Autophagy Regulates the Liver Clock and Glucose Metabolism by Degrading CRY1

The circadian clock coordinates behavioral and circadian cues with availability and utilization of nutrients. Proteasomal degradation of clock repressors, such as cryptochrome (CRY) 1, maintains periodicity. Whether macroautophagy, a quality control pathway, degrades circadian proteins remains unknown. Here we show that circadian proteins BMAL1, CLOCK, REV-ERB alpha, and CRY1 are lysosomal targets, and that macroautophagy affects the circadian clock by selectively degrading CRY1. Autophagic degradation of CRY1, an inhibitor of gluconeogenesis, occurs in a diurnal window when rodents rely on gluconeogenesis, suggesting that CRY1 degradation is timeimprinted to maintenance of blood glucose. High-fat feeding accelerates autophagic CRY1 degradation and contributes to obesity-associated hyperglycemia. CRY1 contains several light chain 3 (LC3)-interacting region (LIR) motifs, which facilitate the interaction of cargo proteins with the autophagosome marker LC3. Using mutational analyses, we identified two distinct LIRs on CRY1 that exert circadian glycemic control by regulating CRY1 degradation, revealing LIRs as potential targets for controlling hyperglycemia.Peer reviewe

Effect of the Monocyte Chemoattractant Protein-1/CC Chemokine Receptor 2 System on Nephrin Expression in Streptozotocin-Treated Mice and Human Cultured Podocytes

OBJECTIVE-Monocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes. RESEARCH DESIGN AND METHODS-Expression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting. RESULTS-In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin. CONCLUSIONS-These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes

Chemokine (C-C Motif) Ligand 2 (CCL2) in Sera of Patients with Type 1 Diabetes and Diabetic Complications

Chemokine (C-C motif) ligand 2 (CCL2), commonly known as monocyte chemoattractant protein-1 (MCP-1), has been implicated in the pathogenesis of many diseases characterized by monocytic infiltration. However, limited data have been reported on MCP-1 in type 1 diabetes (T1D) and the findings are inconclusive and inconsistent.In this study, MCP-1 was measured in the sera from 2,472 T1D patients and 2,654 healthy controls using a Luminex assay. The rs1024611 SNP in the promoter region of MCP-1 was genotyped for a subset of subjects (1764 T1D patients and 1323 controls) using the TaqMan-assay.Subject age, sex or genotypes of MCP-1 rs1024611SNP did not have a major impact on serum MCP-1 levels in either healthy controls or patients. While hemoglobin A1c levels did not have a major influence on serum MCP-1 levels, the mean serum MCP-1 levels are significantly higher in patients with multiple complications (mean = 242 ng/ml) compared to patients without any complications (mean = 201 ng/ml) (p = 3.5×10(-6)). Furthermore, mean serum MCP-1 is higher in controls (mean = 261 ng/ml) than T1D patients (mean = 208 ng/ml) (p<10(-23)). More importantly, the frequency of subjects with extremely high levels (>99(th) percentile of patients or 955 ng/ml) of serum MCP-1 is significantly lower in the T1D group compared to the control group (odds ratio = 0.11, p<10(-33)).MCP-1 may have a dual role in T1D and its complications. While very high levels of serum MCP-1 may be protective against the development of T1D, complications are associated with higher serum MCP-1 levels within the T1D group

Differential activation of Fyn kinase distinguishes saturated and unsaturated fats in mouse macrophages

Diet-induced obesity is associated with increased adipose tissue activated macrophages. Yet, how macrophages integrate fatty acid (FA) signals remains unclear. We previously demonstrated that Fyn deficiency (fynKO) protects against high fat diet-induced adipose tissue macrophage accumulation. Herein, we show that inflammatory markers and reactive oxygen species are not induced in fynKO bone marrow-derived macrophages exposed to the saturated FA palmitate, suggesting that Fyn regulates macrophage function in response to FA signals. Palmitate activates Fyn and re-localizes Fyn into the nucleus of RAW264.7, J774 and wild-type bone marrow-derived macrophages. Similarly, Fyn activity is increased in cells of adipose tissue stromal vascular fraction of high fat-fed control mice, with Fyn protein being located in the nucleus of these cells. We demonstrate that Fyn modulates palmitate-dependent oxidative stress in macrophages. Moreover, Fyn catalytic activity is necessary for its nuclear re-localization and downstream effects, as Fyn pharmacological inhibition abolishes palmitate-induced Fyn nuclear redistribution and palmitate-dependent increase of oxidative stress markers. Importantly, mono-or polyunsaturated FAs do not activate Fyn, and fail to re-localize Fyn to the nucleus. Together these data demonstrate that macrophages integrate nutritional FA signals via a differential activation of Fyn that distinguishes, at least partly, the effects of saturated versus unsaturated fats

A numerical model for NVH analysis of gearboxes employed on agricultural equipment

The aim of this paper is to describe a numerical vibro-acoustic methodology, experimentally assessed, for the estimation of the overall vibratory and acoustic level of a gearbox employed on agricultural equipment. The process is carried out in order to create the NVH digital twin of the real gearbox. The vibro-acoustic model is the combination of three sub-models: a lumped-parameter (LP) model, a structural finite-element (SFE) model and an acoustical finite-element (AFE) model. The LP model is used to obtain the reaction forces on the bearings during working conditions. Reaction forces are employed as an input for the further SFE dynamic model to evaluate the dynamic response of the gearbox’s case, which is the only meshed part. The dynamic response is exploited to set-up an AFE model which allows to estimate the noise generation in terms of overall acoustic pressure. The numerical simulation results are validated using experimental data acquired on a real gearbox. Testing activities have been carried out at Comer Industries facility in Reggiolo, where specimens and test benches have been set. Advantages and limitations of the model are reported

Differential activation of Fyn kinase distinguishes saturated and unsaturated fats in mouse macrophages.

Diet-induced obesity is associated with increased adipose tissue activated macrophages. Yet, how macrophages integrate fatty acid (FA) signals remains unclear. We previously demonstrated that Fyn deficiency (fynKO) protects against high fat diet-induced adipose tissue macrophage accumulation. Herein, we show that inflammatory markers and reactive oxygen species are not induced in fynKO bone marrow-derived macrophages exposed to the saturated FA palmitate, suggesting that Fyn regulates macrophage function in response to FA signals. Palmitate activates Fyn and re-localizes Fyn into the nucleus of RAW264.7, J774 and wild-type bone marrow-derived macrophages. Similarly, Fyn activity is increased in cells of adipose tissue stromal vascular fraction of high fat-fed control mice, with Fyn protein being located in the nucleus of these cells. We demonstrate that Fyn modulates palmitate-dependent oxidative stress in macrophages. Moreover, Fyn catalytic activity is necessary for its nuclear re-localization and downstream effects, as Fyn pharmacological inhibition abolishes palmitate-induced Fyn nuclear redistribution and palmitate-dependent increase of oxidative stress markers. Importantly, mono-or polyunsaturated FAs do not activate Fyn, and fail to re-localize Fyn to the nucleus. Together these data demonstrate that macrophages integrate nutritional FA signals via a differential activation of Fyn that distinguishes, at least partly, the effects of saturated versus unsaturated fats

Alteration of de novo glucose production contributes to fasting hypoglycaemia in fyn deficient mice

Previous studies have demonstrated that glucose disposal is increased in the Fyn knockout (FynKO) mice due to increased insulin sensitivity. FynKO mice also display fasting hypoglycaemia despite decreased insulin levels, which suggested that hepatic glucose production was unable to compensate for the increased basal glucose utilization. The present study investigates the basis for the reduction in plasma glucose levels and the reduced ability for the liver to produce glucose in response to gluconeogenic substrates. FynKO mice had a 5-fold reduction in phosphoenolpyruvate carboxykinase (PEPCK) gene and protein expression and a marked reduction in pyruvate, pyruvate/lactate-stimulated glucose output. Remarkably, de novo glucose production was also blunted using gluconeogenic substrates that bypass the PEPCK step. Impaired conversion of glycerol to glucose was observed in both glycerol tolerance test and determination of the conversion of 13C-glycerol to glucose in the fasted state. α-glycerol phosphate levels were reduced but glycerol kinase protein expression levels were not changed. Fructose-driven glucose production was also diminished without alteration of fructokinase expression levels. The normal levels of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate observed in the FynKO liver extracts suggested normal triose kinase function. Fructose-bisphosphate aldolase (aldolase) mRNA or protein levels were normal in the Fyn-deficient livers, however, there was a large reduction in liver fructose-6-phosphate (30-fold) and fructose-1,6-bisphosphate (7-fold) levels as well as a reduction in glucose-6-phosphate (2-fold) levels. These data suggest a mechanistic defect in the allosteric regulation of aldolase activity

Chemical characterization of cherry (Prunus avium) extract in comparison with commercial mimosa and chestnut tannins

In a growing context of green and circular economy, gaining knowledge of the composition of every crop is crucial, as this will allow for their full exploitation. Cherry (Prunus avium L.) is a widespread tree of particular interest for its fruits and its valuable timber. Its wood is rich in extractives and its characterization will allow to consider other applications for this feedstock. In this study, chipped cherry wood was extracted and chemically analysed to determine its total phenolic content, total condensed tannin, antioxidant capacity, and polysaccharide content through wet chemistry analysis. These investigations were coupled with C-13-NMR and FTIR spectrometry, with HPLC as well as elemental analysis to conduct a comprehensive chemical characterization. Thermogravimetric measurements were also taken to understand the behaviour of the extract when exposed to high temperature. The registered findings were benchmarked against commercial mimosa (Acacia mearnsii De Wild.) and chestnut (Castanea sativa Mill.) tannins which were selected as template for condensed and hydrolysable tannins, respectively. Cherry extract was found to be the poorest in phenolics which are mainly constituted of pyrogallic flavonoids strongly interconnected with significant amounts of polysaccharides