In the current work, we identify T-cadherin in association with insulin granules in pancreatic
-cells, and link T-cadherin functions to the insulin secretory machinery responsible for the second phase of insulin release. We correlate this finding with the development of glucose intolerance in T-cadherin deficient mice fed a normal diet without an apparent impairment of peripheral insulin sensitivity. Assessing insulin resistance and β-cell function prior to the onset of metabolic dysfunction assured that we evaluated the effect of T-cadherin loss-of-function on these parameters rather than recording secondary effects from prolonged hyperglycemia.33-35
Our combined data suggest that T-cadherin primarily affects insulin secretion, which in turn contributes to dysregulation of glucose homeostasis in the Tcad KO mice. Clearly, our work does not exclude the possibility that T-cadherin in peripheral tissues contributes to regulation of insulin sensitivity in other circumstances, such as during high-fat diet induced stress.
Cadherin association with secretory vesicles in β-cells is unprecedented and was asserted through several independent lines of experimentation. First, affinity-purified, highly specific antibodies detected T-cadherin in association with insulin- containing granules in WT, but not in the Tcad-KO mice. Second, T-cadherin immuno-electron microscopy showed T-cadherin in association with the dense-core insulin granules. Third, mRFP-tagged T-cadherin expressed in CHO cells resided on the plasma membrane, while T-cadherin protein transcribed from the same cDNA was targeted to insulin granules in primary Æ-cells. The mRFP-labeled T-cadherin protein expressed in β-cells co-migrated with GFP-labeled Phogrin, an insulin granule marker, upon glucose stimulation. This suggests that T-cadherin, in contrast to its plasma membrane localization elsewhere, is differentially processed and targeted for association with insulin granules in β-cells. Due to limitations of optical microscopy resolution and electron microscopy sample preparation, we are unable to determine if T-cadherin is expressed on all or only a subset of insulin granules (e.g., the reserve pool), which may be assessed in the future by novel super-resolution microscopy techniques (nanoscopy).36
This could further shed light on the function of T-cadherin in the insulin secretory process.
The association of T-cadherin with insulin granules is dispensable during pancreatic development: we detect no overt defects in pancreatic architecture and insulin availability in T-cadherin-deficient adult mice compared with the WT. However, we demonstrate that T-cadherin on insulin granules contributes to the insulin-release properties of β-cells. Insulin release following glucose stimulation of primary Tcad-KO pancreatic islets is more than 50% less effective than from islets isolated from corresponding WT mice.
Insulin granules in β-cells comprise at least two pools based on availability, the readily releasable pool close to the plasma membrane and the reserved pool localized deeper inside the cells.30,37,38
Upon physiological fuel stimulation (glucose, etc.) of Æ-cells, membrane depolarization and concomitant Ca2+
influx lead to the immediate first phase of insulin secretion in which the readily releasable granules fuse with the plasma membrane to release insulin content. Fuel driven secretion is achieved by producing ATP, leading to ATP-dependent closure of KATP
-channels, resulting in membrane depolarization. This is followed by the second, prolonged phase of insulin release, requiring energy-dependent priming and recruitment of the reserved vesicle pool to the cell surface.32,39-44
Dissecting the specific role of T-cadherin in the insulin release process, we show that acute insulin release remains intact after stimulation of primary Tcad-KO β-cells by direct depolarizing action of KCl in vitro and by glucose-driven ATP production in vivo. However, we provide in vitro and in vivo evidence that prolonged glucose-dependent insulin secretion is impaired by the Tcad-KO mutation.
How could T-cadherin associated with insulin granules affect insulin release properties? First, prolonged insulin release is dependent on ATP generation, and T-cadherin may directly or indirectly affect energy availability. We do not favor this interpretation as glucose stimulated first phase insulin secretion, which depends on ATP closure of KATP-channels to cause β-cell depolarization, as described above, is normal in Tcad-KO mice. Moreover, comparative microarray gene expression data from isolated WT and Tcad-KO islets did not identify reductions in genes regulating metabolic pathways (not shown).
Second, T-cadherin associated with insulin granules is a candidate to confer trafficking of the reserved vesicles to the plasma membrane through interactions with protein complexes bound to microtubules. Insulin granule exocytosis depends largely on kinesin motor complexes that traffic along microtubules to the plasma membrane.32,39
Recently, calsyntenins, members of the cadherin superfamily, were localized to secretory granules in the anterior pituitary and the pancreatic α-cell.45
In parallel to calsyntenin functions in excitatory neurons, T-cadherin could promote interactions with kinesin to regulate anteriograde vesicle transport.46
Finally, T-cadherin may serve a role in priming the reserved insulin granules for membrane fusion, a process necessary for glucose-augmented prolongation of insulin release. Different proteins have recently been associated with second phase insulin secretion, including H+
and the Rho family GTPase Cdc42.43
T-cadherin, in the context of cell migration, is engaged in RhoA and Rac signaling,49
thus a plausible function in β-cells is the association with the granular Cdc42-caveolin1-VAMP2 complex that regulates prolonged insulin release.50-52
Alternatively, T-cadherin overexpression alters Akt/Gsk3Æ signaling leading to Æ-catenin redistribution in endothelial cells.53
This may mean that T-cadherin is involved in actin cytoskeleton reorganization in β-cells, which is critical for insulin granule exocytosis through cdc42-dependent mechanisms.54
Future experiments will need to provide mechanistic insights into T-cadherin’s functions in the insulin release process.
Continuously limited availability of insulin may affect the development of progressive glucose intolerance of the Tcad-KO mice. However, a diverse range of other factors also influences the regulation of glucose metabolic control and could contribute to metabolic abnormalities in the Tcad-KO mice. T-cadherin expression in heart, muscle and blood vessels14,15
( and ) could affect overall metabolic regulation by sequestering the adipocyte-secreted metabolic hormone APN.20
Indeed, in Tcad-KO mice, APN fails to associate with the vasculature and insulin-sensitive heart and skeletal muscle, and APN serum levels are more than five-fold increased over WT,14,55
which in theory would make APN abundantly available for interactions with other receptors. Even with a possible overstimulation of other APN-receptors, Tcad-KO mice are, however, glucose intolerant on a normal diet, while APN-KO mice show no deficits under baseline feeding conditions.56
Studies addressing the role of APN in β-cell functions have led to inconsistent results.61-64
In the pancreas in vivo, we detect APN in association with T-cadherin only on the pancreatic vasculature where this interaction may indirectly affect signals regulating β-cell function. Islet vascularization is a critical factor for β-cell function.65
Thus, T-cadherin-APN interactions in the pancreatic vasculature may contribute to metabolic control in addition to direct actions of T-cadherin in insulin release. We note that our in vitro model identified defective insulin secretion from isolated Tcad-KO islets in the absence of vascular or other cellular components or APN. Still, defective endothelium-islet cell interactions could lead to changes in extracellular matrix composition that may have the potential to affect insulin secretion even in vitro.66
Nevertheless, the data presented here suggests that defective insulin secretion reflects a separate function of intracellular T-cadherin that does not seem to require direct interactions with APN. The possible misbalance of T-cadherin/APN functions in islets and other organs will need to be considered in separate studies of the metabolic phenotype of the Tcad-KO mice.
In summary, our work identifies a novel and unexpected localization of T-cadherin on insulin granules that affects second phase insulin release from β-cells, and thus, may contribute to the glucose intolerance observed in Tcad-KO mice. Although the baseline metabolic phenotype of the T-cadherin KO mice somewhat parallels that of mice deficient for APN,56,67
our data do not support a model in which T-cadherin binds APN to regulate pancreatic functions. However, our studies do not rule out the possibility that T-cadherin—APN interactions contribute to metabolic control by regulating insulin release indirectly or confound functions of other organs, such as liver. Defining the molecular interactions of T-cadherin in the pancreas and in other insulin-sensitive tissues will be an important contribution to understanding the role of this unique molecule in metabolic regulation.