A novel 3D imaging approach for quantification of GLUT4 levels across the intact myocardium.

Cellular heterogeneity is a well-accepted feature of tissues, and both transcriptional and metabolic diversity have been revealed by numerous approaches, including optical imaging. However, the high magnification objective lenses needed for high-resolution imaging provides information from only small layers of tissue, which can result in poor cell statistics. There is therefore an unmet need for an imaging modality that can provide detailed molecular and cellular insight within intact tissue samples in 3D. Using GFP-tagged GLUT4 as proof of concept, we present here a novel optical mesoscopy approach that allows precise measurement of the spatial location of GLUT4 within specific anatomical structures across the myocardium in ultrathick sections (5 mm x 5 mm x 3 mm) of intact mouse heart. We reveal distinct GLUT4 distribution patterns across cardiac walls and highlight specific changes in GLUT4 expression levels in response to high fat diet-feeding, and we identify sex-dependent differences in expression patterns. This method is applicable to any target that can be labelled for light microscopy, and to other complex tissues when organ structure needs to be considered simultaneously with cellular detail.

Comparative genomic analyses of multiple backcross mouse populations suggest SGCG as a novel potential obesity-modifier gene.

To nominate novel disease genes for obesity and type 2 diabetes (T2D), we recently generated two mouse backcross populations of the T2D-susceptible New Zealand Obese (NZO/HI) mouse strain and two genetically different, lean and T2D-resistant strains, 129P2/OlaHsd and C3HeB/FeJ. Comparative linkage analysis of our two female backcross populations identified seven novel body fat-associated quantitative trait loci (QTL). Only the locus Nbw14 (NZO body weight on chromosome 14) showed linkage to obesity-related traits in both backcross populations, indicating that the causal gene variant is likely specific for the NZO strain as NZO allele carriers in both crosses displayed elevated body weight and fat mass. To identify candidate genes for Nbw14, we used a combined approach of gene expression and haplotype analysis to filter for NZO-specific gene variants in gonadal white adipose tissue, defined as the main QTL-target tissue. Only two genes, Arl11 and Sgcg, fulfilled our candidate criteria. In addition, expression QTL analysis revealed cis-signals for both genes within the Nbw14 locus. Moreover, retroviral overexpression of Sgcg in 3T3-L1 adipocytes resulted in increased insulin-stimulated glucose uptake. In humans, mRNA levels of SGCG correlated with body mass index and body fat mass exclusively in diabetic subjects, suggesting that SGCG may present a novel marker for metabolically unhealthy obesity. In conclusion, our comparative-cross analysis could substantially improve the mapping resolution of the obesity locus Nbw14. Future studies will throw light on the mechanism by which Sgcg may protect from the development of obesity.

Impact of physical fitness and exercise training on subcutaneous adipose tissue beiging markers in humans with and without diabetes and a high-fat diet-fed mouse model.

AIMS: Exercise training induces white adipose tissue (WAT) beiging and improves glucose homeostasis and mitochondrial function in rodents. This could be relevant for type 2 diabetes in humans, but the effect of physical fitness on beiging of subcutaneous WAT (scWAT) remains unclear. This translational study investigates if beiging of scWAT associates with physical fitness in healthy humans and recent-onset type 2 diabetes and if a voluntary running wheel intervention is sufficient to induce beiging in mice. MATERIALS AND METHODS: Gene expression levels of established beiging markers were measured in scWAT biopsies of humans with (n = 28) or without type 2 diabetes (n = 28), stratified by spiroergometry into low (L-FIT; n = 14 each) and high physical fitness (H-FIT; n = 14 each). High-fat diet-fed FVB/N mice underwent voluntary wheel running, treadmill training or no training (n = 8 each group). Following the training intervention, mitochondrial respiration and content of scWAT were assessed by high-resolution respirometry and citrate synthase activity, respectively. RESULTS: Secreted CD137 antigen (Tnfrsf9/Cd137) expression was three-fold higher in glucose-tolerant H-FIT than in L-FIT, but not different between H-FIT and L-FIT with type 2 diabetes. In mice, both training modalities increased Cd137 expression and enhanced mitochondrial content without changing respiration in scWAT. Treadmill but not voluntary wheel running led to improved whole-body insulin sensitivity. CONCLUSIONS: Higher physical fitness and different exercise interventions associated with higher gene expression levels of the beiging marker CD137 in healthy humans and mice on a high-fat diet. Humans with recent-onset type 2 diabetes show an impaired adipose tissue-specific response to physical activity.

AMPK and Beyond: The Signaling Network Controlling RabGAPs and Contraction-Mediated Glucose Uptake in Skeletal Muscle.

Impaired skeletal muscle glucose uptake is a key feature in the development of insulin resistance and type 2 diabetes. Skeletal muscle glucose uptake can be enhanced by a variety of different stimuli, including insulin and contraction as the most prominent. In contrast to the clearance of glucose from the bloodstream in response to insulin stimulation, exercise-induced glucose uptake into skeletal muscle is unaffected during the progression of insulin resistance, placing physical activity at the center of prevention and treatment of metabolic diseases. The two Rab GTPase-activating proteins (RabGAPs), TBC1D1 and TBC1D4, represent critical nodes at the convergence of insulin- and exercise-stimulated signaling pathways, as phosphorylation of the two closely related signaling factors leads to enhanced translocation of glucose transporter 4 (GLUT4) to the plasma membrane, resulting in increased cellular glucose uptake. However, the full network of intracellular signaling pathways that control exercise-induced glucose uptake and that overlap with the insulin-stimulated pathway upstream of the RabGAPs is not fully understood. In this review, we discuss the current state of knowledge on exercise- and insulin-regulated kinases as well as hypoxia as stimulus that may be involved in the regulation of skeletal muscle glucose uptake.

E96V Mutation in the Kdelr3 Gene Is Associated with Type 2 Diabetes Susceptibility in Obese NZO Mice.

Type 2 diabetes (T2D) represents a multifactorial metabolic disease with a strong genetic predisposition. Despite elaborate efforts in identifying the genetic variants determining individual susceptibility towards T2D, the majority of genetic factors driving disease development remain poorly understood. With the aim to identify novel T2D risk genes we previously generated an N2 outcross population using the two inbred mouse strains New Zealand obese (NZO) and C3HeB/FeJ (C3H). A linkage study performed in this population led to the identification of the novel T2D-associated quantitative trait locus (QTL) Nbg15 (NZO blood glucose on chromosome 15, Logarithm of odds (LOD) 6.6). In this study we used a combined approach of positional cloning, gene expression analyses and in silico predictions of DNA polymorphism on gene/protein function to dissect the genetic variants linking Nbg15 to the development of T2D. Moreover, we have generated congenic strains that associated the distal sublocus of Nbg15 to mechanisms altering pancreatic beta cell function. In this sublocus, Cbx6, Fam135b and Kdelr3 were nominated as potential causative genes associated with the Nbg15 driven effects. Moreover, a putative mutation in the Kdelr3 gene from NZO was identified, negatively influencing adaptive responses associated with pancreatic beta cell death and induction of endoplasmic reticulum stress. Importantly, knockdown of Kdelr3 in cultured Min6 beta cells altered insulin granules maturation and pro-insulin levels, pointing towards a crucial role of this gene in islets function and T2D susceptibility.

AKT/AMPK-mediated phosphorylation of TBC1D4 disrupts the interaction with insulin-regulated aminopeptidase.

TBC1D4 is a 160 kDa multidomain Rab GTPase-activating protein (RabGAP) and a downstream target of the insulin- and contraction-activated kinases AKT and AMPK. Phosphorylation of TBC1D4 has been linked to translocation of GLUT4 from storage vesicles (GSVs) to the cell surface. However, its impact on enzymatic activity is not well understood, as previous studies mostly investigated the truncated GAP domain lacking the known phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using a baculovirus system. Size-exclusion chromatography and coimmunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ∼600 kDa. Compared with the truncated GAP domain, full-length TBC1D4 displayed similar substrate specificity, but had a markedly higher specific GAP activity toward Rab10. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4. We determined Michaelis-Menten kinetics using in vitro phosphorylation assays with purified kinases and stable isotope-labeled γ-[18O4]-ATP. These data revealed that Ser324 (KM ∼6 µM) and Thr649 (KM ∼25 µM) were preferential sites for phosphorylation by AKT, whereas Ser348, Ser577, Ser595 (KM ∼10 µM), Ser711 (KM ∼79 µM), and Ser764 were found to be preferred targets for AMPK. Phosphorylation of TBC1D4 by AKT or AMPK did not alter the intrinsic RabGAP activity, but did disrupt interaction with insulin-regulated aminopeptidase (IRAP), a resident protein of GSVs implicated in GLUT4 trafficking. These findings provide evidence that insulin and contraction may regulate TBC1D4 function primarily by disrupting the recruitment of the RabGAP to GLUT4 vesicles.

Many Ways to Rome: Exercise, Cold Exposure and Diet-Do They All Affect BAT Activation and WAT Browning in the Same Manner?

The discovery of functional brown adipose tissue (BAT) in adult humans and the possibility to recruit beige cells with high thermogenic potential within white adipose tissue (WAT) depots opened the field for new strategies to combat obesity and its associated comorbidities. Exercise training as well as cold exposure and dietary components are associated with the enhanced accumulation of metabolically-active beige adipocytes and BAT activation. Both activated beige and brown adipocytes increase their metabolic rate by utilizing lipids to generate heat via non-shivering thermogenesis, which is dependent on uncoupling protein 1 (UCP1) in the inner mitochondrial membrane. Non-shivering thermogenesis elevates energy expenditure and promotes a negative energy balance, which may ameliorate metabolic complications of obesity and Type 2 Diabetes Mellitus (T2DM) such as insulin resistance (IR) in skeletal muscle and adipose tissue. Despite the recent advances in pharmacological approaches to reduce obesity and IR by inducing non-shivering thermogenesis in BAT and WAT, the administered pharmacological compounds are often associated with unwanted side effects. Therefore, lifestyle interventions such as exercise, cold exposure, and/or specified dietary regimens present promising anchor points for future disease prevention and treatment of obesity and T2DM. The exact mechanisms where exercise, cold exposure, dietary interventions, and pharmacological treatments converge or rather diverge in their specific impact on BAT activation or WAT browning are difficult to determine. In the past, many reviews have demonstrated the mechanistic principles of exercise- and/or cold-induced BAT activation and WAT browning. In this review, we aim to summarize not only the current state of knowledge on the various mechanistic principles of diverse external stimuli on BAT activation and WAT browning, but also present their translational potential in future clinical applications.

Identification of Novel Genes Involved in Hyperglycemia in Mice.

Current attempts to prevent and manage type 2 diabetes have been moderately effective, and a better understanding of the molecular roots of this complex disease is important to develop more successful and precise treatment options. Recently, we initiated the collective diabetes cross, where four mouse inbred strains differing in their diabetes susceptibility were crossed with the obese and diabetes-prone NZO strain and identified the quantitative trait loci (QTL) Nidd13/NZO, a genomic region on chromosome 13 that correlates with hyperglycemia in NZO allele carriers compared to B6 controls. Subsequent analysis of the critical region, harboring 644 genes, included expression studies in pancreatic islets of congenic Nidd13/NZO mice, integration of single-cell data from parental NZO and B6 islets as well as haplotype analysis. Finally, of the five genes (Acot12, S100z, Ankrd55, Rnf180, and Iqgap2) within the polymorphic haplotype block that are differently expressed in islets of B6 compared to NZO mice, we identified the calcium-binding protein S100z gene to affect islet cell proliferation as well as apoptosis when overexpressed in MIN6 cells. In summary, we define S100z as the most striking gene to be causal for the diabetes QTL Nidd13/NZO by affecting ß-cell proliferation and apoptosis. Thus, S100z is an entirely novel diabetes gene regulating islet cell function.

Physical activity attenuates postprandial hyperglycaemia in homozygous TBC1D4 loss-of-function mutation carriers.

AIMS/HYPOTHESIS: The common muscle-specific TBC1D4 p.Arg684Ter loss-of-function variant defines a subtype of non-autoimmune diabetes in Arctic populations. Homozygous carriers are characterised by elevated postprandial glucose and insulin levels. Because 3.8% of the Greenlandic population are homozygous carriers, it is important to explore possibilities for precision medicine. We aimed to investigate whether physical activity attenuates the effect of this variant on 2 h plasma glucose levels after an oral glucose load. METHODS: In a Greenlandic population cohort (n = 2655), 2 h plasma glucose levels were obtained after an OGTT, physical activity was estimated as physical activity energy expenditure and TBC1D4 genotype was determined. We performed TBC1D4-physical activity interaction analysis, applying a linear mixed model to correct for genetic admixture and relatedness. RESULTS: Physical activity was inversely associated with 2 h plasma glucose levels (ß[main effect of physical activity] -0.0033 [mmol/l] / [kJ kg-1 day-1], p = 6.5 × 10-5), and significantly more so among homozygous carriers of the TBC1D4 risk variant compared with heterozygous carriers and non-carriers (ß[interaction] -0.015 [mmol/l] / [kJ kg-1 day-1], p = 0.0085). The estimated effect size suggests that 1 h of vigorous physical activity per day (compared with resting) reduces 2 h plasma glucose levels by an additional ~0.7 mmol/l in homozygous carriers of the risk variant. CONCLUSIONS/INTERPRETATION: Physical activity improves glucose homeostasis particularly in homozygous TBC1D4 risk variant carriers via a skeletal muscle TBC1 domain family member 4-independent pathway. This provides a rationale to implement physical activity as lifestyle precision medicine in Arctic populations. DATA REPOSITORY: The Greenlandic Cardio-Metabochip data for the Inuit Health in Transition study has been deposited at the European Genome-phenome Archive ( https://www.ebi.ac.uk/ega/dacs/EGAC00001000736 ) under accession EGAD00010001428.

Alternative exon splicing and differential expression in pancreatic islets reveals candidate genes and pathways implicated in early diabetes development.

Type 2 diabetes (T2D) has a strong genetic component. Most of the gene variants driving the pathogenesis of T2D seem to target pancreatic ß-cell function. To identify novel gene variants acting at early stage of the disease, we analyzed whole transcriptome data to identify differential expression (DE) and alternative exon splicing (AS) transcripts in pancreatic islets collected from two metabolically diverse mouse strains at 6 weeks of age after three weeks of high-fat-diet intervention. Our analysis revealed 1218 DE and 436 AS genes in islets from NZO/Hl vs C3HeB/FeJ. Whereas some of the revealed genes present well-established markers for ß-cell failure, such as Cd36 or Aldh1a3, we identified numerous DE/AS genes that have not been described in context with ß-cell function before. The gene Lgals2, previously associated with human T2D development, was DE as well as AS and localizes in a quantitative trait locus (QTL) for blood glucose on Chr.15 that we reported recently in our N2(NZOxC3H) population. In addition, pathway enrichment analysis of DE and AS genes showed an overlap of only half of the revealed pathways, indicating that DE and AS in large parts influence different pathways in T2D development. PPARG and adipogenesis pathways, two well-established metabolic pathways, were overrepresented for both DE and AS genes, probably as an adaptive mechanism to cope for increased cellular stress. Our results provide guidance for the identification of novel T2D candidate genes and demonstrate the presence of numerous AS transcripts possibly involved in islet function and maintenance of glucose homeostasis.

Glucose transporters in adipose tissue, liver, and skeletal muscle in metabolic health and disease.

A family of facilitative glucose transporters (GLUTs) is involved in regulating tissue-specific glucose uptake and metabolism in the liver, skeletal muscle, and adipose tissue to ensure homeostatic control of blood glucose levels. Reduced glucose transport activity results in aberrant use of energy substrates and is associated with insulin resistance and type 2 diabetes. It is well established that GLUT2, the main regulator of hepatic hexose flux, and GLUT4, the workhorse in insulin- and contraction-stimulated glucose uptake in skeletal muscle, are critical contributors in the control of whole-body glycemia. However, the molecular mechanism how insulin controls glucose transport across membranes and its relation to impaired glycemic control in type 2 diabetes remains not sufficiently understood. An array of circulating metabolites and hormone-like molecules and potential supplementary glucose transporters play roles in fine-tuning glucose flux between the different organs in response to an altered energy demand.

A collective diabetes cross in combination with a computational framework to dissect the genetics of human obesity and Type 2 diabetes.

To explore the genetic determinants of obesity and Type 2 diabetes (T2D), the German Center for Diabetes Research (DZD) conducted crossbreedings of the obese and diabetes-prone New Zealand Obese mouse strain with four different lean strains (B6, DBA, C3H, 129P2) that vary in their susceptibility to develop T2D. Genome-wide linkage analyses localized more than 290 quantitative trait loci (QTL) for obesity, 190 QTL for diabetes-related traits and 100 QTL for plasma metabolites in the outcross populations. A computational framework was developed that allowed to refine critical regions and to nominate a small number of candidate genes by integrating reciprocal haplotype mapping and transcriptome data. The efficiency of the complex procedure was demonstrated for one obesity QTL. The genomic interval of 35 Mb with 502 annotated candidate genes was narrowed down to six candidates. Accordingly, congenic mice retained the obesity phenotype owing to an interval that contains three of the six candidate genes. Among these the phospholipase PLA2G4A exhibited an elevated expression in adipose tissue of obese human subjects and is therefore a critical regulator of the obesity locus. Together, our broad and complex approach demonstrates that combined- and comparative-cross analysis exhibits improved mapping resolution and represents a valid tool for the identification of disease genes.

Immunity-related GTPase induces lipophagy to prevent excess hepatic lipid accumulation.

BACKGROUND & AIMS: Currently, only a few genetic variants explain the heritability of fatty liver disease. Quantitative trait loci (QTL) analysis of mouse strains has identified the susceptibility locus Ltg/NZO (liver triglycerides from New Zealand obese [NZO] alleles) on chromosome 18 as associating with increased hepatic triglycerides. Herein, we aimed to identify genomic variants responsible for this association. METHODS: Recombinant congenic mice carrying 5.3 Mbp of Ltg/NZO were fed a high-fat diet and characterized for liver fat. Bioinformatic analysis, mRNA profiles and electrophoretic mobility shift assays were performed to identify genes responsible for the Ltg/NZO phenotype. Candidate genes were manipulated in vivo by injecting specific microRNAs into C57BL/6 mice. Pulldown coupled with mass spectrometry-based proteomics and immunoprecipitation were performed to identify interaction partners of IFGGA2. RESULTS: Through positional cloning, we identified 2 immunity-related GTPases (Ifgga2, Ifgga4) that prevent hepatic lipid storage. Expression of both murine genes and the human orthologue IRGM was significantly lower in fatty livers. Accordingly, liver-specific suppression of either Ifgga2 or Ifgga4 led to a 3-4-fold greater increase in hepatic fat content. In the liver of low-fat diet-fed mice, IFGGA2 localized to endosomes/lysosomes, while on a high-fat diet it associated with lipid droplets. Pulldown experiments and proteomics identified the lipase ATGL as a binding partner of IFGGA2 which was confirmed by co-immunoprecipitation. Both proteins partially co-localized with the autophagic marker LC3B. Ifgga2 suppression in hepatocytes reduced the amount of LC3B-II, whereas overexpression of Ifgga2 increased the association of LC3B with lipid droplets and decreased triglyceride storage. CONCLUSION: IFGGA2 interacts with ATGL and protects against hepatic steatosis, most likely by enhancing the binding of LC3B to lipid droplets. LAY SUMMARY: The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease.

AKT and AMP-activated protein kinase regulate TBC1D1 through phosphorylation and its interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP.

In skeletal muscle, the Rab GTPase-activating (GAP) protein TBC1D1 is phosphorylated by AKT and AMP-activated protein kinase (AMPK) in response to insulin and muscle contraction. Genetic ablation of Tbc1d1 or mutation of distinct phosphorylation sites impairs intracellular GLUT4 retention and GLUT4 traffic, presumably through alterations of the activation state of downstream Rab GTPases. Previous studies have focused on characterizing the C-terminal GAP domain of TBC1D1 that lacks the known phosphorylation sites, as well as putative regulatory domains. As a result, it has been unclear how phosphorylation of TBC1D1 would regulate its activity. In the present study, we have expressed, purified, and characterized recombinant full-length TBC1D1 in Sf9 insect cells via the baculovirus system. Full-length TBC1D1 showed RabGAP activity toward GLUT4-associated Rab8a, Rab10, and Rab14, indicating similar substrate specificity as the truncated GAP domain. However, the catalytic activity of the full-length TBC1D1 was markedly higher than that of the GAP domain. Although in vitro phosphorylation of TBC1D1 by AKT or AMPK increased 14-3-3 binding, it did not alter the intrinsic RabGAP activity. However, we found that TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic domain of the insulin-regulated aminopeptidase, a resident protein of GLUT4 storage vesicles, and this binding is disrupted by phosphorylation of TBC1D1 by AKT or AMPK. In summary, our findings suggest that other regions outside the GAP domain may contribute to the catalytic activity of TBC1D1. Moreover, our data indicate that recruitment of TBC1D1 to GLUT4-containing vesicles and not its GAP activity is regulated by insulin and contraction-mediated phosphorylation.

Isoform-specific AMPK association with TBC1D1 is reduced by a mutation associated with severe obesity.

AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis which achieves this through the phosphorylation of a myriad of downstream targets. One target is TBC1D1 a Rab-GTPase-activating protein that regulates glucose uptake in muscle cells by integrating insulin signalling with that promoted by muscle contraction. Ser237 in TBC1D1 is a target for phosphorylation by AMPK, an event which may be important in regulating glucose uptake. Here, we show AMPK heterotrimers containing the α1, but not the α2, isoform of the catalytic subunit form an unusual and stable association with TBC1D1, but not its paralogue AS160. The interaction between the two proteins is direct, involves a dual interaction mechanism employing both phosphotyrosine-binding (PTB) domains of TBC1D1 and is increased by two different pharmacological activators of AMPK (AICAR and A769962). The interaction enhances the efficiency by which AMPK phosphorylates TBC1D1 on its key regulatory site, Ser237 Furthermore, the interaction is reduced by a naturally occurring R125W mutation in the PTB1 domain of TBC1D1, previously found to be associated with severe familial obesity in females, with a concomitant reduction in Ser237 phosphorylation. Our observations provide evidence for a functional difference between AMPK α-subunits and extend the repertoire of protein kinases that interact with substrates via stabilisation mechanisms that modify the efficacy of substrate phosphorylation.

Regulation of RabGAPs involved in insulin action.

Rab (Ras-related proteins in brain) GTPases are key proteins responsible for a multiplicity of cellular trafficking processes. Belonging to the family of monomeric GTPases, they are regulated by cycling between their active GTP-bound and inactive GDP-bound conformations. Despite possessing a slow intrinsic GTP hydrolysis activity, Rab proteins rely on RabGAPs (Rab GTPase-activating proteins) that catalyze GTP hydrolysis and consequently inactivate the respective Rab GTPases. Two related RabGAPs, TBC1D1 and TBC1D4 (=AS160) have been described to be associated with obesity-related traits and type 2 diabetes in both mice and humans. Inactivating mutations of TBC1D1 and TBC1D4 lead to substantial changes in trafficking and subcellular distribution of the insulin-responsive glucose transporter GLUT4, and to subsequent alterations in energy substrate metabolism. The activity of the RabGAPs is controlled through complex phosphorylation events mediated by protein kinases including AKT and AMPK, and by putative regulatory interaction partners. However, the dynamics and downstream events following phosphorylation are not well understood. This review focuses on the specific role and regulation of TBC1D1 and TBC1D4 in insulin action.

Whole-exome sequencing identifies mutations of TBC1D1 encoding a Rab-GTPase-activating protein in patients with congenital anomalies of the kidneys and urinary tract (CAKUT).

Congenital anomalies of the kidneys and urinary tract (CAKUT) are genetically highly heterogeneous leaving most cases unclear after mutational analysis of the around 30 causative genes known so far. Assuming that phenotypes frequently showing dominant inheritance, such as CAKUT, can be caused by de novo mutations, de novo analysis of whole-exome sequencing data was done on two patient-parent-trios to identify novel CAKUT genes. In one case, we detected a heterozygous de novo frameshift variant in TBC1D1 encoding a Rab-GTPase-activating protein regulating glucose transporter GLUT4 translocation. Sequence analysis of 100 further CAKUT cases yielded three novel or rare inherited heterozygous TBC1D1 missense variants predicted to be pathogenic. TBC1D1 mutations affected Ser237-phosphorylation or protein stability and thereby act as hypomorphs. Tbc1d1 showed widespread expression in the developing murine urogenital system. A mild CAKUT spectrum phenotype, including anomalies observed in patients carrying TBC1D1 mutations, was found in kidneys of some Tbc1d1 (-/-) mice. Significantly reduced Glut4 levels were detected in kidneys of Tbc1d1 (-/-) mice and the dysplastic kidney of a TBC1D1 mutation carrier versus controls. TBC1D1 and SLC2A4 encoding GLUT4 were highly expressed in human fetal kidney. The patient with the truncating TBC1D1 mutation showed evidence for insulin resistance. These data demonstrate heterozygous deactivating TBC1D1 mutations in CAKUT patients with a similar renal and ureteral phenotype, and provide evidence that TBC1D1 mutations may contribute to CAKUT pathogenesis, possibly via a role in glucose homeostasis.

Critical illness myopathy and GLUT4: significance of insulin and muscle contraction.

RATIONALE: Critical illness myopathy (CIM) has no known cause and no treatment. Immobilization and impaired glucose metabolism are implicated. OBJECTIVES: We assessed signal transduction in skeletal muscle of patients at risk for CIM. We also investigated the effects of evoked muscle contraction. METHODS: In a prospective observational and interventional pilot study, we screened 874 mechanically ventilated patients with a sepsis-related organ-failure assessment score greater than or equal to 8 for 3 consecutive days in the first 5 days of intensive care unit stay. Thirty patients at risk for CIM underwent euglycemic-hyperinsulinemic clamp, muscle microdialysis studies, and muscle biopsies. Control subjects were healthy. In five additional patients at risk for CIM, we performed corresponding analyses after 12-day, daily, unilateral electrical muscle stimulation with the contralateral leg as control. MEASUREMENTS AND MAIN RESULTS: We performed successive muscle biopsies and assessed systemic insulin sensitivity and signal transduction pathways of glucose utilization at the mRNA and protein level and glucose transporter-4 (GLUT4) localization in skeletal muscle tissue. Skeletal muscle GLUT4 was trapped at perinuclear spaces, most pronounced in patients with CIM, but resided at the sarcolemma in control subjects. Glucose metabolism was not stimulated during euglycemic-hyperinsulinergic clamp. Insulin signal transduction was competent up to p-Akt activation; however, p-adenosine monophosphate-activated protein kinase (p-AMPK) was not detectable in CIM muscle. Electrical muscle stimulation increased p-AMPK, repositioned GLUT4, locally improved glucose metabolism, and prevented type-2 fiber atrophy. CONCLUSIONS: Insufficient GLUT4 translocation results in decreased glucose supply in patients with CIM. Failed AMPK activation is involved. Evoked muscle contraction may prevent muscle-specific AMPK failure, restore GLUT4 disposition, and diminish protein breakdown. Clinical trial registered with http://www.controlled-trials.com (registration number ISRCTN77569430).

Inhibition of proline-rich tyrosine kinase 2 restores cardioprotection by remote ischaemic preconditioning in type 2 diabetes.

BACKGROUND AND PURPOSE: Remote ischaemic preconditioning (rIPC) for cardioprotection is severely impaired in diabetes, and therapeutic options to restore it are lacking. The vascular endothelium plays a key role in rIPC. Given that the activity of endothelial nitric oxide synthase (eNOS) is inhibited by proline-rich tyrosine kinase 2 (Pyk2), we hypothesized that pharmacological Pyk2 inhibition could restore eNOS activity and thus restore remote cardioprotection in diabetes. EXPERIMENTAL APPROACH: New Zealand obese (NZO) mice that demonstrated key features of diabetes were studied. The consequence of Pyk2 inhibition on endothelial function, rIPC and infarct size after myocardial infarction were evaluated. The impact of plasma from mice and humans with or without diabetes was assessed in isolated buffer perfused murine hearts and aortic rings. KEY RESULTS: Plasma from nondiabetic mice and humans, both subjected to rIPC, caused remote tissue protection. Similar to diabetic humans, NZO mice demonstrated endothelial dysfunction. NZO mice had reduced circulating nitrite levels, elevated arterial blood pressure and a larger infarct size after ischaemia and reperfusion than BL6 mice. Pyk2 increased the phosphorylation of eNOS at its inhibitory site (Tyr656), limiting its activity in diabetes. The cardioprotective effects of rIPC were abolished in diabetic NZO mice. Pharmacological Pyk2 inhibition restored endothelial function and rescued cardioprotective effects of rIPC. CONCLUSION AND IMPLICATIONS: Endothelial function and remote tissue protection are impaired in diabetes. Pyk2 is a novel target for treating endothelial dysfunction and restoring cardioprotection through rIPC in diabetes.

Depletion of TBC1D4 Improves the Metabolic Exercise Response by Overcoming Genetically Induced Peripheral Insulin Resistance.

The Rab-GTPase-activating protein (RabGAP) TBC1D4 (AS160) represents a key component in the regulation of glucose transport into skeletal muscle and white adipose tissue (WAT) and is therefore crucial during the development of insulin resistance and type 2 diabetes. Increased daily activity has been shown to be associated with improved postprandial hyperglycemia in allele carriers of a loss-of-function variant in the human TBC1D4 gene. Using conventional Tbc1d4-deficient mice (D4KO) fed a high-fat diet, we show that moderate endurance exercise training leads to substantially improved glucose and insulin tolerance and enhanced expression levels of markers for mitochondrial activity and browning in WAT from D4KO animals. Importantly, in vivo and ex vivo analyses of glucose uptake revealed increased glucose clearance in interscapular brown adipose tissue and WAT from trained D4KO mice. Thus, chronic exercise is able to overcome the genetically induced insulin resistance caused by Tbc1d4 depletion. Gene variants in TBC1D4 may be relevant in future precision medicine as determinants of exercise response.