Werner Waldhäusl

High-Glucose–Triggered Apoptosis in Cultured Endothelial Cells

High ambient glucose concentration, linked to vascular complications in diabetes in vivo, modulates mRNA expression of fibronectin, collagen, tissue-type plasminogen activator, and plasminogen activator inhibitor and induces delayed replication and excess cell death in cultured vascular endothelial cells. To determine the role of high ambient glucose (30 mmol/1) in apoptosis, paired cultures of individual isolates of human umbilical vein endothelial cells (HUVECs) were exposed to both high (30 mmol/1) and low (5 mmol/1) concentrations of glucose for short-term (24, 48, and 72 h) and long-term (13 ± 1 days) experiments. Incubation of HUVECs with high glucose for >48 h increased DNA fragmentation (13.7 ± 6.5% of total DNA, mean ± SD) versus cultures kept in 5 mmol/1 glucose (10.9 ± 5.6%, P < 0.005), as measured by [3H]thymidine assays. Data were confirmed by apoptosis-specific fluorescence-activated cell sorter analysis of confluent HUVEC cultures, which displayed after long-term exposure to 30 mmol/1 glucose a 1.5-fold higher prevalence of apoptosis than control cultures exposed to 5 mmol/1 glucose (P < 0.005). In contrast, no increase in DNA fragmentation in response to 30 mmol/1 glucose was seen for standardized cell lines (K 562, P 815, YT) and fibroblasts. Expression of clusterin mRNA, originally reported to be a molecular marker of apoptosis, was only slightly affected by short-term (24-h) high-glucose exposure but was significantly reduced after long-term incubation in 30 mmol/1 glucose (82.2 ± 13.8% of control) versus 5 mmol/1 glucose, which questions the role of clusterin gene expression as a marker of apoptosis. The results demonstrate that high ambient glucose can promote apoptosis in HUVECs in vitro and suggest potential endothelial damage by hyperglycemia in diabetic patients.

Adipose tissue inflammation induced by high-fat diet in obese diabetic mice is prevented by n−3 polyunsaturated fatty acids

Aims/hypothesisInflammatory alterations in white adipose tissue appear to underlie complications of obesity including diabetes mellitus. Polyunsaturated fatty acids (PUFA), particularly those of the n−3 series, modulate immune responses and may ameliorate insulin sensitivity. In this study, we investigated how PUFA affect white adipose tissue inflammation and gene expression in obese diabetic animals.Materials and methodsWe treated db/db mice as well as lean non-diabetic mice (db/+) with either low-fat standard diet (LF) or high-fat diets rich in (1) saturated/monounsaturated fatty acids (HF/S), (2) n−6 PUFA (HF/6) and (3) the latter including purified marine n−3 PUFA (HF/3).ResultsMany genes involved in inflammatory alterations were upregulated in db/db mice on HF/S compared with LF in parallel with phosphorylation of c-Jun N-terminal kinase (JNK). In parallel, adipose tissue infiltration with macrophages was markedly enhanced by HF/S. When compared with HF/S, HF/6 showed only marginal effects on adipose tissue inflammation. However, inclusion of n−3 PUFA in the diet (HF/3) completely prevented macrophage infiltration induced by high-fat diet and changes in inflammatory gene expression, also tending to reduce JNK phosphorylation (p<0.1) in diabetic mice despite unreduced body weight. Moreover, high-fat diets (HF/S, HF/6) downregulated expression and reduced serum concentrations of adiponectin, but this was not the case with n−3 PUFA.Conclusions/interpretationn−3 PUFA prevent adipose tissue inflammation induced by high-fat diet in obese diabetic mice, thereby dissecting obesity from adipose tissue inflammation. These data suggest that beneficial effects of n−3 PUFA on diabetes development could be mediated by their effect on adipose tissue inflammation.

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.

LAT Displacement from Lipid Rafts as a Molecular Mechanism for the Inhibition of T Cell Signaling by Polyunsaturated Fatty Acids

Polyunsaturated fatty acids (PUFAs) suppress immune responses and inhibit T cell activation through largely unknown mechanisms. The displacement of signaling proteins from membrane lipid rafts has recently been suggested as underlying PUFA-mediated T cell inhibition. We show here that PUFA treatment specifically interferes with T cell signal transduction by blocking tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase Cγ1. A significant fraction of LAT was displaced from rafts by PUFA treatment along with other signaling proteins. However, retaining LAT alone in lipid rafts effectively restored phospholipase Cγ1/calcium signaling in PUFA-treated T cells. These data reveal LAT displacement from lipid rafts as a molecular mechanism by which PUFAs inhibit T cell signaling and underline the predominant importance of LAT localization in rafts for efficient T cell activation.

Severe Hyperglycemia: Effects of Rehydration on Endocrine Derangements and Blood Glucose Concentration

Diabetic ketoacidosis is associated with an excess secretion of counterregulatory hormones. The effect of rehydration on these endocrine derangements before insulin administration is unknown. Therefore, we measured the effect of rehydration with hypoosmolal fluid (220 mosmol/kg) on blood glucose (BG), immunoreactive insulin (IRI), immunoreactive C-peptide (IRCP), immunoreactive glucagon (IRG), human pancreatic polypeptide (hPP), growth hormone (GH), prolactin (PRL), cortisol, aldosterone, renin (PRC), epinephrine, norepinepnrine, and parathyroid hormone (PTH) in ketoacidotic diabetic patients [pH 7.03 ± 0.05 (SEM); n = 8] and in patients (n = 2) with nonketotic hyperglycemia (BG, 29.8 mmol/L and 46.8 mmol/L). The cumulative net fluid balance after rehydration was 4364 ± 690 ml. Basal insulin was inappropriately low, and IRCP was below the normal range (1.5 ± 0.5 ng/ml). Serum osmolality fell during hypoosmolal rehydration (n = 9) from 335 ± 11 to 315 ± 9 mosmol/kg. Rehydration with hypoosmolal fluid with bicarbonate added at a pH of less than 7.2 induced a fall in BG ranging from 6.1 mmol/L to 22.6 mmol/L, or of 16.7% to 79.8% of the initial BG level, as well as a decrease in plasma lactate and urinary glucose. These effects were paralleled by a decrease in IRG, cortisol, epinephrine, norepinephrine, aldosterone, and PRC. No fall in BG was seen in one patient whose dehydrated state was maintained by infusion of isotonic saline. Low dose insulin treatment was initiated in all patients immediately when no further fall in blood glucose levels was achieved. We conclude that rehydration improves the metabolic situation in severe diabetic hyperglycemia and ketoacidosis by reducing (a) the availability of counterregulatory hormones and (b) peripheral insulin resistance on a cellular level. Thus, proper rehydration will support the beneficial action of simultaneous low dose insulin treatment in patients with severe hyperglycemia.

Pronounced Insulin Resistance and Inadequate β-cell Secretion Characterize Lean Gestational Diabetes During and After Pregnancy

OBJECTIVE To evaluate β-cell secretion and glucose metabolism in lean subjects with gestational diabetes mellitus (GDM) compared with that in subjects with normal pregnancy and obesity. RESEARCH DESIGN AND METHODS Insulin secretion, insulin sensitivity (S1), and hepatic insulin extraction were assessed in pregnant women with GDM before and after delivery and in those with normal glucose tolerance (NGT) in comparison to healthy nonpregnant lean and obese women. Kinetic analysis of glucose, insulin, and C-peptide plasma concentrations during oral and intravenous glucose tolerance tests was performed by mathematical modeling. RESULTS S1 was blunted in pregnant women with GDM by 84% and in those with NGT by 66% compared with lean nonpregnant women (P < 0.005 vs. healthy nonpregnant lean control subjects; P < 0.05, GDM vs. pregnant women with NGT), whereas glucose effectiveness was decreased by 33% in both pregnant groups (P < 0.05 vs. healthy nonpregnant lean control subjects). Insulin secretion was 30% higher (P < 0.05) in subjects with GDM than in pregnant women with NGT or in nonpregnant lean women, but decreased (P < 0.005) when compared with obese women with a comparable degree of insulin resistance. Fractional hepatic insulin extraction was similar in both pregnant groups, being lower (P < 0.0001) by 30% versus nonpregnant females. β-cell sensitivity to glucose for insulin release was decreased in subjects with GDM versus pregnant women with NGT as well as nonpregnant women by 40-50% (P < 0.01). Twelve weeks after delivery, GDM returned to normal glucose tolerance, but S1 remained 50% lower than that in lean nonpregnant women, while β-cell sensitivity to glucose did not change (P < 0.01 vs. healthy nonpregnant lean control subjects). CONCLUSIONS Pregnancy is characterized by insulin resistance, diminished hepatic insulin extraction, and glucose effectiveness. Lean subjects with GDM are additionally characterized by having more pronounced insulin resistance and inadequate insulin secretion, which persist after delivery. Compared with other insulin-resistant prediabetic states like impaired glucose tolerance (IGT), defective insulin secretion seems to be a predominant defect in lean GDM subjects, indicating that it might represent a specific prediabetic condition.

Increased plasma leptin in gestational diabetes

Aims/hypothesis. Insulin resistance as well as marked changes in body weight and energy metabolism are associated with pregnancy. Its impact on plasma leptin is not known and was determined in this longitudinal study in both diabetic and normal pregnancy. Methods. At 28 gestational weeks plasma concentrations of leptin and B-cell hormones were measured at fasting and after an oral glucose load (OGTT:75 g) in women with gestational diabetes and pregnant women with normal glucose tolerance and compared with women who were not pregnant (C). Results. Plasma leptin (ng/ml) was higher (p < 0.001) in women with gestational diabetes (24.9 ± 1.6) than in women with normal glucose tolerance (18.2 ± 1.5) and increased in both groups when compared with the non-pregnant women (8.2 ± 1.3; p < 0.0005). No change in plasma leptin concentrations was induced by OGTT in any group. Basal insulin release was higher (p < 0.05) in women with gestational diabetes compared with the pregnant women with normal glucose tolerance. Marked insulin resistance was confirmed by a 20 % lower (p < 0.05) insulin sensitivity in subgroup analysis and a decrease of almost 40 % in fasting glucose/insulin ratio (p < 0.005) in women with gestational diabetes. Leptin correlated in women with gestational diabetes with basal plasma concentrations of glucose (p < 0.02), insulin (p < 0.004) and proinsulin (p < 0.01) as well as with BMI (p < 0.001) and overall pregnancy induced maternal weight gain (p < 0.009). With normalisation of blood glucose 8 weeks after delivery in women with gestational diabetes their plasma leptin decreased (p < 0.0005) to 17.3 ± 1.9 ng/ml but did not completely normalize (p < 0.05 vs non-pregnant women). Conclusion/interpretation. Our data show that women with gestational diabetes without any change in plasma leptin upon oral glucose loading have increased plasma leptin concentrations during and after pregnancy, a clear association of plasma leptin with the respective concentration of glucose and insulin resistance as well as with changes in body weight, and a failure to normalize spontaneously BMI to the same extent as pregnant women with normal glucose tolerance when compared with matched control subjects. [Diabetologia (2001) 44: 164–172]

Insulin production rate following glucose ingestion estimated by splanchnic C-peptide output in normal man.

SummaryInsulin production rate has been estimated in healthy male volunteers (n = 16), and evaluated with respect to splanchnic glucose exchange. Insulin production rate was calculated from splanchnic immunoreactive C-peptide output. C-peptide secretion was estimated by the hepatic venous catheter technique both in the basal state and for 2h following ingestion of various glucose loads (0, 12.5, 25, 50, 75, and 100 g). The results demonstrate a basal insulin production rate of 0.017±0.002 U/min (mean±SEM) or 2.04 U/2 h. Values rose in a dose dependent manner from 2.6±1.1 U/2 h after ingestion of 12.5 g of glucose to 10.8±1.1 U/2 h following a glucose load of 100 g. Insulin retention by the liver was estimated at 0.012±0.001 U/min in the basal state, and ranged from 47–85% (70±2%) of production following an oral glucose load. It was also demonstrated 1) that the relative splanchnic glucose output was inversely related to the amount of ingested glucose, and reached a minimum when glucose in excess of 50 g was ingested; and 2) that hepatic glucose retention was directly proportional to insulin production rate (r=0.83; p<0.001; n= 15). It is suggested that the adaptive capacity of the splanchnic bed to retain glucose depending on the amount of ingested glucose guarantees that splanchnic glucose output fluctuates in healthy man only within a narrow range.

Efficacy of pulsatile versus continuous insulin administration on hepatic glucose production and glucose utilization in type I diabetic humans.

To evaluate the role of pulsatile insulin administration, hepatic glucose production (HGP) and utilization were studied in type I diabetic patients in the fasting state and during a euglycemic insulin (1 mU · kg−1 · min−1 i.v.) clamp with continuous and pulsatile insulin administration. In the latter study, insulin was infused at twice the continuous rate with 3-min-on/7-min-off intervals, thereby reducing total insulin delivery by 40%. The restraining effect of pulsatile insulin on basal HGP (1.91 ± 0.35 mg · kg−1 · min−1) was equipotent to continuous insulin exposure (1.80 ± 0.17 mg · kg−1 min−1). During the insulin-clamp studies, HGP was equally suppressed by pulsed (0.62 ± 0.12 mg · kg−1 min−1) as by continuous insulin infusion (0.63 ± 0.12 mg · kg−1 · min−1). Insulin-stimulated glucose utilization was not significantly altered in either study (2.55 ± 0.27 vs. 2.92 ± 0.23 mg · kg−1 min−1). When in further studies the total insulin dose given during the pulsatile study was infused continuously (0.6 mU · kg−1 · min−1), HGP in the basal state and residual HGP during the insulin-clamp study were 25–30% higher than in the pulsatile experiments, whereas glucose utilization was not significantly different. In conclusion, by reducing total hormone delivery by up to 40%, but given in a pulsatile fashion, insulin is equally potent in controlling HGP as continuous insulin administration. This greater efficacy of pulsatile exposure in suppressing HGP is accompanied by an equipotent effect on glucose utilization. Application of pulsatile insulin substitution in intravenous-pump users may reduce systemic hyperinsulinemia and, in the long run, insulin resistance by reversing downregulation of insulin receptors.

Signal transduction via glycosyl phosphatidylinositol-anchored proteins in T cells is inhibited by lowering cellular cholesterol.

Glycosylphosphatidylinositol (GPI)-anchored proteins can deliver costimulatory signals to lymphocytes, but the exact pathway of signal transduction involved is not yet characterized. GPI-anchored proteins are fixed to the cell surface solely by a phospholipid moiety and are clustered in distinct membrane domains that are formed by an unique lipid composition requiring cholesterol. To elucidate the role of membrane lipids for signal transduction via GPI-anchored proteins, we studied the influence of reduced cellular cholesterol content on calcium signaling via GPI-anchored CD59 and CD48 in Jurkat T cells. Lowering cholesterol by different inhibitors of cellular cholesterol synthesis suppressed calcium response via GPI-anchored proteins by about 50%, whereas stimulation via CD3 was only minimally affected (<10%). The decrease in overall calcium response via GPI-anchored proteins was reflected by inhibition of calcium release from intracellular stores. Cell surface expression of GPI-anchored proteins was not changed quantitatively by lowering cellular cholesterol, and neither was the pattern of immunofluorescence in microscopic examination. In addition, the distribution of GPI-anchored proteins in detergent-insoluble complexes remained unaltered. These results suggest that cellular cholesterol is an important prerequisite for signal transduction via GPI-anchored proteins beyond formation of membrane domains.