Evaluation of insulin secretory capacity is essential to understand the pathophysiologic condition of individuals with diabetes and assess the efficacy of drugs used in the treatment of this disease. The 1-mg i.v. glucagon stimulation test (GST) is widely used to evaluate residual β-cell function; we previously reported that GST assessment of insulin secretory capacity is useful in assessing the efficacy of glucagon-like peptide 1 (GLP-1) receptor agonists (GLP-1RAs). However, recent reports have indicated that pharmacologic concentrations of glucagon stimulate insulin secretion through GLP-1 receptors, confounding the issue. The current studies were undertaken to reassess the reliability of the GST for evaluation of insulin secretory capacity under GLP-1RAs and dipeptidyl peptidase 4 inhibitors (DPP-4is). Our first study included individuals receiving GLP-1RA treatment, evaluated by the GST before and after treatment. Although the fasting C-peptide response (CPR) levels were elevated after treatment, the induction of insulin secretion by glucagon was significantly reduced. Our second study compared glucagon-induced insulin secretion between DPP-4i users and nonusers, assessed by the GST after propensity score matching. Although the fasting CPR levels were similar in the two investigations, glucagon-induced insulin secretion was significantly lower with DPP-4i use. These results suggest that the GST might underestimate insulin secretory capacity under incretin-based therapy.

Pancreatic β-cell dysfunction caused by obesity can be associated with alterations in the levels of miRNAs. However, the role of miRNAs in such processes remains elusive. Here, we show that pancreatic islet miR-27a-5p, which is markedly increased in obese mice and impairs insulin secretion, is mainly delivered by visceral adipocyte-derived extracellular vesicles (EVs). Depleting miR-27a-5p significantly improved insulin secretion and glucose intolerance in db/db mice. Supporting the function of EV miR-27a-5p as a key pathogenic factor, intravenous injection of miR-27a-5p-containing EVs showed their distribution in mouse pancreatic islets. Tracing the injected adeno-associated virus (AAV)-miR-27a-5p (AAV-miR-27a) or AAV-FABP4-miR-27a-5p (AAV-FABP4-miR-27a) in visceral fat resulted in upregulating miR-27a-5p in EVs and serum and elicited mouse pancreatic β-cell dysfunction. Mechanistically, miR-27a-5p directly targeted L-type Ca2+ channel subtype CaV1.2 (Cacna1c) and reduced insulin secretion in β-cells. Overexpressing mouse CaV1.2 largely abolished the insulin secretion injury induced by miR-27a-5p. These findings reveal a causative role of EV miR-27a-5p in visceral adipocyte-mediated pancreatic β-cell dysfunction in obesity-associated type 2 diabetes mellitus.

Diabetic retinopathy (DR) is one of the most common complications of diabetes worldwide and is associated with visual loss and blindness. However, effective treatments for both early- and late-stage DR remain lacking. A streptozotocin-induced diabetic mouse model and high glucose (HG)-treated Müller cell model were established. M1/M2 microglia polarization was assessed by immunofluorescence staining and flow cytometry. Expression of long noncoding RNA (lncRNA) OGRU, cytokines, and other key molecules was detected by quantitative RT-PCR or Western blot. ELISA was used to monitor cytokine secretion. Müller cell-derived exosomes were isolated and characterized by nanopartical tracking analysis, Western blot, and transmission electron microscopy, and exosome uptake assay was used to monitor the intercellular transport of exosomes. Associations among lncRNA-miRNA-mRNA networks were validated by RNA pulldown and RNA immunoprecipitation and dual luciferase assays. Increased M1 polarization but decreased M2 polarization of retinal microglia was observed in DR mice. HG-treated Müller cell-derived exosomes transported OGRU into microglia and promoted microglia polarization toward the M1 phenotype. Mechanistically, OGRU served as a competing endogenous RNA for miR-320-3p, miR-221-3p, and miR-574-5p to regulate aldose reductase (AR), PFKFB3, and glucose transporter 1 (GLUT1) expression in microglia, respectively. Loss of miR-320-3p/miR-221-3p/miR-574-5p or reinforced AR/PFKFB3/GLUT1 abrogated OGRU silencing-mediated microglia polarization in vitro. In vivo studies further showed that OGRU/miR-320-3p/AR, OGRU/miR-221-3p/PFKFB3, and OGRU/miR-574-5p/GLUT1 axes regulated microglia polarization in DR mice. Collectively, Müller cell-derived exosomal OGRU regulated microglia polarization in DR by modulating OGRU/miR-320-3p/AR, OGRU/miR-221-3p/PFKFB3, and OGRU/miR-574-5p/GLUT1 axes.

Leptin is a homeostatic regulatory element that signals the presence of adipocyte energy stores, reduces food intake, and increases energy expenditure. Similarly, serotonin (5-HT), a signaling molecule found in both the central and peripheral nervous systems, also controls food intake. Using neuronal tract tracing, pharmacologic and optogenetic approaches, and in vivo microdialysis, combined with behavioral end points, we tested the hypothesis that leptin controls food intake not only by activating hypothalamic leptin receptors (LepRs) but also through activation of LepRs expressed by serotonergic raphe neurons that send projections to the arcuate (ARC). We showed that microinjection of leptin directly into the dorsal raphe nucleus (DRN) reduced food intake in rats. This effect was mediated by LepR-expressing neurons in the DRN, because selective optogenetic activation of these neurons at either their DRN cell bodies or their ARC terminals reduced food intake. Anatomically, we identified a unique population of serotonergic raphe neurons expressing LepRs that send projections to the ARC. Finally, by using in vivo microdialysis, we showed that leptin administration to the DRN increased 5-HT efflux into the ARC, and specific antagonism of the 5-HT2C receptors in the ARC diminished the leptin anorectic effect. Overall, this study identified a novel circuit for leptin-mediated control of food intake through a DRN-ARC pathway, identifying a new level of interaction between leptin and serotonin to control food intake. Characterization of this new pathway creates opportunities for understanding how the brain controls eating behavior and opens alternative routes for the treatment of eating disorders.

The approval of teplizumab to delay the onset of type 1 diabetes is an important inflection point in the decades-long pursuit to treat the cause of the disease rather than its symptoms. The National Institute of Diabetes and Digestive and Kidney Diseases convened a workshop of the Diabetes Mellitus Interagency Coordinating Committee titled "Evolving Concepts in Pathophysiology, Screening, and Prevention of Type 1 Diabetes" to review this accomplishment and identify future goals. Speakers representing Type 1 Diabetes TrialNet (TrialNet) and the Immune Tolerance Network emphasized that the ability to robustly identify individuals destined to develop type 1 diabetes was essential for clinical trials. The presenter from the U.S. Food and Drug Administration described how regulatory approval relied on data from the single clinical trial of TrialNet with testing of teplizumab for delay of clinical diagnosis, along with confirmatory evidence from studies in patients after diagnosis. The workshop reviewed the etiology of type 1 diabetes as a disease involving multiple immune pathways, highlighting the current understanding of prognostic markers and proposing potential strategies to improve the therapeutic response of disease-modifying therapies based on the mechanism of action. While celebrating these achievements funded by the congressionally appropriated Special Diabetes Program, panelists from professional organizations, nonprofit advocacy/funding groups, and industry also identified significant hurdles in translating this research into clinical care.

The translocon-associated protein-δ (TRAPδ) plays a role in insulin biosynthesis within pancreatic β-cells. However, its pathophysiological significance in maintaining islet β-cell function and glucose homeostasis remains unclear. In this study, we generated a mouse model featuring pancreatic β-cell-specific deletion of TRAPδ (TRAPδ βKO). Our findings revealed that TRAPδ βKO resulted in decreased circulating insulin levels in mice fed either a normal chow diet or a high-fat diet. Multiple independent experiments established that although TRAPδ deletion reduced insulin content in the islets, it had no discernible effect on insulin gene expression, the insulin to proinsulin ratio, or the expression and glycosylation of the prohormone enzymes involved in proinsulin processing. These data suggest that TRAPδ does not play a pivotal role in the transcription of the insulin gene or proinsulin processing. However, untranslocated preproinsulin levels were significantly increased when islets were treated with a proteasomal inhibitor, suggesting that TRAPδ deficiency may hinder preproinsulin translocation, resulting in a rapid degradation of untranslocated preproinsulin that accounts for the decreased insulin production. Remarkably, despite the moderate decrease in circulating insulin levels in TRAPδ βKO mice, their glucose levels remained unaffected, indicating the presence of compensatory mechanisms that help maintain glucose homeostasis. Insulin tolerance tests further revealed improved insulin sensitivity, accompanied by upregulation of phosphorylated AKT in the peripheral tissues of TRAPδ βKO mice. Collectively, these data highlight the important role of TRAPδ in insulin biosynthesis and β-cell function. The moderate reduction in circulating insulin appears to promote insulin sensitivity in insulin target tissues.

Diabetic peripheral neuropathy (DPN) affects ∼50% of the 500 million people with type 2 diabetes worldwide and is considered disabling and irreversible. The current study was undertaken to assess the effect of metformin on peripheral neuropathy outcomes in type 2 diabetes. Participants with type 2 diabetes (n = 69) receiving metformin were recruited and underwent clinical assessment, peripheral nerve ultrasonography, nerve conduction studies, and axonal excitability studies. Also concurrently screened were 318 participants who were not on metformin, and 69 were selected as disease control subjects and matched to the metformin participants for age, sex, diabetes duration, BMI, HbA1c, and use of other diabetes therapies. Medical record data over the previous 20 years were analyzed for previous metformin use. Mean tibial nerve cross-sectional area was lower in the metformin group (metformin 14.1 ± 0.7 mm2, nonmetformin 16.2 ± 0.9 mm2, P = 0.038), accompanied by reduction in neuropathy symptom severity (P = 0.021). Axonal excitability studies demonstrated superior axonal function in the metformin group, and mathematical modeling demonstrated that these improvements were mediated by changes in nodal Na+and K+conductances. Metformin treatment is associated with superior nerve structure and clinical and neurophysiological measures. Treatment with metformin may be neuroprotective in DPN.

Peripheral neuropathy (PN) is a prevalent and debilitating complication of obesity, prediabetes, and type 2 diabetes, which remains poorly understood and lacks disease-modifying therapies. Fortunately, diet and/or exercise have emerged as effective treatment strategies for PN. Here, we examined the impact of caloric restriction (CR) and high-intensity interval training (HIIT) interventions, alone or combined (HIIT-CR), on metabolic and PN outcomes in high-fat diet (HFD) mice. HFD feeding alone resulted in obesity, impaired glucose tolerance, and PN. Peripheral nerves isolated from these mice also developed insulin resistance (IR). CR and HIIT-CR, but not HIIT alone, improved HFD-induced metabolic dysfunction. However, all interventions improved PN to similar extents. When examining the underlying neuroprotective mechanisms in whole nerves, we found that CR and HIIT-CR activate the fuel-sensing enzyme AMPK. We then performed complimentary in vitro work in Schwann cells, the glia of peripheral nerves. Treating primary Schwann cells with the saturated fatty acid palmitate to mimic prediabetic conditions caused IR, which was reversed by the AMPK activator, AICAR. Together, these results enhance our understanding of PN pathogenesis, the differential mechanisms by which diet and exercise may improve PN, and Schwann cell-specific contributions to nerve insulin signaling and PN progression.

Excessive formation of macrophage extracellular trap (MET) has been implicated in several autoimmune disease pathogeneses; however, its impact on type 1 diabetes (T1D) and related mechanisms remains enigmatic. We demonstrated the pivotal role of peptidyl arginine deiminase 4 (PAD4) in driving profuse MET formation and macrophage M1 polarization in intestinal inflammation in NOD mice. Genetic knockout of PAD4 or adoptive transfer of METs altered the proportion of proinflammatory T cells in the intestine, subsequently influencing their migration to the pancreas. Combining RNA sequencing and CUT&Tag analysis, we found activated PAD4 transcriptionally regulated CXCL10 expression. This study comprehensively investigated how excessive PAD4-mediated MET formation in the colon increases the aggravation of intestinal inflammation and proinflammatory T-cell migration and finally is involved in T1D progression, suggesting that inhibition of MET formation may be a potential therapeutic target in T1D.

Obesity-induced lipid overload in cardiomyocytes contributes to profound oxidative stress and cardiomyopathy, culminating in heart failure. In this study, we investigate a novel mechanism whereby lipids accumulate in cardiomyocytes, and seek the relevant treatment strategies. P21-activated kinase 3 (PAK3) was elevated in obese human myocardium, and the murine hearts and cardiomyocytes upon diet- or fatty acid-induced stress, respectively. Mice with cardiac-specific overexpression of PAK3 were more susceptible to the development of cardiac dysfunction upon diet stress, at least partially, because of increased deposition of toxic lipids within the myocardium. Mechanistically, PAK3 promoted the nuclear expression of sterol regulatory element binding protein 1c (SREBP1c) through activation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase β-1 (S6K1) pathway in cardiomyocytes, resulting in abnormal lipid genes profile, accumulation of excessive lipids, and oxidative stress. More importantly, PAK3 knockdown attenuated fatty acid-induced lipotoxicity and cell death in rat and human cardiomyocytes. More importantly, the S6K1 or SREBP1c inhibitor alleviated PAK3-triggered intracellular lipid overload and cardiac dysfunction under obese stress. Collectively, we have demonstrated that PAK3 impairs myocardial lipid homeostasis, while inhibition of cardiac lipotoxicity mitigates cardiac dysfunction. Our study provides a promising therapeutic strategy for ameliorating obesity cardiomyopathy.

We postulated that type 2 diabetes (T2D) predisposes patients to exocrine pancreatic diseases through (epi)genetic mechanisms. We explored the methylome (using MethylationEPIC arrays) of the exocrine pancreas in 141 donors, assessing the impact of T2D. An epigenome-wide association study of T2D identified hypermethylation in an enhancer of the pancreatic lipase-related protein 1 (PNLIPRP1) gene, associated with decreased PNLIPRP1 expression. PNLIPRP1 null variants (found in 191,000 participants in the UK Biobank) were associated with elevated glycemia and LDL cholesterol. Mendelian randomization using 2.5M SNP Omni arrays in 111 donors revealed that T2D was causal of PNLIPRP1 hypermethylation, which in turn was causal of LDL cholesterol. Additional AR42J rat exocrine cell analyses demonstrated that Pnliprp1 knockdown induced acinar-to-ductal metaplasia, a known prepancreatic cancer state, and increased cholesterol levels, reversible with statin. This (epi)genetic study suggests a role for PNLIPRP1 in human metabolism and exocrine pancreatic function, with potential implications for pancreatic diseases.

The aim of this study was to investigate the associations among sex hormone-binding globulin (SHBG), visceral adipose tissue (VAT), liver fat content, and risk of type 2 diabetes (T2D). In the Netherlands Epidemiology of Obesity study, 5,690 women (53%) and men (47%) without preexisting diabetes were included and followed for incident T2D. SHBG concentrations were measured in all participants, VAT was measured using MRI, and liver fat content was measured using proton magnetic resonance spectroscopy in a random subset of 1,822 participants. We examined associations between SHBG and liver fat using linear regression and bidirectional Mendelian randomization analyses and between SHBG and T2D using Cox regression adjusted for confounding and additionally for VAT and liver fat to examine mediation. Mean age was 56 (SD 6) years, mean BMI was 30 (SD 4) kg/m2, median SHBG was 47 (interquartile range [IQR] 34-65) nmol/L in women and 34 (26-43) nmol/L in men, and median liver fat was 3.4% (IQR 1.6-8.2%) in women and 6.0% (2.9-13.5%) in men. Compared with the highest SHBG quartile, liver fat was 2.9-fold (95% CI 2.4, 3.4) increased in women and 1.6-fold (95% CI 1.3, 1.8) increased in men, and the hazard ratio of T2D was 4.9 (95% CI 2.4, 9.9) in women and 1.8 (1.1, 2.9) in men. Genetically predicted SHBG was associated with liver fat content (women: SD -0.45 [95% CI -0.55, -0.35]; men: natural logarithm, -0.25 [95% CI -0.34, -0.16]). VAT and liver fat together mediated 43% (women) and 60% (men) of the SHBG-T2D association. To conclude, in a middle-aged population with overweight, the association between low SHBG and increased risk of T2D was, for a large part, mediated by increased VAT and liver fat.

Type 1 diabetes (T1D) results from β-cell destruction due to autoimmunity. It has been proposed that β-cell loss is relatively quiescent in the early years after seroconversion to islet antibody positivity (stage 1), with accelerated β-cell loss only developing around 6-18 months prior to clinical diagnosis. This construct implies that immunointervention in this early stage will be of little benefit, since there is little disease activity to modulate. Here, we argue that the apparent lack of progression in early-stage disease may be an artifact of the modality of assessment used. When substantial β-cell function remains, the standard assessment, the oral glucose tolerance test, represents a submaximal stimulus and underestimates the residual function. In contrast, around the time of diagnosis, glucotoxicity exerts a deleterious effect on insulin secretion, giving the impression of disease acceleration. Once glucotoxicity is relieved by insulin therapy, β-cell function partially recovers (the honeymoon effect). However, evidence from recent trials suggests that glucose control has little effect on the underlying disease process. We therefore hypothesize that the autoimmune destruction of β-cells actually progresses at a more or less constant rate through all phases of T1D and that early-stage immunointervention will be both beneficial and desirable.

The generation of stem cell-derived β-like cells (sBCs) holds promise as not only an abundant insulin-producing cell source for replacement therapy of type 1 diabetes (T1D) but also as an invaluable model system for investigating human β-cell development, immunogenicity, and function. Several groups have developed methodology to direct differentiate human pluripotent stem cells into pancreatic cell populations that include glucose-responsive sBCs. Nevertheless, the process of generating sBCs poses substantial experimental challenges. It involves lengthy differentiation periods, there is substantial variability in efficiency, and there are inconsistencies in obtaining functional sBCs. Here, we describe a simple and effective cryopreservation approach for sBC cultures that yields homogeneous sBC clusters that are enriched for insulin-expressing cells while simultaneously depleting proliferative progenitors. Thawed sBCs have enhanced glucose-stimulated insulin release compared with controls in vitro and can effectively engraft and function in vivo. Collectively, this approach alleviates current challenges with inefficient and variable sBC generation while improving their functional state. We anticipate that these findings can inform ongoing clinical application of sBCs for the treatment of patients with T1D and serve as an important resource for the wider diabetes field that will allow for accelerated research discoveries.

Persistent enterovirus B infection has been proposed as an important contributor to the etiology of type 1 diabetes. We leveraged extensive bulk RNA-sequencing (RNA-seq) data from α-, β-, and exocrine cells, as well as islet single-cell RNA-seq data from the Human Pancreas Analysis Program (HPAP), to evaluate the presence of enterovirus B sequences in the pancreas of patients with type 1 diabetes and prediabetes (no diabetes but positive for autoantibodies). We examined all available HPAP data for either assay type, including donors without diabetes and with type 1 and type 2 diabetes. To assess the presence of viral reads, we analyzed all reads not mapping to the human genome with the taxonomic classification system Kraken2 and its full viral database augmented to encompass representatives for all 28 enterovirus B serotypes for which a complete genome is available. As a secondary approach, we input the same sequence reads into the STAR aligner using these 28 enterovirus B genomes as the reference. No enterovirus B sequences were detected by either approach in any of the 243 bulk RNA libraries or in any of the 79 single-cell RNA libraries. While we cannot rule out the possibility of a very-low-grade persistent enterovirus B infection in the donors analyzed, our data do not support the notion of chronic viral infection by these viruses as a major driver of type 1 diabetes.

Maturation of postnatal β-cells is regulated in a cell-autonomous manner, and metabolically stressed β-cells regress to an immature state, ensuring defective β-cell function and the onset of type 2 diabetes. The molecular mechanisms connecting the nutritional transition to β-cell maturation remain largely unknown. Here, we report a mature form of miRNA (miR-203)/ZBTB20/MAFA regulatory axis that mediates the β-cell maturation process. We show that the level of the mature form of miRNA (miR-203) in β-cells changes during the nutritional transition and that miR-203 inhibits β-cell maturation at the neonatal stage and under high-fat diet conditions. Using single-cell RNA sequencing, we demonstrated that miR-203 elevation promoted the transition of immature β-cells into CgBHi endocrine cells while suppressing gene expressions associated with β-cell maturation in a ZBTB20/MAFA-dependent manner. ZBTB20 is an authentic target of miR-203 and transcriptionally upregulates MAFA expression. Manipulating the miR-203/ZBTB20/MAFA axis may therefore offer a novel strategy for boosting functional β-cell numbers to alleviate diabetes.

A hallmark of type 2 diabetes (T2D) is endocrine islet β-cell failure, which can occur via cell dysfunction, loss of identity, and/or death. How each is induced remains largely unknown. We used mouse β-cells deficient for myelin transcription factors (Myt TFs; including Myt1, -2, and -3) to address this question. We previously reported that inactivating all three Myt genes in pancreatic progenitor cells (MytPancΔ) caused β-cell failure and late-onset diabetes in mice. Their lower expression in human β-cells is correlated with β-cell dysfunction, and single nucleotide polymorphisms in MYT2 and MYT3 are associated with a higher risk of T2D. We now show that these Myt TF-deficient postnatal β-cells also dedifferentiate by reactivating several progenitor markers. Intriguingly, mosaic Myt TF inactivation in only a portion of islet β-cells did not result in overt diabetes, but this created a condition where Myt TF-deficient β-cells remained alive while activating several markers of Ppy-expressing islet cells. By transplanting MytPancΔ islets into the anterior eye chambers of immune-compromised mice, we directly show that glycemic and obesity-related conditions influence cell fate, with euglycemia inducing several Ppy+ cell markers and hyperglycemia and insulin resistance inducing additional cell death. These findings suggest that the observed β-cell defects in T2D depend not only on their inherent genetic/epigenetic defects but also on the metabolic load.

It is not completely clear which organs are responsible for glucagon elimination in humans, and disturbances in the elimination of glucagon could contribute to the hyperglucagonemia observed in chronic liver disease and chronic kidney disease (CKD). Here, we evaluated kinetics and metabolic effects of exogenous glucagon in individuals with stage 4 CKD (n = 16), individuals with Child-Pugh A-C cirrhosis (n = 16), and matched control individuals (n = 16), before, during, and after a 60-min glucagon infusion (4 ng/kg/min). Individuals with CKD exhibited a significantly lower mean metabolic clearance rate of glucagon (14.0 [95% CI 12.2;15.7] mL/kg/min) compared with both individuals with cirrhosis (19.7 [18.1;21.3] mL/kg/min, P < 0.001) and control individuals (20.4 [18.1;22.7] mL/kg/min, P < 0.001). Glucagon half-life was significantly prolonged in the CKD group (7.5 [6.9;8.2] min) compared with individuals with cirrhosis (5.7 [5.2;6.3] min, P = 0.002) and control individuals (5.7 [5.2;6.3] min, P < 0.001). No difference in the effects of exogenous glucagon on plasma glucose, amino acids, or triglycerides was observed between groups. In conclusion, CKD, but not liver cirrhosis, leads to a significant reduction in glucagon clearance, supporting the kidneys as a primary site for human glucagon elimination.

A ketogenic diet (KD) can induce weight loss and improve glycemic regulation, potentially reducing the risk of type 2 diabetes development. To elucidate the underlying mechanisms behind these beneficial effects of a KD, we investigated the impact of a KD on organ-specific insulin sensitivity (IS) in skeletal muscle, liver, and adipose tissue. We hypothesized that a KD would increase IS in skeletal muscle. The study included 11 individuals with obesity who underwent a randomized, crossover trial with two 3-week interventions: 1) a KD and 2) a standard diet. Skeletal muscle IS was quantified as the increase in glucose disposal during a hyperinsulinemic-euglycemic clamp (HEC). Hepatic IS and adipose tissue IS were quantified as the relative suppression of endogenous glucose production (EGP) and the relative suppression of palmitate flux during the HEC. The KD led to a 2.2-kg weight loss and increased insulin-stimulated glucose disposal, whereas the relative suppression of EGP during the HEC was similar. In addition, the KD decreased insulin-mediated suppression of lipolysis. In conclusion, a KD increased skeletal muscle IS in individuals with obesity.

Growth arrest-specific 6 (GAS6) is a secreted protein that acts as a ligand for TAM receptors (TYRO3, AXL, and MERTK). In humans, GAS6 circulating levels and genetic variations in GAS6 are associated with hyperglycemia and increased risk of type 2 diabetes. However, the mechanisms by which GAS6 influences glucose metabolism are not understood. Here, we show that Gas6 deficiency in mice increases insulin sensitivity and protects from diet-induced insulin resistance. Conversely, increasing GAS6 circulating levels is sufficient to reduce insulin sensitivity in vivo. GAS6 inhibits the activation of the insulin receptor (IR) and reduces insulin response in muscle cells in vitro and in vivo. Mechanistically, AXL and IR form a complex, while GAS6 reprograms signaling pathways downstream of IR. This results in increased IR endocytosis following insulin treatment. This study contributes to a better understanding of the cellular and molecular mechanisms by which GAS6 and AXL influence insulin sensitivity.