Alpha Cells Research Articles

Opposing roles of the entero-pancreatic hormone urocortin-3 in glucose metabolism in rats

Aim/hypothesisUrocortin-3 (UCN3) is a glucoregulatory peptide produced in the gut and pancreatic islets. The aim of this study was to clarify the acute effects of UCN3 on glucose regulation following an oral glucose challenge and to investigate the mechanisms involved.MethodsWe studied the effect of UCN3 on blood glucose, gastric emptying, glucose absorption and secretion of gut and pancreatic hormones in male rats. To supplement these physiological studies, we mapped the expression of UCN3 and the UCN3-sensitive receptor, type 2 corticotropin-releasing factor receptor (CRHR2), by means of fluorescence in situ hybridisation and by gene expression analysis.ResultsIn rats, s.c. administration of UCN3 strongly inhibited gastric emptying and glucose absorption after oral administration of glucose. Direct inhibition of gastrointestinal motility may be responsible because UCN3’s cognate receptor, CRHR2, was detected in gastric submucosal plexus and in interstitial cells of Cajal. Despite inhibited glucose absorption, post-challenge blood glucose levels matched those of rats given vehicle in the low-dose UCN3 group, because UCN3 concomitantly inhibited insulin secretion. Higher UCN3 doses did not further inhibit gastric emptying, but the insulin inhibition progressed resulting in elevated post-challenge glucose and lipolysis. Incretin hormones and somatostatin (SST) secretion from isolated perfused rat small intestine was unaffected by UCN3 infusion; however, UCN3 infusion stimulated secretion of somatostatin from delta cells in the isolated perfused rat pancreas which, unlike alpha cells and beta cells, expressed Crhr2. Conversely, acute antagonism of CRHR2 signalling increased insulin secretion by reducing SST signalling. Consistent with these observations, acute drug-induced inhibition of CRHR2 signalling improved glucose tolerance in rats to a similar degree as administration of glucagon-like peptide-1. UCN3 also powerfully inhibited glucagon secretion from isolated perfused rat pancreas (perfused with 3.5 mmol/l glucose) in a SST-dependent manner, suggesting that UCN3 may be involved in glucose-induced inhibition of glucagon secretion.Conclusions/interpretationOur combined data indicate that UCN3 is an important glucoregulatory hormone that acts through regulation of gastrointestinal and pancreatic functions.Graphical abstract

XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and protects against diabetic beta cell failure during metabolic stress in mice

Aims/hypothesisPancreatic beta cell dedifferentiation, transdifferentiation into other islet cells and apoptosis have been implicated in beta cell failure in type 2 diabetes, although the mechanisms are poorly defined. The endoplasmic reticulum stress response factor X-box binding protein 1 (XBP1) is a major regulator of the unfolded protein response. XBP1 expression is reduced in islets of people with type 2 diabetes, but its role in adult differentiated beta cells is unclear. Here, we assessed the effects of Xbp1 deletion in adult beta cells and tested whether XBP1-mediated unfolded protein response makes a necessary contribution to beta cell compensation in insulin resistance states.MethodsMice with inducible beta cell-specific Xbp1 deletion were studied under normal (chow diet) or metabolic stress (high-fat diet or obesity) conditions. Glucose tolerance, insulin secretion, islet gene expression, alpha cell mass, beta cell mass and apoptosis were assessed. Lineage tracing was used to determine beta cell fate.ResultsDeletion of Xbp1 in adult mouse beta cells led to beta cell dedifferentiation, beta-to-alpha cell transdifferentiation and increased alpha cell mass. Cell lineage-specific analyses revealed that Xbp1 deletion deactivated beta cell identity genes (insulin, Pdx1, Nkx6.1, Beta2, Foxo1) and derepressed beta cell dedifferentiation (Aldh1a3) and alpha cell (glucagon, Arx, Irx2) genes. Xbp1 deletion in beta cells of obese ob/ob or high-fat diet-fed mice triggered diabetes and worsened glucose intolerance by disrupting insulin secretory capacity. Furthermore, Xbp1 deletion increased beta cell apoptosis under metabolic stress conditions by attenuating the antioxidant response.Conclusions/interpretationThese findings indicate that XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and is required for beta cell compensation and prevention of diabetes in insulin resistance states.Graphical abstract

A local insulin reservoir in Drosophila alpha cell homologs ensures developmental progression under nutrient shortage

Insulin/insulin-like growth factor (IGF) signaling (IIS) controls many aspects of development and physiology. In Drosophila, a conserved family of insulin-like peptides called Dilps is produced by brain neurosecretory cells, and it regulates organismal growth and developmental timing. To accomplish these systemic functions, the Dilps are secreted into the general circulation, and they signal to peripheral tissues in an endocrine fashion. Here, we describe the local uptake and storage of Dilps in the corpora cardiaca (CC), an endocrine organ composed of alpha cell homologs known to produce the glucagon-like adipokinetic hormone (AKH). We show that Dilp uptake by the CC relies on the expression of an IGF-binding protein called ImpL2. Following their uptake, immunogold staining demonstrates that Dilps are co-packaged with AKH in dense-core vesicles for secretion. In response to nutrient shortage, this specific Dilp reservoir is released and activates IIS in a paracrine manner in the prothoracic gland. This stimulates the production of the steroid hormone ecdysone and initiates entry into pupal development. We therefore uncover a sparing mechanism whereby insulin stores in CC serve to locally activate IIS and the production of ecdysone in the PG, accelerating developmental progression in adverse food conditions.

The response of three-dimensional pancreatic alpha and beta cell co-cultures to oxidative stress.

The pancreatic islets of Langerhans have low endogenous antioxidant levels and are thus especially sensitive to oxidative stress, which is known to influence cell survival and behaviour. As bioengineered islets are gaining interest for therapeutic purposes, it is important to understand how their composition can be optimized to diminish oxidative stress. We investigated how the ratio of the two main islet cell types (alpha and beta cells) and their culture in three-dimensional aggregates could protect against oxidative stress. Monolayer and aggregate cultures were established by seeding the alphaTC1 (alpha) and INS1E (beta) cell lines in varying ratios, and hydrogen peroxide was applied to induce oxidative stress. Viability, oxidative stress, and the level of the antioxidant glutathione were measured. Both aggregation and an increasing prevalence of INS1E cells in the co-cultures conferred greater resistance to cell death induced by oxidative stress. Increasing the prevalence of INS1E cells also decreased the number of alphaTC1 cells experiencing oxidative stress in the monolayer culture. In 3D aggregates, culturing the alphaTC1 and INS1E cells in a ratio of 50:50 prevented oxidative stress in both cell types. Together, the results of this study lead to new insight into how modulating the composition and dimensionality of a co-culture can influence the oxidative stress levels experienced by the cells.

Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

Function and composition of pancreatic islet cell implants in omentum of type 1 diabetes patients

Intraportal (IP) islet cell transplants can restore metabolic control in type 1 diabetes patients, but limitations raise the need for establishing a functional beta cell mass (FBM) in a confined extrahepatic site. This study reports on function and composition of omental (OM) implants after placement of islet cell grafts with similar beta cell mass as in our IP-protocol (2–5.106 beta cells/kg body weight) on a scaffold. Four of seven C-peptide-negative recipients achieved low beta cell function (hyperglycemic clamp [HGC] 2–8 percent of controls) until laparoscopy, 2–6 months later, for OM-biopsy and concomitant IP-transplant with similar beta cell dose. This IP-transplant increased HGC-values to 15–40 percent. OM-biopsies reflected the composition of initial grafts, exhibiting varying proportions of endocrine-cell-enriched clusters with more beta than alpha cells and leucocyte pole, non-endocrine cytokeratin-positive clusters surrounded by leucocytes, and scaffold remnants with foreign body reaction. OM-implants on a polyglactin-thrombin-fibrinogen-scaffold presented larger endocrine clusters with infiltrating endothelial cells and corresponded to the higher HGC-values. No activation of cellular immunity to GAD/IA2 was measured post-OM-transplant. Establishment of a metabolically adequate FBM in omentum may require a higher beta cell number in grafts but also elimination of their immunogenic non-endocrine components as well as local conditioning that favors endocrine cell engraftment and function.

DNA methylation reveals distinct cells of origin for pancreatic neuroendocrine carcinomas and pancreatic neuroendocrine tumors

BackgroundPancreatic neuroendocrine neoplasms (PanNENs) fall into two subclasses: the well-differentiated, low- to high-grade pancreatic neuroendocrine tumors (PanNETs), and the poorly-differentiated, high-grade pancreatic neuroendocrine carcinomas (PanNECs). While recent studies suggest an endocrine descent of PanNETs, the origin of PanNECs remains unknown.MethodsWe performed DNA methylation analysis for 57 PanNEN samples and found that distinct methylation profiles separated PanNENs into two major groups, clearly distinguishing high-grade PanNECs from other PanNETs including high-grade NETG3. DNA alterations and immunohistochemistry of cell-type markers PDX1, ARX, and SOX9 were utilized to further characterize PanNECs and their cell of origin in the pancreas.ResultsPhylo-epigenetic and cell-type signature features derived from alpha, beta, acinar, and ductal adult cells suggest an exocrine cell of origin for PanNECs, thus separating them in cell lineage from other PanNENs of endocrine origin.ConclusionsOur study provides a robust and clinically applicable method to clearly distinguish PanNECs from G3 PanNETs, improving patient stratification.

The Optimal Maturation of Subcutaneous Pouch Can Improve Pancreatic Islets Engraftment in Rat Model.

Transplantation of pancreatic islets into subcutaneous cavities in diabetic rats may be as or even more effective than transplantation into the portal vein. Identifying the optimal timing of the individual steps in this procedure is critical. Macroporous scaffolds were placed in the subcutaneous tissue of diabetic male Lewis rats for 7 or 28 d and the healing of the tissue inside the scaffolds was monitored. A marginal syngeneic graft comprising 4 islets/g of recipient body weight was transplanted at the best timing focusing mainly on vascularization. Recipients were monitored for blood glucose levels and tolerance tests. Histological examination was performed in all implanted scaffolds. The presence of individual endocrine cells was analyzed in detail. Blood glucose levels remained within the physiological range in all recipients until the end of experiment as well as body weight increase. Coefficients of glucose assimilation were normal or slightly reduced with no statistically significant differences between the groups 40 and 80 d after transplantation. Histological analysis revealed round viable islets in the liver similar to those in pancreas, but alpha cells practically disappeared, whereas islets in the scaffolds formed clusters of cells surrounded by rich vascular network and the alpha cells remained partially preserved. Subcutaneous transplantation of pancreatic islets is considerably less invasive but comparably efficient as commonly used islet transplantation into the portal vein. In consideration of alpha and beta cell ratio, the artificial subcutaneous cavities represent a promising site for future islet transplantation therapy.

Electrical impulse characterization along actin filaments in pathological conditions

We present an interactive Mathematica notebook that characterizes the electrical impulses along actin filaments in both muscle and non-muscle cells for a wide range of physiological and pathological conditions. The simplicity of the theoretical formulation, and high performance of the Mathematica software, enable the analysis of multiple conditions without computational restrictions. The program is based on a multi-scale (atomic → monomer → filament) approach capable of accounting for the atomistic details of a protein molecular structure, its biological environment, and their impact on the travel distance, velocity, and attenuation of monovalent ionic wave packets propagating along microfilaments. The interactive component allows investigators to choose the experimental conditions (intracellular Vs in vitro), nucleotide state (ATP Vs ADP), actin isoform (alpha, gamma, beta, and muscle or non-muscle cell), as well as a conformation model that covers a variety of mutants and wild-type (the control) actin filaments. We used the computational tool to analyze environmental changes such as temperature effects and pH changes of the surrounding solutions, as well as structural changes to an actin monomer due to radius changes. Additionally, we investigated for the first time the electrostatic consequences of actin mutations from different disease conditions. These studies may provide an unprecedented molecular understanding of why and how age, inheritance, and disease conditions induce dysfunctions in the biophysical mechanisms underlying the propagation of electrical signals along actin filaments. Program summaryProgram Title: EIAF (Electrical Impulse along Actin Filaments)CPC Library link to program files:https://doi.org/10.17632/k8vw4j6y75.1Developer's repository link:https://github.com/MarceloMarucho/SignalPropagationPathologicalConditionLicensing provisions: GPLv2Programming language: MathematicaNature of problem: Electrical properties of actin filaments in pathological conditions have yet to be studied, despite previous work showing influential consequences for physiological conditions on wild type actins.Solution method: The interactive Mathematica notebook is based on the Multi-scale Approach for electrical impulses along cytoskeletal filaments and uses non-linear considerations for the Boltzmann statistics of ionic distributions in the electrical double layer (EDL). Using the numerical methods incorporated in Mathematica’s default libraries, and an interactive GUI, the program allows users to conduct their own research by choosing the experimental conditions (intracellular Vs in vitro), nucleotide state (ATP Vs ADP), actin isoform (including muscle and non-muscles cells), as well as a conformation model that covers a variety of mutants and wild-type (the control) actin filament configurations.Additional comments including restrictions and unusual features: The program allows users to adjust pH values, input voltages, as well as study two default electrolyte conditions without rerunning the program. The program is fully editable, such that motivated users can change the default electrolytes to fit specific conditions in their research project in necessary.

Sodium, Glucose and Dysregulated Glucagon Secretion: The Potential of Sodium Glucose Transporters.

Diabetes is defined by hyperglycaemia due to progressive insulin resistance and compromised insulin release. In parallel, alpha cells develop dysregulation of glucagon secretion. Diabetic patients have insufficient glucagon secretion during hypoglycaemia and a lack of inhibition of glucagon secretion at higher blood glucose levels resulting in postprandial hyperglucagonaemia, which contributes to the development of hyperglycaemia. Sodium-glucose co-transporter 2 (SGLT2) inhibitors are an efficient pharmacologic approach for the treatment of hyperglycaemia in type 2 diabetes. While SGLT2 inhibitors aim at increasing glycosuria to decrease blood glucose levels, these inhibitors also increase circulating glucagon concentrations. Here, we review recent advances in our understanding of how SGLTs are involved in the regulation of glucagon secretion. Sodium plays an important role for alpha cell function, and a tight regulation of intracellular sodium levels is important for maintaining plasma membrane potential and intracellular pH. This involves the sodium-potassium pump, sodium-proton exchangers and SGLTs. While the expression of SGLT2 in alpha cells remains controversial, SGLT1 seems to play a central role for alpha cell function. Under hyperglycaemic conditions, SGLT1 mediated accumulation of sodium results in alpha cell dysregulation due to altered cellular acidification and ATP production. Taken together, this suggests that SGLT1 could be a promising, yet highly underappreciated drug target to restore alpha cell function and improve treatment of both type 1 and 2 diabetes.

STING Agonist-Mediated Cytokine Secretion Is Accompanied by Monocyte Apoptosis.

The cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway plays a crucial role in inducing an antiviral and antitumor immune response. We studied the effects of synthetic STING agonists on several immune populations and related cytokine production. In comparison with the toll-like receptor 7 (TLR7) agonist, STING agonists induced secretion of a broader proinflammatory cytokine spectrum. Unlike the TLR7 agonist, the structurally diverse STING agonists partially depleted B and NK cells and completely depleted CD14+ monocytes via induction of apoptosis. The TANK-binding kinase 1 inhibitor efficiently prevented interferon alpha (IFNα) secretion and cell depletion, suggesting their possible dependence on the cGAS-STING pathway activation. Finally, IFNα, tumor necrosis factor alpha, interleukin 6, and interleukin 1 beta secretion and CD14+ monocyte apoptosis were primary responses to STING agonists, whereas IFNγ was secreted secondarily. These findings bring new insights into the cGAS-STING pathway immunomodulation that is of future therapeutic importance.

Differentiation of PANC-1 ductal cells to β-like cells via cellular GABA modulation by Magainin and CPF-7 peptides

Some of the antimicrobial peptides induce insulin release and improve glucose tolerance while their effects on pancreatic cell differentiation have remained unresolved. In this report, we evaluated the effects of two of these peptides, Magainin-II and CPF-7, and also GABA, on PANC-1 ductal cells' differentiation. Based on immunofluorescence and qRT-PCR analyses the expression levels of some of the Epithelial to Mesenchymal transition (EMT)-related factors such as Snai1 and Ngn3, as two biomarkers of alpha and beta cells, were increased. Our findings also revealed a drastic increase in Arx, Pax4, Dnmt-1 and Glucagon expressions associated with dedifferentiation of PANC-1 cells into pancreatic endocrine progenitor cells. Futhermore, Magainin-II and CPF-7 exerted their roles partly via influencing the GABA cellular content. These data would undoubtedly provide a suitable ground for further investigation to guide these cells toward transplantable insulin producing beta cells.

A glycolytic enzyme complex and creatine kinase locally and differentially regulate ATP-sensitive potassium (KATP) channels in pancreatic islet alpha and beta cells

The ATP-sensitive potassium channel (KATP) is a key regulator of membrane potential and islet hormone secretion. Here, using the inside-out excised patch configuration, we isolated the plasma membrane compartment containing KATP on β-cells and α-cells from humans and mice. The characteristic response of KATP to inhibition by ATP and activation by ADP was used to identify the presence of a glycolytic enzyme complex that is functionally coupled to KATP regulation. We found that the phosphofructokinase product fructose-1,6-bisphosphate (FBP), which oscillates in β-cells, can induce KATP closure by its metabolism through the glycolytic chain including aldolase, GAPDH, phosphoglycerate kinase, phosphoglycerate mutase, enolase and finally pyruvate kinase.

Inhibition of SGLT2 Preserves Function and Promotes Proliferation of Human Islets Cells In Vivo in Diabetic Mice.

Dapagliflozin is a sodium-glucose co-transporter 2 (SGLT2) inhibitor used for the treatment of diabetes. This study examines the effects of dapagliflozin on human islets, focusing on alpha and beta cell composition in relation to function in vivo, following treatment of xeno-transplanted diabetic mice. Mouse beta cells were ablated by alloxan, and dapagliflozin was provided in the drinking water while controls received tap water. Body weight, food and water intake, plasma glucose, and human C-peptide levels were monitored, and intravenous arginine/glucose tolerance tests (IVarg GTT) were performed to evaluate islet function. The grafted human islets were isolated at termination and stained for insulin, glucagon, Ki67, caspase 3, and PDX-1 immunoreactivity in dual and triple combinations. In addition, human islets were treated in vitro with dapagliflozin at different glucose concentrations, followed by insulin and glucagon secretion measurements. SGLT2 inhibition increased the animal survival rate and reduced plasma glucose, accompanied by sustained human C-peptide levels and improved islet response to glucose/arginine. SGLT2 inhibition increased both alpha and beta cell proliferation (Ki67+glucagon+ and Ki67+insulin+) while apoptosis was reduced (caspase3+glucagon+ and caspase3+insulin+). Alpha cells were fewer following inhibition of SGLT2 with increased glucagon/PDX-1 double-positive cells, a marker of alpha to beta cell transdifferentiation. In vitro treatment of human islets with dapagliflozin had no apparent impact on islet function. In summary, SGLT2 inhibition supported human islet function in vivo in the hyperglycemic milieu and potentially promoted alpha to beta cell transdifferentiation, most likely through an indirect mechanism.

Artemether and aspterric acid induce pancreatic alpha cells to transdifferentiate into beta cells in zebrafish.

Recently, the antimalarial drug, artemether and the neurotransmitter GABA were identified to convert alpha cells into beta-like cells in vivo. However, some of these observations were challenged by other studies. To help address the controversy, we took advantage of zebrafish as a model to perform this study. First, we performed a small-molecule screening for artemether and its skeleton analogues. Second, we used the Cre-LoxP system for lineage tracing to indicate the conversion of alpha cells into beta cells in vivo. The stable transgenic ins2:eGFP αTC1-6-cell line was used for evaluation of alpha-cell transdifferentiation in vitro. We further used multiple zebrafish transgenic and mutation lines to demonstrate beta-cell differentiation, beta-cell ablation and alpha-cell hyperplasia in this study. We showed that artemether and another sesquiterpene, aspterric acid, induced alpha-cell transdifferentiation into beta cells, both in zebrafish as well as using αTC1-6 cells. Furthermore, these two compounds also converted alpha cells into beta cells when beta cells were lost or alpha cells were hyperplastic in zebrafish. Unlike the previous report, the conversion of alpha cells to beta cells was mediated by increasing Pax4 expression, but not suppression of Arx expression. Our data suggest that in zebrafish and αTC1-6 cells, both artemether and aspterric acid induce alpha-cell transdifferentiation. Our data, along with those of Li et al. (2017), suggested that artemether and aspterric acid were able to induce alpha-cell transdifferentiation, at least in zebrafish and αTC1-6 cells.

Increased alpha cell to beta cell ratio in patients with pancreatic cancer.

The development of pancreatic cancer (PC) is associated with worsening of glucose tolerance. However, there is limited information about the effects of PC on islet morphology. The aim of this study was to elucidate changes in alpha and beta cell mass in patients with PC. We enrolled 30 autopsy cases with death due to PC (9 with diabetes; DM) and 31 age- and BMI-matched autopsy cases without PC (controls, 12 with DM). Tumor-free pancreatic sections were stained for insulin and glucagon, and fractional beta cell (BCA) and alpha cell area (ACA) were quantified. In addition, expression of de-differentiation markers, i.e., ALDH1A3 and UCN3, was qualitatively evaluated. The pancreas of subjects with PC showed atrophic and fibrotic changes. There was no significant difference in BCA in subjects with PC compared to controls (1.53 ± 1.26% vs. 0.95 ± 0.42%, p = 0.07). However, ACA and ACA to BCA ratio were significantly higher in subjects with PC compared to controls (2.48 ± 2.39% vs. 0.53 ± 0.26% and 1.94 ± 1.93 vs. 0.59 ± 0.26, respectively, both p < 0.001). Increased ACA to BCA ratio was observed in subjects with PC irrespective of the presence of DM. Qualitative evaluation of ALDH1A3 and UCN3 expression showed no significant difference between the groups. In conclusion, in subjects with PC, alpha to beta cell mass ratio is increased, which may contribute to the increased risk of worsening glucose metabolism. Further studies are warranted to elucidate the mechanisms of increased alpha to beta cell mass in patients with PC.

The Gene Signatures of Human Alpha Cells in Types 1 and 2 Diabetes Indicate Disease-Specific Pathways of Alpha Cell Dysfunction

The Gene Signatures of Human Alpha Cells in Types 1 and 2 Diabetes Indicate Disease-Specific Pathways of Alpha Cell Dysfunction

Glucose metabolism in amyotrophic lateral sclerosis: it is bitter-sweet.

Glucose metabolism in amyotrophic lateral sclerosis: it is bitter-sweet.

Artificial Pancreas - Bihormonal Closed Loop Glucose Control - versus Current Care after Total Pancreatectomy (APPEL5+): Outpatient Randomized Controlled Crossover Trial

Introduction: Glucose control after total pancreatectomy (TP) is problematic due to complete absence of both alpha and beta cells. Recently, a novel bihormonal (insulin and glucagon) artificial pancreas (AP) for closed loop glucose control showed better glucose control compared to standard insulin pump therapy in patients with diabetes type 1. This AP system might also improve glucose control in patients after TP. The aim is to assess the efficacy and safety of the AP in patients after TP.

Predijabetes - od dijagnoze do prognoze

Pre-diabetes is a condition of intermittent hyperglycemia, insufficient to define diabetes, which is characterized by combination of insulin resistance, alpha and beta cell dysfunction. It carries a risk for type 2 diabetes, often with pre-existing vascular complications, atherosclerotic cardiovascular disease, fatty liver and kidney disease. A reliable diagnosis is made on the basis of the oral glucose tolerance test (oGTT) and the levels of glycosylated hemoglobin, HbA1c. Based on that, the presence of 3 basic disorders within pre-diabetes is determined: elevated fasting glycemia, glucose intolerance or both. However, this is not enough to predict the outcome. Extensive epidemiological studies have defined the minimum tests that should be performed in people with pre-diabetes. These are: calculation of body mass index (BMI), analysis of body composition, oGTT with analysis of glycemia, insulin and c-peptide in 0, 60 and 120 min, or just analysis of insulin and c-peptide with calculation of HOMA IR and HOMA B index, triglycerides, HDL cholesterol, transaminases, gamma GT, albuminuria and glomerular filtration according to CKD-EPI creatinine formula. Based on these parameters, 6 subtypes of pre-diabetes have been proposed that can predict outcomes: 1. Moderately obese with initial disturbance in insulin sensitivity, but normal insulin secretion; 2. Normal body weight with normal insulin sensitivity of tissues, but somewhat reduced insulin secretion; 3. Moderate obesity or grade I obesity with moderately reduced insulin sensitivity and insulin secretion; 4. Grade I obesity, more subcutaneous than visceral fat, with preserved insulin sensitivity and secretion; 5. Grade I, II degree or morbidly obese with fatty liver and low insulin sensitivity and secretion and 6. Obesity I, II degree or morbidly obese with increased visceral adipose tissue and risk of kidney disease. Monitoring of outcomes found that the metabolically healthier subtypes of pre-diabetes are 1, 2 and 4. Type 2 diabetes is most common in subtypes 3 and 5. Phenotype 5 has the highest cardiovascular risk and phenotype 6 has the highest mortality. Chronic kidney disease is most common in subtype 6. The presence of fatty liver increases mortality. Identification of pre-diabetes has preventive therapeutic potential. It is important to make a diagnosis, clarify the prognosis and prevent comorbidity by proper diet and physical activity and drugs to treat type 2 diabetes and obesity.