The Neuroendocrine and Metabolic Outcomes of Bariatric Surgery Depend on Presurgical Control over Eating.

BACKGROUND: The outcomes of bariatric surgery are very irregular and mostly unpredictable. The search for variables of predictive value is encouraged to help preventing therapeutic failures. OBJECTIVE: We aimed to confirm the hypothesis that preexisting eating behaviors could predict neuroendocrine and metabolic outcomes of gastric bypass surgery in morbidly obese subjects. METHODS: Twenty-one morbidly obese patients from the Bariatric Surgery Program of our hospital were selected according to the specific inclusion and exclusion criteria for this study. The subjects filled out a validated questionnaire to quantify the "loss-of-control" (LC) dimension of food craving and provided serum samples at the onset of the study and 1 year after gastric bypass surgery. Hematological, metabolic, and hormonal variables were studied by conventional clinical tests and enzyme immunoassays and checked for correlations with LC both before and after surgery. RESULTS: Those patients that had exhibited worse eating control at the beginning of the study experienced a better metabolic response 1 year after surgery in terms of reduction of serum insulin, HOMA1-IR, HOMA2-IR, and vitamin D1; all these variables were inversely correlated with presurgical LC. Serum brain-derived neurotrophic factor (BDNF) levels showed the same tendency; in fact, BDNF significantly decreased only in those patients with worse eating control. CONCLUSIONS: Problematic eating behaviors may predict a better response of insulin resistance and a specific reduction of serum BDNF in morbidly obese patients after gastric bypass surgery.

Antiproliferative and cytotoxic effects of grape pomace and grape seed extracts on colorectal cancer cell lines.

Grape pomace is the source of bioactive compounds (anthocyanins, flavonols, flavan-3-ols, and stilbenes) which exhibit antiproliferative actions on cell cultures. We have investigated the antitumoral effects of grape pomace and grape seed extracts on colon cancer cells (Caco-2, HT-29) and fibroblasts. Crude extracts prepared from white and red pomace, and grape seeds, reduced the viability and proliferation of Caco-2. HT-29 cells were resistant to these actions. Purified extracts were then prepared from the same sources and compared with the LDH test; again, all three extracts were active and purified extract from grape seed was the most potent and specific on Caco-2 cells. HT-29 cells were more sensitive to these purified extracts. The biological activity resided almost exclusively in the flavonol and flavan-3-ols subfractions, rather than the anthocyanin subfraction. Preliminary results on the mechanisms involved in these effects revealed downregulation of Myc gene expression in HT-29 and upregulation of Ptg2 in Caco-2 cells.

Proteomic Identification of Biomarkers Associated with Eating Control and Bariatric Surgery Outcomes in Patients with Morbid Obesity.

BACKGROUND: The current therapeutics of morbid obesity could be significantly improved after the identification of novel biomarkers associated with the food addiction endophenotype of obesity and with bariatric surgery outcomes. METHODS: We applied differential expression proteomics and enzyme-linked immunosorbent confirmatory assays to identify (a) proteins that varied according to loss of control over eating in morbidly obese patients and (b) proteins that varied between normoweight controls and patients before and 1 year after bariatric surgery. RESULTS: Clusterin was the only protein that consistently varied according to eating control in patients. Patients showed increased levels of serum amyloid P protein, apolipoprotein A4, serotransferrin, complement factors B and C3 and haptoglobin with respect to controls; the levels of all these proteins tended to return to control values 1 year after surgery. In contrast, apolipoprotein A1 and transthyretin were initially downregulated in patients and were scarcely changed by surgery. Leucine-rich alpha-2-glycoprotein was markedly increased in patients only after surgery. CONCLUSIONS: Clusterin could be of interest as a putative biomarker for food addiction diagnosis in people with morbid obesity. In addition, postsurgical normalization of the proteins initially dysregulated in obese subjects might help monitor clinical improvements after surgery, while lasting or newly detected alterations (i.e., those affecting transthyretin and leucine-rich alpha-2-glycoprotein) could reflect partial refractoriness and/or contribute to the early prediction of clinical problems.

Cocaine and amphetamine regulated transcript and brain-derived neurotrophic factor in morbid obesity. One-year follow-up after gastric bypass.

BACKGROUND: The identification of biomarkers associated with obesity and response to treatment could represent an important advance to design more effective and personalized therapeutic strategies. The complexity of morbid obesity could be explained as the combination of genetic, biochemical, cultural, and behavioral factors, among others. The study of biomarkers should be considered a determinant factor taken into account in this equation. OBJECTIVES: The aim of this study was to define better biomarker profiles potentially associated to the short-term outcome of bariatric surgery by paying attention to cocaine and amphetamine regulated transcript and brain-derived neurotrophic factor, 2 neuropeptides related to eating behavior. SETTING: University General Hospital of Ciudad Real, Spain. METHODS: Twenty-seven morbidly obese patients and 30 healthy weight individuals matched by age and sex were selected for the study. RESULTS: Patients underwent bariatric surgery by Roux-en-Y gastric bypass and responded adequately in terms of weight loss and normalization of many biochemical parameters 1 year postsurgery. A multivariate analysis showed that the hormonal/neuropeptidic profile explained 82% of the variability of the weight loss response. The evolution of cocaine and amphetamine regulated transcript paralleled that of insulin and leptin, serum levels of this peptide were initially elevated in patients (4.24 ± .47 ng/mL) with respect to controls (2.94 ± .2 ng/mL), but this difference disappeared 1 year after Roux-en-Y gastric bypass (3.14 ± .26 ng/mL). Brain-derived neurotrophic factor levels were also decreased by Roux-en-Y gastric bypass (11.93 ± .96 ng/mL postsurgery versus 15.3 ± 1.02 ng/mL presurgery), even when this peptide was not elevated in patients before surgery (14.23 ± .86 ng/mL in controls). CONCLUSIONS: The results suggest that cocaine and amphetamine regulated transcript and brain-derived neurotrophic factor could be envisaged as new candidate biomarkers of short-term outcome after surgery.

Cdkn2a deficiency promotes adipose tissue browning.

OBJECTIVES: Genome-wide association studies have reported that DNA polymorphisms at the CDKN2A locus modulate fasting glucose in human and contribute to type 2 diabetes (T2D) risk. Yet the causal relationship between this gene and defective energy homeostasis remains elusive. Here we sought to understand the contribution of Cdkn2a to metabolic homeostasis. METHODS: We first analyzed glucose and energy homeostasis from Cdkn2a-deficient mice subjected to normal or high fat diets. Subsequently Cdkn2a-deficient primary adipose cells and human-induced pluripotent stem differentiated into adipocytes were further characterized for their capacity to promote browning of adipose tissue. Finally CDKN2A levels were studied in adipocytes from lean and obese patients. RESULTS: We report that Cdkn2a deficiency protects mice against high fat diet-induced obesity, increases energy expenditure and modulates adaptive thermogenesis, in addition to improving insulin sensitivity. Disruption of Cdkn2a associates with increased expression of brown-like/beige fat markers in inguinal adipose tissue and enhances respiration in primary adipose cells. Kinase activity profiling and RNA-sequencing analysis of primary adipose cells further demonstrate that Cdkn2a modulates gene networks involved in energy production and lipid metabolism, through the activation of the Protein Kinase A (PKA), PKG, PPARGC1A and PRDM16 signaling pathways, key regulators of adipocyte beiging. Importantly, CDKN2A expression is increased in adipocytes from obese compared to lean subjects. Moreover silencing CDKN2A expression during human-induced pluripotent stem cells adipogenic differentiation promoted UCP1 expression. CONCLUSION: Our results offer novel insight into brown/beige adipocyte functions, which has recently emerged as an attractive therapeutic strategy for obesity and T2D. Modulating Cdkn2a-regulated signaling cascades may be of interest for the treatment of metabolic disorders.

A high-fat diet combined with food deprivation increases food seeking and the expression of candidate biomarkers of addiction.

A mouse model has been developed to study the effect of dietary fat combined with food deprivation periods on palatable food seeking and on the expression of three potential addiction biomarkers in the nucleus accumbens: fumarate hydratase (FH), ATP synthase subunit alpha (ATP5a1) and transketolase (TKT). Forty C57BL/6 J male mice, four-week old, were fed either with a high-fat (HF) diet or standard diet along the experiment. After 3 weeks of differential feeding, animals underwent a two-week training period of two daily sessions where visual cues were paired either to palatable food (chocolate cereals) or no food at all. This training was prolonged one more week with similar, one daily sessions preceded by 12 hours of food deprivation. A behavioural test was finally conducted where mice were confined for 30 minutes either in food unpaired compartments or in compartments previously paired with cereals, but now with empty food trays. Total activity during this behavioural test and serum corticosterone levels right after it were similar in all experimental groups. Mice tested in food-paired compartments showed a marked preference for the empty food tray that gradually disappeared in standard diet-fed individuals but persisted in HF-fed mice. HF-fed mice also overexpressed FH, ATP5a1 and TKT, which positively correlated with the persistence of preference for the empty food tray. It is suggested that HF diets combined with food deprivation may enhance food seeking behaviours while upregulating FH/ATP5a1/TKT, which are further envisaged as biomarkers of addiction.

KAT2B Is Required for Pancreatic Beta Cell Adaptation to Metabolic Stress by Controlling the Unfolded Protein Response.

The endoplasmic reticulum (ER) unfolded protein response (UPR(er)) pathway plays an important role in helping pancreatic ß cells to adapt their cellular responses to environmental cues and metabolic stress. Although altered UPR(er) gene expression appears in rodent and human type 2 diabetic (T2D) islets, the underlying molecular mechanisms remain unknown. We show here that germline and ß cell-specific disruption of the lysine acetyltransferase 2B (Kat2b) gene in mice leads to impaired insulin secretion and glucose intolerance. Genome-wide analysis of Kat2b-regulated genes and functional assays reveal a critical role for Kat2b in maintaining UPR(er) gene expression and subsequent ß cell function. Importantly, Kat2b expression is decreased in mouse and human diabetic ß cells and correlates with UPR(er) gene expression in normal human islets. In conclusion, Kat2b is a crucial transcriptional regulator for adaptive ß cell function during metabolic stress by controlling UPR(er) and represents a promising target for T2D prevention and treatment.

Role of the unfolded protein response in ß cell compensation and failure during diabetes.

Pancreatic ß cell failure leads to diabetes development. During disease progression, ß cells adapt their secretory capacity to compensate the elevated glycaemia and the peripheral insulin resistance. This compensatory mechanism involves a fine-tuned regulation to modulate the endoplasmic reticulum (ER) capacity and quality control to prevent unfolded proinsulin accumulation, a major protein synthetized within the ß cell. These signalling pathways are collectively termed unfolded protein response (UPR). The UPR machinery is required to preserve ER homeostasis and ß cell integrity. Moreover, UPR actors play a key role by regulating ER folding capacity, increasing the degradation of misfolded proteins, and limiting the mRNA translation rate. Recent genetic and biochemical studies on mouse models and human UPR sensor mutations demonstrate a clear requirement of the UPR machinery to prevent ß cell failure and increase ß cell mass and adaptation throughout the progression of diabetes. In this review we will highlight the specific role of UPR actors in ß cell compensation and failure during diabetes.

Role of Ink4a/Arf locus in beta cell mass expansion under physiological and pathological conditions.

The ARF/INK4A (Cdkn2a) locus includes the linked tumour suppressor genes p16INK4a and p14ARF (p19ARF in mice) that trigger the antiproliferative activities of both RB and p53. With beta cell self-replication being the primary source for new beta cell generation in adult animals, the network by which beta cell replication could be increased to enhance beta cell mass and function is one of the approaches in diabetes research. In this review, we show a general view of the regulation points at transcriptional and posttranslational levels of Cdkn2a locus. We describe the molecular pathways and functions of Cdkn2a in beta cell cycle regulation. Given that aging reveals increased p16Ink4a levels in the pancreas that inhibit the proliferation of beta cells and decrease their ability to respond to injury, we show the state of the art about the role of this locus in beta cell senescence and diabetes development. Additionally, we focus on two approaches in beta cell regeneration strategies that rely on Cdkn2a locus negative regulation: long noncoding RNAs and betatrophin.

Assessing addiction vulnerability with different rat strains and place preference procedures: the role of the cocaine and amphetamine-regulated transcript.

Validated biomarkers of addiction vulnerability are unavailable despite their potential value in diagnostics and therapeutics. As cocaine and amphetamine-regulated transcript (CART) peptides can be considered candidates for such biomarkers, we have studied the acute regulation of CART gene expression in the nucleus accumbens of rats with different drug-seeking behaviors. Two subgroups of Sprague-Dawley rats with different persistences of cocaine-induced and morphine-induced place preference showed a similar regulation of CART mRNA irrespective of their behavioral differences: CART gene expression was unaffected by acute cocaine and downregulated by acute morphine to a similar extent in both subgroups. Fischer 344 and Lewis rats, known to exhibit very different drug-seeking behaviors, showed lower basal expression of CART when compared with Sprague-Dawley rats, being almost undetectable in the case of the Lewis strain. Acute morphine downregulated CART in Fischer 344 rats as it did in Sprague-Dawley rats. The results tend to show that CART mRNA regulation by acute morphine or cocaine in the nucleus accumbens does not seem predictive of addiction vulnerability. However, in the particular case of Lewis rats, the pronounced hypoactivity of the CART system could contribute to the high vulnerability of this strain to develop drug-seeking behaviors.

Gene expression analysis of heat shock proteins in the nucleus accumbens of rats with different morphine seeking behaviours.

Heat-shock proteins play functional roles on brain regulatory processes which are deeply involved in drug addiction, such as synaptic plasticity. However, few studies have been focused on gene expression of heat-shock proteins (Hsp) as potential diagnostic tools for addiction risk. This work tries to provide new knowledge on this field by using two rat models of differential vulnerability to morphine addiction in order to study differential gene expression of a selected group of Hsp genes in the nucleus accumbens (NAc). Hsp70-1A, 84, 86 and 105 genes were similarly regulated by an acute injection of morphine in two subpopulations of Sprague Dawley (SD) rats showing different rates of extinction of morphine conditioned preference. However, Lewis and Fischer rats, two strains that differ in many aspects of drug seeking behaviours, exhibited marked differences in their expression patterns of Hsp84, 86 and 105. These results suggest that differential Hsp gene expression could be related to addiction vulnerability and recommend further work to validate these proteins as potential markers for drug addiction risk.

Identification of new drug targets and biomarkers related to obesity and eating disorders: an approach based on reward deficit and addiction.

Current pharmacological treatments for eating disorders and obesity are of limited value and thus the identification of novel targets is highly needed to enhance the development of more effective drugs. Among the bottlenecks limiting the introduction of new medicines is the reported heterogeneity of these diseases, which makes it difficult to find drugs with broad activity and the lack of animal models with translational validity, especially in the case of anorexia nervosa. Some kinds of obesity and eating disorders can be classified within the pathologies affecting the brain reward system together with drug addiction and others, and therefore specific treatments in these cases can be directed to restore normal function in brain reward pathways. Target identification in this field can greatly benefit from the combined application of genomic/proteomic techniques and robust animal models of reward deficits.

Proteomic analysis of the nucleus accumbens of rats with different vulnerability to cocaine addiction.

Vulnerability to the addictive effects of drugs of abuse varies among individuals, but the biological basis of these differences are poorly known. This work tries to increase this knowledge by comparing the brain proteome of animals with different rate of extinction of cocaine-seeking behaviour. To achieve this goal, we used a place-preference paradigm to separate Sprague Dawley rats in two groups: rats that extinguished (E) and rats that did not extinguish (NE) cocaine-seeking behaviour after a five-day period of drug abstinence. Once the phenotype was established, we compared the protein expression in the nucleus accumbens (NAC) of these animals after a single injection of either saline (SAL) or cocaine (COC, 15 mg/kg). The analysis of protein expression was performed by 2-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time of flight mass spectrometry. When comparing E SAL and NE SAL animals we found significant differences in the expression level of 5 proteins: ATP synthase subunit alpha, fumarate hydratase, transketolase, NADH dehydrogenase [ubiquinone] flavoprotein 2 and glutathione transferase omega-1. A single injection of COC differently alters the NAC proteome of E and NE rats; thus in E COC animals there was an alteration in the expression of 6 proteins, including dihydropyrimidinase-related protein 2 and NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10; whereas in NE COC rats 9 proteins were altered (including alpha-synuclein, peroxiredoxin-2 and peroxiredoxin-5). These proteins could be potential biomarkers of individual vulnerability to cocaine abuse and may be helpful in designing new treatments for cocaine addiction.

Behavioral and in vitro evaluation of tetrodotoxin tolerability for therapeutic applications.

Tetrodotoxin (TTX) injection is currently being studied in clinical trials for potential antinociceptive applications. This work tries to increase the knowledge of its biological tolerability by using a behavioral procedure that can detect aversive effects of drug treatments, as well as in vitro cytotoxicity studies in non-excitable cell systems. Place conditioning studies with Sprague-Dawley male rats showed that pharmacologically active TTX injections (2.5 microg/kg, subcutaneous) were devoid of negative reinforcing properties, the drug being able to prevent the aversive effect of the vehicle. Similarly, TTX was not cytotoxic by itself as evaluated with the neutral red test and the MTT assay in HepG2 cells incubated for 24h with TTX concentrations as high as 400 microM. The results support the idea that low doses of TTX can be well tolerated.

Morphine differentially regulates hsp90beta expression in the nucleus accumbens of Lewis and Fischer 344 rats.

We have comparatively studied hsp90beta gene and protein expression in the nucleus accumbens of Lewis and Fischer 344 (F344) rats, two inbred strains that exhibit prominent behavioural differences in drug-seeking behaviours. Phenotypical studies confirmed that Lewis rats developed a higher preference for morphine-paired environments after conditioning. RT-PCR assays did not reveal strain-related differences in hsp90beta gene expression in basal conditions; however, acute morphine treatment provoked an increase of hsp90beta mRNA 2h after injection only in the case of Lewis rats. We also found a significant upregulation of the Hsp90beta protein in both strains 8h after morphine injection, this increase being significantly higher in Lewis rats. Taking into account the suggested roles for Hsp90 in the brain, the data suggest that Lewis and F344 strain differences concerning opioid-seeking behaviours could be related to differential sensitivity to opioid-induced neuronal plasticity within the brain reward system, an effect that could be mediated (at least partially) by stress proteins.