We have shown that deletion of Cd39/Entpd1 results in abnormalities of glucose homeostasis and causes insulin resistance. We show that the plasma insulin levels are higher in Cd39/Entpd1−/− mice than in wild-type mice during a GTT, and islets stain strongly for insulin in Cd39/Entpd1−/− mice tissues. Glucose intolerance in Cd39/Entpd1−/− mice does not appear to be related to an intrinsic abnormality in insulin secretion from β-cells. Significant effects of ATP administration on the GTT in contrast to other nucleotides suggest involvement of P2X receptors that are uniquely activated by ATP in the expression of glucose intolerance.
mice are not obese, but leptin levels are increased. Leptin is an important anorexigenic hormone secreted from adipocytes (24
). Insulin may induce secretion of leptin (27
). Therefore, it is possible that the hyperinsulinemia of Cd39/Entpd1−/−
mice might be a cause for hyperleptinemia. The food intake of Cd39/Entpd1−/−
mice was comparable with those of wild-type mice despite the increased levels of plasma leptin. Leptin is also known to increase energy expenditure. However, calorimetric analysis of Cd39/Entpd1−/−
mice revealed decreased heat generation and energy consumption both during dark and light cycles. Respiratory ratios of Cd39/Entpd1−/−
mice were slightly lower than wild-type mice. Potentially, other tissue-specific mechanisms might exist for the regulation of energy consumption by extracellular nucleotides. Our current data, however, suggest the possibility of both leptin resistance as well as insulin resistance in Cd39/Entpd1−/−
Hyperinsulinemic-euglycemic clamp studies identified the liver as a major target organ for the effects of Cd39/Entpd1 on glucose homeostasis and insulin resistance. Immunohistochemical studies showing Cd39/Entpd1 to be expressed dominantly within the vasculature suggest that observed insulin resistance are secondary to modulation of extracellular and plasma nucleotides levels brought by loss of vascular ecto-nucleotidase activity. This is different mechanistically from the type of insulin resistance induced by a K121Q variant of PC-1 (ectonucleotide pyrophosphatase/phosphodiesterase). The latter requires direct/physical interactions between insulin receptor and PC-1 (28
). Furthermore, PC-1 is expressed in skeletal muscle cells and hepatocytes unlike the unique vascular expression of Cd39/Entpd1.
We next examined aspects of glucose metabolism in primary hepatocyte cultures of Cd39/Entpd1−/−
and wild-type mice. In general, glucose transport by hepatocytes is insulin-independent. However, expression of glucokinase is induced by insulin. We noted that the overall uptake of glucose was significantly decreased in Cd39/Entpd1−/−
mice hepatocytes. Glucose uptake by wild-type primary culture hepatocyte was also significantly decreased by treatment with extracellular ATP, albeit not to the same extent as with Cd39/Entpd1−/−
hepatocytes. ATP is known to induce hepatic gluconeogenesis (30
) and impact upon glucose release via P2X4 receptor–mediated increases in glycogenolysis (31
). Our and these published data suggest that the cellular phenotype seen in the Cd39/Entpd1−/−
mice may be directly dependent upon exposure to extracellular nucleotides. We suggest that these are differential and persistent purinergic responses in these target tissues that are impacted upon by loss of Cd39/Entpd1 in vasculature. We infer that the reduced Rd
values observed in Cd39/Entp1−/−
mice during the hyperinsulinemic-euglycemic studies are secondary due, in substantial part, to the decreased glucose uptake in hepatocytes.
Other contributions of insulin to hepatic glucose homeostasis include the suppression of glucose production. Liver-specific insulin receptor knockout mice exhibit hepatic insulin resistance (i.e., impaired insulin-mediated suppression of hepatic glucose production that is accompanied by elevated plasma insulin levels). These features are also observed in Cd39/Entpd1−/−
mice, suggesting indirect effects on insulin receptor activity (32
mice hepatocytes also show significantly higher basal glucose production than that seen in wild-type mice hepatocytes. Furthermore, the glucose production of Cd39/Entpd1−/−
mice hepatocytes is not suppressed by the treatment of insulin, unlike in the instance of wild-type mice hepatocytes, in which this is suppressed by insulin. Interestingly, ATP treatment of wild-type hepatocytes also induces heightened hepatic glucose production, even in the presence of insulin. These results confirm that extracellular ATP regulates glucose homeostasis in hepatocytes. Furthermore, Cd39/Entpd1 expressed on adjacent cells, such as endothelial cells, is likely to regulate insulin signals in the hepatocyte by modulating extracellular nucleotide levels in the immediate microenvironment in vivo. It is worthy of mention that vascular endothelial-specific insulin receptor knockout mice do not exhibit insulin resistance (33
). We propose that insulin resistance observed in Cd39/Entpd1−/−
mice is not directly caused by modulation of vasculature functions but involves other factors (e.g., paracrine fluxes of extracellular nucleotides that impact target tissues such as the hepatocyte).
Activation of cellular signaling cascades by proinflammatory cytokines such as tumor necrosis factor-α and interleukin-1, free fatty acids, or nucleotides (14
) can also induce cellular activation via inflammatory kinases. Activation of inflammatory kinases, such as c-JNK, also cause aberrant serine phosphorylation of IRS. This specific form of serine phosphorylation prevents consequent tyrosine phosphorylation by insulin receptors and association of phosphoinositide 3-kinases, leading to impaired insulin signal transduction (34
To analyze the molecular mechanisms for insulin resistance of Cd39/Entpd1−/−
mice, we analyzed tyrosine phosphorylation of IRS-1 and -2. Insulin-stimulated tyrosine phosphorylation of IRS-2 was significantly decreased in Cd39/Entpd1−/−
mice liver. In addition, phosphorylation of c-JNK induced by administration of ATP was also significantly increased in Cd39/Entpd1−/−
mice liver. These data suggest that insulin resistance of Cd39/Entpd1−/−
mice is associated with increased activation of hepatic c-JNK/SAP by extracellular ATP. This likely results in aberrant serine phosphorylation and consequently decreased tyrosine phosphorylation of IRS-2. Similar patterns of aberrant IRS-2 phosphorylation have been noted in other insulin resistance states (36
Furthermore, several features observed in Cd39/Entpd1−/−
mice are also seen in IRS-2−/−
mice: both show elevated plasma insulin levels and have normal fasting blood glucose levels with impaired glucose homeostasis in the liver. IRS-2−/−
mice, however, show increases in blood glucose levels with advancing age due to lack of compensatory β-cell hyperplasia (37
Other cellular candidates for mediating insulin resistance of Cd39/Entpd1−/−
mice include immune cells such as macrophages in adipose tissues and the Kupffer cells of the liver, as alluded to above (35
). Activation of purinergic signaling pathways is already known to induce secretion of cytokines from both immune and endothelial cells (38
). We show that serum proinflammatory cytokine levels (interleukin-1β, interleukin-6, interferon-γ, and tumor necrosis factor-α) are significantly higher in Cd39/Entpd1−/−
mice than in wild-type mice. Accordingly, hypersecretion of select cytokines by monocytes and other cells also might contribute, at least in part, to the development of insulin resistance in Cd39/Entpd1−/−
Our data suggest that insulin resistance in Cd39/NTPDase1−/− mice is associated with disordered extracellular nucleotide signaling that directly impacts important metabolic pathways in hepatocytes via aberrant IRS-2 phosphorylation. However, we do not exclude the possibility that deletion of Cd39 might also contribute to insulin resistance in a somewhat more indirect manner via the induction of inflammatory cytokines. The relative contributions of these two nonexclusive mechanisms remain to be determined.
We have already reported on other vascular and immune abnormalities in Cd39/Entpd1−/−
mice. This described phenotype includes disordered thromboregulation, aberrant inflammatory responses, impaired angiogenesis and regeneration of the liver, impaired function of regulatory T-cells, and predisposition to diabetic renal diseases with increased proteinuria and hypertension (3
). To these manifestations of Cd39/Entpd1 deletion, we now add metabolic consequences.
In conclusion, Cd39/Entpd1 expressed by the vasculature and by immune cells serves as an important modulator of hepatic carbohydrate metabolism. The pathogenetic mechanism involves failure to control extracellular nucleotide fluxes that both directly and indirectly impact insulin responsiveness.