Rudolph L. Leibel

VMAT2 quantitation by PET as a biomarker for β-cell mass in health and disease

The common pathology underlying both type 1 and type 2 diabetes (T1DM and T2DM) is insufficient β‐cell mass (BCM) to meet metabolic demands. An important impediment to the more rapid evaluation of interventions for both T1DM and T2DM lack of biomarkers of pancreatic BCM. A reliable means of monitoring the mass and/or function of β‐cells would enable evaluation of the progression of diabetes as well as the monitoring of pharmacologic and other interventions. Recently, we identified a biomarker of BCM that is quantifiable by positron emission tomography (PET). PET is an imaging technique which allows for non‐invasive measurements of radioligand uptake and clearance, is sensitive in the pico‐ to nanomolar range and of which the results can be deconvoluted into measurements of receptor concentration. For BCM estimates, we have identified VMAT2 (vesicular monoamine transporter type 2) as a biomarker and [11C] DTBZ (dihydrotetrabenazine) as the transporter’s ligand. VMAT2 is highly expressed in β‐cells of the human pancreas relative to other cells of the endocrine and exocrine pancreas. Thus measurements of [11C] DTBZ in the pancreas provide an indirect measurement of BCM. Here we summarize our ongoing efforts to validate the clinical utility of this non‐invasive approach to real‐time BCM measurements

A MEMS differential viscometric sensor for affinity glucose detection in continuous glucose monitoring

Micromachined viscometric affinity glucose sensors have been previously demonstrated using vibrational cantilever and diaphragm. These devices featured a single glucose detection module that determines glucose concentrations through viscosity changes of glucose-sensitive polymer solutions. However, fluctuations in temperature and other environmental parameters might potentially affect the stability and reliability of these devices, creating complexity in their applications in subcutaneously implanted continuous glucose monitoring (CGM). To address these issues, we present a MEMS differential sensor that can effectively reject environmental disturbances while allowing accurate glucose detection. The sensor consists of two magnetically driven vibrating diaphragms situated inside microchambers filled with a boronic-acid based glucose-sensing solution and a reference solution insensitive to glucose. Glucose concentrations can be accurately determined by characteristics of the diaphragm vibration through differential capacitive detection. Our in-vitro and preliminary in-vivo experimental data demonstrate the potential of this sensor for highly stable subcutaneous CGM applications.

Cross-sectional and Test-Retest Characterization of PET with [18F]FP-(+)-DTBZ for β Cell Mass Estimates in Diabetes

PurposeThe vesicular monoamine transporter, type 2 (VMAT2) is expressed by insulin producing β cells and was evaluated as a biomarker of β cell mass (BCM) by positron emission tomography (PET) with [18F]fluoropropyl-dihydrotetrabenazine ([18F]FP-(+)-DTBZ).ProceduresWe evaluated the feasibility of longitudinal pancreatic PET VMAT2 quantification in the pancreas in two studies of healthy controls and patients with type 1 or 2 diabetes. VMAT2 binding potential (BPND) was estimated voxelwise using a reference tissue method in a cross-sectional study, followed by assessment of reproducibility using a test-retest paradigm. Metabolic function was evaluated by stimulated c-peptide measurements.ResultsPancreatic BPND was significantly decreased in patients with type 1 diabetes relative to controls and the test-retest variability was 9.4xa0%.ConclusionsPancreatic VMAT2 content is significantly reduced in long-term diabetes patients relative to controls and repeat scans are sufficiently reproducible to suggest the feasibility clinically VMAT2 measurements in longitudinal studies of new onset diabetes.

β-Cell replacement in mice using human type 1 diabetes nuclear transfer embryonic stem cells

β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide–positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide–positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy.

ZNF70, a novel ILDR2-interacting protein, contributes to the regulation of HES1 gene expression

A diabetes susceptibility gene, immunoglobulin-like domain containing receptor 2 (Ildr2), encodes a transmembrane protein localized to the endoplasmic reticulum membrane that is closely related to hepatic lipid metabolism. The livers of ob/ob mice in which Ildr2 is transiently overexpressed are relieved of hepatic steatosis. However, the molecular mechanisms through which ILDR2 affects these changes in hepatic lipid metabolism remain unknown. This study aimed to identify ILDR2-interacting proteins to further elucidate the molecular mechanisms underlying the role of ILDR2 in lipid homeostasis. We purified ILDR2-containing protein complexes using tandem affinity purification tagging and identified ZNF70, a member of the Kruppel C2H2-type zinc finger protein family, as a novel ILDR2-interacting protein. We demonstrated that ZNF70 interacts with ZFP64 and activates HES1 transcription by binding to the HES1 promoter. In addition, HES1 gene expression is increased in ILDR2-knockdown HepG2 cells, in which ZNF70 is translocated from the cytoplasm to the nucleus, suggesting that ZNF70 migration to the nucleus after dissociating from the ILDR2-ZNF70 complex activates HES1 transcription. These results support a novel link between ILDR2 and HES1 gene expression and suggest that ILDR2 is involved in a novel pathway in hepatic steatosis.

Continuous monitoring of glucose in subcutaneous tissue using microfabricated differential affinity sensors.

Objective: We describe miniaturized differential glucose sensors based on affinity binding between glucose and a synthetic polymer. The sensors possess excellent resistance to environmental disturbances and can potentially allow wireless measurements of glucose concentrations within interstitial fluid in subcutaneous tissue for long-term, stable continuous glucose monitoring (CGM). Methods: The sensors are constructed using microelectromechanical systems (MEMS) technology and exploit poly(N-hydroxy-ethyl acrylamide-ran-3-acrylamidophenylboronic acid) (PHEAA-ran-PAAPBA), a glucose-binding polymer with excellent specificity, reversibility, and stability. Two sensing approaches have been investigated, which respectively, use a pair of magnetically actuated diaphragms and perforated electrodes to differentially measure the glucose-binding-induced changes in the viscosity and permittivity of the PHEAA-ran-PAAPBA solution with respect to a reference, glucose-unresponsive polymer solution. Results: In vivo characterization of the MEMS affinity sensors were performed by controlling blood glucose concentrations of laboratory mice by exogenous glucose and insulin administration. The sensors experienced an 8–30 min initialization period after implantation and then closely tracked commercial capillary glucose meter readings with time lags ranging from 0–15 min during rapid glucose concentration changes. Clarke error grid plots obtained from sensor calibration suggest that, for the viscometric and dielectric sensors, respectively, approximately 95% (in the hyperglycemic range) and 84% (ranging from hypoglycemic to hyperglycemic glucose concentrations) of measurement points were clinically accurate, while 5% and 16% of the points were clinically acceptable. Conclusions: The miniaturized MEMS sensors explore differential measurements of affinity glucose recognition. In vivo testing demonstrated excellent accuracy and stability, suggesting that the devices hold the potential to enable long-term and reliable CGM in clinical applications.

The role of Rpgrip1l, a component of the primary cilium, in adipocyte development and function

Genetic variants within the FTO (α‐ketoglutarate‐dependent dioxygenase) gene have been strongly associated with a modest increase in adiposity as a result of increased food intake. These risk alleles are associated with decreased expression of both FTO and neighboring RPGRIP1L (retinitis pigmentosa GTPase regulator‐interacting protein 1 like). RPGRIP1L encodes a protein that is critical to the function of the primary cilium, which conveys extracellular information to the cell. Rpgrip1l+/− mice exhibit increased adiposity, in part, as a result of hyperphagia. Here, we describe the effects of Rpgrip1l in adipocytes that may contribute to the adiposity phenotype observed in these animals and possibly in humans who segregate for FTO risk alleles. Loss of Rpgrip1l in 3T3‐L1 preadipocytes increased the number of cells that are capable of differentiating into mature adipocytes. Knockout of Rpgrip1l in mature adipocytes using Adipoq‐Cre did not increase adiposity in mice that were fed chow or a high‐fat diet. We also did not observe any effects of Rpgrip1l knockdown in mature 3T3‐L1 adipocytes. Thus, to the extent that Rpgrip1l affects cell‐autonomous adipose tissue function, it may do so as a result of the effects conveyed in preadipocytes in which the primary cilium is functionally important. We propose that decreased RPGRIP1L expression in preadipocytes in humans who segregate for FTO obesity risk alleles may increase the storage capacity of adipose tissue.—Martin Carli, J. F., LeDuc, C. A., Zhang, Y., Stratigopoulos, G., Leibel, R. L. The role of Rpgrip1l, a component of the primary cilium, in adipocyte development and function. FASEB J. 32, 3946–3956 (2018). www.fasebj.org

FTO genotype impacts food intake and corticolimbic activation

BackgroundnVariants in the first intron of the fat mass and obesity-associated (FTO) gene increase obesity risk. People with high-risk FTO genotypes exhibit preference for high-fat foods, reduced satiety responsiveness, and greater food intake consistent with impaired satiety.nnnObjectivenWe sought central nervous system mechanisms that might underlie impaired satiety perception in people with a higher risk of obesity based on their FTO genotype.nnnDesignnWe performed a cross-sectional study in a sample that was enriched for obesity and included 20 higher-risk participants with the AA (risk) genotype at the rs9939609 locus of FTO and 94 lower-risk participants with either the AT or TT genotype. We compared subjective appetite, appetite-regulating hormones, caloric intake at a buffet meal, and brain response to visual food cues in an extended satiety network using functional MRI scans acquired before and after a standardized meal.nnnResultsnHigher-risk participants reported less subjective fullness (χ2 = 7.48, P < 0.01), rated calorie-dense food as more appealing (χ2 = 3.92, P < 0.05), and consumed ∼350 more kilocalories than lower-risk participants (β = 348 kcal, P = 0.03), even after adjusting for fat or lean mass. Premeal, the higher-risk group had greater activation by fattening food images (compared with objects) in the medial orbital frontal cortex (β = 11.6; 95% CI: 1.5, 21.7; P < 0.05). Postmeal, the higher-risk subjects had greater activation by fattening (compared with nonfattening) food cues in the ventral tegmental area/substantia nigra (β = 12.8; 95% CI: 2.7, 23.0; P < 0.05), amygdala (β = 10.6; 95% CI: 0.7, 20.5; P < 0.05), and ventral striatum (β = 6.9; 95% CI: 0.2, 13.7; P < 0.05). Moreover, postmeal activation by fattening food cues within the preselected extended satiety network was positively associated with energy intake at the buffet meal (R2 = 0.29, P = 0.04) and this relation was particularly strong in the dorsal striatum (R2 = 0.28, P = 0.01), amygdala (R2 = 0.28, P = 0.03), and ventral tegmental area/substantia nigra (R2 = 0.27, P = 0.01).nnnConclusionnThe findings are consistent with a model in which allelic variants in FTO raise obesity risk through impaired central nervous system satiety processing, thereby increasing food intake. This study is registered at clinicaltrials.gov as NCT02483663.

Resistance Training Reduces Skeletal Muscle Work Efficiency in Weight-Reduced and Non-Weight-Reduced Subjects

The objective of this study is to determine whether resistance training is similarly effective in reducing skeletal muscle efficiency and increasing strength in weight‐reduced and maximal weight subjects.

FTO mediates cell-autonomous effects on adipogenesis and adipocyte lipid content by regulating gene expression via 6mA DNA modifications

SNPs in the first intron of α-ketoglutarate-dependent dioxygenase (FTO) convey effects on adiposity by mechanisms that remain unclear, but appear to include modulation of expression of FTO itself, as well as other genes in cis. FTO expression is lower in fibroblasts and iPSC-derived neurons of individuals segregating for FTO obesity risk alleles. We employed in vitro adipogenesis models to investigate the molecular mechanisms by which Fto affects adipocyte development and function. Fto expression was upregulated during adipogenesis, and was required for the maintenance of CEBPB and Cebpd/CEBPD expression in murine and human adipocytes in vitro. Fto knockdown decreased the number of 3T3-L1 cells that differentiated into adipocytes as well as the amount of lipid per mature adipocyte. This effect on adipocyte programming was conveyed, in part, by modulation of CCAAT enhancer binding protein (C/ebp)β-regulated transcription. We found that Fto also affected Cebpd transcription by demethylating DNA N6-methyldeoxyadenosine in the Cebpd promoter. Fto is permissive for adipogenesis and promotes maintenance of lipid content in mature adipocytes by enabling C/ebpβ-driven transcription and expression of Cebpd. These findings are consistent with the loss of fat mass in mice segregating for a dominant-negative Fto allele.