Digital lifestyle treatment improves long-term metabolic control in type 2 diabetes with different effects in pathophysiological and genetic subgroups.

To address the unmet need for scalable solutions for lifestyle treatment, we developed a new digital method to promote behavioral change. Here we report that patients with type-2 diabetes in Sweden (n = 331) exposed to the intervention have significantly improved HbA1c during a median follow-up of 1038 days (4 mmol/mol compared with matched controls; P = 0.009). This is paralleled by reduced body weight, ameliorated insulin secretion, increased physical activity, and cognitive eating restraints. Participants with high BMI and insulin resistance have an even larger response, as have non-risk allele carriers for the FTO gene. The findings open a new avenue for scalable lifestyle management with sustained efficacy and highlight a previously unrecognized opportunity for digital precision treatment based on genetics and individual pathophysiology. ClinicalTrials.gov NCT04624321.

The highly expressed calcium-insensitive synaptotagmin-11 and synaptotagmin-13 modulate insulin secretion.

AIM: SYT11 and SYT13, two calcium-insensitive synaptotagmins, are downregulated in islets from type 2 diabetic donors, but their function in insulin secretion is unknown. To address this, we investigated the physiological role of these two synaptotagmins in insulin-secreting cells. METHODS: Correlations between gene expression levels were performed using previously described RNA-seq data on islets from 188 human donors. SiRNA knockdown was performed in EndoC-ßH1 and INS-1 832/13 cells. Insulin secretion was measured with ELISA. Patch-clamp was used for single-cell electrophysiology. Confocal microscopy was used to determine intracellular localization. RESULTS: Human islet expression of the transcription factor PDX1 was positively correlated with SYT11 (p = 2.4e-10 ) and SYT13 (p < 2.2e-16 ). Syt11 and Syt13 both co-localized with insulin, indicating their localization in insulin granules. Downregulation of Syt11 in INS-1 832/13 cells (siSYT11) resulted in increased basal and glucose-induced insulin secretion. Downregulation of Syt13 (siSYT13) decreased insulin secretion induced by glucose and K+ . Interestingly, the cAMP-raising agent forskolin was unable to enhance insulin secretion in siSYT13 cells. There was no difference in insulin content, exocytosis, or voltage-gated Ca2+ currents in the two models. Double knockdown of Syt11 and Syt13 (DKD) resembled the results in siSYT13 cells. CONCLUSION: SYT11 and SYT13 have similar localization and transcriptional regulation, but they regulate insulin secretion differentially. While downregulation of SYT11 might be a compensatory mechanism in type-2 diabetes, downregulation of SYT13 reduces the insulin secretory response and overrules the compensatory regulation of SYT11 in a way that could aggravate the disease.

Changes in Skeletal Muscle PAK1 Levels Regulate Tissue Crosstalk to Impact Whole Body Glucose Homeostasis.

Skeletal muscle accounts for ~80% of insulin-stimulated glucose uptake. The Group I p21-activated kinase 1 (PAK1) is required for the non-canonical insulin-stimulated GLUT4 vesicle translocation in skeletal muscle cells. We found that the abundances of PAK1 protein and its downstream effector in muscle, ARPC1B, are significantly reduced in the skeletal muscle of humans with type 2 diabetes, compared to the non-diabetic controls, making skeletal muscle PAK1 a candidate regulator of glucose homeostasis. Although whole-body PAK1 knockout mice exhibit glucose intolerance and are insulin resistant, the contribution of skeletal muscle PAK1 in particular was unknown. As such, we developed inducible skeletal muscle-specific PAK1 knockout (skmPAK1-iKO) and overexpression (skmPAK1-iOE) mouse models to evaluate the role of PAK1 in skeletal muscle insulin sensitivity and glucose homeostasis. Using intraperitoneal glucose tolerance and insulin tolerance testing, we found that skeletal muscle PAK1 is required for maintaining whole body glucose homeostasis. Moreover, PAK1 enrichment in GLUT4-myc-L6 myoblasts preserves normal insulin-stimulated GLUT4 translocation under insulin resistance conditions. Unexpectedly, skmPAK1-iKO also showed aberrant plasma insulin levels following a glucose challenge. By applying conditioned media from PAK1-enriched myotubes or myoblasts to ß-cells in culture, we established that a muscle-derived circulating factor(s) could enhance ß-cell function. Taken together, these data suggest that PAK1 levels in the skeletal muscle can regulate not only skeletal muscle insulin sensitivity, but can also engage in tissue crosstalk with pancreatic ß-cells, unveiling a new molecular mechanism by which PAK1 regulates whole-body glucose homeostasis.

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle.

AIMS/HYPOTHESIS: Skeletal muscle accounts for >80% of insulin-stimulated glucose uptake; dysfunction of this process underlies insulin resistance and type 2 diabetes. Insulin sensitivity is impaired in mice deficient in the double C2 domain ß (DOC2B) protein, while whole-body overexpression of DOC2B enhances insulin sensitivity. Whether insulin sensitivity in the skeletal muscle is affected directly by DOC2B or is secondary to an effect on other tissues is unknown; the underlying molecular mechanisms also remain unclear. METHODS: Human skeletal muscle samples from non-diabetic or type 2 diabetic donors were evaluated for loss of DOC2B during diabetes development. For in vivo analysis, new doxycycline-inducible skeletal-muscle-specific Doc2b-overexpressing mice fed standard or high-fat diets were evaluated for insulin and glucose tolerance, and insulin-stimulated GLUT4 accumulation at the plasma membrane (PM). For in vitro analyses, a DOC2B-overexpressing L6-GLUT4-myc myoblast/myotube culture system was coupled with an insulin resistance paradigm. Biochemical and molecular biology methods such as site-directed mutagenesis, co-immunoprecipitation and mass spectrometry were used to identify the molecular mechanisms linking insulin stimulation to DOC2B. RESULTS: We identified loss of DOC2B (55% reduction in RNA and 40% reduction in protein) in the skeletal muscle of human donors with type 2 diabetes. Furthermore, inducible enrichment of DOC2B in skeletal muscle of transgenic mice enhanced whole-body glucose tolerance (AUC decreased by 25% for female mice) and peripheral insulin sensitivity (area over the curve increased by 20% and 26% for female and male mice, respectively) in vivo, underpinned by enhanced insulin-stimulated GLUT4 accumulation at the PM. Moreover, DOC2B enrichment in skeletal muscle protected mice from high-fat-diet-induced peripheral insulin resistance, despite the persistence of obesity. In L6-GLUT4-myc myoblasts, DOC2B enrichment was sufficient to preserve normal insulin-stimulated GLUT4 accumulation at the PM in cells exposed to diabetogenic stimuli. We further identified that DOC2B is phosphorylated on insulin stimulation, enhancing its interaction with a microtubule motor protein, kinesin light chain 1 (KLC1). Mutation of Y301 in DOC2B blocked the insulin-stimulated phosphorylation of DOC2B and interaction with KLC1, and it blunted the ability of DOC2B to enhance insulin-stimulated GLUT4 accumulation at the PM. CONCLUSIONS/INTERPRETATION: These results suggest that DOC2B collaborates with KLC1 to regulate insulin-stimulated GLUT4 accumulation at the PM and regulates insulin sensitivity. Our observation provides a basis for pursuing DOC2B as a novel drug target in the muscle to prevent/treat type 2 diabetes.

Novel approaches to restore beta cell function in prediabetes and type 2 diabetes.

The World Health Organization estimates that diabetes prevalence has risen from 108 million in 1980 to 422 million in 2014, with type 2 diabetes accounting for more than 90% of these cases. Furthermore, the prevalence of prediabetes (impaired fasting glucose and/or impaired glucose tolerance) is more than 40% in some countries and is associated with a global rise in obesity. Therefore it is imperative that we develop new approaches to reduce the development of prediabetes and progression to type 2 diabetes. In this review, we explore the gains made over the past decade by focused efforts to improve insulin secretion by the beta cell or insulin sensitivity of target tissues. We also describe multitasking candidates, which could improve both beta cell dysfunction and peripheral insulin sensitivity. Moreover, we highlight provocative findings indicating that additional glucose regulatory tissues, such as the brain, may be key therapeutic targets. Taken together, the promise of these new multi-faceted approaches reinforces the importance of understanding and tackling type 2 diabetes pathogenesis from a multi-tissue perspective.

Doc2b Protects ß-Cells Against Inflammatory Damage and Enhances Function.

Loss of functional ß-cell mass is an early feature of type 1 diabetes. To release insulin, ß-cells require soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, as well as SNARE complex regulatory proteins like double C2 domain-containing protein ß (Doc2b). We hypothesized that Doc2b deficiency or overabundance may confer susceptibility or protection, respectively, to the functional ß-cell mass. Indeed, Doc2b+/- knockout mice show an unusually severe response to multiple-low-dose streptozotocin (MLD-STZ), resulting in more apoptotic ß-cells and a smaller ß-cell mass. In addition, inducible ß-cell-specific Doc2b-overexpressing transgenic (ßDoc2b-dTg) mice show improved glucose tolerance and resist MLD-STZ-induced disruption of glucose tolerance, fasting hyperglycemia, ß-cell apoptosis, and loss of ß-cell mass. Mechanistically, Doc2b enrichment enhances glucose-stimulated insulin secretion (GSIS) and SNARE activation and prevents the appearance of apoptotic markers in response to cytokine stress and thapsigargin. Furthermore, expression of a peptide containing the Doc2b tandem C2A and C2B domains is sufficient to confer the beneficial effects of Doc2b enrichment on GSIS, SNARE activation, and apoptosis. These studies demonstrate that Doc2b enrichment in the ß-cell protects against diabetogenic and proapoptotic stress. Furthermore, they identify a Doc2b peptide that confers the beneficial effects of Doc2b and may be a therapeutic candidate for protecting functional ß-cell mass.

MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles.

MicroRNAs contribute to the maintenance of optimal cellular functions by fine-tuning protein expression levels. In the pancreatic ß-cells, imbalances in the exocytotic machinery components lead to impaired insulin secretion and type 2 diabetes (T2D). We hypothesize that dysregulated miRNA expression exacerbates ß-cell dysfunction, and have earlier shown that islets from the diabetic GK-rat model have increased expression of miRNAs, including miR-335-5p (miR-335). Here, we aim to determine the specific role of miR-335 during development of T2D, and the influence of this miRNA on glucose-stimulated insulin secretion and Ca2+-dependent exocytosis. We found that the expression of miR-335 negatively correlated with secretion index in human islets of individuals with prediabetes. Overexpression of miR-335 in human EndoC-ßH1 and in rat INS-1 832/13 cells (OE335) resulted in decreased glucose-stimulated insulin secretion, and OE335 cells showed concomitant reduction in three exocytotic proteins: SNAP25, Syntaxin-binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11). Single-cell capacitance measurements, complemented with TIRF microscopy of the granule marker NPY-mEGFP demonstrated a significant reduction in exocytosis in OE335 cells. The reduction was not associated with defective docking or decreased Ca2+ current. More likely, it is a direct consequence of impaired priming of already docked granules. Earlier reports have proposed reduced granular priming as the cause of reduced first-phase insulin secretion during prediabetes. Here, we show a specific role of miR-335 in regulating insulin secretion during this transition period. Moreover, we can conclude that miR-335 has the capacity to modulate insulin secretion and Ca2+-dependent exocytosis through effects on granular priming.

The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells.

Defects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin-mediated actin polymerization and GLUT4 vesicle translocation.

Elevated miR-130a/miR130b/miR-152 expression reduces intracellular ATP levels in the pancreatic beta cell.

MicroRNAs have emerged as important players of gene regulation with significant impact in diverse disease processes. In type-2 diabetes, in which impaired insulin secretion is a major factor in disease progression, dysregulated microRNA expression in the insulin-secreting pancreatic beta cell has been widely-implicated. Here, we show that miR-130a-3p, miR-130b-3p, and miR-152-3p levels are elevated in the pancreatic islets of hyperglycaemic donors, corroborating previous findings about their upregulation in the islets of type-2 diabetes model Goto-Kakizaki rats. We demonstrated negative regulatory effects of the three microRNAs on pyruvate dehydrogenase E1 alpha (PDHA1) and on glucokinase (GCK) proteins, which are both involved in ATP production. Consequently, we found both proteins to be downregulated in the Goto-Kakizaki rat islets, while GCK mRNA expression showed reduced trend in the islets of type-2 diabetes donors. Overexpression of any of the three microRNAs in the insulin-secreting INS-1 832/13 cell line resulted in altered dynamics of intracellular ATP/ADP ratio ultimately perturbing fundamental ATP-requiring beta cell processes such as glucose-stimulated insulin secretion, insulin biosynthesis and processing. The data further strengthen the wide-ranging influence of microRNAs in pancreatic beta cell function, and hence their potential as therapeutic targets in type-2 diabetes.

Dual Effect of Rosuvastatin on Glucose Homeostasis Through Improved Insulin Sensitivity and Reduced Insulin Secretion.

Statins are beneficial in the treatment of cardiovascular disease (CVD), but these lipid-lowering drugs are associated with increased incidence of new on-set diabetes. The cellular mechanisms behind the development of diabetes by statins are elusive. Here we have treated mice on normal diet (ND) and high fat diet (HFD) with rosuvastatin. Under ND rosuvastatin lowered blood glucose through improved insulin sensitivity and increased glucose uptake in adipose tissue. In vitro rosuvastatin reduced insulin secretion and insulin content in islets. In the beta cell Ca(2+) signaling was impaired and the density of granules at the plasma membrane was increased by rosuvastatin treatment. HFD mice developed insulin resistance and increased insulin secretion prior to administration of rosuvastatin. Treatment with rosuvastatin decreased the compensatory insulin secretion and increased glucose uptake. In conclusion, our data shows dual effects on glucose homeostasis by rosuvastatin where insulin sensitivity is improved, but beta cell function is impaired.

Rosuvastatin Treatment Affects Both Basal and Glucose-Induced Insulin Secretion in INS-1 832/13 Cells.

Rosuvastatin is a member of the statin family. Like the other statins it is prescribed to lower cholesterol levels and thereby reduce the risk of cardiovascular events. Rosuvastatin lowers the cholesterol levels by inhibiting the key enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) in the cholesterol producing mevalonate pathway. It has been recognized that apart from their beneficial lipid lowering effects, statins also exhibit diabetogenic properties. The molecular mechanisms behind these remain unresolved. To investigate the effects of rosuvastatin on insulin secretion, we treated INS-1 832/13 cells with varying doses (20 nM to 20 µM) of rosuvastatin for 48 h. At concentrations of 2 µM and above basal insulin secretion was significantly increased. Using diazoxide we could determine that rosuvastatin did not increase basal insulin secretion by corrupting the KATP channels. Glucose-induced insulin secretion on the other hand seemed to be affected differently at different rosuvastatin concentrations. Rosuvastatin treatment (20 µM) for 24-48 h inhibited voltage-gated Ca(2+) channels, which lead to reduced depolarization-induced exocytosis of insulin-containing granules. At lower concentrations of rosuvastatin (≤ 2 µM) the stimulus-secretion coupling pathway was intact downstream of the KATP channels as assessed by the patch clamp technique. However, a reduction in glucose-induced insulin secretion could be observed with rosuvastatin concentrations as low as 200 nM. The inhibitory effects of rosuvastatin on glucose-induced insulin secretion could be reversed with mevalonate, but not squalene, indicating that rosuvastatin affects insulin secretion through its effects on the mevalonate pathway, but not through the reduction of cholesterol biosynthesis. Taken together, these data suggest that rosuvastatin has the potential to increase basal insulin secretion and reduce glucose-induced insulin secretion. The latter is possibly an unavoidable side effect of rosuvastatin treatment as it occurs through the same mechanisms as the lipid-lowering effects of the drug.

Regulation of Pancreatic Beta Cell Stimulus-Secretion Coupling by microRNAs.

Increased blood glucose after a meal is countered by the subsequent increased release of the hypoglycemic hormone insulin from the pancreatic beta cells. The cascade of molecular events encompassing the initial sensing and transport of glucose into the beta cell, culminating with the exocytosis of the insulin large dense core granules (LDCVs) is termed "stimulus-secretion coupling." Impairment in any of the relevant processes leads to insufficient insulin release, which contributes to the development of type 2 diabetes (T2D). The fate of the beta cell, when exposed to environmental triggers of the disease, is determined by the possibility to adapt to the new situation by regulation of gene expression. As established factors of post-transcriptional regulation, microRNAs (miRNAs) are well-recognized mediators of beta cell plasticity and adaptation. Here, we put focus on the importance of comprehending the transcriptional regulation of miRNAs, and how miRNAs are implicated in stimulus-secretion coupling, specifically those influencing the late stages of insulin secretion. We suggest that efficient beta cell adaptation requires an optimal balance between transcriptional regulation of miRNAs themselves, and miRNA-dependent gene regulation. The increased knowledge of the beta cell transcriptional network inclusive of non-coding RNAs such as miRNAs is essential in identifying novel targets for the treatment of T2D.

Nova1 is a master regulator of alternative splicing in pancreatic beta cells.

Alternative splicing (AS) is a fundamental mechanism for the regulation of gene expression. It affects more than 90% of human genes but its role in the regulation of pancreatic beta cells, the producers of insulin, remains unknown. Our recently published data indicated that the 'neuron-specific' Nova1 splicing factor is expressed in pancreatic beta cells. We have presently coupled specific knockdown (KD) of Nova1 with RNA-sequencing to determine all splice variants and downstream pathways regulated by this protein in beta cells. Nova1 KD altered the splicing of nearly 5000 transcripts. Pathway analysis indicated that these genes are involved in exocytosis, apoptosis, insulin receptor signaling, splicing and transcription. In line with these findings, Nova1 silencing inhibited insulin secretion and induced apoptosis basally and after cytokine treatment in rodent and human beta cells. These observations identify a novel layer of regulation of beta cell function, namely AS controlled by key splicing regulators such as Nova1.