Mammalian Diaphanous1 signalling in neurovascular complications of diabetes.

Over the past few decades, diabetes gradually has become one of the top non-communicable disorders, affecting 476.0 million in 2017 and is predicted to reach 570.9 million people in 2025. It is estimated that 70 to 100% of all diabetic patients will develop some if not all, diabetic complications over the course of the disease. Despite different symptoms, mechanisms underlying the development of diabetic complications are similar, likely stemming from deficits in both neuronal and vascular components supplying hyperglycaemia-susceptible tissues and organs. Diaph1, protein diaphanous homolog 1, although mainly known for its regulatory role in structural modification of actin and related cytoskeleton proteins, in recent years attracted research attention as a cytoplasmic partner of the receptor of advanced glycation end-products (RAGE) a signal transduction receptor, whose activation triggers an increase in proinflammatory molecules, oxidative stressors and cytokines in diabetes and its related complications. Both Diaph1 and RAGE are also a part of the RhoA signalling cascade, playing a significant role in the development of neurovascular disturbances underlying diabetes-related complications. In this review, based on the existing knowledge as well as compelling findings from our past and present studies, we address the role of Diaph1 signalling in metabolic stress and neurovascular degeneration in diabetic complications. In light of the most recent developments in biochemical, genomic and transcriptomic research, we describe current theories on the aetiology of diabetes complications, highlighting the function of the Diaph1 signalling system and its role in diabetes pathophysiology.

In Vivo Regulation of Signal Transduction Pathways by Vitamin D Stabilizes Homeostasis of Human Immune Cells and Counteracts Molecular Stress.

Vitamin D3 is a pre-hormone that regulates hundreds of target genes and dozens of physiological functions, including calcium homeostasis and the activity of the immune system, via its metabolite 1,25-dihydroxyvitamin D3, which is a high-affinity ligand for the transcription factor vitamin D receptor. In this study, we took advantage of data from the VitDHiD vitamin D3 intervention trial (25 healthy individuals) indicating that 442 protein-coding genes were significantly (false discovery rate < 0.05) up- or downregulated in peripheral blood mononuclear cells one day after taking a vitamin D3 bolus. Since more than half of the encoded proteins had "signaling" assigned as a primary biological function, we evaluated their involvement in signal transduction cascades included in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database and found 88 of the vitamin D targets contributing to 16 different pathways. Eight of the pathways show an approximately even contribution of up- and downregulated genes, suggesting that the actions of vitamin D stabilize homeostasis of the physiological processes driven by the respective signaling cascades. Interestingly, vitamin D target genes involved in the signaling pathways of hypoxia-inducible factor 1 (HIF1), tumor necrosis factor (TNF), mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NFκB) are primarily downregulated. This supports the observation that the physiological role of vitamin D in healthy individuals is to tone down certain processes rather than activate them. In conclusion, under in vivo conditions, vitamin D either alleviates the homeostasis of immune cells in healthy individuals or counteracts molecular responses to oxygen deprivation (HIF1), microbe infection (TNF), growth stimulation (MAPKs) and inflammation (NFκB).

Novel insights into the nervous system affected by prolonged hyperglycemia.

Multiple molecular pathways including the receptor for advanced glycation end-products-diaphanous related formin 1 (RAGE-Diaph1) signaling are known to play a role in diabetic peripheral neuropathy (DPN). Evidence suggests that neuropathological alterations in type 1 diabetic spinal cord may occur at the same time as or following peripheral nerve abnormalities. We demonstrated that DPN was associated with perturbations of RAGE-Diaph1 signaling pathway in peripheral nerve accompanied by widespread spinal cord molecular changes. More than 500 differentially expressed genes (DEGs) belonging to multiple functional pathways were identified in diabetic spinal cord and of those the most enriched was RAGE-Diaph1 related PI3K-Akt pathway. Only seven of spinal cord DEGs overlapped with DEGs from type 1 diabetic sciatic nerve and only a single gene cathepsin E (CTSE) was common for both type 1 and type 2 diabetic mice. In silico analysis suggests that molecular changes in spinal cord may act synergistically with RAGE-Diaph1 signaling axis in the peripheral nerve. KEY MESSAGES: Molecular perturbations in spinal cord may be involved in the progression of diabetic peripheral neuropathy. Diabetic peripheral neuropathy was associated with perturbations of RAGE-Diaph1 signaling pathway in peripheral nerve accompanied by widespread spinal cord molecular changes. In silico analysis revealed that PI3K-Akt signaling axis related to RAGE-Diaph1 was the most enriched biological pathway in diabetic spinal cord. Cathepsin E may be the target molecular hub for intervention against diabetic peripheral neuropathy.

Increasing energy expenditure through exercise and low ambient temperature offers oxidative protection to the hypothalamus after high-fat feeding to mice.

The effects of weight loss produced by increased energy expenditure on measures of oxidative stress and mitochondrial damage have not been investigated in the hypothalamus of diet-induced obese mice. The present study aimed to characterize the effects of either a low housing temperature of 17°C or daily exercise on a treadmill on high-fat diet (HFD)-induced abnormalities in the hypothalamic tissue of mice. Exercise and low ambient temperature protocols were designed to produce energy deficit through increased energy expenditure. Forty mice aged 8 weeks were assigned to one of four conditions: chow diet (n = 10), HFD (n = 10), HFD and 5 weeks of either exercise training (ET; n = 10) or an ambient temperature of 17°C (n = 10). Mice were killed at the age of 31 weeks. In comparison with HFD treatment alone, both interventions reduced body adiposity (14.6% and 27.6% reduction for the ET and 17°C groups, respectively). Moreover, exposing obese mice to ET and 17°C restored mitochondrial DNA content (41.3% and 32.6% increase for the ET and 17°C groups, respectively), decreased level of lipid peroxidation as assessed by the detection of 4-hydroxy-nonenal protein adducts (12.8% and 29.4% reduction for the ET and 17°C groups, respectively) and normalized the expression levels of proinflammatory cytokines (Tnfα: 73.9% and 62%; Il1ß: 54.5% and 39.6%; Il6: 33.1% and 35.6% reduction for the ET and 17°C groups, respectively), as well as several proteins associated with mitochondrial respiratory chain (OxPhos Complex I: 75.7% and 53.9%; Complex III: 33% and 36%; Complex V: 42% and 36.9% reduction for the ET and 17°C groups, respectively) in hypothalamic cells. Negative energy balance induced through either lower ambient temperature or exercise resulted in substantial and similar improvements in markers of inflammation and mitochondrial damage in the hypothalamus of mice with diet-induced obesity, potentially by reducing oxidative stress.

Peripheral Neuropathy Presents Similar Symptoms and Pathological Changes in Both High-Fat Diet and Pharmacologically Induced Pre- and Diabetic Mouse Models.

The objective of the study was to compare the effects of experimentally induced type 1 or type 2 diabetes (T1D or T2D) on the functional, structural and biochemical properties of mouse peripheral nerves. Eight-week-old C57BL/6 mice were randomly assigned into three groups, including the control (CTRL, chow-fed), STZ (streptozotocin (STZ)-injected), and HFD (high-fat diet (HFD)-fed) group. After 18-weeks of experimental treatment, HFD mice had higher body weights and elevated levels of plasma lipids, while STZ mice developed hyperglycemia. STZ-treated mice, after an extended period of untreated diabetes, developed motor and sensory nerve conduction-velocity deficits. Moreover, relative to control fibers, pre- and diabetic axons were lower in number and irregular in shape. Animals from both treatment groups manifested a pronounced overexpression of nNOS and a reduced expression of SOD1 proteins in the sciatic nerve, indicating oxidative-nitrosative stress and ineffective antioxidant protection in the peripheral nervous system of these mice. Collectively, STZ- and HFD-treated mice revealed similar characteristics of peripheral nerve damage, including a number of morphological and electrophysiological pathologies in the sciatic nerve. While hyperglycemia is a large component of diabetic neuropathy pathogenesis, the non-hyperglycemic effects of diabetes, including dyslipidemia, may also be of importance in the development of this condition.

Isolation and identification of endogenous RFamide-related peptides 1 and 3 in the mouse hypothalamus.

Although the RFamide-related peptide (RFRP) preproprotein sequence is known in mice, until now, the molecular structure of the mature, functional peptides processed from the target precursor molecule has not been determined. In the present study, we purified endogenous RFRP1 and RFRP3 peptides from mouse hypothalamic tissue extracts using an immunoaffinity column conjugated with specific antibodies against the mouse C-terminus of RFRP-1 and RFRP-3. Employing liquid chromatography coupled with mass spectrometry, we demonstrated that RFRP1 consists of 15 amino acid residues and RFRP3 consists of 10 amino acid residues (ANKVPHSAANLPLRF-NH2 and SHFPSLPQRF-NH2, respectively). To investigate the distribution of RFRPs in the mouse central nervous system, we performed immunohistochemical staining of the brain sections collected from wild-type and Rfrp knockout animals. These data, together with gene expression in multiple tissues, provide strong confidence that RFRP-immunoreactive neuronal cells are localised in the dorsomedial hypothalamic nucleus (DMH) and between the DMH and the ventromedial hypothalamic nuclei. The identification of RFRP1 and RFRP3 peptides and immunohistochemical visualisation of targeting RFRPs neurones in the mice brain provide the basis for further investigations of the functional biology of RFRPs.

Mest and Sfrp5 are biomarkers for healthy adipose tissue.

Obesity depends on a close interplay between genetic and environmental factors. However, it is unknown how these factors interact to cause changes in the obese condition during the progression of obesity from the neonatal to the aged individual. We have utilized Mest and Sfrp5 genes, two genes highly correlated with adipose tissue expansion in diet-induced obesity, to characterize the obese condition during development of 2 genetic models of obesity. A model for the early onset of obesity was presented by leptin-deficient mice (ob/ob), whereas late onset of obesity was induced with high-fat diet (HFD) consumption in C57BL/6J mice with inherent risk of obesity (DIO). We correlated obese and diabetic phenotypes with Mest and Sfrp5 gene expression profiles in subcutaneous fat during pre-weaning, pre-adulthood and adulthood. A rapid development of obesity began in ob/ob mice immediately after weaning at 21 days of age, whereas the obesity of DIO mice was not evident until after 2 months of age. Even after 5 months of HFD treatment, the adiposity index of DIO mice was lower than in ob/ob mice at 2 months of age. In both obesity models, the expression of Mest and Sfrp5 genes increased in parallel with fat mass expansion; however, gene expression proceeded to decrease when the adiposity reached a plateau. The reduction in the expression of genes of caveolae structure and glucose metabolism were also suppressed in the aging adipose tissue. The analysis of fat mass and adipocyte size suggests that reduction in Mest and Sfrp5 is more sensitive to the age of the fat than its morphology. The balance of factors controlling fat deposition can be evaluated in part by the differential expression profiles of Mest and Sfrp5 genes with functions linked to fat deposition as long as there is an active accumulation of fat mass.

Npvf: Hypothalamic Biomarker of Ambient Temperature Independent of Nutritional Status.

The mechanism by which mice, exposed to the cold, mobilize endogenous or exogenous fuel sources for heat production is unknown. To address this issue we carried out experiments using 3 models of obesity in mice: C57BL/6J+/+ (wild-type B6) mice with variable susceptibility to obesity in response to being fed a high-fat diet (HFD), B6. Ucp1-/- mice with variable diet-induced obesity (DIO) and a deficiency in brown fat thermogenesis and B6. Lep-/- with defects in thermogenesis, fat mobilization and hyperphagia. Mice were exposed to the cold and monitored for changes in food intake and body composition to determine their energy balance phenotype. Upon cold exposure wild-type B6 and Ucp1-/- mice with diet-induced obesity burned endogenous fat in direct proportion to their fat reserves and changes in food intake were inversely related to fat mass, whereas leptin-deficient and lean wild-type B6 mice fed a chow diet depended on increased food intake to fuel thermogenesis. Analysis of gene expression in the hypothalamus to uncover a central regulatory mechanism revealed suppression of the Npvf gene in a manner that depends on the reduced ambient temperature and degree of exposure to the cold, but not on adiposity, leptin levels, food intake or functional brown fat.

Effect of dietary supplementation with unprocessed and ethanol-extracted apple pomaces on caecal fermentation, antioxidant and blood biomarkers in rats.

The present 4-week study on growing Wistar rats was aimed at assessing the potential advantages of dietary supplementation with apple pomace containing both fibre and polyphenols, which enables nutritional exploitation of the physiological traits of both compounds. A total of twenty-four rats, assigned to the control (C), group fed with a diet supplemented with 14% of processed apple pomace (A) and group fed with a diet supplemented with 15% unprocessed apple pomace (AP) groups, were fed the following iso-fibrous diets: control, ethanol-extracted and unprocessed apple pomaces (low and high level of dietary polyphenols, i.e. 0·002 and 0·018%, respectively). To measure the animal response, parameters describing the caecal fermentation, antioxidative status and lipoprotein profile of rats were assessed. Both dietary apple pomaces were found to significantly (P≤0·05) decrease caecal pH and ammonia concentration, microbial ß-glucuronidase activity as well as to increase caecal SCFA concentration in comparison to the control diet. The unprocessed pomace did not suppress caecal fermentation. Unlike the extracted one, the dietary apple pomace rich in polyphenols significantly (P≤0·05 v. C group) increased erythrocyte superoxide dismutase activity and serum antioxidant capacity of lipid-soluble substances and significantly (P≤0·05 v. C group) decreased amounts of thiobarbituric acid-reactive substances in liver tissue. Moreover, the 4-week administration of the AP diet to rats evoked a significant decrease in serum glucose concentration (P≤0·05 v. C and A groups). In conclusion, the results demonstrated that the polyphenol-rich fibre complexes from apple pomace exerted positive effects on gastrointestinal physiology and antioxidant status of rats.

Polyphenol-rich strawberry pomace reduces serum and liver lipids and alters gastrointestinal metabolite formation in fructose-fed rats.

This study compared the effects of supplementation with a polyphenol-rich pomace from strawberry (US) and a strawberry pomace without most of these compounds (PS) on gastrointestinal, blood, and tissue biomarkers in rats fed diets differing in carbohydrate contents for 4 wk. The diets were: corn starch (group CS), high fructose (60% by weight; group F), starch with 7.7% of either US or PS (groups CS+US and CS+PS, respectively), and high fructose with 7.7% of either US or PS (groups F+US and F+PS, respectively). An interaction (P < 0.05) was observed between diet type and strawberry preparation, showing that upon fructose feeding, US had a greater effect than PS on lowering serum insulin, liver total cholesterol, and conjugated dienes. Additionally, the F+US group had lower serum FFA than the F+PS group (P < 0.05). The extraction of polyphenols diminished the physiological effect associated with strawberry intake, suggesting that the fiber component of the pomace was also active in reducing metabolic complications following fructose feeding to rats.

Effect of buckwheat sprouts and groats on the antioxidant potential of blood and caecal parameters in rats.

The aim of this study was to investigate whether dietary buckwheat sprouts, cultivated without or with light exposure, exerted different effects on the lower gut, antioxidant status, and lipoprotein profile in rats. For 4 weeks, rats were given a diet containing 30 % expanded buckwheat seeds or 5 % buckwheat sprouts, cultivated with or without light exposure. Buckwheat sprouts that were cultivated under light and dark conditions, and expanded seeds, differed in the levels of total phenolic compounds and Trolox Equivalent Antioxidant Capacity values. All buckwheat products added to the diets decreased pH value and dry matter concentration in the caecal digesta compared with the control group. As compared to the control group, the addition of buckwheat products caused a higher glycolytic activity and the production of short-chain fatty acids in the caecum of the rats. There were no significant differences in the plasma concentrations of glucose, total cholesterol, nor triglycerides between the groups. The concentration of high-density lipoprotein (HDL)-cholesterol was significantly higher, and the atherogenic index of plasma was lower in rats administered buckwheat sprouts cultivated in the light, compared to the group fed sprouts cultivated in the dark. Total plasma antioxidant status, activity of glutathione peroxidase, and superoxide dismutase in whole blood were not affected by dietary treatment. Results of the experiment indicated enhanced benefits of dietary supplementation with buckwheat sprouts cultivated under light, especially in relation to the serum lipoprotein profile.