Final Report Summary - BETAIMAGE (An in vivo imaging approach to understand pancreatic beta-cell signal-transduction)
The central hypothesis of my ERC-AdG is that impaired in vivo pancreatic β-cell function and survival is associated with changes in key signal-transduction events that are manifested as altered Ca2+ dynamics/ oscillations in the β-cell. The overall objective of this research grant was to take an in vivo imaging approach and monitor/characterize quantitatively changes in Ca2+ in the β-cell under normal conditions and in diabetes in the living organism. We developed a novel imaging approach to ultimately understand in vivo β-cell Ca2+ signaling. This approach uses the anterior chamber of the eye (ACE) as a transplantation site for pancreatic islets and the cornea as a natural body window for imaging. When transplanted into the ACE, islets become vascularized and innervated and β-cell function and survival can be readily imaged non-invasively, longitudinally and at single-cell resolution. Our studies clearly show that the engrafted islets in the ACE serve as representative reporters of endogenous islets in the pancreas of the same animal.
We have generated adenovirus-based vectors for transduction of human islets and developed transgenic mice expressing GCaMP3 in β-cells. Our results show that glucose promotes a significant and kinetically uniform response in all β-cells in healthy animals. In contrast, β-cells in glucose intolerant Ob/Ob mice showed only a modest Ca2+ increase in response to the sugar. Mice with diet-induced obesity/insulin resistance showed a change in synchronized Ca2+ oscillations between β-cells within the islet.
Since the ACE as such represents an immune-privileged site, it was of utmost importance to demonstrate that immune responses associated with type 1 diabetes (T1DM) can be monitored in the ACE following transplantation of islets to this site. We demonstrated that the ACE-transplanted islets in NOD-mice develop infiltration and β-cell destruction, recapitulating the autoimmune insulitis of the pancreas, and exemplify this by analyzing reporter cell populations expressing green fluorescent protein under the Cd11c or Foxp3 promoters.
Besides the Ca2+-biosensor we developed a β-cell insulin resistance biosensor based on Fox01-GFP. This allowed us to demonstrate that in young Ob/Ob mice insulin-resistant islets locally express apolipoprotein CIII, a pro-inflammatory factor that leads to hyperactivation of β-cell voltage gated Ca2+-channels through SR-BI/β1 integrin-dependent co-activation of PKA and Src, finally resulting in Ca2+-dependent β-cell death. To study β-cell function in vivo in pancreatic islets transplanted into diabetic mice, we tested different diet-intervention protocols in diabetes-prone male C57Bl/6J mice. While all protocols led to obesity and whole-body insulin resistance, only a High-Fat-High-Sucrose diet led to the development of a diabetic phenotype within 4-8 weeks of treatment, including β-cell insulin resistance and non-compensatory insulin release. Using biosensors for changes in Ca2+ handling, β-cell insulin resistance and for glucose-induced gene transcription we were able to monitor the dynamics of β-cell dysfunction during progression of diabetes development.
We previously showed that the β3 subunit of Cav (Cavβ3) regulates cytosolic Ca2+ oscillation frequency and insulin secretion under physiological conditions. We now reported that islets from diabetic mice show Cavβ3 overexpression, altered Ca2+ dynamics, and impaired insulin secretion upon glucose stimulation. Consequently, in high-fat diet (HFD)-induced diabetes, Cavβ3-deficient (Cavβ3-/-) mice showed improved islet function and enhanced glucose tolerance. Normalization of Cavβ3 expression in ob/ob islets by an antisense oligonucleotide rescued the altered Ca2+ dynamics and impaired insulin secretion. Importantly, transplantation of Cavβ3-/- islets into the ACE improved glucose tolerance in HFD-fed mice. Cavβ3 overexpression in human islets also impaired insulin secretion. We thus suggest that Cavβ3 may serve as a druggable target for diabetes treatment.
Data by us and others have shown that human islets differ from rodent islets in the structure-function-relationship. Therefore it is of utmost importance to clarify signal-transduction processes in human islets in vivo under normal conditions and why these processes do not function properly in diabetes. Our data show that following transplantation of human islets into the ACE of immune compromised mice, these islets not only maintain their cellular organization but also regain their type of intra-islet vascularization and innervation, both parameters being different from those in rodents. Human islets maintained normoglycemia in diabetic immune-deficient mice. Moreover, human islets dictated the glycemic set-point when transplanted to the ACE of mice, i.e. in the transplanted mice the set-point was shifted from ‘mouse’ to ‘human’. By applying our humanized mouse model, we reported on the adverse effects of chronic exposure of mice to liraglutide, which led to the exhaustion of human β-cell function.
We have generated adenovirus-based vectors for transduction of human islets and developed transgenic mice expressing GCaMP3 in β-cells. Our results show that glucose promotes a significant and kinetically uniform response in all β-cells in healthy animals. In contrast, β-cells in glucose intolerant Ob/Ob mice showed only a modest Ca2+ increase in response to the sugar. Mice with diet-induced obesity/insulin resistance showed a change in synchronized Ca2+ oscillations between β-cells within the islet.
Since the ACE as such represents an immune-privileged site, it was of utmost importance to demonstrate that immune responses associated with type 1 diabetes (T1DM) can be monitored in the ACE following transplantation of islets to this site. We demonstrated that the ACE-transplanted islets in NOD-mice develop infiltration and β-cell destruction, recapitulating the autoimmune insulitis of the pancreas, and exemplify this by analyzing reporter cell populations expressing green fluorescent protein under the Cd11c or Foxp3 promoters.
Besides the Ca2+-biosensor we developed a β-cell insulin resistance biosensor based on Fox01-GFP. This allowed us to demonstrate that in young Ob/Ob mice insulin-resistant islets locally express apolipoprotein CIII, a pro-inflammatory factor that leads to hyperactivation of β-cell voltage gated Ca2+-channels through SR-BI/β1 integrin-dependent co-activation of PKA and Src, finally resulting in Ca2+-dependent β-cell death. To study β-cell function in vivo in pancreatic islets transplanted into diabetic mice, we tested different diet-intervention protocols in diabetes-prone male C57Bl/6J mice. While all protocols led to obesity and whole-body insulin resistance, only a High-Fat-High-Sucrose diet led to the development of a diabetic phenotype within 4-8 weeks of treatment, including β-cell insulin resistance and non-compensatory insulin release. Using biosensors for changes in Ca2+ handling, β-cell insulin resistance and for glucose-induced gene transcription we were able to monitor the dynamics of β-cell dysfunction during progression of diabetes development.
We previously showed that the β3 subunit of Cav (Cavβ3) regulates cytosolic Ca2+ oscillation frequency and insulin secretion under physiological conditions. We now reported that islets from diabetic mice show Cavβ3 overexpression, altered Ca2+ dynamics, and impaired insulin secretion upon glucose stimulation. Consequently, in high-fat diet (HFD)-induced diabetes, Cavβ3-deficient (Cavβ3-/-) mice showed improved islet function and enhanced glucose tolerance. Normalization of Cavβ3 expression in ob/ob islets by an antisense oligonucleotide rescued the altered Ca2+ dynamics and impaired insulin secretion. Importantly, transplantation of Cavβ3-/- islets into the ACE improved glucose tolerance in HFD-fed mice. Cavβ3 overexpression in human islets also impaired insulin secretion. We thus suggest that Cavβ3 may serve as a druggable target for diabetes treatment.
Data by us and others have shown that human islets differ from rodent islets in the structure-function-relationship. Therefore it is of utmost importance to clarify signal-transduction processes in human islets in vivo under normal conditions and why these processes do not function properly in diabetes. Our data show that following transplantation of human islets into the ACE of immune compromised mice, these islets not only maintain their cellular organization but also regain their type of intra-islet vascularization and innervation, both parameters being different from those in rodents. Human islets maintained normoglycemia in diabetic immune-deficient mice. Moreover, human islets dictated the glycemic set-point when transplanted to the ACE of mice, i.e. in the transplanted mice the set-point was shifted from ‘mouse’ to ‘human’. By applying our humanized mouse model, we reported on the adverse effects of chronic exposure of mice to liraglutide, which led to the exhaustion of human β-cell function.