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Background:
The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells.
Methods:
Mice with a floxed GC-A gene were bred to Rip-CreTG mice, thereby deleting GC-A selectively in β-cells (β GC-A KO). Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion were monitored in normal diet (ND)- and high-fat diet (HFD)-fed mice. β-cell size and number were measured by immunofluorescence-based islet morphometry.
Results:
In vitro, the insulinotropic and proliferative actions of ANP were abolished in islets isolated from β GC-A KO mice. Concordantly, in vivo, infusion of BNP mildly enhanced baseline plasma insulin levels and glucose-induced insulin secretion in control mice. This effect of exogenous BNP was abolished in β GC-A KO mice, corroborating the efficient inactivation of the GC-A receptor in β-cells. Despite this under physiological, ND conditions, fasted and fed insulin levels, glucose-induced insulin secretion, glucose tolerance and β-cell morphology were similar in β GC-A KO mice and control littermates. However, HFD-fed β GC-A KO animals had accelerated glucose intolerance and diminished adaptative β-cell proliferation.
Conclusions:
Our studies of β GC-A KO mice demonstrate that the cardiac hormones ANP and BNP do not modulate β-cell's growth and secretory functions under physiological, normal dietary conditions. However, endogenous NP/GC-A signaling improves the initial adaptative response of β-cells to HFD-induced obesity. Impaired β-cell NP/GC-A signaling in obese individuals might contribute to the development of type 2 diabetes.
The pancreas and the small intestine are pivotal organs acting in close synergism to regulate glucose metabolism. After absorption and processing of dietary glucose within the small intestine, insulin and glucagon are released from pancreatic islet cells to maintain blood glucose homeostasis. Malfunctions affecting either individual, organ-specific functions or the sophisticated interplay of both organs can result in massive complications and pathologic conditions. One of the most serious metabolic diseases of our society is diabetes mellitus (DM) that is hallmarked by a disturbance of blood glucose homeostasis. Type 1 (T1DM) and type 2 (T2DM) are the main forms of the disease and both are characterized by chronic hyperglycemia, a condition that evokes severe comorbidities in the long-term. In the past, several standard treatment options allowed a more or less adequate therapy for diabetic patients. Albeit there is much effort to develop new therapeutic interventions to treat diabetic patients in a more efficient way, no cure is available so far. In view of the urgent need for alternative treatment options, a more systemic look on whole organ systems, their biological relation and complex interplay is needed when developing new therapeutic strategies for DM.
T1DM is hallmarked by an autoimmune-mediated destruction of the pancreatic β-cell mass resulting in a complete lack of insulin that is in most patients restored by applying a life-long recombinant insulin therapy. Therefore, novel regenerative medicine-based concepts focus on the derivation of bioartificial β-like cells from diverse stem cell sources in vitro that survive and sustain to secrete insulin after implantation in vivo. In this context, the first part of this thesis analyzed multipotent intestinal stem cells (ISCs) as alternative cell source to derive bioartificial, pancreatic β-like cells in vitro. From a translational perspective, intestinal stem cells pose a particularly attractive cell source since intestinal donor tissues could be obtained via minimal invasive endoscopy in an autologous way. Furthermore, intestinal and pancreatic cells both derive from the same developmental origin, the endodermal gut tube, favoring the differentiation process towards functional β-like cells. In this study, pancreas-specific differentiation of ISCs was induced by the ectopic expression of the pancreatic transcription factor 1 alpha (Ptf1a), a pioneer transcriptional regulator of pancreatic fate. Furthermore, pancreatic lineage-specific culture media were applied to support the differentiation process. In general, ISCs grow in vitro in a 3D Matrigel®-based environment. Therefore, a 2D culture platform for ISCs was established to allow delivery and ectopic expression of Ptf1a with high efficiency. Next, several molecular tools were applied and compared with each other to identify the most suitable technology for Ptf1a delivery and expression within ISCs as well as their survival under the new established 2D conditions. Success of differentiation was investigated by monitoring changes in cellular morphology and induction of pancreatic differentiation-specific gene expression profiles. In summary, the data of this project part suggest that Ptf1a harbors the potential to induce pancreatic differentiation of ISCs when applying an adequate differentiation media. However, gene expression analysis indicated rather an acinar lineage-determination than a pancreatic β-cell-like specification. Nevertheless, this study proved ISCs not only as interesting stem cell source for the generation of pancreatic cell types with a potential use in the treatment of T1DM but alsoPtf1a as pioneer factor for pancreatic differentiation of ISCs in general.
Compared to T1DM, T2DM patients suffer from hyperglycemia due to insulin resistance. In T2DM management, the maintenance of blood glucose homeostasis has highest priority and can be achieved by drugs affecting the stabilization of blood glucose levels. Recent therapeutic concepts are aiming at the inhibition of the intestinal glucose transporter Na+-D-Glucose cotransporter 1 (SGLT1). Pharmacological inhibition of SGLT1 results in reduced postprandial blood glucose levels combined with a sustained and increased Glucagon-like peptide 1 (GLP-1) secretion. So far, systemic side effects of this medication have not been addressed in detail. Of note, besides intestinal localization, SGLT1 is also expressed in various other tissues including the pancreas. In context of having a closer look also on the interplay of organs when developing new therapeutic approaches for DM, the second part of this thesis addressed the effects on pancreatic islet integrity after loss of SGLT1. The analyses comprised the investigation of pancreatic islet size, cytomorphology and function by the use of a global SGLT1 knockout (SGLT1-/-) mouse model. As SGLT1-/- mice develop the glucose-galactose malabsorption syndrome when fed a standard laboratory chow, these animals derived a glucose-deficient, fat-enriched (GDFE) diet. Wildtype mice on either standard chow (WTSC) or GDFE (WTDC) allowed the discrimination between diet- and knockout-dependent effects. Notably, GDFE fed mice showed decreased expression and function of intestinal SGLT1, while pancreatic SGLT1 mRNA levels were unaffected. Further, the findings revealed increased isled sizes, reduced proliferation- and apoptosis rates as well as an increased α-cell and reduced β-cell proportion accompanied by a disturbed cytomorphology in islets when SGLT1 function is lost or impaired. In addition, pancreatic islets were dysfunctional in terms of insulin- and glucagon-secretion. Moreover, the release of intestinal GLP-1, an incretin hormone that stimulates insulin-secretion in the islet, was abnormal after glucose stimulatory conditions. In summary, these data show that intestinal SGLT1 expression and function is nutrient dependent. The data obtained from the islet studies revealed an additional and new role of SGLT1 for maintaining pancreatic islet integrity in the context of structural, cytomorphological and functional aspects. With special emphasis on SGLT1 inhibition in diabetic patients, the data of this project indicate an urgent need for analyzing systemic side effects in other relevant organs to prove pharmacological SGLT1 inhibition as beneficial and safe.
Altogether, the findings of both project parts of this thesis demonstrate that focusing on the molecular and cellular relationship and interplay of the small intestine and the pancreas could be of high importance in context of developing new therapeutic strategies for future applications in DM patients.