Abstract Reactive carbonyl species contribute to diabetes and its complications, but how the interconnections of carbonyl-detoxifying enzymes regulate metabolic homeostasis through arginine metabolism remains unclear. Here we generate zebrafish lacking both glyoxalase 1 and aldo-keto reductase 1A1A, two major enzymes that detoxify methylglyoxal and acrolein. Double deficiency causes carbonyl accumulation, suppresses arginine metabolism, and impairs insulin signaling, resulting in elevated glucose levels in larvae and in postprandial hyperglycemia in adult male zebrafish. These metabolic alterations are accompanied by glomerular basement membrane thickening and podocyte effacement, whereas retinal vasculature remains unaffected. Arginine supplementation restores Akt phosphorylation, improves insulin signaling, and attenuates renal pathology, indicating that disrupted arginine metabolism mediates the metabolic consequences of carbonyl stress. Our findings identify glyoxalase 1 and aldo-keto reductase 1A1A as cooperative regulators of carbonyl detoxification and reveal a carbonyl–arginine axis linking reactive carbonyl accumulation to impaired insulin signaling, hyperglycemia, and tissue-specific diabetic injury.

Objective:To determine whether cardiovascular autonomic neuropathy (CAN) and reduced heart rate variability (HRV) predict the development of distal symmetrical polyneuropathy (DSPN) in individuals with diabetes mellitus (DM). Methods:A total of 288 individuals with DM (mean age 58.1 ± 13.4 years, 41.3% women, 78.5% with type 2 diabetes) underwent baseline assessment of CAN using cardiovascular autonomic reflex tests and HRV indices (CVRR, rMSSD, high- and low-frequency power). DSPN was characterized using three definitions: (i) clinical criteria based on symptoms and signs, (ii) the sensory-loss phenotype derived from quantitative sensory testing, and (iii) the Toronto consensus definition requiring abnormal nerve conduction plus symptoms or signs. A total of 194 participants without DSPN at baseline were followed for 3.0 ± 1.3 years to assess incident DSPN. Results:At baseline, CAN prevalence was 16.3%, and low HRV indices were present in 18.6-40.2% of participants. DSPN prevalence was 27.7%, 17.6%, and 20.8% for definitions (i), (ii), and (iii), respectively. Cross-sectionally, CAN and reduced HRV were associated with DSPN (unadjusted ORs 1.8-4.0), with associations largely persisting after adjustment. Incident DSPN occurred at 8.4, 5.7, and 3.3 per 100 person-years. Baseline CAN independently predicted incident DSPN (adjusted HRs 6.20, 3.30, and 7.33 across definitions). Lower rMSSD, HF power, and LF power predicted incident DSPN according to definitions (i) and (iii), whereas reduced CVRR predicted DSPN defined by criteria (ii). Conclusions:Established CAN is a strong predictor of future DSPN. Reduced HRV provides a practical, accessible alternative marker for identifying individuals at elevated neuropathy risk.

Cardiovascular risk management is beneficial, but stringent glycemic control does not prevent the progression of distal sensorimotor polyneuropathy (DSPN). Persistent hyperglycemia-induced alterations and cardiovascular factors may contribute to diabetes-associated nerve damage. This study aimed to evaluate the correlation between skin auto-fluorescence (sAF), an indicator of dermal advanced glycation end-product (AGE) accumulations, cardiovascular risk, and changes in peripheral nerve integrity. Sixty-two individuals with type 2 diabetes (T2D) (20 women and 42 men), including 29 diagnosed with DSPN (7 women and 22 men), and 10 healthy controls (HC) underwent diffusion tensor MR imaging of the sciatic nerve to assess fractional anisotropy (FA), an indicator of nerve structural integrity. sAF measurements were combined with clinical, serological, and electrophysiological evaluations. Arterial stiffness was assessed via pulse wave velocity (PWV). sAF (HC 2.1 ± 0.25 AU, nDSPN 2.3 ± 0.47, DSPN 2.6 ± 0.43; p = 0.005) was higher in individuals with DSPN compared to HC (p = 0.010) and individuals without DSPN (p = 0.035). Within the group of T2D FA correlated negatively with sAF (r = −0.49, p < 0.001), PWV (r = −0.40, p = 0.009) and high-sensitivity troponin T (hsTNT), a marker of microvascular damage (r = −0.39, p < 0.001). In DSPN, sAF correlated positively with hsTNT (r = 0.58, p = 0.005) and with PWV (r = 0.52, p = 0.007), the sciatic nerve’s FA correlated negatively with PWV (r = −0.47, p = 0.010). This study is the first to show close correlations between reduced peripheral nerve integrity and both intradermal AGE deposition and arterial stiffness in individuals with T2D. These findings highlight a mechanistic link between glycation-related vascular injury and neuronal damage emphasizing the importance of cardiovascular risk management in preventing DSPN.

The Heidelberg Study on Diabetes and Complications (HEIST-DiC) is a prospective longitudinal study focused on the development and progression of diabetes-associated complications. Participants with/without diabetes mellitus undergo annual phenotyping of diabetes-associated complications over 11 years. Assessments include: albuminuria, estimated glomerular filtration rate for chronic kidney disease; clinical neuropathy scores, Purdue Pegboard test, electrophysiological examination, transcutaneous electrical nerve fiber stimulation, quantitative sensory testing and high-resolution magnetic resonance neurography for distal sensorimotor polyneuropathy; heart rate variability for cardiovascular autonomic neuropathy; funduscopic examination of undilated pupils for retinopathy; the 6-minute walk test, spirometry, body plethysmography, and carbon monoxide-based diffusing capacity measurements for respiratory lung disease; non-invasive scores, transient elastography and hepatic ultrasound for metabolic dysfunction-associated steatotic liver disease; ankle-brachial index and carotid intima-media thickness for peripheral atherosclerosis; hand grip strength for muscle function; bioelectrical impedance analysis for body composition; skin autofluorescence for measurement of advanced glycation end products. Beta-cell function and tissue-specific insulin sensitivity are evaluated using oral glucose tolerance test or euglycemic hyperinsulinemic clamp. The biobank stores specimens of blood, urine, skeletal muscle, subcutaneous adipose tissue, and skin. Health-related quality of life, physical health, and somatic and depression symptoms are measured via standardized questionnaires. HEIST-DiC explores diabetes onset in high-risk individuals, disease progression and the development of complications, aiming to design personalized strategies to prevent, mitigate, or reverse diabetes-related complications. Trial registration: The study was retrospectively registered at Clinicaltrials.gov (NCT03022721, date of registration 20170112).

Background: Dicarbonyls and advanced glycation end-products (AGEs) contribute to oxidative stress, inflammation, and complications in type 2 diabetes mellitus (T2DM). Menaquinone-7 (MK-7), a vitamin K2 subtype, has shown benefits for glucose tolerance and vascular health in some studies. We evaluated the impact of MK-7 on dicarbonyls, free AGEs, and protein nitration/oxidation adducts in a rat model of T2DM. Methods: Male heterozygous (fa/+, control) and homozygous (fa/fa, diabetic) Zucker Diabetic Fatty rats were fed a diabetogenic diet without or with MK-7 for 12 weeks. After sacrifice, plasma dicarbonyls as well as plasma and urinary levels of free AGEs and protein nitration/oxidation adducts were quantified by isotope dilution tandem mass spectrometry. Results: Diabetic rats showed significantly increased plasma glyoxal, 3-deoxyglucosone, and fructosyl-lysine with non-significant trends toward increased methylglyoxal-derived hydroimidazolone and methionine sulfoxide, as well as reductions in methylglyoxal and dityrosine. Urinary carboxyethyl-lysine, carboxymethyl-lysine, fructosyl-lysine (all significant), and dityrosine (non-significant) were elevated in diabetic rats; glucosepane (non-significant) was reduced. MK-7 supplementation reduced no measured parameter but was associated with non-significant further increases in plasma glyoxal-derived hydroimidazolone, carboxyethyl-lysine, carboxymethyl-lysine, fructosyl-lysine, 3-nitrotyrosine, and methionine sulfoxide, as well as in urinary glyoxal-derived hydroimidazolone, carboxyethyl-lysine, fructosyl-lysine, and 3-nitrotyrosine, in diabetic rats. Correlation analysis revealed significant associations between glucose, dicarbonyls, AGEs, and oxidative markers. Conclusions: High-dose MK-7 supplementation did not improve dicarbonyl stress, AGE burden, or protein nitration/oxidation. With respect to available scientific evidence and our observations, the combination of glycemia-driven amplification of glycation and oxidative stress, as well as MK-7-induced glutathione depletion, were likely causative.

OBJECTIVE:Metabolic inflexibility has been shown to be associated with type 2 diabetes (T2D) and diabetic nephropathy (DN). However, data are lacking, proving that reconstitution of metabolic flexibility by using a 6-month periodic fasting (PF) regimen may improve albuminuria. METHODS:In this post hoc analysis of a randomized-controlled trial, we investigated whether the PF regimen enhanced metabolic flexibility in individuals with T2D and DN showing improvement of albuminuria (responders) compared to non-responders. Participants followed every month either a 5-day fasting-mimicking diet or a Mediterranean diet for 6 months. LC-MS/MS-based comprehensive metabolic profiling was performed in plasma samples before, during, and after the intervention. Changes in metabolomic patterns and enriched signalling pathways were analysed between study groups. RESULTS:PF induced a sustained shift toward enhanced fatty acid oxidation, lipid utilization, and amino acids turnover, particularly in responders. Responders exhibited persistent elevations in short-chain acylcarnitines and cholesteryl esters, indicating more efficient lipid oxidation and tighter integration of lipid metabolism with the tricarboxylic acid cycle. Increased glycine and serine levels suggested enhanced cellular maintenance, a protein-sparing effect, and a metabolic shift favouring lipid over carbohydrate. In contrast, non-responders demonstrated only transient and limited metabolic shifts. Unsupervised clustering identified distinct metabolic response patterns, reinforcing the potential of personalized dietary interventions. CONCLUSIONS:These findings demonstrate that diet-induced restoration of metabolic flexibility is associated with improved albuminuria in T2D, suggesting broader implications for precise nutritional strategies in diabetes management.

Pathological levels of methylglyoxal (MG), a reactive dicarbonyl product of glucose, contribute to major neurological complications associated with type II diabetes, including chronic neuropathic pain. Strategies to target elevated MG have included small molecule MG scavengers, but scavenger deficiencies in proteolytic stability and onset of scavenging activity have precluded clinical translation. To address this gap, we developed a long-lasting and highly reactive cyclic peptide CycK(Myr)R4E, and here evaluated its antihyperalgesic efficacy in the db/db mouse model of type II diabetes painful diabetic neuropathy. To test the hypothesis that CycK(Myr)R4E can reduce behavioral and molecular signs of painful diabetic neuropathy. We assessed heat hypersensitivity as an index of hyperalgesia, and touch-evoked expression of phosphorylated extracellular signal-regulated kinase as a measure of neuronal activity in spinal cord dorsal horn. We report that a single systemic injection of CycK(Myr)R4E (3 mg/kg) reversed heat hypersensitivity. Repeated systemic injection of CycK(Myr)R4E (0.125 mg/kg, 3 times per week, 6-12 weeks of age) prevented heat hypersensitivity and reduced stimulus-evoked phosphorylated extracellular signal-regulated kinase. These studies promote CycK(Myr)R4E as the most promising MG scavenger for the prevention and treatment of hyperalgesia in type 2 diabetic neuropathic pain.

Kidney damage in individuals with methylmalonyl-CoA mutase deficiency (mut0) results from metabolic and oxidative stress, and disrupted mitochondrial homeostasis. Anserine, known for its antioxidant properties and protective effect on cell barrier integrity of endothelial and epithelial kidney and vascular cells, may offer therapeutic potential for chronic disorders. This study explored anserine’s effects on immortalized kidney tubular epithelial cells (iKTEC) from mut0 patients. Compared to healthy controls, iKTEC from mut0 patients showed reduced cell viability, antioxidant capacity, oxygen consumption, and ATP production. Expression of the tight junction scaffolding protein zonula occludens-1 (ZO-1) was increased in mut0 cells compared to control while transepithelial resistance (TER), and dextran transport (10 kDa) remained unchanged. Anserine treatment restored antioxidant capacity and normalized ZO-1 expression but had no effect on TER or dextran transport. Additionally, cell viability, mitochondrial respiration, and ATP production were unaffected by anserine. Metabolic stress induced by high protein load or disease-associated branched-chain amino acids did not worsen mitochondrial dysfunction or epithelial integrity, and anserine exposure showed no further effects. In conclusion, anserine showed promise in restoring antioxidant capacity in iKTEC from mut0 patients, highlighting its potential as a therapeutic agent to mitigate ROS, although its effects on mitochondrial function and epithelial integrity warrant further investigation.

Acrolein (ACR) is an endogenous reactive unsaturated aldehyde that can be detoxified by the aldo-keto reductase (AKR) enzyme system. While it has been shown that accumulation of ACR is associated with several health problems, including inflammation, oxidative stress, and cardiovascular disease the study aimed to analyze whether an endogenous accumulation of ACR is causal for vascular dysfunction in an akr7a3 mutant zebrafish model. Enlargement of the hyaloid and retinal vasculature, as well as alterations in the larval pronephros and thickening of the glomerular basement membrane in the adult kidney were found upon ACR accumulation. Transcriptomic and metabolomic analyses, followed by functional validation, revealed that the up-regulation of genes controlling the arachidonic acid metabolism and activation of the leukotriene pathway are responsible for the observed microvascular changes. In conclusion, the data have identified an intrinsic function of ACR in akr7a3 mutants that activates the arachidonic acid metabolism and subsequently disrupts vascular integrity by promoting an inflammatory response. Thus, ACR is causal in the development of vascular disease.

Introduction:Pathological levels of methylglyoxal (MG), a reactive dicarbonyl product of glucose, contribute to major neurological complications associated with type II diabetes, including chronic neuropathic pain. Strategies to target elevated MG have included small molecule MG scavengers, but scavenger deficiencies in proteolytic stability and onset of scavenging activity have precluded clinical translation. To address this gap, we developed a long-lasting and highly reactive cyclic peptide CycK(Myr)R4E, and here evaluated its antihyperalgesic efficacy in the db/db mouse model of type II diabetes painful diabetic neuropathy. Objectives:To test the hypothesis that CycK(Myr)R4E can reduce behavioral and molecular signs of painful diabetic neuropathy. Methods:We assessed heat hypersensitivity as an index of hyperalgesia, and touch-evoked expression of phosphorylated extracellular signal-regulated kinase as a measure of neuronal activity in spinal cord dorsal horn. Results:We report that a single systemic injection of CycK(Myr)R4E (3 mg/kg) reversed heat hypersensitivity. Repeated systemic injection of CycK(Myr)R4E (0.125 mg/kg, 3 times per week, 6-12 weeks of age) prevented heat hypersensitivity and reduced stimulus-evoked phosphorylated extracellular signal-regulated kinase. Conclusion:These studies promote CycK(Myr)R4E as the most promising MG scavenger for the prevention and treatment of hyperalgesia in type 2 diabetic neuropathic pain.

Fibrosis and persistent inflammation are interconnected processes that inhibit axon regeneration in the mammalian central nervous system (CNS). In zebrafish, by contrast, fibroblast-derived extracellular matrix deposition and inflammation are tightly regulated to facilitate regeneration. However, the regulatory cross-talk between fibroblasts and the innate immune system in the regenerating CNS remains poorly understood. Here, we show that zebrafish fibroblasts possess a dual role in inducing and resolving inflammation, which are both essential for regeneration. We identify a transient, injury-specific cthrc1a+ fibroblast state with an inflammation-associated, less differentiated, and non-fibrotic profile. Induction of this fibroblast state precedes and contributes to the initiation of the inflammatory response. At the peak of neutrophil influx, cthrc1a+ fibroblasts coordinate the resolution of inflammation. Disruption of these inflammation dynamics alters the mechano-structural properties of the lesion environment and inhibits axon regeneration. This establishes the biphasic inflammation control by dedifferentiated fibroblasts as a pivotal mechanism for CNS regeneration. ### Competing Interest Statement The authors have declared no competing interest.

Background: Restrictive lung disease (RLD) is a potential complication in type 2 diabetes (T2D), but its relationship with insulin resistance and liver-related metabolic dysfunction remains unclear. This study evaluated the association between lung function and metabolic markers in T2D and retrospectively assessed whether metabolic improvements from dietary intervention were accompanied by changes in lung function. Methods: This cross-sectional analysis included 184 individuals (101 with T2D, 33 with prediabetes, and 50 glucose-tolerant individuals). Lung function parameters—vital capacity (VC), total lung capacity by plethysmography (TLC-B), and diffusion capacity for carbon monoxide (TLCO)—were assessed alongside metabolic markers including HOMA2-IR, fatty liver index (FLI), NAFLD score, and Fibrosis-4 index (FIB-4). In a subset of 54 T2D participants, lung function was reassessed after six months following either a fasting-mimicking diet (FMD, n = 14), Mediterranean diet (n = 13), or no dietary intervention (n = 27). Results: T2D participants had significantly lower VC and TLC-B compared to glucose-tolerant and prediabetic individuals, with 18–21% falling below clinical thresholds for RLD. Lung volumes were negatively correlated with HOMA2-IR, FLI, NAFLD score, and FIB-4 across the cohort and within the T2D group. Although the FMD intervention led to significant improvements in HOMA2-IR and FLI, no corresponding changes in lung function were observed over the six-month period. Conclusions: Restrictive lung impairment in T2D is associated with insulin resistance and markers of liver steatosis and fibrosis. While short-term dietary interventions can improve metabolic parameters, their effect on lung function may require a longer duration or additional interventions and targeted follow-up. These findings highlight the relevance of pulmonary assessment in individuals with metabolic dysfunction.