Cannabinoids Inhibit Insulin Receptor Signaling in Pancreatic β-Cells

Wook Kim; Máire E. Doyle; Zhuo Liu; Qizong Lao; Yu-Kyong Shin; Olga D. Carlson; Hee Seung Kim; Sam Thomas; Joshua K. Napora; Eun Kyung Lee; Ruin Moaddel; Yan Wang; Stuart Maudsley; Bronwen Martin; Rohit N. Kulkarni; Josephine M. Egan

Disclosures

Diabetes. 2011;60(4):1198-1209. 

In This Article

Discussion

In contrast to adipose tissue, liver, and muscle, the presence and effects of CB1Rs in β-cells have been inconclusive. Here, we demonstrate that β-cells contain all of the components of a self-contained ECS: CB1R, the necessary enzymes for catalyzing EC biosynthesis and degradation, and the capacity to generate ECs in response to glucose stimulation and depolarization, even when isolated from the pancreas. We used several antibodies,[33,34] including the L15 antibody to the C-terminus of CB1R that exhibits the expression pattern most consistent with in-situ hybridization[33] as well as qRT-PCR of laser-captured β-cells from islets, to affirm that CB1Rs, but not CB2Rs, are present on β-cells of mice and men. Bermúdez-Silva et al.[14] have very recently written that using the L15 antibody they also found a CB1R signal with insulin costaining; after using a commercially available CB1R antibody, they reported that CB1Rs were mainly expressed in α-cells.[10] CB2Rs are absent from α- and β-cells. Most reports, including this study, found that β-cells contain EC synthetic and degrading enzymes.[9–11,14]

Since at least 1979, insulin mediators, also referred to as insulin second messengers, are known to be generated from lipid precursors present on plasma membranes in response to IR activation and consequent downstream phospholipase activation.[35] We are now suggesting that ECs, also generated from lipid precursors in β-cells, influence IR activation (Supplementary Fig. 3). This is especially relevant because insulin concentrations would be expected to be at their highest levels surrounding β-cells and therefore the islet ECS potentially evolved to prevent an over-exuberant β-cell IR signaling cascade. Favoring this view are our findings that in various β-cell lines, isolated islets and a mouse model of diabetes CB1R signaling counteracts the effects of insulin on β-cells by preventing IR autophosphorylation and downstream signals. This finding was not unique to pancreatic β-cells because activation of CB1Rs also impeded exogenous insulin-stimulated IR autophosphorylation in non-insulin-secreting cells.

We also found that Gαi, which is involved in the regulation of insulin secretion[36] and β-cell proliferation,[37] mediates the inhibitory effect of CB1R activation on IR activity by its association with IR. CB1R activation increased Gαi3 activity and Gαi3/IR association was strengthened by CB1R activation and substitution of Tyr1158/1162/1163 residues of IR with Ala, which, conversely, was weakened by suppression of CB1R activity and by insulin. Furthermore, knockdown of Gαi3 by siRNA abolished the ability of CB1R to inhibit exogenous insulin-stimulated IR autophosphorylation and β-cell proliferation.

These results suggest a functional and physical crosstalk between CB1R and IR signaling upon IR autophosphorylation in a Gαi-dependent manner. Given that binding of insulin to the extracellular α-chains of IR causes a change within the quaternary structure of IR that places the phosphorylation sites of one β-chain within reach of the active site of the other β-chain and that results in autophosphorylation at Tyr1158/1162/1163 residues in the activation loop of the β-chains,[38,39] we propose that Gαi3 activated by CB1R associates with unphosphorylated IR at the Tyr1158/1162/1163 residues, preventing a conformational change that secures the activation loop in a catalytically competent configuration upon ligand binding.

Because these receptors are found to be present within caveolae, a cholesterol-rich microdomain that performs a number of signaling functions,[40] it is possible that the closeness of the CB1Rs causes them to be involved in modulating IR-mediated signaling. Although the detailed molecular mechanism underlying CB1R and Gαi3 as regulatory components in the IR signaling pathway awaits further exploration, collectively, our results imply that the alteration in IR activity by CB1Rs is a reflection of a direct inhibition of IR autophosphorylation in a Gαi-dependent manner and that ECs directly regulate proliferation through activation of CB1Rs expressed in β-cells. Through these actions, CB1Rs are likely to set a threshold level for IR-mediated responses, which depends on the level of expression or activation of CB1R. Alteration in IR activity by CB1Rs may additionally be due to the change in autocrine activation because of altered insulin secretion. CB1R-mediated suppression of insulin secretion in a Ca2+-dependent manner has been reported;[12,41] however, there are also reports to the contrary.[9,10,15,42,43]

We demonstrate the therapeutic advantage of CB1R modulation in a type 2 diabetic condition. Inhibition of CB1R activity in db/db mice led to reduced blood glucose and increased β-cell proliferation, coupled with enhanced IR signaling. There is also evidence that insulin itself reduces glucose-stimulated EC synthesis in β-cells, which would serve as a negative feedback loop to reduce intraislet EC levels.[9] This would logically mean that when IR function is reduced, as in type 2 diabetes, such a robust feedback would also be impaired, leading to nonphysiologic EC levels in islets (in addition to in fat and liver) and consequent CB1R-mediated β-cell dysfunction through further impeding IR activity. Blocking CB1Rs would therefore be expected to improve β-cell function in db/db mice, as we found. CB2R antagonism had no such effects.

An inadequate expansion of β-cell mass or failure of the existing β-cell mass to compensate for the changing insulin demand are hallmarks of type 2 diabetes, and these prominent features may result from defective IR signaling.[1–4,27–29] Therefore, our data should result in the resumption of attention being paid to ECS as a key factor in β-cell physiology and may lead to development of a new therapeutic strategy aiming to preserve better functioning β-cells.

EC levels, not only in the circulating blood but also in the pancreas, are said to be elevated in diabetes and obesity,[9,11,44,45] and elevated EC levels are associated with increased DAGLα and decreased FAAH levels in β-cells.[11] Thus, it is possible that increased EC tone (due to increased EC synthesis, receptor expression or activity) affects the well-described glucose-unresponsiveness of β-cells and the development of insulin resistance by impeding IR autophosphorylation in insulin-sensitive tissues. Indeed, AEA was recently found to impair insulin-stimulated AKT phosphorylation and decrease glucose uptake in skeletal muscle cells,[7] and CB1R antagonism enhanced insulin responsiveness of skeletal muscle.[8]

In addition, pharmacologic blockade of CB1R in obese fa/fa Zucker rats decreased blood glucose levels and preserved β-cell mass,[46] and eliminating CB1Rs in liver protected against fatty liver and improved glucose tolerance and insulin sensitivity in high-fat diet–fed mice;[23] IR function in those mice was not investigated. Peripheral, but not central, blockade of CB1R was recently reported to improve overall insulin sensitivity and glucose homeostasis[26] and a non-brain-penetrant CB1R antagonist improved glucose homeostasis, insulin sensitivity, and fatty liver in a weight-independent manner.[25] This is a very important point, because a centrally acting CB1R antagonist, rimonabant, used for treating obesity, was removed from patient use because of potentially life-threatening psychiatric problems.[47] Therefore, CB1R antagonists with poor brain penetrance might be useful therapies in type 2 diabetes where they would be expected to lessen insulin resistance in skeletal muscle and liver, ameliorate or prevent fatty liver, and improve β-cell function/proliferation.

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