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This article explores the relationship between insulin hypersecretion and impaired insulin action on glucose metabolism. It presents two competing hypotheses regarding the role of insulin hypersecretion in promoting obesity and insulin resistance. The study conducted by Tricò et al. focuses on individuals with insulin hypersecretion and finds that they have higher rates of de novo lipogenesis and increased adipose tissue. The article also discusses the limitations of current methods used to assess insulin sensitivity and highlights the complexity of glucose homeostasis. Overall, this research provides important insights into the regulation of plasma glucose concentration and raises questions about the traditional understanding of the relationship between insulin sensitivity, insulin secretion, and plasma glucose levels. Further research is needed to better understand the mechanisms underlying insulin hypersecretion and its impact on metabolic health. [Extracted from the article]

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease (ESKD). Prognostic biomarkers reflective of underlying molecular mechanisms are critically needed for effective management of DKD. A three-marker panel was derived from a proteomics analysis of plasma samples by an unbiased machine learning approach from participants (N = 58) in the Clinical Phenotyping and Resource Biobank study. In combination with standard clinical parameters, this panel improved prediction of the composite outcome of ESKD or a 40% decline in glomerular filtration rate. The panel was validated in an independent group (N = 68), who also had kidney transcriptomic profiles. One marker, plasma angiopoietin 2 (ANGPT2), was significantly associated with outcomes in cohorts from the Cardiovascular Health Study (N = 3,183) and the Chinese Cohort Study of Chronic Kidney Disease (N = 210). Glomerular transcriptional angiopoietin/Tie (ANG-TIE) pathway scores, derived from the expression of 154 ANG-TIE signaling mediators, correlated positively with plasma ANGPT2 levels and kidney outcomes. Higher receptor expression in glomeruli and higher ANG-TIE pathway scores in endothelial cells corroborated potential functional effects in the kidney from elevated plasma ANGPT2 levels. Our work suggests that ANGPT2 is a promising prognostic endothelial biomarker with likely functional impact on glomerular pathogenesis in DKD. [ABSTRACT FROM AUTHOR]

The article "Response to Comment on Mittendorfer et al. Insulin Hypersecretion as Promoter of Body Fat Gain and Hyperglycemia" discusses the complexities of insulin signaling and insulin resistance. It highlights the role of spare receptors in achieving a maximal response to insulin, even when the number of insulin receptors on the cell surface is low. The text emphasizes that spare receptors do not protect against impaired insulin sensitivity, which is commonly observed in obesity. The authors, Bettina Mittendorfer, James D. Johnson, Giovanni Solinas, and Per-Anders Jansson, provide insights into the mechanisms of insulin resistance and the importance of understanding insulin signaling in metabolic disorders. [Extracted from the article]

Glucagon hypersecretion from pancreatic islet α-cells exacerbates hyperglycemia in type 1 diabetes (T1D) and type 2 diabetes. Still, the underlying mechanistic pathways that regulate glucagon secretion remain controversial. Among the three complementary main mechanisms (intrinsic, paracrine, and juxtacrine) proposed to regulate glucagon release from α-cells, juxtacrine interactions are the least studied. It is known that tonic stimulation of α-cell EphA receptors by ephrin-A ligands (EphA forward signaling) inhibits glucagon secretion in mouse and human islets and restores glucose inhibition of glucagon secretion in sorted mouse α-cells, and these effects correlate with increased F-actin density. Here, we elucidate the downstream target of EphA signaling in α-cells. We demonstrate that RhoA, a Rho family GTPase, plays a key role in this pathway. Pharmacological inhibition of RhoA disrupts glucose inhibition of glucagon secretion in islets and decreases cortical F-actin density in dispersed α-cells and α-cells in intact islets. Quantitative FRET biosensor imaging shows that increased RhoA activity follows directly from EphA stimulation. We show that in addition to modulating F-actin density, EphA forward signaling and RhoA activity affect α-cell Ca2+ activity in a novel mechanistic pathway. Finally, we show that stimulating EphA forward signaling restores glucose inhibition of glucagon secretion from human T1D donor islets. [ABSTRACT FROM AUTHOR]

Dipeptidyl peptidase 4 (DPP-4) degrades the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide (GIP). DPP-4 inhibitors improve glycemic control in type 2 diabetes, but the importance of protecting GIP from degradation for their clinical effects is unknown. We included 12 patients with type 2 diabetes (mean ± SD BMI 27 ± 2.6 kg/m2, HbA1c 7.1 ± 1.4% [54 ± 15 mmol/mol]) in this double-blind, placebo-controlled, crossover study to investigate the contribution of endogenous GIP to the effects of the DPP-4 inhibitor sitagliptin. Participants underwent two randomized, 13-day treatment courses of sitagliptin (100 mg/day) and placebo, respectively. At the end of each treatment period, we performed two mixed-meal tests with infusion of the GIP receptor antagonist GIP(3-30)NH2 (1,200 pmol/kg/min) or saline placebo. Sitagliptin lowered mean fasting plasma glucose by 1.1 mmol/L compared with placebo treatment. During placebo treatment, postprandial glucose excursions were increased during GIP(3-30)NH2 compared with saline (difference in area under the curve ± SEM 7.3 ± 2.8%) but were unchanged during sitagliptin treatment. Endogenous GIP improved β-cell function by 37 ± 12% during DPP-4 inhibition by sitagliptin. This was determined by the insulin secretion rate/plasma glucose ratio. We calculated an estimate of the absolute sitagliptin-mediated impact of GIP on β-cell function as the insulinogenic index during sitagliptin treatment plus saline infusion minus the insulinogenic index during sitagliptin plus GIP(3-30)NH2. This estimate was expressed relative to the maximal potential contribution of GIP to the effect of sitagliptin (100%), defined as the difference between the full sitagliptin treatment effect, including actions mediated by GIP (sitagliptin + saline), and the physiological response minus any contribution by GIP [placebo treatment + GIP(3-30)NH2]. We demonstrate insulinotropic and glucose-lowering effects of endogenous GIP in patients with type 2 diabetes and that endogenous GIP contributes to the improved β-cell function observed during DPP-4 inhibition. [ABSTRACT FROM AUTHOR]

Chronic, nonhealing skin wounds, such as diabetic foot ulcers (DFUs), are common in patients with type 2 diabetes. Here, we investigated the role of chemokine (C-C motif) ligand 28 (CCL28) and its receptor C-C chemokine receptor type 10 (CCR10) in downregulation of endothelial nitric (NO) oxide synthase (eNOS) in association with delayed skin wound healing in the db/db mouse model of type 2 diabetes. We observed reduced eNOS expression and elevated CCL28/CCR10 levels in dorsal skin of db/db mice and subdermal leg biopsy specimens from human subjects with type 2 diabetes. Further interrogation revealed that overexpression of CCR10 reduced eNOS expression, NO bioavailability, and tube formation of human dermal microvascular endothelial cells (HDMVECs) in vitro, which was recapitulated in mouse dorsal skin. In addition, incubation of HDMVECs with CCL28 led to internalization of the CCR10/eNOS complex and colocalization with lysosome-associated membrane protein 1. Finally, topical application of myristoylated CCR10 binding domain 7 amino acid (Myr-CBD7) peptide prevented CCR10-eNOS interaction and subsequent eNOS downregulation, enhanced eNOS/NO levels, eNOS/VEGF-R2+ microvessel density, and blood perfusion, reduced inflammatory cytokine levels, and importantly, decreased wound healing time in db/db mice. Thus, endothelial cell CCR10 activation in genetically obese mice with type 2 diabetes promotes eNOS depletion and endothelial dysfunction, and targeted disruption of CCR10/eNOS interaction improves wound healing. [ABSTRACT FROM AUTHOR]

Long-term glucagon receptor (GCGR) agonism is associated with hyperglycemia and glucose intolerance, while acute GCGR agonism enhances whole-body insulin sensitivity and hepatic AKTSer473 phosphorylation. These divergent effects establish a critical gap in knowledge surrounding GCGR action. mTOR complex 2 (mTORC2) is composed of seven proteins, including RICTOR, which dictates substrate binding and allows for targeting of AKTSer473. We used a liver-specific Rictor knockout mouse (RictorΔLiver) to investigate whether mTORC2 is necessary for insulin receptor (INSR) and GCGR cross talk. RictorΔLiver mice were characterized by impaired AKT signaling and glucose intolerance. Intriguingly, RictorΔLiver mice were also resistant to GCGR-stimulated hyperglycemia. Consistent with our prior report, GCGR agonism increased glucose infusion rate and suppressed hepatic glucose production during hyperinsulinemic-euglycemic clamp of control animals. However, these benefits to insulin sensitivity were ablated in RictorΔLiver mice. We observed diminished AKTSer473 and GSK3α/βSer21/9 phosphorylation in RictorΔLiver mice, whereas phosphorylation of AKTThr308 was unaltered in livers from clamped mice. These signaling effects were replicated in primary hepatocytes isolated from RictorΔLiver and littermate control mice, confirming cell-autonomous cross talk between GCGR and INSR pathways. In summary, our study reveals the necessity of RICTOR, and thus mTORC2, in GCGR-mediated enhancement of liver and whole-body insulin action. [ABSTRACT FROM AUTHOR]

Net synthesis of pancreatic β-cells peaks before 2 years of life. β-Cell mass is set within the first 5 years of life. In-frame translational readthrough of the NRP1 gene exon 9 into intron 9 generates a truncated neuropilin-1 protein lacking downstream sequence necessary for binding VEGF that stimulates β-cell replication. VEGF is critical for developing but not adult islet neogenesis. Herein we show that cells in human pancreatic islets containing the full-length neuropilin-1 possess insulin but cells that contain the truncated neuropilin-1 are devoid of insulin. Decreased insulin cells increases susceptibility to onset of type 1 diabetes at a younger age. We also show that the frequency of a genetic marker in NRP1 intron 9 is higher among patients with onset of type 1 diabetes before age 4 years (31.8%), including those with onset at 0.67-2.00 and 2-4 years, compared with that in patients with onset at 4-8 years, at 8-12 years, and after 16 years (16.1%) with frequency equal to that in subjects without diabetes (16.0%). Decreased insulin cells plus the genetic data are consistent with a low effect mechanism that alters the onset of type 1 diabetes to a very young age in some patients, thus supporting the endotype concept that type 1 diabetes is a heterogeneous disease. [ABSTRACT FROM AUTHOR]

There is increasing evidence that dopamine (DA) functions as a negative regulator of glucose-stimulated insulin secretion; however, the underlying molecular mechanism remains unknown. Using total internal reflection fluorescence microscopy, we monitored insulin granule exocytosis in primary islet cells to dissect the effect of DA. We found that D1 receptor antagonists rescued the DA-mediated inhibition of glucose-stimulated calcium (Ca2+) flux, thereby suggesting a role of D1 in the DA-mediated inhibition of insulin secretion. Overexpression of D2, but not D1, alone exerted an inhibitory and toxic effect that abolished the glucose-stimulated Ca2+ influx and insulin secretion in β-cells. Proximity ligation and Western blot assays revealed that D1 and D2 form heteromers in β-cells. Treatment with a D1-D2 heteromer agonist, SKF83959, transiently inhibited glucose-induced Ca2+ influx and insulin granule exocytosis. Coexpression of D1 and D2 enabled β-cells to bypass the toxic effect of D2 overexpression. DA transiently inhibited glucose-stimulated Ca2+ flux and insulin exocytosis by activating the D1-D2 heteromer. We conclude that D1 protects β-cells from the harmful effects of DA by modulating D2 signaling. The finding will contribute to our understanding of the DA signaling in regulating insulin secretion and improve methods for preventing and treating diabetes. [ABSTRACT FROM AUTHOR]

The induction of nausea and emesis is a major barrier to maximizing the weight loss profile of obesity medications, and therefore, identifying mechanisms that improve tolerability could result in added therapeutic benefit. The development of peptide YY (PYY)-based approaches to treat obesity are no exception, as PYY receptor agonism is often accompanied by nausea and vomiting. Here, we sought to determine whether glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) agonism reduces PYY-induced nausea-like behavior in mice. We found that central and peripheral administration of a GIPR agonist reduced conditioned taste avoidance (CTA) without affecting hypophagia mediated by a PYY analog. The receptors for GIP and PYY (Gipr and Npy2r) were found to be expressed by the same neurons in the area postrema (AP), a brainstem nucleus involved in detecting aversive stimuli. Peripheral administration of a GIPR agonist induced neuronal activation (cFos) in the AP. Further, whole-brain cFos analyses indicated that PYY-induced CTA was associated with augmented neuronal activity in the parabrachial nucleus (PBN), a brainstem nucleus that relays aversive/emetic signals to brain regions that control feeding behavior. Importantly, GIPR agonism reduced PYY-mediated neuronal activity in the PBN, providing a potential mechanistic explanation for how GIPR agonist treatment reduces PYY-induced nausea-like behavior. Together, the results of our study indicate a novel mechanism by which GIP-based therapeutics may have benefit in improving the tolerability of weight loss agents. [ABSTRACT FROM AUTHOR]

Long-chain fatty acids (LCFAs) are not only energy sources but also serve as signaling molecules. GPR120, an LCFA receptor, plays key roles in maintaining metabolic homeostasis. However, whether endogenous ligand-GPR120 circuits exist and how such circuits function in pancreatic islets are unclear. Here, we found that endogenous GPR120 activity in pancreatic δ-cells modulated islet functions. At least two unsaturated LCFAs, oleic acid (OA) and linoleic acid (LA), were identified as GPR120 agonists within pancreatic islets. These two LCFAs promoted insulin secretion by inhibiting somatostatin secretion and showed bias activation of GPR120 in a model system. Compared with OA, LA exerted higher potency in promoting insulin secretion, which is dependent on β-arrestin2 function. Moreover, GPR120 signaling was impaired in the diabetic db/db model, and replenishing OA and LA improved islet function in both the db/db and streptozotocin-treated diabetic models. Consistently, the administration of LA improved glucose metabolism in db/db mice. Collectively, our results reveal that endogenous LCFA-GPR120 circuits exist and modulate homeostasis in pancreatic islets. The contributions of phenotype differences caused by different LCFA-GPR120 circuits within islets highlight the roles of fine-tuned ligand-receptor signaling networks in maintaining islet homeostasis. [ABSTRACT FROM AUTHOR]

Cutaneous wound healing in diabetes is impaired and would develop into nonhealing ulcerations. However, the molecular mechanism underlying the wound-healing process remains largely obscure. Here, we found that cutaneous PDGFRα+ fibroblast-expressing lncRNA-H19 (lncH19) accelerates the wound-healing process via promoting dermal fibroblast proliferation and macrophage infiltration in injured skin. PDGFRα+ cell-derived lncH19, which is lower in contents in the wound-healing cutaneous tissue of patients and mice with type 2 diabetes, is required for wound healing through promoting proliferative capacity of dermis fibroblasts as well as macrophage recruitments. Mechanistically, lncH19 relieves the cell cycle arrest of fibroblasts and increases macrophage infiltration in injured tissues via inhibiting p53 activity and GDF15 releasement. Furthermore, exosomes derived from adipocyte progenitor cells efficiently restore the impaired diabetic wound healing via delivering lncH19 to injured tissue. Therefore, our study reveals a new role for lncRNA in regulating cutaneous tissue repair and provides a novel promising insight for developing clinical treatment of diabetes. [ABSTRACT FROM AUTHOR]

Pancreatic α-cells are important in maintaining metabolic homeostasis, but their role in regulating maternal metabolic adaptations to pregnancy has not been studied. The objective of this study was to determine whether pancreatic α-cells respond to pregnancy and their contribution to maternal metabolic adaptation. With use of C57BL/6 mice, the findings of our study showed that pregnancy induced a significant increase of α-cell mass by promoting α-cell proliferation that was associated with a transitory increase of maternal serum glucagon concentration in early pregnancy. Maternal pancreatic GLP-1 content also was significantly increased during pregnancy. Using the inducible Cre/loxp technique, we ablated the α-cells (α-null) before and during pregnancy while maintaining enteroendocrine L-cells and serum GLP-1 in the normal range. In contrast to an improved glucose tolerance test (GTT) before pregnancy, significantly impaired GTT and remarkably higher serum glucose concentrations in the fed state were observed in α-null dams. Glucagon receptor antagonism treatment, however, did not affect measures of maternal glucose metabolism, indicating a dispensable role of glucagon receptor signaling in maternal glucose homeostasis. However, the GLP-1 receptor agonist improved insulin production and glucose metabolism of α-null dams. Furthermore, GLP-1 receptor antagonist Exendin (9-39) attenuated pregnancy-enhanced insulin secretion and GLP-1 restored glucose-induced insulin secretion of cultured islets from α-null dams. Together, these results demonstrate that α-cells play an essential role in controlling maternal metabolic adaptation to pregnancy by enhancing insulin secretion. [ABSTRACT FROM AUTHOR]

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) augments glucose-dependent insulin secretion through its receptor expressed on islet β-cells. GIP also acts on adipose tissue; yet paradoxically, both enhanced and reduced GIP receptor (GIPR) signaling reduce adipose tissue mass and attenuate weight gain in response to nutrient excess. Moreover, the precise cellular localization of GIPR expression within white adipose tissue (WAT) remains uncertain. We used mouse genetics to target Gipr expression within adipocytes. Surprisingly, targeting Cre expression to adipocytes using the adiponectin (Adipoq) promoter did not produce meaningful reduction of WAT Gipr expression in Adipoq-Cre:Giprflx/flx mice. In contrast, adenoviral expression of Cre under the control of the cytomegalovirus promoter, or transgenic expression of Cre using nonadipocyte-selective promoters (Ap2/Fabp4 and Ubc) markedly attenuated WAT Gipr expression. Analysis of single-nucleus RNA-sequencing, adipose tissue data sets localized Gipr/GIPR expression predominantly to pericytes and mesothelial cells rather than to adipocytes. Together, these observations reveal that adipocytes are not the major GIPR+ cell type within WAT-findings with mechanistic implications for understanding how GIP and GIP-based co-agonists control adipose tissue biology. [ABSTRACT FROM AUTHOR]

Accumulating evidence supports a critical role for posttranslationally modified (PTM) islet neoantigens in type 1 diabetes. However, our understanding regarding thymic development and peripheral activation of PTM autoantigen-reactive T cells is still limited. Using HLA-DR4 humanized mice, we observed that deamidation of GAD65115-127 generates a more immunogenic epitope that recruits T cells with promiscuous recognition of both the deamidated and native epitopes and reduced frequency of regulatory T cells. Using humanized HLA/T-cell receptor (TCR) mice, we observed that TCRs reactive to the native or deamidated GAD65115-127 led to efficient development of CD4+ effector T cells; however, regulatory T-cell development was reduced in mice expressing the PTM-reactive TCR, which was partially restored with exogenous PTM peptide. Upon priming, both the native-specific and the deamidated-specific T cells accumulated in pancreatic islets, suggesting that both specificities can recognize endogenous GAD65 and contribute to anti-β-cell responses. Collectively, our observations in polyclonal and single TCR systems suggest that while effector T-cell responses can exhibit cross-reactivity between native and deamidated GAD65 epitopes, regulatory T-cell development is reduced in response to the deamidated epitope, pointing to regulatory T-cell development as a key mechanism for loss of tolerance to PTM antigenic targets. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptor 40 (GPR40) is a promising target to support glucose-induced insulin release in patients with type 2 diabetes. We studied the role of GPR40 in the regulation of blood-nerve barrier integrity and its involvement in diabetes-induced neuropathies. Because GPR40 modulates insulin release, we used the streptozotocin model for type 1 diabetes, in which GPR40 functions can be investigated independently of its effects on insulin release. Diabetic wild-type mice exhibited increased vascular endothelial permeability and showed epineural microlesions in sciatic nerves, which were also observed in naïve GPR40-/- mice. Fittingly, expression of vascular endothelial growth factor-A (VEGF-A), an inducer of vascular permeability, was increased in diabetic wild-type and naïve GPR40-/- mice. GPR40 antagonists increased VEGF-A expression in murine and human endothelial cells as well as permeability of transendothelial barriers. In contrast, GPR40 agonists suppressed VEGF-A release and mRNA expression. The VEGF receptor inhibitor axitinib prevented diabetes-induced hypersensitivities and reduced endothelial and epineural permeability. Importantly, the GPR40 agonist GW9508 reverted established diabetes-induced hypersensitivity, an effect that was blocked by VEGF-A administration. Thus, GPR40 activation suppresses VEGF-A expression, thereby reducing diabetes-induced blood-nerve barrier permeability and reverting diabetes-induced hypersensitivities. [ABSTRACT FROM AUTHOR]

Recent advances in genetic analysis have significantly helped in progressively attenuating the heritability gap of obesity and have brought into focus monogenic variants that disrupt the melanocortin signaling. In a previous study, next-generation sequencing revealed a monogenic etiology in ∼50% of the children with severe obesity from a consanguineous population in Pakistan. Here we assess rare variants in obesity-causing genes in young adults with severe obesity from the same region. Genomic DNA from 126 randomly selected young adult obese subjects (BMI 37.2 ± 0.3 kg/m2; age 18.4 ± 0.3 years) was screened by conventional or augmented whole-exome analysis for point mutations and copy number variants (CNVs). Leptin, insulin, and cortisol levels were measured by ELISA. We identified 13 subjects carrying 13 different pathogenic or likely pathogenic variants in LEPR, PCSK1, MC4R, NTRK2, POMC, SH2B1, and SIM1. We also identified for the first time in the human, two homozygous stop-gain mutations in ASNSD1 and IFI16 genes. Inactivation of these genes in mouse models has been shown to result in obesity. Additionally, we describe nine homozygous mutations (seven missense, one stop-gain, and one stop-loss) and four copy-loss CNVs in genes or genomic regions previously linked to obesity-associated traits by genome-wide association studies. Unexpectedly, in contrast to obese children, pathogenic mutations in LEP and LEPR were either absent or rare in this cohort of young adults. High morbidity and mortality risks and social disadvantage of children with LEP or LEPR deficiency may in part explain this difference between the two cohorts. [ABSTRACT FROM AUTHOR]

Studies of monogenic diabetes are particularly useful because we can gain insight into the molecular events of pancreatic β-cell failure. Maturity-onset diabetes of the young 1 (MODY1) is a form of monogenic diabetes caused by a mutation in the HNF4A gene. Human-induced pluripotent stem cells (hiPSCs) provide an excellent tool for disease modeling by subsequently directing differentiation toward desired pancreatic islet cells, but cellular phenotypes in terminally differentiated cells are notoriously difficult to detect. Re-creating a spatial (three-dimensional [3D]) environment may facilitate phenotype detection. We studied MODY1 by using hiPSC-derived pancreatic β-like patient and isogenic control cell lines in two different 3D contexts. Using size-adjusted cell aggregates and alginate capsules, we show that the 3D context is critical to facilitating the detection of mutation-specific phenotypes. In 3D cell aggregates, we identified irregular cell clusters and lower levels of structural proteins by proteome analysis, whereas in 3D alginate capsules, we identified altered levels of glycolytic proteins in the glucose sensing apparatus by proteome analysis. Our study provides novel knowledge on normal and abnormal function of HNF4A, paving the way for translational studies of new drug targets that can be used in precision diabetes medicine in MODY. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2D) causes an increased risk of morbidity and mortality in response to viral infection. T2D is characterized by hyperglycemia and is typically associated with insulin resistance and compensatory hyperinsulinemia. CD8 T cells express the insulin receptor, and previously, we have shown that insulin is able to directly modulate effector CD8 T-cell function. We therefore hypothesized that memory CD8 T-cell responsiveness in the context of T2D is negatively impacted by hyperinsulinemia or hyperglycemia. Using a mouse model for T2D, we could show that memory CD8 T-cell function was significantly reduced in response to rechallenge by viral infection or with melanoma cells. Basal insulin injection of mice increased GLUT-1 expression and glucose uptake in memory CD8 T-cell precursors early after infection, which was prevented when these cells were deficient for the insulin receptor. However, neither insulin injection nor insulin receptor deficiency resulted in a difference in metabolism, memory formation, cytokine production, or recall responses of memory CD8 T cells compared with controls. Importantly, in context of obesity, insulin receptor deficiency on CD8 T cells did not affect the functional capacity of memory CD8 T cells. In contrast, we could show in vitro and in vivo that hyperglycemia significantly impairs the antiviral capacity of memory CD8 T cells. Our findings indicate that obesity impairs the memory CD8 T-cell response against viral infection and cancer through the detrimental effects of hyperglycemia rather than hyperinsulinemia. [ABSTRACT FROM AUTHOR]

In the endoplasmic reticulum (ER), the translocation-associated protein complex (TRAP), also called signal sequence receptor (SSR), includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2, and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPβ/SSR2, or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels, leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here, we report that diminished TRAPγ/SSR3 expression in pancreatic β-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting cotranslational and posttranslational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels, as observed in cultured rodent β-cell lines and confirmed in human islets. Strikingly, pancreatic β-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkably, overexpression of TRAPγ/SSR3 in control β-cells raises proinsulin levels, even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis. [ABSTRACT FROM AUTHOR]

We previously showed that treating NOD mice with an agonistic monoclonal anti-TLR4/MD2 antibody (TLR4-Ab) reversed acute type 1 diabetes (T1D). Here, we show that TLR4-Ab reverses T1D by induction of myeloid-derived suppressor cells (MDSCs). Unbiased gene expression analysis after TLR4-Ab treatment demonstrated upregulation of genes associated with CD11b+Ly6G+ myeloid cells and downregulation of T-cell genes. Further RNA sequencing of purified, TLR4-Ab-treated CD11b+ cells showed significant upregulation of genes associated with bone marrow-derived CD11b+ cells and innate immune system genes. TLR4-Ab significantly increased percentages and numbers of CD11b+ cells. TLR4-Ab-induced CD11b+ cells, derived ex vivo from TLR4-Ab-treated mice, suppress T cells, and TLR4-Ab-conditioned bone marrow cells suppress acute T1D when transferred into acutely diabetic mice. Thus, the TLR4-Ab-induced CD11b+ cells, by the currently accepted definition, are MDSCs able to reverse T1D. To understand the TLR4-Ab mechanism, we compared TLR4-Ab with TLR4 agonist lipopolysaccharide (LPS), which cannot reverse T1D. TLR4-Ab remains sequestered at least 48 times longer than LPS within early endosomes, alters TLR4 signaling, and downregulates inflammatory genes and proteins, including nuclear factor-κB. TLR4-Ab in the endosome, therefore, induces a sustained, attenuated inflammatory response, providing an ideal "second signal" for the activation/maturation of MDSCs that can reverse acute T1D. [ABSTRACT FROM AUTHOR]

Fructosamine is a measure of short-term glycemic control, which has been suggested as a useful complement to glycated hemoglobin (HbA1c) for the diagnosis and monitoring of diabetes. To date, a single genome-wide association study (GWAS) including 8,951 U.S. White and 2,712 U.S. Black individuals without a diabetes diagnosis has been published. Results in Whites and Blacks yielded different association loci, near RCN3 and CNTN5, respectively. In this study, we performed a GWAS on 20,731 European-ancestry blood donors and meta-analyzed our results with previous data from U.S. White participants from the Atherosclerosis Risk in Communities (ARIC) study (Nmeta = 29,685). We identified a novel association near GCK (rs3757840, βmeta = 0.0062; minor allele frequency [MAF] = 0.49; Pmeta = 3.66 × 10-8) and confirmed the association near RCN3 (rs113886122, βmeta = 0.0134; MAF = 0.17; Pmeta = 5.71 × 10-18). Colocalization analysis with whole-blood expression quantitative trait loci data suggested FCGRT as the effector transcript at the RCN3 locus. We further showed that fructosamine has low heritability (h2 = 7.7%), has no significant genetic correlation with HbA1c and other glycemic traits in individuals without a diabetes diagnosis (P > 0.05), but has evidence of shared genetic etiology with some anthropometric traits (Bonferroni-corrected P < 0.0012). Our results broaden knowledge of the genetic architecture of fructosamine and prioritize FCGRT for downstream functional studies at the established RCN3 locus. [ABSTRACT FROM AUTHOR]

Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes, where it acts by engaging its ligand, CD47. SIRPG, which encodes SIRPγ, contains a nonsynonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. SIRPG produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of SIRPG transcript isoforms. All of these transcript isoforms produced protein upon transient expression in vitro. However, CRISPR/Cas9 targeting of one of the alternatively spliced exons in SIRPG eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild-type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling, and had significantly increased levels of cell-surface CD47. In primary CD4+ and CD8+ T cells, cell-surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that SIRPG is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide 1 receptor (GLP-1R) agonists decrease body weight and improve glycemic control in obesity and diabetes. Patient compliance and maximal efficacy of GLP-1 therapeutics are limited by adverse side effects, including nausea and emesis. In three different species (i.e., mice, rats, and musk shrews), we show that glucose-dependent insulinotropic polypeptide receptor (GIPR) signaling blocks emesis and attenuates illness behaviors elicited by GLP-1R activation, while maintaining reduced food intake, body weight loss, and improved glucose tolerance. The area postrema and nucleus tractus solitarius (AP/NTS) of the hindbrain are required for food intake and body weight suppression by GLP-1R ligands and processing of emetic stimuli. Using single-nuclei RNA sequencing, we identified the cellular phenotypes of AP/NTS cells expressing GIPR and GLP-1R on distinct populations of inhibitory and excitatory neurons, with the greatest expression of GIPR in γ-aminobutyric acid-ergic neurons. This work suggests that combinatorial pharmaceutical targeting of GLP-1R and GIPR will increase efficacy in treating obesity and diabetes by reducing nausea and vomiting. [ABSTRACT FROM AUTHOR]

Activating transcription factor 3 (ATF3) has been shown to play an important role in HDL metabolism; yet, the role of hepatocytic ATF3 in the development of steatohepatitis remains elusive. Here we show that adenoassociated virus-mediated overexpression of human ATF3 in hepatocytes prevents diet-induced steatohepatitis in C57BL/6 mice and reverses steatohepatitis in db/db mice. Conversely, global or hepatocyte-specific loss of ATF3 aggravates diet-induced steatohepatitis. Mechanistically, hepatocytic ATF3 induces hepatic lipolysis and fatty acid oxidation and inhibits inflammation and apoptosis. We further show that hepatocyte nuclear factor 4α (HNF4α) is required for ATF3 to improve steatohepatitis. Thus, the current study indicates that ATF3 protects against steatohepatitis through, at least in part, hepatic HNF4α. Targeting hepatic ATF3 may be useful for treatment of steatohepatitis. [ABSTRACT FROM AUTHOR]

There is considerable interest in GIPR agonism to enhance the insulinotropic and extrapancreatic effects of GIP, thereby improving glycemic and weight control in type 2 diabetes (T2D) and obesity. Recent genetic epidemiological evidence has implicated higher GIPR-mediated GIP levels in raising coronary artery disease (CAD) risk, a potential safety concern for GIPR agonism. We therefore aimed to quantitatively assess whether the association between higher GIPR-mediated fasting GIP levels and CAD risk is mediated via GIPR or is instead the result of linkage disequilibrium (LD) confounding between variants at the GIPR locus. Using Bayesian multitrait colocalization, we identified a GIPR missense variant, rs1800437 (G allele; E354), as the putatively causal variant shared among fasting GIP levels, glycemic traits, and adiposity-related traits (posterior probability for colocalization [PPcoloc] > 0.97; PP explained by the candidate variant [PPexplained] = 1) that was independent from a cluster of CAD and lipid traits driven by a known missense variant in APOE (rs7412; distance to E354 ∼770 Kb; R2 with E354 = 0.004; PPcoloc > 0.99; PPexplained = 1). Further, conditioning the association between E354 and CAD on the residual LD with rs7412, we observed slight attenuation in association, but it remained significant (odds ratio [OR] per copy of E354 after adjustment 1.03; 95% CI 1.02, 1.04; P = 0.003). Instead, E354's association with CAD was completely attenuated when conditioning on an additional established CAD signal, rs1964272 (R2 with E354 = 0.27), an intronic variant in SNRPD2 (OR for E354 after adjustment for rs1964272: 1.01; 95% CI 0.99, 1.03; P = 0.06). We demonstrate that associations with GIP and anthropometric and glycemic traits are driven by genetic signals distinct from those driving CAD and lipid traits in the GIPR region and that higher E354-mediated fasting GIP levels are not associated with CAD risk. These findings provide evidence that the inclusion of GIPR agonism in dual GIPR/GLP1R agonists could potentiate the protective effect of GLP-1 agonists on diabetes without undue CAD risk, an aspect that has yet to be assessed in clinical trials. [ABSTRACT FROM AUTHOR]

During the past decade, pharmaceutical engineering of unimolecular agents has revealed the therapeutic potential of glucose-dependent insulinotropic polypeptide receptor (GIPR) agonism. From this work, one of the most intriguing findings is that engagement of GIPR enhances the weight loss profile of glucagon-like peptide 1 (GLP-1)-based therapeutics. Consequently, this pharmacological approach, in combination with novel Gipr mouse models, has provided evidence indicating that activation of GIPR in certain areas of the brain that regulate energy balance is required for the synergistic weight loss of dual GIPR and GLP-1 receptor (GLP-1R) agonism. This has led to significant interest in understanding how GIPR activity in the brain functions to reduce caloric intake, induce negative energy balance, and drive weight loss. Herein, we review key findings in this field and provide a novel perspective explaining how GIP may act in the brain to affect energy balance both alone and in concert with GLP-1R agonism. [ABSTRACT FROM AUTHOR]

The dorsal vagal complex (DVC) in the hindbrain, composed of the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus, plays a critical role in modulating satiety. The incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) act directly in the brain to modulate feeding, and receptors for both are expressed in the DVC. Given the impressive clinical responses to pharmacologic manipulation of incretin signaling, understanding the central mechanisms by which incretins alter metabolism and energy balance is of critical importance. Here, we review recent single-cell approaches used to detect molecular signatures of GLP-1 and GIP receptor-expressing cells in the DVC. In addition, we discuss how current advancements in single-cell transcriptomics, epigenetics, spatial transcriptomics, and circuit mapping techniques have the potential to further characterize incretin receptor circuits in the hindbrain. [ABSTRACT FROM AUTHOR]

We previously reported genotype-phenotype correlations in 12 missense variants causing severe insulin resistance, located in the second and third fibronectin type III (FnIII) domains of the insulin receptor (INSR), containing the α-β cleavage and part of insulin-binding sites. This study aimed to identify genotype-phenotype correlations in FnIII domain variants of IGF1R, a structurally related homolog of INSR, which may be associated with growth retardation, using the recently reported crystal structures of IGF1R. A structural bioinformatics analysis of five previously reported disease-associated heterozygous missense variants and a likely benign variant in the FnIII domains of IGF1R predicted that the disease-associated variants would severely impair the hydrophobic core formation and stability of the FnIII domains or affect the α-β cleavage site, while the likely benign variant would not affect the folding of the domains. A functional analysis of these variants in CHO cells showed impaired receptor processing and autophosphorylation in cells expressing the disease-associated variants but not in those expressing the wild-type form or the likely benign variant. These results demonstrated genotype-phenotype correlations in the FnIII domain variants of IGF1R, which are presumably consistent with those of INSR and would help in the early diagnosis of patients with disease-associated IGF1R variants. [ABSTRACT FROM AUTHOR]

Regulatory T lymphocytes expressing the forkhead/winged helix transcription factor Foxp3 (Treg) play a vital role in the protection of the organism from autoimmune disease and other immunopathologies. The antigen specificity of Treg plays an important role in their in vivo activity. We therefore assessed the diversity of the T-cell receptors (TCRs) for antigen expressed by Treg newly developed in the thymus of autoimmune type 1 diabetes-prone NOD mice and compared it to the control mouse strain C57BL/6. Our results demonstrate that use of the TCRα and TCRβ variable (V) and joining (J) segments, length of the complementarity determining region (CDR) 3, and the diversity of the TCRα and TCRβ chains are comparable between NOD and C57BL/6 mice. Genetic defects affecting the diversity of the TCR expressed by newly developed Treg therefore do not appear to be involved in the etiology of type 1 diabetes in the NOD mouse. [ABSTRACT FROM AUTHOR]

Targeting of the glucose-dependent insulinotropic polypeptide receptor (GIPR) is an emerging strategy in antidiabetic drug development. The aim of this study was to develop a positron emission tomography (PET) radioligand for the GIPR to enable the assessment of target distribution and drug target engagement in vivo. The GIPR-selective peptide S02-GIP was radiolabeled with 68Ga. The resulting PET tracer [68Ga]S02-GIP-T4 was evaluated for affinity and specificity to human GIPR (huGIPR). The in vivo GIPR binding of [68Ga]S02-GIP-T4 as well as the occupancy of a drug candidate with GIPR activity were assessed in nonhuman primates (NHPs) by PET. [68Ga]S02-GIP-T4 bound with nanomolar affinity and high selectivity to huGIPR in overexpressing cells. In vivo, pancreatic binding in NHPs could be dose-dependently inhibited by coinjection of unlabeled S02-GIP-T4. Finally, subcutaneous pretreatment with a high dose of a drug candidate with GIPR activity led to a decreased pancreatic binding of [68Ga]S02-GIP-T4, corresponding to a GIPR drug occupancy of almost 90%. [68Ga]S02-GIP-T4 demonstrated a safe dosimetric profile, allowing for repeated studies in humans. In conclusion, [68Ga]S02-GIP-T4 is a novel PET biomarker for safe, noninvasive, and quantitative assessment of GIPR target distribution and drug occupancy. [ABSTRACT FROM AUTHOR]

Regulatory T cell (Treg) insufficiency licenses the destruction of insulin-producing pancreatic β-cells by autoreactive effector T cells (Teffs), causing spontaneous autoimmune diabetes in NOD mice. We investigated the contribution to diabetes of the T-cell receptor (TCR) repertoires of naive regulatory T cells (nTregs), activated/memory Tregs (amTregs), and CD4+ Teffs from prediabetic NOD mice and normal C57BL/6 (B6) mice. NOD mice amTreg and Teff repertoire diversity was unexpectedly higher than that of B6 mice. This was due to the presence of highly expanded clonotypes in B6 amTregs and Teffs that were largely lost in their NOD counterparts. Interleukin-2 (IL-2) administration to NOD mice restored such amTreg clonotype expansions and prevented diabetes development. In contrast, IL-2 administration only led to few or no clonotype expansions in nTregs and Teffs, respectively. Noteworthily, IL-2-expanded amTreg and nTreg clonotypes were markedly enriched in islet-antigen specific TCRs. Altogether, our results highlight the link between a reduced clonotype expansion within the activated Treg repertoire and the development of an autoimmune disease. They also indicate that the repertoire of amTregs is amenable to rejuvenation by IL-2. [ABSTRACT FROM AUTHOR]

The prevalence of type 2 diabetes in youth has increased substantially, yet the genetic underpinnings remain largely unexplored. To identify genetic variants predisposing to youth-onset type 2 diabetes, we formed ProDiGY, a multiethnic collaboration of three studies (TODAY, SEARCH, and T2D-GENES) with 3,006 youth case subjects with type 2 diabetes (mean age 15.1 ± 2.9 years) and 6,061 diabetes-free adult control subjects (mean age 54.2 ± 12.4 years). After stratifying by principal component-clustered ethnicity, we performed association analyses on ∼10 million imputed variants using a generalized linear mixed model incorporating a genetic relationship matrix to account for population structure and adjusting for sex. We identified seven genome-wide significant loci, including the novel locus rs10992863 in PHF2 (P = 3.2 × 10-8; odds ratio [OR] = 1.23). Known loci identified in our analysis include rs7903146 in TCF7L2 (P = 8.0 × 10-20; OR 1.58), rs72982988 near MC4R (P = 4.4 × 10-14; OR 1.53), rs200893788 in CDC123 (P = 1.1 × 10-12; OR 1.32), rs2237892 in KCNQ1 (P = 4.8 × 10-11; OR 1.59), rs937589119 in IGF2BP2 (P = 3.1 × 10-9; OR 1.34), and rs113748381 in SLC16A11 (P = 4.1 × 10-8; OR 1.04). Secondary analysis with 856 diabetes-free youth control subjects uncovered an additional locus in CPEB2 (P = 3.2 × 10-8; OR 2.1) and consistent direction of effect for diabetes risk. In conclusion, we identified both known and novel loci in the first genome-wide association study of youth-onset type 2 diabetes. [ABSTRACT FROM AUTHOR]

Patients with diabetes often experience visual defects before any retinal pathologies are detected. The molecular mechanism for the visual defects in early diabetes has not been elucidated. Our previous study reported that in early diabetic retinopathy (DR), rhodopsin levels were reduced due to impaired 11-cis-retinal regeneration. Interphotoreceptor retinol-binding protein (IRBP) is a visual cycle protein and important for 11-cis-retinal generation. IRBP levels are decreased in the vitreous and retina of DR patients and animal models. To determine the role of IRBP downregulation in the visual defects in early DR, we induced diabetes in transgenic mice overexpressing IRBP in the retina. IRBP overexpression prevented diabetes-induced decline of retinal function. Furthermore, IRBP overexpression also prevented decreases of rhodopsin levels and 11-cis-retinal generation in diabetic mice. Diabetic IRBP transgenic mice also showed ameliorated retinal oxidative stress, inflammation, apoptosis, and retinal degeneration compared with diabetic wild-type mice. These findings suggest that diabetes-induced IRBP downregulation impairs the regeneration of 11-cis-retinal and rhodopsin, leading to retinal dysfunction in early DR. Furthermore, increased 11-cis-retinal-free opsin constitutively activates the phototransduction pathway, leading to increased oxidative stress and retinal neurodegeneration. Therefore, restored IRBP expression in the diabetic retina may confer a protective effect against retinal degeneration in DR. [ABSTRACT FROM AUTHOR]

Renal epidermal growth factor receptor (EGFR) signaling is activated in models of diabetic nephropathy (DN), and inhibition of the EGFR signaling pathway protects against the development of DN. We have now determined that in cultured podocytes, high glucose led to increases in activation of EGFR signaling but decreases in autophagy activity as indicated by decreased beclin-1 and inhibition of LC3B autophagosome formation as well as increased rubicon (an autophagy inhibitor) and SQSTM1 (autophagy substrate). Either genetic (small interfering [si]EGFR) or pharmacologic (AG1478) inhibition of EGFR signaling attenuated the decreased autophagy activity. In addition, rubicon siRNA knockdown prevented high glucose-induced inhibition of autophagy in podocytes. We further examined whether selective EGFR deletion in podocytes affected the progression of DN in type 2 diabetes. Selective podocyte EGFR deletion had no effect on body weight or fasting blood sugars in either db/db mice or nos3-/-; db/db mice, a model of accelerated type 2 DN. However selective podocyte EGFR deletion led to relative podocyte preservation and marked reduction in albuminuria and glomerulosclerosis, renal proinflammatory cytokine/chemokine expression, and decreased profibrotic and fibrotic components in nos3-/-; db/db mice. Podocyte EGFR deletion led to decreased podocyte expression of rubicon, in association with increased podocyte autophagy activity. Therefore, activation of EGFR signaling in podocytes contributes to progression of DN at least in part by increasing rubicon expression, leading to subsequent autophagy inhibition and podocyte injury. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide 1 receptor (GLP-1R) agonists effectively improve glycemia and body weight in patients with type 2 diabetes and obesity but have limited weight-lowering efficacy and minimal insulin sensitizing action. In preclinical models, peripherally restricted cannabinoid receptor type 1 (CB1R) inhibitors, which are devoid of the neuropsychiatric adverse effects observed with brain-penetrant CB1R blockers, ameliorate obesity and its multiple metabolic complications. Using mouse models with genetic loss of CB1R or GLP-1R, we demonstrate that these two metabolic receptors modulate food intake and body weight via reciprocal functional interactions. In diet-induced obese mice, the coadministration of a peripheral CB1R inhibitor with long-acting GLP-1R agonists achieves greater reduction in body weight and fat mass than monotherapies by promoting negative energy balance. This cotreatment also results in larger improvements in systemic and hepatic insulin action, systemic dyslipidemia, and reduction of hepatic steatosis. Thus, peripheral CB1R blockade may allow safely potentiating the antiobesity and antidiabetic effects of currently available GLP-1R agonists. [ABSTRACT FROM AUTHOR]

Toll-like receptor 9 (TLR9) is highly expressed in B cells, and B cells are important in the pathogenesis of type 1 diabetes (T1D) development. However, the intrinsic effect of TLR9 in B cells on β-cell autoimmunity is not known. To fill this knowledge gap, we generated NOD mice with a B-cell-specific deficiency of TLR9 (TLR9fl/fl/CD19-Cre+ NOD). The B-cell-specific deletion of TLR9 resulted in near-complete protection from T1D development. Diabetes protection was accompanied by an increased proportion of interleukin-10 (IL-10)-producing B cells. We also found that TLR9-deficient B cells were hyporesponsive to both innate and adaptive immune stimuli. This suggested that TLR9 in B cells modulates T1D susceptibility in NOD mice by changing the frequency and function of IL-10-producing B cells. Molecular analysis revealed a network of TLR9 with matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, and CD40, all of which are interconnected with IL-10. Our study has highlighted an important connection of an innate immune molecule in B cells to the immunopathogenesis of T1D. Thus, targeting the TLR9 pathway, specifically in B cells, may provide a novel therapeutic strategy for T1D treatment. [ABSTRACT FROM AUTHOR]

The aim for this study was to elucidate how the hypothalamic hunger-inducing hormone acyl-ghrelin (AG), which is also produced in the pancreas, affects β-cell function, with particular attention to the role of ATP-sensitive K+ (KATP) channels and the exact site of action of the hormone. AG hyperpolarized the membrane potential and decreased cytoplasmic calcium concentration [Ca2+]c and glucose-stimulated insulin secretion (GSIS). These effects were abolished in β-cells from SUR1-knockout (KO) mice. AG increased KATP current but only in a configuration with intact metabolism. Unacylated ghrelin counteracted the effects of AG. The influence of AG on membrane potential and GSIS could only be averted in the combined presence of a ghrelin receptor (GHSR1a) antagonist and an inverse agonist. The inhibition of GSIS by AG could be prevented by dibutyryl cyclic-cAMP or 3-isobutyl-1-methylxanthine and the somatostatin (SST) receptor 2-5 antagonist H6056. These data indicate that AG indirectly opens KATP channels probably by interference with the cAMP/cAMP-dependent protein kinase pathway, resulting in a decrease of [Ca2+]c and GSIS. The experiments with SUR1-KO β-cells point to a direct effect of AG on β-cells and not, as earlier suggested, to an exclusive effect by AG-induced SST release from δ-cells. Nevertheless, SST receptors may be involved in the effect of AG, possibly by heteromerization of AG and SST receptors. [ABSTRACT FROM AUTHOR]

Type 2 diabetes has become a pandemic and leads to late diabetic complications of organs, including kidney and eye. Lowering hyperglycemia is the typical therapeutic goal in clinical medicine. However, hyperglycemia may only be a symptom of diabetes but not the sole cause of late diabetic complications; instead, other diabetes-related alterations could be causative. Here, we studied the role of CaM kinase II-δ (CaMKIIδ), which is known to be activated through diabetic metabolism. CaMKIIδ is expressed ubiquitously and might therefore affect several different organ systems. We crossed diabetic leptin receptor-mutant mice to mice lacking CaMKIIδ globally. Remarkably, CaMKIIδ-deficient diabetic mice did not develop hyperglycemia. As potential underlying mechanisms, we provide evidence for improved insulin sensing with increased glucose transport into skeletal muscle and also reduced hepatic glucose production. Despite normoglycemia, CaMKIIδ-deficient diabetic mice developed the full picture of diabetic nephropathy, but diabetic retinopathy was prevented. We also unmasked a retina-specific gene expression signature that might contribute to CaMKII-dependent retinal diabetic complications. These data challenge the clinical concept of normalizing hyperglycemia in diabetes as a causative treatment strategy for late diabetic complications and call for a more detailed analysis of intracellular metabolic signals in different diabetic organs. [ABSTRACT FROM AUTHOR]

Hypoadiponectinemia is a risk factor of gestational diabetes mellitus (GDM). Our previous study reported that adiponectin gene knockout mice (Adipoq-/- ) develop GDM due to insulin insufficiency. The main objective of this study was to elucidate the underlying mechanism through which adiponectin controls islet expansion during pregnancy. A significant reduction in β-cell proliferation rates, β-cell areas, and blood insulin concentrations was detected in Adipoq-/- mice at midpregnancy. Surprisingly, conditionally knocking down adiponectin receptor 1 (AdipoR1) or AdipoR2 genes in β-cells during pregnancy did not reduce β-cell proliferation rates or blood insulin concentrations. In vitro adiponectin treatment also failed to show any effect on β-cell proliferation of isolated pancreatic islets. It was reported that placental lactogen (PL) plays a crucial role in pregnancy-induced maternal β-cell proliferation. A significant decrease in phosphorylation of signal transducer and activator of transcription 5, a downstream molecule of PL signaling, was observed in islets from Adipoq-/- dams. The mRNA levels of mouse PL genes were robustly decreased in the placentas of Adipoq-/- dams. In contrast, adiponectin treatment increased PL expression in human placenta explants and JEG3 trophoblast cells. Most importantly, bovine PL injection restored β-cell proliferation and blood insulin concentrations in Adipoq-/- dams. Together, these results demonstrate that adiponectin plays a vital role in pregnancy-induced β-cell proliferation by promoting PL expression in trophoblast cells. [ABSTRACT FROM AUTHOR]

An increasing number of studies suggest that the renal proximal tubule is a site of injury in diabetic nephropathy (DN), and progressive renal tubulointerstitial fibrosis is an important mediator of progressive kidney dysfunction in DN. In this study, we observed increased expression and activation of YAP (yes-associated protein) in renal proximal tubule epithelial cells (RPTC) in patients with diabetes and in mouse kidneys. Inducible deletion of Yap specifically in RPTC or administration of the YAP inhibitor verteporfin significantly attenuated diabetic tubulointerstitial fibrosis. EGFR-dependent activation of RhoA/Rock and PI3K-Akt signals and their reciprocal interaction were upstream of proximal tubule YAP activation in diabetic kidneys. Production and release of CTGF in culture medium were significantly augmented in human embryonic kidney (HEK)-293 cells transfected with a constitutively active YAP mutant, and the conditioned medium collected from these cells activated and transduced fibroblasts into myofibroblasts. This study demonstrates that proximal tubule YAP-dependent paracrine mechanisms play an important role in diabetic interstitial fibrogenesis; therefore, targeting Hippo signaling may be a therapeutic strategy to prevent the development and progression of diabetic interstitial fibrogenesis. [ABSTRACT FROM AUTHOR]

Diabetic kidney disease (DKD) is a major complication of diabetes and the leading cause of end-stage renal failure. Epigenetics has been associated with metabolic memory in which prior periods of hyperglycemia enhance the future risk of developing DKD despite subsequent glycemic control. To understand the mechanistic role of such epigenetic memory in human DKD and to identify new therapeutic targets, we profiled gene expression, DNA methylation, and chromatin accessibility in kidney proximal tubule epithelial cells (PTECs) derived from subjects with and without type 2 diabetes (T2D). T2D-PTECs displayed persistent gene expression and epigenetic changes with and without transforming growth factor-β1 treatment, even after culturing in vitro under similar conditions as nondiabetic PTECs, signified by deregulation of fibrotic and transport-associated genes (TAGs). Motif analysis of differential DNA methylation and chromatin accessibility regions associated with genes differentially regulated in T2D revealed enrichment for SMAD3, HNF4A, and CTCF transcription factor binding sites. Furthermore, the downregulation of several TAGs in T2D (including CLDN10, CLDN14, CLDN16, SLC16A2, and SLC16A5) was associated with promoter hypermethylation, decreased chromatin accessibility, and reduced enrichment of HNF4A, histone H3-lysine-27-acetylation, and CTCF. Together, these integrative analyses reveal epigenetic memory underlying the deregulation of key target genes in T2D-PTECs that may contribute to sustained renal dysfunction in DKD. [ABSTRACT FROM AUTHOR]

Loss-of-function mutations in both alleles of the human insulin receptor gene (INSR) cause extreme insulin resistance (IR) and usually death in childhood, with few effective therapeutic options. Bivalent antireceptor antibodies can elicit insulin-like signaling by mutant INSR in cultured cells, but whether this translates into meaningful metabolic benefits in vivo, wherein the dynamics of insulin signaling and receptor recycling are more complex, is unknown. To address this, we adopted a strategy to model human insulin receptoropathy in mice, using Cre recombinase delivered by adeno-associated virus to knockout endogenous hepatic Insr acutely in floxed Insr mice (liver insulin receptor knockout [L-IRKO] + GFP), before adenovirus-mediated add back of wild-type (WT) or mutant human INSR Two murine anti-INSR monoclonal antibodies, previously shown to be surrogate agonists for mutant INSR, were then tested by intraperitoneal injections. As expected, L-IRKO + GFP mice showed glucose intolerance and severe hyperinsulinemia. This was fully corrected by add back of WT but not with either D734A or S350L mutant INSR. Antibody injection improved glucose tolerance in D734A INSR-expressing mice and reduced hyperinsulinemia in both S350L and D734A INSR-expressing animals. It did not cause hypoglycemia in WT INSR-expressing mice. Antibody treatment also downregulated both WT and mutant INSR protein, attenuating its beneficial metabolic effects. Anti-INSR antibodies thus improve IR in an acute model of insulin receptoropathy, but these findings imply a narrow therapeutic window determined by competing effects of antibodies to stimulate receptors and induce their downregulation. [ABSTRACT FROM AUTHOR]

Low-dose interleukin-2 (IL-2) represents a new therapeutic approach to regulate immune homeostasis to promote immune tolerance in patients with autoimmune diseases, including type 1 diabetes. We have developed a new IL-2-based biologic, an IL-2/CD25 fusion protein, with greatly improved pharmacokinetics and pharmacodynamics when compared with recombinant IL-2 to enhance this type of immunotherapy. In this study, we show that low-dose mouse IL-2/CD25 (mIL-2/CD25), but not an equivalent amount of IL-2, prevents the onset of diabetes in NOD mice and controls diabetes in hyperglycemic mice. mIL-2/CD25 acts not only to expand regulatory T cells (Tregs) but also to increase their activation and migration into lymphoid tissues and the pancreas. Lower incidence of diabetes is associated with increased serum levels of IL-10, a cytokine readily produced by activated Tregs. These effects likely act in concert to lower islet inflammation while increasing Tregs in the remaining inflamed islets. mIL-2/CD25 treatment is also associated with lower anti-insulin autoantibody levels in part by inhibition of T follicular helper cells. Thus, long-acting mIL-2/CD25 represents an improved IL-2 analog that persistently elevates Tregs to maintain a favorable Treg/effector T cell ratio that limits diabetes by expansion of activated Tregs that readily migrate into lymphoid tissues and the pancreas while inhibiting autoantibodies. [ABSTRACT FROM AUTHOR]

NKG2D is implicated in autoimmune diabetes. However, the role of this receptor in diabetes pathogenesis is unclear owing to conflicting results with studies involving global inhibition of NKG2D signaling. We found that NKG2D and its ligands are present in human pancreata, with expression of NKG2D and its ligands increased in the islets of patients with type 1 diabetes. To directly assess the role of NKG2D in the pancreas, we generated NOD mice that express an NKG2D ligand in β-islet cells. Diabetes was reduced in these mice. The reduction corresponded with a decrease in the effector to central memory CD8+ T-cell ratio. Further, NKG2D signaling during in vitro activation of both mouse and human CD8+ T cells resulted in an increased number of central memory CD8+ T cells and diabetes protection by central memory CD8+ T cells in vivo. Taken together, these studies demonstrate that there is a protective role for central memory CD8+ T cells in autoimmune diabetes and that this protection is enhanced with NKG2D signaling. These findings stress the importance of anatomical location when determining the role NKG2D signaling plays, as well as when developing therapeutic strategies targeting this pathway, in type 1 diabetes development. [ABSTRACT FROM AUTHOR]

α-Klotho is a circulating factor with well-documented antiaging properties. However, the central role of α-klotho in metabolism remains largely unexplored. The current study investigated the potential role of central α-klotho to modulate neuropeptide Y/agouti-related peptide (NPY/AgRP)-expressing neurons, energy balance, and glucose homeostasis. Intracerebroventricular administration of α-klotho suppressed food intake, improved glucose profiles, and reduced body weight in mouse models of type 1 and 2 diabetes. Furthermore, central α-klotho inhibition via an anti-α-klotho antibody impaired glucose tolerance. Ex vivo patch clamp electrophysiology and immunohistochemical analysis revealed that α-klotho suppresses NPY/AgRP neuron activity, at least in part, by enhancing miniature inhibitory postsynaptic currents. Experiments in hypothalamic GT1-7 cells observed that α-klotho induces phosphorylation of AKTser473, ERKthr202/tyr204, and FOXO1ser256 as well as blunts AgRP gene transcription. Mechanistically, fibroblast growth factor receptor 1 (FGFR1) inhibition abolished the downstream signaling of α-klotho, negated its ability to modulate NPY/AgRP neurons, and blunted its therapeutic effects. Phosphatidylinositol 3 kinase (PI3K) inhibition also abolished α-klotho's ability to suppress food intake and improve glucose clearance. These results indicate a prominent role of hypothalamic α-klotho/FGFR1/PI3K signaling in the modulation of NPY/AgRP neuron activity and maintenance of energy homeostasis, thus providing new insight into the pathophysiology of metabolic disease. [ABSTRACT FROM AUTHOR]

Monogenic forms of obesity have been identified in ≤10% of severely obese European patients. However, the overall spectrum of deleterious variants (point mutations and structural variants) responsible for childhood severe obesity remains elusive. In this study, we genetically screened 225 severely obese children from consanguineous Pakistani families through a combination of techniques, including an in-house-developed augmented whole-exome sequencing method (CoDE-seq) that enables simultaneous detection of whole-exome copy number variations (CNVs) and point mutations in coding regions. We identified 110 (49%) probands carrying 55 different pathogenic point mutations and CNVs in 13 genes/loci responsible for nonsyndromic and syndromic monofactorial obesity. CoDE-seq also identified 28 rare or novel CNVs associated with intellectual disability in 22 additional obese subjects (10%). Additionally, we highlight variants in candidate genes for obesity warranting further investigation. Altogether, 59% of cases in the studied cohort are likely to have a discrete genetic cause, with 13% of these as a result of CNVs, demonstrating a remarkably higher prevalence of monofactorial obesity than hitherto reported and a plausible overlapping of obesity and intellectual disabilities in several cases. Finally, inbred populations with a high prevalence of obesity provide unique, genetically enriched material in the quest of new genes/variants influencing energy balance. [ABSTRACT FROM AUTHOR]

Diabetic keratopathy, a sight-threatening corneal disease, comprises several symptomatic conditions including delayed epithelial wound healing, recurrent erosions, and sensory nerve (SN) neuropathy. We investigated the role of neuropeptides in mediating corneal wound healing, including epithelial wound closure and SN regeneration. Denervation by resiniferatoxin severely impaired corneal wound healing and markedly upregulated proinflammatory gene expression. Exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP) partially reversed resiniferatoxin's effects, with VIP specifically inducing interleukin-10 expression. Hence, we focused on VIP and observed that wounding induced VIP and VIP type 1 receptor (VIPR1) expression in normal (NL) corneas, but not corneas from mice with diabetes mellitus (DM). Targeting VIPR1 in NL corneas attenuated corneal wound healing, dampened wound-induced expression of neurotrophic factors, and exacerbated inflammatory responses, while exogenous VIP had the opposite effects in DM corneas. Remarkably, wounding and diabetes also affected the expression of Sonic Hedgehog (Shh) in a VIP-dependent manner. Downregulating Shh expression in NL corneas decreased while exogenous Shh in DM corneas increased the rates of corneal wound healing. Furthermore, inhibition of Shh signaling dampened VIP-promoted corneal wound healing. We conclude that VIP regulates epithelial wound healing, inflammatory response, and nerve regeneration in the corneas in an Shh-dependent manner, suggesting a therapeutic potential for these molecules in treating diabetic keratopathy. [ABSTRACT FROM AUTHOR]

Mitochondrial protein FAM3A suppresses hepatic gluconeogenesis and lipogenesis. This study aimed to screen drug(s) that activates FAM3A expression and evaluate its effect(s) on hyperglycemia and steatosis. Drug-repurposing methodology predicted that antidepressive drug doxepin was among the drugs that potentially activated FAM3A expression. Doxepin was further validated to stimulate the translocation of transcription factor HNF4α from the cytoplasm into the nucleus, where it promoted FAM3A transcription to enhance ATP synthesis, suppress gluconeogenesis, and reduce lipid deposition in hepatocytes. HNF4α antagonism or FAM3A deficiency blunted doxepin-induced suppression on gluconeogenesis and lipid deposition in hepatocytes. Doxepin administration attenuated hyperglycemia, steatosis, and obesity in obese diabetic mice with upregulated FAM3A expression in liver and brown adipose tissues (BAT). Notably, doxepin failed to correct dysregulated glucose and lipid metabolism in FAM3A-deficient mice fed on high-fat diet. Doxepin's effects on ATP production, Akt activation, gluconeogenesis, and lipogenesis repression were also blunted in FAM3A-deficient mouse livers. In conclusion, FAM3A is a therapeutic target for diabetes and steatosis. Antidepressive drug doxepin activates FAM3A signaling pathways in liver and BAT to improve hyperglycemia and steatosis of obese diabetic mice. Doxepin might be preferentially recommended as an antidepressive drug in potential treatment of patients with diabetes complicated with depression. [ABSTRACT FROM AUTHOR]

Hypovascularized diabetic nonhealing wounds are due to reduced number and impaired physiology of endogenous endothelial progenitor cell (EPC) population that limits their recruitment and mobilization at the wound site. For enrichment of the EPC repertoire from nonendothelial precursors, abundantly available mesenchymal stromal cells (MSC) were reprogrammed into induced endothelial cells (iEC). We identified cell signaling molecular targets by meta-analysis of microarray data sets. BMP-2 induction leads to the expression of inhibitory Smad 6/7-dependent negative transcriptional regulation of ID1, rendering the latter's reduced binding to TWIST1 during transdifferentiation of Wharton jelly-derived MSC (WJ-MSC) into iEC. TWIST1, in turn, regulates endothelial gene transcription, positively of proangiogenic KDR and negatively, in part, of antiangiogenic SFRP4 Twist1 reprogramming enhanced the endothelial lineage commitment of WJ-MSC and increased the vasculogenic potential of reprogrammed endothelial cells (rEC). Transplantation of stable TWIST1 rEC into a type 1 and 2 diabetic full-thickness splinted wound healing murine model enhanced the microcirculatory blood flow and accelerated the wound tissue regeneration. An increased or decreased colocalization of GFP with KDR/SFRP4 and CD31 in the regenerated diabetic wound bed with TWIST1 overexpression or silencing (piLenti-TWIST1-shRNA-GFP), respectively, further confirmed improved neovascularization. This study depicted the reprogramming of WJ-MSC into rEC using unique transcription factor TWIST1 for an efficacious cell transplantation therapy to induce neovascularization-mediated diabetic wound tissue regeneration. [ABSTRACT FROM AUTHOR]