Alan Fischman

Reprogramming of Intestinal Glucose Metabolism and Glycemic Control in Rats After Gastric Bypass

Glucose Control Goes Out on a Limb Roux-en-Y gastric bypass, a surgical procedure used to induce weight loss in morbidly obese patients, often leads to permanent remission of diabetes, even when patients regain weight. Studying a rat model, Saeidi et al. (p. 406; see Perspective by Berthoud) found that the surgically reconfigured intestinal segment (the Roux limb) underwent an adaptive response characterized by increased glucose uptake and utilization, apparently triggered by exposure to undigested nutrients. As a result of this change, the intestine provided a major tissue for whole-body glucose control. Whether the same adaptive response occurs in the human intestine remains to be examined. The intestine can adopt a role in glucose control after surgery, possibly explaining why the surgery cures diabetes. [Also see Perspective by Berthoud] The resolution of type 2 diabetes after Roux-en-Y gastric bypass (RYGB) attests to the important role of the gastrointestinal tract in glucose homeostasis. Previous studies in RYGB-treated rats have shown that the Roux limb displays hyperplasia and hypertrophy. Here, we report that the Roux limb of RYGB-treated rats exhibits reprogramming of intestinal glucose metabolism to meet its increased bioenergetic demands; glucose transporter-1 is up-regulated, basolateral glucose uptake is enhanced, aerobic glycolysis is augmented, and glucose is directed toward metabolic pathways that support tissue growth. We show that reprogramming of intestinal glucose metabolism is triggered by the exposure of the Roux limb to undigested nutrients. We demonstrate by positron emission tomography–computed tomography scanning and biodistribution analysis using 2-deoxy-2-[18F]fluoro-d-glucose that reprogramming of intestinal glucose metabolism renders the intestine a major tissue for glucose disposal, contributing to the improvement in glycemic control after RYGB.

Dopamine release during human emotional processing

Involvement of dopamine neurotransmission in human emotional processing is unclear but animal studies have indicated that it is critical for processing of fear response. In this experiment we examined dopaminergic involvement in the processing of human emotions. We used a novel dynamic molecular imaging technique to detect and map dopamine released during presentation of emotional stimuli. The technique exploited the competition between endogenously released dopamine and its ligand for receptor occupancy and involved dynamic voxel-wise measurement of the rate at which a dopamine receptor ligand ((18)F-Fallypride) was displaced from receptor sites during emotional processing. An increase in the rate indicated dopamine release. We found that the rate of ligand displacement increased significantly in the left amygdala, left medial temporal lobe (MTL) and left inferior frontal gyrus. The results provide the first direct evidence of dopaminergic modulation of human emotional processing and suggest that the modulation occurs at multiple levels of processing. This finding indicates that the neurocognitive models of human emotion should take into account dopaminergic effects, and that, there is a need to investigate whether manipulation of the dopaminergic system could be an alternate strategy for treatment of conditions in which emotional processing is impaired.

Simvastatin treatment improves survival in a murine model of burn sepsis: Role of interleukin 6.

Infection is the most common and most serious complication of a major burn related to burn size. Recent studies have demonstrated that statin treatment can decrease mortality in murine or human sepsis. In the current study mice were anesthetized and subjected to a dorsal 30% TBSA scald burn. Simvastatin or placebo were administered by intraperitoneal injection once daily or every 12h. On post burn day 7 cecal ligation and puncture with a 21-gauge needle (CLP) was performed under ketamine/xylazine anesthesia, the two different dosing schedules were continued and survival was monitored. In other groups of mice, interleukin-6 (IL-6) levels in blood were measured in mice at 7 days after injury. A simvastatin dependent improvement in survival was observed in the burn sepsis model. This protection was found to be dose and time dependent. In addition, statin treatment reduced the elevation in IL-6 levels of mice burned 7 days previously. However, IL-6 levels in burned mice with or without statin treatment were elevated by CLP to the same degree. The results of these studies suggest that statin treatment reduces mortality in mice with burns and CLP and that this effect may not be mediated via IL-6 levels.

Effects of burn injury, cold stress and cutaneous wound injury on the morphology and energy metabolism of murine brown adipose tissue (BAT) in vivo.

AIMS Cold stress has been shown to produce dramatic increases in 2-fluoro-2-deoxy-D-Glucose ((18)FDG) accumulation by brown adipose tissue (BAT) in rodents. However, neither the effects of other types of stress on (18)FDG accumulation nor the effects of stressors on the accumulation of tracers of other aspects of energy metabolism have been evaluated. In this report we studied the effects of cold stress, burn injury and cutaneous wounds on murine BAT at the macroscopic, microscopic and metabolic level. MAIN METHODS Glucose metabolism was studied with (18)FDG, fatty acid accumulation was evaluated with trans-9(RS)-(18)F-fluoro-3,4(RS,RS)-methyleneheptadecanoic acid (FCPHA) and tricarboxcylic acid cycle (TCA) activity was evaluated with (3)H acetate. KEY FINDINGS All three stressors produced dramatic changes in BAT at the macroscopic and microscopic level. Macroscopically, BAT from the stressed animals appeared to be a much darker brown in color. Microscopically BAT of stressed animals demonstrated significantly fewer lipid droplets and an overall decrease in lipid content. Accumulation of (18)FDG by BAT was significantly (p<0.01) increased by all 3 treatments (Cold: ~16 fold, burn ~7 Fold and cutaneous wound ~14 fold) whereas uptake of FDG by white fat was unchanged. This effect was also demonstrated non invasively by μPET imaging. Although less prominent than with (18)FDG, BAT uptake of FCPHA and acetate were also significantly increased by all three treatments. These findings suggest that in addition to cold stress, burn injury and cutaneous wounds produce BAT activation in mice. SIGNIFICANCE This study demonstrates brown fat activated by several stressors leads to increased uptake of various substrates.

Nitric oxide activates intradomain disulfide bond formation in the kinase loop of Akt1/PKBα after burn injury

Severe burn injury is an acute inflammatory state with massive alterations in gene expression and levels of growth factors, cytokines and free radicals. During the catabolic processes, changes in insulin sensitivity and skeletal muscle wasting (unintended loss of 5–15% of lean body mass) are observed clinically. Here, we reveal a novel molecular mechanism of Akt1/protein kinase Bα (Akt1/PKBα) regulated via cross-talking between dephosphorylation of Thr308 and S-nitrosylation of Cys296 post severe burn injury, which were characterized using nano-LC interfaced with tandem quadrupole time-of-fight mass spectrometry (Q-TOF)micro tandem mass spectrometry in both in vitro and in vivo studies. For the in vitro studies, Akt1/PKBα was S-nitrosylated with S-nitrosoglutathione and derivatized by three methods. The derivatives were isolated by SDS-PAGE, trypsinized and analyzed by the tandem MS. For the in vivo studies, Akt1/PKBα in muscle lysates from burned rats was immuno-precipitated, derivatized with HPDP-Biotin and analyzed as above. The studies demonstrated that the NO free radical reacts with the free thiol of Cys296 to produce a Cys296-SNO intermediate which accelerates interaction with Cys310 to form Cys296-Cys310 in the kinase loop. MS/MS sequence analysis indicated that the dipeptide, linked via Cys296-Cys310, underwent dephosphorylation at Thr308. These effects were not observed in lysates from sham animals. As a result of this dual effect of burn injury, the loose conformation that is slightly stabilized by the Lys297-Thr308 salt bridge may be replaced by a more rigid structure which may block substrate access. Together with the findings of our previous report concerning mild IRS-1 integrity changes post burn, it is reasonable to conclude that the impaired Akt1/PKBα has a major impact on FOXO3 subcellular distribution and activities.

SILAM for quantitative proteomics of liver Akt1/PKBα after burn injury

Akt1/protein kinase Bα (Akt1/PKBα) is a downstream mediator of the insulin signaling system. In this study we explored mechanism(s) for its role in burn injury. Akt1/PKBα in liver extracts from mice with burn injury fed with (2H7)-L-Leu was immunoprecipitated and isolated with SDS-PAGE. Two tryptic peptides, one in the kinase loop and a control peptide just outside of the loop were sequenced via nano-LC interfaced with quadruple time-of-flight tandem mass spectrometry (Q-TOF tandem MS). Their relative isotopologue abundances were determined by stable isotope labeling by amino acids in mammalians (SILAM). Relative quantifications based on paired heavy/light peptides were obtained in 3 steps. The first step included homogenization of mixtures of equal amounts of tissue from burned and sham-treated animals (i.e., isotope dilution) and acquisition of uncorrected data based on parent monoisotopic MS ion ratios. The second step included determination of isotopic enrichment of the kinase from burned mice on Day 7 and the third step enrichment correction of partially labeled heavy and light monoisotopic MS ion ratios for relative quantification of bioactivity (loop peptide) and expression level (control peptide). Protein synthesis and enrichment after injury were found to be dependent on tissue and turnover of individual proteins. Three heavy and light monoisotopic ion ratios for albumin peptides from burned mice indicated ~55% enrichment and ~16.7-fold downregulation. In contract, serum amyloid P had ~66% enrichment and was significantly upregulated. Akt1/PKBα had ~56% enrichment and kinase level in response to the burn injury was upregulated compared with the control peptide. However, kinase bioactivity, represented by the Cys296 peptide, was significantly reduced. Overall, we demonstrated that i) quantitative proteomics can be performed without completely labeled mice; ii) measurement of enrichment of acyl-tRNAs is unnecessary and iii) Cys296 plays an important role in kinase activity after burn injury.

Role of Protein Farnesylation in Burn-Induced Metabolic Derangements and Insulin Resistance in Mouse Skeletal Muscle

Objective Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury) and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase) inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle. Methods A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated. Results Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277. Conclusions Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn-induced metabolic dysfunction and inflammatory response. Our study identifies FTase as a novel potential molecular target to reverse or ameliorate metabolic derangements in burn patients.

Increased uncoupling protein 1 mRNA expression in mice brown adipose tissue after burn injury.

Brown adipose tissue (BAT) contains numerous mitochondria and is characterized by the presence of uncoupling protein 1 (UCP1). UCP1 is the main mediator of thermogenesis that plays an important role in the modulation of energy balance. The authors hypothesize that alterations in the expression of UCP1 might be involved in the major metabolic disorders occurring during burn trauma. The present study is designed to explore the potential role of the UCP1 in metabolic disorders after burn injury. The authors have used the real-time reverse transcription-polymerase chain reaction to quantify UCP1 mRNA expression in the mice BAT and white adipose tissue (WAT). UCP1 mRNA expression was up-regulated in BAT, especially at 24 hours after burn. UCP1 mRNA expression was detectable and also up-regulated by burn injury in WAT. The authors provide evidence that one of the mechanisms mediating hypermetabolism and increased energy expenditure in burn injury is a pronounced increase in thermogenic capacity, as illustrated by robust gene expression of UCP1 in BAT and WAT.

Evaluation of intragastric vs intraperitoneal glucose tolerance tests in the Evaluation of insulin resistance in a rodent model of burn injury and glucagon-like polypeptide-1 treatment

Evaluation of glucose tolerance in rodent models is usually performed after intraperitroneal administration of glucose (intraperitoneal glucose tolerance test [IPGTT]), whereas in humans the test is performed with oral glucose. Hyperglycemia is a major clinical manifestation of burn injury. Our previous studies using IPGTT have demonstrated burn injury–induced insulin resistance and the beneficial effects of glucagon-like polypeptide-1 (GLP-1) in improving insulin resistance. The goal of the present study is to compare the results of these two procedures under 1) burn injury–induced insulin resistance and 2) GLP-1 treatment after burn. Male CD rats were divided into three groups: sham burn, burn, and burn with GLP-1. Blood glucose and plasma insulin levels were measured during intragastric glucose tolerance test (IGGTT) on day 6 after 40% of full-thickness burn injury. The results were compared with our previous IPGTT. Blood glucose curves for IGGTT and IPGTT showed a similar pattern. However, IGGTT demonstrated a significant lower level of maximal blood glucose when compared with IPGTT. This was accompanied by higher peak insulin levels in sham burn and burn groups. In contrast, peak insulin levels of each burn with GLP-1 group were similar. 1) Both IPGTT and IGGTT demonstrated burn injury–induced insulin resistance and the efficacy of GLP-1 for reducing hyperglycemia after burn injury. 2) The observed differences in the plasma glucose and insulin levels between IGGTT and IPGTT suggest that endogenously produced GLP-1 during the IGGTT may play a role in ameliorating insulin resistance after burn injury.

Functional Imaging of Neurotransmission

Functional neurotransmitter imaging (fNTI) is an evolving technique that uses molecular imaging to detect neurotransmitters released during a task performance. This technique provides a tool to study neurochemistry of human cognition and involves dynamic measurement of the concentration of a specific radioligand during the task performance. Since ligands are competitively displaced by endogenously released neurotransmitters, a reduction in ligand concentration during task performance indicates task-induced release of endogenous neurotransmitter. Most of the fNTI experiments have used a specific dopamine receptor ligand 11 C-raclopride, which is suitable only for detection of dopamine released in the striatum. Ligands such as 18 F-fallypride and 11 C-FLB456 are potential candidates for detection of extrastriatal dopamine release. Using this technique, we have studied striatal and extrastriatal dopamine neurotransmission during performance of a variety of cognitive and behavioral tasks. These tasks include, motor planning, conscious and non- conscious motor memory, cured-recall, response inhibition and emotional memory. Since, fNTI is an emerging technique, it has so far been used to study only dopaminergic neurotransmission. Its utility in the study of human brain and cognition depends critically on the development of appropriate ligands for other neurotransmitters.