Silicon-nitride microring resonators for nonlinear optical and biosensing applications.

We discuss the design, fabrication, and characterization of silicon-nitride microring resonators for nonlinear-photonic and biosensing device applications. The first part presents new theoretical and experimental results that overcome highly normal dispersion of silicon-nitride microresonators by adding a dispersive coupler. The latter parts review our work on highly efficient second-order nonlinear interaction in a hybrid silicon-nitride slot waveguide with nonlinear polymer cladding and silicon-nitride microring application as a biosensor for human stress indicator neuropeptide Y at the nanomolar level.

Quantification of Neuropeptide Y with Picomolar Sensitivity Enabled by Guided-Mode Resonance Biosensors.

Assessing levels of neuropeptide Y (NPY) in the human body has many medical uses. Accordingly, we report the quantitative detection of NPY biomarkers applying guided-mode resonance (GMR) biosensor methodology. The label-free sensor operates in the near-infrared spectral region exhibiting distinctive resonance signatures. The interaction of NPY with bioselective molecules on the sensor surface causes spectral shifts that directly identify the binding event without additional processing. In the experiments described here, NPY antibodies are attached to the sensor surface to impart specificity during operation. For the low concentrations of NPY of interest, we apply a sandwich NPY assay in which the sensor-linked anti-NPY molecule binds with NPY that subsequently binds with anti-NPY to close the sandwich. The sandwich assay achieves a detection limit of ~0.1 pM NPY. The photonic sensor methodology applied here enables expeditious high-throughput data acquisition with high sensitivity and specificity. The entire bioreaction is recorded as a function of time, in contrast to label-based methods with single-point detection. The convenient methodology and results reported are significant, as the NPY detection range of 0.1-10 pM demonstrated is useful in important medical circumstances.

Efficient broadband energy detection from the visible to near-infrared using a plasmon FET.

Plasmon based field effect transistors (FETs) can be used to convert energy induced by incident optical radiation to electrical energy. Plasmonic FETs can efficiently detect incident light and amplify it by coupling to resonant plasmonic modes thus improving selectivity and signal to noise ratio. The spectral responses can be tailored both through optimization of nanostructure geometry as well as constitutive materials. In this paper, we studied various plasmonic nanostructures using gold for a wideband spectral response from visible to near-infrared. We show, using empirical data and simulation results, that detection loss exponentially increases as the volume of metal nanostructure increases and also a limited spectral response is possible using gold nanostructures in a plasmon to electric conversion device. Finally, we demonstrate a plasmon FET that offers a broadband spectral response from visible to telecommunication wavelengths.

Branched-chain amino acids alter neurobehavioral function in rats.

Recently, we have described a strong association of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) with obesity and insulin resistance. In the current study, we have investigated the potential impact of BCAA on behavioral functions. We demonstrate that supplementation of either a high-sucrose or a high-fat diet with BCAA induces anxiety-like behavior in rats compared with control groups fed on unsupplemented diets. These behavioral changes are associated with a significant decrease in the concentration of tryptophan (Trp) in brain tissues and a consequent decrease in serotonin but no difference in indices of serotonin synaptic function. The anxiety-like behaviors and decreased levels of Trp in the brain of BCAA-fed rats were reversed by supplementation of Trp in the drinking water but not by administration of fluoxetine, a selective serotonin reuptake inhibitor, suggesting that the behavioral changes are independent of the serotonergic pathway of Trp metabolism. Instead, BCAA supplementation lowers the brain levels of another Trp-derived metabolite, kynurenic acid, and these levels are normalized by Trp supplementation. We conclude that supplementation of high-energy diets with BCAA causes neurobehavioral impairment. Since BCAA are elevated spontaneously in human obesity, our studies suggest a potential mechanism for explaining the strong association of obesity and mood disorders.

Leptin therapy in insulin-deficient type I diabetes.

In nonobese diabetic mice with uncontrolled type 1 diabetes, leptin therapy alone or combined with low-dose insulin reverses the catabolic state through suppression of hyperglucagonemia. Additionally, it mimics the anabolic actions of insulin monotherapy and normalizes hemoglobin A1c with far less glucose variability. We show that leptin therapy, like insulin, normalizes the levels of a wide array of hepatic intermediary metabolites in multiple chemical classes, including acylcarnitines, organic acids (tricarboxylic acid cycle intermediates), amino acids, and acyl CoAs. In contrast to insulin monotherapy, however, leptin lowers both lipogenic and cholesterologenic transcription factors and enzymes and reduces plasma and tissue lipids. The results imply that leptin administration may have multiple short- and long-term advantages over insulin monotherapy for type 1 diabetes.

Genetic networks of liver metabolism revealed by integration of metabolic and transcriptional profiling.

Although numerous quantitative trait loci (QTL) influencing disease-related phenotypes have been detected through gene mapping and positional cloning, identification of the individual gene(s) and molecular pathways leading to those phenotypes is often elusive. One way to improve understanding of genetic architecture is to classify phenotypes in greater depth by including transcriptional and metabolic profiling. In the current study, we have generated and analyzed mRNA expression and metabolic profiles in liver samples obtained in an F2 intercross between the diabetes-resistant C57BL/6 leptin(ob/ob) and the diabetes-susceptible BTBR leptin(ob/ob) mouse strains. This cross, which segregates for genotype and physiological traits, was previously used to identify several diabetes-related QTL. Our current investigation includes microarray analysis of over 40,000 probe sets, plus quantitative mass spectrometry-based measurements of sixty-seven intermediary metabolites in three different classes (amino acids, organic acids, and acyl-carnitines). We show that liver metabolites map to distinct genetic regions, thereby indicating that tissue metabolites are heritable. We also demonstrate that genomic analysis can be integrated with liver mRNA expression and metabolite profiling data to construct causal networks for control of specific metabolic processes in liver. As a proof of principle of the practical significance of this integrative approach, we illustrate the construction of a specific causal network that links gene expression and metabolic changes in the context of glutamate metabolism, and demonstrate its validity by showing that genes in the network respond to changes in glutamine and glutamate availability. Thus, the methods described here have the potential to reveal regulatory networks that contribute to chronic, complex, and highly prevalent diseases and conditions such as obesity and diabetes.

High heritability of metabolomic profiles in families burdened with premature cardiovascular disease.

Integration of genetic and metabolic profiling holds promise for providing insight into human disease. Coronary artery disease (CAD) is strongly heritable, but the heritability of metabolomic profiles has not been evaluated in humans. We performed quantitative mass spectrometry-based metabolic profiling in 117 individuals within eight multiplex families from the GENECARD study of premature CAD. Heritabilities were calculated using variance components. We found high heritabilities for amino acids (arginine, ornithine, alanine, proline, leucine/isoleucine, valine, glutamate/glutamine, phenylalanine and glycine; h(2)=0.33-0.80, P=0.005-1.9 x 10(-16)), free fatty acids (arachidonic, palmitic, linoleic; h(2)=0.48-0.59, P=0.002-0.00005) and acylcarnitines (h(2)=0.23-0.79, P=0.05-0.0000002). Principal components analysis was used to identify metabolite clusters. Reflecting individual metabolites, several components were heritable, including components comprised of ketones, beta-hydroxybutyrate and C2-acylcarnitine (h(2)=0.61); short- and medium-chain acylcarnitines (h(2)=0.39); amino acids (h(2)=0.44); long-chain acylcarnitines (h(2)=0.39) and branched-chain amino acids (h(2)=0.27). We report a novel finding of high heritabilities of metabolites in premature CAD, establishing a possible genetic basis for these profiles. These results have implications for understanding CAD pathophysiology and genetics.

Chemical knockout of pantothenate kinase reveals the metabolic and genetic program responsible for hepatic coenzyme A homeostasis.

Coenzyme A (CoA) is the major acyl group carrier in intermediary metabolism. Hopantenate (HoPan), a competitive inhibitor of the pantothenate kinases, was used to chemically antagonize CoA biosynthesis. HoPan dramatically reduced liver CoA and mice developed severe hypoglycemia. Insulin was reduced, glucagon and corticosterone were elevated, and fasting accelerated hypoglycemia. Metabolic profiling revealed a large increase in acylcarnitines, illustrating the role of carnitine in buffering acyl groups to maintain the nonesterified CoASH level. HoPan triggered significant changes in hepatic gene expression that substantially increased the thioesterases, which liberate CoASH from acyl-CoA, and increased pyruvate dehydrogenase kinase 1, which prevents the conversion of CoASH to acetyl-CoA. These results identify the metabolic rearrangements that maintain the CoASH pool which is critical to mitochondrial functions, including gluconeogenesis, fatty acid oxidation, and the tricarboxylic acid and urea cycles.

Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism.

Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet ß cells, heralds the onset of type 2 diabetes (T2D). To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13 and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP) and an increase in adenylosuccinate (S-AMP), suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS). Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human ß cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1). S-AMP also overcomes the defect in glucose-induced exocytosis in ß cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing ß cell dysfunction in T2D.

Factors that affect ion trap data-dependent MS/MS in proteomics.

Quadrupole ion trap scanning parameters for performing bottom-up proteomics in a data-dependent fashion were evaluated on a Finnigan LCQ Deca mass spectrometer. Evaluation of parameters such as the number of averaged full scans, the number of averaged MS/MS scans, and ion injection times were necessary for acquiring high quality MS/MS spectra that yield favorable b and y ion coverage and high correlation to proteins using database searching algorithms. In this study, we demonstrated how the duty cycle of the mass spectrometer affects the number of peptides that can be successfully identified by SEQUEST using a model system of tryptic BSA peptides to mimic a typical complex mixture associated with bottom-up proteomics. The number of averaged scans and the duration of ion accumulation in the trap had a significant effect on the quality of acquired MS/MS spectra. For example, by increasing the ion injection time from 500 ms to 600 ms, peptide HLVDEPQNLIK improved from being improperly identified to being correctly identified with a SEQUEST cross-correlation score of 3.60. As a result of these experiments, we have devised the following set of ion trap parameters for performing bottom-up proteomics analysis in our laboratory: Three averaged full scans, five averaged MS/MS scans, and a maximum ion injection time of 600 ms.

Research resource: tissue- and pathway-specific metabolomic profiles of the steroid receptor coactivator (SRC) family.

The rapidly growing family of transcriptional coregulators includes coactivators that promote transcription and corepressors that harbor the opposing function. In recent years, coregulators have emerged as important regulators of metabolic homeostasis, including the p160 steroid receptor coactivator (SRC) family. Members of the SRC family have been ascribed important roles in control of gluconeogenesis, fat absorption and storage in the liver, and fatty acid oxidation in skeletal muscle. To provide a deeper and more granular understanding of the metabolic impact of the SRC family members, we performed targeted metabolomic analyses of key metabolic byproducts of glucose, fatty acid, and amino acid metabolism in mice with global knockouts (KOs) of SRC-1, SRC-2, or SRC-3. We measured amino acids, acyl carnitines, and organic acids in five tissues with key metabolic functions (liver, heart, skeletal muscle, brain, plasma) isolated from SRC-1, -2, or -3 KO mice and their wild-type littermates under fed and fasted conditions, thereby unveiling unique metabolic functions of each SRC. Specifically, SRC-1 ablation revealed the most significant impact on hepatic metabolism, whereas SRC-2 appeared to impact cardiac metabolism. Conversely, ablation of SRC-3 primarily affected brain and skeletal muscle metabolism. Surprisingly, we identified very few metabolites that changed universally across the three SRC KO models. The findings of this Research Resource demonstrate that coactivator function has very limited metabolic redundancy even within the homologous SRC family. Furthermore, this work also demonstrates the use of metabolomics as a means for identifying novel metabolic regulatory functions of transcriptional coregulators.

Atypical antipsychotics rapidly and inappropriately switch peripheral fuel utilization to lipids, impairing metabolic flexibility in rodents.

Patients taking atypical antipsychotics are frequented by serious metabolic (eg, hyperglycemia, obesity, and diabetes) and cardiac effects. Surprisingly, chronic treatment also appears to lower free fatty acids (FFAs). This finding is paradoxical because insulin resistance is typically associated with elevated not lower FFAs. How atypical antipsychotics bring about these converse changes in plasma glucose and FFAs is unknown. Chronic treatment with olanzapine, a prototypical, side effect prone atypical antipsychotic, lowered FFA in Sprague-Dawley rats. Olanzapine also lowered plasma FFA acutely, concomitantly impairing in vivo lipolysis and robustly elevating whole-body lipid oxidation. Increased lipid oxidation was evident from accelerated losses of triglycerides after food deprivation or lipid challenge, elevated FFA uptake into most peripheral tissues (∼2-fold) except heart, rises in long-chain 3-hydroxylated acyl-carnitines observed in diabetes, and rapid suppression of the respiratory exchange ratio (RER) during the dark cycle. Normal rises in RER following refeeding, a sign of metabolic flexibility, were severely blunted by olanzapine. Increased lipid oxidation in muscle could be explained by ∼50% lower concentrations of the negative cytoplasmic regulator of carnitine palmitoyltransferase I, malonyl-CoA. This was associated with loss of anapleurotic metabolites and citric acid cycle precursors of malonyl-CoA synthesis rather than adenosine monophosphate-activated kinase activation or direct ACC1/2 inhibition. The ability of antipsychotics to lower dark cycle RER in mice corresponded to their propensities to cause metabolic side effects. Our studies indicate that lipocentric mechanisms or altered intermediary metabolism could underlie the FFA lowering and hyperglycemia (Randle cycle) as well as some of the other side effects of atypical antipsychotics, thereby suggesting strategies for alleviating them.

Caloric restriction alters the metabolic response to a mixed-meal: results from a randomized, controlled trial.

OBJECTIVES: To determine if caloric restriction (CR) would cause changes in plasma metabolic intermediates in response to a mixed meal, suggestive of changes in the capacity to adapt fuel oxidation to fuel availability or metabolic flexibility, and to determine how any such changes relate to insulin sensitivity (S(I)). METHODS: Forty-six volunteers were randomized to a weight maintenance diet (Control), 25% CR, or 12.5% CR plus 12.5% energy deficit from structured aerobic exercise (CR+EX), or a liquid calorie diet (890 kcal/d until 15% reduction in body weight)for six months. Fasting and postprandial plasma samples were obtained at baseline, three, and six months. A targeted mass spectrometry-based platform was used to measure concentrations of individual free fatty acids (FFA), amino acids (AA), and acylcarnitines (AC). S(I) was measured with an intravenous glucose tolerance test. RESULTS: Over three and six months, there were significantly larger differences in fasting-to-postprandial (FPP) concentrations of medium and long chain AC (byproducts of FA oxidation) in the CR relative to Control and a tendency for the same in CR+EX (CR-3 month P = 0.02; CR-6 month P = 0.002; CR+EX-3 month P = 0.09; CR+EX-6 month P = 0.08). After three months of CR, there was a trend towards a larger difference in FPP FFA concentrations (P = 0.07; CR-3 month P = 0.08). Time-varying differences in FPP concentrations of AC and AA were independently related to time-varying S(I) (P<0.05 for both). CONCLUSIONS: Based on changes in intermediates of FA oxidation following a food challenge, CR imparted improvements in metabolic flexibility that correlated with improvements in S(I). TRIAL REGISTRATION: ClinicalTrials.gov NCT00099151.

Ablation of steroid receptor coactivator-3 resembles the human CACT metabolic myopathy.

Oxidation of lipid substrates is essential for survival in fasting and other catabolic conditions, sparing glucose for the brain and other glucose-dependent tissues. Here we show Steroid Receptor Coactivator-3 (SRC-3) plays a central role in long chain fatty acid metabolism by directly regulating carnitine/acyl-carnitine translocase (CACT) gene expression. Genetic deficiency of CACT in humans is accompanied by a constellation of metabolic and toxicity phenotypes including hypoketonemia, hypoglycemia, hyperammonemia, and impaired neurologic, cardiac and skeletal muscle performance, each of which is apparent in mice lacking SRC-3 expression. Consistent with human cases of CACT deficiency, dietary rescue with short chain fatty acids drastically attenuates the clinical hallmarks of the disease in mice devoid of SRC-3. Collectively, our results position SRC-3 as a key regulator of ß-oxidation. Moreover, these findings allow us to consider platform coactivators such as the SRCs as potential contributors to syndromes such as CACT deficiency, previously considered as monogenic.

Exercise-induced changes in metabolic intermediates, hormones, and inflammatory markers associated with improvements in insulin sensitivity.

OBJECTIVE: To understand relationships between exercise training-mediated improvements in insulin sensitivity (S(I)) and changes in circulating concentrations of metabolic intermediates, hormones, and inflammatory mediators. RESEARCH DESIGN AND METHODS: Targeted mass spectrometry and enzyme-linked immunosorbent assays were used to quantify metabolic intermediates, hormones, and inflammatory markers at baseline, after 6 months of exercise training, and 2 weeks after exercise training cessation (n = 53). A principal components analysis (PCA) strategy was used to relate changes in these intermediates to changes in S(I). RESULTS: PCA reduced the number of intermediates from 90 to 24 factors composed of biologically related components. With exercise training, improvements in S(I) were associated with reductions in by-products of fatty acid oxidation and increases in glycine and proline (P < 0.05, R² = 0.59); these relationships were retained 15 days after cessation of exercise training (P < 0.05, R² = 0.34). CONCLUSIONS: These observations support prior observations in animal models that exercise training promotes more efficient mitochondrial ß-oxidation and challenges current hypotheses regarding exercise training and glycine metabolism.

Effect of caloric restriction with and without exercise on metabolic intermediates in nonobese men and women.

OBJECTIVES: The objective of the study was to evaluate whether serum concentrations of metabolic intermediates are related to adiposity and insulin sensitivity (Si) in overweight healthy subjects and compare changes in metabolic intermediates with similar weight loss achieved by diet only or diet plus exercise. DESIGN: This was a randomized controlled trial. PARTICIPANTS AND INTERVENTION: The cross-sectional study included 46 (aged 36.8 ± 1.0 yr) overweight (body mass index 27.8 ± 0.7 kg/m(2)) subjects enrolled in a 6-month study of calorie restriction. To determine the effect of diet only or diet plus exercise on metabolic intermediates, 35 subjects were randomized to control (energy intake at 100% of energy requirements); CR (25% calorie restriction), or CR+EX: (12.5% CR plus 12.5% increase in energy expenditure by exercise). MAIN OUTCOME MEASURES: Serum concentrations of eight fatty acids, 15 amino acids, and 45 acylcarnitines (ACs) measured by targeted mass spectrometry. RESULTS: In overweight subjects, the concentrations of C2 AC and long-chain ACs were positively associated with percent fat (R(2) = 0.75, P = 0.0001) and Si (R(2) = 0.12, P = 0.05). The percent fat (R(2) = 0.77, P < 0.0001), abdominal visceral fat (R(2) = 0.64, P < 0.0001), and intrahepatic fat (R(2) = 0.30, P = 0.0002) were positively associated with fatty acid concentrations. There was a significant increase in an AC factor (comprised of C2 and several medium chain ACs) in the CR group (P = 0.01). CONCLUSION: In nonobese subjects, fasted serum ACs are associated with Si and fat mass. Despite similar weight loss, serum ACs increase with CR alone but not CR+EX. A greater improvement in Si with weight loss during CR+EX interventions may be related to improved coupling of ß-oxidation and tricarboxylic acid cycle flux induced by exercise.

Differential metabolic impact of gastric bypass surgery versus dietary intervention in obese diabetic subjects despite identical weight loss.

Glycemic control is improved more after gastric bypass surgery (GBP) than after equivalent diet-induced weight loss in patients with morbid obesity and type 2 diabetes mellitus. We applied metabolomic profiling to understand the mechanisms of this better metabolic response after GBP. Circulating amino acids (AAs) and acylcarnitines (ACs) were measured in plasma from fasted subjects by targeted tandem mass spectrometry before and after a matched 10-kilogram weight loss induced by GBP or diet. Total AAs and branched-chain AAs (BCAAs) decreased after GBP, but not after dietary intervention. Metabolites derived from BCAA oxidation also decreased only after GBP. Principal components (PC) analysis identified two major PCs, one composed almost exclusively of ACs (PC1) and another with BCAAs and their metabolites as major contributors (PC2). PC1 and PC2 were inversely correlated with pro-insulin concentrations, the C-peptide response to oral glucose, and the insulin sensitivity index after weight loss, whereas PC2 was uniquely correlated with levels of insulin resistance (HOMA-IR). These data suggest that the enhanced decrease in circulating AAs after GBP occurs by mechanisms other than weight loss and may contribute to the better improvement in glucose homeostasis observed with the surgical intervention.

The coactivator SRC-1 is an essential coordinator of hepatic glucose production.

Gluconeogenesis makes a major contribution to hepatic glucose production, a process critical for survival in mammals. In this study, we identify the p160 family member, SRC-1, as a key coordinator of the hepatic gluconeogenic program in vivo. SRC-1-null mice displayed hypoglycemia secondary to a deficit in hepatic glucose production. Selective re-expression of SRC-1 in the liver restored blood glucose levels to a normal range. SRC-1 was found induced upon fasting to coordinate in a cell-autonomous manner, the gene expression of rate-limiting enzymes of the gluconeogenic pathway. At the molecular level, the main role of SRC-1 was to modulate the expression and the activity of C/EBPα through a feed-forward loop in which SRC-1 used C/EBPα to transactivate pyruvate carboxylase, a crucial gene for initiation of the gluconeogenic program. We propose that SRC-1 acts as a critical mediator of glucose homeostasis in the liver by adjusting the transcriptional activity of key genes involved in the hepatic glucose production machinery.

The STEDMAN project: biophysical, biochemical and metabolic effects of a behavioral weight loss intervention during weight loss, maintenance, and regain.

The Study of the Effects of Diet on Metabolism and Nutrition (STEDMAN) Project uses comprehensive metabolic profiling to probe biochemical mechanisms of weight loss in humans. Measurements at baseline, 2 and 4 weeks, 6 and 12 months included diet, body composition, metabolic rate, hormones, and 80 intermediary metabolites measured by mass spectrometry. In 27 obese adults in a behavioral weight loss intervention, median weight decreased 13.9 lb over the first 6 months, then reverted towards baseline by 12 months. Insulin resistance (HOMA) was partially ameliorated in the first 6 months and showed sustained improvement at 12 months despite weight regain. Ghrelin increased with weight loss and reverted to baseline, whereas leptin and PYY fell at 6 months and remained persistently low. NPY levels did not change. Factors possibly contributing to sustained improvement in insulin sensitivity despite weight regain include adiponectin (increased by 12 months), IGF-1 (increased during weight loss and continued to increase during weight regain), and visceral fat (fell at 6 months but did not change thereafter). We observed a persistent reduction in free fatty acids, branched chain amino acids, and related metabolites that may contribute to improved insulin action. These findings provide evidence for sustained benefits of weight loss in obese humans and insights into mechanisms.

A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance.

Metabolomic profiling of obese versus lean humans reveals a branched-chain amino acid (BCAA)-related metabolite signature that is suggestive of increased catabolism of BCAA and correlated with insulin resistance. To test its impact on metabolic homeostasis, we fed rats on high-fat (HF), HF with supplemented BCAA (HF/BCAA), or standard chow (SC) diets. Despite having reduced food intake and a low rate of weight gain equivalent to the SC group, HF/BCAA rats were as insulin resistant as HF rats. Pair-feeding of HF diet to match the HF/BCAA animals or BCAA addition to SC diet did not cause insulin resistance. Insulin resistance induced by HF/BCAA feeding was accompanied by chronic phosphorylation of mTOR, JNK, and IRS1Ser307 and by accumulation of multiple acylcarnitines in muscle, and it was reversed by the mTOR inhibitor, rapamycin. Our findings show that in the context of a dietary pattern that includes high fat consumption, BCAA contributes to development of obesity-associated insulin resistance.