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

Results

DIO Mice Exhibit Insulin Resistance

We measured fasting serum insulin, leptin, glucagon, and IGF-I levels in mice fed an HFD and in chow-fed control littermates. Insulin and leptin levels were elevated in DIO mice relative to controls as reported previously (Fig. 1A and B).[2,7] DIO mice had lower serum glucagon levels than did controls (Fig. 1C); IGF-I levels were unchanged in DIO mice relative to those in lean controls. (Fig. 1D). DIO mice exhibited glucose intolerance after D-dextrose injection in as little as 15 min (Fig. 1E).

Figure 1.

WT lean and DIO female mice were fasted overnight, and biochemical data are shown. A: Insulin. B: Leptin. C: Glucagon. D: IGF-I. E: Glucose tolerance test (2 g/kg i.p. D-glucose injected; glucose levels measured at different time points). n = 4–6/group. *,a,bSignificant differences (P < 0.05). NS, nonsignificant. GTT, glucose tolerance test.

Blood Glucose Levels After Insulin Injection

We measured the time course of the response of circulating glucose levels to insulin in order to identify a time at which we could measure early insulin-signaling events independent of insulin-induced hypoglycemia. We injected overnight-fasted lean mice with insulin (0.5, 1, 1.5, and 3 units/kg body wt) and measured glucose at 10, 30, and 45 min after injection. Glucose levels declined, as expected, at the 30- and 45-min time points for all doses of insulin. No significant decrease in glucose levels was observed at 10 min for any of the doses of insulin (Fig. 2). Additionally, the highest levels of pAKT in the pituitary and ovary were observed at this time (Supplementary Fig. 1). Therefore, for all subsequent studies (except those specifically addressed) a 10-min time point was used to avoid the effects of hypoglycemia on signaling pathway activity.

Figure 2.

Time course of response to different doses of insulin in overnight-fasted lean female mice. Glucose was measured at 0, 10, 30, and 45 min. No significant decline in glucose levels was observed at any dose at 10 min. (A high-quality color representation of this figure is available in the online issue.)

Pituitary and Ovary Are Less Insulin Sensitive Than Classic Energy Storage Tissues

For determination of whether reproductive tissues (hypothalamus, pituitary, and ovary) are comparable in insulin sensitivity with classical energy storage tissues such as liver, muscle, and fat, insulin (0.5, 1, or 1.5 units/kg body wt) was injected intraperitoneally into fasted lean WT female mice. At 10 min, tissues were collected, and pAKT, pERK, and total AKT levels were quantified by Luminex assays. Results are expressed as fold change compared with data from saline-injected littermates. Liver and muscle showed significantly increased pAKT levels at 0.5 units/kg body wt insulin (Fig. 3A and C), demonstrating that intraperitoneal insulin administration achieves circulating insulin levels at 10 min adequate for inducing signaling in these tissues. However, the pituitary and ovary first demonstrated AKT phosphorylation at the higher insulin dose of 1.5 units/kg body wt (Fig. 3E and G). No change in pAKT levels was observed in the hypothalamus at any of the doses (Fig. 3I). There was no significant increase in pERK in pituitary, ovary, or hypothalamus in response to insulin (Fig. 3F, H, and J). Muscle and liver exhibited increased pERK levels at 1 and 1.5 units/kg body wt insulin but not 0.5 units/kg body wt insulin (Fig. 3B and D). Because no changes in ERK signaling were observed in the reproductive tissues at these doses, subsequent analyses focused on changes in the phosphatidylinositol (PI) 3-kinase signaling pathway. Meanwhile, total AKT levels were also measured, with no observed difference between insulin- and vehicle-injected lean mice (Fig. 3K, L, and M).

Figure 3.

Fasted lean female mice were injected with saline or 0.5, 1, or 1.5 units/kg body wt insulin, and total AKT, pAKT, and pERK1/2 levels were measured in tissue homogenates using the Luminex analyzer. pAKT and pERK values are displayed relative to those of saline-injected mice and are corrected for total AKT. Total AKT values are expressed as relative light units. Different letters indicate significant differences between groups (P < 0.05). A and B: Liver. C and D: Muscle. E and F: Pituitary. G and H: Ovary. I and J: Hypothalamus. K: 0.5 units–total AKT. L: 1 unit–total AKT. M: 1.5 units–total AKT. NS, nonsignificant. pit, pituitary. hypo, hypothalamus.

In addition to Luminex assays of insulin signaling, we conducted Western blot analysis to explore insulin-signaling pathways in reproductive tissues and the energy storage tissues (Fig. 4A). We found similar tissue-specific phosphorylation patterns in response to insulin using Western blot analysis. We analyzed insulin signaling in the DIO mice by Western blot, and both the pituitary and ovary responded to insulin with increased phosphorylation of AKT. Furthermore, higher basal levels of pAKT were observed in the pituitary and ovary of DIO female mice compared with those in lean WT mice (Fig. 4B). However, insulin resistance was clearly observed in liver in DIO mice with dramatic reduction in the levels of insulin-stimulated pAKT compared with those seen in lean mice (Fig. 4B).

Figure 4.

Western blot identifies differences in insulin-signaling pathways between reproductive tissues and the energy storage tissues. A: Female lean mice were fasted overnight and injected with insulin (1.5 units/kg body wt). B: pAKT levels of lean and DIO female mice were compared before or after insulin stimulation. (A high-quality color representation of this figure is available in the online issue.)

Pituitary and Ovary Express Higher Basal Levels of pAKT and Maintain Insulin Sensitivity in WT DIO Mice

Although pituitary and ovary were less sensitive to insulin than muscle and liver, we observed that they maintain insulin sensitivity in obese mice in which insulin resistance was demonstrated in the energy storage tissues. To examine the differences between the tissues in pAKT signaling with higher throughput, accuracy, and sensitivity, we used four to eight animals per tissue per treatment and applied each individual protein into 96-well microplates to perform Luminex signaling assays. Since AKT phosphorylation in the reproductive tissues only increased at higher insulin doses in the lean mice, 3 units/kg body wt insulin were also included in our study. Pituitary-specific insulin receptor conditional KO mice were examined as a control to verify that the pAKT signaling is mediated through the insulin receptor. After 12 weeks of HFD, WT DIO mice showed a blunted response to insulin in liver and muscle compared with that in the lean WT mice (Fig. 5). Basal levels of pAKT in the pituitary and ovary were also significantly elevated in DIO mice relative to lean mice, and insulin treatment increased levels of pAKT to equal magnitudes in both lean and DIO mice (Fig. 5A and B). The increased basal level of pAKT and the response to insulin in pituitary were confirmed to be mediated by the insulin receptor by using PITIRKO-DIO mice that had no significant increase of basal or insulin-stimulated pAKT levels in the pituitary relative to lean mice. Since the insulin receptor is intact in the liver, muscle, and ovary, these tissues respond to insulin nearly identically to WT DIO mice (Fig. 5B, D, and E). The hypothalamus did not respond to insulin at even the 3 units/kg body wt dose (Fig. 5C).

Figure 5.

Luminex analysis of pAKT signaling after overnight-fasted mice were injected with insulin. WT lean, WT DIO, and PITIRKO-DIO mice were compared with regard to reproductive and energy storage tissues. n = 4–8/group. Bars with different letters are significantly different (P < 0.05). A: Pituitary. B: Ovary. C: Hypothalamus. D: Liver. E: Muscle.

Pituitary and Ovary Have Tissue-specific Differences in Insulin Signaling Through the IRS Proteins

IRS1 and IRS2 are scaffolding proteins that transduce signals from the insulin receptor to the PI 3-kinase and mitogen-activated protein kinase (MAPK) signaling pathways. They are also proposed as loci of protein modifications that produce insulin resistance.[8] pTyr is a marker of insulin sensitivity,[9] and pTyr-IRS1 levels were measured. We observed that pituitary pTyr-IRS1 levels do not change in response to insulin treatment in either lean or DIO mice (Fig. 6A). As for pAKT, the basal levels of pTyr-IRS1 in the ovary are elevated in DIO mice relative to those in lean mice (Fig. 6B). Additionally, insulin stimulated an increase in pTyr-IRS1 in the ovary in both lean and DIO mice in a dose-dependent manner. Liver and muscle exhibited insulin resistance at the level of IRS1 in DIO mice compared with lean mice (Fig. 6C and D). The insulin receptors remained intact in the ovary, liver, and muscle in the PITIRKO mice; therefore, insulin-induced IRS1 signaling in these tissues closely resembles that seen in WT mice.

Figure 6.

Analysis of pTyr-IRS1 and pTyr-IRS2 signaling and IRS1 expression levels. AH: pTyr-IRS1 (AD) and pTyr-IRS2 (EH) values of insulin-injected fasted mice are displayed relative to those of saline-injected mice. WT lean, WT DIO, and PITIRKO-DIO mice were compared with regard to reproductive and energy storage tissues. n = 4–8/group. A and E: Pituitary. B and F: Ovary. C and G: Liver. D and H: Muscle. I: irs1 mRNA level was measured in pituitary and ovary by real-time PCR in fed mice. J: IRS1 protein level shown by Western blot. K: IRS1 basal protein level was measured by Luminex assay in fed mice. Bars with different letters are significantly different (P < 0.05). (A high-quality color representation of this figure is available in the online issue.)

Both IRS1 and IRS2 Contribute to Insulin Signaling in Ovary

Since IRS1 does not mediate insulin signaling in the pituitary, we also measured pTyr-IRS2 levels. Both the pituitary and the ovary showed higher basal levels of pTyr-IRS2 in DIO mice compared with lean controls (Fig. 6E and F). Furthermore, pTyr-IRS2 levels were increased in both the ovary and pituitary in response to insulin in both lean and WT DIO mice. In contrast, while pTyr-IRS2 levels were increased in liver and muscle in response to insulin in lean controls, there was no increase observed in DIO mice (Fig. 6G and H). Both the basal increase in pTyr-IRS2 levels in DIO mice and the insulin-stimulated increase in pTyr-IRS2 levels were absent in the pituitaries of PITIRKO mice, confirming that both are mediated by the insulin receptor. As expected, there was no difference in the responses of WT DIO and PITIRKO-DIO mice in liver, muscle, or ovary. Since we did not observe any change in pTyr-IRS1 in pituitary but did in ovary, we examined the mRNA and protein level of IRS1 in pituitary and ovary and found that both were significantly lower in the pituitary (Fig. 6IK).

Pituitary and Ovary Have No Obesity-related Changes in Macrophage Infiltration or pJNK

Others have shown that HFD increased macrophage infiltration in energy storage tissues,[10,11] which can induce pJNK activation of liver and muscle in DIO mice.[7,12,13] pJNK activation is a major contributor to insulin resistance.[7] Since basal pAKT levels of pituitary and ovary were elevated in DIO versus lean mice, we chose to investigate whether macrophage infiltration and JNK phosphorylation were different between reproductive tissues and the energy storage tissues. Using immunofluorescence and Western blot analysis, we observed that the pituitary and ovary of DIO mice had levels of macrophage infiltration and pJNK similar to those of their lean littermates (Fig. 7A and B). In contrast, adipose tissue exhibited dramatically increased staining of the macrophage-specific protein F4/80 (Fig. 7A) in the DIO mice. Another macrophage-specific protein, Mac2, showed a distribution similar to that of F4/80 (data not shown). Liver and adipose tissue expressed strong staining for pJNK in the DIO mice relative to the lean controls (Fig. 7B). No change in total JNK was observed in examined tissues with HFD conditions (Fig. 7B).

Figure 7.

HFD-induced inflammation in different tissues. A: Macrophage infiltration was detected by immunohistochemistry using antibody F4/80. B: pJNK was examined by Western blot in different tissues. (A high-quality digital representation of this figure is available in the online issue.)

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