The integrin beta1 modulator Tirofiban prevents adipogenesis and obesity by the overexpression of integrin-linked kinase: a pre-clinical approach in vitro and in vivo.

BACKGROUND: Obesity is caused by the enlargement of the white adipose tissue (WAT) depots, characterized by the hypertrophic enlargement of malfunctioning adipocytes within WAT which increases the storage of triglycerides (TG) in the lipid droplets (LD). Adipogenesis pathways as well as the expression and activity of some extracellular matrix receptors integrins are upregulated. Integrinß1 (INTB1) is the main isoform involved in WAT remodeling during obesity and insulin resistance-related diseases. We recently described Integrin Linked Kinase (ILK), a scaffold protein recruited by INTB1, as an important mediator of WAT remodeling and insulin resistance. As the few approved drugs to fight obesity have brought long-term cardiovascular side effects and given that the consideration of INTB1 and/or ILK modulation as anti-obesogenic strategies remains unexplored, we aimed to evaluate the anti-obesogenic capacity of the clinically approved anticoagulant Tirofiban (TF), stated in preclinical studies as a cardiovascular protector. METHODS: Fully differentiated adipocytes originating from C3H10T1/2 were exposed to TF and were co-treated with specific INTB1 blockers or with siRNA-based knockdown ILK expression. Lipid-specific dyes were used to determine the TG content in LD. The genetic expression pattern of ILK, pro-inflammatory cytokines (MCP1, IL6), adipogenesis (PPARγ, Leptin), thermogenesis (UCP1), proliferation (PCNA), lipid metabolism (FASN, HSL, ATGL), and metabolite transporters (FABP4, FAT, AQP7) were detected using quantitative PCR. Cytoskeletal actin polymerization was detected by confocal microscopy. Immunoblotting was performed to detect INTB1 phosphorylation at Thr788/9 and ILK activity as phosphorylation levels of protein kinase B (AKT) in Ser473 and glycogen synthase kinase 3ß (GSK3ß) at Ser9. TF was intraperitoneally administered once per day to wildtype and ILK knockdown mice (cKDILK) challenged with a high-fat diet (HFD) or control diet (STD) for 2 weeks. Body and WAT weight gains were compared. The expression of ILK and other markers was determined in the visceral epididymal (epi) and inguinal subcutaneous (sc) WAT. RESULTS: TF reduced TG content and the expression of adipogenesis markers and transporters in adipocytes, while UCP-1 expression was increased and the expression of lipases, cytokines or PCNA was not affected. Mechanistically, TF rapidly increased and faded the intracellular phosphorylation of INTB1 but not AKT or GSK3ß. F-actin levels were rapidly decreased, and INTB1 blockade avoided the TF effect. After 24 h, ILK expression and phosphorylation rates of AKT and GSK3ß were upregulated, while ILK silencing increased TG content. INTB1 blockade and ILK silencing avoided TF effects on the TG content and the transcriptional expression of PPARγ and UCP1. In HFD-challenged mice, the systemic administration of TF for several days reduced the weight gain on WAT depots. TF reduced adipogenesis and pro-inflammatory biomarkers and increased lipolysis markers HSL and FAT in epiWAT from HFD, while increased UCP1 in scWAT. In both WATs, TF upregulated ILK expression and activity, while no changes were observed in other tissues. In HFD-fed cKDILK, the blunted ILK in epiWAT worsened weight gain and avoided the anti-obesogenic effect of in vivo TF administration. CONCLUSIONS: ILK downregulation in WAT can be considered a biomarker of obesity establishment. Via an INTB1-ILK axis, TF restores malfunctioning hypertrophied WAT by changing the expression of adipocyte-related genes, increasing ILK expression and activity, and reducing TG storage. TF prevents obesity, a property to be added to its anticoagulant and cardiovascular protective advantages.

A role for the outer retina in development of the intrinsic pupillary light reflex in mice.

Mice do not require the brain in order to maintain constricted pupils. However, little is known about this intrinsic pupillary light reflex (iPLR) beyond a requirement for melanopsin in the iris and an intact retinal ciliary marginal zone (CMZ). Here, we study the mouse iPLR in vitro and examine a potential role for outer retina (rods and cones) in this response. In wild-type mice the iPLR was absent at postnatal day 17 (P17), developing progressively from P21-P49. However, the iPLR only achieved ∼ 30% of the wild-type constriction in adult mice with severe outer retinal degeneration (rd and rdcl). Paradoxically, the iPLR increased significantly in retinal degenerate mice >1.5 years of age. This was accompanied by an increase in baseline pupil tone in the dark to levels indistinguishable from those in adult wild types. This rejuvenated iPLR response was slowed by atropine application, suggesting the involvement of cholinergic neurotransmission. We could find no evidence of an increase in melanopsin expression by quantitative PCR in the iris and ciliary body of aged retinal degenerates and a detailed anatomical analysis revealed a significant decline in melanopsin-positive intrinsically photosensitive retinal ganglion cells (ipRGCs) in rdcl mice >1.5 years. Adult mice lacking rod function (Gnat1(-/-)) also had a weak iPLR, while mice lacking functional cones (Cpfl5) maintained a robust response. We also identify an important role for pigmentation in the development of the mouse iPLR, with only a weak and transient response present in albino animals. Our results show that the iPLR in mice develops unexpectedly late and are consistent with a role for rods and pigmentation in the development of this response in mice. The enhancement of the iPLR in aged degenerate mice was extremely surprising but may have relevance to behavioral observations in mice and patients with retinitis pigmentosa.

Number and spatial distribution of intrinsically photosensitive retinal ganglion cells in the adult albino rat.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light and are responsible of the synchronization of the circadian rhythm with the photic stimulus and for the pupillary light reflex. To quantify the total population of rat-ipRGCs and to assess their spatial distribution we have developed an automated routine and used neighbour maps. Moreover, in all analysed retinas we have studied the general population of RGCs - identified by their Brn3a expression - and the population of ipRGCs - identified by melanopsin immunodetection - thus allowing the co-analysis of their topography. Our results show that the total mean number ± standard deviation of ipRGCs in the albino rat is 2047 ± 309. Their distribution in the retina seems to be complementary to that of Brn3a(+)RGCs, being denser in the periphery, especially in the superior retina where their highest densities are found in the temporal quadrant, above the visual streak. In addition, by tracing the retinas from both superior colliculi, we have also determined that 90.62% of the ipRGC project to these central targets.