Origins of mitochondrial thymidine triphosphate: dynamic relations to cytosolic pools

Nuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments and on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear DNA are synthesized de novo from ribonucleotides and by salvage of thymidine in the cytosol. Mitochondria contain specific kinases for salvage of deoxyribonucleosides that may provide a compartmentalized synthesis of dNTPs. Here we investigate the source of intra-mt thymidine phosphates and their relationship to cytosolic pools by isotope-flow experiments with [3H]thymidine in cultured human and mouse cells by using a rapid method for the clean separation of mt and cytosolic dNTPs. In the absence of the cytosolic thymidine kinase, the cells (i) phosphorylate labeled thymidine exclusively by the intra-mt kinase, (ii) export thymidine phosphates rapidly to the cytosol, and (iii) use the labeled dTTP for nuclear DNA synthesis. The specific radioactivity of dTTP is highly diluted, suggesting that cytosolic de novo synthesis is the major source of mt dTTP. In the presence of cytosolic thymidine kinase dilution is 100-fold less, and mitochondria contain dTTP with high specific radioactivity. The rapid mixing of the cytosolic and mt pools was not expected from earlier data. We propose that in proliferating cells dNTPs for mtDNA come largely from import of cytosolic nucleotides, whereas intra-mt salvage of deoxyribonucleosides provides dNTPs in resting cells. Our results are relevant for an understanding of certain genetic mitochondrial diseases.     

Leukocyte Activation in Obese Patients: Effect of Bariatric Surgery

The rising prevalence of obesity is a major global health problem. In severe obesity, bariatric surgery (BS) allows to obtain a significant weight loss and comorbidities improvement, among them one of the factors is the thrombotic risk. In this observational study, we measured indices of leukocyte activation in severely obese patients as markers of increased thrombotic risk in relation with serum markers of inflammation before and after BS.Frequency of polymorphonuclear neutrophil-platelet (PLT) and monocyte (MONO)-PLT aggregates as well as of tissue factor (TF) expressing MONOs was measured in the peripheral blood of 58 consecutive obese patients and 30 healthy controls. In 31 of the 58 obese patients, data obtained at the enrollment were compared with those obtained at 3, 6, and 12 months after BS.Compared with healthy controls, obese patients showed a higher frequency of polymorphonuclear leukocyte (PMNL)-PLT aggregates (7.47 ± 2.45 [6.82–8.11]% vs 5.85 ± 1.89 [5.14–6.55]%, P = 0.001), MONO-PLT aggregates (12.31 ± 7.33 [10.38–14.24]% vs 8.14 ± 2.22 [7.31–8.97]%, P < 0.001), and TF expressing MONOs (4.01 ± 2.11 [3.45–4.56]% vs 2.64 ± 1.65 [2.02–3.25]%, P = 0.002). PMNL-PLT and MONO-PLT aggregate frequency was positively correlated with TF expressing MONOs (R² = 0.260, P = 0.049 and R² = 0.318, P = 0.015, respectively).BS was performed in 31 patients and induced a significant reduction of the body mass index, and waist and hip circumferences. These effects were associated with a significant decrease of PMNL-PLT aggregates at 12 months (7.58 ± 2.27 [6.75–8.42]% vs 4.47 ± 1.11 [3.93–5.01]%, P < 0.001), and a reduction of TF expressing MONOs at 6 (3.82 ± 2.04 [3.07–4.57]% vs 1.60 ± 1.69 [0.30–2.90]%, P = 0.008) and 12 months (3.82 ± 2.04 [3.07–4.57]% vs 1.71 ± 0.54 [1.45–1.97]%, P = 0.001) after BS.These data suggest that leukocyte-PLT aggregate formation and MONO activation represent an important mechanism underlying the increased thrombotic risk of obese patients. We also show that BS is effective in normalizing these inflammatory indices.     

A Surgical Option for Familial Chylomicronemia Associated with Insulin-Resistant Diabetes Mellitus

Background: The goal of the present work is to present an effective surgical approach for the treatment of a medically-resisitant form of hyperlipidemia. Methods: Two siblings with familial lipoprotein-lipase deficiency and subsequent hyperchylomicronemia, widespread skin xanthomas and severe insulin-resistant diabetes mellitus came to our observation after several unsuccessful attempts at medical treatment. In order to lower plasma lipids through lipid malabsorption, a modified bilio-pancreatic diversion operation was employed. The rationale in deciding to use this surgical approach was based also on the likely hypothesis that diabetes, in these subjects, was secondary to high circulating and tissue levels of lipids. Insulin sensitivity in the two treated subjects, as well as in 24 healthy volunteers constituting the control group, was assessed by euglycemic hyperinsulinemic clamp and indirect calorimetry, obtaining total end-clamp glucose uptake (M) and end-clamp glucose oxidation (ECGO) rates. Results: Within 3 weeks of surgery, plasma triglycerides and cholesterol levels had decreased from 4500 and 500 mg/dl (with dietary restrictions) to lower than 450 and 150 mg/dl (on a free, lipid-rich diet) respectively. Fasting plasma glucose levels had decreased from above 300 (under daily repeated subcutaneous injections of insulin) to 80-100 mg/dl (without administration of insulin or oral hypoglycemic agents). Body weight and fat free mass were maintained in both subjects after surgery. In both patients, before surgery M and ECGO were significantly lower than in normal subjects, while after surgery they were not significantly different from normal subjects, confirming the positive metabolic effect of the operation. Conclusion: The surgical option used in these patients may represent an interesting and effective new possibility for treatment of those severe cases of hyperlipemia leading otherwise to metabolic complications and a low quality of life.     

How to Choose the Best Metabolic Procedure?

Bariatric/metabolic surgery has proven to be effective in inducing and maintaining diabetes remission—although with a percentage of patients undergoing hyperglycemia relapse—weight loss, and improvement of the cardiovascular risk. It is, however, associated with mortality, although low, and early and late complications. In particular, metabolic complications are related to vitamin deficiency due to the erratic absorption of the supplemented vitamins and to the unpredictable compliance of patients to vitamin and trace element supplementation. In addition, often, the general practitioners and even the specialists are unaware of the clinical effects of metabolic dysfunction following malabsorptive surgery. The choice of the surgical procedure should be the balance between benefits and risks. Our review addresses this important question trying to give some suggestions.     

Surfactant Protein D, a Marker of Lung Innate Immunity, Is Positively Associated With Insulin Sensitivity

OBJECTIVE

Impaired lung function and innate immunity have both attracted growing interest as a potentially novel risk factor for glucose intolerance, insulin resistance, and type 2 diabetes. We aimed to evaluate whether surfactant protein D (SP-D), a lung-derived innate immune protein, was behind these associations.

RESEARCH DESIGN AND METHODS

Serum SP-D was evaluated in four different cohorts. The cross-sectional associations between SP-D and metabolic and inflammatory parameters were evaluated in two cohorts, the cross-sectional relationship with lung function in one cohort, and the longitudinal effects of weight loss on fasting and circadian rhythm of serum SP-D and cortisol concentrations in one prospective cohort.

RESULTS

In the cross-sectional studies, serum SP-D concentration was significantly decreased in subjects with obesity and type 2 diabetes (P = 0.005) and was negatively associated with fasting and postload serum glucose. SP-D was also associated with A1C, serum lipids, insulin sensitivity, inflammatory parameters, and plasma insulinase activity. Smoking subjects with normal glucose tolerance, but not smoking patients with type 2 diabetes, showed significantly higher serum SP-D concentration than nonsmokers. Serum SP-D concentration correlated positively with end-tidal carbon dioxide tension (r = 0.54, P = 0.034). In the longitudinal study, fasting serum SP-D concentration decreased significantly after weight loss (P = 0.02). Moreover, the main components of cortisol and SP-D rhythms became synchronous after weight loss.

CONCLUSIONS

These findings suggest that lung innate immunity, as inferred from circulating SP-D concentrations, is at the cross-roads of inflammation, obesity, and insulin resistance.Impaired lung function has attracted growing interest in association with metabolic disorders (1–6). Decreased lung function has been proposed as a potential novel risk factor for glucose intolerance, insulin resistance, and type 2 diabetes (1–6). In prospective studies of middle-aged men and women without known lung disease, lower vital capacity predicted the subsequent development of type 2 diabetes. Lower forced vital capacity and forced expiratory volume in 1 s at baseline predicted hyperinsulinemia and estimated insulin resistance over 20 years of follow-up, independent of age, adiposity, and smoking (1).Possible mechanisms for the hypothesized link include direct effects of hypoxemia on glucose and insulin regulation (7), adverse early-life exposures and their effects on organ development (8), and lung-related inflammatory mediators and their effects on insulin signaling (9). In fact, nuclear factor interleukin-6, early growth response-1, and hypoxia-inducible factor-1 mediate inflammatory responses to chronic hypoxia in macrophages, pulmonary vascular endothelium, and smooth muscle (6,9). Cigarette smoking, an independent predictor of type 2 diabetes (10), provokes an inflammatory response (11) and is inversely associated with vital capacity. However, the link between lower vital capacity and diabetes risk was completely independent of cigarette exposure and was stronger in never-smokers (6).Reduced vital capacity is a common residual effect of lower respiratory tract infections, including those in childhood and infancy (8), that might provoke an inflammatory response. A reduced ability to sense and eradicate pathogens could thus cause frequent respiratory tract infections, reduced vital capacity, and chronic inflammation resulting in insulin resistance and type 2 diabetes (12). The total incidence rate of infections needing hospitalization in diabetic patients was 41/1,000 persons-years compared with 16/1,000 person-years of follow-up in the general population. Roughly half of the infections were severe lung infections, suggesting impaired lung immunity in patients with type 2 diabetes (13).Pulmonary surfactant is a complex mixture of lipids (90%) and proteins (5–10%) that constitutes the mobile liquid phase covering the large surface area of the alveolar epithelium. It maintains minimal surface tension within the lungs to avoid lung collapse during respiration. The innate immune system, by upregulating SP-D synthesis, can immediately respond to intrusion of foreign agents by helping to prevent further invasion (14). This recognition is important in the day-to-day physiology. Each day, we breathe >7,000 liters of air, laden with inorganic and organic particles and an array of microbes. Secreted primarily by alveolar epithelial type II pneumocytes, plasma SP-D appears to increase early in the clinical course of lung injury, and its concentration is thought to reflect pulmonary epithelial injury (15).Subtle deficiencies in proteins of the sensing arm of the innate immune system have been found to be associated with alterations of glucose metabolism. These deficiencies run in parallel with inflammation and impaired insulin action (16).We hypothesized that SP-D could be behind the association of lung function with impaired insulin action. For that reason, we aimed to evaluate SP-D according to metabolic and inflammatory parameters. As SP-D was associated with obesity status and impaired glucose metabolism, we evaluated the influence of weight loss on both fasting and circadian serum SP-D concentration. As glucocorticoids seem to regulate SP-D production in in vitro studies (17), we investigated the influence of circadian cortisol rhythm on serum SP-D concentration. Finally, we also studied the association of SP-D with lung function tests.     

One-Hour Plasma Glucose Identifies Insulin Resistance and ��-Cell Dysfunction in Individuals With Normal Glucose Tolerance: Cross-sectional data from the Relationship between Insulin Sensitivity and Cardiovascular Risk (RISC) study

OBJECTIVE

Some individuals with normal glucose tolerance (NGT) exhibit a 1-h excursion of plasma glucose during oral glucose tolerance testing as high as that of individuals with impaired glucose tolerance (IGT). The aim of this study was to characterize their metabolic phenotype.

RESEARCH DESIGN AND METHODS

A total of 1,205 healthy volunteers (aged 29–61 years) underwent assessment of 1) oral glucose tolerance and 2) insulin sensitivity (standardized euglycemic-hyperinsulinemic clamp), as part of the Relationship between Insulin Sensitivity and Cardiovascular Risk (RISC) study.

RESULTS

One-hour plasma glucose correlated better than 2-h plasma glucose with total insulin secretion (r = 0.43), β-cell glucose sensitivity (r = −0.46), and β-cell rate sensitivity (r = −0.18). Receiver operating characteristic analysis identified 8.95 mmol/l as the best cutoff value for prediction of IGT from 1-h plasma glucose (sensitivity 77% and specificity 80%). Participants with NGT with 1-h plasma glucose >8.95 mmol/l had larger waist circumference, higher BMI, lower insulin sensitivity, higher fasting glucose, and higher insulin secretion than their counterparts with 1-h plasma glucose ≤8.95 mmol/l (P < 0.001 for all comparisons). Moreover, they exhibited lower β-cell glucose sensitivity (P < 0.001), β-cell rate sensitivity (P < 0.001), and potentiation factor (P = 0.026). When compared with conventionally defined IGT, they were not different in waist circumference and BMI, hepatic insulin extraction, β-cell glucose sensitivity, β-cell rate sensitivity, and potentiation factor but did have greater insulin sensitivity along with reduced basal (P = 0.001) and total insulin secretion (P = 0.002).

CONCLUSIONS

Higher values of 1-h plasma glucose may identify an intermediate condition between NGT and IGT characterized by greater insulin resistance, reduced β-cell glucose sensitivity, and reduced β-cell rate sensitivity.Impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) are states of carbohydrate metabolism intermediate between normal glucose tolerance (NGT) and type 2 diabetes, which represent two partially overlapping conditions with distinct metabolic characteristics (1,2). In IFG, there is marked hepatic insulin resistance with near-normal muscle insulin sensitivity, whereas this pattern is reversed in IGT (2). Although both conditions are characterized by reduced early-phase insulin secretion, there is an additional impairment of late-phase insulin secretion in IGT. Accordingly, individuals with IGT have a rapid early (30 min) rise in plasma glucose during an oral glucose tolerance test (OGTT) which continues to rise until 60 min (1-h plasma glucose) and thereafter remains ≥7.8 mmol/l (140 mg/dl) at 120 min (2-h plasma glucose).As longitudinal studies have demonstrated that 40% of patients who develop type 2 diabetes after 10 years have NGT at baseline (1), there may be additional information beyond conventional IFG/IGT categories that may better discriminate future progression to type 2 diabetes (3). We have noted a subset of individuals with NGT who have early glucose excursions during an OGTT as high as those observed in individuals with IGT. However, because plasma glucose concentrations decline adequately by 2 h, due to preservation of late-phase insulin secretion, these individuals do not have, by current definitions, any form of disordered carbohydrate metabolism (4). Data from the San Antonio Study have shown that β-cell glucose sensitivity and insulin sensitivity contribute to values of 2-h plasma glucose independently of each other (5); thus, we hypothesized that individuals with NGT with 1-h plasma glucose levels as high as in those with IGT might represent an intermediate phenotype of abnormal carbohydrate metabolism with either impaired insulin sensitivity or β-cell glucose sensitivity, who are potentially at increased risk of progression to type 2 diabetes.To investigate this hypothesis we analyzed cross-sectional data from the European Relationship between Insulin Sensitivity and Cardiovascular Risk (RISC) study (6), examining the metabolic phenotype of individuals with NGT who had high 1-h plasma glucose excursions. We aimed to identify a new glucose tolerance subgroup who might benefit from targeted lifestyle advice and/or pharmacological intervention.