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Background Caveolin‐1 is a structural protein of caveolae which forms a signaling platform for several cell surface receptors including leptin and insulin receptor. Caveolin‐1 expression is increased with obesity which is implicated in impaired adipose tissue leptin signaling. Considering the overlap between insulin and leptin cellular signaling pathways, we investigated known insulin‐sensitizing drugs which may reduce caveolin‐1 expression in adipose tissue and consequently improve leptin signaling. Methods We used an in‐vitro approach to identify insulin‐sensitizing drugs which may reduce caveolin‐1 expression. Differentiated human white preadipocytes (dHWP) were treated with pioglitazone (5–30 μM), troglitazone (10–30 μM), metformin (1–5 mM), or glimepiride (20–100 μM) for 24 hours. Caveolin‐1 expression was determined by Western blot. We further tested the responses in human adipose tissue from obese individuals (n=12, age: 45.4 ± 9.2 years, BMI: 48.9 ± 7.3 kg/m 2 ) undergoing surgery. Tissue was incubated with drugs for 24 h and caveolin‐1 expression was determined. Furthermore, the effect of drugs on leptin‐dependent activation of ERK and STAT3 was examined by western blot. Results Among the drugs investigated, only treatment with increasing concentrations of metformin resulted in a dose‐dependent decease in caveolin‐1 protein (p=0.01) expression in dHWP. Treatment with increasing concentrations of troglitazone upregulated caveolin‐1 (p=0.01), while pioglitazone (p=0.50) and glimepiride (p=0.09) had no effect of caveolin‐1 protein expression. Given these findings, only metformin was tested in human adipose tissue. Overall, metformin did not alter caveolin‐1 expression in adipose tissue from obese individuals (n=12, control: 0.040 ± 0.056, metformin: 0.024 ± 0.030, p=0.57), although a metformin dependent decrease in caveolin‐1 was evident in adipose tissue from 7 of the 12 obese subjects (control: 0.062 ± 0.066, metformin: 0.014 ± 0.013, p=0.02). Notably, changes in caveolin‐1 expression induced by metformin treatment were positively correlated with BMI (p=0.05, r 2 =0.33, n=12). Also, leptin dependent activation of ERK (control: 0.26 ± .098, leptin: 0.28 ± .08) and STAT3 (control: 0.028 ± 0.027, leptin: 0.032 ± 0.023) was impaired in obese adipose tissue and did not change with metformin treatment even in the 7 individuals with metformin associated decreases in caveolin‐1. Conclusions Metformin reduces caveolin‐1 expression in differentiated HWP. However, metformin decreases caveolin‐1 expression in adipose tissue of some but not all obese subjects, and does not have any effects on leptin signaling. The heterogeneous effects of metformin on caveolin‐1 expression in adipose tissue from obese individuals may be related to BMI and needs further investigation. Support or Funding Information MAP and KRS were supported by the American Physiological Society Stride Summer Undergraduate Research Fellowship NHLBI 1 R25 HL115473‐01. PS was supported by AHA scientist development grant 11SDG7260046.

Background We have previously shown that increased caveolin‐1 expression impairs leptin‐dependent activation of cellular signaling pathways and may be responsible for attenuating leptin‐mediated increases in adiponectin expression in obesity. Further, insulin and leptin cellular signaling mechanisms are closely related such that molecules known to impair insulin signaling also alter leptin signaling. Conversely, known insulin‐sensitizing drugs may improve leptin‐signaling via reducing caveolin‐1 expression. We therefore tested the hypothesis that insulin‐sensitizing drugs decrease caveolin‐1 expression. Methods We used an in‐vitro approach to examine the effects of different insulin‐sensitizing drugs on caveolin‐1 expression. Human white preadipocytes were cultured to confluence and differentiated for 15 days. After differentiation, cells were then treated with increasing concentrations of pioglitazone (5–30 μM), troglitazone (10–30 μM), metformin (1–5 mM), or glimepiride (20–100 μM) for 24 hours. Caveolin‐1 protein expression was determined by Western blot analysis. Results Among the insulin‐sensitizers used in our experiment, only treatment with increasing concentrations of metformin resulted in a dose‐dependent decease in caveolin‐1 expression (P =0.007). Treatment with increasing concentration of troglitazone upregulated caveolin‐1 expression (p=0.005), while pioglitazone (p=0.5020) and glimepiride (p=0.0965) had no effect of caveolin‐1 protein expression. Conclusions Metformin reduces caveolin‐1 expression in differentiated human white preadipocytes. In obesity, caveolin‐1 expression is increased which may contribute to impaired leptin signaling. Therefore, metformin may be used to decrease caveolin‐1 expression and consequently improve leptin signaling and leptin‐dependent increases in adiponectin expression in obese individuals. Support or Funding Information Michaela Pfeifer was supported by the American Physiological Society Stride Summer Undergraduate Research Fellowship NHLBI 1 R25 HL115473‐01; Prachi Singh was supported by the American Heart Association Scientist Development Grant 11SDG7260046.

AbstractMitogen‐activated protein kinase 8‐interacting protein 3 gene (MAPK8IP3) encodes the c‐Jun‐amino‐terminal kinase‐interacting protein 3 (JIP3) and is involved in retrograde axonal transport. Heterozygous de novo pathogenic variants in MAPK8IP3 result in a neurodevelopmental disorder with or without brain abnormalities and possible axonal peripheral neuropathy. Whole‐exome sequencing was performed on an individual presenting with severe congenital muscle hypotonia of neuronal origin mimicking lethal spinal muscular atrophy. Compound heterozygous rare variants (a splice and a missense) were detected in MAPK8IP3, inherited from the healthy parents. Western blot analysis in a muscle biopsy sample showed a more than 60% decrease in JIP3 expression. Here, we suggest a novel autosomal recessive phenotype of a lower motor neuron disease caused by JIP3 deficiency.

Background. Danlou tablet (DLT), the traditional Chinese medicine has been commonly used for dyslipidemia, atherosclerosis, and coronary heart disease. Whether it was effective against vascular injury caused by CIH has remained unknown. The aim of the current study was to observe the effects of DLT on chronic intermittent hypoxia (CIH)-induced vascular injury via regulation of blood lipids and to explore potential mechanisms. Methods. Sixteen 12-week-old male ApoE−/− mice were randomly divided into four groups. The sham group was exposed to normal room air, whereas the other three groups were exposed to CIH. Mice in the CIH + normal saline (NS) group were gavaged with NS. Mice in the CIH + Angptl4-ab group were intraperitoneally injected with Angptl4-antibody. Mice in the CIH + DLT group were gavaged with DLT. After four weeks of intervention, serum lipid concentrations, and serum lipoprotein lipase (LPL) activity were detected. The changes in atherosclerosis in vascular tissue were detected by hematoxylin and eosin (H&E) staining. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were applied to detect the expression levels of hypoxia-induciblefactor-1 (HIF-1), factor-inhibiting HIF-1 (FIH-1), angiopoietin-like 4 (Angptl4), and LPL in different tissues. Results. CIH exposure increases serum lipid levels, decreases serum LPL activity, and exacerbates atherosclerosis. Both Angptl4-ab and DLT treatment reversed the changes in lipid concentration, LPL activity, and atherosclerosis caused by CIH. In the epididymal fat pad, CIH exposure decreased the expression of FIH-1 and increased the expression of HIF-1, whereas DLT treatment increased the expression of FIH-1 and LPL and inhibited the expression of HIF-1 and Angptl4. In heart tissue, the expression levels of LPL and Angptl4 were not affected by modeling or treatment. Conclusions. DLT improved vascular damage by improving the increase in blood lipids induced by CIH, potentially by upregulating FIH-1 and downregulating HIF-1 and Angptl4 in adipose tissue. Therefore, DLT may be a promising agent for the prevention and treatment of CIH-induced vascular injury.

Background Abdominal (ABD) and lower‐body (LB) fat depots exert opposing effects on cardiometabolic risk factors. However, molecular mechanisms underlying these differences are not completely understood. Studies suggest an inflammatory role of CD95 in obesity‐associated adipose tissue inflammation and dysfunction. Also, leptin deficiency is associated with increased CD95 expression. Objective To examine the expression of CD95 in the ABD and LB fat depots and to investigate leptin‐dependent CD95 regulation. Methods Transcription of CD95 and other adipokines was determined in paired subcutaneous abdominal (ABD) and lower‐body (femoral, LB) fat samples obtained from 29 healthy individuals (10 females, age: 29.4 ± 7.6 years, BMI: 24 ± 4.1 kg/m 2 ). The underlying mechanisms and down‐stream effects of altered CD95 expression were evaluated using differentiated human white preadipocytes (dHWP). Results Reduced mRNA expression of CD95 was apparent in adipose tissue obtained from LB fat depot [ABD: 4.30 (13.91 – 0.06), LB: 2.49 (10.88 – 0.04), p = 0.01]. mRNA expression of MCP1 [ABD: 0.51 (1.35 – 0.34), LB: 0.51 (1.21 – 0.28), p = 0.51], CCL5 [ABD: 6.60 (43.45 – 1.40), LB: 4.44 (11.28 – 1.27), p = 0.20], TNFα [ABD: 1.10 (6.14 – 0.03), LB: 0.07 (5.84 – 0.03), p = 0.30], and leptin [ABD: 21.86 (31.33 – 14.6), LB: 27.23 (41.69 – 20.35), p = 0.08] was not different between the two fat depots. A positive relationship between CD95 mRNA and other inflammatory cytokines was evident in both ABD (MCP1: ρ = 0.79, p < 0.001; CCL5: ρ = 0.83, p < 0.001; TNFα: ρ = 0.87, p < 0.001) and LB fat depot (MCP1: ρ = 0.87, p < 0.001; CCL5: ρ = 0.78, p < 0.001; TNFα: ρ = 0.83, p < 0.001). Furthermore, leptin‐dependent decreases in CD95 mRNA (p = 0.04) and protein expression (p = 0.03) was seen in dHWP. Also, 24 hour pre‐treatment of dHWP with leptin protected cells from FasL (p = 0.03) induced apoptosis. Conclusions Compared to ABD adipose tissue, LB fat has reduced transcription of CD95. Additionally, expression of CD95 mRNA was associated with increased expression of inflammatory cytokines in both ABD and LB subcutaneous fat. Moreover, we show that leptin may mediate decreases in CD95 expression thereby protecting adipose tissue from FasL mediated apoptosis. Our studies suggest that the decreased CD95 mRNA expression in LB fat depot may be related to higher localized leptin expression and possibly contribute to depot specific alterations in adipose tissue function and consequent cardiometabolic risk. Support or Funding Information Prachi Singh is supported by AHA grant‐in‐Aid 17GRNT33660138. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Intra-acinar trypsinogen activation occurs in the earliest stages of pancreatitis and is believed to play important roles in pancreatitis pathogenesis. However, the exact role of intra-acinar trypsin activity in pancreatitis remains elusive. Here, we aimed to examine the specific effects of intra-acinar trypsin activity on the development of pancreatitis using a transgenic mouse model. This transgenic mouse model allowed for the conditional expression of a mutant trypsinogen that can be activated specifically inside pancreatic acinar cells. We found that expression of this active mutated trypsin had no significant effect on triggering spontaneous pancreatitis. Instead, several protective compensatory mechanisms, including SPINK1 and heat shock proteins, were upregulated. Notably, these transgenic mice developed much more severe acute pancreatitis, compared with control mice, when challenged with caerulein. Elevated tissue edema, serum amylase, inflammatory cell infiltration and acinar cell apoptosis were dramatically associated with increased trypsin activity. Furthermore, chronic pathological changes were observed in the pancreas of all transgenic mice, including inflammatory cell infiltration, parenchymal atrophy and cell loss, fibrosis, and fatty replacement. These changes were not observed in control mice treated with caerulein. The alterations in pancreata from transgenic mice mimicked the histological changes common to human chronic pancreatitis. Taken together, we provided in vivo evidence that increased intra-acinar activation of trypsinogen plays an important role in the initiation and progression of both acute and chronic pancreatitis. NEW & NOTEWORTHY Trypsinogen is activated early in pancreatitis. However, the roles of trypsin in the development of pancreatitis have not been fully addressed. Using a genetic approach, we showed trypsin activity is critical for the severity of both acute and chronic pancreatitis.

Background: Leptin contributes to obesity-related cardiometabolic pathology. While the role of adipose tissue dysfunction in altering metabolic profile in obesity is recognized, the direct effects of leptin signaling in adipose tissue are not completely understood. Among factors impacting adipose tissue function in obesity, adipocyte death is associated with increased inflammation with consequent adipose tissue insulin-resistance. Furthermore, apoptosis of adipocyte progenitor cells (preadipocytes) is important as it lowers adipose tissue capacity to maintain and expand fat storage. Objective: To determine the effects of leptin on regulation of proteins important to apoptotic pathways in cultured human white preadipocytes (HWP) and differentiated HWP (dHWP). Methods: Cells were exposed to increasing concentrations of leptin to evaluate the role of leptin on regulation of anti-apoptotic Bcl2, and pro-apoptotic Bax and CD95 expression. Next, we examined if pre-exposure to leptin attenuated TNF-α and FasL induced apoptosis. Results: Leptin upregulated transcription of Bcl2 (p=0.02) and down-regulated transcription of Bax (p=0.02) and CD95 (p=0.04) in a leptin-concentration dependent manner in dHWP. Leptin dependent increases in Bcl2 (p=0.004) and decreases in Bax (p=0.003) and CD95 (P=0.03) protein expression were also observed. Furthermore, 24 hour pre-exposure to leptin protected cells from TNF-α (p=0.01; p=0.006) and FasL (p=0.03; p=0.007) induced apoptosis in dHWP and HWP respectively. Additionally, leptin attenuated TNF-mediated activation of the p53 pathway, suggesting another mechanism by which leptin mediates its inhibitory effects on TNF-induced apoptosis. Conclusion: Leptin regulates the expression of proteins involved in the apoptotic pathway such that it decreases the apoptotic potential of adipose tissue by increasing anti-apoptotic Bcl2 and decreasing pro-apoptotic Bax and CD95 protein expression. In contrast to our in-vitro findings, prior studies in adipose tissue from obese humans report decreased Bcl2 and increased Bax protein expression, along with high leptin expression. This suggests that increased adipose tissue apoptosis in obesity may at least in part be secondary to altered leptin signaling.

ABSTRACT Dimethylarginine dimethylaminohydrolases (DDAHs) are known to degrade asymmetric dimethylarginine, an endogenous inhibitor of NOS, and maintain vascular homeostasis; however, the regulatory pathways of DDAHs remain unclear. In this study, we aimed to define the role of transmembrane glycoprotein neuropilin‐1 (NRP1) in the expression of DDAHs and investigate the potential roles of NRP1 in regulation of blood pressure. Short hairpin RNA–mediated knockdown of NRP1 reduced the level and mRNA stability of DDAH1 but not DDAH2 in HUVECs, whereas overexpression of NRP1 increased the mRNA stability of DDAH1. Meanwhile, mesenteric arteries and lung vascular endothelial cells of tamoxifen‐inducible endothelial cell–specific NRP1 knockout mice exhibited decreased expression of DDAH1 and slightly increased expression of DDAH2. Mechanistically, the regulation of NRP1 on DDAH1 expression is mediated by a posttranscriptional mechanism involving miR‐219‐5p in HUVECs. Although the endothelial cell–specific NRP1 knockout mice did not exhibit any significant change in blood pressure at the basal level, they were more sensitive to low‐dose angiotensin II infusion–induced increases in blood pressure. Our results show that NRP1 is required for full expression of DDAH1 in endothelial cells and that NRP1 contributes to protection from low‐dose angiotensin II–induced increases in blood pressure.—Wang, Y., Wang, E., Zhang, Y., Madamsetty, V. S., Ji, B., Radisky, D. C., Grande, J. P., Misra, S., Mukhopadhyay, D. Neuropilin‐1 maintains dimethylarginine dimethylaminohydrolase 1 expression in endothelial cells, and contributes to protection from angiotensin II–induced hypertension. FASEB J. 33, 494–500 (2019). www.fasebj.org