Peter Schadewaldt

Mechanisms Underlying the Onset of Oral Lipid–Induced Skeletal Muscle Insulin Resistance in Humans

Several mechanisms, such as innate immune responses via Toll-like receptor-4, accumulation of diacylglycerols (DAG)/ceramides, and activation of protein kinase C (PKC), are considered to underlie skeletal muscle insulin resistance. In this study, we examined initial events occurring during the onset of insulin resistance upon oral high-fat loading compared with lipid and low-dose endotoxin infusion. Sixteen lean insulin-sensitive volunteers received intravenous fat (iv fat), oral fat (po fat), intravenous endotoxin (lipopolysaccharide [LPS]), and intravenous glycerol as control. After 6 h, whole-body insulin sensitivity was reduced by iv fat, po fat, and LPS to 60, 67, and 48%, respectively (all P < 0.01), which was due to decreased nonoxidative glucose utilization, while hepatic insulin sensitivity was unaffected. Muscle PKCθ activation increased by 50% after iv and po fat, membrane Di-C18:2 DAG species doubled after iv fat and correlated with PKCθ activation after po fat, whereas ceramides were unchanged. Only after LPS, circulating inflammatory markers (tumor necrosis factor-α, interleukin-6, and interleukin-1 receptor antagonist), their mRNA expression in subcutaneous adipose tissue, and circulating cortisol were elevated. Po fat ingestion rapidly induces insulin resistance by reducing nonoxidative glucose disposal, which associates with PKCθ activation and a rise in distinct myocellular membrane DAG, while endotoxin-induced insulin resistance is exclusively associated with stimulation of inflammatory pathways.

Liver transplantation in maple syrup urine disease.

Abstract Maple syrup urine disease (MSUD) is an autosomal recessive disorder. Impaired activity of the branched-chain 2-oxoacid dehydrogenase complex (BCOA-DH) causes accumulation of branched-chain L-amino (BCAA) and 2-oxoacids (BCOA) which may exert neurotoxic effects. Treatment comprises dietary management with strictly reduced quantities of protein and BCAA as well as aggressive intervention during acute neonatal and subsequent metabolic complications. MSUD is regarded as a metabolic disorder with potentially favourable outcome when the patients are kept on a carefully supervised long-term therapy. Up to now, three MSUD patients, exhibiting the classical form of the disease, have received orthotopic whole liver transplantation (OLT). Liver replacement resulted in a clear increase in whole body BCOA-DH activity to at least the level of very mild MSUD variants. These patients no longer require protein restricted diets and the risk of metabolic decompensation during catabolic events is apparently abolished. Conclusion Considering the overall expenses, risks, and outcome, however, the benefit of OLT, even in the most severe form of MSUD, may not be significantly different from that of a classical strict dietary management. Thus, OLT appears not to represent a specific option in the treatment in MSUD.

Impact of longitudinal plasma leucine levels on the intellectual outcome in patients with classic MSUD.

Maple syrup urine disease (MSUD) is an inherited deficiency of branched chain α-ketoacid dehydrogenase (BCKDH) activity impairing the degradation of the branched chain amino acids valine, leucine, and isoleucine. Classic MSUD may lead to severe neonatal encephalopathy including coma and impaired cognitive outcome in later life. Early start of dietary treatment and careful metabolic control may improve the outcome of patients with classic MSUD. The aim of this study was to investigate the impact of long-term metabolic control assessed by plasma leucine levels on cognitive outcome in patients with classic MSUD. Plasma leucine levels of 24 patients were obtained retrospectively for the first 6 y of life and yearly medians of mean plasma leucine levels were calculated. At the age of 6 y, IQ tests were performed. Yearly medians of mean plasma leucine levels yielded three homogeneous clusters (low, intermediate, high). Patients of the low cluster showed statistically significant higher IQ scores compared with those of those of intermediate and high clusters. Long-term plasma leucine levels are associated with impaired cognitive outcome in patients with classic MSUD. To achieve the best possible intellectual outcome for affected individuals, we recommend that in infants and preschool children the target range for plasma leucine should not exceed 200 μmol/L.

Variant maple syrup urine disease (MSUD)—The entire spectrum

SummaryBackground: In the rare inborn autosomal recessive disorder maple syrup urine disease (MSUD) the accumulation of the branched-chain amino acids (BCAAs) and their metabolic products results in acute and chronic brain dysfunction. About 20% of the patients suffer from non-classic variant forms of MSUD of different clinical severity. Aim: Up to now variant cases have mostly been published as individual case reports; the aim of this study was to give a comparative description of 16 individuals (aged 6–30 years) with different forms of variant MSUD. Methods: Laboratory data, information on clinical course and treatment as well as aspects of developmental, intellectual and social outcome were obtained retrospectively. Data from in vitro and in vivo methods measuring the degree of enzyme deficiency were included. Results: In addition to a mild phenotype, which fits well into the so-called intermittent variant, and a more severe phenotype with a wider range from a mild variant to an almost classic form, which fits well into the so-called intermediate variant, we assume the existence of an asymptomatic, non-disease variant of MSUD. These clinical phenotypes are not unambiguously differentiable on the basis of biochemical parameters. Conclusion: A continuum of clinical severity from asymptomatic to very severe (border to classic) exists in variant MSUD. Apart from newborns with classic MSUD, also those with variant forms benefit from early diagnosis and start of adequate treatment.

Longitudinal assessment of intellectual achievement in patients with classical galactosemia.

OBJECTIVE: To conduct a longitudinal assessment of long-term cognitive outcome in patients with classical galactosemia. METHODS: Inclusion criteria were (1) previous assessment of IQ dating back >10 years with tests being comparable with the recent German tests HAWIK-III and HAWIE-R, (2) absence of illnesses other than galactosemia, (3) absence of foreign language problems, (4) enzymatic-metabolic proof of classical galactosemia, (5) compliance with dietary therapy, and (6) written informed consent. Twenty-three patients who fulfilled these criteria were found. They underwent the first IQ test at a mean age of 11 ± 5 years and the second 13.6 to 15.5 years later at a mean age of 26 ± 5 years. Results were corrected for the obsolescence of test norms (Flynn effect). RESULTS: Mean total IQ scores on the first and second tests were 78 ± 14 and 73 ± 15, respectively, and not significantly different. IQ scores in the average range were observed for 7 patients on the first test and for 5 patients on the second test. For 17 patients, the intraindividual IQ scores remained essentially unchanged. Five patients showed a decrease and 1 an increase of the IQ score over time. No consistent pattern of change was found with respect to performance or verbal IQ subscores or in achievements in the individual subtest. CONCLUSIONS: The results confirm the presence of reduced cognitive ability in classical galactosemia and present evidence for an absence of substantial galactosemia-induced aggravation of this impairment with increasing age, at least in patients from 4 to 40 years of age. It remains to be clarified whether a reduction of cognitive function in galactosemia may be initiated by an in utero toxicity of endogenously formed galactose and which role such a process may play in the development of intellectual deficiencies that are later maintained throughout life.

Differential Glycomics of Epithelial Membrane Glycoproteins from Urinary Exovesicles Reveals Shifts toward Complex-Type N-Glycosylation in Classical Galactosemia

A variety of genetic variations in the galactose-1-phosphate uridyltransferase (GALT) gene cause profound activity loss of the enzyme and acute toxic effects mediated by accumulating metabolic intermediates of galactose in newborns induced by dietary galactose. However, even on a severely galactose-restricted diet, patients develop serious long-term complications of the CNS and ovaries, which may result from damaging perturbations in cell biology caused by endogenously synthezised galactose. Under galactose stress, the cosubstrate of GALT, galactose-1-phosphate, accumulates and disturbs catabolic and anabolic pathways of the carbohydrate metabolism with potential effects on protein glycosylation and membrane localization of glycoprotein receptors, like the epidermal growth factor receptor. To address this issue in view of a cellular pathomechanism, we performed a differential semiquantitative N-glycomics study of membrane proteins. A suitable noninvasive cellular material derived from epithelial plasma membranes was found in urinary exovesicles and in the shed Tamm-Horsfall protein. By applying matrix-assisted laser ionization mass spectrometry on permethylated, PNGaseF released N-glycans, we demonstrate that GALT deficiency is associated with dramatic shifts from prevalent high-mannose-type glycans found in healthy subjects toward complex-type N-linked glycosylation in patients. These N-glycosylation shifts were observed on exosomal N-glycoproteins but not on the Tamm-Horsfall glycoprotein, which showed predominant high-mannose-type glycosylation with M6.

Indirect calorimetry in humans: a postcalorimetric evaluation procedure for correction of metabolic monitor variability

BACKGROUND Indirect calorimetry (IC) with metabolic monitors is widely used for noninvasive assessment of energy expenditure and macronutrient oxidation in health and disease. OBJECTIVE To overcome deficiencies in validity and reliability of metabolic monitors, we established a procedure that allowed correction for monitor-specific deviations. DESIGN Randomized comparative IC (canopy mode) with the Deltatrac MBM-100 (Datex) and Vmax Encore 29n (SensorMedix) was performed in postabsorptive (overnight fast >8 h) healthy subjects (n = 40). In vitro validation was performed by simulation of oxygen consumption (VO2) and carbon dioxide output (VCO2) rates by using mass-flow regulators and pure gases. A simulation-based postcalorimetric calibration of cart readouts [individual calibration control evaluation (ICcE)] was established in adults (n = 24). RESULTS The comparison of carefully calibrated monitors showed marked differences in VCO2 and VO2 (P < 0.01) and derived metabolic variables [resting energy expenditure (REE), respiratory quotient (RQ), glucose/carbohydrate oxidation (Gox), and fat oxidation (Fox); P < 0.001]. Correlations appeared to be acceptable for breath gas rates and REE (R(2) ~ 0.9) but were unacceptable for RQ (R(2) = 0.3), Gox, and Fox (R(2) = 0.2). In vitro simulation experiments showed monitor-dependent interferences for VCO2 and VO2 as follows: 1) within series, nonlinear and variable deviations of monitor readouts at different exchange rates; 2) between series, differences and unsteady variability; and 3) differences in individual monitor characteristics (eg, rate dependence, stability, imprecision). The introduction of the postcalorimetric recalibration by ICcE resulted in an adjustment of gas exchange rates and the derived metabolic variables with reasonable correlations (R(2) > 0.9). CONCLUSIONS Differential, metabolic, monitor-specific deviations are the primary determinants for lack of accuracy, comparability, and transferability of results. This problem can be overcome by the present postcalorimetric ICcE procedure.

Significance of Diagnostic Parameters in [13C]Octanoic Acid Gastric Emptying Breath Tests*

Abstract Two novel characteristic parameters, the latency time (t (lat)) and the ascension time (t (asc)), are proposed for evaluation of non-invasive [(13)C]octanoic acid breath tests for assessment of the gastric emptying of solids. In breath tests performed in control subjects (n = 30) and diabetic patients (n = 100), the usefulness of these parameters was compared to conventional parameters, i.e., gastric half emptying-time t (1/2,b )) and lag phase (t (lag,b )). The proposed parameters were only loosely correlated (controls, r = 0.199; diabetics, 0.616). A strong correlation was found between the conventional parameters (controls, r = 0.891; diabetics, r = 0.962). Based on the conventional method, 36 patients were suspicious of delayed gastric emptying including 24 patients which exhibited a simultaneous delay in both parameters. Using the new parameters, a total of 46 patients were suspicious of delayed gastric emptying with 15 and 20 having isolated delay in t (lat) and t (asc), respectively. We conclude that the novel parameters may be more appropriate for examination of the different phases of gastric emptying and for evaluation of gastric emptying disturbances in diabetic patients than the parameters conventionally used for this purpose.

Branched-chain L-amino acid metabolism in classical maple syrup urine disease after orthotopic liver transplantation.

We characterized the effect of orthotopic liver transplantation on the catabolism of branched-chain L-amino acids in a female patient with classical form of maple syrup urine disease. Transplantation was performed at the age of 7.4 years due to a terminal liver failure triggered by a hepatitis A infection. Since then, the patient is on an unrestricted diet and plasma concentrations of branched-chain L-amino and 2-oxo acids are stable, yet at moderately increased levels (2- to 3-fold of control). L-Alloisoleucine concentrations, however, remained remarkably elevated (>5-fold of control). In vivo catabolism was investigated by measuring the metabolic L-alloisoleucine clearance and whole-body leucine oxidation in the postabsorptive state. In an oral loading test with 580 μmol alloisoleucine per kg body wt, the L-alloisoleucine elimination rate constant (0.067 h−1) was in the normal range (0.069±0.012 h−1, n=4). In an oral L-[1-13C]leucine load (38 μmol/kg body wt), 19.5% of the tracer dose applied was recovered in exhaled 13CO2 versus 18.9±3.6% in healthy subjects (n=10). Thus, the patient exhibited obviously normal whole-body catabolic rates although branched-chain L-amino acid oxidation was confined to the liver transplant. Most likely, the enhanced substrate supply from extrahepatic sources led to an elevation of the plasma concentrations and thus induced a compensatory enhancement of the metabolic flux through the branched-chain 2-oxo acid dehydrogenase complex in the intact liver tissue.

On the mechanism ofl-alloisoleucine formation: Studies on a healthy subject and in fibroblasts from normals and patients with maple syrup urine disease

Summaryl-Alloisoleucine formation froml-isoleucine was studiedin vitro andin vivo. When a healthy subject was loaded withl-isoleucine, plasma levels ofl-isoleucine and 3-methyl-2-oxopentanoate (KMV), as well asl-alloisoleucine, increased. Peak values were reached successively and were in the orderl-isoleucine > > KMV > >l-alloisoleucine. Metabolic clearance ofl-isoleucine and KMV was rapid; clearance ofl-alloisoleucine was considerably delayed. When human skin fibroblast cultures were challenged withl-isoleucine, KMV accumulated at a gradually decreased rate, whereasl-alloisoleucine accumulated at a gradually accelerated rate. KMV andl-alloisoleucine formation were related and depended on thel-isoleucine concentration applied. In cell lines derived from MSUD patients (classical form), metabolite formation was only about 2-fold higher than in control strains. The relatively small difference between normal and MSUD fibroblastsin vitro as opposed to the striking differences between healthy subjects and MSUD patientsin vivo are discussed with respect to the significance of physiological mechanisms participating in the formation and degradation ofl-alloisoleucine in man.