Expression and activation of the ketone body receptor HCAR2/GPR109A promotes preservation of retinal endothelial cell barrier function.

Preservation of retinal barrier function is critical to maintenance of retinal health. Therefore, it is not surprising that loss of barrier integrity is a pathologic feature common to degenerative retinal diseases such as diabetic retinopathy. Our prior studies demonstrate the importance of hydroxycarboxylic acid receptor 2/GPR109A (HCAR2/GPR109A) expression in the retinal pigment epithelium (RPE) to outer retinal barrier integrity. However, whether HCAR2/GPR109A is expressed in retinal endothelial cells and has a similar relationship to inner blood retinal barrier regulation is not known. In the current study, we examined relevance of receptor expression to endothelial cell dependent-blood retinal barrier integrity. siRNA technology was used to modulate HCAR2/GPR109A expression in human retinal endothelial cells (HRECs). Cells were cultured in the presence or absence of VEGF, a pro-inflammatory stimulus, and/or various concentrations of the HCAR2/GPR109A-specific agonist beta-hydyroxybutyrate (BHB). HCAR2/GPR109A expression was monitored by qPCR and electrical cell impedance sensing (ECIS) was used to evaluate barrier function. Complementary in vivo studies were conducted in wildtype and HCAR2/GPR109A knockout mice treated intraperitoneally with lipopolysaccharide and/or BHB. Vascular leakage was monitored using fluorescein angiography and Western blot analyses of albumin extravasation. Additionally, retinal function was evaluated by OptoMotry. Decreased (siRNA knockdown) or absent (gene knockout) HCAR2/GPR109A expression was associated with impaired barrier function both in vitro and in vivo. BHB treatment provided some protection, limiting disruptions in retinal barrier integrity and function; an effect that was found to be receptor (HCAR2/GPR109A)-dependent. Collectively, the present studies support a key role for HCAR2/GPR109A in regulating blood-retinal barrier integrity and highlight the therapeutic potential of the receptor toward preventing and treating retinal diseases such as diabetic retinopathy in which compromised barrier function is paramount.

Autonomous regulation of retinal insulin biosynthesis in diabetes.

Diabetic retinopathy (DR) is a neurodegenerative disease that results as a complication of dysregulated glucose metabolism, or diabetes. The signaling of insulin is lost or dampened in diabetes, but this hormone has also been shown to be an important neurotrophic factor which supports neurons of the brain. The role of local insulin synthesis and secretion in the retina, however, is unclear. We have investigated whether changes in local insulin synthesis occur in the diabetic retina and in response to stressors known to initiate retinal neurodegenerative processes. The expression of insulin and its cleavage product, c-peptide, were examined in retinas of a Type I diabetes animal model and human postmortem donors with DR. We detected mRNAs for insulin I (Ins1), insulin II (Ins2) and human insulin (Ins) by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. Using an ex-vivo system, isolated neuroretinas and retinal pigmented epithelium (RPE) layers were exposed to glycemic, oxidative and inflammatory environments to measure insulin gene transcripts produced de novo in the retina under disease-relevant conditions. The expression of insulin in the retina was altered with the progression of diabetes in STZ mice and donors with DR. Transcription factors for insulin, were simultaneously expressed in a pattern matching insulin genes. Furthermore, de novo insulin mRNA in isolated retinas was induced by acute stress. RPE explants displayed the most pronounced changes in Ins1 and Ins2. This data reveals that the retina, like the brain, is an organ capable of producing local insulin and this synthesis is altered in diabetes.

Inhibiting microRNA-144 potentiates Nrf2-dependent antioxidant signaling in RPE and protects against oxidative stress-induced outer retinal degeneration.

The retinal pigment epithelium (RPE) is consistently exposed to high levels of pro-oxidant and inflammatory stimuli. As such, under normal conditions the antioxidant machinery in the RPE cell is one of the most efficient in the entire body. However, antioxidant defense mechanisms are often impacted negatively by the process of aging and/or degenerative disease leaving RPE susceptible to damage which contributes to retinal dysfunction. Thus, understanding better the mechanisms governing antioxidant responses in RPE is critically important. Here, we evaluated the role of the redox sensitive microRNA miR-144 in regulation of antioxidant signaling in human and mouse RPE. In cultured human RPE, miR-144-3p and miR-144-5p expression was upregulated in response to pro-oxidant stimuli. Likewise, overexpression of miR-144-3p and -5p using targeted miR mimics was associated with reduced expression of Nrf2 and downstream antioxidant target genes (NQO1 and GCLC), reduced levels of glutathione and increased RPE cell death. Alternately, some protection was conferred against the above when miR-144-3p and miR-144-5p expression was suppressed using antagomirs. Expression analyses revealed a higher conservation of miR-144-3p expression across species and additionally, the presence of two potential Nrf2 binding sites in the 3p sequence compared to only one in the 5p sequence. Thus, we evaluated the impact of miR-144-3p expression in the retinas of mice in which a robust pro-oxidant environment was generated using sodium iodate (SI). Subretinal injection of miR-144-3p antagomir in SI mice preserved retinal integrity and function, decreased oxidative stress, limited apoptosis and enhanced antioxidant gene expression. Collectively, the present work establishes miR-144 as a potential target for preventing and treating degenerative retinal diseases in which oxidative stress is paramount and RPE is prominently affected (e.g., age-related macular degeneration and diabetic retinopathy).

Loss of GPR109A/HCAR2 induces aging-associated hepatic steatosis.

GPR109A agonists have been used for the treatment of obesity however, the role of GPR109A in regulating aging-associated alterations in lipid metabolism is unknown. In this study we used Gpr109a-/- mice to investigate the effect of aging in the regulation of lipid accumulation. We observed that in mouse and human livers, in addition to Kupffer cells, GPR109A is expressed in hepatocytes. Over 12 months, compared to wild type (WT), Gpr109a-/- mice gained significantly more weight. Food intake and levels of serum lipids were similar among both groups. Compared to age-matched WT mice, 12-months old Gpr109a-/- mice had significantly increased liver weight, hepatic steatosis and serum markers of liver injury. The fatty liver phenotype in Gpr109a-/- mice was associated with increased hepatic expression of lipogenesis genes and decreased expression of lipolysis genes. Gpr109a-/- mice had significantly increased fat tissues, which was associated with significant increase in adipocyte diameter and surface area. Adipose tissue from Gpr109a-/- mice had increased expression of lipogenesis genes; however, expression of lipolytic genes was similar in both groups. Collectively, these results indicate that during aging, GPR109A modulates de novo lipid accumulation in liver and adipose tissue, and its dysregulation can lead to age-associated obesity and hepatic steatosis.

Loss of NAMPT in aging retinal pigment epithelium reduces NAD+ availability and promotes cellular senescence.

Retinal pigment epithelium (RPE) performs numerous functions critical to retinal health and visual function. RPE senescence is a hallmark of aging and degenerative retinal disease development. Here, we evaluated the temporal expression of key nicotinamide adenine dinucleotide (NAD+)-biosynthetic genes and associated levels of NAD+, a principal regulator of energy metabolism and cellular fate, in mouse RPE. NAD+ levels declined with age and correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression, increased expression of senescence markers (p16INK4a, p21Waf/Cip1, ApoJ, CTGF and ß-galactosidase) and significant reductions in SIRT1 expression and activity. We simulated in vitro the age-dependent decline in NAD+ and the related increase in RPE senescence in human (ARPE-19) and mouse primary RPE using the NAMPT inhibitor FK866 and demonstrated the positive impact of NAD+-enhancing therapies on RPE cell viability. This, we confirmed in vivo in the RPE of mice injected sub-retinally with FK866 in the presence or absence of nicotinamide mononucleotide. Our data confirm the importance of NAD+ to RPE cell biology normally and in aging and demonstrate the potential utility of therapies targeting NAMPT and NAD+ biosynthesis to prevent or alleviate consequences of RPE senescence in aging and/or degenerative retinal diseases in which RPE dysfunction is a crucial element.