Intracerebral matrix metalloproteinase 9 in fatal diabetic ketoacidosis.

There is increasing awareness that in addition to the metabolic crisis of diabetic ketoacidosis (DKA) caused by severe insulin deficiency, the immune inflammatory response is likely an active multicomponent participant in both the acute and chronic insults of this medical crisis, with strong evidence of activation for both the cytokine and complement system. Recent studies report that the matrix metalloproteinase enzymes and their inhibitors are systemically activated in young Type 1 diabetes mellitus (T1D) patients during DKA and speculate on their involvement in blood-brain barrier (BBB) disruption. Based on our previous studies, we address the question if matrix metalloproteinase 9 (MMP9) is expressed in the brain in the fatal brain edema (BE) of DKA. Our data show significant expression of MMP9 on the cells present in brain intravascular areas. The presence of MMP9 in intravascular cells and that of MMP+ cells seen passing the BBB indicates a possible role in tight junction protein disruption of the BBB, possibly leading to neurological complications including BE. We have also shown that MMP9 is expressed on neurons in the hippocampal areas of both BE/DKA cases investigated, while expression of tissue inhibitor of metalloproteinases 1 (TIMP1) was reduced in the same areas. We can speculate that intraneuronal MMP9 can be a sign of neurodegeneration. Further studies are necessary to determine the role of MMP9 in the pathogenesis of the neurologic catastrophe of the brain edema of DKA. Inhibition of MMP9 expression might be helpful in preserving neuronal function and BBB integrity during DKA.

Markers of immune-mediated inflammation in the brains of young adults and adolescents with type 1 diabetes and fatal diabetic ketoacidosis. Is there a difference?

Due to the limited data on diabetic ketoacidosis and brain edema (DKA/BE) in children/adolescents and the lack of recent data on adults with type 1 diabetes (T1D), we addressed the question of whether neuroinflammation was present in the fatal DKA of adults. We performed immunohistochemistry (IHC) studies on the brains of two young adults with T1D and fatal DKA and compared them with two teenagers with poorly controlled diabetes and fatal DKA. C5b-9, the membrane attack complex (MAC) had significantly greater deposits in the grey and white matter of the teenagers than the young adults (p=0.03). CD59, a MAC assembly inhibitory protein was absent, possibly suppressed by the hyperglycemia in the teenagers but was expressed in the young adults despite comparable average levels of hyperglycemia. The receptor for advanced glycation end products (RAGE) had an average expression in the young adults significantly greater than in the teenagers (p=0.02). The autophagy marker Light Chain 3 (LC3) A/B was the predominant form of programmed cell death (PCD) in the teenage brains. The young adults had high expressions of both LC3A/B and TUNEL, an apoptotic cell marker for DNA fragmentation. BE was present in the newly diagnosed young adult with hyperglycemic hyperosmolar DKA and also in the two teenagers. Our data indicate that significant differences in neuroinflammatory components, initiated by the dysregulation of DKA and interrelated metabolic and immunologic milieu, are likely present in the brains of fatal DKA of teenagers when compared with young adults.

MCP-1and IL-1ß expression in the myocardia of two young patients with Type 1 diabetes mellitus and fatal diabetic ketoacidosis.

Convincing evidence exists for the early onset of diabetic cardiomyopathy and coronary artery disease (CAD) as distinct forms of cardiac disease in young patients with Type 1 diabetes mellitus (T1DM) and the pre-stages of T2DM, forms of dysregulated insulin signaling. Progression of both chronic cardiac conditions is mediated by oxidative stress and low grade inflammation. This study reports the expression of monocyte chemotactic protein-1 (MCP-1) chemokine and the interleukin (IL)-1ß inflammatory cytokine in two young patients with suboptimal metabolic control and fatal diabetic ketoacidosis (DKA), two age-matched overweight/obesity cases and two age-matched controls. In addition, markers of oxidative stress, apoptosis, collagen deposition and cardiomyocyte hypertrophy were studied. Significant expression of MCP-1 and IL-1ß was seen in the myocardia of the T1DM/DKA cases, with lesser amounts expressed in the overweight/obesity myocardia. All of the other markers except cardiomyocyte hypertrophy were expressed to a significantly greater extent in the T1DM/DKA and overweight/obesity cases in comparison to the age-matched controls. Cardiomyocyte hypertrophy was significantly greater in the overweight/obesity cases than in the T1DM/DKA or the control cases.

Autophagy in the brains of young patients with poorly controlled T1DM and fatal diabetic ketoacidosis.

Semi-quantitative neuroradiologic studies, quantitative neuron density studies and immunocytochemistry markers of oxidative stress and neuroinflammation indicate neuronal injury and deficits in young patients with chronic poorly controlled type 1 diabetes mellitus (T1DM). Present data suggest that pathogenesis of the neuronal deficits in young patients, who die as the result of diabetic ketoacidosis (DKA) and brain edema (BE), does not involve apoptosis, a prominent form of regulated cell death in many disease states. To further address this we studied mediators of macroautophagy, endoplasmic reticulum (ER) stress and apoptosis. In all areas studied we demonstrated increased levels of macroautophagy-associated proteins including light chain-3 (LC3) and autophagy related protein-4 (Atg4), as well as increased levels of the ER-associated glucose-regulated protein78/binding immunoglobulin protein (GRP78/BiP) in T1DM. In contrast, cleaved caspase-3 was rarely detected in any T1DM brain regions. These results suggest that chronic metabolic instability and oxidative stress may cause alterations in the autophagy-lysosomal pathway but not apoptosis, and macroautophagy-associated molecules may serve as useful candidates for further study in the pathogenesis of early neuronal deficits in T1DM.

Tryptophan, kynurenine pathway, and diabetic ketoacidosis in type 1 diabetes.

Diabetic ketoacidosis (DKA) is a serious complication of complete insulin deficiency and insulin resistance in Type 1 diabetes (T1D). This results in the body producing high levels of serum ketones in an attempt to compensate for the insulin deficiency and decreased glucose utilization. DKA's metabolic and immunologic dysregulation results in gradual increase of systemic and cerebral oxidative stress, along with low grade systemic and cerebral inflammation and the development of pretreatment subclinical BE. During treatment the early progression of oxidative stress and inflammation is hypothesized to advance the possibility of occurrence of crisis of clinical brain edema (BE), which is the most important cause of morbidity and mortality in pediatric DKA. Longitudinal neurocognitive studies after DKA treatment show progressive and latent deficits of cognition and emphasize the need for more effective DKA treatment of this long-standing conundrum of clinical BE, in the presence of systemic osmotic dehydration, metabolic acidosis and immune dysregulation. Candidate biomarkers of several systemic and neuroinflammatory pathways prior to treatment also progress during treatment, such as the neurotoxic and neuroprotective molecules in the well-recognized tryptophan (TRP)/kynurenine pathway (KP) that have not been investigated in DKA. We used LC-MS/MS targeted mass spectrometry analysis to determine the presence and initiation of the TRP/KP at three time points: A) 6-12 hours after initiation of treatment; B) 2 weeks; and C) 3 months following DKA treatment to determine if they might be involved in the pathogenesis of the acute vasogenic complication of DKA/BE. The Trp/KP metabolites TRP, KYN, quinolinic acid (QA), xanthurnenic acid (XA), and picolinic acid (PA) followed a similar pattern of lower levels in early treatment, with subsequent increases. Time point A compared to Time points B and C were similar to the pattern of sRAGE, lactate and pyruvic acid. The serotonin/melatonin metabolites also followed a similar pattern of lower quantities at the early stages of treatment compared to 3 months after treatment. In addition, glutamate, n-acetylglutamate, glutamine, and taurine were all lower at early treatment compared to 3 months, while the ketones 3-hydroxybutaric acid and acetoacetate were significantly higher in the early treatment compared to 3 months. The two major fat metabolites, L-carnitine and acetyl-L-carnitine (ALC) changed inversely, with ALC significantly decreasing at 2 weeks and 3 months compared to the early stages of treatment. Both anthranilic acid (AA) and 3-OH-anthranilic acid (3OH-AA) had overall higher levels in the early stages of treatment (A) compared to Time points (B and C). Interestingly, the levels of AA and 3OH-AA early in treatment were higher in Caucasian females compared to African American females. There were also differences in the metabolite levels of QA and kynurenic acid (KA) between genders and between races that may be important for further development of custom targeted treatments. We hypothesize that the TRP/KP, along with the other inflammatory pathways, is an active participant in the metabolic and immunologic pathogenesis of DKA's acute and chronic insults.

Soluble Receptor for Glycation End-products Concentration Increases Following the Treatment of Severe Diabetic Ketoacidosis

Objective: To determine the time relationships of soluble receptor for glycation end-products (sRAGE), [a decoy of the advanced glycation end-products (AGE)-RAGE axis] and D-lactate, (a metabolite of methylglyoxal) in the inflammatory response to diabetic ketoacidosis (DKA). Methods: Sixteen children and adolescents with type 1 diabetes (T1D) had blood samples obtained, 6-12 hours into treatment, at three weeks and three months post start of treatment. sRAGE and D-lactate concentrations at three months were considered baseline. Expression of RAGE was investigated in the myocardium of a newly diagnosed and untreated young person with fatal T1D/DKA. Results: sRAGE 6-12 hours after the start of treatment was 39% lower than the values at two weeks (p=0.0036) and at three months (p=0.0023) post treatment. D-lactate was higher during treatment than at three weeks (p=0.04) and at three months (p=0.035). Conclusion: sRAGE concentration was decreased during treatment, compared to concentrations at two weeks and three months after treatment. The increased D-lactate during treatment was in keeping with the known increase in dicarbonyls at this time. The finding of RAGE expression in a young myocardium prior to DKA treatment suggested cardiovascular inflammation pre-treatment and at a young age.

Heritability of levels of autoantibodies to thyroid antigens using the method of plotting regression of offspring on midparent (ROMP).

Only a few methods can be applied in a simple manner to estimate the genetic control of autoimmunity in humans. Here we examined the heritability of autoantibodies to two thyroid antigens; thyroglobulin (Tg) and thyroperoxidase (TPO, formerly known as thyroid microsomal antigen), using methods of regression of offspring on mid-parental values (ROMP). With the data sets available, affected and unaffected siblings were compared by this rapid screening method using results determined by hemagglutination (HA). The presence of both types of autoantibodies showed positive heritability in patients with Graves' thyrotoxicosis (TT), but it was not observed in chronic lymphocytic or Hashimoto's thyroiditis (CLT) patients. Since these assays have been extensively used over the years by most diagnostic and research laboratories, they should provide some insight as to which quantifiable parameters may be usefully accumulated to help select groups of patients and their families for further genetic study. ROMP may also be useful to determine the sequential appearance of different types of antibody in predicting disease onset in other family members, and in distinguishing maternal and paternal effects on imprinting. The method may be extended to study epitope spreading and other measures of disease progression.

Cardiac antibody production to self-antigens in children and adolescents during and following the correction of severe diabetic ketoacidosis.

Diabetic cardiomyopathy (DC) is an independent phenotype of diabetic cardiovascular disease. The understanding of the pathogenesis of DC in young patients with type 1 diabetes (T1D) is limited. The cardiac insults of diabetic ketoacidosis (DKA) and progression of DC could include development of antibodies (Abs) to cardiac self-antigens (SAgs) such as: myosin (M), vimentin (V) and k-alpha 1 tubulin (Kα1T). The goal of this study is to determine if the insults of severe DKA and its inflammatory cascade are associated with immune responses to SAgs. Development of Abs to the SAgs were determined by an ELISA using sera collected at three time points in relation to severe DKA (pH < 7.2). Results demonstrate significant differences between the development of Abs to VIM and a previously reported diastolic abnormality (DA) during DKA and its treatment and a NDA group at 2-3 months post DKA (p = 0.0452). A significant association is present between T1D duration (<3 years) and Abs to Kα1T (p = 0.0134). Further, Abs to MYO and VIM are associated with inflammatory cytokines. We propose that severe DKA initiates the synthesis of Abs to cardiac SAgs that are involved in the early immunopathogenesis of DC in young patients with T1D.

Diabetic ketoacidosis increases extracellular levels of the major inducible 70-kDa heat shock protein.

OBJECTIVES: Diabetic ketoacidosis (DKA) represents a metabolic stress whose treatment induces a systemic proinflammatory cytokine profile and accentuates life-threatening acute complications. The present study determined whether serum levels of the major inducible 70-kDa heat shock protein (Hsp72), a modulator of cytokine expression, were influenced by DKA and its treatment. DESIGN AND METHODS: Serum levels of Hsp72 and glucose were measured in five adolescents with type 1 diabetes mellitus (T1DM) prior to, during and following correction of severe DKA. Samples from nine relatively euglycemic T1DM patients served as controls. RESULTS: DKA pre-treatment samples showed significant elevation in Hsp72 (40.8 +/- 6.9 ng/ml) relative to euglycemic T1DM controls (33.6 +/- 3.2 ng/ml) (P < 0.05). Treatment resulted in a decline in Hsp72 to control levels within 24 h, with Hsp72 and glucose levels being tightly correlated (r = 0.9258). CONCLUSION: Extracellular Hsp72 is increased by DKA, paralleling changes in serum glucose levels.

Retinal vascular integrity following correction of diabetic ketoacidosis in children and adolescents.

OBJECTIVE: Increased permeability of the cerebral microvasculature occurs during the treatment of diabetic ketoacidosis (DKA). Microvascular changes consistent with diabetic retinopathy have been reported prior to and after the treatment of DKA. This study evaluated the structural and functional aspects of the retina immediately following the correction of DKA. METHODS: Seven young patients had comprehensive ophthalmologic examinations, including fluorescein angiography, within 24 h after the correction of severe DKA (pH <7.2). RESULTS: None of the patients had clinical, photographic, or angiographic evidence of a retinal abnormality. CONCLUSION: The blood-retinal barrier (BRB) does not experience the same degree of perturbation as the blood-brain barrier (BBB) does and may be a protected site during the insult of DKA and its treatment. The greater stability of the retinal microvasculature may be due to the increased number of pericytes in the BRB in comparison with the BBB.

Effects of Diabetic Ketoacidosis on Visual and Verbal Neurocognitive Function in Young Patients Presenting with New-Onset Type 1 Diabetes.

OBJECTIVE: To evaluate the effects of diabetic ketoacidosis (DKA) on neurocognitive functions in children and adolescents presenting with new-onset type 1 diabetes. METHODS: Newly diagnosed patients were divided into two groups: those with DKA and those without DKA (non-DKA). Following metabolic stabilization, the patients took a mini-mental status exam prior to undergoing a baseline battery of cognitive tests that evaluated visual and verbal cognitive tasks. Follow-up testing was performed 8-12 weeks after diagnosis. Patients completed an IQ test at follow-up. RESULTS: There was no statistical difference between the DKA and non-DKA groups neither in alertness at baseline testing nor in an IQ test at follow-up. The DKA group had significantly lower baseline scores than the non-DKA group for the visual cognitive tasks of design recognition, design memory and the composite visual memory index (VMI). At follow-up, Design Recognition remained statistically lower in the DKA group, but the design memory and the VMI tasks returned to statistical parity between the two groups. No significant differences were found in verbal cognitive tasks at baseline or follow-up between the two groups. Direct correlations were present for the admission CO2 and the visual cognitive tasks of VMI, design memory and design recognition. Direct correlations were also present for admission pH and VMI, design memory and picture memory. CONCLUSION: Pediatric patients presenting with newly diagnosed type 1 diabetes and severe but uncomplicated DKA showed a definite trend for lower cognitive functioning when compared to the age-matched patients without DKA.

Diabetic ketoacidosis and its treatment increase plasma 3-deoxyglucosone.

OBJECTIVES: Highly reactive dicarbonyl compounds are known to be increased by hyperglycemia, ketone bodies and lipid peroxidation. This study was carried out to investigate the effect of diabetic ketoacidosis (DKA) and its treatment on the plasma concentration of 3 deoxyglucosone (3-DG) one of the dicarbonyl compounds. DESIGN AND METHODS: 3-DG was measured in 7 children before, during and following correction of severe DKA. 3-DG was elevated before treatment (610 nmol/L +or/- 70) in comparison to baseline (120 h) (200 nmol/L+/or- 17) (p < 0.05). At 6 to 24 h into treatment 3-DG was further elevated (1080 nmol/L +or/- 80) in comparison to both pretreatment (p < 0.05) and baseline (p < 0.05). CONCLUSION: 3-DG is significantly elevated before the treatment of DKA and increases further during the treatment of DKA. The time course of the increase of 3-DG coincides with the time of progression of subclinical brain edema, which occurs in DKA.

Acetoacetate increases expression of intercellular adhesion molecule-1 (ICAM-1) in human brain microvascular endothelial cells.

It has been hypothesized that ketone bodies cause activation of brain endothelial cells and that this is a factor in the intracerebral crises of diabetic ketoacidosis (DKA). In this study we used cultured human brain microvascular endothelial cells (HBMEC) to investigate the effect of beta hydroxybutyrate (BOHB) and acetoacetate (AcAc) on the expression of the adhesion molecule, intercellular adhesion molecule-1 (ICAM-1). Increasing concentrations of AcAc, but not BOHB, caused a significant upregulation of ICAM-1 in comparison to unstimulated cells. Glucose concentrations of 10 and 30 mM, but not 50 mM, also resulted in increased expression of ICAM-1. These results support the hypothesis that activation of HBMEC is involved in the acute complications of DKA, and that ketone bodies and hyperglycemia are factors in the perturbed membrane function.

Lipid peroxidation and antioxidant vitamins prior to, during, and after correction of diabetic ketoacidosis.

Oxidative stress plays an important role in the chronic complications of insulin-dependent diabetes mellitus (IDDM). Hyperketonemia, as well as hyperglycemia, is involved in the generation of oxygen-free radicals. We have studied the degree of oxidative stress in six patients before, during, and after correction of diabetic ketoacidosis (DKA) by determining the plasma ratios of C20 and C18 fatty acids to C16 fatty acids in the cholesteryl esters of the lipoproteins as well as in the plasma concentrations of the antioxidant vitamins A, C, and E. Lipid peroxidation was slightly increased prior to treatment. However, the C20/C16 ratio at 120 h was significantly decreased in comparison to the ratio at pretreatment (P<.025), at 6-8 h (P<.005), and at 24 h (P<.025). The C18/16 ratio at 120 h was also decreased in comparison to the ratio at 6-8 h (P<.025), indicating an increase in lipid peroxidation after correction of DKA. Vitamin A was below normal at pretreatment and was increased at 120 h (P<.05). Vitamin C was normal at pretreatment and decreased to low normal at 24 h (P<.005). Vitamin E was normal at pretreatment and decreased to below normal at 24 and 120 h, although the changes were not statistically significant. These data demonstrate that there is an increase in lipid peroxidation after the correction of DKA and therefore support the position that administering antioxidant vitamins during the treatment of DKA could be beneficial in minimizing oxidative stress and possibly both the acute and chronic complications of IDDM.

Plasma C-reactive protein levels in severe diabetic ketoacidosis.

Elevated plasma levels of C-reactive protein (CRP) and IL-6 have been reported to be sensitive indicators of infection in adults with diabetic ketoacidosis (DKA). However, both CRP and the pro-inflammatory cytokines, which regulate CRP, can be elevated without infection. Our hypothesis was that CRP is increased in young patients with severe DKA, even in the absence of an infection, and may serve as a marker for systemic inflammatory response syndrome (SIRS). In 7 patients with severe DKA without infection, we measured plasma CRP, IL-6, IL-1beta and TNF-alpha levels prior to, during, and following correction of DKA. CRP was significantly but transiently elevated in 4 of the patients prior to or during treatment of DKA, compared to their baseline values (96 hr after correction of DKA). There were significant positive relationships between CRP and both IL-6 and IL-1beta prior to treatment (p <0.05); between CRP and IL-6, IL-1beta, and TNF-alpha at 6 hr (p <0.05); and between CRP and IL-1beta at 24 hr (p <0.05). The results support the hypothesis that CRP is increased in some patients by severe DKA and its treatment, and that DKA can be associated with a non-infectious form of SIRS.

Increased systemic Th17 cytokines are associated with diastolic dysfunction in children and adolescents with diabetic ketoacidosis.

Diastolic dysfunction suggestive of diabetic cardiomyopathy is established in children with T1DM, but its pathogenesis is not well understood. We studied the relationships of systemic inflammatory cytokines/chemokines and cardiac function in 17 children with T1DM during and after correction of diabetic ketoacidosis (DKA). Twenty seven of the 39 measured cytokines/chemokines were elevated at 6-12 hours into treatment of DKA compared to values after DKA resolution. Eight patients displayed at least one parameter of diastolic abnormality (DA) during acute DKA. Significant associations were present between nine of the cytokine/chemokine levels and the DA over time. Interestingly, four of these nine interactive cytokines (GM-CSF, G-CSF, IL-12p40, IL-17) are associated with a Th17 mediated cell response. Both the DA and CCL7 and IL-12p40, had independent associations with African American patients. Thus, we report occurrence of a systemic inflammatory response and the presence of cardiac diastolic dysfunction in a subset of young T1DM patients during acute DKA.

Oxidative damage is present in the fatal brain edema of diabetic ketoacidosis.

Oxidative stress is implicated as a pathogenic factor in a spectrum of chronic diseases, notably, neurodegenerative disease. Noteworthy in this regard is that type 1 diabetes mellitus (T1DM) results in oxidative stress, leading to systemic complications of T1DM. We hypothesized that oxidative stress associated with diabetic ketoacidosis (DKA) of T1DM might have measurable brain sequelae. Consistent with this hypothesis are neurohistology and neuroradiologic studies of T1DM that suggest oxidative insults are involved in the chronic complications of diabetic encephalopathy. To further address the role of oxidative stress in an acute setting, specifically in fatal brain edema (BE) associated with DKA, we studied neuronal localization and levels of oxidative stress markers reported to be increased in other neurodegenerative conditions. We demonstrated increased levels of 8-hydroxyguanosine (8OHG), 4-hydroxynonenal (HNE), and heme oxygenase-1 (HO-1) in the pyramidal neurons of the hippocampus of DKA BE in comparison to controls. However, in the cerebellum, only 8OHG was increased in the Purkinje cells and other cells of the molecular layer. These results indicate a role for oxidative stress in the pathogenesis of T1DM encephalopathy.

Autoantibody heritability in thyroiditis: IgG subclass contributions.

Using a simple screening technique called regression of offspring on mid-parent (ROMP) to examine the role of IgG subclasses in affected and unaffected siblings of children and adolescents with autoimmune thyroid disease and their parents, both total-restricted and subclass-restricted autoantibodies to thyroglobulin (Tg) were assayed quantitatively for each of the IgG subclasses. There was a significant correlation of anti-Tg titer of probands with parental titers in thyrotoxicosis (TT), (R(2) = 0.569, p = 0.001), but not in chronic lymphocytic thyroiditis. The most striking correlation was in TT patients of African-American ancestry, (R(2) = 0.9863, p = 0.0007). Additional insight is provided by examining the contributions of the IgG subclasses individually, particularly those whose concentrations appear not to have direct influence on the total IgG titers. Thus, using small numbers of patients, and assaying the IgG subclass distributions, as well as any other immunoglobulin isotypes that are significantly altered in autoantibody assays, ROMP can be performed rapidly to ascertain which quantifiable parameters may be usefully extended to predict disease onset and progression.

Insulin and IGF-1 receptors, nitrotyrosin and cerebral neuronal deficits in two young patients with diabetic ketoacidosis and fatal brain edema.

Gray and white matter structural deficits may accompany type 1 diabetes. Earlier experimental studies have demonstrated neuronal deficits associated with impaired neurotrophic support, inflammation and oxidative stress. In this study we demonstrate in two patients with histories of poorly controlled type 1 diabetes and fatal brain edema of ketoacidosis neuronal deficits associated with a decreased presence of insulin and IGF-1 receptors and accumulation of nitrotyrosin in neurons of affected areas and the choroid plexus. The findings add support to the suggested genesis of T1DM encephalopathy due to compromised neurotrophic protection, oxidative stress, inflammation and neuronal deficits, as demonstrated in T1DM encephalopathy in the BB/Wor-rat.