Martin Krssak

IMPAIRED GLUCOSE TRANSPORT AS A CAUSE OF DECREASED INSULIN- STIMULATED MUSCLE GLYCOGEN SYNTHESIS IN TYPE 2 DIABETES

BACKGROUND Insulin resistance, a major factor in the pathogenesis of type 2 diabetes mellitus, is due mostly to decreased stimulation of glycogen synthesis in muscle by insulin. The primary rate-controlling step responsible for the decrease in muscle glycogen synthesis is not known, although hexokinase activity and glucose transport have been implicated. METHODS We used a novel nuclear magnetic resonance approach with carbon-13 and phosphorus-31 to measure intramuscular glucose, glucose-6-phosphate, and glycogen concentrations under hyperglycemic conditions (plasma glucose concentration, approximately 180 mg per deciliter [10 mmol per liter]) and hyperinsulinemic conditions in six patients with type 2 diabetes and seven normal subjects. In vivo microdialysis of muscle tissue was used to determine the gradient between plasma and interstitial-fluid glucose concentrations, and open-flow microperfusion was used to determine the concentrations of insulin in interstitial fluid. RESULTS The time course and concentration of insulin in interstitial fluid were similar in the patients with diabetes and the normal subjects. The rates of whole-body glucose metabolism and muscle glycogen synthesis and the glucose-6-phosphate concentrations in muscle were approximately 80 percent lower in the patients with diabetes than in the normal subjects under conditions of matched plasma insulin concentrations. The mean (+/-SD) intracellular glucose concentration was 2.0+/-8.2 mg per deciliter (0.11+/-0.46 mmol per liter) in the normal subjects. In the patients with diabetes, the intracellular glucose concentration was 4.3+/-4.9 mg per deciliter (0.24+/-0.27 mmol per liter), a value that was 1/25 of what it would be if hexokinase were the rate-controlling enzyme in glucose metabolism. CONCLUSIONS Impaired insulin-stimulated glucose transport is responsible for the reduced rate of insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes mellitus.

Muscle mitochondrial ATP synthesis and glucose transport/phosphorylation in type 2 diabetes.

Background Muscular insulin resistance is frequently characterized by blunted increases in glucose-6-phosphate (G-6-P) reflecting impaired glucose transport/phosphorylation. These abnormalities likely relate to excessive intramyocellular lipids and mitochondrial dysfunction. We hypothesized that alterations in insulin action and mitochondrial function should be present even in nonobese patients with well-controlled type 2 diabetes mellitus (T2DM). Methods and Findings We measured G-6-P, ATP synthetic flux (i.e., synthesis) and lipid contents of skeletal muscle with 31P/1H magnetic resonance spectroscopy in ten patients with T2DM and in two control groups: ten sex-, age-, and body mass-matched elderly people; and 11 younger healthy individuals. Although insulin sensitivity was lower in patients with T2DM, muscle lipid contents were comparable and hyperinsulinemia increased G-6-P by 50% (95% confidence interval [CI] 39%–99%) in all groups. Patients with diabetes had 27% lower fasting ATP synthetic flux compared to younger controls (p = 0.031). Insulin stimulation increased ATP synthetic flux only in controls (younger: 26%, 95% CI 13%–42%; older: 11%, 95% CI 2%–25%), but failed to increase even during hyperglycemic hyperinsulinemia in patients with T2DM. Fasting free fatty acids and waist-to-hip ratios explained 44% of basal ATP synthetic flux. Insulin sensitivity explained 30% of insulin-stimulated ATP synthetic flux. Conclusions Patients with well-controlled T2DM feature slightly lower flux through muscle ATP synthesis, which occurs independently of glucose transport /phosphorylation and lipid deposition but is determined by lipid availability and insulin sensitivity. Furthermore, the reduction in insulin-stimulated glucose disposal despite normal glucose transport/phosphorylation suggests further abnormalities mainly in glycogen synthesis in these patients.

Effects of obesity, diabetes and exercise on Fndc5 gene expression and irisin release in human skeletal muscle and adipose tissue: in vivo and in vitro studies

Considerable controversy exists regarding the role of irisin, a putative exercise‐induced myokine, in human metabolism. We therefore studied irisin and its precursor Fndc5 in obesity, type 2 diabetes and exercise. Complex clinical studies combined with cell culture work revealed that Fndc5/irisin was decreased in type 2 diabetes in vivo, but not in muscle cells in vitro, indicating that diabetes‐related factor(s) regulate Fndc5/irisin in vivo. Several attributes of type 2 diabetes, such as hyperglycaemia, triglyceridaemia, visceral adiposity and extramyocellular lipid deposition were negatively associated with adipose tissue Fndc5 mRNA and circulating irisin. Moreover, mimicking diabetic status in vitro by treating muscle cells with palmitate and glucose lowered Fndc5 mRNA. Neither exercise training nor an acute exercise bout modulated circulating irisin or muscle Fndc5 expression. However, the associations between intensity of habitual physical activity, muscle volume, strength, contractility and circulating irisin provide a link between irisin and positive outcomes of increased physical activity.

In utero tractography of fetal white matter development.

Diffusion tensor imaging (DTI) and tractography are noninvasive tools that enable the study of three-dimensional diffusion characteristics and their molecular, cellular, and microstructural correlates in the human brain. To date, these techniques have mainly been limited to postnatal MR studies of premature infants and newborns. The primary aim of this cross-sectional study was to assess the potential of in utero DTI and tractography to visualize the main projection and commissural pathways in 40 living, non-sedated human fetuses between 18 and 37 gestational weeks (GW) of age, with no structural brain pathologies. During a mean time of 1 min and 49 s, an axial, single-shot, echo planar DT sequence, with 32 diffusion gradient encoding directions and a reconstructed voxel size of 1.44 mm/1.45 mm/4.5 mm, was acquired. Most (90%) of the fetuses were imaged in the cephalic presentation. In 40% of examined fetuses, DTI measurements were robust enough to successfully calculate and visualize bilateral, craniocaudally oriented (mainly sensorimotor), and callosal trajectories in utero. Furthermore, fiber lengths, ADC, FA, and eigenvalues (lambda(1), lambda(2) and lambda(3)) were determined at different anatomically defined areas. FA values and the axial eigenvalue (lambda(1)) showed a characteristic distribution, with the highest values for the splenium, followed by the genu, the right, and the left posterior limb of the internal capsule. The right-sided sensorimotor trajectories were found to be significantly longer than on the left side (p=0.007), reflecting higher right-sided lambda(1) values (14 cases vs. 9 cases). Based on the good correlation of these initial in utero tractography results with prior documented postmortem and ex utero DTI data, this new imaging technique promises new insights into the normal and pathological development of the unborn child.

Abnormal hepatic energy homeostasis in type 2 diabetes

Increased hepatocellular lipids relate to insulin resistance and are typical for individuals with type 2 diabetes mellitus (T2DM). Steatosis and T2DM have been further associated with impaired muscular adenosine triphosphate (ATP) turnover indicating reduced mitochondrial fitness. Thus, we tested the hypothesis that hepatic energy metabolism could be impaired even in metabolically well‐controlled T2DM. We measured hepatic lipid volume fraction (HLVF) and absolute concentrations of γATP, inorganic phosphate (Pi), phosphomonoesters and phosphodiesters using noninvasive 1H/ 31P magnetic resonance spectroscopy in individuals with T2DM (58 ± 6 years, 27 ± 3 kg/m 2), and age‐matched and body mass index–matched (mCON; 61 ± 4 years, 26 ± 4 kg/m 2) and young lean humans (yCON; 25 ± 3 years, 22 ± 2 kg/m 2, P < 0.005, P < 0.05 versus T2DM and mCON). Insulin‐mediated whole‐body glucose disposal (M) and endogenous glucose production (iEGP) were assessed during euglycemic‐hyperinsulinemic clamps. Individuals with T2DM had 26% and 23% lower γATP (1.68 ± 0.11; 2.26 ± 0.20; 2.20 ± 0.09 mmol/L; P < 0.05) than mCON and yCON individuals, respectively. Further, they had 28% and 31% lower Pi than did individuals from the mCON and yCON groups (0.96 ± 0.06; 1.33 ± 0.13; 1.41 ± 0.07 mmol/L; P < 0.05). Phosphomonoesters, phosphodiesters, and liver aminotransferases did not differ between groups. HLVF was not different between those from the T2DM and mCON groups, but higher (P = 0.002) than in those from the yCON group. T2DM had 13‐fold higher iEGP than mCON (P < 0.05). Even after adjustment for HLVF, hepatic ATP and Pi related negatively to hepatic insulin sensitivity (iEGP) (r =‐0.665, P = 0.010, r =‐0.680, P = 0.007) but not to whole‐body insulin sensitivity. Conclusion: These data suggest that impaired hepatic energy metabolism and insulin resistance could precede the development of steatosis in individuals with T2DM. (HEPATOLOGY 2009.)

5-Aminolevulinic acid is a promising marker for detection of anaplastic foci in diffusely infiltrating gliomas with nonsignificant contrast enhancement.

Because of intratumoral heterogeneity, diffusely infiltrating gliomas that lack significant contrast enhancement on magnetic resonance imaging are prone to tissue sampling error. Subsequent histologic undergrading may delay adjuvant treatments. 5‐Aminolevulinic acid (5‐ALA) leads to accumulation of fluorescent porphyrins in malignant glioma tissue, and is currently used for resection of malignant gliomas. The aim of this study was to clarify whether 5‐ALA might serve as marker for visualization of anaplastic foci in diffusely infiltrating gliomas with nonsignificant contrast enhancement for precise intraoperative tissue sampling.

Liver ATP Synthesis Is Lower and Relates to Insulin Sensitivity in Patients With Type 2 Diabetes

OBJECTIVE Steatosis associates with insulin resistance and may even predict type 2 diabetes and cardiovascular complications. Because muscular insulin resistance relates to myocellular fat deposition and disturbed energy metabolism, we hypothesized that reduced hepatic ATP turnover (fATP) underlies insulin resistance and elevated hepatocellular lipid (HCL) contents. RESEARCH DESIGN AND METHODS We measured hepatic fATP using 31P magnetic resonance spectroscopy in patients with type 2 diabetes and age- and body mass–matched controls. Peripheral (M and M/I) and hepatic (suppression of endogenous glucose production) insulin sensitivity were assessed with euglycemic-hyperinsulinemic clamps. RESULTS Diabetic individuals had 29% and 28% lower peripheral and hepatic insulin sensitivity as well as 42% reduced fATP than controls. After adjusting for HCL, fATP correlated positively with peripheral and hepatic insulin sensitivity but negatively with waist circumference, BMI, and fasting plasma glucose. Multiple regression analysis identified waist circumference as an independent predictor of fATP and inorganic phosphate (PI) concentrations, explaining 65% (P = 0.001) and 56% (P = 0.003) of the variations. Hepatocellular PI primarily determined the alterations in fATP. CONCLUSIONS In patients with type 2 diabetes, insulin resistance relates to perturbed hepatic energy metabolism, which is at least partly accounted for by fat depots.

Lipid-dependent control of hepatic glycogen stores in healthy humans

Aims/hypothesis. Non-esterified fatty acids and glycerol could stimulate gluconeogenesis and also contribute to regulating hepatic glycogen stores. We examined their effect on liver glycogen breakdown in humans.¶Methods. After an overnight fast healthy subjects participated in three protocols with lipid/heparin (plasma non-esterified fatty acids: 2.2 ± 0.1 mol/l; plasma glycerol: 0.5 ± 0.03 mol/l; n = 7), glycerol (0.4 ± 0.1 mol/l; 1.5 ± 0.2 mol/l; n = 5) and saline infusion (control; 0.5 ± 0.1 mol/l; 0.2 ± 0.02 mol/l; n = 7). Net rates of glycogen breakdown were calculated from the decrease of liver glycogen within 9 h using 13C nuclear magnetic resonance spectroscopy. Endogenous glucose production was measured with infusion of D-[6,6-2H2]glucose.¶Results. Endogenous glucose production decreased by about 25 % during lipid and saline infusion (p < 0.005) but not during glycerol infusion (p < 0.001 vs lipid, saline). An increase of plasma non-esterified fatty acids or glycerol reduced the net glycogen breakdown by about 84 % to 0.6 ± 0.3 μmol · kg–1· min–1 (p < 0.001 vs saline: 3.7 ± 0.5 μmol · kg–1· min–1) and by about 46 % to 2.0 ± 0.4 μmol · kg–1· min–1 (p < 0.01 vs saline and lipid), respectively. Rates of gluconeogenesis increased to 11.5 ± 0.8 μmol · kg–1· min–1 (p < 0.01) and 12.8 ± 1.0 μmol · kg–1· min–1 (p < 0.01 vs saline: 8.2 ± 0.7 μmol · l–1· min–1), respectively.¶Conclusion/interpretation: An increase of non-esterified fatty acid leads to a pronounced inhibition of net hepatic glycogen breakdown and increases gluconeogenesis whereas glucose production does not differ from the control condition. We suggest that this effect is not due to increased availability of glycerol alone but rather to lipid-dependent control of hepatic glycogen stores. [Diabetologia (2001) 44: 48–54]

Contributions of net hepatic glycogenolysis and gluconeogenesis to glucose production in cirrhosis

Net hepatic glycogenolysis and gluconeogenesis were examined in normal ( n = 4) and cirrhotic ( n = 8) subjects using two independent methods [13C nuclear magnetic resonance spectroscopy (NMR) and a2H2O method]. Rates of net hepatic glycogenolysis were calculated by the change in hepatic glycogen content before (∼11:00 PM) and after (∼7:00 AM) an overnight fast using13C NMR and magnetic resonance imaging. Gluconeogenesis was calculated as the difference between the rates of glucose production determined with an infusion of [6,6-2H2]glucose and net hepatic glycogenolysis. In addition, the contribution of gluconeogenesis to glucose production was determined by the2H enrichment in C-5/C-2 of blood glucose after intake of2H2O (5 ml/kg body water). Plasma levels of total and free insulin-like growth factor I (IGF-I) and IGF-I binding proteins-1 and -3 were significantly decreased in the cirrhotic subjects ( P < 0.01 vs. controls). Postprandial hepatic glycogen concentrations were 34% lower in the cirrhotic subjects ( P = 0.007). Rates of glucose production were similar between the cirrhotic and healthy subjects [9.0 ± 0.9 and 10.0 ± 0.8 μmol ⋅ kg body wt-1 ⋅ min-1, respectively]. Net hepatic glycogenolysis was 3.5-fold lower in the cirrhotic subjects ( P = 0.01) and accounted for only 13 ± 6% of glucose production compared with 40 ± 10% ( P = 0.03) in the control subjects. Gluconeogenesis was markedly increased in the cirrhotic subjects and accounted for 87 ± 6% of glucose production vs. controls: 60 ± 10% ( P = 0.03). Gluconeogenesis in the cirrhotic subjects, as determined from the2H enrichment in glucose C-5/C-2, was also increased and accounted for 68 ± 3% of glucose production compared with 54 ± 2% ( P = 0.02) in the control subjects. In conclusion, cirrhotic subjects have increased rates of gluconeogenesis and decreased rates of net hepatic glycogenolysis compared with control subjects. These alterations are likely important contributing factors to their altered carbohydrate metabolism.

Reduced NAA-Levels in the NAWM of Patients with MS Is a Feature of Progression. A Study with Quantitative Magnetic Resonance Spectroscopy at 3 Tesla

Background Reduced N-acetyl-aspartate (NAA) levels in magnetic resonance spectroscopy (MRS) may visualize axonal damage even in the normal appearing white matter (NAWM). Demyelination and axonal degeneration are a hallmark in multiple sclerosis (MS). Objective To define the extent of axonal degeneration in the NAWM in the remote from focal lesions in patients with relapsing-remitting (RRMS) and secondary progressive MS (SPMS). Material and Methods 37 patients with clinical definite MS (27 with RRMS, 10 with SPMS) and 8 controls were included. We used 2D 1H-MR-chemical shift imaging (TR = 1500ms, TE = 135ms, nominal resolution 1ccm) operating at 3Tesla to assess the metabolic pattern in the fronto–parietal NAWM. Ratios of NAA to creatine (Cr) and choline (Cho) and absolute concentrations of the metabolites in the NAWM were measured in each voxel matching exclusively white matter on the anatomical T2 weighted MR images. Results No significant difference of absolute concentrations for NAA, Cr and Cho or metabolite ratios were found between RRMS and controls. In SPMS, the NAA/Cr ratio and absolute concentrations for NAA and Cr were significantly reduced compared to RRMS and to controls. Conclusions In our study SPMS patients, but not RRMS patients were characterized by low NAA levels. Reduced NAA-levels in the NAWM of patients with MS is a feature of progression.