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Objective: Markers reliably identifying vascular damage and risk in diabetic patients are rare, and reports on associations of serum adiponectin with macrovascular disease have been inconsistent. In contrast to existing data on serum adiponectin, this study assesses whether urinary adiponectin excretion might represent a more consistent vascular damage marker in type 2 diabetes.Research Design and Methods: Adiponectin distribution in human kidney biopsies was assessed by immunohistochemistry, and urinary adiponectin isoforms were characterized by Western blot analysis. Total urinary adiponectin excretion rate was measured in 156 patients with type 2 diabetes who had a history of diabetic nephropathy and 40 healthy control subjects using enzyme-linked immunosorbent assay. Atherosclerotic burden was assessed by common carotid artery intima-media-thickness (IMT).Results: A homogenous staining of adiponectin was found on the endothelial surface of glomerular capillaries and intrarenal arterioles in nondiabetic kidneys, whereas staining was decreased in diabetic nephropathy. Low-molecular adiponectin isoforms ( approximately 30-70 kDa) were detected in urine by Western blot analysis. Urinary adiponectin was significantly increased in type 2 diabetes (7.68 +/- 14.26 vs. control subjects: 2.91 +/- 3.85 microg/g creatinine, P = 0.008). Among type 2 diabetic patients, adiponectinuria was associated with IMT (r = 0.479, P < 0.001) and proved to be a powerful independent predictor of IMT (beta = 0.360, P < 0.001) in multivariable regression analyses. In a risk prediction model including variables of the UK Prospective Diabetes Study coronary heart disease risk engine urinary adiponectin, but not the albumin excretion rate, added significant value for the prediction of increased IMT (P = 0.007).Conclusions: Quantification of urinary adiponectin excretion appears to be an independent indicator of vascular damage potentially identifying an increased risk for vascular events. [ABSTRACT FROM AUTHOR]

Lesioned fascicles (LF) in the sciatic nerves of individuals with diabetic neuropathy (DN) correlate with clinical symptom severity. This study aimed to characterize the structural and molecular composition of these lesions to better understand DN pathogenesis. Sciatic nerves from amputees with and without type 2 diabetes (T2D) were examined using ex vivo magnetic resonance neurography, in vitro imaging, and proteomic analysis. Lesions were only found in T2D donors and exhibited significant structural abnormalities, including axonal degeneration, demyelination, and impaired blood nerve barrier (BNB). While non-lesioned fascicles from T2D donors showed activation of neuroprotective pathways, lesioned fascicles lacked this response and instead displayed increased complement activation via the classical pathway. The detection of liver-derived acute-phase proteins suggests that BNB disruption facilitates harmful inter-organ communication between the liver and nerves. These findings reveal key molecular mechanisms contributing to DN and highlight potential targets for therapeutic intervention., (© 2024 by the American Diabetes Association.)

Lifestyle intervention (LI) can prevent type 2 diabetes, but response to LI varies depending on risk subphenotypes. We tested whether individuals with prediabetes with low risk (LR) benefit from conventional LI and individuals with high risk (HR) benefit from an intensification of LI in a multicenter randomized controlled intervention over 12 months with 2 years' follow-up. A total of 1,105 individuals with prediabetes based on American Diabetes Association glucose criteria were stratified into an HR or LR phenotype based on previously described thresholds of insulin secretion, insulin sensitivity, and liver fat content. LR individuals were randomly assigned to conventional LI according to the Diabetes Prevention Program (DPP) protocol or control (1:1) and HR individuals to conventional or intensified LI with doubling of required exercise (1:1). A total of 908 (82%) participants completed the study. In HR individuals, the difference between conventional and intensified LI in postchallenge glucose change was -0.29 mmol/L [95% CI -0.54; -0.04], P = 0.025. Liver fat (-1.34 percentage points [95% CI -2.17; -0.50], P = 0.002) and cardiovascular risk (-1.82 percentage points [95% CI -3.13; -0.50], P = 0.007) underwent larger reductions with intensified than with conventional LI. During a follow-up of 3 years, intensified compared with conventional LI had a higher probability of normalizing glucose tolerance ( P = 0.008). In conclusion, it is possible in HR individuals with prediabetes to improve glycemic and cardiometabolic outcomes by intensification of LI. Individualized, risk phenotype-based LI may be beneficial for the prevention of diabetes., (© 2021 by the American Diabetes Association.)

Type 2 diabetes has become a pandemic and leads to late diabetic complications of organs, including kidney and eye. Lowering hyperglycemia is the typical therapeutic goal in clinical medicine. However, hyperglycemia may only be a symptom of diabetes but not the sole cause of late diabetic complications; instead, other diabetes-related alterations could be causative. Here, we studied the role of CaM kinase II-δ (CaMKIIδ), which is known to be activated through diabetic metabolism. CaMKIIδ is expressed ubiquitously and might therefore affect several different organ systems. We crossed diabetic leptin receptor-mutant mice to mice lacking CaMKIIδ globally. Remarkably, CaMKIIδ-deficient diabetic mice did not develop hyperglycemia. As potential underlying mechanisms, we provide evidence for improved insulin sensing with increased glucose transport into skeletal muscle and also reduced hepatic glucose production. Despite normoglycemia, CaMKIIδ-deficient diabetic mice developed the full picture of diabetic nephropathy, but diabetic retinopathy was prevented. We also unmasked a retina-specific gene expression signature that might contribute to CaMKII-dependent retinal diabetic complications. These data challenge the clinical concept of normalizing hyperglycemia in diabetes as a causative treatment strategy for late diabetic complications and call for a more detailed analysis of intracellular metabolic signals in different diabetic organs., (© 2020 by the American Diabetes Association.)

Clinical studies have suggested that changes in peripheral nerve microcirculation may contribute to nerve damage in diabetic polyneuropathy (DN). High-sensitivity troponin T (hsTNT) assays have been recently shown to provide predictive values for both cardiac and peripheral microangiopathy in type 2 diabetes (T2D). This study investigated the association of sciatic nerve structural damage in 3 Tesla (3T) magnetic resonance neurography (MRN) with hsTNT and N-terminal pro-brain natriuretic peptide serum levels in patients with T2D. MRN at 3T was performed in 51 patients with T2D (23 without DN, 28 with DN) and 10 control subjects without diabetes. The sciatic nerve's fractional anisotropy (FA), a marker of structural nerve integrity, was correlated with clinical, electrophysiological, and serological data. In patients with T2D, hsTNT showed a negative correlation with the sciatic nerve's FA ( r = -0.52, P < 0.001), with a closer correlation in DN patients ( r = -0.66, P < 0.001). hsTNT further correlated positively with the neuropathy disability score ( r = 0.39, P = 0.005). Negative correlations were found with sural nerve conduction velocities (NCVs) ( r = -0.65, P < 0.001) and tibial NCVs ( r = -0.44, P = 0.002) and amplitudes ( r = -0.53, P < 0.001). This study is the first to show that hsTNT is a potential indicator for structural nerve damage in T2D. Our results indirectly support the hypothesis that microangiopathy contributes to structural nerve damage in T2D., (© 2020 by the American Diabetes Association.)

Studies on magnetic resonance neurography (MRN) in diabetic polyneuropathy (DPN) have found proximal sciatic nerve lesions. The aim of this study was to evaluate the functional relevance of sciatic nerve lesions in DPN, with the expectation of correlations with the impairment of large-fiber function. Sixty-one patients with type 2 diabetes (48 with and 13 without DPN) and 12 control subjects were enrolled and underwent MRN, quantitative sensory testing, and electrophysiological examinations. There were differences in mechanical detection (Aβ fibers) and mechanical pain (Aδ fibers) but not in thermal pain and thermal detection clusters (C fibers) among the groups. Lesion load correlated with lower Aα-, Aβ-, and Aδ-fiber but not with C-fiber function in all participants. Patients with lower function showed a higher load of nerve lesions than patients with elevated function or no measurable deficit despite apparent DPN. Longer diabetes duration was associated with higher lesion load in patients with DPN, suggesting that nerve lesions in DPN may accumulate over time and become clinically relevant once a critical amount of nerve fascicles is affected. Moreover, MRN is an objective method for determining lower function mainly in medium and large fibers in DPN., (© 2019 by the American Diabetes Association.)

Hyperglycemia causes micro- and macrovascular complications in diabetic patients. Elevated glucose concentrations lead to increased formation of the highly reactive dicarbonyl methylglyoxal (MG), yet the early consequences of MG for development of vascular complications in vivo are poorly understood. In this study, zebrafish were used as a model organism to analyze early vascular effects and mechanisms of MG in vivo. High tissue glucose increased MG concentrations in tg(fli:EGFP) zebrafish embryos and rapidly induced several additional malformed and uncoordinated blood vessel structures that originated out of existing intersomitic blood vessels (ISVs). However, larger blood vessels, including the dorsal aorta and common cardinal vein, were not affected. Expression silencing of MG-degrading enzyme glyoxalase (glo) 1 elevated MG concentrations and induced a similar vascular hyperbranching phenotype in zebrafish. MG enhanced phosphorylation of vascular endothelial growth factor (VEGF) receptor 2 and its downstream target Akt/protein kinase B (PKB). Pharmacological inhibitors for VEGF receptor 2 and Akt/PKB as well as MG scavenger aminoguanidine and glo1 activation prevented MG-induced hyperbranching of ISVs. Taken together, MG acts on smaller blood vessels in zebrafish via the VEGF receptor signaling cascade, thereby describing a new mechanism that can explain vascular complications under hyperglycemia and elevated MG concentrations., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Objective: Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE(-/-) model of accelerated atherosclerosis., Research Design and Methods: ApoE(-/-) and RAGE(-/-)/apoE(-/-) double knockout mice were rendered diabetic with streptozotocin and followed for 20 weeks, at which time plaque accumulation was assessed by en face analysis., Results: Although diabetic apoE(-/-) mice showed increased plaque accumulation (14.9 +/- 1.7%), diabetic RAGE(-/-)/apoE(-/-) mice had significantly reduced atherosclerotic plaque area (4.9 +/- 0.4%) to levels not significantly different from control apoE(-/-) mice (4.3 +/- 0.4%). These beneficial effects on the vasculature were associated with attenuation of leukocyte recruitment; decreased expression of proinflammatory mediators, including the nuclear factor-kappaB subunit p65, VCAM-1, and MCP-1; and reduced oxidative stress, as reflected by staining for nitrotyrosine and reduced expression of various NADPH oxidase subunits, gp91phox, p47phox, and rac-1. Both RAGE and RAGE ligands, including S100A8/A9, high mobility group box 1 (HMGB1), and the advanced glycation end product (AGE) carboxymethyllysine were increased in plaques from diabetic apoE(-/-) mice. Furthermore, the accumulation of AGEs and other ligands to RAGE was reduced in diabetic RAGE(-/-)/apoE(-/-) mice., Conclusions: This study provides evidence for RAGE playing a central role in the development of accelerated atherosclerosis associated with diabetes. These findings emphasize the potential utility of strategies targeting RAGE activation in the prevention and treatment of diabetic macrovascular complications.

The initial phase of wound repair involves inflammation, induction of tissue factor (TF), formation of a fibrin matrix, and growth of new smooth muscle actin (alpha-SMA)-positive vessels. In diabetes, TF induction in response to cutaneous wounding, which ordinarily precedes increased expression of vascular endothelial growth factor (VEGF) and alpha-SMA transcription, is diminished, though not to a degree causing excessive local bleeding. Enhanced TF expression in wounds of diabetic mice caused by somatic TF gene transfer increased VEGF transcription and translation and, subsequently, enhanced formation of new blood vessels and elevated blood flow. Furthermore, increased levels of TF in wounds of diabetic mice enhanced wound healing; the time to achieve 50% wound closure was reduced from 5.5 days in untreated diabetic mice to 4.1 days in animals undergoing TF gene transfer (this was not statistically different from wound closure in nondiabetic mice). Thus, cutaneous wounds in diabetic mice display a relative deficiency of TF compared with nondiabetic controls, and this contributes to delayed wound repair. These data establish TF expression as an important link between the early inflammatory response to cutaneous wounding and reparative processes.

Twenty-three nondiabetic volunteers were divided into three groups. In group A (n = 9), the glucose infusion was adjusted to maintain blood glucose at 5 mmol/l (euglycemic clamp). In group B (n = 9), the glucose infusion was adjusted to maintain blood glucose at 10 mmol/l (hyperglycemic clamp) over 2 h. Group C consisted of five volunteers who were studied as the control group. Peripheral blood mononuclear cells (PBMCs) were isolated before and at the end of a 2-h clamp. In group C, PBMCs were isolated before and after 2 h without performing a clamp. The euglycemic clamp as well as "no clamp" had no effects on all parameters studied. In contrast, a significant increase in carboxymethyllysine (CML) content and p21(ras) and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation was observed at the end of a 2-h hyperglycemic clamp. The nuclear factor (NF)-kappaB (but not Oct-1) binding activity increased significantly in the hyperglycemic clamp. Western blots confirmed NF-kappaB-p65-antigen translocation into the nucleus. IkappaBalpha did not change significantly in both groups. Hyperglycemia-mediated NF-kappaB activation and increase of CML content, p21(ras), and p42/44 MAPK phosphorylation was also seen in ex vivo-isolated PBMCs stimulated with 5 or 10 mmol/l glucose. Addition of insulin did not influence the results. Inhibition of activation of ras, MAPK, or protein kinase C blocked hyperglycemia-mediated NF-kappaB activation in ex vivo-isolated PBMCs stimulated with 10 mmol/l glucose. Similar data were obtained using an NF-kappaB-luciferase reporter plasmid. Therefore, we can conclude that an acute hyperglycemia-mediated mononuclear cell activation is dependent on activation of ras, p42/p44 MAPK phosphorylation, and subsequent NF-kappaB activation and results in transcriptional activity in PBMCs.

Previous studies have shown that renal function in type 2 diabetes correlates better with tubular changes than with glomerular pathology. Since advanced glycation end products (AGEs; AGE-albumin) and in particular carboxymethyllysine (CML) are known to play a central role in diabetic nephropathy, we studied the activation of nuclear factor kappaB (NF-kappaB) in tubular epithelial cells in vivo and in vitro by AGE-albumin and CML. Urine samples from healthy control subjects (n = 50) and type 2 diabetic patients (n = 100) were collected and tested for excretion of CML and the presence of proximal tubular epithelial cells (pTECs). CML excretion was significantly higher in diabetic patients than in healthy control subjects (P < 0.0001) and correlated with the degree of albuminuria (r = 0.7, P < 0.0001), while there was no correlation between CML excretion and HbA(1c) (r = 0.03, P = 0.76). Urine sediments from 20 of 100 patients contained pTECs, evidenced by cytokeratin 18 positivity, while healthy control subjects (n = 50) showed none (P < 0.0001). Activated NF-kappaB could be detected in the nuclear region of excreted pTECs in 8 of 20 patients with pTECs in the urine sediment (40%). Five of eight NF-kappaBp65 antigen-positive cells stained positive for interleukin-6 (IL-6) antigen (62%), while only one of the NF-kappaB-negative cells showed IL-6 positivity. pTECs in the urine sediment correlated positively with albuminuria (r = 0.57, P < 0.0001) and CML excretion (r = 0.55, P < 0.0001). Immunohistochemistry in diabetic rat kidneys and a human diabetic kidney confirmed strong expression of NF-kappaB in tubular cells. To further prove an AGE/CML-induced NF-kappaB activation in pTECs, NF-kappaB activation was studied in cultured human pTECs by electrophoretic mobility shift assays (EMSAs) and Western blot. Stimulation of NF-kappaB binding activity was dose dependent and was one-half maximal at 250 nmol/l AGE-albumin or CML and time dependent at a maximum of activation after 4 days. Functional relevance of the observed NF-kappaB activation was demonstrated in pTECs transfected with a NF-kappaB-driven luciferase reporter plasmid and was associated with an increased release of IL-6 into the supernatant. The AGE- and CML-dependent activation of NF-kappaBp65 and NF-kappaB-dependent IL-6 expression could be inhibited using the soluble form of the receptor for AGEs (RAGE) (soluble RAGE [sRAGE]), RAGE-specific antibody, or the antioxidant thioctic acid. In addition transcriptional activity and IL-6 release from transfected cells could be inhibited by overexpression of the NF-kappaB-specific inhibitor kappaBalpha. The findings that excreted pTECs demonstrate activated NF-kappaB and IL-6 antigen and that AGE-albumin and CML lead to a perpetuated activation of NF-kappaB in vitro infer that a perpetuated increase in proinflammtory gene products, such as IL-6, plays a role in damaging the renal tubule.

Lipoprotein lipase (LPL) plays a key role in lipid metabolism by hydrolyzing triglycerides in circulating lipoproteins. Low LPL activity has been linked to coronary artery disease (CAD), but the factors influencing LPL expression are not completely understood. Peroxisome proliferator--activated receptor (PPAR)-gamma is a nuclear receptor regulating lipid and glucose metabolism, and a PPAR-responsive element is present in the LPL promoter. We determined the Pro12Ala polymorphism in the PPAR-gamma2 gene in 194 male CAD patients because this allele is associated with decreased PPAR activity and reduced LPL promoter activity in vitro. Presence of 12Ala was associated with 20% lower LPL activity in postheparin plasma (141 +/- 58 vs. 177 +/- 77 nmol.ml(-1).min(-1), P < 0.005). Remarkably, the influence of 12Ala on LPL was greater than that of the frequent polymorphisms (HindIII +9%, PvuII +/- 0%, 447stop +12%) in the LPL gene itself. To confirm these results in a different group of patients, we analyzed 100 diabetic patients in whom the 12Ala allele was also associated with lower LPL activity (12Ala: 132 +/- 88 vs. 190 +/- 129 nmol.ml(-1).min(-1), P < 0.05). Our data demonstrate that the Pro12Ala substitution in PPAR-gamma2 is associated with lower LPL activity in vivo and provides a new target for the analysis of genetic influences on LPL activity and CAD risk.

Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) has been suggested to participate in chronic disorders, such as diabetes and its complications. In contrast to the short and transient activation of NF-kappaB in vitro, we observed a long-lasting sustained activation of NF-kappaB in the absence of decreased IkappaBalpha in mononuclear cells from patients with type 1 diabetes. This was associated with increased transcription of NF-kappaBp65. A comparable increase in NF-kappaBp65 antigen and mRNA was also observed in vascular endothelial cells of diabetic rats. As a mechanism, we propose that binding of ligands such as advanced glycosylation end products (AGEs), members of the S100 family, or amyloid-beta peptide (Abeta) to the transmembrane receptor for AGE (RAGE) results in protein synthesis-dependent sustained activation of NF-kappaB both in vitro and in vivo. Infusion of AGE-albumin into mice bearing a beta-globin reporter transgene under control of NF-kappaB also resulted in prolonged expression of the reporter transgene. In vitro studies showed that RAGE-expressing cells induced sustained translocation of NF-kappaB (p50/p65) from the cytoplasm into the nucleus for >1 week. Sustained NF-kappaB activation by ligands of RAGE was mediated by initial degradation of IkappaB proteins followed by new synthesis of NF-kappaBp65 mRNA and protein in the presence of newly synthesized IkappaBalpha and IkappaBbeta. These data demonstrate that ligands of RAGE can induce sustained activation of NF-kappaB as a result of increased levels of de novo synthesized NF-kappaBp65 overriding endogenous negative feedback mechanisms and thus might contribute to the persistent NF-kappaB activation observed in hyperglycemia and possibly other chronic diseases.

Incubation of bovine aortic endothelial cells (BAECs) with erythrocytes from patients with type 2 diabetes induced an increase in endothelin 1 (ET-1) production. The effect of erythrocytes on ET-1 synthesis was dependent on glycemic control. ET-1 levels after incubation with erythrocytes derived from patients with HbA(1c) levels <6% were just half the levels observed after incubation with erythrocytes from patients with HbA(1c) levels >8%. Nepsilon-(carboxymethyl)lysine (CML)-containing protein isolated from patients' erythrocytes induced ET-1, and CML-containing protein-dependent ET-1 induction was blocked by the recombinant decoy peptide soluble receptor for advanced glycation end products (AGEs), which comprises the NH2-terminal Ig domain of the receptor for AGEs. In vitro-generated AGEs induced ET-1 mRNA transcription (nuclear run-on assay and Northern blot) in a time- and dose-dependent manner. Transient transfection of BAECs with a chimeric construct containing the 5' promoter region of the ET-1 gene linked to a reporter gene confirmed that AGE induced ET-1 promoter activity. Electrophoretic mobility shift assay confirmed AGE-inducible binding of members of the nuclear factor-kappab (NF-kappaB) family to a potential binding site at -2,090 bp. Binding was functionally significant because overexpression of the cytoplasmic inhibitor of NF-kappaB or deletion of the NF-kappaB binding site reduced ET-1 induction, whereas overexpression of NF-kappaB p65 induced ET-1 even in the absence of AGEs. Thus, ET-1 transcription is controlled by the AGE-inducible redox-sensitive transcription factor NF-kappaB.

Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxidative stress led to the activation of the transcription factor NF-kappaB and thus promoted the upregulation of various NF-kappaB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antioxidative defense with the natural occurring antioxidant alpha-lipoic acid before AGE albumin induction completely prevented the AGE albumin-dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-kappaB activation was also reduced in a time- and dose-dependent manner as long as alpha-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since alpha-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-kappaB. Western blots further demonstrated that alpha-lipoic acid inhibited the release and translocation of NF-kappaB from the cytoplasm into the nucleus. As a consequence, alpha-lipoic acid reduced AGE albumin-induced NF-kappaB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered alpha-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.