Glucose-stimulated insulin secretion depends on FFA1 and Gq in neonatal mouse islets.

AIMS/HYPOTHESIS: After birth, the neonatal islets gradually acquire glucose-responsive insulin secretion, a process that is subjected to maternal imprinting. Although NEFA are major components of breastmilk and insulin secretagogues, their role for functional maturation of neonatal beta cells is still unclear. NEFA are the endogenous ligands of fatty acid receptor 1 (FFA1, encoded by Ffar1 in mice), a Gq-coupled receptor with stimulatory effect on insulin secretion. This study investigates the role of FFA1 in neonatal beta cell function and in the adaptation of offspring beta cells to parental high-fat feeding. METHODS: Wild-type (WT) and Ffar1-/- mice were fed high-fat (HFD) or chow diet (CD) for 8 weeks before mating, and during gestation and lactation. Blood variables, pancreas weight and insulin content were assessed in 1-, 6-, 11- and 26-day old (P1-P26) offspring. Beta cell mass and proliferation were determined in P1-P26 pancreatic tissue sections. FFA1/Gq dependence of insulin secretion was evaluated in isolated islets and INS-1E cells using pharmacological inhibitors and siRNA strategy. Transcriptome analysis was conducted in isolated islets. RESULTS: Blood glucose levels were higher in CD-fed Ffar1-/- P6-offspring compared with CD-fed WT P6-offspring. Accordingly, glucose-stimulated insulin secretion (GSIS) and its potentiation by palmitate were impaired in CD Ffar1-/- P6-islets. In CD WT P6-islets, insulin secretion was stimulated four- to fivefold by glucose and five- and sixfold over GSIS by palmitate and exendin-4, respectively. Although parental HFD increased blood glucose in WT P6-offspring, it did not change insulin secretion from WT P6-islets. In contrast, parental HFD abolished glucose responsiveness (i.e. GSIS) in Ffar1-/- P6-islets. Inhibition of Gq by FR900359 or YM-254890 in WT P6-islets mimicked the effect of Ffar1 deletion, i.e. suppression of GSIS and of palmitate-augmented GSIS. The blockage of Gi/o by pertussis toxin (PTX) enhanced (100-fold) GSIS in WT P6-islets and rendered Ffar1-/- P6-islets glucose responsive, suggesting constitutive activation of Gi/o. In WT P6-islets, FR900359 cancelled 90% of PTX-mediated stimulation, while in Ffar1-/- P6-islets it completely abolished PTX-elevated GSIS. The secretory defect of Ffar1-/- P6-islets did not originate from insufficient beta cells, since beta cell mass increased with the offspring's age irrespective of genotype and diet. In spite of that, in the breastfed offspring (i.e. P1-P11) beta cell proliferation and pancreatic insulin content had a genotype- and diet-driven dynamic. Under CD, the highest proliferation rate was reached by the Ffar1-/- P6 offspring (3.95% vs 1.88% in WT P6), whose islets also showed increased mRNA levels of genes (e.g. Fos, Egr1, Jun) typically high in immature beta cells. Although parental HFD increased beta cell proliferation in both WT (4.48%) and Ffar1-/- (5.19%) P11 offspring, only the WT offspring significantly increased their pancreatic insulin content upon parental HFD (5.18 µg under CD to 16.93 µg under HFD). CONCLUSIONS/INTERPRETATION: FFA1 promotes glucose-responsive insulin secretion and functional maturation of newborn islets and is required for adaptive offspring insulin secretion in the face of metabolic challenge, such as parental HFD.

Brain Insulin Resistance at the Crossroads of Metabolic and Cognitive Disorders in Humans.

Ever since the brain was identified as an insulin-sensitive organ, evidence has rapidly accumulated that insulin action in the brain produces multiple behavioral and metabolic effects, influencing eating behavior, peripheral metabolism, and cognition. Disturbances in brain insulin action can be observed in obesity and type 2 diabetes (T2D), as well as in aging and dementia. Decreases in insulin sensitivity of central nervous pathways, i.e., brain insulin resistance, may therefore constitute a joint pathological feature of metabolic and cognitive dysfunctions. Modern neuroimaging methods have provided new means of probing brain insulin action, revealing the influence of insulin on both global and regional brain function. In this review, we highlight recent findings on brain insulin action in humans and its impact on metabolism and cognition. Furthermore, we elaborate on the most prominent factors associated with brain insulin resistance, i.e., obesity, T2D, genes, maternal metabolism, normal aging, inflammation, and dementia, and on their roles regarding causes and consequences of brain insulin resistance. We also describe the beneficial effects of enhanced brain insulin signaling on human eating behavior and cognition and discuss potential applications in the treatment of metabolic and cognitive disorders.

Acylated- and unacylated ghrelin during an oral glucose tolerance test in humans at risk for type 2 diabetes mellitus.

BACKGROUND/OBJECTIVES: The orexigenic peptide hormone ghrelin has been implicated in the pathophysiology of obesity and type 2 diabetes mellitus through its effects on nutrient homeostasis. Ghrelin is subject to a unique post-translational acyl modification regulating its biochemical activity. SUBJECTS/METHODS: In this study we aimed to investigate the relation of acylated (AcG) as well as unacylated ghrelin (UnG) with body weight and insulin resistance in the fasting (n = 545) and post-oral glucose tolerance test (oGTT) state (n = 245) in a metabolically well characterized cohort covering a broad range of BMI (17.95 kg/m²-76.25 kg/m²). RESULTS: Fasting AcG (median 94.2 pg/ml) and UnG (median 175.3 pg/ml) were negatively and the AcG/UnG ratio was positively correlated with BMI (all p < 0.0001). Insulin sensitivity (ISI) correlated positively with AcG (p = 0.0014) and UnG (p = 0.0004) but not with the AcG/UnG ratio. In a multivariate analysis, including ISI and BMI, only BMI, but not ISI was independently associated with AcG and UnG concentrations. Significant changes of AcG and UnG concentrations were detectable after oGTT stimulation, with slight decreases after 30 min and increases after 90-120 min. Subject stratification into BMI-divergent groups revealed more pronounced AcG increases in the two groups with BMI < 40 kg/m². CONCLUSION: Our data demonstrate lower concentrations for both AcG and UnG with increasing BMI as well as an increased proportion of the biologically active, acylated form of ghrelin giving point to pharmacologic intervention in ghrelin acylation and/or increase in UnG for treatment of obesity despite decreased absolute AcG levels.

Brain insulin responsiveness is linked to age and peripheral insulin sensitivity.

AIMS: Insulin action in the brain influences cognitive processes, peripheral metabolism and eating behaviour. However, the influence of age and peripheral insulin sensitivity on brain insulin action remains unclear. MATERIALS AND METHODS: We used intranasal administration of insulin and functional magnetic resonance imaging in a randomized, placebo-controlled within-subject design in 110 participants (54 women, body mass index 18-49 kg/m2 , age 21-74 years). Cerebral blood flow was measured before and after nasal spray application to assess brain insulin action. Peripheral insulin sensitivity was assessed by a five-point oral glucose tolerance test. Linear regressions were used to investigate associations between age and peripheral insulin sensitivity with brain insulin action in predefined region of interests (i.e. insulin-sensitive brain regions). RESULTS: We found significant negative associations between age and insulin action in the hippocampus (ß = -0.215; p = .017) and caudate nucleus (ß = -0.184; p = .047); and between peripheral insulin sensitivity and insulin action in the amygdala (ß = -0.190, p = .023). Insulin action in the insular cortex showed an interaction effect between age and peripheral insulin sensitivity (ß = -0.219 p = .005). Furthermore, women showed the strongest negative association between age and hippocampal insulin action, while men showed the strongest associations with peripheral insulin sensitivity and age in reward-related brain regions. CONCLUSION: We could show a region-specific relationship between brain insulin responsiveness, age and peripheral insulin sensitivity. Our findings underline the need to study brain insulin action in both men and women and further substantiate that brain insulin sensitivity is a possible link between systemic metabolism and neurocognitive functions.

Identification of MicroRNAs Associated with Prediabetic Status in Obese Women.

MicroRNAs (miRNAs) recently emerged as means of communication between insulin-sensitive tissues to mediate diabetes development and progression, and as such they present a valuable proxy for epigenetic alterations associated with type 2 diabetes. In order to identify miRNA markers for the precursor of diabetes called prediabetes, we applied a translational approach encompassing analysis of human plasma samples, mouse tissues and an in vitro validation system. MiR-652-3p, miR-877-5p, miR-93-5p, miR-130a-3p, miR-152-3p and let-7i-5p were increased in plasma of women with impaired fasting glucose levels (IFG) compared to those with normal fasting glucose and normal glucose tolerance (NGT). Among these, let-7i-5p and miR-93-5p correlated with fasting blood glucose levels. Human data were then compared to miRNome data obtained from islets of Langerhans and adipose tissue of 10-week-old female New Zealand Obese mice, which differ in their degree of hyperglycemia and liver fat content. Similar to human plasma, let-7i-5p was increased in adipose tissue and islets of Langerhans of diabetes-prone mice. As predicted by the in silico analysis, overexpression of let-7i-5p in the rat ß-cell line INS-1 832/12 resulted in downregulation of insulin signaling pathway components (Insr, Rictor, Prkcb, Clock, Sos1 and Kcnma1). Taken together, our integrated approach highlighted let-7i-5p as a potential regulator of whole-body insulin sensitivity and a novel marker of prediabetes in women.

Extracellular Matrix Expression in Human Pancreatic Fat Cells of Patients with Normal Glucose Regulation, Prediabetes and Type 2 Diabetes.

Previously, we found that human pancreatic preadipocytes (PPAs) and islets influence each other and that the crosstalk with the fatty liver via the hepatokine fetuin-A/palmitate induces inflammatory responses. Here, we examined whether the mRNA-expression of pancreatic extracellular matrix (ECM)-forming and -degrading components differ in PPAs from individuals with normal glucose regulation (PPAs-NGR), prediabetes (PPAs-PD), and type 2 diabetes (PPAs-T2D), and whether fetuin-A/palmitate impacts ECM-formation/degradation and associated monocyte invasion. Human pancreatic resections were analyzed (immuno)histologically. PPAs were studied for mRNA expression by real-time PCR and protein secretion by Luminex analysis. Furthermore, co-cultures with human islets and monocyte migration assays in Transwell plates were conducted. We found that in comparison with NGR-PPAs, TIMP-2 mRNA levels were lower in PPAs-PD, and TGF-ß1 mRNA levels were higher in PPAs-T2D. Fetuin-A/palmitate reduced fibronectin, decorin, TIMP-1/-2 and TGF-ß1 mRNA levels. Only fibronectin was strongly downregulated by fetuin-A/palmitate independently of the glycemic status. Co-culturing of PPAs with islets increased TIMP-1 mRNA expression in islets. Fetuin-A/palmitate increased MMP-1, usherin and dermatopontin mRNA-levels in co-cultured islets. A transmigration assay showed increased monocyte migration towards PPAs, which was enhanced by fetuin-A/palmitate. This was more pronounced in PPAs-T2D. The expression of distinct ECM components differs in PPAs-PD and PPAs-T2D compared to PPAs-NGR, suggesting that ECM alterations can occur even in mild hyperglycemia. Fetuin-A/palmitate impacts on ECM formation/degradation in PPAs and co-cultured islets. Fetuin-A/palmitate also enhances monocyte migration, a process which might impact on matrix turnover.

Sex differences in central insulin action: Effect of intranasal insulin on neural food cue reactivity in adults with normal weight and overweight.

BACKGROUND/OBJECTIVES: Central insulin action influences cognitive processes, peripheral metabolism, and eating behavior. However, the contribution of obesity and sex on central insulin-mediated neural food cue processing still remains unclear. SUBJECTS/METHODS: In a randomized within-participant design, including two visits, 60 participants (30 women, BMI 18-32 kg/m2, age 21-69 years) underwent a functional MRI task measuring blood oxygen level-dependent (BOLD) signal in response to visual food cues after intranasal insulin or placebo spray administration. Central insulin action was defined as the neural BOLD response to food cues after insulin compared to placebo administration. Afterwards, participants were asked to rate the food cues for desire to eat (i.e., wanting rating). For statistical analyses, participants were grouped according to BMI and sex. RESULTS: Food cue reactivity in the amygdala showed higher BOLD activation in response to central insulin compared to placebo. Furthermore, women with overweight and obesity and men of normal weight showed higher BOLD neural food cue responsivity to central insulin compared to placebo. Higher central insulin action in the insular cortex was associated with better peripheral insulin sensitivity and higher cognitive control. Moreover, central insulin action in the dorsolateral prefrontal cortex (DLPFC) revealed significant sex differences. In response to central insulin compared to placebo, men showed lower DLPFC BOLD activity, whereas women showed higher DLPFC activity in response to highly desired food cues. On behavioral level, central insulin action significantly reduced hunger, whereas the desire to eat, especially for low caloric food cues was significantly higher with central insulin than with placebo. CONCLUSIONS: Obesity and sex influenced the central insulin-mediated neural BOLD activity to visual food cues in brain regions implicated in reward and cognitive control. These findings show that central insulin action regulates food response differentially in men and women, which may have consequences for metabolism and eating behavior.

Identification of proliferative and mature ß-cells in the islets of Langerhans.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of ß-cells. Pancreatic ß-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature ß-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger ß-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for ß-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional ß-cell heterogeneity and induce ß-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional ß-cell mass in diabetic patients.

Pancreatic fat cells of humans with type 2 diabetes display reduced adipogenic and lipolytic activity.

Obesity, especially visceral fat accumulation, increases the risk of type 2 diabetes (T2D). The purpose of this study was to investigate the impact of T2D on the pancreatic fat depot. Pancreatic fat pads from 17 partial pancreatectomized patients (PPP) were collected, pancreatic preadipocytes isolated, and in vitro differentiated. Patients were grouped using HbA1c into normal glucose tolerant (NGT), prediabetic (PD), and T2D. Transcriptome profiles of preadipocytes and adipocytes were assessed by RNAseq. Insulin sensitivity was estimated by quantifying AKT phosphorylation on Western blots. Lipogenic capacity was assessed with oil red O staining, lipolytic activity via fatty acid release. Secreted factors were measured using ELISA. Comparative transcriptome analysis of preadipocytes and adipocytes indicates defective upregulation of genes governing adipogenesis (NR1H3), lipogenesis (FASN, SCD, ELOVL6, and FADS1), and lipolysis (LIPE) during differentiation of cells from T2D-PPP. In addition, the ratio of leptin/adiponectin mRNA was higher in T2D than in NGT-PPP. Preadipocytes and adipocytes of NGT-PPP were more insulin sensitive than T2D-PPP cells in regard to AKT phosphorylation. Triglyceride accumulation was similar in NGT and T2D adipocytes. Despite a high expression of the receptors NPR1 and NPR2 in NGT and T2D adipocytes, lipolysis was stimulated by ANP 1.74-fold in NGT cells only. This stimulation was further increased by the PDE5 inhibitor dipyridamole (3.09-fold). Dipyridamole and forskolin increased lipolysis receptor independently 1.88-fold and 1.48-fold, respectively, solely in NGT cells. In conclusion, the metabolic status persistently affects differentiation and lipolysis of pancreatic adipocytes. These alterations could aggravate the development of T2D.

Liver-targeting drugs and their effect on blood glucose and hepatic lipids.

The global epidemic of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) and the high prevalence among individuals with type 2 diabetes has attracted the attention of clinicians specialising in liver disorders. Many drugs are in the pipeline for the treatment of NAFLD/NASH, and several glucose-lowering drugs are now being tested specifically for the treatment of liver disease. Among these are nuclear hormone receptor agonists (e.g. peroxisome proliferator-activated receptor agonists, farnesoid X receptor agonists and liver X receptor agonists), fibroblast growth factor-19 and -21, single, dual or triple incretins, sodium-glucose cotransporter inhibitors, drugs that modulate lipid or other metabolic pathways (e.g. inhibitors of fatty acid synthase, diacylglycerol acyltransferase-1, acetyl-CoA carboxylase and 11ß-hydroxysteroid dehydrogenase type-1) or drugs that target the mitochondrial pyruvate carrier. We have reviewed the metabolic effects of these drugs in relation to improvement of diabetic hyperglycaemia and fatty liver disease, as well as peripheral metabolism and insulin resistance.

The hepatokine fetuin-A disrupts functional maturation of pancreatic beta cells.

AIMS/HYPOTHESIS: Neonatal beta cells carry out a programme of postnatal functional maturation to achieve full glucose responsiveness. A partial loss of the mature phenotype of adult beta cells may contribute to a reduction of functional beta cell mass and accelerate the onset of type 2 diabetes. We previously found that fetuin-A, a hepatokine increasingly secreted by the fatty liver and a determinant of type 2 diabetes, inhibits glucose-stimulated insulin secretion (GSIS) of human islets. Since fetuin-A is a ubiquitous fetal glycoprotein that declines peripartum, we examined here whether fetuin-A interferes with the functional maturity of beta cells. METHODS: The effects of fetuin-A were assessed during in vitro maturation of porcine neonatal islet cell clusters (NICCs) and in adult human islets. Expression alterations were examined via microarray, RNA sequencing and reverse transcription quantitative real-time PCR (qRT-PCR), proteins were analysed by western blotting and immunostaining, and insulin secretion was quantified in static incubations. RESULTS: NICC maturation was accompanied by the gain of glucose-responsive insulin secretion (twofold stimulation), backed up by mRNA upregulation of genes governing beta cell identity and function, such as NEUROD1, UCN3, ABCC8 and CASR (Log2 fold change [Log2FC] > 1.6). An active TGFß receptor (TGFBR)-SMAD2/3 pathway facilitates NICC maturation, since the TGFBR inhibitor SB431542 counteracted the upregulation of aforementioned genes and de-repressed ALDOB, a gene disallowed in mature beta cells. In fetuin-A-treated NICCs, upregulation of beta cell markers and the onset of glucose responsiveness were suppressed. Concomitantly, SMAD2/3 phosphorylation was inhibited. Transcriptome analysis confirmed inhibitory effects of fetuin-A and SB431542 on TGFß-1- and SMAD2/3-regulated transcription. However, contrary to SB431542 and regardless of cMYC upregulation, fetuin-A inhibited beta cell proliferation (0.27 ± 0.08% vs 1.0 ± 0.1% Ki67-positive cells in control NICCs). This effect was sustained by reduced expression (Log2FC ≤ -2.4) of FOXM1, CENPA, CDK1 or TOP2A. In agreement, the number of insulin-positive cells was lower in fetuin-A-treated NICCs than in control NICCs (14.4 ± 1.2% and 22.3 ± 1.1%, respectively). In adult human islets fetuin-A abolished glucose responsiveness, i.e. 1.7- and 1.1-fold change over 2.8 mmol/l glucose in control- and fetuin-A-cultured islets, respectively. In addition, fetuin-A reduced SMAD2/3 phosphorylation and suppressed expression of proliferative genes. Of note, in non-diabetic humans, plasma fetuin-A was negatively correlated (p = 0.013) with islet beta cell area. CONCLUSIONS/INTERPRETATION: Our results suggest that the perinatal decline of fetuin-A relieves TGFBR signalling in islets, a process that facilitates functional maturation of neonatal beta cells. Functional maturity remains revocable in later life, and the occurrence of a metabolically unhealthy milieu, such as liver steatosis and elevated plasma fetuin-A, can impair both function and adaptive proliferation of beta cells. DATA AVAILABILITY: The RNAseq datasets and computer code produced in this study are available in the Gene Expression Omnibus (GEO): GSE144950; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144950.

Insulin Action in the Hypothalamus Increases Second-Phase Insulin Secretion in Humans.

BACKGROUND: Animal studies and initial correlative data in humans indicate that insulin action in the brain may affect pancreatic insulin secretion. An important brain region for this process is the hypothalamus, an area that can develop insulin resistance. METHODS: Fifteen young, healthy men (27 ± 3 years) with a wide BMI spectrum (20-30 kg/m2) underwent 2 hyperglycemic clamps (target blood glucose: 10 mmol/L). In this double-blind study, subjects received 160 U of insulin or placebo as a nasal spray on 2 days in randomized order. On another day, insulin sensitivity of the hypothalamus was determined by functional magnetic resonance imaging. RESULTS: Glucose levels were comparable on both study days. In the whole group, C-peptide levels were not significantly different between conditions. Though, there was a significant interaction between insulin sensitivity of the hypothalamus × nasal spray × time on C-peptide levels (p = 10-6). The group was therefore divided according to median hypothalamic insulin sensitivity. C-peptide concentrations were higher after intranasal insulin compared to placebo spray in the group with a strong hypothalamic insulin response (p < 0.0001, ß = 6.00 ± 1.24) and lower in the brain insulin-resistant group (p = 0.005, ß = -2.68 ± 0.95). Neither somatostatin nor glucagon kinetics was altered by the nasal spray. CONCLUSIONS: In participants with high hypothalamic insulin sensitivity, insulin action in the brain enhanced second-phase insulin secretion from pancreatic beta cells. This reaction could, for example, contribute to late postprandial glucose regulation by suppressing hepatic glucose production by portal venous insulin.

Monitoring of low dabigatran concentrations: diagnostic performance at clinically relevant decision thresholds.

The direct oral anticoagulant dabigatran does not require therapeutic drug monitoring, however emergency measurements are gaining importance. Current assays feature good performance at intermediate and high dabigatran concentrations but show limited accuracy at low concentrations. This area requires more attention as clinical decision threshold values currently lie at 30 and 50 ng/ml. The objective of the study was to evaluate and compare diagnostic performance of dabigatran assays at these thresholds. Dabigatran concentrations of 293 plasma samples taken from 50 patients were measured with the INNOVANCE direct thrombin inhibitor assay (DTI) from Siemens, the Biophen direct thrombin inhibitor assay (BDTI), the BDTI using a low range calibrator (BDTI-low), the Hemoclot direct thrombin inhibitor assay (HTI) and an ecarin clotting time assay (ECT). Assay results were compared to ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and test characteristics were calculated for thresholds of 30 and 50 ng/ml. DTI, BDTI-low and ECT showed very strong correlation and high agreement with UPLC-MS/MS and an improved determination of low dabigatran concentrations. ROC curve analyses revealed very high accuracy at the 30/50 ng/ml thresholds for DTI (AUC = 0.989/0.995), BDTI-low (AUC = 0.980/0.991) and ECT (AUC = 0.990/0.996) measurements. Sensitivity and specificity in detecting were calculated for DTI (98/92%), BDTI-low (87/95%), ECT (97/96%), BDTI (99/82%) and HTI (86/89%) measurements. Compared to the previously available HTI and BDTI, both novel assays, DTI and BDTI-low, reliably determine low dabigatran plasma concentrations around the clinical decision thresholds with very high sensitivity and specificity.

Linking bioenergetic function of mitochondria to tissue-specific molecular fingerprints.

Mitochondria are dynamic organelles with diverse functions in tissues such as liver and skeletal muscle. To unravel the mitochondrial contribution to tissue-specific physiology, we performed a systematic comparison of the mitochondrial proteome and lipidome of mice and assessed the consequences hereof for respiration. Liver and skeletal muscle mitochondrial protein composition was studied by data-independent ultra-high-performance (UHP)LC-MS/MS-proteomics, and lipid profiles were compared by UHPLC-MS/MS lipidomics. Mitochondrial function was investigated by high-resolution respirometry in samples from mice and humans. Enzymes of pyruvate oxidation as well as several subunits of complex I, III, and ATP synthase were more abundant in muscle mitochondria. Muscle mitochondria were enriched in cardiolipins associated with higher oxidative phosphorylation capacity and flexibility, in particular CL(18:2)4 and 22:6-containing cardiolipins. In contrast, protein equipment of liver mitochondria indicated a shuttling of complex I substrates toward gluconeogenesis and ketogenesis and a higher preference for electron transfer via the flavoprotein quinone oxidoreductase pathway. Concordantly, muscle and liver mitochondria showed distinct respiratory substrate preferences. Muscle respired significantly more on the complex I substrates pyruvate and glutamate, whereas in liver maximal respiration was supported by complex II substrate succinate. This was a consistent finding in mouse liver and skeletal muscle mitochondria and human samples. Muscle mitochondria are tailored to produce ATP with a high capacity for complex I-linked substrates. Liver mitochondria are more connected to biosynthetic pathways, preferring fatty acids and succinate for oxidation. The physiologic diversity of mitochondria may help to understand tissue-specific disease pathologies and to develop therapies targeting mitochondrial function.

Effect of SGLT2 inhibitors on body composition, fluid status and renin-angiotensin-aldosterone system in type 2 diabetes: a prospective study using bioimpedance spectroscopy.

BACKGROUND: SGLT2-inhibitors are potent antihyperglycemic drugs for patients with type 2 diabetes and have been shown to reduce body weight. However, it is unclear which body compartments are reduced and to what extent. METHODS: In this longitudinal observational study, we analyzed the body composition of 27 outpatients with type 2 diabetes mellitus during the first week and up to 6 months after initiation of treatment with SGLT2-inhibitors (n = 18 empagliflozin, n = 9 dapagliflozin) using bioimpedance spectroscopy (BCM, Fresenius). Fluid status of hypertensive patients taking medication with hydrochlorothiazide (n = 14) and healthy persons (n = 16) were analyzed for comparison. RESULTS: At 6 months, HbA1c decreased by 0.8% (IQR 2.3; 0.4), body weight and BMI by 2.6 kg (1.5; 9.3) and 0.9 kg/m2 (0.4; 3.3), respectively. Bioimpedance spectroscopy revealed significant decrease in adipose tissue mass and fat tissue index while lean tissue parameters remained stable. Overhydration (OH) and extracellular water (ECW) decreased by - 0.5 L/1.73 m2 (- 0.1; - 0.9) and - 0.4 L/1.73 m2 (- 0.1; - 0.8) at day 3, respectively, and returned to the initial value after 3 and 6 months. Plasma renin activity increased by 2.1-fold (0.5; 3.6) at 1 month and returned to the initial level at month 3 and 6. Fluid status of patients with SGLT2 inhibitors after 6 months showed no difference from that of hypertensive patients taking hydrochlorothiazide or healthy persons. CONCLUSIONS: Body weight reduction under the treatment with SGLT2-inhibitors is caused by reduction of adipose tissue mass and transient loss of extracellular fluid, which is accompanied by upregulation of renin-angiotensin-aldosterone system (RAAS). Permanent loss of extracellular water does not occur under SGLT2 inhibition.

Nasal insulin administration does not affect hepatic glucose production at systemic fasting insulin levels.

AIMS: To evaluate the effects of brain insulin on endogenous glucose production in fasting humans, with a focus on hepatic glucose release by performing a randomized, placebo-controlled, blinded, crossover experiment. MATERIALS AND METHODS: On two separate days, 2 H2 -glucose was infused to nine healthy lean men, and blood was sampled from the hepatic vein and a radial artery. On day 1, participants received 160 U human insulin through nasal spray, and on day 2 they received placebo spray, together with an intravenous insulin bolus to mimic spillover of nasal insulin to the circulation. Hepatic glucose fluxes and endogenous glucose production were calculated. RESULTS: Plasma insulin concentrations were similar on the two study days, and no differences in whole-body endogenous glucose production or hepato-splanchnic glucose turnover were detected. CONCLUSIONS: Nasal administration of insulin does not influence whole-body or hepatic glucose production in fasting humans. By contrast, pharmacological delivery of insulin to the brain might modulate insulin effectiveness in glucose-producing tissue when circulating insulin levels are elevated; therefore, the metabolic consequences of brain insulin action appear to be dependent on metabolic prandial status.

Performance of a novel high sensitivity cardiac troponin I assay in asymptomatic hemodialysis patients - evidence for sex-specific differences.

Background High sensitivity assays for the determination of cardiac troponin I (cTnI) are able to reliably measure cTnI far below the 99th percentile of healthy persons (hs-cTnI) and display sex-specific differences. There is uncertainty regarding the clinical utility of hs-cTnI in asymptomatic hemodialysis (HD) patients and if sex-specific differences also apply in this cohort. Methods In this multicenter study we measured hs-cTnI and sensitive cTnI (s-TnI) concentrations (both on Siemens Centaur) in 215 HD patients from a predialytic sample to determine the prevalence of elevated concentrations above the 99th percentile, the association with baseline characteristics, prognostic accuracy for death, and sex-specific differences. Results Hs-cTnI and s-cTnI concentrations were below the 99th percentile in 93% and 85% of patients with a median concentration of 12 ng/L (interquartile range 7-66) and 19 ng/L (12; 31, p < 0.0001). Hs-cTnI and s-cTnI concentrations were independently associated with age (p < 0.05) and ischemic cardiac disease (p < 0.05), but not with residual renal function. Both hs-cTnI and s-cTnI were predictors of death after median follow-up of 2.6 years with an AUC of 0.733 and 0.744, respectively (both p < 0.0001). Important sex-differences emerged for hs-cTnI, but not for s-cTnI: first, women had significantly lower hs-cTnI concentrations than men (p = 0.03); second, hs-cTnI had significantly higher prognostic accuracy for death in women than for men (AUC 0.824 vs. 0.674, p = 0.04). Conclusions The majority of HD patients have (h)s-cTnI concentrations below the 99th percentile. High normal values are predictive of death. Hs-cTnI allows to elucidate important sex-differences in HD patients with lower concentrations and higher prognostic accuracy in women.

T-helper-1-cell cytokines drive cancer into senescence.

Cancer control by adaptive immunity involves a number of defined death and clearance mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation. Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence, we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control. When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo, Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1(-/-)Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.

Homeostatic nuclear RAGE-ATM interaction is essential for efficient DNA repair.

The integrity of genome is a prerequisite for healthy life. Indeed, defects in DNA repair have been associated with several human diseases, including tissue-fibrosis, neurodegeneration and cancer. Despite decades of extensive research, the spatio-mechanical processes of double-strand break (DSB)-repair, especially the auxiliary factor(s) that can stimulate accurate and timely repair, have remained elusive. Here, we report an ATM-kinase dependent, unforeseen function of the nuclear isoform of the Receptor for Advanced Glycation End-products (nRAGE) in DSB-repair. RAGE is phosphorylated at Serine376 and Serine389 by the ATM kinase and is recruited to the site of DNA-DSBs via an early DNA damage response. nRAGE preferentially co-localized with the MRE11 nuclease subunit of the MRN complex and orchestrates its nucleolytic activity to the ATR kinase signaling. This promotes efficient RPA2S4-S8 and CHK1S345 phosphorylation and thereby prevents cellular senescence, IPF and carcinoma formation. Accordingly, loss of RAGE causatively linked to perpetual DSBs signaling, cellular senescence and fibrosis. Importantly, in a mouse model of idiopathic pulmonary fibrosis (RAGE-/-), reconstitution of RAGE efficiently restored DSB-repair and reversed pathological anomalies. Collectively, this study identifies nRAGE as a master regulator of DSB-repair, the absence of which orchestrates persistent DSB signaling to senescence, tissue-fibrosis and oncogenesis.

Acquired arteriovenous fistula of a finger.

Arteriovenous fistulae are defined as congenital or acquired abnormal direct communications between an artery and a vein leading to abnormal blood circulation. This report describes an unusual manifestation of an acquired peripheral arteriovenous fistula with a high shunt volume of 410 ml/min following a fracture of the 5th finger.