta cells are impaired and dysfunctional, and have lower GLP1R levels.

In the Diabetes study, Shalev and colleagues found that overexpression of miR-204 decreased expression of GLP1R in rat beta cells and in mouse and human pancreatic islets. Conversely, knock-down of miR-204 increased expression of GLP1R in those cells and pancreatic islets.

Greater GLP1R expression is beneficial because it helps transfer a signal to the beta cell to secrete more insulin, such as after a meal. Also, many of the newer diabetes drugs act as agonists to activate GLP1R. Higher expression can allow use of a lower-drug dose to treat diabetes, thus reducing dose-dependent side effects.

In mice, the UAB researchers found that a deletion of miR-204 caused enhanced GLP1R expression, and also better insulin secretion and glucose control. Furthermore, the knockout mice were more responsive to a GLP1R agonist in glucose tolerance tests. When the GLP1R knockout mice were used in a model of diabetes, where beta cells are damaged by low doses of the toxin streptozotocin, the diabetic mice showed improved glucose control and increased serum insulin levels.

These results suggest that downregulating miR-204, now revealed as an upstream regulator of GLP1R, could lead to better treatment of diabetes.

One key fact about miR-204 may further aid improved treatment. This microRNA is highly expressed in beta cells, but it is not highly expressed in the rest of the pancreas or in cells of the gastrointestinal tract that also express GLP1R and therefore respond to GLP1R agonists. Thus, an inhibitor of miR-204 would be relatively selective for beta cells.

“This novel concept of inhibiting a microRNA in a non-targeted manner, but taking advantage of its restricted tissue distribution and thereby selectively upregulating its target genes in that tissue, may have far reaching implications for microRNA biology and tissue-specific gene targeting in general,” Shalev said.

“Since miR-204 is expressed primarily in pancreatic beta cells, manipulating its levels allows for preferential upregulation of GLP1R in the beta cell, where it helps secrete insulin, rather than in the gastrointestinal system, where it can cause nausea and impaired gastric emptying, or in the pancreas, where it can increase the risk for pancreatitis,” Shalev said. “So by inhibiting miR-204, one could increase the effects of GLP1R agonist drugs on insulin secretion, thereby lowering the necessary dose and avoiding some of the dose-dependent adverse effects.”

The mechanism by which miR-204 downregulates expression of GLP1R is binding of the microRNA to the 3-prime-untranslated region of GLP1R messenger RNA. Such binding is a known method to control gene expression by microRNAs. The UAB researchers discovered this specific binding using microRNA target prediction software. They found two binding sites for miR-204 in the messenger RNA for human GLP1R and one binding site in the messenger RNA for mouse GLP1R. When they mutated those binding sites, it eliminated the regulatory effect of miR-204.

Additionally, the Shalev group showed a novel link between TXNIP and GLP1R signaling. Mice with a beta cell-specific knockout of the protein TXNIP had lower miR-204 levels and higher GLP1R expression, and the mice showed enhanced insulin secretion and glucose control in response to an agonist of GLP1R. Thus, through both control of insulin production and regulation of GLP1R, as well as regulation of the unfolded protein response and beta cell apoptosis, miR-204 appears to play a linchpin role to control the function of beta cells in the pancreas.

Cancer therapy shows promise for psoriasis treatment

HDAC inhibitors, already widely used to treat cancer, may be an effective therapy for psoriasis as well, scientists report.

They have shown that HDAC3 inhibitors are particularly adept at increasing expression of aquaporin-3, or AQP3, a channel that transports glycerin, a natural alcohol and water attractor, which helps skin look better and aids healthy production and maturation of high-turnover skin cells.

“We’ve found that HDAC3 normally inhibits expression of AQP3 and we think we can use this knowledge to treat patients with psoriasis,” said Dr. Vivek Choudhary, molecular biologist and physiologist in the Department of Physiology at the Medical College of Georgia at Augusta University.

MCG scientists knew that AQP3 levels were lower in psoriasis than in healthy skin, said Choudhary, corresponding author of the study in the Journal of Investigative Dermatology. The protein helps skin cells proliferate, differentiate into the right kind of cells and get to the right location in the body. It also aids the skin’s hydration, wound recovery and elasticity. Histone deacetylase, which they found suppresses AQP3, helps regulate gene expression and protein function.

Since the immune system is believed to play a key role in psoriasis, many current treatments generally suppress the immune response, which increases the risk of infections, even cancer. MCG scientists hope they can one day instead directly enhance the presence of AQP3 or maybe its key cargo glycerin.

Psoriasis is one of the most common skin disorders, with red, flaky patches most often erupting on the elbows, knees, scalp and back, said Dr. Wendy B. Bollag, cell physiologist in the MCG Department of Physiology and the study’s senior author.

Like cancer, inflammation and excessive proliferation of cells are a psoriasis hallmark. That common ground and other emerging clues got the scientists thinking about the treatment potential of HDAC inhibitors. But first they had to establish a relationship.

When they introduced a broad-acting HDAC inhibitor to normal skin cells, or keratinocytes, – both mouse and human – they found expression of AQP3 went up within 24 hours, the first time the relationship had been noted.

They reiterated that AQP3 was critical because when it was missing, there was no commensurate increase in glycerin. AQP3 knockout mice also further clarified AQP3’s role in skin hydration, elasticity and wound healing and that it is glycerin – rather than water – that is most key to these healthy functions.

They also found that p53, a known, natural tumor suppressor that also supports cell differentiation, helps the HDAC inhibitors enable more AQP3 and ultimately more glycerin, Choudhary said. HDACs also are known to inhibit p53 activity. However overexpressing p53 by itself did not result in increased functional levels of AQP3, the scientists found.

The MCG scientists first used the HDAC inhibitor, suberoylanilide hydroxamic acid, or SAHA, which was approved by the Food and Drug Administration more than a decade ago to treat cutaneous T cell lymphoma, which has symptoms that can include dry, itchy skin as well as enlarged lymph nodes.

“We think this is one of the ways it works,” Bollag said of SAHA and their new findings. They also used several other HDAC inhibitors and found the ones that suppressed HDAC3 were also most effective at increasing AQP3.

AQP3 is adept at hauling glycerin, the backbone of many lipids and typically a key ingredient in skin lotion. Bollag’s lab reported in the Journal of Investigative Dermatology in 2003 that glycerin helps skin cells mature properly. Inside skin cells, phospholipase D – an enzyme that converts fats or lipids in the external protective cell membrane into cell signals – and AQP3 interact. AQP3 hands off glycerin, which produces phosphatidylglycerol, which, in turn, aids skin cell differentiation.

“We think phosphatidylglycerol is the key,” Bollag said of the positive results. “If you don’t have enough, you may have psoriasis.”

The Bollag lab and others also had found that AQP3, which is present in psoriasis, appears rather immature and out of place, largely inside the cell cytoplasm instead of on the protective, outer cell membrane. The inner location puts quite a damper on its normal mature function of transporting glycerin, water and other substances through the membrane.

“If you use antibodies to visualize where AQP3 is in the keratinocytes, you will see it nicely outlining the cells because it’s right there on the plasma membrane,” Bollag said. “So clearly it’s normally expressed in keratinocytes but the fact that we can upregulate it even more with an HDAC3 inhibitor suggests that normally HDAC3 keeps it in check.”

Cambridge, Massachusetts-based biotech company Shape Pharmaceuticals Inc., currently has a topical version of an HDAC inhibitor in clinical trials for cutaneous T cell lymphoma. If psoriasis patients end up taking HDAC inhibitors, low doses or a topical application likely would help avoid some side effects, including nausea, Bollag said.

One way HDAC inhibitors help fight cancer is by temporarily loosening DNA, increasing the expression of tumor-suppressing genes and making the tumor more vulnerable. HDAC inhibitors also are being explored for their potential in treating neurological diseases such as Huntington’s.

Others have provided evidence that dysregulation of AQP3 contributes to psoriasis and AQP3 is linked to other skin diseases as well like atopic dermatitis – the most common type of eczema and vitiligo, which results in white patches on the skin.

Interestingly, even though psoriatic cells are known for their propensity to replicate, it’s hard to grow an adequate number of cells for scientific study: they increase a certain amount then go quiet. There also is no real animal model of psoriasis. Moving forward, the MCG scientists may try developing a model using a topical drug for genital warts since some patients who take it develop psoriasis.