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Am J Physiol Regul Integr Comp Physiol 292: R2225-R2233, 2007. First published March 1, 2007; doi:10.1152/ajpregu.00902.2006
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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Postpartum glycemic homeostasis in early lactating rats is accompanied by transient and specific increase of soleus insulin response through IRS2/AKT pathway

Gabriel F. Anhê, Sandro M. Hirabara, Tatiana C. Turrer, Luciana C. Caperuto, Fernando F. Anhê, Luciene M. Ribeiro, Anderson C. Marçal, Carla R. O. Carvalho, Rui Curi, Ubiratan F. Machado, and Silvana Bordin

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil

Submitted 27 December 2006 ; accepted in final form 28 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It is known that at the moment of delivery immediate lost of conceptus (main site of glucose disposal in late pregnancy) is not able to disturb glucose homeostasis in early lactating mothers. However, the mechanism by which this adaptation takes place in early lactation is still unknown. Most studies concerning insulin sensitivity in lactating rats were carried out at 11–13 days postpartum and did not describe functional changes in insulin response in early lactation. Here we show that lactation hypersensitivity to insulin is observed as early as 3 days after delivery (L3). We show that the oxidative soleus muscle displays a transient increased maximal insulin-induced glucose uptake and CO2 production, which is temporally limited to L3. Response of soleus muscle was accompanied by an increase in glucose transporter 4 (GLUT4) content at L3. This adaptive response was not detected in the glycolytic plantaris muscle, which displayed lower content of GLUT4. We also found that soleus muscle from early lactating rats have higher insulin receptor expression and tyrosine phosphorylation. Downstream steps of insulin signaling pathway; e.g., insulin receptor substrate 2 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase were also upregulated in soleus muscle. In parallel, protein tyrosine phosphatase 1B expression, a negative regulator of insulin signal, was reduced. Importantly, all of these molecular alterations were time limited to L3 and were not observed in plantaris muscle. These results suggest that improved insulin action in oxidative, but not in glycolytic muscle might contribute to achievement of glucose homeostasis postpartum.

soleus muscle; insulin signaling; insulin receptor substrate 2; murine thymoma viral oucogene

AT LATE PREGNANCY, THE GLUCOSE utilization rate of the conceptus represents 23% of maternal glucose utilization rate in the basal state and, under insulin stimulation, placental glucose utilization displays an additional increase of 30% (31). Additionally, this representative flux of glucose to the conceptus is also guaranteed by insulin resistance in adipose tissue (18) and skeletal muscles, particularly in those predominantly composed by glycolytic fibers (e.g., extensor digitorum longus and epitrochlearis) (31). In contrast, muscles that are predominantly oxidative (soleus) have a normal insulin response at late pregnancy (30).

Just after the delivery, glycemic homeostasis is maintained even without the presence of conceptus. Lower postabsorptive blood glucose concentrations and increased metabolic clearance rate of glucose are observed as early as 3 days of lactation (8). However, the mechanism underlying the adaptation of the maternal organism during the switch from an insulin resistance state (pregnancy) to a hypersensitivity (lactation) period, after the lost of the main target of glucose disposal, is not completely clarified.

Our question is raised mainly because most of the studies concerning insulin sensitivity in lactating rats were carried out at 11–13 days postpartum. This period is characterized as the peak of lactation in rats (9–12), and the molecular changes that occur in insulin response during early lactation remain to be investigated. At the peak of lactation, an overall increase in insulin sensitivity is observed, and the mammary gland and liver display increased insulin sensitivity (9), while glucose uptake is reduced in white adipose tissue and the glycolytic muscle epitrochlearis (11).

Insulin stimulates glucose uptake in muscle and adipose cells by a well-characterized intracellular mechanism, which culminates with the translocation of glucose transporter 4 (GLUT4) vesicles to the cell surface. The serial events begin with the binding to and the activation of its cell surface transmembrane receptor, transmitting a signal that activates the tyrosine kinase domain of the beta-subunit within the insulin receptor (IR). Once activated, the insulin receptor phosphorylates proteins that belong to the IR substrate (IRS) family 1–4. Tyrosine-phosphorylated IRS proteins became able to recognize and bind to the regulatory subunit of the phosphatidylinositol 3-kinase (PI3-kinase). Activation of PI3-kinase targets downstream activates AKT, an essential step for insulin-stimulated GLUT4 translocation. In addition to tyrosine phosphorylation, the insulin receptor and IRS proteins are also targeted by several protein tyrosine phosphatases (PTPases) that can downregulate insulin signal transmission. Two relevant PTPases that have been implicated in the negative regulation of the insulin signaling are PTP1B and leukocyte antigen-related (LAR) (4, 36) proteins.

Attempting to clarify the alterations in insulin action that occur to maintain glucose homeostasis in early lactation, the present work analyses the glucose metabolism and the key steps of insulin signaling during the entire period of lactation in soleus and plantaris skeletal muscles. Soleus was chosen as representative of the oxidative muscles (~87% of oxidative fibers) and plantaris was chosen as representative of the glycolytic muscles (~91% of glycolytic fibers) (3).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. The general chemical reagents were obtained from Synth (Diadema, São Paulo, Brazil). The apparatus for SDS-PAGE and electrotransfer, and the nitrocellulose membranes were from Bio-Rad (Hercules, CA). Tris, PMSF, aprotinin, dithiothreitol, luminol. and p-coumaric acid were from Sigma-Aldrich (St. Louis, MO). Sodium thiopental was purchased from Cristália (Itapira, São Paulo, Brazil) and regular insulin was from Biobrás (M. Claros, Minas Gerais, Brazil). [D-U-14C]Glucose, and 2-deoxy-[D-2,6-3H]-glucose were from New England Nuclear Life Sciences (Boston, MA). Protein A Sepharose 6 MB and the horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies were from Amersham-Pharmacia Biotech (Buckinghamshire, UK). Anti-IR, anti-IRS1, anti-IRS2, anti-phospho-Tyr and anti-PTP1B antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PI3-kinase was from Upstate Biotechnology (Lake Place, NY). Phospho-AKT (Ser473) antibody was from New England BioLabs (Beverly, MA). X-ray-sensitive films and chemical developer were from IBF (Rio de Janeiro, Brazil). GoTaq DNA polymerase and ImProm-II Reverse Transcriptase were from Promega (Madison, WI). Random primers, dNTP set, agarose, and Trizol reagent were from Invitrogen (Carlsbad, CA). Primers for GLUT4 and RPL37a were synthesized by Integrated DNA Technologies (Coralville, IA).

Animals. Adult female Wistar rats at 8 wk of age (250–300 g) were used in all experiments in accordance with the guidelines of the Brazilian College for Animal Experimentation. They were kept at 24°C with lights on from 7:00 AM to 7:00 PM. Standard chow and water were provided ad libitum. After being habituated to the experimental conditions, groups of two female rats each were housed with one male for 5 days. The presence of spermatozoa in the vaginal lavage indicated day 0 of gestation, and then the pregnant rats were isolated in a separate cage. On the day of the delivery, the number of pups was adjusted to 8 per lactating mother. After an overnight fast, the rats were anaesthetized with sodium thiopental (5 mg/100 g body wt ip) and used for experimental procedures. Lactating rats were killed at 3 (L3), 8 (L8), 13 (L13) and 21 (L21) days postpartum. At 21 days after delivery, the pups were removed, and 7 days after the end of lactation (PL7) the rats were also used for intraperitoneal insulin tolerance test (ITT) experiments. Virgin age-matched rats were used as control group.

Intraperitoneal ITT. Insulin (2 IU/kg) was administered by intraperitoneal injection, and blood samples were collected from the tail at 0, 5, 10, 15, 20, 25, and 30 min for measurement of serum glucose. The constant rate for glucose disappearance (Kitt) was calculated using the formula 0.693/t1/2. The glucose t1/2 was calculated from the slope of the least-squares analysis of the plasma glucose concentrations during the linear decay phase (5).

Glucose metabolism in isolated soleus muscle from lactating rats. Soleus muscles were isolated and incubated as previously described (23). Rats were killed by cervical dislocation, and soleus muscles were rapidly and carefully isolated, split longitudinally in portions weighing 25 to 35 mg and preincubated for 30 min at 37° C in Krebs-Ringer bicarbonate buffer pregassed for 30 min with 95% O2-5% CO2, containing 5.6 mM glucose, pH 7.4, with agitation at 120 rpm. After this period, the muscles were transferred to other vials containing the same buffer, but 0.3 µCi/ml [D-U-14C]-glucose, 0.2 µCi/ml 2-deoxy-[D-2,6-3H]-glucose and glucose 5.6 mM were added. Incubation was then performed for 1 h under similar conditions, in the absence or presence of 0.1 or 10 mU/ml insulin. Phenylethylamine (0.3 ml solution 1:1 in methanol) was added into a separate compartment to 14CO2 adsorption for the analysis of [D-14C]-glucose oxidation. For determination of the extracellular space, some muscles were incubated in the presence of 0.1 µCi/ml [L-114C]-glucose. After the incubation period, the muscles were briefly washed in saline at 4°C and frozen in liquid N2. [14C]-glycogen synthesis (as estimated by [D-14C]-glucose incorporation into glycogen) was determined as described by Espinal et al. (17). 2-Deoxy-[D-2,6-3H]-glucose uptake was measured as previously described (24).

Immunoprecipitation and immunoblotting. After the loss of pedal reflex as an effect of anesthesia, one of the soleus and the plantaris hind limb muscles were removed and kept in dry ice. The abdominal cavity was opened, and the portal vein was exposed and injected with 0.5 ml of a 10–5 M insulin solution. After 90 s, the remaining soleus and plantaris muscles were removed. The muscles were homogenized immediately in ~10 volume of extraction solubilization buffer in ice-cold bath by using a Polytron-Aggregate (Luzern, Switzerland) operated at maximum speed for 10 s. The extracts were centrifuged at 12,000 g at 4°C for 30 min to remove insoluble material. The supernatant was either used for immunoprecipitation with anti-IRS1 or anti-IRS2 and protein A-Sepharose 6 MB (for soleus samples) or treated as whole extract for immunoblotting as previously described (2). Similar-sized aliquots from whole extract tissue (75 µg of protein) and the immunoprecipitated beads of Sepharose were treated with Laemmli sample buffer, subjected to SDS-PAGE and electrotransferred to nitrocellulose membranes in a semi-dry system for 60 min at 15 V (Bio-Rad). To reduce nonspecific protein binding to the nitrocellulose, the filter was preincubated overnight at 4°C in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl and 0.02% Tween 20). The nitrocellulose membranes were incubated for 4 h at 22°C with specific antibodies, as described in the figure legends, followed by 1-h incubation with a HRP-conjugated secondary antibody (1:10,000 in 1% nonfat dry milk). Chemiluminescence reagents were used to visualize the autoradiogram, which was later exposed to photographic film. Quantitative analysis of blots was done using Scion Image software (Scion, Frederick, MD).

Membrane preparation for GLUT4 detection. Total membrane extracts from soleus and plantaris skeletal muscles were performed as previously described (35). Briefly, the muscles from control and L3 rats were homogenized by using a Polytron (Brinkmann Instruments, Westbury, NY) for 30 s at 4°C in 10 mM Tris·HCl, 1 mM EDTA, and 250 mM sucrose, pH 7.4, buffer. Homogenates were centrifuged at 700 g at 4°C for 10 min. The supernatants were transferred to a new tube, and the pellets were submitted to the same procedure one more time to assure a more efficient protein recovery. The two supernatants were put together in a new tube and submitted to a 90,000 g centrifugation at 4°C for 60 min. The final pellet was resuspended as a total membrane fraction. The total protein concentration of membrane samples was assayed by the Bradford method and used for GLUT4 protein analysis by Western blotting as described in Immunoprecipitation and immunoblotting.

RNA extraction and RT-PCR analysis. Total RNA was extracted from ~100 mg of soleus muscle using Trizol reagent. Conventional RT-PCR analysis was performed as previously described (6). The amplification products were run on a 1.2% agarose gel containing ethidium bromide, and the band intensities were determined by digital scanning followed by quantification by using the Scion Image analysis software. The result was expressed as a ratio of the target gene to the housekeeping RPL37a. The primer sequences used for RT-PCR analysis with their respective melting point and lengths were as follows: GLUT4 sense, 5'-GCTGTGCCATCTTGATGACGG-3' and antisense, 5'-TGAAGAAGCCAAGCAGGAGGAC-3', 58.6°C, 300 bp; RPL37a sense, 5'-CAAGAAGGTCGGGATCGTCG-3' and antisense, 5'-ACCAGGCAAGTCTCAGGAGGTG-3', 57°C, 289 bp.

Statistical analysis. All values were reported as means ± SE. The results were analyzed by unpaired Student's t-test or one-way ANOVA, followed by Bonferroni post hoc testing when appropriate. P values <0.05 indicated a significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Increased insulin sensitivity in lactating rats was observed 3 days after delivery and returned to control levels 7 days after the end of lactation. To characterize the beginning and the temporal extension of the increased insulin sensitivity, we performed intraperitoneal ITT in lactating rats starting at the 3rd day of lactation. The glucose disappearance constant is increased at the 3rd day of lactation (~2.4-fold compared with control; P < 0.05). This hypersensitivity remained at similar levels until the 21st day (~1.8-fold in L8, ~2.6-fold in L13, and ~2.2-fold in L21 compared with control; P < 0.05). Seven days after the end of the lactation period (PL7), insulin sensitivity is similar to that observed in control rats (Fig. 1).


Figure 1
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  		Fig. 1. Increased insulin sensitivity in lactating rats starts 3 days after delivery and returns to control (CTL) levels 7 days after lactation. Virgin (CTL), lactating rats at 3 (L3), 8 (L8), 13 (L13), and 21 (L21) days postpartum and 7 days after the end of lactation (PL7) received insulin (2 UI/kg) by intraperitoneal injection and blood samples were collected from the tail at 0, 5, 10, 15, 20, 25, and 30 min for measurement of serum glucose. The constant rate for glucose disappearance (Kitt) was calculated using the formula 0.693/t1/2. The bars represent means ± SE of Kit obtained from 6 independent experiments. *P < 0.05 vs. CTL.

 
Increased maximal insulin-induced glucose uptake and CO2 production in isolated soleus muscle from early lactating rats. Insulin-induced glucose uptake (at 0.1 mU/ml) was similar to control in L3, L8, and L13 (Fig. 2A). The same was observed for basal glucose uptake in isolated soleus muscle. However, the maximal insulin-induced glucose uptake was higher in early lactation (L3) than in control (~1.4-fold; P < 0.05), and returned to values similar to control at L8 and L13.


Figure 2
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  		Fig. 2. Increased maximal insulin-induced glucose uptake and CO2 production in isolated soleus muscle from L3 rats. Virgin (CTL) and lactating rats at L3, L8, and L13 were killed by cervical dislocation and soleus muscles were rapidly and carefully isolated and processed for 2-deoxy-[D-2,6-3H]-glucose uptake (A), [14C]-glycogen synthesis (B), and 14CO2 production (C) as described in MATERIALS AND METHODS. Bars represent means ± SE obtained from 3 independent experiments where the white bars are the muscles incubated in absence of insulin (basal), grey bars are the soleus incubated in insulin 0.1 mU/ml, and black bars are the soleus incubated in insulin 10 mU/ml. *P < 0.05 vs. basal within the same group; #P < 0.05 vs. insulin 0.1 mU/ml within the same group; &P < 0.05 vs. CTL incubated with insulin 10 mU/ml.

 
The fate of glucose metabolism in soleus muscle was assessed by measurement of glycogen synthesis and CO2 production. Insulin-induced glycogen synthesis was increased to similar levels in control and lactating rats (Fig. 2B). Maximal insulin-induced CO2 production reached higher values in soleus muscles isolated from rats in early lactation (L3) than in control (~1.44-fold; P < 0.05, Fig. 2C). This temporal pattern of insulin-induced CO2 production in soleus from lactating rats is similar to that observed for insulin-induced glucose uptake.

GLUT4 expression is increased in soleus and decreased in plantaris muscle from L3 rats. To investigate the mechanism involved in the increased glucose uptake in early lactation we first determined the expression of GLUT4. The relative quantity of GLUT4 mRNA was increased in soleus 3 days after delivery (~1.23-fold; P < 0.05) and returned to control levels 8 days postpartum (Fig. 3A). GLUT4 protein content at L3 was increased (~1.85-fold; P < 0.05) in soleus muscle (Fig. 3B) and decreased (~0.40-fold; P < 0.05) in plantaris muscle (Fig. 3C).


Figure 3
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  		Fig. 3. Glucose transporter 4 (GLUT4) expression is increased in soleus and decreased in plantaris muscle from L3 rats. Virgin (CTL) and lactating rats at L3, L8, and L13 were killed by cervical dislocation, and soleus muscle were rapidly isolated and processed for RNA extraction and used for RT-PCR analysis of GLUT4 mRNA expression (A). Soleus (B) and plantaris (C) muscle from CTL and L3 rats were also processed for total membrane protein extraction and Western blot analysis detection of GLUT4. The bars represent the relative expression of GLUT4 as determined by optical densitometry and are presented as means ± SE. *P < 0.05 vs. CTL.

 
Increased IR expression and insulin-induced tyrosine phosphorylation in soleus muscle from early lactating rats (L3). The band migrating at 95 kDa corresponds to the beta-subunit of the IR in a protein extract resolved in SDS-PAGE (25). The ability of insulin to induce IR-beta subunit tyrosine phosphorylation was significantly higher in soleus muscle from L3 than in control rats (1.58-fold; P < 0.05, Fig. 4A). In contrast, IR tyrosine phosphorylation in plantaris muscle at L3 was similar to that of control rats (Fig. 4B). Not only the insulin-induced tyrosine phosphorylation but also the IR expression was increased 3 days after delivery (1.70-fold; P < 0.05) and returned to control levels 8 and 13 days postpartum (Fig. 4C). IR expression is not altered in plantaris muscle at L3 (Fig. 4D).


Figure 4
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  		Fig. 4. Increased insulin receptor (IR) expression and insulin-induced tyrosine phosphorylation in soleus muscle from L3 rats. Virgin (CTL) and lactating rats at L3, L8, and L13 were anesthetized and the soleus muscle was removed (basal = –, white bars). Plantaris muscle was removed from CTL and L3 rats. The abdominal cavity was opened, and the portal vein was injected with 0.5 ml of a 10–5 M insulin solution. After 90 s, the remaining soleus muscle was removed (black bars). The remaining plantaris was removed from CTL and L3-stimulated rats. Stimulated and unstimulated muscles were processed for protein extraction, resolved in 6.5% SDS-PAGE, and the membranes with soleus (A) and plantaris (B) samples were used for immunoblotting (IB) with anti-pTyr antibody. Separated gels containing exclusively the basal samples from soleus (C)- and plantaris (D)-originated membranes that were probed with anti-IR antibody. The bars represent the IR tyrosine phosphorylation and protein content determined by optical densitometry and are presented as means ± SE. *P < 0.05 vs. basal within the same group; &P < 0.05 vs. stimulated CTL; #P < 0.05 vs. CTL.

 
Unchanged IRS1 and IRS2 expression and insulin-induced pp185 tyrosine phosphorylation in soleus muscle from lactating rats. Upon activation, the insulin receptor catalyzes the tyrosine phosphorylation of several intracellular substrates, including the IRS1 and IRS2. IRS1 and IRS2 migrate at the region of the 165–185 kDa proteins (pp185). Insulin induced a similar increase in pp185 tyrosine phosphorylation in both soleus and plantaris muscle from control and lactating rats (P < 0.05 vs. basal, Figs. 5, A and B). Additionally, IRS1 and IRS2 protein levels are not altered during lactation in soleus muscle ( Figs. 5, C and E. respectively) and plantaris muscle (Figs. 5, D and F, respectively).


Figure 5
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  		Fig. 5. Unchanged IR substrate (IRS)1 and IRS2 expression and insulin-induced pp185 tyrosine phosphorylation in soleus from L3 rats. Virgin (CTL) and lactating rats at L3, L8, and L13 were anesthetized, and the soleus muscle was removed (basal = –, white bars). Plantaris muscle was removed from CTL and L3 rats. The abdominal cavity was opened, and the portal vein was injected with 0.5 ml of a 10–5 M insulin solution. After 90 s, the remaining soleus muscle was removed (black bars). The remaining plantaris was removed from CTL and L3-stimulated rats. Stimulated and unstimulated muscles were processed for protein extraction, resolved in 6.5% SDS-PAGE, and the membranes with soleus (A) and plantaris (B) samples were probed with anti-pTyr antibody. Separated gels containing exclusively the basal soleus and plantaris samples originated membranes that were probed with anti-IRS1 (C and D, respectively) and anti-IRS2 (E and F, respectively) antibodies. The bars represent the pp185 tyrosine phosphorylation and IRS1 and IRS2 protein content determined by optical densitometry and are presented as means ± SE. *P < 0.05 vs. basal within the same group.

 
Increased IRS2 but not IRS1 tyrosine phosphorylation and association with PI3-kinase in soleus muscle from early lactating rats (L3). Because the main IR targets for tyrosine phosphorylation are IRS1 and IRS2 and we did not find any change in pp185 tyrosine phosphorylation and IRS1 and IRS2 content in L3, we then used the immunoprecipitation approach to investigate whether the changes were specific for IRS1 or IRS2 phosphorylation. The present results show that insulin-induced IRS1 tyrosine phosphorylation is similar in control and L3 rats (Fig. 6A). However, insulin-induced IRS2 tyrosine phosphorylation is significantly higher in L3 compared with control rats (1.88-fold; P < 0.05; Fig. 6B). As demonstrated herein, the same pattern of results was obtained for the association of these substrates with the regulatory subunit of PI3-kinase. Insulin-induced IRS1/PI3-kinase association is similar for control and L3 rats (Fig. 6C) but stimulated formation of the IRS2/PI3-kinase complex was increased in L3 rats (2.00-fold; P < 0.05; Fig. 6D).


Figure 6
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  		Fig. 6. Increased IRS2 but not IRS1 tyrosine phosphorylation and association with PI3-kinase (PI3K) in soleus muscle from L3 rats. Virgin (CTL) and lactating rats at L3 were anesthetized and the soleus muscle was removed (basal = –, white bars). The abdominal cavity was opened and the portal vein was injected with 0.5 ml of a 10–5 M insulin solution. After 90 s, the remaining soleus muscle was removed (black bars). Stimulated and unstimulated muscles were processed for immunoprecipitation (IP) with anti-IRS1 and anti-IRS2 antibodies. The precipitated proteins were resolved in 6.5% SDS-PAGE and transferred to different nitrocellulose membranes. Both IRS1- and IRS2-containing membranes were probed with anti-pTyr (A and B, respectively) and anti-PI3-kinase (C and D, respectively). The bars represent the IRS1 and IRS2 tyrosine phosphorylation and association with PI3-kinase determined by optical densitometry and are presented as means ± SE. *P < 0.05 vs. basal within the same group; &P < 0.05 vs. stimulated CTL.

 
Increased insulin-induced AKT serine phosphorylation in soleus muscle from early lactating rats (L3). Although PI3-kinase activity is clearly necessary for insulin-stimulated glucose uptake, additional downstream signals are also required for the stimulation of GLUT4 translocation. One of the PI3-kinase targets is the serine/threonine kinase known as PKB or AKT (36). Insulin induced serine phosphorylation of AKT in control and lactating rats (P < 0.05 vs. basal levels, Fig. 7). However, insulin-induced AKT serine phosphorylation reached higher values in L3 (1.49-fold vs. control; P < 0.05) and returned to levels similar to that of control in L8 and L13.


Figure 7
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  		Fig. 7. Increased insulin-induced AKT serine phosphorylation (pAKT-Ser) in soleus muscle from L3 rats. Virgin (CTL) and lactating rats at L3, L8, and L13 were anesthetized, and the soleus muscle was removed (basal = –, white bars). The abdominal cavity was opened, and the portal vein was injected with 0.5 ml of a 10–5 M insulin solution. After 90 s, the remaining soleus muscle was removed (black bars). Stimulated and unstimulated muscles were processed for protein extraction, resolved in 6.5% SDS-PAGE, and the membranes were probed with anti-pAKT antibody. The bars represent the serine AKT phosphorylation determined by optical densitometry and are presented as means ± SE. *P < 0.05 vs. basal within the same group; &P < 0.05 vs. stimulated CTL.

 
Decreased PTP1B expression in soleus muscle from early lactating rats (L3). Two PTPases have been implicated in the negative regulation of the insulin signaling, namely PTP1B and LAR (36). We have found a decrease in PTP1B expression in early lactating rats (L3), as showed in Fig. 8A (0.17-fold vs. control; P < 0.05). Additionally, PTP1B levels returned to the levels observed in control at L8 and L13 (Fig. 8). In contrast to soleus, plantaris muscle did not exhibit any change in PTP1B expression at L3 (Fig. 8B). We have also assessed the mRNA levels of LAR but it was not altered during lactation (data not shown).


Figure 8
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  		Fig. 8. Decreased protein tyrosine phosphatases 1B (PTP1B) expression in soleus muscle from L3 rats. Virgin (CTL) and lactating rats at L3, L8, and L13 were killed by cervical dislocation, and soleus muscles were rapidly isolated and processed for protein extraction. Plantaris muscle was removed from CTL and L3 rats. The samples were resolved in 8% SDS-PAGE and the membranes containing soleus (A) and plantaris (B) samples were probed with anti-PTP1B antibody. The bars represent the PTP1B protein content determined by optical densitometry and are presented as means ± SE. *P < 0.05 vs. CTL.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Stuebe et al. (40) recently demonstrated that the longer duration of breast feeding was associated with the reduced incidence of type 2 diabetes in women. Lactation may reduce risk of type 2 diabetes in young and middle-aged women by improving glucose homeostasis that was independent of the body weight. Considering that the latest projection from the International Diabetes Federation suggests that 190 million people worldwide currently have type 2 diabetes, a clear understanding of the mechanisms underlying the glucose homeostasis during lactation might help to develop new strategies to prevent what is considered, in terms of glucose intolerance, the largest epidemia in human history.

In 1986, Burnol et al. (9) demonstrated that the lactation period is characterized by increased insulin sensitivity. This data was achieved by the use of hyperinsulinemic euglycemic clamp applied to lactating rats at 11–13 day postpartum. A former study from this group, however, collected important data suggesting that improved response to insulin could take place in early lactation (3 days postpartum) (8). Therefore, we first focused on the search for temporal limits of the increased insulin sensitivity. By using an insulin tolerance test, we determined that lactation is an entirely insulin-hypersensitive period. Thus, insulin resistance observed in late pregnant rats is rapidly switched to hypersensitivity at L3. This feature is no longer observed 1 wk after the end of lactation.

Skeletal muscles represent the most important tissues responsible for glucose clearance after an intravenous glucose or insulin administration. Skeletal muscles that are predominantly composed by glycolytic fibers like epitrochlearis and extensor digitorum longus display reduced insulin-dependent glucose uptake during late pregnancy (31, 38), and, in the case of the epitrochlearis, insulin resistance is also detectable at 11–13 day postpartum (11). Similarly, the adipose tissue, another major territory for glucose disposal, is also insulin resistant in late pregnant and midlactating rats (11, 18, 31). In contrast, the soleus skeletal muscle, which is predominantly composed by oxidative fibers, does not present resistance to insulin action during late pregnancy (30). Therefore, soleus muscle was chosen as a candidate to participate in the increased insulin sensitivity in early lactation and therefore contributes to glycemic control in the postpartum period. In fact, our results showed that maximal insulin-induced glucose uptake and CO2 production in isolated soleus is increased in early lactating rats.

Although glycolytic fibers account for ~75% of hindlimb skeletal muscle mass, the blood flow through predominantly oxidative muscles is 3 to 4 times higher than that of predominantly glycolytic muscles (3). Therefore, the relative small increase in the insulin-induced glucose uptake obtained in 1-h-stimulated soleus muscles from L3 rats may become markedly significant in a chronic situation. As a consequence, the phenomenon observed in oxidative muscles might account for an important part in the twofold increase in whole body insulin sensitivity during early lactation, as observed in vivo during the ITT test.

This feature of the oxidative skeletal muscle is noticed in a critical moment when the conceptus is no longer present and, therefore, a considerable amount of glucose from the bloodstream must be driven to an alternative territory. Thus, our results led us to postulate that the increased responsiveness to insulin of oxidative muscles (e.g., soleus muscle) might contribute to normal glucose homeostasis of the maternal organism in the postpartum. This phenomenon is temporally restricted to the early lactation, and, as the lactation proceeds, other tissues, such as the mammary gland, will certainly assume its role in the hypersensitivity to insulin and glucose disposal. Indeed, at 11–13 days of lactation on, the fraction of glucose taken up by the mammary gland to ensure milk synthesis represents a large percentage of the total glucose turnover rate in fed rats (42).

Oxidative soleus muscle, rather than the glycolytic epitrochlearis muscle, is notably more susceptible to the effect of insulin sensitizing agents in pathological situations of insulin resistance. This was demonstrated by upregulation of glucose uptake and postreceptor signaling steps selectively in soleus muscle from obese Zucker rats (14, 22). In accordance with these observations, the present study describes a physiological situation where the transition from an overall insulin resistance (pregnancy) to hypersensitivity condition (lactation) is marked by an increased insulin response of the oxidative soleus muscle. The reason for this specific response of oxidative muscles may be explained by the high expression and activity of the insulin signaling proteins (39) and GLUT4 (21) compared with the glycolytic fibers. In accordance with these studies, we found that GLUT4 protein content and mRNA levels are increased in the oxidative soleus muscle but, in the case of GLUT4 protein, are decreased in the glycolytic plantaris muscle.

Some of the molecular alterations in soleus muscle that we described herein are observed in skeletal muscle from other experimental situations that display increased insulin sensitivity. For instance, caloric restriction promotes an increase in IR activity (44), and exercise training upregulates GLUT4 expression (29) and insulin-induced AKT phosphorylation (33). It is important to note that, like GLUT4 expression, other key proteins of the insulin signaling pathway are upregulated in soleus muscle exclusively during early lactation (e.g., IR expression and tyrosine phosphorylation). Corroborating the idea that participation in increased insulin sensitivity during early lactation is restricted to oxidative muscles, no changes regarding expression of IR, GLUT4, IRS1, IRS2, and phosphorylation of AKT were detected in gastrocnemius muscles from early lactating rats (data not shown). Taken together, these results suggest that increased responsiveness to insulin at the postpartum is attributed exclusively to muscles that are predominantly composed by oxidative fibers.

A remarkable observation of the present study was that the improved insulin action in soleus muscle at L3 lies in the fact that IRS2, but not IRS1, displayed higher insulin-induced tyrosine phosphorylation and association with the regulatory subunit of PI3-kinase. This seems to be a unique feature of early lactation since the majority of the published studies in this field suggest that IRS1, rather than IRS2, is the major substrate leading to stimulation of glucose transport in skeletal muscle (41). The latest studies concerning this issue have demonstrated, by the use of interference RNA, that IRS1 knockdown impairs insulin-induced glucose uptake in skeletal muscle cell lineages, which is not observed for IRS2 knockdown (7, 27).

The results presented herein, however, are in accordance with other reports that have pointed out for the participation of IRS2 in skeletal muscle insulin response. In humans and rodents, specific upregulation of insulin-induced IRS2 association with PI3-kinase accounts, at least in part, for the sensitizing effect of a single bout of exercise (25, 26). Furthermore, a classic study of Withers et al. (43) demonstrated that IRS2-deficient mice show progressive deterioration of glucose homeostasis due to insulin resistance in the liver and skeletal muscle and a lack of beta-cell compensation for this insulin resistance.

Together with the positive regulation of the IRS2/PI3-kinase/AKT pathway, we have found a reduction in PTP1B protein content in soleus muscle from L3 rats. This result is in accordance with previous studies that show elevated expression of this phosphatase in conditions of insulin resistance (1, 20), and its abrogation in conditions of enhanced insulin sensitivity (15). Again, PTP1B reduction is temporally limited to the early stage of lactation and is not observed in plantaris muscle.

Insulin resistance during pregnancy is attributed to a complex change in plasma hormonal levels. In humans, there is strong evidence that high values of placental growth hormone (GH) exert a major role in insulin resistance during late gestation (19). In rats, a marked increase in circulating levels of GH during late pregnancy has already been observed (13, 16); however, the pituitary and/or placental origin of this hormone has not been clearly established. In addition to GH, other hormones have been suggested as participating in the genesis of insulin resistance in pregnant rats. Progesterone has the prominent ability to decrease insulin-stimulated glucose metabolism in isolated rat muscles (32). On the other hand, the participation of placental lactogens is controversial. Leturque and colleagues have demonstrated that placental lactogens do not interfere in insulin-induced glucose utilization by isolated muscle (32), but a former study from Ryan and Enns (37) suggested that placental lactogens and prolactin impair insulin action in adipose cells by affecting postreceptor binding events. Additionally, it is unlikely that placental lactogens and prolactin participate in the skeletal muscle insulin resistance from pregnant rats, because this tissue contains a reduced number of mRNA copies that encode for the long functional form of the prolactin receptor (34). Finally, the plasma levels of prolactin remain elevated on the 3rd day after the delivery (28) when an increased soleus muscle response to insulin is observed.

It was not the purpose of the present study to determine which factors during the transition from pregnancy to lactation generate the phenomenon described herein; however, we can speculate on potential candidates. After the delivery, it is notable that rats exhibit a rapid fall in progesterone and GH circulating levels, reaching the control levels in 3 days (16, 28), which might play an important role for the increased insulin response of the soleus muscle in early lactation.

In summary, we demonstrated herein that the increased insulin response in soleus muscle at 3rd day postpartum participates in the overall insulin sensitivity to the hormone observed in early lactation. This phenomenon is an exclusive feature of oxidative skeletal muscles and is associated with increased phosphorylation of IR, IRS2, AKT, and PI3-kinase association with IRS2, GLUT4, and IR expression, and decreased content of PTP1B. These results point to a new mechanism by which early lactating rats might maintain glycemic homeostasis postpartum, a critical moment when the conceptus, the most important territory responsible for the maternal glucose disposal, is no longer present.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Brazilian foundations Fundaçáo de Amparo à Pesquisa do Estado de São Paulo, Coordenaçáo de Aperfeiçoamento de Pessoal de Nivel Superior, and Conselho Nacional de Desenvoluimento Científico e Tecnológico.


   ACKNOWLEDGMENTS
 
The authors thank M. A. S. Lima for technical assistance and M. T. Nunes for helpful discussion.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Bordin, Prof. Lineu Prestes Av. 1524. São Paulo, SP, Brazil 05508-900 (e-mail: sbordin{at}icb.usp.br)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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