Michaelis-Menten kinetic modeling of hemoglobin A1c status facilitates personalized glycemic control.

INTRODUCTION: Discrepancy between measured HbA1c and HbA1c calculated from plasma glucose is associated with higher risk for diabetic complications. However, quantification of this difference is inaccurate due to the imperfect linear conversion models. We propose to introduce a mathematical formula that correlates with the observational data and supports individualized glycemic control. METHODS: We analysed 175,437 simultaneous plasma glucose and HbA1c records stored in our laboratory database. Employing the Michaelis-Menten (MM) equation, we compared the calculated HbA1c levels to the measured HbA1c levels. Data from patients with multiple records were used to establish the patients' glycemic status and to assess the predictive power of our MM model. RESULTS: HbA1c levels calculated with the MM equation closely matched the population's average HbA1c levels. The Michaelis constant (Km) had a negative correlation with HbA1c (r2 = 0.403). Using personalized Km values in the MM equation, 85.1% of HbA1c predictions were within 20% error (ADAG calculation: 78.4%). MM prediction also performed better in predicting pathologic HbA1c levels (0.904 AUC vs. 0.849 AUC for ADAG). CONCLUSION: MM equation is an improvement over linear models and could be readily employed in routine diabetes management. Km is a reliable and quantifiable marker to characterize variations in glucose tolerance.

Oncodiabetology II. Antidiabetics and cancer prevention / Onkodiabetológia II. Antidiabetikumok és rákmegelozés

The number of patients with type 2 diabetes is increasing worldwide. In Hungary, the prevalence of known diabetic adults exceeds 9.1%, causing increased economical and medical burden to the society. It is obvious that there is a considerable urge to develop novel, safer and more efficient antidiabetic drugs. Therefore, studies have been focusing on the beneficial or detrimental side effects of antidiabetic drugs besides their general metabolic effects. Every anti-diabetic agent has an indirect anti-tumor effect as a consequence of lowering blood glucose levels and controlling carbohydrate, protein and lipid metabolism. In addition, most agents have their own direct antitumor effects, on the other hand, some may play a negligible role in cancer promotion. While the latter possibility is based mainly on pre-clinical, experimental data or on short-duration clinical studies, the informations about the safety of antidiabetic drugs are verified by large-scale, randomized, multicenter, placebo-controlled trials. Nowadays, metformin is the only drug that has been shown to reduce cancer risk in a variety of tumor localizations in monotherapy or in combination with other antidiabetic agents and insulins, and even in combination with certain cytostatics and biological therapies. The available data about the role of other antidiabetics in tumor prevention are less clear or insufficient. Here, we review the available ­ sometimes contradictory ­ literature about the relationship of tumor and antidiabet ics, verifying the safety of antidiabetics. Here, we propose that in the future tumor-specifically optimized antidiabetic treatment may play a role in tumor prevention or even in specific oncotherapy in patients with or without diabetes.

Oncodiabetology III. The relationship of antineoplastic therapies and carbohydrate metabolism / Onkodiabetológia III. Az antineoplasztikus terápiák és a szénhidrát-anyagcsere viszonya

The epidemiological indicators of malignant diseases and diabetes are changing similarly, as lately both have been dynamically increasing worldwide. They occur usually in the same patient synchronously or metachronously, because of their common metabolic and molecular background. Consequently, in more and more cases they require common treatment. That has led to a new science, called oncodiabetology, the main purpose of which is to optimize the combination of antineoplastic and antidiabetic therapies. Regarding the antineoplastic agents, their complex influence on metabolism has to be considered, especially diabetogenic side effects inducing insulin-resistance and decreasing insulin production. According to antidiabetic agents' role in preventing tumors, diminishing toxicity of cytostatic drugs, and promoting the breakthrough of chemoresistance should be considered. In this study, we investigate the contexts of antineoplastic agents' efficiency and the glucometabolism of the organization, the characteristics of oncotherapy in patients suffering from malignant disease and diabetes, and review those cytostatic agents, having massive diabetogenic adverse effects. We describe the properties and subtypes of secondary diabetes, thoroughly discuss the specific characteristics of hyperglycaemia and diabetes caused by malignant diseases and antineoplastic treatments, especially pancreatic diabetes. In the end, we attempt to determine the proper place and role of oncodiabetology in the treatment of patients suffering from malignancies. During our investigation, we assessed the effects on glucometabolism of the recently used classic cytostatics, molecularly targeted therapies and different endocrine manipulations treating malignancies. We reviewed the schedules and scientific background of almost 300 medicines for this aim. We established that every third antineoplastic agent influenced glucometabolism adversely. We report our further observations in our next reviews.

Oncodiabetology I. Metabolic and molecular relationships between cancer and diabetes / Onkodiabetológia I. Metabolikus és molekuláris összefüggések a rosszindulatú daganatok és a cukorbetegség között

In the recent decades, numerous studies have investigated the metabolic and molecular links between carbohydrate metabolic disorders and cancer, raising potential anti-tumor therapies. Based on epidemiological, preclinical, and clinical studies, now we know that advanced diabetes is a distinct risk factor of the development of many tumors, and even prediabetes may lead to the increased risk of developing cancer. Nowadays we can also state that the relationship is also present vice versa. It is a well-known fact that malignancies cause metabolic and molecular changes in the host over time resulting in an insulin-resistant state, characteristic of early diabetes. The tumor-induced insulin resistance may lead to the development of secondary diabetes in some patients with cancer. Furthermore, the diabetogenic ef-fect of the present anticancer therapies may worsen the metabolic condition. In recent years, research exploring the molecular causes of the correlation between malignancies and type 2 diabetes mellitus has highlighted the central role of RAS and PI3K signaling pathways. The altered function of these pathways significantly effects cell cycle, cellular metabolism, cell growth and proliferation, thus modifying cell survival, leading to tumorigenesis and tumor progres-sion and to insulin resistance as well. Without understanding the correlations between IGF receptors, RAS and PI3K signaling pathways the underlying molecular mechanism cannot be understood. Therefore, here we focus on these molecular mechanisms after a brief description of the most important metabolic connections between cancer and diabetes.

ECG alterations suggestive of hyperkalemia in normokalemic versus hyperkalemic patients.

BACKGROUND: In periarrest situations and during resuscitation it is essential to rule out reversible causes. Hyperkalemia is one of the most common, reversible causes of periarrest situations. Typical electrocardiogram (ECG) alterations may indicate hyperkalemia. The aim of our study was to compare the prevalence of ECG alterations suggestive of hyperkalemia in normokalemic and hyperkalemic patients. METHODS: 170 patients with normal potassium (K+) levels and 135 patients with moderate (serum K+ = 6.0-7.0 mmol/l) or severe (K+ > 7.0 mmol/l) hyperkalemia, admitted to the Department of Emergency Medicine at the Somogy County Kaposi Mór General Hospital, were selected for this retrospective, cross-sectional study. ECG obtained upon admission were analyzed by two emergency physicians, independently, blinded to the objectives of the study. Statistical analysis was performed using SPSS22 software. χ2 test and Fischer exact tests were applied. RESULTS: 24% of normokalemic patients and 46% of patients with elevated potassium levels had some kind of ECG alteration suggestive of hyperkalemia. Wide QRS (31.6%), peaked T-waves (18.4%), Ist degree AV-block (18.4%) and bradycardia (18.4%) were the most common and significantly more frequent ECG alterations suggestive of hyperkalemia in severely hyperkalemic patients compared with normokalemic patients (8.2, 4.7, 7.1 and 6.5%, respectively). There was no significant difference between the frequency of ECG alterations suggestive of hyperkalemia in normokalemic and moderately hyperkalemic patients. Upon examining ECG alterations not typically associated with hyperkalemia, we found that prolonged QTc was the only ECG alteration which was significantly more prevalent in both patients with moderate (17.5%) and severe hyperkalemia (21.1%) compared to patients with normokalemia (5.3%). CONCLUSIONS: A minority of patients with normal potassium levels may also exhibit ECG alterations considered to be suggestive of hyperkalemia, while more than half of the patients with hyperkalemia do not have ECG alterations suggesting hyperkalemia. These results imply that treatment of hyperkalemia in the prehospital setting should be initiated with caution. Multiple ECG alterations, however, should draw attention to potentially life threatening conditions.

Role of O-linked N-acetylglucosamine modification in diabetic nephropathy.

Increased O-linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) is a known contributor to diabetes; however, its relevance in diabetic nephropathy (DN) is poorly elucidated. Here, we studied the process and enzymes of O-GlcNAcylation with a special emphasis on Akt-endothelial nitric oxide synthase (eNOS) and heat shock protein (HSP)72 signaling. Since tubular injury is the prominent site of DN, the effect of hyperglycemia was first measured in proximal tubular (HK2) cells cultured in high glucose. In vivo O-GlcNAcylation and protein levels of O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), phosphorylated (p)Akt/Akt, peNOS/eNOS, and HSP72 were assessed in the kidney cortex of streptozotocin-induced diabetic rats. The effects of various renin-angiotensin-aldosterone system (RAAS) inhibitors were also evaluated. In proximal tubular cells, hyperglycemia-induced OGT expression led to increased O-GlcNAcylation, which was followed by a compensatory increase of OGA. In parallel, peNOS and pAkt levels decreased, whereas HSP72 increased. In diabetic rats, elevated O-GlcNAcylation was accompanied by decreased OGT and OGA. RAAS inhibitors ameliorated diabetes-induced kidney damage and prevented the elevation of O-GlcNAcylation and the decrement of pAkt, peNOS, and HSP72. In conclusion, hyperglycemia-induced elevation of O-GlcNAcylation contributes to the progression of DN via inhibition of Akt/eNOS phosphorylation and HSP72 induction. RAAS blockers successfully inhibit this process, suggesting a novel pathomechanism of their renoprotective action in the treatment of DN.

Acute regulation of cardiac metabolism by the hexosamine biosynthesis pathway and protein O-GlcNAcylation.

OBJECTIVE: The hexosamine biosynthesis pathway (HBP) flux and protein O-linked N-acetyl-glucosamine (O-GlcNAc) levels have been implicated in mediating the adverse effects of diabetes in the cardiovascular system. Activation of these pathways with glucosamine has been shown to mimic some of the diabetes-induced functional and structural changes in the heart; however, the effect on cardiac metabolism is not known. Therefore, the primary goal of this study was to determine the effects of glucosamine on cardiac substrate utilization. METHODS: Isolated rat hearts were perfused with glucosamine (0-10 mM) to increase HBP flux under normoxic conditions. Metabolic fluxes were determined by (13)C-NMR isotopomer analysis; UDP-GlcNAc a precursor of O-GlcNAc synthesis was assessed by HPLC and immunoblot analysis was used to determine O-GlcNAc levels, phospho- and total levels of AMPK and ACC, and membrane levels of FAT/CD36. RESULTS: Glucosamine caused a dose dependent increase in both UDP-GlcNAc and O-GlcNAc levels, which was associated with a significant increase in palmitate oxidation with a concomitant decrease in lactate and pyruvate oxidation. There was no effect of glucosamine on AMPK or ACC phosphorylation; however, membrane levels of the fatty acid transport protein FAT/CD36 were increased and preliminary studies suggest that FAT/CD36 is a potential target for O-GlcNAcylation. CONCLUSION/INTERPRETATION: These data demonstrate that acute modulation of HBP and protein O-GlcNAcylation in the heart stimulates fatty acid oxidation, possibly by increasing plasma membrane levels of FAT/CD36, raising the intriguing possibility that the HBP and O-GlcNAc turnover represent a novel, glucose dependent mechanism for regulating cardiac metabolism.

Evidence of O-linked N-acetylglucosamine in diabetic nephropathy.

AIMS: There is increasing evidence that O-linked N-acetylglucosamine (O-GlcNAc) plays an important role in cell signaling pathways. It has also been reported that increases in O-GlcNAc contribute to the development of diabetes and diabetic complications; however, little is known about O-GlcNAc levels in diabetic nephropathy (DNP). Therefore the goal of this study was to determine whether O-GlcNAc could be detected in human kidney biopsy specimens, and if so to examine whether O-GlcNAc levels were increased in the kidneys of patients with DNP compared to the non-diabetic individuals. MAIN METHODS: Kidney biopsy specimens were obtained from type-2 diabetic patients (n=6) and patients diagnosed with thin basement membrane nephropathy (n=7) were used as non-diabetic controls. O-GlcNAc levels were assessed by immunohistochemistry using the anti-O-GlcNAc antibody CTD110.6. KEY FINDINGS: We show that O-GlcNAc modification of proteins can be detected in the human kidney biopsy specimens. Furthermore, in diabetic patients, we found significantly increased numbers of O-GlcNAc positive cells in the glomeruli and significantly elevated staining in the tubuli (both in the nucleus and in the cytosol). In addition we also observed an intense, granular O-GlcNAc staining specifically in diabetic tubuli. SIGNIFICANCE: In light of the increase in O-GlcNAc staining in the diabetic patients, we propose that increased O-GlcNAc levels might contribute to the development of diabetic nephropathy.

Aging leads to increased levels of protein O-linked N-acetylglucosamine in heart, aorta, brain and skeletal muscle in Brown-Norway rats.

Changes in the levels of O-linked N-acetyl-glucosamine (O-GlcNAc) on nucleocytoplasmic protein have been associated with a number of age-related diseases such as Alzheimer's and diabetes; however, there is relatively little information regarding the impact of age on tissue O-GlcNAc levels. Therefore, the goal of this study was to determine whether senescence was associated with alterations in O-GlcNAc in heart, aorta, brain and skeletal muscle and if so whether there were also changes in the expression of enzymes critical in regulating O-GlcNAc levels, namely, O-GlcNAc transferase (OGT), O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase (GFAT). Tissues were harvested from 5- and 24-month old Brown-Norway rats; UDP-GlcNAc, a precursor of O-GlcNAc was assessed by HPLC, O-GlcNAc and OGT levels were assessed by immunoblot analysis and GFAT1/2, OGT, O-GlcNAcase mRNA levels were determined by RT-PCR. In the 24-month old animals serum insulin and triglyceride levels were significantly increased compared to the 5-month old group; however, glucose levels were unchanged. Protein O-GlcNAc levels were significantly increased with age (30-107%) in all tissues examined; however, paradoxically the expression of OGT, which catalyzes O-GlcNAc formation, was decreased by approximately 30% in the heart, aorta and brain. In the heart increased O-GlcNAc was associated with increased UDP-GlcNAc levels and elevated GFAT mRNA while in other tissues we found no difference in UDP-GlcNAc or GFAT mRNA levels. These results demonstrate that senescence is associated with increased O-GlcNAc levels in multiple tissues and support the notion that dysregulation of pathways leading to O-GlcNAc formation may play an important role in the development of age-related diseases.

Hexosamine biosynthesis and protein O-glycosylation: the first line of defense against stress, ischemia, and trauma.

An early and rapid response to severe injury or trauma is the development of hyperglycemia, which has long been thought to be an essential survival response by providing fuel for vital organ systems and facilitating mobilization of interstitial fluid reserves by increasing osmolarity. However, glucose can also be metabolized via the hexosamine biosynthesis pathway (HBP), leading to the synthesis of uridine diphosphate N-acetyl-glucosamine(UDP-GlcNAc). UDP-GlcNAc is a substrate for the addition, via an O-linkage, of a single N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins (O-glycosylation, O-GlcNAc). There is increasing appreciation that protein O-glycosylation is a highly dynamic posttranslational modification that plays a key role in signal transduction pathways. Sustained increases in O-GlocNAc have been implicated in the development of diabetes and diabetic complications; however, recent studies have demonstrated that stress leads to a transient increase in O-GlcNAc levels that is associated with increased tolerance to stress. Indeed, activation of pathways leading to O-GlcNAc formation improves cell survival after I/R injury, whereas inhibition of O-GlcNAc formation decreases cell survival. In addition, in rodent models of trauma-hemorrhage, increasing O-GlcNAc levels during resuscitation improves cardiac function and organ perfusion and attenuates the inflammatory response. At the cellular level, increasing O-GlcNAc levels attenuates nuclear factor-kappaB activation. It is noteworthy that other metabolic-based treatments for severe injury such as glucose-insulin-potassium and glutamine also lead to increased HBP flux and O-GlcNAc levels. The goal of this review is to summarize our current understanding of the role of the HBP and O-GlcNAc on the regulation of cell function and survival and to present evidence to support the notion that activation of these pathways represents a novel treatment strategy for severe injury and trauma.

Glucosamine administration improves survival rate after severe hemorrhagic shock combined with trauma in rats.

We have previously shown that glucosamine administration resulted in higher cardiac output and improved tissue perfusion after trauma-hemorrhage with resuscitation in rats, which was associated with the increased levels of protein O-linked-N-acetylglucosamine (O-GlcNAc). The purpose of the study was to evaluate the effect of glucosamine on the survival, without resuscitation, in rats. Adult male rats underwent midline laparotomy and 55% of total blood volume was withdrawn for 25 min under isoflurane anesthesia. At the end of the hemorrhage period, 2.5 mL of 150 mM glucosamine or equivalent osmolarity of mannitol solution was injected intravenously for 10 min. The survival time, mean blood pressure, heart rate, and central body temperature were monitored continuously; then, the O-GlcNAc levels in heart, brain, liver, and muscle were measured by means of Western blot analysis. Glucosamine administration significantly increased the survival rate in comparison with mannitol administration (percentage of survival after 2 h, 47% vs. 20%; P < 0.05). The mean arterial pressure was significantly higher in the glucosamine group for 18 min after treatment. The protein O-GlcNAc levels, assessed 30 min after glucosamine treatment, were significantly increased in the heart, brain, and liver. These data demonstrate that i.v. glucosamine administration improves the survival rate after trauma-hemorrhage without resuscitation; this effect may be related to the glucosamine-induced increase in protein O-glycosylation. Furthermore, the increase in mean arterial pressure may suggest a vasoactive and/or positive inotropic effect of glucosamine in hypovolemic shock.

Glucosamine cardioprotection in perfused rat hearts associated with increased O-linked N-acetylglucosamine protein modification and altered p38 activation.

We have shown that, in the perfused heart, glucosamine improved functional recovery following ischemia and that this appeared to be mediated via an increase in O-linked N-acetylglucosamine (O-GlcNAc) levels on nucleocytoplasmic proteins. Several kinase pathways, specifically Akt and the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2, which have been implicated in ischemic cardioprotection, have also been reported to be modified in response to increased O-GlcNAc levels. Therefore, the goals of this study were to determine the effect of ischemia on O-GlcNAc levels and to evaluate whether the cardioprotection resulting from glucosamine treatment could be attributed to changes in ERK1/2, Akt, and p38 phosphorylation. Isolated rat hearts were perfused with or without 5 mM glucosamine and were subjected to 5, 10, or 30 min of low-flow ischemia or 30 min of low-flow ischemia and 60 min of reperfusion. Glucosamine treatment attenuated ischemic contracture and improved functional recovery at the end of reperfusion. Glucosamine treatment increased flux through the hexosamine biosynthesis pathway and increased O-GlcNAc levels but had no effect on ATP levels. Glucosamine did not alter the response of either ERK1/2 or Akt to ischemia-reperfusion; however, it significantly attenuated the ischemia-induced increase in p38 phosphorylation and paradoxically increased p38 phosphorylation at the end of reperfusion. These data support the notion that O-GlcNAc may play an important role as an internal stress response and that glucosamine-induced cardioprotection may be mediated via the p38 MAPK pathway.

Role of protein O-linked N-acetyl-glucosamine in mediating cell function and survival in the cardiovascular system.

There is growing recognition that the O-linked attachment of N-acetyl-glucosamine (O-GlcNAc) on serine and threonine residues of nuclear and cytoplasmic proteins is a highly dynamic post-translational modification that plays a key role in signal transduction pathways. Numerous proteins have been identified as targets of O-GlcNAc modifications including kinases, phosphatases, transcription factors, metabolic enzymes, chaperons, and cytoskeletal proteins. Modulation of O-GlcNAc levels has been shown to modify DNA binding, enzyme activity, protein-protein interactions, the half-life of proteins, and subcellular localization. The level of O-GlcNAc is regulated in part by the metabolism of glucose via the hexosamine biosynthesis pathway (HBP), and the metabolic abnormalities associated with insulin resistance and diabetes, such as hyperglycemia, hyperlipidemia, and hyperinsulinemia, are all associated with increased flux through the HBP and elevated O-GlcNAc levels. Increased HBP flux and O-GlcNAc levels have been implicated in the impaired relaxation of isolated cardiomyocytes, blunted response to angiotensin II and phenylephrine, hyperglycemia-induced cardiomyocyte apoptosis, and endothelial and vascular cell dysfunction. In contrast to these adverse effects, recent studies have also shown that O-GlcNAc levels increase in response to acute stress and that this is associated with increased cell survival. Thus, while the relationship between O-GlcNAc levels and cellular function is complex and not well-understood, it is clear that these pathways play a critical role in the regulation of cell function and survival in the cardiovascular system and may be implicated in the adverse effects of metabolic disease on the heart.

Impact of Type 2 diabetes and aging on cardiomyocyte function and O-linked N-acetylglucosamine levels in the heart.

Increased levels of O-linked attachment of N-acetylglucosamine (O-GlcNAc) on nucleocytoplasmic proteins are implicated in the development of diabetic cardiomyopathy and are regulated by O-GlcNAc transferase (OGT) expression and its substrate UDP-GlcNAc. Therefore, the goal of this study was to determine whether the development of diabetes in the Zucker diabetic fatty (ZDF) rat, a model of Type 2 diabetes, results in defects in cardiomyocyte mechanical function and, if so, whether this is associated with increased levels of O-GlcNAc and increased OGT expression. Six-week-old ZDF rats were hyperinsulinemic but normoglycemic, and there were no differences in cardiomyocyte mechanical function, UDP-GlcNAc, O-GlcNAc, or OGT compared with age-matched lean control rats. Cardiomyocytes isolated from 22-wk-old hyperglycemic ZDF rats exhibited significantly impaired relaxation, compared with both age-matched lean control and 6-wk-old ZDF groups. There was also a significant increase in O-GlcNAc levels in high-molecular-mass proteins in the 22-wk-old ZDF group compared with age-matched lean control and 6-wk-old ZDF groups; this was associated with increased UDP-GlcNAc levels but not increased OGT expression. Surprisingly, there was a significant decrease in overall O-GlcNAc levels between 6 and 22 wk of age in lean, ZDF, and Sprague-Dawley rats that was associated with decreased OGT expression. These results support the notion that an increase in O-GlcNAc on specific proteins may contribute to impaired cardiomyocyte function in diabetes. However, this study also indicates that in the heart the level of O-GlcNAc on proteins appears to be differentially regulated by age and diabetes.