Search

Hundreds of millions of people are affected by hyperinsulinaemia, insulin resistance, obesity and the dysglycaemia that mark a common progression from metabolic health to type 2 diabetes. Although the relative contribution of these features and the order in which they appear may differ between individuals, the common clustering and seemingly progressive nature of type 2 diabetes aetiology has guided research and clinical practice in this area for decades. At the same time, lively debate around the causal relationships between these features has continued, as new data from human trials and highly controlled animal studies are presented. This 'For debate' article was prompted by the review in Diabetologia by Esser, Utzschneider and Kahn ( https://doi.org/10.1007/s00125-020-05245-x ), with the purpose of reviewing established and emerging data that provide insight into the relative contributions of hyperinsulinaemia and impaired glucose-stimulated insulin secretion in progressive stages between health, obesity and diabetes. It is concluded that these beta cell defects are not mutually exclusive and that they are both important, but at different stages.

Aims/hypothesis Hyperinsulinaemia is associated with obesity but its causal role in the onset of obesity remains controversial. In this study, we tested the hypothesis that transient attenuation of diet-induced insulin hypersecretion in young mice can provide sustained protection against obesity throughout adult life. Methods Using ‘genetically humanised’ mice lacking both alleles of rodent-specific Ins1, we compared mice heterozygous for the ancestral insulin gene Ins2 with Ins2+/+ controls. Female Ins1−/−:Ins2+/− and Ins1−/−:Ins2+/+ littermates were fed chow or high-fat diet (HFD). Insulin secretion, metabolic health variables and body mass/composition were tracked for over 1 year. We examined islet function and adipose transcript levels of adipogenic, lipogenic and lipolytic genes at two time points. Results In control Ins1−/−:Ins2+/+ mice, HFD resulted in elevated fasting and glucose-stimulated insulin secretion between 8 weeks and 27 weeks of age. Hyperinsulinaemia was reduced by nearly 50% in Ins1−/−:Ins2+/− mice during this period, without lasting adverse effects on glucose homeostasis. This corresponded with attenuated weight gain and adiposity. White adipose tissue from Ins1−/−:Ins2+/− mice had fewer large lipid droplets, although transcriptional changes were not detected. Importantly, Ins1−/−:Ins2+/− mice remained lighter than Ins1−/−:Ins2+/+ littermates despite reaching an equivalent degree of hyperinsulinaemia on HFD by 52 weeks. Conclusions/interpretation These data demonstrate that attenuation of hyperinsulinaemia in young, growing female mice provides a long-lasting protection against obesity. This protection persists despite a late-onset emergence of hyperinsulinaemia in HFD-fed Ins1−/−:Ins2+/− mice. Given the evolutionary conserved roles of insulin, it is possible that suppressing hyperinsulinaemia early in life may have far-reaching consequences on obesity in full-grown adult humans. Electronic supplementary material The online version of this article (doi:10.1007/s00125-015-3676-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

The production of fully functional insulin-secreting cells to treat diabetes is a major goal of regenerative medicine. In this article, I review progress towards this goal over the last 15 years from the perspective of a beta cell biologist. I describe the current state-of-the-art, and speculate on the general approaches that will be required to identify and achieve our ultimate goal of producing functional beta cells. The need for deeper phenotyping of heterogeneous cultures of stem cell derived islet-like cells in parallel with a better understanding of the heterogeneity of the target cell type(s) is emphasised. This deep phenotyping should include high-throughput single-cell analysis, as well as comprehensive ’omics technologies to provide unbiased characterisation of cell products and human beta cells. There are justified calls for more detailed and well-powered studies of primary human pancreatic beta cell physiology, and I propose online databases of standardised human beta cell responses to physiological stimuli, including both functional and metabolomic/proteomic/transcriptomic profiles. With a concerted, community-wide effort, including both basic and applied scientists, beta cell replacement will become a clinical reality for patients with diabetes.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is associated with neurodegenerative diseases and has been suggested to have roles in pancreatic beta cells. Our proteomic analysis revealed that UCHL1 was the most increased protein in MIN6 cells exposed to palmitate. The present study used a genetic loss-of-function model to test the hypothesis that UCHL1 is required for normal beta cell function and fate under lipotoxic conditions.Human islets, mouse islets and MIN6 cells were used to analyse UCHL1 protein levels and regulation of UCHL1 by palmitate. The levels of free mono-ubiquitin and poly-ubiquitinated proteins were assessed. Gracile axonal dystrophy (GAD) mutant mice lacking UCHL1 were fed a normal or lipotoxic high-fat diet. Glucose tolerance, insulin tolerance and insulin secretion were assessed in vivo. Beta cell death and proliferation were assessed by TUNEL and proliferating cell nuclear antigen (PCNA) staining. Insulin secretion, calcium signalling, endoplasmic reticulum (ER) stress, apoptosis and SNARE protein levels were assessed in vitro.UCHL1 protein, which was highly specific to beta cells, was increased by palmitate at basal glucose, but not in the context of hyperglycaemia associated with frank diabetes. Although islet development and function were initially normal in Uchl1 (-/-) mice, a 4-week high-fat diet caused glucose intolerance and impaired insulin secretion. Uchl1 (-/-) mice had increased ER stress and beta cell apoptosis. The levels of SNARE proteins were dysregulated in Uchl1 (-/-) islets. Palmitate-stimulated vesicle-associated membrane protein 2 (VAMP2) ubiquitination was modulated by a chemical UCHL1 inhibitor.Together, these data suggest that UCHL1 has essential functional and anti-apoptotic roles in beta cells under stress conditions associated with lipotoxicity.

The functional maturity of pancreatic beta cells is impaired in diabetes mellitus. We sought to define factors that can influence adult beta cell maturation status and function.MIN6 cells labelled with a Pdx1 monomeric red fluorescent protein-Ins1 enhanced green fluorescent protein dual reporter lentivirus were used to screen candidate growth and/or differentiation factors using image-based approaches with confirmation by real-time RT-PCR and assays of beta cell function using primary mouse islets.Activin A strikingly decreased the number of mature beta cells and increased the number of immature beta cells. While activins are critical for pancreatic morphogenesis, their role in adult beta cells remains controversial. In primary islets and MIN6 cells, activin A significantly decreased the expression of insulin and several genes associated with beta cell maturity (e.g. Pdx1, Mafa, Glut2 [also known as Slc2a2]). Genes found in immature beta cells (e.g. Mafb) tended to be upregulated by activin A. Insulin secretion was also reduced by activin A. In addition, activin A-treated MIN6 cells proliferated faster than non-treated cells. The effects of endogenous activin A on beta cells were completely reversed by exogenous follistatin.These results suggest that autocrine and/or paracrine activin A signalling exerts a suppressive effect on adult beta cell maturation and function. Thus, the maturation state of adult beta cells can be modulated by external factors in culture. Interventions inhibiting activin or its signalling pathways may improve beta cell function. Understanding of maturation and plasticity of adult pancreatic tissue has significant implications for islet regeneration and for in vitro generation of functional beta cells.

The pathogenesis of diabetes and the success of islet transplantation depend on the control of pancreatic beta cell fate. The Notch signalling pathway is essential for normal prenatal pancreatic development, but the presence and function of this gene network in adult islets has received much less attention. The presence of Notch signalling components was assessed in vitro using RT-PCR, western blotting and immunofluorescence. The functional consequences of altering Notch signalling on insulin secretion and programmed cell death were examined. Adult mouse islets, human islets and mouse insulinoma MIN6 cells possess key components of the Notch pathway. RT-PCR, western blotting and immunofluorescence indicated that the Notch target gene, neurogenin3 (Ngn3, also known as Neurog3), is also present in adult islet cells. Inhibiting Notch signalling with N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) increased Ngn3 mRNA expression and protein levels in adult islets. The activated notch homologue 1 (NOTCH1) protein level was decreased upon serum withdrawal, as well as after treatment with a phosphatidylinositol 3-kinase inhibitor, or hydroxy-2-naphthalenylmethylphosphonic acid, an insulin receptor inhibitor. While islets cultured in DAPT did not exhibit defects in insulin secretion, indicating that differentiation is unaltered, inhibiting gamma-secretase-dependent Notch activation led to a dose-dependent increase in caspase-3-dependent apoptosis in both MIN6 cells and human islets. Conversely, gamma-secretase overactivity resulted in an accumulation of cleaved NOTCH1 and protection from apoptosis. Together these results show that the Notch/Ngn3 signalling network is intact and functional in adult islets. This pathway represents an attractive target for modulating beta cell fate in diabetes, islet transplantation and efforts to derive beta cell surrogates in vitro.

Beta cell death is a hallmark of diabetes. It is not known whether specific cellular stresses associated with type 1 or type 2 diabetes require specific factors to protect pancreatic beta cells. No systematic comparison of endogenous soluble factors in the context of multiple pro-apoptotic conditions has been published.Primary mouse islet cells were cultured in conditions mimicking five type 1 or type 2 diabetes-related stresses: basal 5 mmol/l glucose, cytokine cocktail (25 ng/ml TNF-α, 10 ng/ml IL-1β, 10 ng/ml IFN-γ), 1 μmol/l thapsigargin, 1.5 mmol/l palmitate and 20 mmol/l glucose (all in the absence of serum). We surveyed the effects of a library of 206 endogenous factors (selected based on islet expression of their receptors) on islet cell survival through multi-parameter, live-cell imaging.Our survey pointed to survival factors exhibiting generalised protective effects across conditions meant to model different types of diabetes and stages of the diseases. For example, our survey and follow-up experiments suggested that OLFM1 is a novel protective factor for mouse and human beta cells across multiple conditions. Most strikingly, we also found specific protective survival factors for each model stress condition. For example, semaphorin4A (SEMA4A) was toxic to islet cells in the serum-free baseline and serum-free 20 mmol/l glucose conditions, but protective in the context of lipotoxicity. Rank product testing supported the consistency of our observations.Collectively, our survey reveals previously unidentified islet cell survival factors and suggest their potential utility in individualised medicine.

Adult pancreatic islets contain multiple cell types that produce and secrete well characterised hormones, including insulin, glucagon and somatostatin. Although it is increasingly apparent that islets release and respond to more secreted factors than previously thought, systematic analyses are lacking. We therefore sought to identify potential autocrine and/or paracrine islet growth factor loops, and to characterise the function of the netrin family of islet-secreted factors and their receptors, which have been previously unreported in adult islets.Gene expression databases, islet-specific tag sequencing libraries and microarray datasets of FACS purified beta cells were used to compile a list of secreted factors and receptors present in mouse or human islets. Netrins and their receptors were further assessed using RT-PCR, Western blot analysis and immunofluorescence staining. The roles of netrin-1 and netrin-4 in beta cell function, apoptosis and proliferation were also examined.We identified 233 secreted factors and 234 secreted factor receptors in islets. The presence of netrins and their receptors was further confirmed. Downregulation of caspase-3 activation was observed when MIN6 cells were exposed to exogenous netrin-1 and netrin-4 under hyperglycaemic conditions. Reduction in caspase-3 cleavage was linked to the decrease in dependence receptors, neogenin and unc-5 homologue A, as well as the activation of Akt and extracellular signal-regulated protein kinase (ERK) signalling.Our results highlight the large number of potential islet growth factors and point to a context-dependent pro-survival role for netrins in adult beta cells. Since diabetes results from a deficiency in functional beta cell mass, these studies are important steps towards developing novel therapies to improve beta cell survival.