The role of CART in islet function

Diminished insulin secretion and dysregulated glucagon secretion are key features of type 2 diabetes (T2D). The overall aim of this thesis was to study the role of cocaine- and amphetamine-regulated transcript (CART) in islet cell function and how CART regulates glucose homeostasis. We found that CART is expressed in human islet cells and that its expression is increased in patients with T2D, as well as in mouse models of T2D. Rat islet CART expression was regulated by glucose and beta cell CART expression was normalised by insulin treatment in diabetic rats. Furthermore, CART increased insulin secretion from both mouse and human islets in a glucose-dependent fashion. This could partly be explained by increased beta cell exocytosis, altered intracellular Ca2+ oscillation pattern as well as improved synchronisation of Ca2+ oscillations between islet cells. Importantly, CART increased insulin secretion and glucose elimination in vivo in mice. We also showed that CART decreased glucagon secretion in mouse and human islets, as well as in vivo in mice, and that CART reduced exocytosis in alpha cells. To mimic the situation in patients, with increased beta cell CART expression, we generated transgenic mice with beta cell-specific CART overexpression (CARTtg) and studied the impact of increased beta cell CART expression on glucose homeostasis in vivo. Under basal conditions, CARTtg mice were normoglycaemic and normoinsulinemic, but when challenged by streptozotocin treatment or a high fat diet, and in ageing, CARTtg mice displayed increased insulin secretion compared with wild-type littermates. This was accompanied by improved glucose elimination in streptozotocin-treated and aged mice, but not in high fat diet-fed mice, which instead displayed mild insulin resistance. Moreover, viral overexpression of CART in INS-1 (832/13) cells increased insulin secretion. Finally, we found that adipocytes from CARTtg mice had altered metabolism, suggesting that CART may be a mediator of cross-talk between beta cells and adipose tissue. Collectively, our data imply that CART has an important role in beta cell function and in regulation of glucose homeostasis. Hence, the potential for CART-based therapies in T2D should be evaluated

The role of CART in islet biology

Cocaine- and amphetamine-regulated transcript (CART) is mostly known for its appetite regulating effects in the central nervous system. However, CART is also highly expressed in the peripheral nervous system as well as in certain endocrine cells. Our group has dedicated more than 20 years to understand the role of CART in the pancreatic islets and in this review we summarize what is known to date about CART expression and function in the islets. CART is expressed in both islet cells and nerve fibers innervating the islets. Large species differences are at hand and CART expression is highly dynamic and increased during development, as well as in Type 2 Diabetes and certain endocrine tumors. In the human islets CART is expressed in alpha cells and beta cells and the expression is increased in T2D patients. CART increases insulin secretion, reduces glucagon secretion, and protects against beta cell death by reducing apoptosis and increasing proliferation. It is still not fully understood how CART mediates its effects or which receptors that are involved. Nevertheless, CART is endowed with several properties that are beneficial in a T2D perspective. Many of the described effects of CART resemble those of GLP-1, and interestingly CART has been found to potentiate some of the effects of GLP-1, paving the way for CART-based treatments in combination with GLP-1-based drugs

GK-rats respond to gastric bypass surgery with improved glycemia despite unaffected insulin secretion and beta cell mass

Roux-en-Y gastric bypass (RYGB) is the most effective treatment for morbid obesity and results in rapid remission of type 2 diabetes (T2D), before significant weight loss occurs. The underlying mechanisms for T2D remission are not fully understood. To gain insight into these mechanisms we used RYGB-operated diabetic GK-rats and Wistar control rats. Twelve adult male Wistar- and twelve adult male GK-rats were subjected to RYGB- or sham-operation. Oral glucose tolerance tests (OGTT) were performed six weeks after surgery. RYGB normalized fasting glucose levels in GK-rats, without affecting fasting insulin levels. In both rat strains, RYGB caused increased postprandial responses in glucose, GLP-1, and GIP. RYGB caused elevated postprandial insulin secretion in Wistar-rats, but had no effect on insulin secretion in GK-rats. In agreement with this, RYGB improved HOMA-IR in GK-rats, but had no effect on HOMA-β. RYGB-operated GK-rats had an increased number of GIP receptor and GLP-1 receptor immunoreactive islet cells, but RYGB had no major effect on beta or alpha cell mass. Furthermore, in RYGB-operated GK-rats, increased Slc5a1, Pck2 and Pfkfb1 and reduced Fasn hepatic mRNA expression was observed. In summary, our data shows that RYGB induces T2D remission and enhanced postprandial incretin hormone secretion in GK-rats, without affecting insulin secretion or beta cell mass. Thus our data question the dogmatic view of how T2D remission is achieved and instead point at improved insulin sensitivity as the main mechanism of remission

Overexpressed beta cell CART increases insulin secretion in mouse models of insulin resistance and diabetes

Impaired beta cell function and beta cell death are key features of type 2 diabetes (T2D). Cocaine- and amphetamine-regulated transcript (CART) is necessary for normal islet function in mice. CART increases glucose-stimulated insulin secretion in vivo in mice and in vitro in human islets and CART protects beta cells against glucotoxicity-induced cell death in vitro in rats. Furthermore, beta cell CART is upregulated in T2D patients and in diabetic rodent models as a consequence of hyperglycaemia. The aim of this study was to assess the impact of upregulated beta cell CART on islet hormone secretion and glucose homeostasis in a transgenic mouse model. To this end, mice with beta cell-specific overexpression of CART (CARTtg mice) were generated. CARTtg mice challenged by aging, high fat diet feeding or streptozotocin treatment were phenotyped with respect to in vivo and in vitro insulin and glucagon secretion, glucose homeostasis, and beta cell mass. In addition, the impact of adenoviral overexpression of CART on insulin secretion was studied in INS-1 832/13 cells. CARTtg mice had a normal metabolic phenotype under basal conditions. On the other hand, with age CARTtg mice displayed increased insulin secretion and improved glucose elimination, compared with age-matched WT mice. Furthermore, compared with WT controls, CARTtg mice had increased insulin secretion after feeding a high fat diet, as well as lower glucose levels and higher insulin secretion after streptozotocin treatment. Viral overexpression of CART in INS-1 832/13 cells resulted in increased glucose-stimulated insulin secretion. Together, these results imply that beta cell CART acts to increase insulin secretion when beta cell function is challenged. We propose that the increase in beta cell CART is part of a compensatory mechanisms trying to counteract the hyperglycaemia in T2D

Mitochondrial transcription factor B2 is essential for mitochondrial and cellular function in pancreatic β-cells

Objective: Insulin release from pancreatic β-cells is controlled by plasma glucose levels via mitochondrial fuel metabolism. Therefore, insulin secretion is critically dependent on mitochondrial DNA (mtDNA) and the genes it encodes. Mitochondrial transcription factor B2 (TFB2M) controls transcription of mitochondrial-encoded genes. However, its precise role in mitochondrial metabolism in pancreatic β-cells and, consequently, in insulin secretion remains unknown. Methods: To elucidate the role of TFB2M in mitochondrial function and insulin secretion in vitro and in vivo, mice with a β-cell specific homozygous or heterozygous knockout of Tfb2m and rat clonal insulin-producing cells in which the gene was silenced were examined with an array of metabolic and functional assays. Results: There was an effect of gene dosage on Tfb2m expression and function. Loss of Tfb2m led to diabetes due to disrupted transcription of mitochondrial DNA (mtDNA) and reduced mtDNA content. The ensuing mitochondrial dysfunction activated compensatory mechanisms aiming to limit cellular dysfunction and damage of β-cells. These processes included the mitochondrial unfolded protein response, mitophagy, and autophagy. Ultimately, however, these cell-protective systems were overridden, leading to mitochondrial dysfunction and activation of mitochondrial-dependent apoptotic pathways. In this way, β-cell function and mass were reduced. Together, these perturbations resulted in impaired insulin secretion, progressive hyperglycemia, and, ultimately, development of diabetes. Conclusions: Loss of Tfb2m in pancreatic β-cells results in progressive mitochondrial dysfunction. Consequently, insulin secretion in response to metabolic stimuli is impaired and β-cell mass reduced. Our findings indicate that TFB2M plays an important functional role in pancreatic β-cells. Perturbations of its actions may lead to loss of functional β-cell mass, a hallmark of T2D

Defective exocytosis and processing of insulin in a cystic fibrosis mouse model

Cystic fibrosis-related diabetes (CFRD) is a common complication for patients with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The cause of CFRD is unclear, but a commonly observed reduction in first-phase insulin secretion suggests defects at the beta cell level. Here we aimed to examine beta- and alpha-cell function in the Cftrtm1EUR/F508del mouse model (C57BL/6J), which carries the most common human mutation in CFTR, the F508del mutation. CFTR expression, beta cell mass, insulin granule distribution, hormone secretion and single cell capacitance changes were evaluated using islets (or beta cells) from F508del mice and age-matched wild-type mice aged 7-10 weeks. Granular pH was measured with DND-189 fluorescence. Serum glucose, insulin and glucagon levels were measured in vivo, and glucose tolerance was assessed using IPGTT. We show increased secretion of proinsulin and concomitant reduced secretion of C-peptide in islets from F508del mice compared to WT mice. Exocytosis and number of docked granules was reduced. We confirmed reduced granular pH by CFTR stimulation. We detected decreased pancreatic beta cell area, but unchanged beta cell number. Moreover, the F508del mutation caused failure to suppress glucagon secretion leading to hyperglucagonemia. In conclusion, F508del mice have beta cell defects resulting in 1) reduced number of docked insulin granules and reduced exocytosis, and 2) potential defective proinsulin cleavage and secretion of immature insulin. These observations provide insight into the functional role of CFTR in pancreatic islets and contribute to increased understanding of the pathogenesis of CFRD

Loss of TFB1M results in mitochondrial dysfunction that leads to impaired insulin secretion and diabetes.

We have previously identified Transcription Factor B1 Mitochondrial (TFB1M) as a Type 2 Diabetes (T2D) risk gene, using human and mouse genetics. To further understand the function of TFB1M and how it is associated with T2D we created a β-cell specific knockout of Tfb1 m, which gradually developed diabetes. Prior to the onset of diabetes, β-Tfb1 m(-/-) mice exhibited retarded glucose clearance due to impaired insulin secretion. β-Tfb1 m(-/-) islets released less insulin in response to fuels, contained less insulin and secretory granules, and displayed reduced β-cell mass. Moreover, mitochondria in Tfb1 m-deficient β-cells were more abundant with disrupted architecture. TFB1M is known to control mitochondrial protein translation by adenine-dimethylation of 12S ribosomal RNA (rRNA). Here, we found that levels of TFB1M and mitochondrial encoded proteins, mitochondrial 12S rRNA methylation, ATP production and oxygen consumption were reduced in β-Tfb1 m(-/-) islets. Furthermore, levels of reactive oxygen species in response to cellular stress were increased while induction of defense mechanisms was attenuated. We also show increased apoptosis and necrosis as well as infiltration of macrophages and CD4(+)-cells in the islets. Taken together, our findings demonstrate that Tfb1 m-deficiency in β-cells caused mitochondrial dysfunction and subsequently diabetes due to combined loss of β-cell function and mass. These observations reflect pathogenetic processes in human islets: using RNA sequencing, we found that the TFB1M risk variant exhibited a negative gene-dosage effect on islet TFB1M mRNA levels, as well as insulin secretion. Our findings highlight the role of mitochondrial dysfunction in impairments of β-cell function and mass, the hallmarks of T2D

CART is overexpressed in human type 2 diabetic islets and inhibits glucagon secretion and increases insulin secretion

Aims/hypothesis: Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart−/− mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. Methods: CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca2+ oscillation patterns and exocytosis were studied in mouse islets. Results: We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca2+ signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. Conclusions/interpretation: We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes