Reproductive Tissues Maintain Insulin Sensitivity in Diet-induced Obesity

Sheng Wu; Sara Divall; Fredric Wondisford; Andrew Wolfe

Disclosures

Diabetes. 2012;61(1):114-123. 

In This Article

Discussion

That states of extreme positive energy balance and peripheral insulin resistance result in reproductive dysfunction is becoming increasingly clear. To investigate the response of reproductive tissues to hyperinsulinemia, we used a mouse model of infertility associated with DIO.

Initial analysis demonstrated that reproductive tissues required higher doses of insulin to activate downstream signaling pathways than do the classic energy storage tissues, suggesting that reproductive tissues were significantly less sensitive to insulin than classical energy storage tissues (Fig. 3). These initial studies were performed in lean female mice and suggest that fundamental tissue-specific differences in insulin sensitivity exist. Under normal physiologic conditions, insulin signaling in the gonadotropin-releasing hormone neuron and pituitary does not play an important role in reproductive function, since deletion of the insulin receptor in these neurons have no impact on fertility.[2,14] A role for pathologic insulin signaling in states of insulin excess could occur, however, since insulin signaling was activated in the reproductive tissues at higher doses (Fig. 3). Consistent with this assertion, mice on 60% HFD for 12 weeks develop hyperinsulinemia associated with infertility and elevated luteinizing hormone and testosterone levels.[2]

Curiously, the hypothalamus exhibited no change in AKT or IRS1 phosphorylation after insulin at any time point examined (Fig. 3I and Supplementary Figs. 1and 4). The hypothalamus is a putative target of insulin in feeding behavior and discrete nuclei, or whole hypothalami, as previously shown in male rats and mice, are activated by insulin.[15–17] However, these studies generally used intracerebroventricular infusions of insulin or used much higher peripheral doses of insulin than were used in our studies performed in female mice. Alternatively, the effects of insulin in the hypothalamus may have been masked by an abundance of nonresponse cells in the fragment. In DIO mice, there was a blunted response to insulin signaling at the level of AKT in both liver and muscle demonstrating classical insulin resistance (Fig. 5D and E). In contrast, the pituitary and ovary exhibited elevated basal levels of pAKT and were not resistant to insulin activation of AKT in the DIO mice (Fig. 5A and B). In the pituitary, the significance of AKT activation is not known. While insulin administration causes a robust stimulation of AKT in a gonadotrope cell line,[18] whether AKT signaling plays a role in luteinizing hormone secretion remains to be determined. In the ovary, AKT activation has been found to play a role in insulin induction of 5α-reductase gene expression[19] and 17α-hydroxylase activity,[20] leading to an increase in ovarian androgen biosynthesis. Interestingly, polymorphisms in the Akt2 gene have been found to be associated with PCOS in women.[21] Insulin and IGF-I–induced progesterone production via MAPK pathways in the human ovary have also been described,[22] with insulin stimulating the p38 family of MAPKs to induce this effect. We did not see an effect of insulin on ERK activation in the ovary but did not investigate for activation of the p38 family of MAPKs in the mice; thus, lack of activation of the entire MAPK pathway cannot be excluded by our studies.

To explore why the pituitary and the ovary maintain insulin sensitivity in the obese state, we measured activation of the IRS proteins (IRS1 and IRS2). Pituitary and ovary exhibited an elevation in basal pTyr-IRS2 levels in DIO mice compared with lean mice (Fig. 6E and F) that was similar to the tissue-specific pattern of AKT phosphorylation. In contrast with the liver and muscle, insulin was able to induce a further increase in pTyr-IRS2 levels in the pituitary and ovary from DIO mice (Fig. 6). Phosphorylation events were mediated by the insulin receptor—not the IGF-I receptor—since increases in pituitary pAKT or pTyr-IRS2 did not occur in PITIRKO mice (Figs. 5A and 6E). However, signaling characteristics in the ovary, liver, and muscle were nearly identical to those seen in WT mice. Our study is in agreement with phosphorylation patterns seen using a paradigm of chronic treatment with either insulin or human chorionic gonadotropin/insulin in rats to mimic chronic hyperinsulinemia.[23,24] An increase in the IRS–PI 3-kinase/pAKT signaling pathway in whole ovary extracts was observed, while the liver and muscle became insulin resistant as indicated by attenuated insulin-induced pAKT levels. Others have reported a female rat model of hyperinsulinemia that exhibited a distinctly exhibited a distinctly PCOS appearance that was similar to our model of DIO-related infertility with high luteinizing hormone levels.[23,24]

Interestingly, pTyr-IRS1 levels in the pituitary were not changed by insulin treatment in lean or DIO mice (Fig. 6A and Supplementary Fig. 2), although this may be a function of the paucity of IRS1 in the pituitary as assessed by Western blot or Luminex assay (Fig. 6I, J, and K). Analysis of the IRS2 KO mice has also identified a similar predominant role of IRS2 in the pituitary without any detection of pTyr-IRS1 using pooled pituitaries.[3] At the 10-unit dose of insulin, activation of IRS1 signaling was observed in the pituitary of both WT and PITIRKO mice, suggesting IGF-I receptor activation (Supplementary Fig. 3).

While there were significant differences in the pituitary in the phosphorylation of IRS1 and IRS2 to insulin, in the ovary, the two IRS proteins exhibited similar responses to insulin in both lean and DIO mice (Fig. 6A, B, E, and F). Insulin stimulated an increase in ovarian pTyr-IRS1 and pTyr-IRS2 in lean mice. Compared with those in lean mice, basal levels of ovarian pTyr-IRS1 and pTyr-IRS2 were elevated in DIO mice, with a further increase in levels of ovarian pTyr-IRS1 and pTyr-IRS2 after insulin administration. This preservation of insulin signaling in the setting of peripheral insulin resistance suggests that ovarian IRS is not susceptible to the mechanisms proposed to underlie tissue resistance to insulin, including serine phosphorylation and degradation of IRS.[8] These data suggest that differences at this proximal level of the insulin-signaling pathway between the ovary and pituitary and the energy storage tissues underlie the retained sensitivity to insulin in obese mice.

The DIO mice are chronically hyperinsulinemic, which differs from studies that used acute or chronic insulin stimulation as a model.[25,26] In the latter study, lean rats were injected with insulin for 22 days. However, these rats retained cyclicity, which may be a result of unchanged gonadotropin-releasing hormone sensitivity of the pituitary observed in this model. It is not clear whether this is a species-specific difference or due to the differences between the models of hyperinsulinemia (DIO versus insulin injection). However, recent work in a DIO rat model[27] showed that 120 days of HFD was associated with elevated circulating luteinizing hormone levels, elevated basal pAKT levels, and a retained responsiveness to acute insulin treatment—all of which were observed in our mouse DIO model (Figs. 4B and 5).[2] In contrast to our model, insulin resistance in the rat ovary developed after 180 days on HFD and luteinizing hormone levels normalized. The authors of the study also measured interleukin-1B, tumor necrosis factor-α, and pJNK expression in the ovary and found no increase in DIO versus lean females at 120 days but a significant increase after 180 days in DIO rats.[27] These obesity-induced stress responses have been proposed as mechanisms for the development of insulin resistance in other tissues,[8] and suppression of JNK signaling in liver and muscle has been shown to ameliorate insulin resistance in obese mice.[28,29] The time course of cytokine expression and insulin resistance in the ovary observed by Akamine et al.[27] supports our contention that a lack of macrophage infiltration combined with a lack of increase in cytokine signaling in the pituitary and ovary may contribute to retained insulin sensitivity in the DIO mouse (Fig. 7). As our model exhibits retained insulin signaling with estrous acyclicity, this difference suggests that excessive insulin signaling may lead to anovulation and associated lack of estrous cycling. The development of ovarian insulin resistance observed by Akamine et al. may be a function of species differences (rat versus mouse). Alternatively, there are clearly time-dependent changes that occur between 120 and 180 days in the rat that could emerge with extended analysis of the DIO mice. We favor the former hypothesis, since we have observed retained insulin signaling in the ovaries and pituitaries of mice on an HFD for over 6 months (data not shown), suggesting that the protection from insulin resistance in these tissues may be more durable in the mouse than in the rat.

The lack of elevated JNK signaling in DIO mice relative to controls (Fig. 7B) differs from the findings of a recent study that proposed a role for pituitary JNK signaling in the regulation of glucose homeostasis.[30] These investigators noted an increase in JNK-induced c-Jun phosphorylation in DIO male mice relative to chow-fed controls. These differences are likely due to strain and sex differences between our studies.

We focused on PI 3-kinase AKT, which is thought to more closely mediate metabolic signaling, whereas the MAPK pathway mediates growth and development,[31] and because our studies did not show significant changes in ERK phosphorylation between conditions. This does not preclude important roles for ERK signaling in reproductive tissues, and in fact, ERK mediates essential signaling events in both the pituitary[32] and ovary[33] as a result of G-protein receptor activation. In addition, a role for non-ERK MAPK signaling has been implicated in ovarian progesterone synthesis.[22] Impaired glucose uptake and diminished accumulation of lactate were observed in granulosa cells of women with anovulatory PCOS compared with control women, suggesting that granulosa cells in women with PCOS exhibit resistance to the metabolic effects of insulin.[34] While our studies did not distinguish between signaling in theca and granulosa cells, our results would be consistent with retained signaling in the theca cells. Therefore, the retained insulin responsiveness in DIO females (Figs. 5 and 6) may be manifest at the level of the theca cell even in the face of resistance in the granulosa cells, resulting in increased steroidogenesis.[35–37]

Persistent ovarian sensitivity to insulin in the context of peripheral insulin resistance has been implicated in the pathophysiology of PCOS. Human studies indicate that ovarian steroidogenesis involves AKT activation[18,19] and that steroid metabolism is preserved in women with hyperinsulinemia and PCOS.[2,38,39] Our results, which demonstrate maintained insulin-induced Akt phosphorylation in the ovary in the setting of hyperinsulinemia, suggest that persistent AKT phosphorylation may be contributing to the pathologic steroidogenesis in women with PCOS. Furthermore, women with type 1 diabetes, who are often treated with supraphysiologic doses of insulin on a chronic basis, have a two- to threefold increased risk of development of PCOS. It is noteworthy that this subset of women with PCOS has hyperandrogenemia, implying abnormal ovarian steroid metabolism, without evidence of elevated antimullerian hormone (produced by small ovarian follicles), luteinizing hormone-to-follicle-stimulating hormone ratio, or sulfated dihydroepiandrosterone as seen in PCOS.[40]

Hence, it is likely that a hormonal milieu that differs between PCOS and type 1 diabetes is responsible for disruption of ovarian and adrenal function and that persistent AKT signaling cannot alone account for the ovarian dysfunction seen in PCOS. As a further indication of the link between hyperinsulinemia and ovarian steroidogenesis, hyperinsulinemic states induced by hyperinsulinemic-euglycemic clamps lead to increased ovarian androgen production in lean women with PCOS, demonstrating an acute induction of ovarian steroidogenesis by insulin.[41]

In summary, we have shown that the insulin responsiveness of the pituitary and ovary is uniquely preserved in mice with peripheral insulin resistance (Fig. 8). Our study is the first to suggest that the preservation of insulin signaling in the pituitary and ovary of the DIO mouse occurs at a proximal point in insulin signaling, i.e., at the level of the IRS proteins. Further delineation of these pathways will provide insight into the role of hyperinsulinemia in the development of PCOS.

Figure 8.

A model summarizing the insulin-signaling pathways in the liver, muscle, and pituitary of lean or DIO mice. Arrows indicate active signaling pathways, and an X over the arrow indicates blocked signaling pathways. PI3K, PI 3-kinase.

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