Meal-induced increases in C-reactive protein, interleukin-6 and tumour necrosis factor α are attenuated by prandial + basal insulin in patients with Type 2 diabetes.

AIM: To determine if a regimen with prandial + basal insulin compared with basal insulin attenuates post-meal inflammatory and glycative biomarkers in patients with Type 2 diabetes. METHODS: This test-meal sub-study in the USA is from a previously reported clinical trial comparing the effect on glycaemic control of 24 weeks of thrice-daily pre-meal insulin lispro mix 50 (50% insulin lispro, 50% insulin lispro protamine suspension) or bedtime insulin glargine, both plus metformin. In the sub-study, glucose, insulin, triglycerides, high-sensitivity C-reactive protein, tumour necrosis factor α, interleukin-6, methylglyoxal and 3-deoxyglucosone were measured during the post-meal period of a mixed-meal breakfast at the final visit. Prandial + basal (n = 25) and basal (n = 21) insulin were administered at the same times as during the previous 24 weeks. RESULTS: Post-meal, the prandial + basal insulin group had significantly higher insulin, lower glucose and triglycerides, as well as lower high-sensitivity C-reactive protein, tumour necrosis factor α and interleukin-6, than the basal insulin group. Glucose incremental area under the concentration curve significantly correlated with high-sensitivity C-reactive protein, tumour necrosis factor α, interleukin-6, methylglyoxal and 3-deoxyglucosone incremental area under the concentration curve. Insulin incremental area under the concentration curve correlated inversely with high-sensitivity C-reactive protein and tumour necrosis factor α incremental area under the concentration curve. However, after adjusting for glucose incremental area under the concentration curve, these inverse correlations were no longer significant. Triglyceride incremental area under the concentration curve was not correlated with any biomarker incremental area under the concentration curve. CONCLUSIONS: Controlling post-meal hyperglycaemia with prandial + basal insulin in patients with Type 2 diabetes attenuates meal-induced increases in high-sensitivity C-reactive protein, interleukin-6 and tumour necrosis factor α compared with basal insulin. The rise in post-meal glucose, but not triglycerides, significantly correlated with the rise in post-meal inflammatory and glycative biomarkers.

Degradation products of proteins damaged by glycation, oxidation and nitration in clinical type 1 diabetes.

AIMS/HYPOTHESIS: Hyperglycaemia in diabetes is associated with increased glycation, oxidative stress and nitrosative stress. Proteins modified consequently contain glycation, oxidation and nitration adduct residues, and undergo cellular proteolysis with release of corresponding free adducts. These free adducts leak into blood plasma for eventual renal excretion. The aim of this study was to perform a comprehensive quantitative analysis of protein glycation, oxidation and nitration adduct residues in plasma protein and haemoglobin as well as of free adducts in plasma and urine to quantify increased protein damage and flux of proteolytic degradation products in diabetes. METHODS: Type 1 diabetic patients (n=21) and normal healthy control subjects (n=12) were studied. Venous blood samples, with heparin anticoagulant, and 24-h urine samples were taken. Samples were analysed for protein glycation, oxidation and nitration adducts by a quantitative comprehensive screening method using liquid chromatography with triple quadrupole mass spectrometric detection. RESULTS: In type 1 diabetic patients, the concentrations of protein glycation, oxidation and nitration adduct residues increased up to three-fold in plasma protein and up to one-fold in haemoglobin, except for decreases in pentosidine and 3-nitrotyrosine residues in haemoglobin when compared with normal control subjects. In contrast, the concentrations of protein glycation and oxidation free adducts increased up to ten-fold in blood plasma, and urinary excretion increased up to 15-fold in diabetic patients. CONCLUSIONS/INTERPRETATION: We conclude that there are profound increases in proteolytic products of glycated and oxidised proteins in diabetic patients, concurrent with much lower increases in protein glycation and oxidation adduct residues.

Alpha-oxoaldehyde metabolism and diabetic complications.

The factors responsible for variable susceptibility to diabetic nephropathy are not clear. According to the non-enzymatic glycation hypothesis, diabetes-related tissue damage occurs due to a complex mixture of toxic products, including alpha-oxoaldehydes, which are inherently toxic as well as serving as precursors for advanced glycation end-products. Protective mechanisms exist to control this unavoidable glycation, and these are determined by genetic or environmental factors that can regulate the concentrations of the reactive sugars or end-products. In diabetes these protective mechanisms become more important, since glycation stress increases, and less efficient defence systems against this stress could lead to diabetic complications. Some of these enzymatic control mechanisms, including those that regulate alpha-oxoaldehydes, have been identified. We have observed significant increases in production of the alpha-oxoaldehydes methylglyoxal and 3-deoxyglucosone in three human populations with biopsy-proven progression of nephropathy. The increase in methylglyoxal could be secondary to defects in downstream glycolytic enzymes (such as glyceraldehyde-3-phosphate dehydrogenase) that regulate its production, or in detoxification mechanisms such as glyoxalase. Other mechanisms, however, appear to be responsible for the observed increase in 3-deoxyglucosone levels. We present results of our studies on the mechanisms responsible for variable production of alpha-oxoaldehydes by measuring the activity and characteristics of these enzymes in cells from complication-prone and -resistant diabetic patients. New therapeutic interventions designed to control these endogenous mechanisms could potentially enhance protection against excessive glycation and prevent or reverse complications of long-term diabetes.

Enzymatic deglycation--a new paradigm or an epiphenomenon?

Following the discovery of FN3K (fructosamine 3-kinase), and more recently of FN3KRP (FN3K-related protein), research in our laboratory has been focused on testing the enzymatic deglycation hypothesis and investigating the roles of FN3K and FN3KRP. Thus far, using human erythrocytes as a model system, we have obtained the following evidence of enzymatic deglycation: (a) production of GHb (glycated haemoglobin) by D-glucose in intact erythrocytes is 5-fold lower than in haemolysates; (b) glycation of GHb by D-glucose in intact erythrocytes is 5-fold lower than that by L-glucose; (c) inhibition of ATP production in erythrocytes leads to an acceleration in the rate of GHb production by D-glucose; and (d) inhibition of FN3K in erythrocytes by a competitive inhibitor increases the accumulation of GHb. In spite of these data supporting the enzymatic deglycation hypothesis, some outstanding issues remain. These include, among others, the fact that while the apparent deglycation mechanism does not operate on L-glucose, semi-purified FN3K appears to be able to use both D- and L-fructosamines as substrates. Moreover, analysis of the fructoselysine 3-phosphate content of haemoglobin from diabetic subjects suggests that, in addition to FN3K, another deglycating mechanism may be operative in human erythrocytes. Elucidation of these issues is a challenge in the evolving field of deglycation research. Most important, however, is the question of whether enzymatic deglycation is truly an important defence mechanism or merely an epiphenomenon.

Human fructosamine-3-kinase: purification, sequencing, substrate specificity, and evidence of activity in vivo.

Nonenzymatic glycation appears to be an important factor in the pathogenesis of diabetic complications. Key early intermediates in this process are fructosamines, such as protein-bound fructoselysines. In this report, we describe the purification and characterization of a mammalian fructosamine-3-kinase (FN3K), which phosphorylates fructoselysine (FL) residues on glycated proteins, to FL-3-phosphate (FL3P). This phosphorylation destabilizes the FL adduct and leads to its spontaneous decomposition, thereby reversing the nonenzymatic glycation process at an early stage. FN3K was purified to homogeneity from human erythrocytes and sequenced by means of electrospray tandem mass spectrometry. The protein thus identified is a 35-kDa monomer that appears to be expressed in all mammalian tissues. It has no significant homology to other known proteins and appears to be encoded by genomic sequences located on human chromosomes 1 and 17. The lability of FL3P, the high affinity of FN3K for FL, and the wide distribution of FN3K suggest that the function of this enzyme is deglycation of nonenzymatically glycated proteins. Because the condensation of glucose and lysine residues is an ubiquitous and unavoidable process in homeothermic organisms, a deglycation system mediated by FN3K may be an important factor in protecting cells from the deleterious effects of nonenzymatic glycation. Our sequence data of FN3K are in excellent agreement with a recent report on this enzyme by Delpierre et al. (Diabetes 49:1627-1634, 2000).

Glycated proteins in diabetes.

The term nonenzymatic glycation (of protein) refers to a wide variety of spontaneous reactions between reducing sugars and protein-bound amines. This reaction has been documented in humans and plays a role in the development of diabetic complications and perhaps in some of the degenerative processes of aging. In addition to the monocarbonyl sugars and their derivatives, an additional source of glycation is alpha-dicarbonyls. Over time, nonenzymatic glycation leads to the formation of irreversible terminal products known collectively as advanced glycation end-products (AGE) and extensive data on the role of AGEs in the etiology of diabetic complications exist. Our improved ability to measure alpha-dicarbonyls and specific AGEs may provide new and more powerful tools to monitor diabetes and predict diabetic complications in the future.

alpha-Dicarbonyls increase in the postprandial period and reflect the degree of hyperglycemia.

OBJECTIVE: Chronic hyperglycemia is known to increase tissue glycation and diabetic complications, but controversy exists regarding the independent role of increased postprandial glucose excursions. To address this question, we have studied the effect of postprandial glycemic excursions (PPGEs) on levels of methylglyoxal (MG) and 3-deoxyglucosone (3-DG), two highly reactive precursors of advanced glycation end products (AGEs). RESEARCH DESIGN AND METHODS: We performed 4-month crossover studies on 21 subjects with type 1 diabetes and compared the effect of premeal insulin lispro or regular insulin on PPGEs and MG/3-DG excursions. PPGE was determined after standard test meal (STMs) and by frequent postprandial glucose monitoring. HbA1c and postprandial MG and D-lactate were measured by HPLC, whereas 3-DG was determined by gas chromatography/mass spectroscopy. RESULTS: Treatment with insulin lispro resulted in a highly significant reduction in PPGEs relative to the regular insulin-treated group (P = 0.0005). However, HbA1c levels were similar in the two groups, and no relationship was observed between HbA1c and PPGE (P = 0.93). Significant postprandial increases in MG, 3-DG, and D-lactate occurred after the STM. Excursions of MG and 3-DG were highly correlated with levels of PPGE (R = 0.55, P = 0.0002 and R = 0.61, P = 0.0004; respectively), whereas a significant inverse relationship was seen between PPGE and D-lactate excursions (R = 0.40, P = 0.01). Conversely, no correlation was observed between HbAlc and postprandial MG, 3-DG, or D-lactate levels. CONCLUSIONS: Increased production of MG and 3-DG occur with greater PPGE, whereas HbA1c does not reflect these differences. Reduced PPGE also leads to increased production of D-lactate, indicating a role for increased detoxification in reducing MG levels. The higher postprandial levels of MG and 3-DG observed with greater PPGE may provide a partial explanation for the adverse effects of glycemic lability and support the value of agents that reduce glucose excursions.

Metformin reduces systemic methylglyoxal levels in type 2 diabetes.

Methylglyoxal (MG) is a reactive alpha-dicarbonyl that is thought to contribute to diabetic complications either as a direct toxin or as a precursor for advanced glycation end products. It is produced primarily from triose phosphates and is detoxified to D-lactate (DL) by the glyoxalase pathway. Because guanidino compounds can block dicarbonyl groups, we have investigated the effects of the diamino biguanide compound metformin and of hyperglycemia on MG and its detoxification products in type 2 diabetes. MG and DL were measured by high-performance liquid chromatography in plasma from 57 subjects with type 2 diabetes. Of these subjects, 27 were treated with diet, sulfonylureas, or insulin (nonmetformin), and 30 were treated with metformin; 28 normal control subjects were also studied. Glycemic control was determined by HbA1c. MG was significantly elevated in diabetic subjects versus the normal control subjects (189.3 +/- 38.7 vs. 123.0 +/- 37 nmol/l, P = 0.0001). MG levels were significantly reduced by high-dosage (1,500-2,500 mg/day) metformin (158.4 +/- 44.2 nmol/l) compared with nonmetformin (189.3 +/- 38.7 nmol/l, P = 0.03) or low-dosage (< or = 1,000 mg/day) metformin (210.98 +/- 51.0 nmol/l, P = 0.001), even though the groups had similar glycemic control. Conversely, DL levels were significantly elevated in both the low- and high-dosage metformin groups relative to the nonmetformin group (13.8 +/- 7.7 and 13.4 +/- 4.6 vs. 10.4 +/- 3.9 micromol/l, P = 0.03 and 0.06, respectively). MG correlated with rising HbA1c levels (R = 0.4, P = 0.03, slope = 13.2) in the nonmetformin subjects but showed no increase with worsening glycemic control in the high-dosage metformin group (R = 0.0004, P = 0.99, slope = 0.02). In conclusion, MG is elevated in diabetes and relates to glycemic control. Metformin reduces MG in a dose-dependent fashion and minimizes the effect of worsening glycemic control on MG levels. To the extent that elevated MG levels lead to their development, metformin treatment may protect against diabetic complications by mechanisms independent of its antihyperglycemic effect.

Quantitation of 3-deoxyglucosone levels in human plasma.

3-Deoxyglucosone (3DG), a reactive dicarbonyl, is an important intermediate in the formation of advanced glycation end products (AGEs). The AGEs are particularly important in diabetes since they have been correlated with the development of diabetic complications. Consequently, measurements of 3DG are likely to provide valuable insights into the role of this metabolite in the etiology of diabetic complications. While several methods of 3DG quantitation in human plasma have been previously published, a significant discrepancy (over 30-fold) exists in the reported values. Knecht et al. (Arch. Biochem. Biophys. 294, 130-137, 1992) have reported the levels of plasma 3DG in normoglycemics to be 61 nM, using a GC/MS procedure. In contrast to this, Niwa et al. (Biochem. Biophys. Res. Commun. 196, 837-843, 1993) reported 3DG levels to be 1800 nM in normoglycemics, using a totally independent GC/MS method. To resolve this disagreement and fill the need for a robust assay for this dicarbonyl, suitable for absolute quantitation, a GC/MS procedure was devised for its measurement. Plasma samples were deproteinized either by ultrafiltration or by addition of ethanol as described by Niwa et al. (Biochem. Biophys. Res. Commun. 196, 837-843, 1993). 3DG in the ultrafiltrate or the supernatant was conjugated with 2,3-diamino-naphthalene to produce a stable adduct which was then converted to a silyl ether and analyzed by GC/MS. The analyte was monitored by selected ion monitoring at an m/z of 295 and 306 and quantitated using an internal standard of [U-13C]3DG. Using this approach, 3DG levels in plasma deproteinized by ultrafiltration were found to be significantly elevated from 58.5 +/- 14 (SD) nM in normoglycemics to 98.5 +/- 34 (SD) nM in type I diabetics. When deproteinization of the plasma was carried out using ethanol, the levels of 3DG from normoglycemic plasma were similar to those reported by Niwa et al. (1710 +/- 750 (SD) nM). These results suggest that 3DG levels measured by ultrafiltration may represent the free circulating 3DG and those obtained by ethanol extraction may represent aform of 3DG bound to a macromolecule (presumbaly protein).

Formation of immunochemical advanced glycosylation end products precedes and correlates with early manifestations of renal and retinal disease in diabetes.

Elevated levels of advanced glycosylation end products (AGEs) have been found in multiple tissues in association with diabetic vascular complications and during the microalbuminuric phase of diabetic nephropathy. In this study, we have used an AGE-specific enzyme-linked immunosorbent assay (ELISA) to measure skin AGEs to determine whether elevated levels can be detected before the onset of overt microangiopathy. Subjects with type I diabetes (n = 48) were graded for the degree of nephropathy (normal [23], microalbuminuria [12], or macroalbuminuria [12]) and retinopathy (none [13], background [20], or proliferative [15]). Subgroups with a premicroalbuminuric phase of albumin excretion (< or = 28 mg/24 h, n = 27) or with the earliest stages of retinopathy (n = 27) were identified. A significant increase in tissue AGEs was found as urinary albumin increased during the premicroalbuminuric phase of nephropathy even when the data were adjusted for age and duration of diabetes (P = 0.005). Immunoreactive AGEs also increased as normal renal status advanced to microalbuminuria and macroalbuminuria (P = 0.0001 across groups). Significant elevation of AGEs was also found in association with the earliest stages of clinically evident retinopathy (early background versus minimal grades). In addition, higher AGE levels were found in subjects with proliferative retinopathy when compared with those with less severe retinopathy (P < 0.004 across groups). In contrast, no significant differences were found in tissue AGE levels between groups with or without early retinopathy based on pentosidine or fluorescent AGE measurements, although fluorescent AGEs correlated with albumin excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycosylated serum proteins and glycosylated hemoglobin in the assessment of glycemic control in insulin-dependent and non-insulin-dependent diabetes mellitus.

To evaluate the relative value of glycosylated serum proteins (GSPs) versus glycosylated hemoglobin (HbA1c) in assessing glycemic control in diabetes mellitus, we performed regular monitoring of GSPs and HbA1c in 30 subjects with insulin-dependent diabetes mellitus (IDDM) or non-insulin-dependent diabetes mellitus (NIDDM) who performed frequent self-glucose monitoring. Analysis of the relationship between patterns of glycemic control and GSPs and HbA1c demonstrated that subjects with IDDM and NIDDM appeared similar when the more traditional indicators of glycemic control such as mean blood glucose level (166.9 +/- 20.9 v 177.4 +/- 39.6 mg/dL) or HbA1c (83.57 +/- 12.8 v 80.24 +/- 15.7 mmol hydroxymethyl furfuraldehyde [HMF]/mol hemoglobin [Hgb]) were used. However, when GSP levels or the standard deviation of mean glucose levels (SDMG) were used to assess glycemic control, higher levels were found in subjects with IDDM (52 +/- 10.3 mg/g protein and 28.59 +/- 7.60 mg/dL) versus NIDDM (44.6 +/- 15.2 mg/g protein and 21.6 +/- 15.9 mg/dL). Using multivariate analysis, GSPs were predictive of SDMG (P = .046), whereas HbA1c added no significant further information (P = .27). Our results suggest that GSPs may be more sensitive than HbA1c assay to the greater fluctuations in blood glucose levels generally associated with IDDM.

Increased collagen-linked pentosidine levels and advanced glycosylation end products in early diabetic nephropathy.

RATIONALE: Advanced glycosylation end products (AGEs) may play an important role in the development of diabetic vascular sequelae. An AGE cross-link, pentosidine, is a sensitive and specific marker for tissue levels of AGEs. OBJECTIVES: To evaluate the role of AGEs in the development of diabetic nephropathy and retinopathy, we studied pentosidine levels and the clinical characteristics of 48 subjects with insulin-dependent diabetes mellitus. Diabetic nephropathy was classified as normal, microalbuminuria, or gross proteinuria, and retinopathy was graded as none, background, or proliferative. NEWLY OBSERVED FINDINGS: Significant elevation of pentosidine (P = 0.025) was found in subjects with microalbuminuria or gross proteinuria (73.03 +/- 9.47 vs 76.46 +/- 6.37 pmol/mg col) when compared with normal (56.96 +/- 3.26 pmol/mg col). Multivariate analysis to correct for age, duration of diabetes, and gender did not modify the results. Elevated pentosidine levels were also found in those with proliferative when compared with those with background retinopathy (75.86 +/- 5.66 vs 60.42 +/- 5.98 pmol/mg col) (P < 0.05). CONCLUSIONS: Microalbuminuria is associated with elevated levels of pentosidine similar to those found in overt diabetic nephropathy suggesting that elevated AGE levels are already present during the earliest detectable phase of diabetic nephropathy.

Relationship between glycemic control and collagen-linked advanced glycosylation end products in type I diabetes.

OBJECTIVE: To evaluate the relationship between glycemic control over a 3-yr period and tissue levels of advanced glycosylation end products. The development of renal failure, blindness, and generalized vascular occlusion continue to be the most serious ravages of diabetes. Tissue glycosylation and AGEs are felt to play an important role in the development of these sequelae, but no data are available on the relationship between AGEs and long-term glycemic control. RESEARCH DESIGN AND METHODS: We studied 48 subjects with type I diabetes. Glycemic control was determined by mean levels of HbA1, and AGEs were determined on collagenase digests of skin collagen by fluorescence at excitation/emission readings of 335/385 and 370/440 nm. RESULTS: To evaluate the relationship between glycemic control and AGE levels, control was classified as good (< or = 8.5%), fair (> 8.5% but < or = 10%), or poor (> 10%) on the basis of mean HbA1 levels during 1- and 3-yr periods. Analysis of the mean AGE levels for each level of glycemic control over 1-3 yr showed that AGEs differed significantly across categories of glycemic control (P = 0.04 and 0.003), with the lowest AGE levels associated with good and the highest with poor glycemic control. The relationship also was highly significant when adjusted for age, sex, and duration of diabetes, and when examined by Pearson's correlation coefficients (P = 0.02 and 0.008). CONCLUSIONS: Finding a relationship between glycemic control over 1-3 yr and tissue levels of AGEs suggests that AGEs can be modified and possibly reversed by improved glucose levels.

Evaluation of first-generation sulfonylureas and glipizide in non-insulin-dependent diabetes mellitus.

The current study examines the impact of glipizide, a second-generation sulfonylurea, on diabetes control in patients in whom adequate control was not achieved while receiving treatment with first-generation agents. The interim results of this multicenter study are presented in which patients in whom euglycemia was not achieved based on fasting and two-hour postprandial plasma glucose criteria with first-generation sulfonylureas were given glipizide therapy for 24 weeks. Twenty-three percent of the patients who had only fair results with first-generation sulfonylureas (fasting plasma glucose level of more than 140 mg/dl, postprandial plasma glucose level of more than 175 mg/dl) had good to excellent results with glipizide therapy alone (fasting plasma glucose level of less than 140 mg/dl or less than 115 mg/dl, respectively). Overall, 15.4 percent of patients with fair to poor control using first-generation agents had good to excellent control using glipizide. At least a 25-mg/dl decrease in fasting plasma glucose and postprandial plasma glucose levels was also seen in 47 and 46 percent of patients given treatment with glipizide who had poor control (fasting plasma glucose level of more than 200 mg/dl, postprandial plasma glucose level of more than 235 mg/dl) with first-generation agents. Glipizide was extremely well tolerated, with no significant side effects. Preliminary data indicate that glipizide may provide a significant advantage in terms of safety and efficacy over first-generation agents.

Glomerular basement membrane: biosynthesis and chemical composition in the streptozotocin diabetic rat.

To study the effect of streptozotocin induced diabetes on glomerular basement membrane (GBM) synthesis, an isolated rat glomerular preparation has been developed, and its metabolic properties have been defined. The chemical composition of normal rat GBM isolated from this preparation closely resembles human GBM. Incubation with [U-14C] lysine leads to prompt incorporation of label into GBM and the subsequent appearance of labeled hydroxylysine. A 1-h lag before detection of labeled hydroxylysine in GBM suggests a delay in the release of GBM precursors. Significantly lower counts appeared in the nondialyzable fraction of the medium than in insoluble GBM during pulse-chase experiments, and labeled hydroxylysine accounted for a lower portion of the total counts in the medium (0.85%) than in the GBM (1.98%). Isolated glomeruli were prepared from streptozotocin diabetic rats of 4-6 wks duration. After incubation with [ U-14C] lysine recovery of label in diabetic GBM (88.98+/-8.26 nmol/g GBM) did not differ from age matched controls (82.52 +/- 8.26 nmol/g GBM). In pulse-chase experiments recovery of label in hydroxylysine of diabetic GBM (o.473 +/- 0.082 nmol/g GBM) did not differ from age matched controls (0567+/-0.065 nmol/g GBM). These findings indicate normal rates of GBM synthesis and hydroxylation of lysine residues in animals with streptozotocin diabetes.

Neuropathic and vascular complications occurring in diabetes.

Vascular and neuropathic complications of diabetes are a significant cause of morbidity and mortality. Symmetric polyneuropathy is the most common diabetic neuropathy. Treatment of the mononeuropathies consists of pain control and physical therapy to maintain muscle tone. Prognosis for recovery is excellent. Renal and retinal microangiopathy produce most of the clinically significant mortality and morbidity in diabetes. Recent advances in chronic hemodialysis and renal transplantation have improved the outlook for diabetics with end-stage nephropathy. The poor prognosis for retention of vision in diabetic malignant retinopathy has led to exploration of various forms of palliative therapy, including pituitary ablation, xenon arc coagulation, and laser treatment. Cardiovascular disease is more prevalent among diabetics than among the general population, according to a recent study, and mortality from this cause is three times higher. Animal studies linking aortic wall metabolism and atherosclerotic changes with hyperglycemia suggest that poor control of diabetes may play a role in the development of vascular lesions.