Microbiota analysis in individuals with type two diabetes mellitus and end­stage renal disease: A pilot study.

Chronic kidney disease (CKD) is a widespread health concern, which affects ~9.1% of the global population and 12-15% of individuals in upper-middle income countries. Notably, ~2% of patients with CKD progress to end-stage renal disease (ESRD), which leads to a substantial decline in the quality of life, an increased risk of mortality and significant financial burden. Patients with ESRD often still suffer from uremia and uremic syndromes, due to the accumulation of toxins between dialysis sessions and the inadequate removal of protein-bound toxins during dialysis. A number of these toxins are produced by the gut microbiota through the fermentation of dietary proteins or cholines. Furthermore, the gut microbial community serves a key role in maintaining metabolic and immune equilibrium in individuals. The present study aimed to investigate the gut microbiota patterns in individuals with type 2 diabetes mellitus (T2DM) and ESRD via quantitative PCR analysis of the 16S and 18S ribosomal RNA of selected members of the gut microbiota. Individuals affected by both T2DM and ESRD displayed distinctive features within their intestinal microbiota. Specifically, there were increased levels of Gammaproteobacteria observed in these patients, and all subjects exhibited a notably increased presence of Enterobacteriaceae compared with healthy individuals. This particular microbial community has established connections with the presence of inflammatory processes in the colon. Moreover, the elevated levels of Enterobacteriaceae may serve as an indicator of an imbalance in the intestinal microbiota, a condition known as dysbiosis. In addition, the Betaproteobacteria phylum was significantly more prevalent in the stool samples of patients with both T2DM and ESRD when compared with the control group. In conclusion, the present pilot study focused on gut microbiome alterations in T2DM and ESRD. Understanding the relationship between dysbiosis and CKD may identify new areas of research and therapeutic interventions aimed at modulating the gut microbiota to improve the health and outcomes of individuals with CKD and ESRD.

Impact of COVID-19 on the Microbiome and Inflammatory Status of Type 2 Diabetes Patients.

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) pandemic has advanced our understanding of the host-microbiome-virus interplay. Several studies in various geographical regions report that SARS-CoV-2 infection disrupts the intestinal microbiota, allowing pathogenic bacteria such as Enterobacteriaceae to thrive, and triggering more severe disease outcomes. Here, we profile the microbiota of 30 individuals, 15 healthy controls and 15 type 2 diabetes (T2D) patients, before and after coronavirus disease 2019 (COVID-19). Despite similar viral loads in both patients and controls, SARS-CoV-2 infection led to exacerbated microbiome changes in T2D patients, characterized by higher levels of Enterobacteriaceae, loss of butyrate producers and an enrichment in fungi such as Candida spp. and Aspergillus spp. Several members of the microbiota were associated with more severe clinical and inflammatory (IL-8 and IL-17) parameters. Future studies to delineate the connection between cytokine release and microbiota disturbances will enhance our understanding of whether these microbial shifts directly impact the cytokine storm in COVID-19 patients or whether they are consecutive to the critical disease.

Snapshot into the Type-2-Diabetes-Associated Microbiome of a Romanian Cohort.

The prevalence of type 2 diabetes mellitus (T2D) is alarmingly increasing worldwide, urgently calling for a better understanding of the underlying mechanisms in order to step up prevention and improve therapeutic approaches. It is becoming evident that the gut microbiota seem to have an endless capacity to impact T2D. In this study, we profile the gut microbiome patterns in T2D patients from Romania, by using quantitative Real-Time PCR and next generation sequencing. We enrolled a total of 150 individuals (105 T2D patients, 50 of them without metformin treatment and 45 healthy volunteers). The levels of potentially beneficial butyrate-producing bacteria were significantly reduced, while potentially pathogenic microorganisms such as Enterobacteriaceae and Fusobacterium were enriched in T2D patients. We evaluated the correlation between clinical parameters and gut microbiota and identified the genera Bacteroides, Alistipes, Dialister, Bilophila and Sutterella as possible detrimental factors in T2D. Our findings suggest that the gut microbiota may be a potential target in novel approaches to halt the development of T2D-associated complications.

Dysbiosis in the Development of Type I Diabetes and Associated Complications: From Mechanisms to Targeted Gut Microbes Manipulation Therapies.

Globally, we are facing a worrying increase in type 1 diabetes mellitus (T1DM) incidence, with onset at younger age shedding light on the need to better understand the mechanisms of disease and step-up prevention. Given its implication in immune system development and regulation of metabolism, there is no surprise that the gut microbiota is a possible culprit behind T1DM pathogenesis. Additionally, microbiota manipulation by probiotics, prebiotics, dietary factors and microbiota transplantation can all modulate early host-microbiota interactions by enabling beneficial microbes with protective potential for individuals with T1DM or at high risk of developing T1DM. In this review, we discuss the challenges and perspectives of translating microbiome data into clinical practice. Nevertheless, this progress will only be possible if we focus our interest on developing numerous longitudinal, multicenter, interventional and double-blind randomized clinical trials to confirm their efficacy and safety of these therapeutic approaches.

Evolution of Inflammatory and Oxidative Stress Markers in Romanian Obese Male Patients with Type 2 Diabetes Mellitus after Laparoscopic Sleeve Gastrectomy: One Year Follow-Up.

Geography is one of the key drivers of the significant variation in the etiopathogenic profile and prevalence of type 2 diabetes mellitus (T2DM) and obesity, therefore geographically based data are fundamental for implementing the appropriate interventions. Presently, the selection criteria of T2DM and obesity patients for laparoscopic sleeve gastrectomy (LSG) have not reached a worldwide consensus-highlighting the need for sharing experts' guidance in the preoperative evaluation, choice of the interventional procedure, perioperative management and patient long-term care. The aim of the current study was to evaluate the impact of LSG on T2DM (T2DM) remission in Romanian obese male patients, based on a multiparametric, prospective investigation. We have conducted a randomized controlled study on 41 obese male participants with the body mass index (BMI) ≥ 30 kg/m2, aged 30-65 years, which were randomly divided in two study groups: one receiving conventional treatment and the second undergoing LSG. The clinical and anthropometrical parameters, resting metabolic rate, general biochemical status, adipocytes profile, gastrointestinal hormones levels, proinflammatory, oxidant and antioxidant profiles were determined at three time points: V1 (baseline), V2 (after six months) and V3 (after 12 months). Glycated hemoglobin (HbA1c), blood glucose levels, BMI, weight, visceral fat level, HDL-cholesterol, incretin hormones, proinflammatory and the oxidative stress status were significantly improved in the LSG versus conventional treatment group. This is the first study reporting on the evaluation of metabolic surgery impact on Romanian obese male patients with T2DM. Our results confirm that LSG could contribute to T2DM remission in patients with diabesity, but this beneficial effect seems to be critically influenced by the duration of T2DM rather than by the obesity status. Our results show that, in addition to the parameters included in the prediction algorithm, the proinsulin levels, proinsulin/insulin ratio and the visceral fat percentage could bring added value to the assessment of metabolic status.

Mono-epoxy-tocotrienol-α enhances wound healing in diabetic mice and stimulates in vitro angiogenesis and cell migration.

Diabetes mellitus is characterized by hyperglycemia and capillary hypoxia that causes excessive production of free radicals and impaired antioxidant defense, resulting in oxidative stress and diabetes complications such as impaired wound healing. We have previously shown that modified forms of tocotrienols possess beneficial effects on the biosynthesis of the mevalonate pathway lipids including increase in mitochondrial CoQ. The aim of this study is to investigate the effects of mono-epoxy-tocotrienol-α on in vitro and in vivo wound healing models as well as its effects on mitochondrial function. Gene profiling analysis and gene expression studies on HepG2 cells and human dermal fibroblasts were performed by microarray and qPCR, respectively. In vitro wound healing using human fibroblasts was studied by scratch assay and in vitro angiogenesis using human dermal microvascular endothelial cells was studied by the tube formation assay. In vivo wound healing was performed in the diabetic db/db mouse model. For the study of mitochondrial functions and oxygen consumption rate Seahorse XF-24 was employed. In vitro, significant increase in wound closure and cell migration (p<0.05) both in normal and high glucose and in endothelial tube formation (angiogenesis) (p<0.005) were observed. Microarray profiling analysis showed a 20-fold increase of KIF26A gene expression and 11-fold decrease of lanosterol synthase expression. Expression analysis by qPCR showed significant increase of the growth factors VEGFA and PDGFB. The epoxidated compound induced a significantly higher basal and reserve mitochondrial capacity in both HDF and HepG2 cells. Additionally, in vivo wound healing in db/db mice, demonstrated a small but significant enhancement on wound healing upon local application of the compound compared to treatment with vehicle alone. Mono-epoxy-tocotrienol-α seems to possess beneficial effects on wound healing by increasing the expression of genes involved in cell growth, motility and angiogenes as well as on mitochondrial function.

The impact of basal insulin analogues on glucose variability in patients with type 2 diabetes undergoing renal replacement therapy for end-stage renal disease.

PURPOSE: We aimed to analyze the impact of basal insulin analogues on glucose variability (GV) in patients with type 2 diabetes (DM) undergoing renal replacement therapy. METHODS: Fourteen subjects on insulin therapy for at least 6 months (detemir, n = 7 vs. glargine, n = 7) were sequentially enrolled in this prospective study. Continuous glucose monitoring system (CGMS Gold, Dex Com 7+) was applied for 5 days, over 3 consecutive sessions of hemodialysis (HD). Various glycemic profiles (coefficient of variation-CV of mean glucose) were compared between the day on (HD-on) and the day off (HD-off) dialysis. The CV of at least 3 values of HbA1c (HPLC) since replacement therapy has been applied to assay the long-term GV. Endogenous insulin and insulin resistance (HOMA using fasting glucose and C-peptide levels), fasting lipid profile, quantitative C-reactive protein (CRP) and ferritin (values adjusted for Hb) were measured in serum at inclusion. RESULTS: The overnight HD-off and HD-on short-term (CV CGMS) GV, overall long-term (CV of HbA1c) GV, CRP and ferritin were reduced in subjects treated with detemir (paired t test, p = 0.0001, 0.0011, 0.036, <0.001, and <0.001 between groups). All participants were insulin-resistant (HOMA-IR > 3). CONCLUSIONS: Insulin-resistant patients with type 2 diabetes undergoing hemodialysis for end-stage renal disease on insulin detemir exhibit lower glycemic variability and pro-inflammatory profile than with insulin glargine.

L-arginine catabolism is driven mainly towards nitric oxide synthesis in the erythrocytes of patients with type 2 diabetes at first clinical onset.

BACKGROUND: We investigated the l-arginine (l-Arg)-nitric oxide (NO) metabolic pathway in the erythrocytes (RBCs) and plasma of subjects with type 2 diabetes at first clinical onset. METHODS: RBCs and plasma were collected from 26 patients with type 2 diabetes at first clinical onset and 19 age-matched non-diabetes subjects as controls. l-Arg content was assayed by capillary electrophoresis. We measured arginase activity and nitrate/nitrite concentrations by spectrophotometry, and glycosylated haemoglobin (HbA1c) by standardized immunoturbidimetry. RESULTS: We found that, when compared with controls, l-Arg content was similar in RBCs while decreased in the plasma of patients with type 2 diabetes. Interestingly, arginase activity was lower in RBCs and increased in plasma of patients with diabetes. NO production was higher in RBCs in patients with type 2 diabetes, while no difference was found in the plasma of our subjects. CONCLUSIONS: l-Arg catabolism is driven mainly towards NO synthesis in RBCs of patients with type 2 diabetes at first clinical onset. The decreased RBC arginase activity could be considered a potential mechanism of increased RBC NO production in early diabetes. Therefore, the RBC pool would represent a potentially compensatory intravascular compartment for endothelial dysfunction in diabetes.

Paraoxonase lactonase activity, inflammation and antioxidant status in plasma of patients with type 1 diabetes mellitus.

OBJECTIVES: To investigate paraoxonase-1 (PON1) lactonase activity, myeloperoxidase (MPO) activity (as a marker of inflammation) and antioxidant status in plasma of patients with type 1 diabetes mellitus. METHODS: Whole blood and plasma samples were collected from patients with diabetes and healthy control subjects. PON1 lactonase and MPO activities and total antioxidant capacity (TEAC) were determined in plasma. Glycosylated haemoglobin (HbA1c) was quantified in whole blood. RESULTS: Plasma PON1 lactonase and MPO activities were significantly higher and TEAC was significantly lower in patients with diabetes (n = 18) compared with healthy control subjects (n = 20). There were significant positive correlations between PON1 lactonase activity and MPO activity and HbA1c level, and plasma MPO and HbA1c. There were significant negative correlations between PON1 lactonase activity and TEAC, and MPO activity and TEAC. CONCLUSIONS: Increased lactonase activity may inefficiently compensate for the high level of chronic inflammation and low antioxidant capacity in the plasma of patients with type 1 diabetes mellitus.

Erythrocyte caspase-3 and antioxidant defense is activated in red blood cells and plasma of type 2 diabetes patients at first clinical onset.

OBJECTIVES: We studied erythrocyte (RBC) caspase-3 activity and oxidative status in plasma and RBCs of 33 patients with type 2 diabetes at first clinical onset and 23 age-matched non-diabetes control subjects. METHODS: Caspase-3 activity was assayed during the life span of RBCs; lipid peroxides and total antioxidant capacity (TEAC) were assessed in plasma and RBCs as indicators of oxidative stress and non-enzymatic antioxidant defense; and superoxide dismutase, catalase, and glutathione peroxidase activity were measured in RBCs as enzymatic antioxidants. RESULTS: We found that, compared to controls, RBCs caspase-3 is activated early in type 2 diabetes (P < 0.05); TEAC and malondialdehyde increased in plasma of patients with early diabetes, even when hypertension and macroangiopathy were present (P < 0.01); and RBCs TEAC, malondialdehyde (P < 0.01), superoxide dismutase, and glutathione peroxidase (P < 0.05) exhibited similar behavior in patients with diabetes and hypertensive patients with diabetes. DISCUSSION: Increased antioxidant defense in plasma and RBCs of early type 2 diabetes patients is a potential mechanism that can overcome oxidative damage induced by reactive oxygen species overproduction, and occurs even in RBCs with a decreased life span. This observation could provide a possible explanation for the controversial effects of antioxidant supplementation in diabetes patients.

Epigenetic DNA methylation in the promoters of the Igf1 receptor and insulin receptor genes in db/db mice.

We have investigated promoter methylation of the Insr, Igf1 and Igf1r genes in skeletal and cardiac muscles of normal and diabetic db/db mice. No differences in Insr promoter methylation were found in the heart and skeletal muscles and no methylation was detected in the Igf1 promoter in skeletal muscle. In skeletal muscle, db/db males exhibited a 7.4-fold increase in Igf1r promoter methylation, which was accompanied by a 1.8-fold decrease in Igf1r mRNA levels, compared with controls. More than 50% of the detected methylation events were concentrated within an 18 bp sequence that includes one of the Sp1 binding sites. We conclude that the methylation level and pattern of the Igf1r promoter in skeletal muscle is related to gender and the diabetic state.

Stability of mitochondrial DNA against reactive oxygen species (ROS) generated in diabetes.

BACKGROUND: Increased production of reactive oxygen species (ROS) in mitochondria has been proposed as the pathogenic mechanism for chronic complications of diabetes. Mitochondrial DNA (mtDNA) is more vulnerable to reactive oxygen species. However, there are few data on the mitochondrial DNA damage in diabetes and these are available only from patients with different duration of the disease and tissues not relevant to the chronic complications of diabetes. We therefore proposed to study the stability of mitochondrial DNA under controlled experimental conditions, to understand its contribution to chronic complications of diabetes. METHODS: The mitochondrial DNA damage was evaluated by long-fragment polymerase chain reaction in human dermal fibroblasts exposed to high glucose level and hypoxia (an additional source of reactive oxygen species) or in organs from diabetic animals (db/db mice) at different ages. Reactive oxygen species production was assessed in vitro by fluorescence and in vivo by nitrosylation of the proteins. The antioxidant enzymes were assessed by enzyme activity and by quantitative real-time polymerase chain reaction while the mitochondrial repair activity (base excision repair) was determined by using abasic site-containing oligonucleotides as substrates. RESULTS: Hyperglycaemia, when combined with hypoxia, is able to induce mitochondrial DNA damage in human dermal fibroblasts. The deleterious effect is mediated by mitochondrial reactive oxygen species, being abolished when the mitochondria electron transport is blocked. The accumulation of mitochondrial DNA damage in vivo is, however, decreased in 'old' diabetic animals (db/db) despite higher reactive oxygen species levels. This mitochondrial DNA protection might be conferred by an increased base excision repair activity. CONCLUSION: Increased base excision repair activity in tissues affected by the chronic complications of diabetes is a potential mechanism that can overcome mitochondrial DNA damage induced by hyperglycaemia-related reactive oxygen species overproduction.

Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice.

Relative hypoxia is essential in wound healing since it normally plays a pivotal role in regulation of all the critical processes involved in tissue repair. Hypoxia-inducible factor (HIF) 1alpha is the critical transcription factor that regulates adaptive responses to hypoxia. HIF-1alpha stability and function is regulated by oxygen-dependent soluble hydroxylases targeting critical proline and asparaginyl residues. Here we show that hyperglycemia complexly affects both HIF-1alpha stability and activation, resulting in suppression of expression of HIF-1 target genes essential for wound healing both in vitro and in vivo. However, by blocking HIF-1alpha hydroxylation through chemical inhibition, it is possible to reverse this negative effect of hyperglycemia and to improve the wound healing process (i.e., granulation, vascularization, epidermal regeneration, and recruitment of endothelial precursors). Local adenovirus-mediated transfer of two stable HIF constructs demonstrated that stabilization of HIF-1alpha is necessary and sufficient for promoting wound healing in a diabetic environment. Our findings outline the necessity to develop specific hydroxylase inhibitors as therapeutic agents for chronic diabetes wounds. In conclusion, we demonstrate that impaired regulation of HIF-1alpha is essential for the development of diabetic wounds, and we provide evidence that stabilization of HIF-1alpha is critical to reverse the pathological process.

Hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha are expressed in kaposi sarcoma and modulated by insulin-like growth factor-I.

PURPOSE: Neoangiogenesis is essential for tumor development. Hypoxia-inducible factor (HIF), a transcriptional factor composed of two subunits (alpha and beta), plays a key role in this process, activating proangiogenic factors such as vascular endothelial growth factor (VEGF). The HIF alpha subunits are critically regulated by oxygen and are also modulated by growth factors. Kaposi sarcoma (KS) is a highly vascular tumor that releases large amounts of VEGF and for which we have recently described an essential role for the insulin-like growth factor (IGF) system. We therefore investigated the expression of HIF alpha subunits in biopsies from KS tumors and their modulation by IGF-I in KSIMM, a KS cell line. RESULTS: Both HIF-1alpha and HIF-2alpha were expressed in KS biopsies in all tumoral stages. HIF-1alpha immunopositivity increased through the tumor development with highest expression in the late nodular stages. In KSIMM cells, IGF-I induced accumulation of both HIF alpha subunits. The induction suggests a translation mechanism as documented by cycloheximide chase experiment coupled with constant RNA levels as evaluated by quantitative real-time PCR. IGF-I-induced HIF alpha accumulation was followed by an increase in HIF function as assessed both by reporter gene assay and by induction of endogenous target gene expression (VEGF-A). Specific blockade of IGF-I receptor with alphaIR3 antibody or with picropodophyllin, a specific IGF-IR tyrosine kinase inhibitor, diminishes the basal and IGF-I-dependent induction of both HIF alpha congeners. CONCLUSION: These novel findings show the coupling between the IGF and HIF signaling in KS and suggest a coordinated contribution by these pathways to the characteristic vascular phenotype of this tumor.