Direct and carry-over effect of grassland herbage allowance on metabolic hormones and reproduction in primiparous beef cows undergoing temporary weaning and flushing.

Grazing management significantly contributes to low beef production in cow-calf systems within the Rio de la Plata native grasslands. An herbage allowance (HA) of 4 kg DM/kg BW increased the productive response of primiparous cows grazing shallow soils compared to 2.5. However, the impact of HA on metabolic changes and its association with productive response were not studied. We studied two levels of native grassland HA from -150 days relative to calving (DC) to weaning (195 DC) in spring-calving primiparous beef cows undergoing temporary weaning (TW) and flushing at 86 ±â€¯12 DC on herbage intake (HI), body condition score (BCS), BW, milk yield, calf weight, concentrations of metabolic hormones, and the probability of ovulation and pregnancy. Thirty-one heifers were assigned to HA treatments that fluctuated throughout seasons: autumn (-150 to -90 DC) at 5 and 3 kg DM/kg BW, winter (-90 to 0 DC) at 3 and 3 kg DM/kg BW, and spring-summer (0 to 195 DC) at 4 and 2 kg DM/kg BW for High and Low HA, respectively. Data were analysed using linear models and generalised linear models for continuous and categorical variables, respectively. During the autumn period, HI, insulin, IGF-I, BCS, and BW were higher in High HA than Low, despite small differences in herbage mass between HA. Throughout the winter, spring, and summer, HI, insulin, leptin, and BCS changes did not differ between HA. However, IGF-I concentrations were greater at -65 and -40 DC (84 vs 55 ±â€¯8.6 ng/mL; P < 0.05) and tended to be greater after TW in High HA than Low. The probability of ovulation did not reach significance (0.94 vs 0.75 ±â€¯0.11 for High and Low HA, respectively; P = 0.125), while the probability of pregnancy was greater in High HA than in Low HA (0.9 vs 0.61 ±â€¯0.10; P = 0.07). Ovulation probability exhibited a positive association with IGF-I concentrations at -90 and -40 DC (P < 0.05), but not postpartum. Milk yield did not differ between treatments, while calf weight was heavier at weaning in High HA cows (194 vs 178 ±â€¯3.3 kg; P < 0.05). High HA enhances autumn HI and BCS and generates a carry-over effect on IGF-I concentrations throughout winter and after TW ("metabolic memory"), explaining the better reproductive response. Moderate changes in cows' nutrition during autumn contribute to changes in metabolic status and reproductive outcomes in primiparous cows grazing moderate herbage production native grasslands.

The role of metabolic memory in diabetic kidney disease: identification of key genes and therapeutic targets.

Diabetic kidney disease (DKD) is characterized by complex pathogenesis and poor prognosis; therefore, an exploration of novel etiological factors may be beneficial. Despite glycemic control, the persistence of transient hyperglycemia still induces vascular complications due to metabolic memory. However, its contribution to DKD remains unclear. Using single-cell RNA sequencing data from the Gene Expression Omnibus (GEO) database, we clustered 12 cell types and employed enrichment analysis and a cell‒cell communication network. Fibrosis, a characteristic of DKD, was found to be associated with metabolic memory. To further identify genes related to metabolic memory and fibrosis in DKD, we combined the above datasets from humans with a rat renal fibrosis model and mouse models of metabolic memory. After overlapping, NDRG1, NR4A1, KCNC4 and ZFP36 were selected. Pharmacology analysis and molecular docking revealed that pioglitazone and resveratrol were possible agents affecting these hub genes. Based on the ex vivo results, NDRG1 was selected for further study. Knockdown of NDRG1 reduced TGF-ß expression in human kidney-2 cells (HK-2 cells). Compared to that in patients who had diabetes for more than 10 years but not DKD, NDRG1 expression in blood samples was upregulated in DKD patients. In summary, NDRG1 is a key gene involved in regulating fibrosis in DKD from a metabolic memory perspective. Bioinformatics analysis combined with experimental validation provided reliable evidence for identifying metabolic memory in DKD patients.

Update: the role of epigenetics in the metabolic memory of diabetic complications.

Diabetes, a chronic disease characterized by hyperglycemia, is associated with significantly accelerated complications, including diabetic kidney disease (DKD), which increases morbidity and mortality. Hyperglycemia and other diabetes-related environmental factors such as overnutrition, sedentary lifestyles, and hyperlipidemia can induce epigenetic changes. Working alone or with genetic factors, these epigenetic changes that occur without alterations in the underlying DNA sequence, can alter the expression of pathophysiological genes and impair functions of associated target cells/organs, leading to diabetic complications like DKD. Notably, some hyperglycemia-induced epigenetic changes persist in target cells/tissues even after glucose normalization, leading to sustained complications despite glycemic control, so-called metabolic memory. Emerging evidence from in vitro and in vivo animal models and clinical trials with subjects with diabetes identified clear associations between metabolic memory and epigenetic changes including DNA methylation, histone modifications, chromatin structure, and noncoding RNAs at key loci. Targeting such persistent epigenetic changes and/or molecules regulated by them can serve as valuable opportunities to attenuate, or erase metabolic memory, which is crucial to prevent complication progression. Here, we review these cell/tissue-specific epigenetic changes identified to-date as related to diabetic complications, especially DKD, and the current status on targeting epigenetics to tackle metabolic memory. We also discuss limitations in current studies, including the need for more (epi)genome-wide studies, integrative analysis using multiple epigenetic marks and Omics datasets, and mechanistic evaluation of metabolic memory. Considering the tremendous technological advances in epigenomics, genetics, sequencing, and availability of genomic datasets from clinical cohorts, this field is likely to see considerable progress in the upcoming years.

Regulation role of miR-204 on SIRT1/VEGF in metabolic memory induced by high glucose in human retinal pigment epithelial cells.

AIM: To examine the regulatory role of microRNA-204 (miR-204) on silent information regulator 1 (SIRT1) and vascular endothelial growth factor (VEGF) under high-glucose-induced metabolic memory in human retinal pigment epithelial (hRPE) cells. METHODS: Cells were cultured with either normal (5 mmol/L) or high D-glucose (25 mmol/L) concentrations for 8d to establish control and high-glucose groups, respectively. To induce metabolic memory, cells were cultured with 25 mmol/L D-glucose for 4d followed by culture with 5 mmol/L D-glucose for 4d. In addition, exposed in 25 mmol/L D-glucose for 4d and then transfected with 100 nmol/L miR-204 control, miR-204 inhibitor or miR-204 mimic in 5 mmol/L D-glucose for 4d. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect miR-204 mRNA levels. SIRT1 and VEGF protein levels were assessed by immunohistochemical and Western blot. Flow cytometry was used to investigate apoptosis rate. RESULTS: It was found that high glucose promoted miR-204 and VEGF expression, and inhibited SIRT1 activity, even after the return to normal glucose culture conditions. Upregulation of miR-204 promoted apoptosis inhibiting SIRT1 and increasing VEGF expression. However, downregulation of miR-204 produced the opposite effects. CONCLUSION: The study identifies that miR-204 is the upstream target of SIRT1 and VEGF, and that miR-204 can protect hRPE cells from the damage caused by metabolic memory through increasing SIRT1 and inhibiting VEGF expression.

Screening of natural epigenetic modifiers for managing glycemic memory and diabetic nephropathy.

Short hyperglycaemic episodes trigger metabolic memory (MM) in which managing hyperglycaemia alone is not enough to tackle the progression of Diabetic nephropathy on the epigenetic axis. We used a structural similarity search approach to identify phytochemicals similar to natural epigenetic modifiers and docked with SIRT1 protein and did ADME studies. We found that UMB was 84.3% similar to esculetin. Upon docking, we found that UMB had a binding energy of -9.2 kcal/mol while the standard ligand had -11.8 kcal/mol. ADME showed UMB to be a good lead. 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay showed it to be a good antioxidant with IC50 of 107 µg/mL and MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) showed that it does not promote cell death. Oxidative biomarkers in vitro showed UMB was able to ameliorate glycemic memory induced by high glucose. Western blot revealed decreased histone acetylation under hyperglycaemic conditions and upon treatment with UMB along with DR, its levels increased. This led us to check our hypothesis of whether concomitant diet reversal (DR) together with UMB can alleviate high-fat diet-induced metabolic memory and diabetic nephropathy (DN) in SD rats. UMB was able to decrease blood glucose, lipid, renal, and liver profile concluding UMB was able to ameliorate DN and MM by increasing the histone acetylation level.

An update on chronic complications of diabetes mellitus: from molecular mechanisms to therapeutic strategies with a focus on metabolic memory.

Diabetes mellitus, a chronic metabolic disease, often leads to numerous chronic complications, significantly contributing to global morbidity and mortality rates. High glucose levels trigger epigenetic modifications linked to pathophysiological processes like inflammation, immunity, oxidative stress, mitochondrial dysfunction, senescence and various kinds of cell death. Despite glycemic control, transient hyperglycemia can persistently harm organs, tissues, and cells, a latent effect termed "metabolic memory" that contributes to chronic diabetic complications. Understanding metabolic memory's mechanisms could offer a new approach to mitigating these complications. However, key molecules and networks underlying metabolic memory remain incompletely understood. This review traces the history of metabolic memory research, highlights its key features, discusses recent molecules involved in its mechanisms, and summarizes confirmed and potential therapeutic compounds. Additionally, we outline in vitro and in vivo models of metabolic memory. We hope this work will inform future research on metabolic memory's regulatory mechanisms and facilitate the development of effective therapeutic compounds to prevent diabetic complications.

The Impact of Metabolic Memory on Immune Profile in Young Patients with Uncomplicated Type 1 Diabetes.

Metabolic memory refers to the long-term effects of achieving early glycemic control and the adverse implications of high blood glucose levels, including the development and progression of diabetes complications. Our study aimed to investigate whether the phenomenon of metabolic memory plays a role in the immune profile of young patients with uncomplicated type 1 diabetes (T1D). The study group included 67 patients with uncomplicated type 1 diabetes with a mean age of 15.1 ± 2.3 years and a minimum disease duration of 1.2 years. The control group consisted of 27 healthy children and adolescents with a mean age of 15.1 ± 2.3 years. Patients were divided into three groups according to their HbA1c levels at the onset of T1D, and the average HbA1c levels after one and two years of disease duration. The subgroup A1 had the lowest initial HbA1c values, while the subgroup C had the highest initial HbA1c values. Cytokine levels (including TNF-α, IL-35, IL-4, IL-10, IL-18, and IL-12) were measured in all study participants. Our data analysis showed that subgroup A1 was characterized by significantly higher levels of IL-35 and IL-10 compared to all other groups, and significantly higher levels of IL-4 compared to group B. Additionally, a comparative analysis of cytokine levels between the groups of diabetic patients and healthy controls demonstrated that subgroup A1 had significantly higher levels of anti-inflammatory cytokines. The lipid profile was also significantly better in subgroup A1 compared to all other patient groups. Based on our findings, it appears that an inflammatory process, characterized by an imbalance between the pro- and anti-inflammatory cytokines, is associated with poor glycemic control at the onset of diabetes and during the first year of disease duration. These findings also suggest that both metabolic memory and inflammation contribute to the abnormal lipid profile in patients with type 1 diabetes.

Donor fat-to-muscle ratio and kidney transplant outcomes: A proposition of metabolic memory.

AIMS: The impact of donor abdominal fat-to-muscle ratio (FMR) on kidney transplant (KT) outcomes was assessed. Given the transient nature of the donor's metabolic environment in transplant recipients, this study investigated the capacity of body composition to induce metabolic memory effects. MATERIALS AND METHODS: KT patients (n = 895) who received allografts from living donors (2003-2013) were included. Donor fat and muscle were quantified using pre-KT abdominal computed tomography scans. Patients were categorised into donor FMR tertiles and followed up for graft outcomes. Additionally, genome-wide DNA methylation analysis was performed on 28 kidney graft samples from KT patients in the low- and high-FMR groups. RESULTS: Mean recipient age was 42.9 ± 11.4 years and 60.9% were males. Donor FMR averaged 1.67 ± 0.79. Over a median of 120.9 ± 42.5 months, graft failure (n = 127) and death-censored graft failure (n = 109) were more frequent in the higher FMR tertiles. Adjusted hazard ratios for the highest versus lowest FMR tertile were 1.71 (95% CI, 1.06-2.75) for overall graft failure and 1.90 (95% CI, 1.13-3.20) for death-censored graft failure. Genome-wide DNA methylation analysis identified 58 differentially methylated regions (p < 0.05, |Δß| > 0.2) and 35 genes showed differential methylation between the high- (FMR >1.91) and low-FMR (FMR <1.27) groups. CONCLUSIONS: Donors with increased fat and reduced muscle composition may negatively impact kidney allograft survival in recipients, possibly through the transmission of epigenetic changes, implying a body-composition-related metabolic memory effect.

Impaired Removal of the Damaged Mitochondria in the Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy.

Retinopathy fails to halt even after diabetic patients in poor glycemic control try to institute tight glycemic control, suggesting a "metabolic memory" phenomenon, and the experimental models have demonstrated that mitochondria continue to be damaged/dysfunctional, fueling into the vicious cycle of free radicals. Our aim was to investigate the role of removal of the damaged mitochondria in the metabolic memory. Using human retinal endothelial cells (HRECs), incubated in 20 mM D-glucose for 4 days, followed by 5 mM D-glucose for 4 additional days, mitochondrial turnover, formation of mitophagosome, and mitophagy flux were evaluated. Mitophagy was confirmed in a rat model of metabolic memory where the rats were kept in poor glycemic control (blood glucose ~ 400 mg/dl) for 3 months soon after induction of streptozotocin-induced diabetes, followed by 3 additional months of good control (BG < 150 mg/dl). Reversal of high glucose by normal glucose had no effect on mitochondrial turnover and mitophagosome formation, and mitophagy flux remained compromised. Similarly, 3 months of good glycemic control in rats, which had followed 3 months of poor glycemic control, had no effect on mitophagy flux. Thus, poor turnover/removal of the damaged mitochondria, initiated during poor glycemic control, does not benefit from the termination of hyperglycemic insult, and the damaged mitochondria continue to produce free radicals, suggesting the importance of mitophagy in the metabolic memory phenomenon associated with the continued progression of diabetic retinopathy.

Construction and effect of a high glucose induced hippocampal neuron metabolic memory cell model in HT-22 mice / 天津医药

Objective To construct an in vitro"metabolic memory"cell model of HT-22 mouse hippocampal neurons induced by high glucose,and to investigate the effect of"metabolic memory"on apoptosis and histone acetylation in HT-22 cells.Methods HT-22 cells were cultured in high glucose medium(glucose concentration was 55 mmol/L)and conventional glucose medium(glucose concentration was 25 mmol/L),and cells were divided into the control group(NG 4,6 and 8 groups,25 mmol/L glucose was cultured for 4,6 and 8 days,respectively),the high glucose group(HG 4,6 and 8 groups,respectively)and the metabolic memory group(HG2NG2,HG2NG4,HG2NG6,HG4NG2 and HG4NG4 groups,high glucose culture for 2 days to 25 mmol/L glucose culture for 2,4 or 6 days,high glucose culture for 4 days to 25 mmol/L glucose culture for 2 or 4 days).Cell viability was detected by CCK-8 method.The release of lactate dehydrogenase(LDH)in cell culture supernatant was detected,and the optimal time to establish a"metabolic memory"model was selected.Subsequently,cells were divided into the NG4 group,the NG8 group,the HG4 group,the HG4NG4 group and the HG8 group,and the cell morphology of each group was observed by optical microscope.The apoptosis rate was detected by flow cytometry.The activities of deacetylase(HDAC)and histone acetyltransferase(HAT)were detected by enzyme-linked immunosorbent assay(ELISA).Western blot assay was used to detect expression levels of histone deacetylase 4(HDAC4),B lymphocyte tumor 2(Bcl-2),Bcl-2 related X protein(Bax)and Caspase-3 protein.Results The HG4NG4 group was the ideal cell model with high glucose metabolic memory.Cells of the NG4 group and the NG8 group were interwoven into a dense network,growing well,with spindle shaped cells and distinct synaptic structures.However,in the HG4 group and the HG8 group,the cell body became round,synaptic structure disappeared and growth was inhibited.In the HG4NG4 group,the number of cells increased but their morphology was damaged.Results of flow cytometry showed that compared with the NG8 group,the apoptosis rates were significantly increased in the HG8 group and the HG4NG4 group(P＜0.05).ELISA results showed that compared with the NG8 group,the expression levels of HDAC4,Bax,and Caspase-3 proteins increased in the HG8 group and the HG4NG4 group,while the expression level of Bcl-2 protein significantly decreased(P＜0.05).Compared with the HG8 group,there were no significant differences in protein expression levels of HAT and HDAC in the HG4NG4 group.Western blot reslts showed that compared with the NG8 group,the levels of HDAC4,Bax and Caspase-3 protein increased in the HG8 group and the HG4NG4 group(P＜0.05).Compared with the HG8 group,there were no significant differences in protein expression levels in the HG4NG4 group.Conclusion HT-22 mouse hippocampal neurons cultured with 55mmol/L high glucose for 4 days,and then cultured with 25 mmol/L glucose for 4 days are the ideal"metabolic memory"cell model.The mechanism may be related to the increased activity of HDAC,HAT and HDAC4 expression in the hyperglycemic model.

The legacy effect of hyperglycemia and early use of SGLT-2 inhibitors: a cohort study with newly-diagnosed people with type 2 diabetes.

Background: A delay in reaching HbA1c targets in patients with newly-diagnosed type 2 diabetes (T2D) is associated with an increased long-term risk of developing cardiovascular diseases (CVD), a phenomenon referred to as legacy effect. Whether an early introduction of glucose-lowering drugs with proven benefit on CVD can attenuate this phenomenon is unknown. Methods: Using data derived from a large Italian clinical registry, i.e. the AMD Annals, we identified 251,339 subjects with newly-diagnosed T2D and without CVD at baseline. Through Cox regressions adjusted for multiple risk factors, we examined the association between having a mean HbA1c between 7.1 and 8% or >8%, compared with ≤7%, for various periods of early exposure (0-1, 0-2, 0-3 years) and the development of later (mean subsequent follow-up 4.6 ± 2.9 years) CVD, evaluated as a composite of myocardial infarction, stroke, coronary or peripheral revascularization, and coronary or peripheral bypass. We performed this analysis in the overall cohort and then splitting the population in two groups of patients: those that introduced sodium-glucose transport protein 2 inhibitors (SGLT-2i) during the exposure phase and those not treated with these drugs. Findings: Considering the whole cohort, subjects with both a mean HbA1c between 7.1 and 8% and >8%, compared with patients attaining a mean HbA1c ≤ 7%, showed an increased risk of developing the outcome in all the three early exposure periods assessed, with the highest risk observed in patients with mean HbA1c > 8% in the 3 years exposure period (hazard ratio [HR]1.33; 95% confidence interval [CI] 1.063-1.365). The introduction of SGLT-2i during the exposure periods of 0-1 and 0-2 years eliminated the association between poor glycemic control and the outcome (p for interaction 0.006 and 0.003, respectively, vs. patients with the same degree of glycemic control but not treated with these drugs). Interpretation: Among patients with newly diagnosed T2D and free of CVD at baseline, a poor glycemic control in the first three years after diagnosis is associated with an increased subsequent risk of CVD. This association is no longer evident when SGLT-2i are introduced in the first two years, suggesting that these drugs attenuate the phenomenon of legacy effect. An early treatment with these drugs might thus promote a long-lasting benefit in patients not attaining proper glycemic control after T2D diagnosis. Funding: This work was supported, in part, by the Italian Ministry of Health (Ricerca Corrente) to IRCCS MultiMedica.

A Narrative Review of Signaling Pathway and Treatment Options for Diabetic Nephropathy.

BACKGROUND: Diabetic nephropathy is a progressive kidney disease that frequently results in end-stage renal disorders and is characterized by proteinuria, albuminuria, decreased filtration, and renal fibrosis. Despite the fact that there are a number of therapeutic alternatives available, DN continues to be the main contributor to end-stage renal disease. Therefore, significant innovation is required to enhance outcomes in DN patients. MATERIAL AND METHODS: Information was collected from online search engines like, Google Scholar, Web of Science, PubMed, Scopus, and Sci-Hub databases using keywords like diabetes, nephropathy, kidney disease, autophagy, etc. Result: Natural compounds have anti-inflammatory and antioxidant properties and impact various signaling pathways. They ameliorate kidney damage by decreasing oxidative stress, inflammatory process, and fibrosis and enhance the antioxidant system, most likely by activating and deactivating several signaling pathways. This review focuses on the role of metabolic memory and various signaling pathways involved in the pathogenesis of DN and therapeutic approaches available for the management of DN. Special attention is given to the various pathways modulated by the phytoconstituents.

Epigenetics of the Pathogenesis and Complications of Type 2 Diabetes Mellitus.

Epigenetics of type 2 diabetes mellitus (T2DM) has widened our knowledge of various aspects of the disease. The aim of this review is to summarize the important epigenetic changes implicated in the disease risks, pathogenesis, complications and the evolution of therapeutics in our current understanding of T2DM. Studies published in the past 15 years, from 2007 to 2022, from three primary platforms namely PubMed, Google Scholar and Science Direct were included. Studies were searched using the primary term 'type 2 diabetes and epigenetics' with additional terms such as 'risks', 'pathogenesis', 'complications of diabetes' and 'therapeutics'. Epigenetics plays an important role in the transmission of T2DM from one generation to another. Epigenetic changes are also implicated in the two basic pathogenic components of T2DM, namely insulin resistance and impaired insulin secretion. Hyperglycaemia-i nduced permanent epigenetic modifications of the expression of DNA are responsible for the phenomenon of metabolic memory. Epigenetics influences the development of micro-and macrovascular complications of T2DM. They can also be used as biomarkers in the prediction of these complications. Epigenetics has expanded our understanding of the action of existing drugs such as metformin, and has led to the development of newer targets to prevent vascular complications. Epigenetic changes are involved in almost all aspects of T2DM, from risks, pathogenesis and complications, to the development of newer therapeutic targets.

Metabolic memory in diabetic foot syndrome (DFS): MICRO-RNAS, single nucleotide polymorphisms (SNPs) frequency and their relationship with indices of endothelial function and adipo-inflammatory dysfunction.

BACKGROUND: Diabetic foot is a significant cause of morbidity in diabetic patients, with a rate that is approximately twice that of patients without foot ulcers. "Metabolic memory" represents the epigenetic changes induced by chronic hyperglycaemia, despite the correction of the glucose levels themselves. These epigenetic modifications appear to perpetuate the damage caused by persistently elevated glucose levels even in their absence, acting at various levels, mostly affecting the molecular processes of diabetic ulcer healing. METHODS: The aim of our cross-sectional study was to analyse a cohort of patients with diabetes with and without lower limb ulcers. We examined the effects of epigenetic changes on miRNA 126, 305, and 217 expression and the frequency of the SNPs of genes encoding inflammatory molecules (e.g., IL-6 and TNF-alpha) and their correlations with serum levels of proangiogenic molecules (e.g., ENOS, VEGF and HIF-1alpha) and several adipokines as well as with endothelial dysfunction, assessed noninvasively by reactive hyperaemia peripheral artery tonometry. Between March 2021 and June 2022, 110 patients were enrolled into the study: 50 diabetic patients with diabetic foot injuries, 40 diabetic patients without ulcerative complications and 20 nondiabetic patients as the control group. RESULTS: Diabetic subjects with lower limb ulcerative lesions exhibited higher levels of inflammatory cytokines, such as VEGF (191.40 ± 200 pg/mL vs. 98.27 ± 56.92 pg/mL vs. 71.01 ± 52.96 pg/mL; p = 0.22), HIF-1alpha (40.18 ± 10.80 ng/mL vs. 33.50 ± 6.16 ng/mL vs. 33.85 ± 6.84 ng/mL; p = 0.10), and Gremlin-1 (1.72 ± 0.512 ng/mL vs. 1.31 ± 0.21 ng/mL vs. 1.11 ± 0.19 ng/mL; p < 0.0005), than those without lower limb ulcers and healthy controls. Furthermore, we observed that miR-217-5p and miR-503-5p were 2.19-fold (p < 0.05) and 6.21-fold (p = 0.001) more highly expressed in diabetic foot patients than in healthy controls, respectively. Additionally, diabetic patients without lower limb ulcerative complications showed 2.41-fold (p = 0) and 2.24-fold (p = 0.029) higher expression of miR-217-5p and miR-503-5p, respectively, than healthy controls. Finally, diabetic patients with and without ulcerative complications of the lower limbs showed higher expression of the VEGFC2578A CC polymorphism (p = 0.001) and lower expression of the VEGFC2578A AC polymorphism (p < 0.005) than the healthy control population. We observed a significant increase in Gremlin-1 levels in patients with diabetic foot, suggesting that this inflammatory adipokine may serve as a predictive marker for the diagnosis of diabetic foot. CONCLUSIONS: Our results highlighted that patients with diabetic foot showed predominant expression of the VEGF C2578A CC polymorphism and reduced expression of the AC allele. Additionally, we found an overexpression of miR-217-5p and miR-503-5p in diabetic patients with and without diabetic foot syndrome compared with healthy controls. These results align with those reported in the literature, in which the overexpression of miR-217-5p and miR-503-5p in the context of diabetic foot is reported. The identification of these epigenetic modifications could therefore be helpful in the early diagnosis of diabetic foot and the treatment of risk factors. However, further studies are necessary to confirm this hypothesis.

Endogenous Biological Drivers in Diabetic Lower Limb Wounds Recurrence: Hypothetical Reflections.

An impaired healing response underlies diabetic foot wound chronicity, frequently translating to amputation, disability, and mortality. Diabetics suffer from underappreciated episodes of post-epithelization ulcer recurrence. Recurrence epidemiological data are alarmingly high, so the ulcer is considered in "remission" and not healed from the time it remains epithelialized. Recurrence may result from the combined effects of behavioral and endogenous biological factors. Although the damaging role of behavioral, clinical predisposing factors is undebatable, it still remains elusive in the identification of endogenous biological culprits that may prime the residual scar tissue for recurrence. Furthermore, the event of ulcer recurrence still waits for the identification of a molecular predictor. We propose that ulcer recurrence is deeply impinged by chronic hyperglycemia and its downstream biological effectors, which originate epigenetic drivers that enforce abnormal pathologic phenotypes to dermal fibroblasts and keratinocytes as memory cells. Hyperglycemia-derived cytotoxic reactants accumulate and modify dermal proteins, reduce scar tissue mechanical tolerance, and disrupt fibroblast-secretory activity. Accordingly, the combination of epigenetic and local and systemic cytotoxic signalers induce the onset of "at-risk phenotypes" such as premature skin cell aging, dysmetabolism, inflammatory, pro-degradative, and oxidative programs that may ultimately converge to scar cell demise. Post-epithelialization recurrence rate data are missing in clinical studies of reputed ulcer healing therapies during follow-up periods. Intra-ulcer infiltration of epidermal growth factor exhibits the most consistent remission data with the lowest recurrences during 12-month follow-up. Recurrence data should be regarded as a valuable clinical endpoint during the investigational period for each emergent healing candidate.

Mitochondrial Quality Control and Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy.

Diabetic retinopathy continues to progress even when hyperglycemia is terminated, suggesting a 'metabolic memory' phenomenon. Mitochondrial dysfunction is closely associated with the development of diabetic retinopathy, and mitochondria remain dysfunctional. Quality control of mitochondria requires a fine balance between mitochondrial fission-fusion, removal of the damaged mitochondria (mitophagy) and formation of new mitochondria (biogenesis). In diabetes, while mitochondrial fusion protein (Mfn2) is decreased, fission protein (Drp1) is increased, resulting in fragmented mitochondria. Re-institution of normal glycemia fails to reverse mitochondrial fragmentation, and dysfunctional mitochondria continue to accumulate. Our aim was to investigate the direct effect of regulation of the mitochondrial fusion process during normal glycemia that follows a high glucose insult on mitochondrial quality control in the 'metabolic memory' phenomenon. Human retinal endothelial cells, incubated in 20 mM glucose for four days, followed by 5 mM glucose for four additional days, with or without the Mfn2 activator leflunomide, were analyzed for mitochondrial fission (live cell imaging), mitophagy (flow cytometry and immunofluorescence microscopy), and mitochondrial mass (mitochondrial copy numbers and MitoTracker labeling). Mitochondrial health was determined by quantifying mitochondrial reactive oxygen species (ROS), respiration rate (Seahorse XF96) and mitochondrial DNA (mtDNA) damage. Addition of leflunomide during normal glucose exposure that followed high glucose prevented mitochondrial fission, facilitated mitophagy and increased mitochondrial mass. Glucose-induced decrease in mitochondrial respiration and increase in ROS and mtDNA damage were also prevented. Thus, direct regulation of mitochondrial dynamics can help maintain mitochondrial quality control and interfere with the metabolic memory phenomenon, preventing further progression of diabetic retinopathy.

Diabetic retinopathy and the role of Omega-3 PUFAs: A narrative review.

Diabetes mellitus has been a major cause of concern for the past few decades. As the number of diabetic patients increases, so too does the occurrence of its complications. Diabetic retinopathy (DR) is one of these and constitutes the most common cause of blindness amongst working-age individuals. Chronic exposure to a hyperglycaemic environment remains the driving force of a cascade of molecular events that disrupt the microvasculature of the retina and if left untreated can lead to blindness. In this review, we identify oxidative stress as a major implication in the pathway to the development of DR and speculate that it plays a central role especially in the early stages of the disease. Cells lose their antioxidant capacity under a hyperglycaemic state, free radicals are formed and eventually apoptosis ensues. The polyol pathway; advanced glycation end-product formation; the protein kinase C pathway, and the hexosamine pathway are found to contribute to the increase in oxidative stress observed in diabetic patients. We also investigate the use of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in DR. These molecules possess antioxidant and anti-inflammatory properties and have been previously investigated for use in other ocular pathologies with promising results. In this review we present the latest findings in pre-clinical and clinical studies for the use of ω-3 PUFAs in DR. We hypothesise that ω-3 PUFAs could be beneficial for DR in ways of reducing the oxidative stress and limiting the progression of the disease that threatens the eyesight of the patient, in conjunction with conventional therapy.

Profile analysis of differentially expressed long non­coding RNAs in metabolic memory induced by high glucose in human umbilical vein endothelial cells.

Numerous long non-coding RNAs (lncRNAs) are dysregulated in the hyperglycemia-induced phenomenon of metabolic memory (MM). In the present study, the significance of these lncRNAs in MM was explored by screening for MM-involved differentially expressed lncRNAs (MMDELs) in human umbilical vein endothelial cells (HUVECs) induced by high glucose. A total of nine HUVEC samples were divided into three groups to mimic conditions of low and high glucose environments, as well as induce the state of metabolic memory. The expression of lncRNAs was profiled using RNA sequencing. Bioinformatic analysis was performed using the Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes databases to explore the parental genes from which the lncRNAs are transcribed and target genes of the MMDELs and generate enrichment datasets. Reverse transcription-quantitative PCR was performed to validate the expression levels of the selected lncRNAs. The present study identified 308 upregulated and 157 downregulated MMDELs, which were enriched in numerous physiologic processes. Key functional enrichment terms included 'cell cycle', 'oocyte meiosis' and 'p53 signaling pathway'. In conclusion, certain MMDELs may regulate the expression level of highly associated mRNAs through various mechanisms and pathways, thereby interfering with several processes, such as the regulation of the cell cycle, and affecting vascular endothelial cell function. Furthermore, the disorders of these lncRNAs can be retained in MM, further investigation into the functions of these lncRNAs may result in novel insights and treatments, which could help control MM in patients with diabetes.

Mitochondrial and metabolic features of salugenesis and the healing cycle.

Pathogenesis and salugenesis are the first and second stages of the two-stage problem of disease production and health recovery. Salugenesis is the automatic, evolutionarily conserved, ontogenetic sequence of molecular, cellular, organ system, and behavioral changes that is used by living systems to heal. It is a whole-body process that begins with mitochondria and the cell. The stages of salugenesis define a circle that is energy- and resource-consuming, genetically programmed, and environmentally responsive. Energy and metabolic resources are provided by mitochondrial and metabolic transformations that drive the cell danger response (CDR) and create the three phases of the healing cycle: Phase 1-Inflammation, Phase 2-Proliferation, and Phase 3-Differentiation. Each phase requires a different mitochondrial phenotype. Without different mitochondria there can be no healing. The rise and fall of extracellular ATP (eATP) signaling is a key driver of the mitochondrial and metabolic reprogramming required to progress through the healing cycle. Sphingolipid and cholesterol-enriched membrane lipid rafts act as rheostats for tuning cellular sensitivity to purinergic signaling. Abnormal persistence of any phase of the CDR inhibits the healing cycle, creates dysfunctional cellular mosaics, causes the symptoms of chronic disease, and accelerates the process of aging. New research reframes the rising tide of chronic disease around the world as a systems problem caused by the combined action of pathogenic triggers and anthropogenic factors that interfere with the mitochondrial functions needed for healing. Once chronic pain, disability, or disease is established, salugenesis-based therapies will start where pathogenesis-based therapies end.