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Long-term studies comparing the mechanisms of different bariatric techniques for T2DM remission are scarce. We aimed to compare type 2 diabetes (T2DM) remission after a gastric bypass with a 200-cm biliopancreatic limb (mRYGB), sleeve gastrectomy (SG), and greater curvature plication (GCP), and to assess if the initial secretion of gastrointestinal hormones may predict metabolic outcomes at 5 years. Forty-five patients with mean BMI of 39.4(1.9)kg/m2 and T2DM with HbA1c of 7.7(1.9)% were randomized to mRYGB, SG, or GCP. Anthropometric and biochemical parameters, fasting concentrations of PYY, ghrelin, glucagon, and AUC of GLP-1 after SMT were determined prior to and at months 1 and 12 after surgery. At 5-year follow-up, anthropometrical and biochemical parameters were determined. Total weight loss percentage (TWL%) at year 1 and GLP-1 AUC at months 1 and 12 were higher in the mRYGB than in the SG and GCP. TWL% remained greater at 5 years in mRYGB group − 27.32 (7.8) vs. SG − 18.00 (10.6) and GCP − 14.83 (7.8), p = 0.001. At 5 years, complete T2DM remission was observed in 46.7% after mRYGB vs. 20.0% after SG and 6.6% after GCP, p

Introduction: Tacrolimus is the backbone immunosuppressant after solid organ transplantation. Tacrolimus has a narrow therapeutic window with large intra- and inter-patient pharmacokinetic variability leading to frequent over- and under-immunosuppression. While routine therapeutic drug monitoring (TDM) remains the standard of care, tacrolimus pharmacokinetic variability may be influenced by circadian rhythms. Our aim was to analyze tacrolimus pharmacokinetic/pharmacodynamic profiles on circadian rhythms comparing morning and night doses of a twice-daily tacrolimus formulation.Methods: This is a post-hoc analysis from a clinical trial to study the area under curve (AUC) and the area under effect (AUE) profiles of calcineurin inhibition after tacrolimus administration in twenty-five renal transplant patients. Over a period of 24 h, an intensive sampling (0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 12.5, 13, 13.5, 14, 15, 20, and 24 h) was carried out. Whole blood and intracellular tacrolimus concentrations and calcineurin activity were measured by UHPLC-MS/MS.Results: Whole blood and intracellular AUC12–24 h and Cmax achieved after tacrolimus night dose was significantly lower than after morning dose administration (AUC0–12 h) (p < 0.001 for both compartments). AUE0–12 h and AUE12–24 h were not statistically different after morning and night doses. Total tacrolimus daily exposure (AUC0–24 h), in whole blood and intracellular compartments, was over-estimated when assessed by doubling the morning AUC0–12 h data.Conclusion: The lower whole blood and intracellular tacrolimus concentrations after night dose might be influenced by a distinct circadian clock. This significantly lower tacrolimus exposure after night dose was not translated into a significant reduction of the pharmacodynamic effect. Our study may provide conceptual bases for better understanding the TDM of twice-daily tacrolimus formulation.

Tacrolimus (Tac) is the cornerstone calcineurin inhibitor in transplantation. Extended-release Meltdose formulation (Tac-LCP) offers better bioavailability compared with immediate-release formulation (Tac-IR). We postulated that the less fluctuating pharmacokinetic (PK) profile of Tac-LCP might maintain a sustained inhibition of calcineurin activity (CNA) between dose intervals. Higher concentrations (peak plasma concentration (Cmax )) after Tac-IR may not result in a more potent CNA inhibition due to a capacity-limited effect. This study was aimed at evaluating the pharmacodynamic (PD)/PK profiles of Tac-IR compared with Tac-LCP. An open-label, prospective, nonrandomized, investigator-driven study was conducted. Twenty-five kidney transplant recipients receiving Tac-IR were switched to Tac-LCP. Before and 28 days after conversion, intensive CNA-PD and PK sampling were conducted using ultra-high-performance liquid chromatography-tandem accurate mass spectrometry. PD nonlinear mixed effects model was performed in Phoenix-WinNonlin. Statistically significant higher Cmax (P < 0.001) after Tac-IR did not result in lower CNA as compared with after Tac-LCP (P = 0.860). Tac-LCP showed a statistically more maintained CNA inhibition between dose intervals (area under the effect-time curve from 0 to 24 hours (AUE0-24h )) compared with Tac-IR, in which CNA returned to predose levels after 4 hours of drug intake (373.8 vs. 290.5 pmol RII·h/min·mg prot, Tac-LCP vs. Tac-IR; P = 0.039). No correlation was achieved between any PD and PK parameters in any formulations. Moreover, Tac concentration to elicit a 50% of the maximum response (half-maximal inhibitory concentration) was 9.24 ng/mL. The higher Cmax after Tac-IR does not result in an additional CNA inhibition compared with Tac-LCP attributable to a capacity-limited effect. Tac-LCP may represent an improvement of the PD of Tac due to the more sustained CNA inhibition during dose intervals.

There is scant evidence of the long-term effects of bariatric surgery on bone mineral density (BMD). We compared BMD changes in patients with severe obesity and type 2 diabetes (T2D) 5 years after randomization to metabolic gastric bypass (mRYGB), sleeve gastrectomy (SG) and greater curvature plication (GCP). We studied the influence of first year gastrointestinal hormone changes on final bone outcomes. Forty-five patients, averaging 49.4 (7.8) years old and body mass index (BMI) 39.4 (1.9) kg/m2, were included. BMD at lumbar spine (LS) was lower after mRYGB compared to SG and GCP: 0.89 [0.82, 0.94] vs. 1.04 [0.91, 1.16] vs. 0.99 [0.89, 1.12], p = 0.020. A higher percentage of LS osteopenia was present after mRYGB 78.6% vs. 33.3% vs. 50.0%, respectively. BMD reduction was greater in T2D remitters vs. non-remitters. Weight at fifth year predicted BMD changes at the femoral neck (FN) (adjusted R2: 0.3218, p = 0.002), and type of surgery (mRYGB) and menopause predicted BMD changes at LS (adjusted R2: 0.2507, p &lt, 0.015). In conclusion, mRYGB produces higher deleterious effects on bone at LS compared to SG and GCP in the long-term. Women in menopause undergoing mRYGB are at highest risk of bone deterioration. Gastrointestinal hormone changes after surgery do not play a major role in BMD outcomes.

Background Therapeutic drug monitoring of calcineurin inhibitor, tacrolimus (TAC), is routinely used in post-transplantation. Currently, measurement of calcineurin activity has been proposed as a promising clinical tool to evaluate efficacy and to optimize drug dosing. The main aim of our study was to develop a method to measure phosphatase calcineurin activity (CNA) in peripheral blood mononuclear cells (PBMCs) by ultra-high performance liquid chromatography-tandem mass spectrometry and to validate it following FDA and EMA guidelines. Methods This methodology is based on monitoring the Ca2+-dependent dephosphorylation of a phosphopeptide substrate. CNA was evaluated in 5 healthy volunteers and in 5 renal transplant patients receiving twice-daily formulation of TAC before drug intake. Moreover, we studied pharmacodynamic effect of TAC and blood concentrations of TAC in different drug dose intervals (0, 1, 3, 6 and 12 h). Results Our results showed linearity in the range 0.04–2.00 μM with a lower limit of quantification of 0.04 μM. Coefficients of variation and absolute relative biases were Conclusions This method improves the extraction phase of PBMCs and achieves faster determination compared to other techniques, bringing us closer to be applied in daily laboratory practice.

Cardiovascular disease is the leading cause of morbidity and mortality in kidney transplant recipients. Several single-nucleotide polymorphisms (SNPs) in the ANRIL gene pathway have been associated with cardiovascular events (CE). The main objective was to ascertain whether ANRIL (rs10757278) and CARD8 (rs2043211) SNPs could mediate susceptibility to CE. This was an observational follow-up cohort study of renal transplant recipients at Bellvitge University Hospital (Barcelona) from 2000 to 2014. A total of 505 recipients were followed up until achievement of a CE. Patients who did not achieve the endpoint were followed up until graft loss, lost to follow-up or death. Survival analysis was used to ascertain association between genetic markers, clinical data, and outcome. Fifty-three patients suffered a CE after renal transplantation. Results showed a significant association between ANRIL SNP and CE. Homozygous GG for the risk allele showed higher risk for CE than A carriers for the protective allele [HR = 2.93(1.69-5.11), P < 0.0001]. This effect was maintained when it was analyzed in combination with CARD8, suggesting that CARD8 SNP could play a role in the ANRIL mechanism. However, our study does not clarify the molecular mechanism for the CARD8 SNP regulation by ANRIL. ANRIL SNP may predispose to the development of CE after successful kidney transplantation.

Control of energy metabolism is crucial for optimal functioning of organs and tissues. Amongst all nutrients, glucose is the principal energy source for most cells and, therefore, minimum blood glucose levels must be guaranteed. Alterations in glycaemia can lead to hyperglycaemic states (producing protein glycosylation and toxicity in glucose-sensitive cells) or hypoglycaemic states (that can affect brain function), both harmful. Therefore, mechanisms must exist to keep glycaemia in a narrow physiological range (4-8 mM) independently of the nutritional state. To achieve control of blood glucose levels, our body has a complex, interorgan signaling system using nutrients (glucose, lipids, amino acids), hormones (insulin, glucagon, ghrelin, etc.) and the autonomic nervous system. In response to these signals, organs and tissues (mainly intestine, endocrine pancreas, liver, skeletal muscle, adipose tissue, brain and adrenal glands) adapt their function to energetic requirements. The liver plays a pivotal role in the maintenance of glucose homeostasis by continuously adapting its metabolism to energetic needs. In the fed state, when blood glucose levels are high and there is insulin, liver takes-up part glucose to replenish glycogen stores. Besides, when glucose stores are full, the liver has the capacity to synthesize lipids de novo from glucose for-long term energy storage. Lipids are packaged in very low-density lipoprotein (VLDL) particles and then transported to the adipose tissue. Conversely during starvation, when glycaemia falls and glucagon increases, the liver produces glucose to maintain circulating glucose levels by breaking down glycogen stores or by synthesizing glucose de novo through gluconeogenesis. Gluconeogenesis, as an energy-consuming pathway, is linked to ┚-oxidation of fatty acids (fuel supplier pathway). From this introduction on the regulation of glucose homeostasis, one can appreciate the close relation that exists between carbohydrate metabolism and lipid metabolism in the liver. Therefore, alterations in hepatic carbohydrate metabolic pathways may directly affect hepatic and/or blood lipid levels. Particularly, this chapter will focus on evaluating the incidence of glucokinase (GK) –the first enzyme of the glycolytic pathway in the liveron lipidemia and on hepatic lipid content. But first, an introductory overview of the physiology behind the first-pass metabolism of dietary glucose in the liver will be presented.

OBJECTIVE—Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; encoded by Pck1) catalyzes the first committed step in gluconeogenesis. Extensive evidence demonstrates a direct correlation between PEPCK-C activity and glycemia control. Therefore, we aimed to evaluate the metabolic impact and their underlying mechanisms of knocking down hepatic PEPCK-C in a type 2 diabetic model. RESEARCH DESIGN AND METHODS—PEPCK-C gene targeting was achieved using adenovirus-transduced RNAi. The study assessed several clinical symptoms of diabetes and insulin signaling in peripheral tissues, in addition to changes in gene expression, protein, and metabolites in the liver. Liver bioenergetics was also evaluated. RESULTS—Treatment resulted in reduced PEPCK-C mRNA and protein. After treatment, improved glycemia and insulinemia, lower triglyceride, and higher total and HDL cholesterol were measured. Unsterified fatty acid accumulation was observed in the liver, in the absence of de novo lipogenesis. Despite hepatic lipidosis, treatment resulted in improved insulin signaling in the liver, muscle, and adipose tissue. O2 consumption measurements in isolated hepatocytes demonstrated unaltered mitochondrial function and a consequent increased cellular energy charge. Key regulatory factors (FOXO1, hepatocyte nuclear factor-4α, and peroxisome proliferator–activated receptor-γ coactivator [PGC]-1α) and enzymes (G6Pase) implicated in gluconeogenesis were downregulated after treatment. Finally, the levels of Sirt1, a redox-state sensor that modulates gluconeogenesis through PGC-1α, were diminished. CONCLUSIONS—Our observations indicate that silencing PEPCK-C has direct impact on glycemia control and energy metabolism and provides new insights into the potential significance of the enzyme as a therapeutic target for the treatment of diabetes.

Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) is the rate-controlling enzyme in gluconeogenesis. In diabetic individuals, altered rates of gluconeogenesis are responsible for increased hepatic glucose output and sustained hyperglycemia. Liver-specific inhibition of PEPCK has not been assessed to date as a treatment for diabetes. We have designed a therapeutic, vector-based RNAi approach to induce posttranscriptional gene silencing of hepatic PEPCK using nonviral gene delivery. A transient reduction of PEPCK enzymatic activity (7.6 +/- 0.6 vs 9.7 +/- 1.1 mU/mg, P < 0.05) that correlated with decreased protein content of up to 50% was achieved using this strategy in diabetic mice. PEPCK partial silencing was sufficient to demonstrate lowered blood glucose (218 +/- 26 vs 364 +/- 33 mg/dl, P < 0.001) and improved glucose tolerance together with decreased circulating FFA (0.89 +/- 0.10 vs 1.44 +/- 0.11 mEq/dl, P < 0.001) and TAG (65 +/- 11 vs 102 +/- 16 mg/dl, P < 0.01), in the absence of liver steatosis or lactic acidosis. SREBP1c was down-regulated in PEPCK-silenced animals, suggesting a role for this pathway in the alterations of lipid metabolism. These data reinforce the significance of PEPCK in sustaining diabetes-induced hyperglycemia and validate liver-specific intervention at the level of PEPCK for diabetes gene therapy.

Recent reports point out the importance of the complex GK-GKRP in controlling glucose and lipid homeostasis. Several GK mutations affect GKRP binding, resulting in permanent activation of the enzyme. We hypothesize that hepatic overexpression of a mutated form of GK, GKA456V, described in a patient with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) and could provide a model to study the consequences of GK-GKRP deregulationin vivo. GKA456Vwas overexpressed in the liver of streptozotocin diabetic mice. Metabolite profiling in serum and liver extracts, together with changes in key components of glucose and lipid homeostasis, were analyzed and compared to GK wild-type transfected livers. Cell compartmentalization of the mutant but not the wild-type GK was clearly affectedin vivo,demonstrating impaired GKRP regulation. GKA456Voverexpression markedly reduced blood glucose in the absence of dyslipidemia, in contrast to wild-type GK-overexpressing mice. Evidence in glucose utilization did not correlate with increased glycogen nor lactate levels in the liver. PEPCK mRNA was not affected, whereas the mRNA for the catalytic subunit of glucose-6-phosphatase was upregulated ~4 folds in the liver of GKA456V-treated animals, suggesting that glucose cycling was stimulated. Our results provide new insights into the complex GK regulatory network and validate liver-specific GK activation as a strategy for diabetes therapy.