Irisin Ameliorate Acute Pancreatitis and Acinar Cell Viability through Modulation of the Unfolded Protein Response (UPR) and PPARγ-PGC1α-FNDC5 Pathways.

Acute pancreatitis (AP) entails pancreatic inflammation, tissue damage and dysregulated enzyme secretion, including pancreatic lipase (PL). The role of irisin, an anti-inflammatory and anti-apoptotic cytokine, in AP and exocrine pancreatic stress is unclear. We have previously shown that irisin regulates PL through the PPARγ-PGC1α-FNDC5 pathway. In this study, we investigated irisin and irisin's pathway on AP in in vitro (AR42J-B13) and ex vivo (rat primary acinar) models using molecular, biochemical and immunohistochemistry methodology. Pancreatitis induction (cerulein (cer)) resulted in a significant up-regulation of the PPARγ-PGC1α-FNDC5 axis, PL expression and secretion and endoplasmic reticulum (ER) stress unfolded protein response (UPR) signal-transduction markers (CHOP, XBP-1 and ATF6). Irisin addition in the cer-pancreatitis state resulted in a significant down-regulation of the PPARγ-PGC1α-FNDC5 axis, PPARγ nucleus-translocation and inflammatory state (TNFα and IL-6) in parallel to diminished PL expression and secretion (in vitro and ex vivo models). Irisin addition up-regulated the expression of pro-survival UPR markers (ATF6 and XBP-1) and reduced UPR pro-apoptotic markers (CHOP) under cer-pancreatitis and induced ER stress (tunicamycin), consequently increasing cells viability. Irisin's pro-survival effect under cer-pancreatitis state was abolished under PPARγ inhibition. Our findings suggest irisin as a potential therapeutic option for AP via its ability to up-regulate pro-survival UPR signals and activate the PPARγ-PGC1α-FNDC5 pathway.

BBS genes are involved in accelerated proliferation and early differentiation of BBS-related tissues.

Bardet-Biedl syndrome (BBS) is an inherited disorder primarily ciliopathy with pleiotropic multi-systemic phenotypic involvement, including adipose, nerve, retinal, kidney, Etc. Consequently, it is characterized by obesity, cognitive impairment and retinal, kidney and cutaneous abnormalities. Initial studies, including ours have shown that BBS genes play a role in the early developmental stages of adipocytes and ß-cells. However, this role in other BBS-related tissues is unknown. We investigated BBS genes involvement in the proliferation and early differentiation of different BBS cell types. The involvement of BBS genes in cellular proliferation were studied in seven in-vitro and transgenic cell models; keratinocytes (hHaCaT) and Ras-transfected keratinocytes (Ras-hHaCaT), neuronal cell lines (hSH-SY5Y and rPC-12), silenced BBS4 neural cell lines (siBbs4 hSH-SY5Y and siBbs4 rPC-12), adipocytes (m3T3L1), and ex-vivo transformed B-cells obtain from BBS4 patients, using molecular and biochemical methodologies. RashHaCaT cells showed an accelerated proliferation rate in parallel to significant reduction in the transcript levels of BBS1, 2, and 4. BBS1, 2, and 4 transcripts linked with hHaCaT cell cycle arrest (G1 phase) using both chemical (CDK4 inhibitor) and serum deprivation methodologies. Adipocyte (m3T3-L1) Bbs1, 2 and 4 transcript levels corresponded to the cell cycle phase (CDK4 inhibitor and serum deprivation). SiBBS4 hSH-SY5Y cells exhibited early cell proliferation and differentiation (wound healing assay) rates. SiBbs4 rPC-12 models exhibited significant proliferation and differentiation rate corresponding to Nestin expression levels. BBS4 patients-transformed B-cells exhibited an accelerated proliferation rate (LPS-induced methodology). In conclusions, the BBS4 gene plays a significant, similar and global role in the cellular proliferation of various BBS related tissues. These results highlight the universal role of the BBS gene in the cell cycle, and further deepen the knowledge of the mechanisms underlying the development of BBS.

Adipose Tissue Hyperplasia and Hypertrophy in Common and Syndromic Obesity-The Case of BBS Obesity.

Obesity is a metabolic state generated by the expansion of adipose tissue. Adipose tissue expansion depends on the interplay between hyperplasia and hypertrophy, and is mainly regulated by a complex interaction between genetics and excess energy intake. However, the genetic regulation of adipose tissue expansion is yet to be fully understood. Obesity can be divided into common multifactorial/polygenic obesity and monogenic obesity, non-syndromic and syndromic. Several genes related to obesity were found through studies of monogenic non-syndromic obesity models. However, syndromic obesity, characterized by additional features other than obesity, suggesting a more global role of the mutant genes related to the syndrome and, thus, an additional peripheral influence on the development of obesity, were hardly studied to date in this regard. This review summarizes present knowledge regarding the hyperplasia and hypertrophy of adipocytes in common obesity. Additionally, we highlight the scarce research on syndromic obesity as a model for studying adipocyte hyperplasia and hypertrophy, focusing on Bardet-Biedl syndrome (BBS). BBS obesity involves central and peripheral mechanisms, with molecular and mechanistic alternation in adipocyte hyperplasia and hypertrophy. Thus, we argue that using syndromic obesity models, such as BBS, can further advance our knowledge regarding peripheral adipocyte regulation in obesity.

Fndc5/irisin is regulated by myogenesis stage, irisin, muscle type and training.

OBJECTIVES: Irisin, a novel myokine that responds to exercise, was initially identified as a regulator of fat tissue metabolism. We aimed to investigate fibronectin type III domain-containing protein 5 (Fndc5)/irisin, auto/para-crine role in different muscle fibers, different activities, and muscle cell differentiation. METHODS: Using in-vitro, ex-vivo, and in-vivo muscle models, Fndc5 was studied at the physiological and molecular levels. RESULTS: Following training, C57BL/6 mice (n=10) were subject to fast and slow-twitch muscles dissection and molecular analysis. Isolated mice (C57BL/6, n=14) slow and fast-twitch muscles were subject to electrical aerobic and anaerobic pulses stimulation (EPS). L6 muscle cells differentiation was characterized by Fndc5 differentiation-depended expression pattern parallel with significant hypertrophy, Myogenin elevation, and overlapping Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (Pgc-1α) expression pattern. Exogenous irisin significantly altered Fndc5 expression; augmented at early differentiation (3-4-fold, P<0.05) and decreased (2-fold, P<0.05) at late differentiation. Training induced a significant elevation in Fndc5/irisin and Pgc-1α expression levels in all muscle types compared to the sedentary state, where soleus muscle (slow) Fndc5 expression levels were significantly higher compared to levels in all other fast muscles (3-140-fold, P<0.001). Similarly, following EPS, Fndc5 expression levels were significantly augmented in the soleus slow muscle following both aerobic and anaerobic activity (3-3.5-fold, P<0.05) compared to extensor digitorum longus (fast) muscle. CONCLUSIONS: Muscle cell's Fndc5 expression has a differentiation-depended pattern paralleling Pgc-1α expression and hypertrophy. Irisin autocrinally and significantly regulate Fndc5 and Pgc-1α in a differentiation-depended manner. Muscle Fndc5 expression levels are dependent on fiber type and activity type.

BBS4 Is Essential for Nuclear Transport of Transcription Factors Mediating Neuronal ER Stress Response.

Bardet-Biedl syndrome (BBS) is an autosomal recessive syndrome presenting with retinal dystrophy, cognitive impairment, and obesity. BBS is characterized by elevated endoplasmic reticulum (ER) stress in the early stages of adipocyte and retinal development. BBS expression in the CNS and indications of hippocampal dysgenesis suggest neural development abnormalities. However, the role of BBS in ER stress in neuronal cells has not yet been studied. Therefore, we aimed at studying the role of BBS4 in neuronal development under normal and ER stress conditions. ER stress and unfolded protein response (UPR) were studied in BBS4-silenced (SiBBS4) SH-SY5Y cells during differentiation under normal and stress states, using molecular and biochemical markers. ER stress was demonstrated at early neural differentiation, with significantly augmented expression of UPR markers corresponding to BBS4 expression. In the undifferentiated state, BBS4 silencing resulted in significantly reduced ER-stress markers' expression under normal and ER-stress states. Independent of ER stress, SiBBS4 cells demonstrated significant reduction in activated phospho-IRE1α. Under BBS4 silencing, both sXBP-1 and activated ATF6α p50 failed to translocate to the nucleus. Transcript levels of apoptosis markers were upregulated under BBS4 depletion and ER-stress induction, corresponding to decreased viability. BBS4 depletion in neuronal cells results in reduced sensitivity to ER stress during differentiation and under ER-stress induction, partly due to failure in translocation of ER-transcription factors (TF) sXBP-1 and ATF6α p50 to the nucleus. Hence, BBS4 is essential for nuclear transport under ER-stress response in neuronal cells during early differentiation. Our studies shed light on molecular mechanisms through which BBS4 malfunction alters neuronal ER stress response.

Endocrine and exocrine pancreas pathologies crosstalk: Insulin regulates the unfolded protein response in pancreatic exocrine acinar cells.

Exocrine pancreas insufficiency is common in diabetic mellitus (DM) patients. Cellular stress is a prerequisite in the development of pancreatic pathologies such as acute pancreatitis (AP). The molecular mechanisms underlying exocrine pancreatic ER-stress in DM are largely unknown. We studied the effects of insulin and glucose (related to DM) alone and in combination with cerulein (CER)-induced stress (mimicking AP) on ER-stress unfolded protein response (UPR) in pancreatic acinar cells. Exocrine pancreas cells (AR42J) were exposed to high glucose (Glu, 25 mM) and insulin (Ins, 100 nM) levels with or without CER (10 nM). ER-stress UPR activation was analyzed at the transcript, protein, immunocytochemistry, western blotting, quantitative RT-PCR and XBP1 splicing, including; XBP1, sXBP1, ATF6, cleaved ATF6, IRE1-p, CHOP, Caspase-12 and Bax. Exocrine acinar cells exposed to high Ins or Ins+Glu concentrations (but not Glu alone) exhibited ER-stress UPR, demonstrated by significant increase of transcript and protein levels of downstream markers in the ATF6 and IRE1 transduction arms, including: sXBP1, cleaved ATF6, XBP1, CHOP, IRE1-p and caspase-12. UPR activation resulted in IRE1-p aggregation and nuclear trans-localization of cleaved activated ATF6 and sXBP1. Ins further aggravated UPR when cells were co-challenged with CER-induced stress, exacerbating the effects of CER alone. High Ins levels, typical to type-2-DM, activate the ER-stress UPR in pancreatic acinar cells, through the ATF6 and IRE1 pathways. This effect of Ins in naïve acinar cells further augments CER-induced UPR. Our data highlight molecular pathways through which DM enhances exocrine pancreas pathologies.