Acute knockdown of the insulin receptor or its substrates Irs1 and 2 in 3T3-L1 adipocytes suppresses adiponectin production

Loss of function of the insulin receptor (INSR) in humans produces severe insulin resistance. Unlike “common” insulin resistance, this is associated with elevated plasma levels of the insulin-sensitising, adipose-derived protein adiponectin. The underlying mechanism for this paradox is unclear, and it is at odds with the acute stimulation of adiponectin secretion reported on insulin treatment of cultured adipocytes. Given recent evidence for ligand-independent actions of the INSR, we used a lentiviral system to knock down Insr or its substrates Irs1 and Irs2 conditionally in 3T3-L1 murine preadipocytes/adipocytes to assess whether acute loss of their expression has different consequences to withdrawal of insulin. Efficient knockdown of either Insr or Irs1/2 was achieved by conditional shRNA expression, severely attenuating insulin-stimulated AKT phosphorylation and glucose uptake. Dual knockdown of Irs1 and Irs2 but not Insr in preadipocytes impaired differentiation to adipocytes. Acute knockdown of Insr or both Irs1 and Irs2 in adipocytes increased Adipoq mRNA expression but reduced adiponectin secretion, assessed by immunoassay. Knockdown sustained for 14 days also reduced immunoassay-detected adiponectin secretion, and moreover induced delipidation of the cells. These findings argue against a distinct effect of Insr deficiency to promote adiponectin secretion as the explanation for paradoxical insulin receptoropathy-related hyperadiponectinaemia.


Results and Discussion
3T3-L1 preadipocyte lines were generated allowing knockdown of Insr or Irs1/2 by doxycycline-dependent expression of shRNA. Clonal cell lines were screened for knockdown efficiency, and subsequent studies undertaken using the most efficient lines. After differentiation to adipocytes highly efficient knockdown of Insr or Irs1/2 mRNA and protein was induced by 72 hours of doxycycline treatment (Fig. 1A,B). Knockdown after differentiation did not affect cellular lipid content (Fig. 1I) but severely attenuated insulin-induced Akt phosphorylation (Fig. 1C) and 2-deoxyglucose uptake (Fig. 1D). Insulin-dependent glucose uptake depends upon Akt, and half maximal uptake requires an Akt phosphorylation level of only 5-22% of its maximum 11 . Thus severe blunting of this response confirms potent Insr and Irs1/2 knockdown.
One challenge when using shRNA to study gene function in adipocytes is that some genes of interest are also involved in preadipocyte differentiation, and their stable knockdown precludes efficient adipocyte generation. Early studies using genetically engineered 3T3 cells suggested that Insr function is required for adipogenesis, although prolonged passage of cells may have reduced the differentiative capacity of the cells 12 . Recent studies using cre-mediated gene deletion in murine primary brown preadipocytes have instead suggested that Insr and Igf1r play redundant roles in early adipogenesis, and that the Insr is thus dispensable for the process 13 .
Our cellular model of inducible Insr knockdown enabled us to re-address this question in 3T3-L1 cells. In keeping with previous reports (e.g. 14 ) Insr expression was up-regulated during differentiation (Fig. 1E), while Igf1r expression decreased (Fig. 1E). Moreover, expression of Igf1r was not detectable after fractionation of lipid-rich cells to remove residual undifferentiated cells (Fig. 1F). On doxycycline treatment of preadipocytes Insr protein was reduced after 12 hours, near complete knockdown being achieved at 72 hours (Fig. 1G). Knockdown induced between day − 3 and day 6 of differentiation only modestly impaired triglyceride accumulation (Fig. 1H), while Irs1/2 knockdown impaired lipidation more severely (Fig. 1H). Insr knockdown for 14 days after adipocyte differentiation led to striking delipidation of the cells (Fig. 1J).
These findings suggest that in the 3T3-L1 adipocyte cell line, as in murine brown primary preadipocytes 9 , Insr plays a predominant role only in the later phase of adipogenesis, when it is highly expressed relative to Igf1r. Indeed, although Insr knockout mice die before day 3 of postnatal life with reduced fat cell mass, adipocytes are detectable, indicating that the role of the Insr in adipogenesis in vivo, too, is not obligate 12 . The more deleterious effect of Irs1/2 knockdown is consistent with previous findings 15 , and may be accounted for by their involvement in both Insulin and IGF1 signalling.
Our study was primarily motivated by the unexplained discordance in patients with loss of Insr function between elevated adiponectin and severe insulin resistance 5,6 . We thus sought to use our model of conditional Insr deficiency to test whether non ligand-dependent actions of the Insr may be important for regulation of adiponectin secretion. After inducing knockdown in differentiated 3T3-L1 adipocytes, secreted adiponectin was measured over 24 hours using a DELFIA assay and immunoblotting. Both Insr and Irs1/2 knockdown reduced adiponectin secretion assessed by immunoassay ( Fig. 2A), although the effect was not apparent in non-denaturing, non-reducing immunoblots, where the complex higher order structure of adiponectin renders interpretation more complex (Fig. 2B). AdipoQ mRNA, encoding adiponectin, was increased in adipocytes by Insr knockdown, however the difference between Irs1/2 knockdown cells and doxycycline-free controls was not significant (Fig. 2C).
Some previous data suggest that the acute increase in adiponectin secretion seen on insulin treatment is transient and induced by altered endoplasmic reticulum redox tone 8 . It thus remains possible that increased AdipoQ mRNA is more relevant to the in vivo setting, in keeping with reports that in humans low plasma adiponectin corresponds to low adipose ADIPOQ mRNA 16,17 . Knockdown of either Insr or Irs1/2 for 2 weeks in adipocytes once again decreased adiponectin secretion as assessed by immunoassay (Fig. 2D), with no difference discerned by immunoblotting (Fig. 2E). AdipoQ mRNA expression showed no significant response to Insr knockdown, but was modestly increased by Irs1/2 knockdown (Fig. 2F). These findings argue against the hypothesis that insulin has divergent acute and long-term effects on adiponectin secretion.
Our findings do not support the notion that the hyperadiponectinaemia of insulin receptoropathy is explained by consequences of INSR deficiency on adipocyte-autonomous adiponectin expression or secretion, however are in keeping with a preponderance of prior studies assessing the consequences of insulin stimulation of adipocytes. The human biochemical paradox thus remains unexplained. Culture conditions used may not adequately mimic the in vivo cellular milieu, or the adipocytes studied may not represent the adipose depot driving the in vivo phenomenon 22 . Alternatively, loss of INSR function may affect adiponectin levels indirectly through alteration of adipocyte turnover. Further insights may require study of different adipose depots from patients with loss of INSR function.
Cell culture. 3T3-L1 preadipocyte maintainance and differentiation were performed as previously described 19 . Experiments were undertaken at day 7 of differentiation unless otherwise indicated. Insulin stimulation studies and glucose uptake assays. For insulin stimulation studies, cells were washed twice with warm PBS and serum starved in serum-free DMEM medium containing 0.5% bovine serum albumin 16 hours before insulin stimulation. Deoxyglucose uptake was assessed on day 10 of differentiation in 12-well plates essentially as previously described 20 .
Statistical Analysis. Data are presented as mean ± standard error of the mean (SEM) from three independent experiments. Paired two-tailed Student's t-test were used to determine statistical significance and were calculated using GraphPad Prism.