Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

Modulating Phase Behavior in Fatty Acid-Modified Elastin-like Polypeptides (FAMEs): Insights into the Impact of Lipid Length on Thermodynamics and Kinetics of Phase Separation.

Although post-translational lipidation is prevalent in eukaryotes, its impact on the liquid-liquid phase separation of disordered proteins is still poorly understood. Here, we examined the thermodynamic phase boundaries and kinetics of aqueous two-phase system (ATPS) formation for a library of elastin-like polypeptides modified with saturated fatty acids of different chain lengths. By systematically altering the physicochemical properties of the attached lipids, we were able to correlate the molecular properties of lipids to changes in the thermodynamic phase boundaries and the kinetic stability of droplets formed by these proteins. We discovered that increasing the chain length lowers the phase separation temperature in a sigmoidal manner due to alterations in the unfavorable interactions between protein and water and changes in the entropy of phase separation. Our kinetic studies unveiled remarkable sensitivity to lipid length, which we propose is due to the temperature-dependent interactions between lipids and the protein. Strikingly, we found that the addition of just a single methylene group is sufficient to allow tuning of these interactions as a function of temperature, with proteins modified with C7-C9 lipids exhibiting non-Arrhenius dependence in their phase separation, a behavior that is absent for both shorter and longer fatty acids. This work advances our theoretical understanding of protein-lipid interactions and opens avenues for the rational design of lipidated proteins in biomedical paradigms, where precise control over the phase separation is pivotal.

Adaptive Recombinant Nanoworms from Genetically Encodable Star Amphiphiles.

Recombinant nanoworms are promising candidates for materials and biomedical applications ranging from the templated synthesis of nanomaterials to multivalent display of bioactive peptides and targeted delivery of theranostic agents. However, molecular design principles to synthesize these assemblies (which are thermodynamically favorable only in a narrow region of the phase diagram) remain unclear. To advance the identification of design principles for the programmable assembly of proteins into well-defined nanoworms and to broaden their stability regimes, we were inspired by the ability of topologically engineered synthetic macromolecules to acess rare mesophases. To test this design principle in biomacromolecular assemblies, we used post-translational modifications (PTMs) to generate lipidated proteins with precise topological and compositional asymmetry. Using an integrated experimental and computational approach, we show that the material properties (thermoresponse and nanoscale assembly) of these hybrid amphiphiles are modulated by their amphiphilic architecture. Importantly, we demonstrate that the judicious choice of amphiphilic architecture can be used to program the assembly of proteins into adaptive nanoworms, which undergo a morphological transition (sphere-to-nanoworms) in response to temperature stimuli.

Short-Range Forecasting of COVID-19 During Early Onset at County, Health District, and State Geographic Levels Using Seven Methods: Comparative Forecasting Study.

BACKGROUND: Forecasting methods rely on trends and averages of prior observations to forecast COVID-19 case counts. COVID-19 forecasts have received much media attention, and numerous platforms have been created to inform the public. However, forecasting effectiveness varies by geographic scope and is affected by changing assumptions in behaviors and preventative measures in response to the pandemic. Due to time requirements for developing a COVID-19 vaccine, evidence is needed to inform short-term forecasting method selection at county, health district, and state levels. OBJECTIVE: COVID-19 forecasts keep the public informed and contribute to public policy. As such, proper understanding of forecasting purposes and outcomes is needed to advance knowledge of health statistics for policy makers and the public. Using publicly available real-time data provided online, we aimed to evaluate the performance of seven forecasting methods utilized to forecast cumulative COVID-19 case counts. Forecasts were evaluated based on how well they forecast 1, 3, and 7 days forward when utilizing 1-, 3-, 7-, or all prior-day cumulative case counts during early virus onset. This study provides an objective evaluation of the forecasting methods to identify forecasting model assumptions that contribute to lower error in forecasting COVID-19 cumulative case growth. This information benefits professionals, decision makers, and the public relying on the data provided by short-term case count estimates at varied geographic levels. METHODS: We created 1-, 3-, and 7-day forecasts at the county, health district, and state levels using (1) a naïve approach, (2) Holt-Winters (HW) exponential smoothing, (3) a growth rate approach, (4) a moving average (MA) approach, (5) an autoregressive (AR) approach, (6) an autoregressive moving average (ARMA) approach, and (7) an autoregressive integrated moving average (ARIMA) approach. Forecasts relied on Virginia's 3464 historical county-level cumulative case counts from March 7 to April 22, 2020, as reported by The New York Times. Statistically significant results were identified using 95% CIs of median absolute error (MdAE) and median absolute percentage error (MdAPE) metrics of the resulting 216,698 forecasts. RESULTS: The next-day MA forecast with 3-day look-back length obtained the lowest MdAE (median 0.67, 95% CI 0.49-0.84, P<.001) and statistically significantly differed from 39 out of 59 alternatives (66%) to 53 out of 59 alternatives (90%) at each geographic level at a significance level of .01. For short-range forecasting, methods assuming stationary means of prior days' counts outperformed methods with assumptions of weak stationarity or nonstationarity means. MdAPE results revealed statistically significant differences across geographic levels. CONCLUSIONS: For short-range COVID-19 cumulative case count forecasting at the county, health district, and state levels during early onset, the following were found: (1) the MA method was effective for forecasting 1-, 3-, and 7-day cumulative case counts; (2) exponential growth was not the best representation of case growth during early virus onset when the public was aware of the virus; and (3) geographic resolution was a factor in the selection of forecasting methods.

Considerations for best practices in studies of fiber or other dietary components and the intestinal microbiome.

A 2-day workshop organized by the National Institutes of Health and U.S. Department of Agriculture included 16 presentations focused on the role of diet in alterations of the gastrointestinal microbiome, primarily that of the colon. Although thousands of research projects have been funded by U.S. federal agencies to study the intestinal microbiome of humans and a variety of animal models, only a minority addresses dietary effects, and a small subset is described in sufficient detail to allow reproduction of a study. Whereas there are standards being developed for many aspects of microbiome studies, such as sample collection, nucleic acid extraction, data handling, etc., none has been proposed for the dietary component; thus this workshop focused on the latter specific point. It is important to foster rigor in design and reproducibility of published studies to maintain high quality and enable designs that can be compared in systematic reviews. Speakers addressed the influence of the structure of the fermentable carbohydrate on the microbiota and the variables to consider in design of studies using animals, in vitro models, and human subjects. For all types of studies, strengths and weaknesses of various designs were highlighted, and for human studies, comparisons between controlled feeding and observational designs were discussed. Because of the lack of published, best-diet formulations for specific research questions, the main recommendation is to describe dietary ingredients and treatments in as much detail as possible to allow reproduction by other scientists.

N-linked glycan stabilization of the VWF A2 domain.

Shear forces in the blood trigger a conformational transition in the von Willebrand factor (VWF) A2 domain, from its native folded to an unfolded state, in which the cryptic scissile bond (Y1605-M1606) is exposed and can then be proteolysed by ADAMTS13. The conformational transition depends upon a Ca(2+)binding site and a vicinal cysteine disulfide bond. Glycosylation at N1574 has previously been suggested to modulate VWF A2 domain interaction with ADAMTS13 through steric hindrance by the bulky carbohydrate structure. We investigated how the N-linked glycans of the VWF A2 domain affect thermostability and regulate both the exposure of the ADAMTS13 binding sites and the scissile bond. We show by differential scanning fluorimetry that the N-linked glycans thermodynamically stabilize the VWF A2 domain. The essential component of the glycan structure is the first sugar residue (GlcNAc) at the N1574 attachment site. From its crystal structures, N1574-GlcNAc is predicted to form stabilizing intradomain interactions with Y1544 and nearby residues. Substitution of the surface-exposed Y1544 to aspartic acid is able to stabilize the domain in the absence of glycosylation and protect against ADAMTS13 proteolysis in both the VWF A2 domain and FLVWF. Glycan stabilization of the VWF A2 domain acts together with the Ca(2+)binding site and vicinal cysteine disulfide bond to control unfolding and ADAMTS13 proteolysis.

Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF.

Rheological shear forces in the blood trigger von Willebrand factor (VWF) unfolding which exposes the Y1605-M1606 scissile bond within the VWF A2 domain for cleavage by ADAMTS13. The VWF A2 domain contains 2 structural features that provide it with stability: a vicinal disulphide bond and a Ca(2+)-binding site (CBS). We investigated how these 2 structural features interplay to determine stability and regulate the exposure of the scissile bond in full-length VWF. We have used differential scanning fluorimetry together with site-directed mutagenesis of residues involved in both the vicinal disulphide bond and the CBS to demonstrate that both of these sites contribute to stability against thermal unfolding of the isolated VWF A2 domain. Moreover, we show that the combination of site mutations can result in increased susceptibility of FL-VWF to proteolysis by ADAMTS13, even in the absence of an agent (such as urea) required to induce unfolding. These studies demonstrate that VWF A2 domain stability provided by its 2 structural elements (vicinal disulphide bond and CBS) is a key protective determinant against FL-VWF cleavage by ADAMTS13. They suggest a 2-step mechanism for VWF A2 domain unfolding.

Global deletion of BCATm increases expression of skeletal muscle genes associated with protein turnover.

Consumption of a protein-containing meal by a fasted animal promotes protein accretion in skeletal muscle, in part through leucine stimulation of protein synthesis and indirectly through repression of protein degradation mediated by its metabolite, α-ketoisocaproate. Mice lacking the mitochondrial branched-chain aminotransferase (BCATm/Bcat2), which interconverts leucine and α-ketoisocaproate, exhibit elevated protein turnover. Here, the transcriptomes of gastrocnemius muscle from BCATm knockout (KO) and wild-type mice were compared by next-generation RNA sequencing (RNA-Seq) to identify potential adaptations associated with their persistently altered nutrient signaling. Statistically significant changes in the abundance of 1,486/∼39,010 genes were identified. Bioinformatics analysis of the RNA-Seq data indicated that pathways involved in protein synthesis [eukaryotic initiation factor (eIF)-2, mammalian target of rapamycin, eIF4, and p70S6K pathways including 40S and 60S ribosomal proteins], protein breakdown (e.g., ubiquitin mediated), and muscle degeneration (apoptosis, atrophy, myopathy, and cell death) were upregulated. Also in agreement with our previous observations, the abundance of mRNAs associated with reduced body size, glycemia, plasma insulin, and lipid signaling pathways was altered in BCATm KO mice. Consistently, genes encoding anaerobic and/or oxidative metabolism of carbohydrate, fatty acids, and branched chain amino acids were modestly but systematically reduced. Although there was no indication that muscle fiber type was different between KO and wild-type mice, a difference in the abundance of mRNAs associated with a muscular dystrophy phenotype was observed, consistent with the published exercise intolerance of these mice. The results suggest transcriptional adaptations occur in BCATm KO mice that along with altered nutrient signaling may contribute to their previously reported protein turnover, metabolic and exercise phenotypes.

Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity.

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

Adipose transplant for inborn errors of branched chain amino acid metabolism in mice.

Liver transplantation appears to be quite beneficial for treatment of maple syrup urine disease (MSUD, an inherited disorder of branched chain amino acid metabolism); however, there is a limited availability of donor livers worldwide and the first year costs of liver transplants are quite high. Recent studies have suggested that intact adipose tissue, already widely used in reconstructive surgery, may have an underappreciated high capacity for branched chain amino acid (BCAA) metabolism. Here we examined the potential for adipose tissue transplant to lower circulating BCAAs in two models of defective BCAA metabolism, BCATm and PP2Cm [branched chain keto acid dehydrogenase complex (BCKDC) phosphatase] knockout (KO) mice. After 1-2g fat transplant, BCATm and PP2Cm KO mice gained or maintained body weight 3weeks after surgery and consumed similar or more food/BCAAs the week before phlebotomy. Transplant of fat into the abdominal cavity led to a sterile inflammatory response and nonviable transplanted tissue. However when 1-2g of fat was transplanted subcutaneously into the back, either as small (0.1-0.3g) or finely minced pieces introduced with an 18-ga. needle, plasma BCAAs decreased compared to Sham operated mice. In two studies on BCATm KO mice and one study on PP2Cm KO mice, fat transplant led to 52-81% reductions in plasma BCAAs compared to baseline plasma BCAA concentrations of untreated WT type siblings. In PP2Cm KO mice, individual BCAAs in plasma were also significantly reduced by fat transplant, as were the alloisoleucine/Phe ratios. Therefore, subcutaneous fat transplantation may have merit as an adjunct to dietary treatment of MSUD. Additional studies are needed to further refine this approach.

Quantification of branched-chain keto acids in tissue by ultra fast liquid chromatography-mass spectrometry.

Branched-chain keto acids (BCKAs) are associated with increased susceptibility to several degenerative diseases. However, BCKA concentrations in tissues or the amounts of tissue available are frequently at the limit of detection for standard plasma methods. To accurately and quickly determine tissue BCKAs, we have developed a sensitive ultra fast liquid chromatography-mass spectrometry (UFLC-MS) method. BCKAs from deproteinized tissue extractions were o-phenylenediamine (OPD) derivatized, ethyl acetate extracted, lyophilized in a vacuum centrifuge, and reconstituted in 200 mM ammonium acetate. Samples were injected onto a Shimadzu UFLC system coupled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument that detected masses of the OPD BCKA products using a multiple reaction monitoring method. An OPD-derivatized (13)C-labeled keto acid was used as an internal standard. Application of the method for C57BL/6J (wild-type) and PP2Cm knockout mouse tissues, including kidney, adipose tissue, liver, gastrocnemius, and hypothalamus, is shown. The lowest tissue concentration measured by this method was 20 nM, with the standard curve covering a wide range (7.8-32,000 nM). Liquid chromatography-mass spectrometry run times for this assay were less than 5 min, facilitating high throughput, and the OPD derivatives were found to be stable over several days.

Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle.

Leucine is recognized as a nutrient signal; however, the long-term in vivo consequences of leucine signaling and the role of branched-chain amino acid (BCAA) metabolism in this signaling remain unclear. To investigate these questions, we disrupted the BCATm gene, which encodes the enzyme catalyzing the first step in peripheral BCAA metabolism. BCATm(-/-) mice exhibited elevated plasma BCAAs and decreased adiposity and body weight, despite eating more food, along with increased energy expenditure, remarkable improvements in glucose and insulin tolerance, and protection from diet-induced obesity. The increased energy expenditure did not seem to be due to altered locomotor activity, uncoupling proteins, sympathetic activity, or thyroid hormones but was strongly associated with food consumption and an active futile cycle of increased protein degradation and synthesis. These observations suggest that elevated BCAAs and/or loss of BCAA catabolism in peripheral tissues play an important role in regulating insulin sensitivity and energy expenditure.

Some cannabinoid receptor ligands and their distomers are direct-acting openers of SUR1 K(ATP) channels.

Here, we examined the chronic effects of two cannabinoid receptor-1 (CB1) inverse agonists, rimonabant and ibipinabant, in hyperinsulinemic Zucker rats to determine their chronic effects on insulinemia. Rimonabant and ibipinabant (10 mg·kg⁻¹·day⁻¹) elicited body weight-independent improvements in insulinemia and glycemia during 10 wk of chronic treatment. To elucidate the mechanism of insulin lowering, acute in vivo and in vitro studies were then performed. Surprisingly, chronic treatment was not required for insulin lowering. In acute in vivo and in vitro studies, the CB1 inverse agonists exhibited acute K channel opener (KCO; e.g., diazoxide and NN414)-like effects on glucose tolerance and glucose-stimulated insulin secretion (GSIS) with approximately fivefold better potency than diazoxide. Followup studies implied that these effects were inconsistent with a CB1-mediated mechanism. Thus effects of several CB1 agonists, inverse agonists, and distomers during GTTs or GSIS studies using perifused rat islets were unpredictable from their known CB1 activities. In vivo rimonabant and ibipinabant caused glucose intolerance in CB1 but not SUR1-KO mice. Electrophysiological studies indicated that, compared with diazoxide, 3 µM rimonabant and ibipinabant are partial agonists for K channel opening. Partial agonism was consistent with data from radioligand binding assays designed to detect SUR1 K(ATP) KCOs where rimonabant and ibipinabant allosterically regulated ³H-glibenclamide-specific binding in the presence of MgATP, as did diazoxide and NN414. Our findings indicate that some CB1 ligands may directly bind and allosterically regulate Kir6.2/SUR1 K(ATP) channels like other KCOs. This mechanism appears to be compatible with and may contribute to their acute and chronic effects on GSIS and insulinemia.

The Ithaca Free Clinic: a multidisciplinary health services delivery model that includes complementary and alternative medicine practitioners.

Inadequate access to health care services poses a considerable threat to public health and has significant national economic consequences. In the following report, the authors describe a financially stable, multidisciplinary free community health clinic that has operated successfully in Ithaca, New York, since 2006. The clinic provides a diverse collection of conventional and complementary and alternative medicine (CAM) services. The establishment of multidisciplinary free community health clinics in other geographical areas with large uninsured and underinsured populations and the integration of CAM services into them may be a viable strategy to improve access to health services and thereby improve other communities' health.

Temperature-Responsive Nano-Biomaterials from Genetically Encoded Farnesylated Disordered Proteins.

Despite broad interest in understanding the biological implications of protein farnesylation in regulating different facets of cell biology, the use of this post-translational modification to develop protein-based materials and therapies remains underexplored. The progress has been slow due to the lack of accessible methodologies to generate farnesylated proteins with broad physicochemical diversities rapidly. This limitation, in turn, has hindered the empirical elucidation of farnesylated proteins' sequence-structure-function rules. To address this gap, we genetically engineered prokaryotes to develop operationally simple, high-yield biosynthetic routes to produce farnesylated proteins and revealed determinants of their emergent material properties (nano-aggregation and phase-behavior) using scattering, calorimetry, and microscopy. These outcomes foster the development of farnesylated proteins as recombinant therapeutics or biomaterials with molecularly programmable assembly.

Estimating the Health Effects of Adding Bicycle and Pedestrian Paths at the Census Tract Level: Multiple Model Comparison.

BACKGROUND: Adding additional bicycle and pedestrian paths to an area can lead to improved health outcomes for residents over time. However, quantitatively determining which areas benefit more from bicycle and pedestrian paths, how many miles of bicycle and pedestrian paths are needed, and the health outcomes that may be most improved remain open questions. OBJECTIVE: Our work provides and evaluates a methodology that offers actionable insight for city-level planners, public health officials, and decision makers tasked with the question "To what extent will adding specified bicycle and pedestrian path mileage to a census tract improve residents' health outcomes over time?" METHODS: We conducted a factor analysis of data from the American Community Survey, Center for Disease Control 500 Cities project, Strava, and bicycle and pedestrian path location and use data from two different cities (Norfolk, Virginia, and San Francisco, California). We constructed 2 city-specific factor models and used an algorithm to predict the expected mean improvement that a specified number of bicycle and pedestrian path miles contributes to the identified health outcomes. RESULTS: We show that given a factor model constructed from data from 2011 to 2015, the number of additional bicycle and pedestrian path miles in 2016, and a specific census tract, our models forecast health outcome improvements in 2020 more accurately than 2 alternative approaches for both Norfolk, Virginia, and San Francisco, California. Furthermore, for each city, we show that the additional accuracy is a statistically significant improvement (P<.001 in every case) when compared with the alternate approaches. For Norfolk, Virginia (n=31 census tracts), our approach estimated, on average, the percentage of individuals with high blood pressure in the census tract within 1.49% (SD 0.85%), the percentage of individuals with diabetes in the census tract within 1.63% (SD 0.59%), and the percentage of individuals who had >2 weeks of poor physical health days in the census tract within 1.83% (SD 0.57%). For San Francisco (n=49 census tracts), our approach estimates, on average, that the percentage of individuals who had a stroke in the census tract is within 1.81% (SD 0.52%), and the percentage of individuals with diabetes in the census tract is within 1.26% (SD 0.91%). CONCLUSIONS: We propose and evaluate a methodology to enable decision makers to weigh the extent to which 2 bicycle and pedestrian paths of equal cost, which were proposed in different census tracts, improve residents' health outcomes; identify areas where bicycle and pedestrian paths are unlikely to be effective interventions and other strategies should be used; and quantify the minimum amount of additional bicycle path miles needed to maximize health outcome improvements. Our methodology shows statistically significant improvements, compared with alternative approaches, in historical accuracy for 2 large cities (for 2016) within different geographic areas and with different demographics.

Functional proteomic analysis reveals sex-dependent differences in structural and energy-producing myocardial proteins in rat model of alcoholic cardiomyopathy.

Long-term ethanol exposure leads to a sexually dimorphic response in both the susceptibility to cardiac pathology (protective effect of the female heart) and the expression of selected myocardial proteins. The purpose of the present study was to use proteomics to examine the effect of chronic alcohol consumption on a broader array of cardiac proteins and how these were affected between the sexes. Male and female rats were maintained for 18 wk on a 40% ethanol-containing diet in which alcohol was provided in drinking water and agar blocks. Differences in the content of specific cardiac proteins in isopycnic centrifugal fractions were determined using mass spectrometry on iTRAQ-labeled tryptic fragments. A random effects model of meta-analysis was developed to combine the results from multiple iTRAQ experiments. Analysis of a network of proteins involved in cardiovascular system development and function showed that troponins were oppositely regulated by alcohol exposure in females (upregulated) vs. males (downregulated), and this effect was validated by Western blot analysis. Pathway analysis also revealed that alcohol-consuming males showed increased expression of proteins involved in various steps of oxidative phosphorylation including complexes I, III, IV, and V, whereas females showed no change or decreased content. One implication from these findings is that females may be protected from the toxic effects of alcohol due to their ability to maintain contractile function, maintain efficiency of force generation, and minimize oxidative stress. However, the alcohol-induced insult may lead to increased production of reactive oxygen species and structural abnormalities in male myocardium.

Transamination is required for {alpha}-ketoisocaproate but not leucine to stimulate insulin secretion.

It remains unclear how α-ketoisocaproate (KIC) and leucine are metabolized to stimulate insulin secretion. Mitochondrial BCATm (branched-chain aminotransferase) catalyzes reversible transamination of leucine and α-ketoglutarate to KIC and glutamate, the first step of leucine catabolism. We investigated the biochemical mechanisms of KIC and leucine-stimulated insulin secretion (KICSIS and LSIS, respectively) using BCATm(-/-) mice. In static incubation, BCATm disruption abolished insulin secretion by KIC, D,L-α-keto-ß-methylvalerate, and α-ketocaproate without altering stimulation by glucose, leucine, or α-ketoglutarate. Similarly, during pancreas perfusions in BCATm(-/-) mice, glucose and arginine stimulated insulin release, whereas KICSIS was largely abolished. During islet perifusions, KIC and 2 mM glutamine caused robust dose-dependent insulin secretion in BCATm(+/+) not BCATm(-/-) islets, whereas LSIS was unaffected. Consistently, in contrast to BCATm(+/+) islets, the increases of the ATP concentration and NADPH/NADP(+) ratio in response to KIC were largely blunted in BCATm(-/-) islets. Compared with nontreated islets, the combination of KIC/glutamine (10/2 mM) did not influence α-ketoglutarate concentrations but caused 120 and 33% increases in malate in BCATm(+/+) and BCATm(-/-) islets, respectively. Although leucine oxidation and KIC transamination were blocked in BCATm(-/-) islets, KIC oxidation was unaltered. These data indicate that KICSIS requires transamination of KIC and glutamate to leucine and α-ketoglutarate, respectively. LSIS does not require leucine catabolism and may be through leucine activation of glutamate dehydrogenase. Thus, KICSIS and LSIS occur by enhancing the metabolism of glutamine/glutamate to α-ketoglutarate, which, in turn, is metabolized to produce the intracellular signals such as ATP and NADPH for insulin secretion.

Adipose tissue branched chain amino acid (BCAA) metabolism modulates circulating BCAA levels.

Whereas the role of adipose tissue in glucose and lipid homeostasis is widely recognized, its role in systemic protein and amino acid metabolism is less well-appreciated. In vitro and ex vivo experiments suggest that adipose tissue can metabolize substantial amounts of branched chain amino acids (BCAAs). However, the role of adipose tissue in regulating BCAA metabolism in vivo is controversial. Interest in the contribution of adipose tissue to BCAA metabolism has been renewed with recent observations demonstrating down-regulation of BCAA oxidation enzymes in adipose tissue in obese and insulin-resistant humans. Using gene set enrichment analysis, we observe alterations in adipose-tissue BCAA enzyme expression caused by adipose-selective genetic alterations in the GLUT4 glucose-transporter expression. We show that the rate of adipose tissue BCAA oxidation per mg of tissue from normal mice is higher than in skeletal muscle. In mice overexpressing GLUT4 specifically in adipose tissue, we observe coordinate down-regulation of BCAA metabolizing enzymes selectively in adipose tissue. This decreases BCAA oxidation rates in adipose tissue, but not in muscle, in association with increased circulating BCAA levels. To confirm the capacity of adipose tissue to modulate circulating BCAA levels in vivo, we demonstrate that transplantation of normal adipose tissue into mice that are globally defective in peripheral BCAA metabolism reduces circulating BCAA levels by 30% (fasting)-50% (fed state). These results demonstrate for the first time the capacity of adipose tissue to catabolize circulating BCAAs in vivo and that coordinate regulation of adipose-tissue BCAA enzymes may modulate circulating BCAA levels.

Molecular characterization of skeletal muscle atrophy in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD), a neurodegenerative disorder caused by mutant huntingtin, is characterized by a catabolic phenotype. To determine the mechanisms underlying muscle wasting, we examined key signal transduction pathways governing muscle protein metabolism, apoptosis, and autophagy in R6/2 mice, a well-characterized transgenic model of HD. R6/2 mice exhibited increased adiposity, elevated energy expenditure, and decreased body weight and lean mass without altered food intake. Severe skeletal muscle wasting accounted for a majority of the weight loss. Protein synthesis was unexpectedly increased 19% in gastrocnemius muscle, which was associated with overactivation of basal and refeeding-stimulated mammalian target of rapamycin (mTOR) signaling, elevated Akt expression and Ser(473) phosphorylation, and decreased AMPK Thr(172) phosphorylation. Moreover, mRNA abundance of atrogenes muscle ring finger-1 and atrophy F-box, was markedly attenuated during fasting and refeeding, and the urinary excretion of 3-methylhistidine was decreased, arguing against a role for the ubiquitin proteasome-mediated proteolysis in the atrophy. In contrast, mRNA expression of several caspase genes and genes involved in the extrinsic or intrinsic apoptotic pathway, caspase-3/7, -8, and -9 activity, protein abundance of caspase-3 and -9, Fas, and Fadd, and cytochrome c release were elevated. Protein expressions of LC3B-I and -II, beclin-I, and atg5 and -7 in muscle were upregulated. Thus, mutant huntingtin in skeletal muscle results in increased protein synthesis and mTOR signaling, which is countered by activation of the apoptotic and autophagic pathways, contributing to an overall catabolic phenotype and the severe muscle wasting.