Effect of liraglutide 3.0 mg in individuals with obesity and moderate or severe obstructive sleep apnea: the SCALE Sleep Apnea randomized clinical trial.

BACKGROUND: Obesity is strongly associated with prevalence of obstructive sleep apnea (OSA), and weight loss has been shown to reduce disease severity. OBJECTIVE: To investigate whether liraglutide 3.0 mg reduces OSA severity compared with placebo using the primary end point of change in apnea-hypopnea index (AHI) after 32 weeks. Liraglutide's weight loss efficacy was also examined. SUBJECTS/METHODS: In this randomized, double-blind trial, non-diabetic participants with obesity who had moderate (AHI 15-29.9 events h(-1)) or severe (AHI ⩾30 events h(-1)) OSA and were unwilling/unable to use continuous positive airway pressure therapy were randomized for 32 weeks to liraglutide 3.0 mg (n=180) or placebo (n=179), both as adjunct to diet (500 kcal day(-1) deficit) and exercise. Baseline characteristics were similar between groups (mean age 48.5 years, males 71.9%, AHI 49.2 events h(-1), severe OSA 67.1%, body weight 117.6 kg, body mass index 39.1 kg m(-2), prediabetes 63.2%, HbA1c 5.7%). RESULTS: After 32 weeks, the mean reduction in AHI was greater with liraglutide than with placebo (-12.2 vs -6.1 events h(-1), estimated treatment difference: -6.1 events h(-1) (95% confidence interval (CI), -11.0 to -1.2), P=0.0150). Liraglutide produced greater mean percentage weight loss compared with placebo (-5.7% vs -1.6%, estimated treatment difference: -4.2% (95% CI, -5.2 to -3.1%), P<0.0001). A statistically significant association between the degree of weight loss and improvement in OSA end points (P<0.01, all) was demonstrated post hoc. Greater reductions in glycated hemoglobin (HbA1c) and systolic blood pressure (SBP) were seen with liraglutide versus placebo (both P<0.001). The safety profile of liraglutide 3.0 mg was similar to that seen with doses ⩽1.8 mg. CONCLUSIONS: As an adjunct to diet and exercise, liraglutide 3.0 mg was generally well tolerated and produced significantly greater reductions than placebo in AHI, body weight, SBP and HbA1c in participants with obesity and moderate/severe OSA. The results confirm that weight loss improves OSA-related parameters.

Exposure-response analyses of liraglutide 3.0 mg for weight management.

AIMS: Liraglutide 3.0 mg, an acylated GLP-1 analogue approved for weight management, lowers body weight through decreased energy intake. We conducted exposure-response analyses to provide important information on individual responses to given drug doses, reflecting inter-individual variations in drug metabolism, absorption and excretion. METHODS: We report efficacy and safety responses across a wide range of exposure levels, using data from one phase II (liraglutide doses 1.2, 1.8, 2.4 and 3.0 mg), and two phase IIIa [SCALE Obesity and Prediabetes (3.0 mg); SCALE Diabetes (1.8; 3.0 mg)] randomized, placebo-controlled trials (n = 4372). RESULTS: There was a clear exposure-weight loss response. Weight loss increased with greater exposure and appeared to level off at the highest exposures associated with liraglutide 3.0 mg in most individuals, but did not fully plateau in men. In individuals with overweight/obesity and comorbid type 2 diabetes, there was a clear exposure-glycated haemoglobin (HbA1c) relationship. HbA1c reduction increased with higher plasma liraglutide concentration (plateauing at ∼21 nM); however, for individuals with baseline HbA1c >8.5%, HbA1c reduction did not fully plateau. No exposure-response relationship was identified for any safety outcome, with the exception of gastrointestinal adverse events (AEs). Individuals with gallbladder AEs, acute pancreatitis or malignant/breast/benign colorectal neoplasms did not have higher liraglutide exposure compared with the overall population. CONCLUSIONS: These analyses support the use of liraglutide 3.0 mg for weight management in all subgroups investigated; weight loss increased with higher drug exposure, with no concomitant deterioration in safety/tolerability besides previously known gastrointestinal side effects.

Effects of the once-daily GLP-1 analog liraglutide on gastric emptying, glycemic parameters, appetite and energy metabolism in obese, non-diabetic adults.

INTRODUCTION: Mechanisms for liraglutide-induced weight loss are poorly understood. OBJECTIVE: We investigated the effects of liraglutide on gastric emptying, glycemic parameters, appetite and energy metabolism in obese non-diabetic individuals. DESIGN: Participants (N=49, 18-75 years, body mass index: 30-40 kg m(-2)) were randomized to two of three treatments: liraglutide 1.8 mg, 3.0 mg, or placebo in a double-blind, incomplete crossover trial. After 5 weeks, 24-h energy expenditure (EE) and substrate oxidation were measured in a respiratory chamber. Gastric emptying (acetaminophen absorption method), glycemic parameters and appetite were assessed during a 5-h meal test. Ad libitum energy intake during a subsequent lunch was also assessed. RESULTS: Five-hour gastric emptying (AUC(0-300 min)) was found to be equivalent for liraglutide 1.8 versus 3.0 mg (primary end point), and for both liraglutide doses versus placebo, as 90% confidence intervals for the estimated treatment ratios were contained within the prespecified interval (0.80-1.25). However, 1-h gastric emptying was 23% lower than placebo with liraglutide 3.0 mg (P=0.007), and a nonsignificant 13% lower than placebo with liraglutide 1.8 mg (P=0.14). Both liraglutide doses similarly reduced fasting glucose (0.5-0.6 mmol l(-1) versus placebo, P<0.0001), glucose Cmax and 1-h AUC versus placebo; only liraglutide 3.0 mg reduced iAUC(0-300 min) (by ∼26% versus placebo, P=0.02). Glucagon iAUC(0-300 min) decreased by ∼30%, and iAUC(0-60 min) for insulin and C-peptide was ∼20% lower with both liraglutide doses versus placebo. Liraglutide doses similarly increased mean postprandial satiety and fullness ratings, reduced hunger and prospective food consumption and decreased ad libitum energy intake by ∼16%. Liraglutide-associated reductions in EE were partly explained by a decrease in body weight. A relative shift toward increased fat and reduced carbohydrate oxidation was observed with liraglutide. Clinicaltrials.gov ID:NCT00978393. FUNDING: Novo Nordisk. CONCLUSION: Gastric emptying AUC(0-300 min) was equivalent for liraglutide 1.8 and 3.0 mg, and for liraglutide versus placebo, whereas reductions in 1-h gastric emptying of 23% with liraglutide 3.0 mg and 13% with 1.8 mg versus placebo were observed. Liraglutide 3.0 mg improved postprandial glycemia to a greater extent than liraglutide 1.8 mg. Liraglutide-induced weight loss appears to be mediated by reduced appetite and energy intake rather than increased EE.

The T allele of rs7903146 TCF7L2 is associated with impaired insulinotropic action of incretin hormones, reduced 24 h profiles of plasma insulin and glucagon, and increased hepatic glucose production in young healthy men.

AIMS/HYPOTHESIS: We studied the physiological, metabolic and hormonal mechanisms underlying the elevated risk of type 2 diabetes in carriers of TCF7L2 gene. METHODS: We undertook genotyping of 81 healthy young Danish men for rs7903146 of TCF7L2 and carried out various beta cell tests including: 24 h glucose, insulin and glucagon profiles; OGTT; mixed meal test; IVGTT; hyperglycaemic clamp with co-infusion of glucagon-like peptide (GLP)-1 or glucose-dependent insulinotropic polypeptide (GIP); and a euglycaemic-hyperinsulinaemic clamp combined with glucose tracer infusion to study hepatic and peripheral insulin action. RESULTS: Carriers of the T allele were characterised by reduced 24 h insulin concentrations (p < 0.05) and reduced insulin secretion relative to glucose during a mixed meal test (beta index: p < 0.003), but not during an IVGTT. This was further supported by reduced late-phase insulinotropic action of GLP-1 (p = 0.03) and GIP (p = 0.07) during a 7 mmol/l hyperglycaemic clamp. Secretion of GLP-1 and GIP during the mixed meal test was normal. Despite elevated hepatic glucose production, carriers of the T allele had significantly reduced 24 h glucagon concentrations (p < 0.02) suggesting altered alpha cell function. CONCLUSIONS/INTERPRETATION: Elevated hepatic glucose production and reduced insulinotropic effect of incretin hormones contribute to an increased risk of type 2 diabetes in carriers of the rs7903146 risk T allele of TCF7L2.

Leukocyte telomere length is associated with elevated plasma glucose and HbA1c in young healthy men independent of birth weight.

Telomeres are protein-bound regions of repetitive nucleotide sequences (TTAGGG) at the end of human chromosomes, and their length is a marker of cellular aging. Intrauterine growth restriction is associated with shorter blood cell telomeres at birth and individuals with type 2 diabetes have shorter telomeres. Individuals with a low birth weight (LBW) have an increased risk of metabolic disease and type 2 diabetes. Therefore, we aimed to investigate the relationship between birth weight and telomere length and the association between birth weight, telomere length and cardiometabolic phenotype in adulthood. Young, healthy men with LBW (n = 55) and normal birth weight (NBW) (n = 65) were examined including blood pressure, blood samples and body composition. Leukocyte telomere length was determined using a high-throughput qPCR method. The LBW men were more insulin resistant as determined by the HOMA-IR index. There was no difference in telomere length between LBW and NBW subjects. When adjusting for birth weight and cohort effect, significant negative associations between telomere length and fasting glucose (P = 0.003) and HbA1c (P = 0.0008) were found. In conclusion, no significant difference in telomere length was found between LBW and NBW men. The telomere length was negatively associated with glucose concentrations and HbA1c levels within the normal non-diabetic range independent of birth weight.

Insulin signal transduction in skeletal muscle from glucose-intolerant relatives of type 2 diabetic patients [corrected].

To determine whether defects in the insulin signal transduction cascade are present in skeletal muscle from prediabetic individuals, we excised biopsies from eight glucose-intolerant male first-degree relatives of patients with type 2 diabetes (IGT relatives) and nine matched control subjects before and during a euglycemic-hyperinsulinemic clamp. IGT relatives were insulin-resistant in oxidative and nonoxidative pathways for glucose metabolism. In vivo insulin infusion increased skeletal muscle insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation (P = 0.01) and phosphatidylinositide 3-kinase (PI 3-kinase) activity (phosphotyrosine and IRS-1 associated) in control subjects (P < 0.02) but not in IGT relatives (NS). The incremental increase in insulin action on IRS-1 tyrosine phosphorylation was lower in IGT relatives versus control subjects (P < 0.05). The incremental defects in signal transduction noted for IRS-1 and PI 3-kinase may be attributed to elevated basal phosphorylation/activity of these parameters, because absolute phosphorylation/activity under insulin-stimulated conditions was similar between IGT relatives and control subjects. Insulin increased Akt serine phosphorylation in control subjects and IGT relatives, with a tendency for reduced phosphorylation in IGT relatives (P = 0.12). In conclusion, aberrant phosphorylation/activity of IRS-1, PI 3-kinase, and Akt is observed in skeletal muscle from relatives of patients with type 2 diabetes with IGT. However, the elevated basal activity of these signaling intermediates and the lack of a strong correlation between these parameters to glucose metabolism suggests that other defects of insulin signal transduction and/or downstream components of glucose metabolism may play a greater role in the development of insulin resistance in skeletal muscle from relatives of patients with type 2 diabetes.

Prenatal exposure to vitamin-D from fortified margarine and milk and body size at age 7 years.

BACKGROUND/OBJECTIVES: Prenatal vitamin-D deficiency may be associated with increased risk of obesity later in life. Using two national vitamin-D fortification programs as the setting for a societal experiment, we investigated whether exposure to vitamin-D from fortified margarine and low-fat milk during foetal life was associated with body size at 7 years of age. SUBJECTS/METHODS: Vitamin-D fortification of margarine was mandatory in Denmark from 1961 to 1985, and voluntary fortification of low-fat milk was permitted from 1972 to 1976. Using information on body mass index (BMI) Z-score at the age of 7 years of 54,270 children, who were measured during the mandatory Copenhagen School Health examination, we compared children according to whether the mothers were pregnant during the fortification programs or not. The comparisons were performed for children born just before and after initiation or termination of margarine and milk fortification periods, respectively. In total four sets of analyses were performed. RESULTS: We observed no difference in mean BMI Z-score between children exposed to vitamin-D fortification in utero and non-exposed children. Similar results were observed for overweight and obesity. CONCLUSIONS: Prenatal exposure to vitamin-D from fortification of margarine and low-fat milk showed no association with body size at 7 years.

Normal insulin-stimulated endothelial function and impaired insulin-stimulated muscle glucose uptake in young adults with low birth weight.

Low birth weight has been linked to insulin resistance and cardiovascular disease. We hypothesized that insulin sensitivity of both muscle and vascular tissues were impaired in young men with low birth weight. Blood flow was measured by venous occlusion plethysmography during dose-response studies of acetylcholine and sodium nitroprusside in the forearm of fourteen 21-yr-old men with low birth weight and 16 controls of normal birth weight. Glucose uptake was measured during intraarterial insulin infusion. Dose-response studies were repeated during insulin infusion. The maximal blood flow during acetylcholine infusion was 14.1 +/- 2.7 and 14.4 +/- 2.1 [ml x (100 ml forearm)(-1) x min(-1)] in low and normal birth weight subjects, respectively. Insulin coinfusion increased acetylcholine-stimulated flow in both groups: 18.0 +/- 3.1 vs. 17.9 +/- 3.1 [ml x (100 ml forearm)(-1) x min(-1)], NS. Insulin infusion increased glucose uptake significantly in the normal birth weight group, compared with the low birth weight group: 0.40 +/- 0.09 to 1.00 +/- 0.16 vs. 0.44 +/- 0.09 to 0.59 +/- 0.1 [ micro mol glucose x (100 ml forearm)(-1) x min(-1)], P = 0.04. Young men with low birth weight have normal insulin-stimulated endothelial function and impaired insulin-stimulated forearm glucose uptake. Thus, endothelial dysfunction does not necessarily coexist with metabolic alterations in subjects with low birth weight.

Two ubiquitin-long-tail fusion genes arranged as closely spaced direct repeats in barley.

Ubiquitin (Ubi) genes encode two types of fusion proteins: polyUbi with a varying number of direct repeats of Ubi, and Ubi-tail fusions with long or short basic C-terminal extensions. A barley (Hordeum vulgare) genomic clone has been isolated with two very similar, intronless genes encoding monoUbi-long-tail fusion peptides. The genes are arranged as direct repeats separated by 3 kb of DNA and account for two of the probable three long-tail genes in the haploid barley genome. Both genes are active and give rise to messengers about 800 nt long. The sequence of the encoded Ubi moieties is identical to the sequence of Ubi repeats of polyUbi precursors from barley and other plants. The basic tails of the peptides are 79 aa long and 71-72% homologous to corresponding sequences from yeast and man. Recently, it was found that the long and short tails are ribosomal proteins in yeast [Finley et al., Nature 338 (1989) 394-401] and the evolutionary conservation of the structure of the Ubi-tail fusion genes suggests that they serve the same function in plants. The similarity between yeast and barley Ubi-long-tail fusion genes may extend to the regulatory regions, since upstream activating sites characteristic of ribosomal protein-encoding genes in yeast (UASrpg) were found in the barley genes.

Nafenopin-induced peroxisome proliferation in vitamin A deficient rats.

Induction of peroxisome proliferator responsive genes is thought to be mediated through binding of a peroxisome proliferator-activated receptor (PPAR) to specific peroxisome proliferator response elements in the upstream region of these genes. Binding of PPAR to the acyl-CoA oxidase promoter requires heterodimerization with the retinoid X receptor (RXR), and subsequent transactivation is strongest when ligands for both PPAR and RXR are present. Therefore, we hypothesized that depletion of ligand for the retinoid receptor would limit the induction of peroxisome proliferation in rats. Hepatic retinol content was reduced by more than 90% by feeding weanling rats a vitamin A deficient (VAD) diet for approximately 3 months. Nafenopin treatment for 7 days induced peroxisomal beta-oxidation 18-fold in VAD rats compared with 16-fold in rats fed a vitamin A sufficient (VAS) diet. Nafenopin induced microsomal laurate hydroxylase and mitochondrial beta-oxidation to comparable rates of specific activity in both VAD and VAS rats. However, the activities in VAD controls were significantly lower than in VAS controls, so the magnitude of the nafenopin-induced increases was greater in the VAD rats. Relative liver weights were increased nearly 2-fold in both VAS and VAD rats treated with nafenopin. Ultrastructural examination of the livers demonstrated that nafenopin increased the number and size of peroxisomes in both VAD and VAS rats. These data demonstrate that rats with severely depleted vitamin A stores remained responsive to the peroxisome proliferator nafenopin. Whether critical retinoid pools that supply RXR ligand (9-cis-retinoic acid) are spared in the vitamin A deficient rats remains to be determined.

The effects of antirhino- and enteroviral vinylacetylene benzimidazoles on cytochrome P450 function and hepatic porphyrin levels in mice.

In an ongoing effort to identify an orally bioavailable compound for the treatment of rhino- and enteroviral infections, a series of vinylacetylene benzimidazoles was recently examined. Previous studies demonstrated the potential for these compounds to possess both good in vitro antiviral activity as well as acceptable oral plasma concentrations in mice. Optimization of these properties led to four compounds as candidates for further evaluation. In view of the recognized potential for certain acetylenic drugs both to inhibit cytochrome P450 enzymes by mechanism-based inactivation and to possibly perturb heme metabolism, information regarding drug effects on cytochromes P450 and hepatic porphyrin levels was sought. In an initial single-dose pharmacokinetic study, the four selected compounds were given orally to mice, and both plasma concentrations and porphyrin levels were determined. Two of the compounds, 4 and 5, caused a pronounced increase in liver porphyrin levels whereas compounds 6 and 7 exhibited almost no effect on porphyrin levels. Analysis of plasma concentrations showed that only 4 and 5 gave significant exposure and that 6 and 7 produced negligible levels of drug in the plasma even at the highest dose tested (500 mg/kg). A multiple dose study was then initiated in which compounds 4 and 5 were given for 1 week in daily oral doses to mice. Upon completion of dosing, liver was analyzed for cytochrome P450-dependent 7-ethoxyresorufin O-deethylase (EROD) and benzphetamine N-demethylase (BND) activities, total cytochrome P450 content, and porphyrin levels. Both vinylacetylenes showed dose dependent inhibitory and induction effects on EROD and BND activities. In addition, these compounds caused a marked increase in hepatic porphyrin levels. Therefore, while all four selected compounds displayed potent antiviral activity and two of the compounds exhibited acceptable pharmacokinetic properties, the hepatic effects of these latter two compounds suggest the potential for drug induced porphyria with multidose therapeutic use.

Determination of liothyronine and levothyroxine in thyroid preparations by liquid chromatography.

Liothyronine and levothyroxine were quantitatively determined in samples of commercial thyroid tablets and bulk powders. Samples were first hydrolyzed using a bacterial protease and then analyzed by high-performance liquid chromatography. Various hydrolysis conditions were investigated. The liothyronine and levothyroxine contents of commercial tablets and bulk powders were found to be approximately 8-11 micrograms and 25-43 micrograms, respectively, per 65 mg of thyroid. The stability of the iodothyronines in thyroid tablets was also investigated.

The 'Seamless Web': the development of the electronic patient record in Aarhus region, Denmark.

OBJECTIVES: The article surveys the organization of the current project to develop an electronic patient record in the Aarhus Region, Denmark. METHODS: The article is based on various policy documents and reports as well as a number of semi-structured interviews with project managers from the EPR organization in Aarhus County and with participants in the development process at local hospitals. This material is used to present and discuss the framing of the project in a 'discourse coalition'. RESULTS: The stabilization of a specific discourse coalition has been an important factor in ensuring the success of the development project up to the present moment. This coalition became relatively stable by integrating a diverse set of actors in a story-line about the relationships between co-operation, management and technology in the medial sector, and has influenced the modular organization of the project. CONCLUSIONS: The successful maintenance of the discourse coalition allows the project to appear 'seamless' from the outside. Conversely, the project is likely to be continually reviewed as successful only to the extent that it is able to flexibly keep the fluctuating set of relevant actors in alignment. If the practical work of keeping a coalition in place remains invisible it becomes easy to imagine an ideal way of planning large socio-technical projects, like developing an ECR. But practical success is more likely to be achieved if one takes seriously the thorough intertwining of discursive, organizational and technical aspects of development projects.

Improved method for determination of cellular thiols, disulfides and protein mixed disulfides using HPLC with electrochemical detection.

The role of glutathione in cellular responses to toxic insult is well established in the literature and is largely based on chemical determinations of GSH. Although these methods have proved to be adequate to demonstrate the relationships between cellular levels of GSH and xenobiotic toxicity, they may be inadequate for careful investigations regarding the regulation of cellular concentrations of GSH, GSSG and GSSProt. Enzymatic and chromatographic methods provide increased specificity and sensitivity, but the problems of variable accuracy or tedious derivitization steps remain. The HPLC-EC method constitutes a significant improvement over previous methods by satisfying the requirements for selectivity, sensitivity, accuracy and convenience. The utility of the HPLC-EC method has been described in two systems of toxicological interest, particularly with regard to the determination of GSSProt. In further studies, the HPLC-EC method has been useful in investigating the mechanism(s) of alternate nucleophiles, such as cysteamine. The capability to determine various thiols simultaneously in a given biological sample greatly facilitates these investigations. In view of its power as an analytical tool, the HPLC-EC method has the potential for widespread application to the study of cellular thiols.

Increased nocturnal fat oxidation in young healthy men with low birth weight: results from 24-h whole-body respiratory chamber measurements.

OBJECTIVE: Low birth weight (LBW), a marker of disturbed fetal growth, is associated with adiposity and increased risk of type 2 diabetes (T2D). The aim of the study was to investigate whether LBW is associated with changes in 24-h energy expenditure (EE) and/or substrate utilization rates, potentially contributing to the development of adiposity and/or T2D compared to matched control subjects. MATERIALS/METHODS: Forty-six young, healthy men were included in the study; 20 with LBW (≤ 10th percentile) and 26 control subjects with normal birth weight (NBW) (50th-90th percentile). The subjects were fed a weight maintenance diet and 24-h energy expenditure (EE), respiratory quotient (RQ), and substrate oxidation were assessed in a respiratory chamber. RESULTS: No differences in 24-h EE, RQ or substrate oxidation were observed between LBW and controls. Interestingly, the LBW group exhibited lower nocturnal RQ compared to controls (0.81 ± 0.01 vs. 0.85 ± 0.01 (mean ± SE), P = 0.01), and hence higher nocturnal fat oxidation (2.55 ± 0.13 vs. 2.09 ± 0.12 kJ/min (mean ± SE), P = 0.02). CONCLUSIONS: Young LBW men do not exhibit reductions in 24-h EE. However, LBW subjects display increased nocturnal fat oxidation at the expense of reduced glucose oxidation. We speculate that this may be associated with insufficient capability to retain fat in subcutaneous adipose tissue after meals during day time, with an increased rate of nocturnal and morning lipolysis, and potentially with subtle elevations of gluconeogenesis and of fasting glucose levels in the LBW subjects.

Consequences of low birthweight on urinary excretion of DNA markers of oxidative stress in young men.

OBJECTIVE: Low birthweight (LBW) has been associated with an increased risk of development of type 2 diabetes in adult life. Both type 1 and type 2 diabetes mellitus are characterized by increased oxidative stress. The purpose of this study was to investigate whether young healthy adults born with LBW showed differences in oxidative stress under normal conditions and during the added challenge of a physiological Intralipid infusion. MATERIAL AND METHODS: Urinary excretion of DNA markers of oxidative stress were analyzed by LC-MS/MS in 19 men (aged 19 years) with LBW and in 19 age matched, normal birthweight (NBW) controls pre- and post a 3-fold increase of plasma free fatty acids. RESULTS: Mean excretion rates of 8-oxo-guanine (8oxoGua), 8-oxo-guanosine (8oxoGuo), 8-oxo-2'deoxyguanosine (8oxodG), and 1,N6-ethenodeoxyadenosine (epsilon dA) did not statistically differ between subjects with LBW and NBW (66.9 versus 73.9 nmol/15 h, 17.8 versus 18.5 nmol/15 h, 11.9 versus 14.4 nmol/15 h and 44.0 versus 43.2 pmol/15 h, respectively). Furthermore, Intralipid infusion did not affect excretion of DNA adducts in LBW or NBW subjects. Statistically significant correlations were found between body mass index and urinary excretion of 8oxoGua (r = 0.64, p = 0.003) and 8oxoGuo (r = 0.64, p = 0.003) in the LBW group only. CONCLUSIONS: These findings suggest that oxidative stress may be a consequence of diabetes and is not, or at least only partly, involved in the early pathogenesis of type 2 diabetes.

Decreased protein levels of key insulin signalling molecules in adipose tissue from young men with a low birthweight: potential link to increased risk of diabetes?

AIMS/HYPOTHESIS: Individuals with low birthweight are at increased risk of type 2 diabetes mellitus. However, the underlying molecular mechanisms are unknown. Previously we have shown that low birthweight is associated with changes in muscle insulin signalling proteins. Here we determined whether low birthweight is associated with changes in insulin signalling proteins in adipose tissue. METHODS: Men (age 23 years) with either a low (bottom 10th percentile) (n = 17) or a normal (50th-90th percentile) (n = 17) birthweight were recruited from the Danish Medical Birth Registry and subcutaneous adipose biopsies were taken. RESULTS: Between the two groups there was no difference in protein level of the insulin receptor, protein kinase C zeta, glycogen synthase kinase-3 (GSK3) alpha, GSK3 beta, protein kinase B alpha and beta, peroxisome proliferative activated receptor gamma coactivator 1 or Src-homology-2-containing protein. However, the levels of GLUT4 (also known as solute carrier family 2 [facilitated glucose transporter], member 4 [SLC2A4]) (52 +/- 10.9% reduction, p < 0.01), p85alpha subunit of phosphoinositide 3-kinase (PI3K) (45 +/- 9% reduction, p < 0.01), p110ss subunit of PI3K (48 +/- 17% reduction, p = 0.06) and IRS1 (59 +/- 24% reduction, p < 0.05) were reduced in men of low birthweight. CONCLUSIONS/INTERPRETATION: These findings show that low birthweight is associated with reduced levels of adipose insulin signalling proteins, thus providing a potential molecular framework to explain why people with low birthweight are at increased risk of developing type 2 diabetes. These differences precede the development of diabetes and thus may help predict disease risk.

Low birthweight is associated with specific changes in muscle insulin-signalling protein expression.

AIMS/HYPOTHESIS: People with low birthweight have an increased risk of developing type 2 diabetes mellitus in adulthood. The mechanistic basis of this phenomenon is not known. Here we investigate the effect of early growth restriction on the expression of insulin-signalling proteins in skeletal muscle in a human cohort and a rat model. METHODS: We recruited 20 young men with low birthweight (mean birthweight 2702+/-202 g) and 20 age-matched control subjects (mean birthweight 3801+/-99 g). Biopsies were obtained from the vastus lateralis muscle and protein expression of selected insulin-signalling proteins was determined. Rats used for this study were male offspring born to dams fed a standard (20%) protein diet or a low (8%) protein diet during pregnancy and lactation. Protein expression was determined in soleus muscle from adult offspring. RESULTS: Low-birthweight subjects showed reduced muscle expression of protein kinase C (PKC)zeta, p85alpha, p110beta and GLUT4. PKCzeta, GLUT4 and p85 were also reduced in the muscle of rats fed a low-protein diet. Other proteins studied were unchanged in low-birthweight humans and in rats fed a low-protein diet when compared with control groups. CONCLUSIONS/INTERPRETATION: We found decreased expression of specific insulin-signalling proteins in low-birthweight subjects compared to controls. These changes precede the onset of impaired glucose tolerance. The similarity of protein expression profile in the men with low birthweight compared to that of the offspring of rats fed a low-protein diet suggests that the rodent model is an accurate representation of the human situation. It also provides a potential mechanistic explanation as to why the fetal environment plays an important role in determining risk of developing type 2 diabetes.

The role of N-hydroxyphenetidine in phenacetin-induced hemolytic anemia.

Phenacetin is well known to cause hemolytic anemia and methemoglobinemia in humans. Early mechanistic studies clearly established a causal role for active/reactive drug metabolites in the process but did not unequivocally identify these metabolite(s) or resolve the question of whether these two hemotoxicities are mechanistically linked. As part of ongoing studies on the mechanism underlying arylamine-induced hemotoxicities, we have recently shown that the arylhydroxylamine metabolites of aniline and dapsone mediate the hemolytic activity of aniline and dapsone, respectively. The present study was undertaken to determine if N-hydroxyphenetidine (PNOH), the known arylhydroxylamine metabolite of phenacetin, is responsible for phenacetin-induced hemolytic anemia. As measured by decreased survival of 51Cr-labeled erythrocytes in rats, phenacetin, p-phenetidine, and PNOH were all hemolytic in vivo, with PNOH being significantly the most potent of the three. In vitro exposure of 51Cr-tagged erythrocytes to PNOH, followed by transfusion into isologous rats, resulted in a concentration-dependent reduction in erythrocyte survival, indicating that PNOH is a direct-acting hemolytic agent. Phenacetin and p-phenetidine were inactive. Phenacetin, p-phenetidine, and PNOH all produced dose-dependent methemoglobinemia in rats. In parallel in vitro studies, PNOH elevated methemoglobin levels, p-phenetidine and phenacetin did not. However, attempts to identify PNOH in the blood of phenacetin- and p-phenetidine-treated rats were unsuccessful, despite the use of a highly sensitive analytical method. Hemotoxic concentrations of PNOH were found to be highly unstable in the presence of red cells, though relatively stable in the buffer vehicle alone. Inhibitors of acetylation (p-aminobenzoic acid [PABA]) and deacetylation (bis-[p-nitrophenyl]phosphate [BNPP]), used to alter the cyclic interconversion of phenacetin and p-phenetidine, caused changes in phenacetin hemotoxicity that indicated the hemotoxin was a deacetylated metabolite distal to p-phenetidine. These data are consistent with the hypothesis that PNOH, formed during the metabolic clearance of phenacetin, mediates phenacetin-induced hemolytic anemia and methemoglobinemia through direct toxic actions in the erythrocyte.