Amino Acid Signatures to Evaluate the Beneficial Effects of Weight Loss.

Aims. We investigated the relationship between circulating amino acid levels and obesity; to what extent weight loss followed by weight maintenance can correct amino acid abnormalities; and whether amino acids are related to weight loss. Methods. Amino acids associated with waist circumference (WC) and BMI were studied in 804 participants from the Malmö Diet and Cancer Cardiovascular Cohort (MDC-CC). Changes in amino acid levels were analyzed after weight loss and weight maintenance in 12 obese subjects and evaluated in a replication cohort (n = 83). Results. Out of the eight identified BMI-associated amino acids from the MDC-CC, alanine, isoleucine, tyrosine, phenylalanine, and glutamate decreased after weight loss, while asparagine increased after weight maintenance. These changes were validated in the replication cohort. Scores that were constructed based on obesity-associated amino acids and known risk factors decreased in the ≥10% weight loss group with an associated change in BMI (R2 = 0.16-0.22, p < 0.002), whereas the scores increased in the <10% weight loss group (p < 0.0004). Conclusions. Weight loss followed by weight maintenance leads to differential changes in amino acid levels associated with obesity. Treatment modifiable scores based on epidemiological and interventional data may be used to evaluate the potential metabolic benefit of weight loss.

Changes in glucose-elicited blood metabolite responses following weight loss and long term weight maintenance in obese individuals with impaired glucose tolerance.

AIMS: Weight loss improves insulin sensitivity and glucose tolerance in obese subjects with impaired glucose tolerance (IGT), but the long term dynamic effects on blood metabolites other than glucose during an oral glucose tolerance test (OGTT), are largely unknown. Here, we studied changes in OGTT-elicited metabolite patterns in obese subjects during a diet-induced weight loss study. METHODS: Blood samples from 14 obese individuals with IGT were collected at 0, 30 and 120 min during a standard 75 g OGTT at baseline (BMI 44 ± 2 kg/m(2)), after weight loss (BMI 36 ± 2 kg/m(2)) and after weight maintenance (BMI 35 ± 2 kg/m(2)). Serum metabolite levels were analyzed by gas chromatography/mass spectrometry and compared to a lean glucose tolerant group. RESULTS: Changes in the OGTT-elicited metabolite patterns occurred differentially during weight loss and weight maintenance. Enhanced suppression of aromatic amino acids were associated with decreased insulinogenic index observed after weight loss (tyrosine: r=0.72, p=0.013; phenylalanine: r=0.63, p=0.039). The OGTT-elicited suppression and/or lack of increase in levels of glutamate, glutamine, isoleucine, leucine, and the fatty acids laurate, oleate and palmitate, improved towards the lean profile after weight maintenance, paralleling an improvement in glucose tolerance. The greater heterogeneity in the response before and after weight loss in the obese, compared to lean subjects, was markedly reduced after weight maintenance. CONCLUSIONS: Diet-induced weight loss followed by weight maintenance results in changes in metabolite profiles associated with either hepatic insulin sensitivity or peripheral glucose tolerance. Our results highlight the importance of evaluating the effects of weight loss and weight maintenance separately.

Metabolite profile deviations in an oral glucose tolerance test-a comparison between lean and obese individuals.

OBJECTIVE: While impaired glucose tolerance diagnosed by the oral glucose tolerance test (OGTT) is a common trait in obese individuals, less is known about changes in levels of other metabolites. The aim was to reveal the complex alterations in metabolite levels provoked by an OGTT and its perturbation in obese individuals. METHODS: Gas chromatography/mass spectrometry was used to profile metabolite levels in serum from 14 obese participants (body mass index [BMI] of 43.6 ± 1.5 kg m(-2) [mean ± SEM]) at 0, 30, and 120 min during a standard 2-h 75 g OGTT. Metabolite profiles from six lean individuals (BMI of 22.4 ± 2.4 kg m(-2) ), collected from a previous study, were included for comparison. RESULTS: In the obese group, 59 metabolite profiles were determined. Among these, 16 deviated from profiles in the lean group. Deviating metabolites were categorized into three groups. Delayed reduction in levels of five fatty acids. Increased levels at 30 min of five amino acids, including isoleucine and leucine. A blunted increase at 30 min of six metabolites. CONCLUSIONS: Metabolomics analysis revealed distinct differences in alterations of metabolite levels during an OGTT in obese and lean subjects. To this end, our data suggests a disrupted regulation of ketogenesis, lipolysis and proteolysis in obese individuals.

Coordinate changes in histone modifications, mRNA levels, and metabolite profiles in clonal INS-1 832/13 ß-cells accompany functional adaptations to lipotoxicity.

Lipotoxicity is a presumed pathogenetic process whereby elevated circulating and stored lipids in type 2 diabetes cause pancreatic ß-cell failure. To resolve the underlying molecular mechanisms, we exposed clonal INS-1 832/13 ß-cells to palmitate for 48 h. We observed elevated basal insulin secretion but impaired glucose-stimulated insulin secretion in palmitate-exposed cells. Glucose utilization was unchanged, palmitate oxidation was increased, and oxygen consumption was impaired. Halting exposure of the clonal INS-1 832/13 ß-cells to palmitate largely recovered all of the lipid-induced functional changes. Metabolite profiling revealed profound but reversible increases in cellular lipids. Glucose-induced increases in tricarboxylic acid cycle intermediates were attenuated by exposure to palmitate. Analysis of gene expression by microarray showed increased expression of 982 genes and decreased expression of 1032 genes after exposure to palmitate. Increases were seen in pathways for steroid biosynthesis, cell cycle, fatty acid metabolism, DNA replication, and biosynthesis of unsaturated fatty acids; decreases occurred in the aminoacyl-tRNA synthesis pathway. The activity of histone-modifying enzymes and histone modifications of differentially expressed genes were reversibly altered upon exposure to palmitate. Thus, Insig1, Lss, Peci, Idi1, Hmgcs1, and Casr were subject to epigenetic regulation. Our analyses demonstrate that coordinate changes in histone modifications, mRNA levels, and metabolite profiles accompanied functional adaptations of clonal ß-cells to lipotoxicity. It is highly likely that these changes are pathogenetic, accounting for loss of glucose responsiveness and perturbed insulin secretion.

Time-resolved metabolomics analysis of ß-cells implicates the pentose phosphate pathway in the control of insulin release.

Insulin secretion is coupled with changes in ß-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 ß-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the ß-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 ß-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in ß-cell stimulus-secretion coupling.

Differential gene expression in adipose tissue from obese human subjects during weight loss and weight maintenance.

BACKGROUND: Differential gene expression in adipose tissue during diet-induced weight loss followed by a weight stability period is poorly characterized. Markers of these processes may provide a deeper understanding of underlying mechanisms. OBJECTIVE: We aimed to identify differentially expressed genes in human adipose tissue during weight loss and weight maintenance after weight loss. DESIGN: RNA from subcutaneous abdominal adipose tissue from 9 obese subjects was analyzed by using a complementary DNA microarray at baseline after weight loss on a low-calorie diet and after weight maintenance. RESULTS: Subjects lost 18.8 ± 1.8% of weight and maintained this loss during weight maintenance (1.1 ± 2.1%; range: -9.3 to 10.6%). Most differentially expressed genes exhibited a reciprocal regulation and returned to baseline after weight loss (2163 genes) and weight maintenance (3175 genes). CETP and ABCG1, both of which participate in the HDL-mediated reverse cholesterol transport (RCT), were among the most upregulated of the 750 genes that were differentially expressed after both processes. Several genes involved in inflammation were downregulated. The use of real-time polymerase chain reaction confirmed or partially confirmed the previously implicated genes TNMD and MMP9 (both downregulated), PNPLA3 (upregulated), and CIDEA and SCD (both reciprocally regulated). CONCLUSIONS: The beneficial effects of weight loss should be investigated after long-term weight maintenance. The processes of weight loss and weight maintenance should be viewed as biologically distinct. CETP and ABCG1 may be important mediators of these effects through HDL-mediated RCT.

Development and optimization of a metabolomic method for analysis of adherent cell cultures.

In this investigation, a gas chromatography/mass spectrometry (GC/MS)-based metabolomic protocol for adherent cell cultures was developed using statistical design of experiments. Cell disruption, metabolite extraction, and the GC/MS settings were optimized aiming at a gentle, unbiased, sensitive, and high-throughput metabolomic protocol. Due to the heterogeneity of the metabolome and the inherent selectivity of all analytical techniques, development of unbiased protocols is highly complex. Changing one parameter of the protocol may change the response of many groups of metabolites. In this investigation, statistical design of experiments and multivariate analysis also allowed such interaction effects to be taken into account. The protocol was validated with respect to linear range, precision, and limit of detection in a clonal rat insulinoma cell line (INS-1 832/13). The protocol allowed high-throughput profiling of metabolites covering the major metabolic pathways. The majority of metabolites displayed a linear range from a single well in a 96-well plate up to a 10 cm culture dish. The method allowed a total of 47 analyses to be performed in 24h.